Advances in Immunology Volume 11

ADVANCES IN Immunology VOLUME 1 1 CONTRIBUTORS TO THIS VOLUME BARUJBENACERRAF IVORN. BROWN ROBERTR. DOURMASHKIN N. ...

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ADVANCES IN

Immunology VOLUME 1 1

CONTRIBUTORS TO THIS VOLUME BARUJBENACERRAF IVORN. BROWN ROBERTR. DOURMASHKIN

N. MICHAELGREEN G ~ R A HOLM N JOHN

H. HUMPHREY

H. S. LAWRENCE HUGH0. MCDEVITT PETERPERLMANN

ADVANCES IN

Immunology EDITED BY

F. J. DIXON, JR.

H E N R Y G. K U N K E L

Division of Experirnenfol Pathology Scripps Clinic ond Research Foundofion l a lolla, California

The Rockefeller University New York, New York

V O L U M E

1 1

1969

ACADEMIC PRESS

New York and London

COPYRICHTO 1969,

BY

ACADEMIC PRESS,INC.

ALL RIGHTS RESERVED

NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,

13Y PHOTOSTAT, MICROFILM, RETRlGVAL S l S T E M , OR ANY OTHER MEANS, WITHOUT WRITTEN PERhlISSlON FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 121 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London WlX6BA

LIBRARYOF CONGRESS CATALOG C A ~ NUMBER: D 61-17057

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

BARUJ BENACERRAF, Division of Immunology, Department of Medicine, Stanford University School of Medicine, Stanford, California ( 31) IVORN. BROWN, Division of Parasitology, National Institute for Medical Research, London, England (267) ROBERTR. DOURMASHKIN, National Institute for Medical Research and Imperial Cancer Research Fund Laboratories, Mill Hill, London, England ( 7 5 )

N. MICHAELGREEN,National Institute for Medical Research, Mill Hill, London, England (1) GORANHOLM,Department of Immunology, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden ( 117)

H. HUMPHREY, National Institute for Medical Research and Imperial Cancer Research Fund Laboratories, Mill Hill, London, England (75)

JOHN

H. S. LAWRENCE, Infectious Disease and Immunology Division, Department of Medicine, New York University School of Medicine, New York, New York (195)

HUGH0. MCDEVITT, * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesdu, Maryland ( 31) PETERPERLMANN, Department of Immunology, The Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden ( 117) Present address: Division of Immunology, School of Medicine, Stanford University Medical Center, Stanford, California.

V

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PREFACE The diversity of fields in which exponents of immunology may be found continues to surprise even its most enthusiastic adherents. It is not just the exquisite sensitivity and precision of immunological techniques that have served as the impetus for this diffusion. More significant appears to be the recognition of the broad biological impact of the relevant events that precede and follow an immune stimulus. Volume 11 exhibits such diversity unusually well, with physicist to parasitologist represented. The volume also includes three chapters by English scientists illustrating once again the continuing strong contribution of this group to immunology. The first chapter deals with the exciting contributions of electron microscopy to the analysis of antibody moIecuIes. Dr. Green initiated the use of small bivalent haptenes to link antibody inolecules which has proven so successful in their characterization. The concept of a threearmed molecule with n flexible hinge region is well documented for yG globulin through the surprisingly clear electron micrographs obtained by this procedure. The ameboid appearance of yM molecules is most esthetically satisfying. Dr. McDevitt and Dr. Renacerraf review the recent important findings concerning “immune response genes” in the second chapter. The fact that genetic factors are involved in the response to antigenic stimulus has Iong been known. However, the credit for establishing this on a firm scientific basis in terms of modern genetics must be given to the authors of this chapter. The use of synthetic polypeptide antigens played a major role in elucidating the multiple genes which are described. The intriguing question of at what level in the immune response these genes act remains to be determined. It appears clear that they do not represent the structural genes for the antibody molecule. The third chapter has been contributed by Dr. Humphrey and Dr. Dourniashkin and deals with that most important of all complement questions, the terminal phase of cellular injury. Their most elegant electron microscope pictures of the holes in the cell membrane produced by complement have intrigued all immunologists. Considerable progress in the understanding of the underlying mechanism involved has been gained although the final answer is not yet in. Is an enzyme attacking lipid Vi i

viii

PREFACE

moieties in the membrane primarily involved? Many unpublished studies of the authors relating to these questions are included in this fascinating review. The fourth chapter by Dr. Perlmann and Dr. Holm deals with the complex problem of different types of cytotoxic effects of lymphoid cells. These outstanding workers in the field have managed to present a cohesive picture of the various effects on target cells. The role of “nonspecific” factors is particularly wcll clarified. The interrelationships among contact lysis, release of pharmacologically active substances, and the terminal components of the complement system are given special consideration. There is little question that significant developments conccrning in vim events will stem from these in vitro findings. In another chapter Dr. H. S. Lawrence reviews the extensive and confusing literature on various factors involved in cellular immunity. Transfer factor, which h e first described, is placed in perspective with the various substances under active current investigation in the guinea pig. This is a very enlightening review of an area of immunology from which much will be heard in the future. The methodology has been partially worked out for obtaining transfer factor, as well as some of the other materials, in sufficient purity for chemical analysis, and further results in this area are awaited with great interest. The assay systems remain difficult but the shift to in vitro systems has been a major achievement. The last chapter by Dr. Ivor Brown deals with immunity in malaria, an old subject that has suddenly become of considerable current interest. New methods for the study of the relevant antibodies and a new appreciation for a role for cell-mediated immunity are responsible for this development. The very diverse contributions to this subject present unusual difficulties for a reviewer. However, a clear and interesting summary of the subject has emerged which should prove of considerable value as a reference for all immunologists. The complete cooperation of the publishers in all aspcbcts of the work involved in the production of Volume 11 is gratefully acknowledged.

H. G. KUNKEL F. J. DIXON

CONTENTS

. PREFACE . . . . . . CONTENTS OF PREVIOUS VOLUMES. LIST OF CONTFXBUrORS

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V

vii

xi

Electron Microscopy of the Immunoglobulins

N. MICHAELGREEN

I. Introduction . . . . . . . 11. Electron Microscopy at the Molecular Level 111. Electron Microscopy of IgG . . . . IV. Electron Microscopy of IgM . . . . V. Comments and Conclusions . . . . References . . . . . . .

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1 2 G 17 26 28

Genetic Control of Specific Immune Responses

HUGH0. MCDEVITTAND BARUJBENACEHRAF I. Introduction . . . . . . , . . . . . 11. Constitutional Differences in Individual Responses to Complex Multi. . . . . . . . . , determinant Antigens . 111. Analysis of the Mechanism of Gene Action . . . . . . IV. Genetic Differences in Imiriune Response to Defincd Protein Antigens . V. Genetic Differences in Immune Response.; to Synthetic Polypeptide Antigens . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . .

31 33 37 38 39 69 71

The lesions in Cell Membranes Caused by Complement JOHN

H. HUMPHREY AND ROBERT R. DOURMASHKIN

I. Introduction . . . . . . . . . . . . . 11. Description of Holes Produced by the Action of C‘ . . . . . 111. Holes Produced by C‘ in Substrates Other than Erythrocyte Membranes . IV. The Relationship of Holes t o Sites of Damage on the Cell Surface . V. Occurrence of Multiple Holes (Clusters) at Single Sites of Damage . VI. The Number of Antibody Molecules Required to Produce a Lesion VII. The Stage of C’ Action at Which Holes Are Formed . . . . VIII. The Nature of C’ Holes . . . . . . . . . . IX. Arti6ciaI Membrane ModeIs . . . . . . . . . . X. Biological Significance of the Terminal C’ Lesion . . . . . References . . . . . . . . . . . . . ix

75 77 85 88 92 95 98 101 108 110 113

X

CONTENTS

Cytotoxic Effects of Lymphoid Cells in Vifro

PETERPERLMANN AND GORANHoLhr

.

I Introduction . . . . . . . . I1. Methods . . . . . . . . . I11. Different in Vitro Models . . . . . 1V Some in Vivo Implications of the in Vitro Models V Summary . . . . . . . . References . . . . . . . .

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117 119 127 172 183 185

I. Introduction . . . . . . . . . . . . . I1. Definitions and General Principles . . . . . . . . I11. Transfer of Delayed Hypersensitivity with Viable Blood Leukocytes . 1V. Transfer Factor-Characterization and Mechanism of Action . . . V Nature and Properties of Dialyzable Transfer Factor . . . . VI . Transfer Factor and in Vitro Correlates of Cellular Immunity . . VII . Mechanism of Action of Transfer Factor in Viuo and in Vitro . . VIII . Transfer Factor and Mechanisms of Cellular Immune Deficiency Diseases IX . Transfer Factor and Reconstitution of Cellular Immune Deficiency . X . Transfer Factor. Immunological Surveillance. and Tumor Immunity . XI . Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . .

196 199 202 217 229 234 245 248 252 258 259 261

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Transfer Factor

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13 S. LAWRENCE

.

Immunological Aspects of Malaria Infection

IVOR N . BROWN

I . Introduction . . . . . . . I1. The Life Cycle of Malaria Parasites . . I11. Innate and Nonspecific Immunity to Malaria IV . Immunity Acquired through Infection . V . Relapses and Antigenic Variation . . VI . Cellular Factors in Malaria Infection . . VII . Antigens of Malaria Parasites . . . VIII . Humoral Factors in Malarial Immunity . IX. Active Immunization to Malaria . . . . X . Experimental Modification of Immunity XI . Immunopathology . . . . . . XI1. Discussion . . . . . . . XI11. Summary . . . . . . . References . . . . . . . AUTHORINDEX.

SUBJEW INDEX .

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268 269 275 278 284 288 296 303 323 329 331 338 339 340

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351 368

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance

M. HA~EK, A. LENCEROV~, AND T. HRABA Immunological Tolerance of Nonliving Antigens

RICHARDT. SMITH Functions of the Complement System

ABRAHAM G. OSLER In Vitro Studies o f the Antibody Response

ABRAMB. STAVITSKY Duration of Immunity in Virus Diseases

J. H. HALE Fate and Biological Action of Antigen-Antibody Complexes

WILLIAM0. WEIGLE Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. GELLAND B. BENACERRAF The Antigenic Structure of Tumors

P. A. GORER AUTHORINDEX-SUBJ

E

INDEX ~

Volume 2 Immunologic Specificity and Molecular Structure

FREDKARUSH Heterogeneity of y-Globulins JOHN

L. FAHEY

The Immunological Significance o f the Thymus

J. F. A. P. MILLER,A. H. E. MARSHALL, AND R. G. WHITE Cellular Genetics of Immune Responses

G. J. V. NOSSAL Antibody Production by Transferred Cells

CHARLES G. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY xi

CONTENTS OF PREVIOUS VOLUMES

Xii

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOULSBY Embryological Development of Antigens

REEDA. FLICKINGER AUTHOR INDEX-SUB JECT INDEX Volume 3 In Vifro Studies o f the Mechanism of Anaphylaxis

K. FRANK AUSTENAND JOHN H. HUMPHREY The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON Immune Adherence

D. S. NELSON Reaginic Antibodies

D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms

DAN€1. CAMPBELL AND JUSTINE S. GARVEY Blood Groups in Animals Other Than Man

W. H. STONEAND M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R.

JENKIN

AUTHOR INDEX-SUB JECX INDEX Volume 4 Ontogeny and Phylogeny o f Adoptive Immunity

ROBERTA. GOODAND BENW. PAPERMASTER Cellular Reactions in Infection

EMANUEL SUTERAND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH

D. FELDMAN

Cell W a l l Antigens of Gram-Positive Bacteria

MACLYNMCCARTY AND STEPHENI. MORSE Structure and Biological Activity of Immunoglobulins

SYDNEY COHENAND RODNEY R. PORTER

COXTENTS OF PREVIOUS VOLUMES

Autoantibodies and Disease

H. G. KUNKELAND E. M. TAN Effect of Bacteria and Bacterial Products on Antibody Response

J. MUNOZ AUTHORINDEX-SUBJECT IXDEX Volume 5 Natural Antibodies and the Immune Response

STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIPY. PATERSON The Immunology of Insulin

C. G . POPE Tissue-Specific Antigens

D.

c. D U h I O N D E

AUTHORINDEX-SUBJECT INDEX Volume 6 Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR. UNANUE AND FRANK J. DIXOX Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSON AND ROBERTA . GOOD Nucleic Acids as Antigens OTTO

J. PLESCIA

AND If’ERiXER

BRAVN

In Vifro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DUTTON Developmental Aspects of Immunity J A R O S L ~ VSTERZL AND

ARTHURM. SILVERSTEIN

Anti-antibodies

PHILIPG. H. GELLAXD ANDREWS. KELUS Conglutinin and lmmunoconglutinins P . J. LACHMANN AIJTHOR IKDEX-SITB JECT INDEX

...

Xlll

xiv

CONTENTS OF PREVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins

SYDNEY COHENAND CESAR MILSTEIN Genetics of Immunoglobulins i n the Mouse

MICHAELPOITERAND ROSELIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues

JOHNB. ZABRISKIE lymphocytes and Transplantation Immunity

DARCY B. WILSON AND R. E. BILLINGHAM Human Tissue Transplantation

JOHNP. MERRILL AUTHORINDEX-SUBJECT INDEX Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. M~~LLER-EBERHARD Regulatory Effect of Antibody on the Immune Response JONATHAN

W. UHRAND GORANMOLLER

The Mechanism of Immunological Paralysis

D. W. DRESSER AND N. A. MITCHISON In Vifro Studies of Human Reaginic Allergy

ABRAHAM G. OSLER,LAWRENCE M. LICHTENSTEIN, AND DAVID A. LEVY AUTHOR INDEX-SUBJECT INDEX Volume 9 Secretory Immunoglobulins

THOMAS B. TOMASI, JR., AND JOHNBIENENSTOCK Immunologic Tissue Injury Mediated by Neutrophilic leukocytes

CHARLESG. COCHRANE The Structure and Function of Monocytes and Macrophages

ZANVILA. COHEN The Immunology and Pathology of NZB Mice

J. B. HOWIEAND B. J. HELYER

AUTHOR INDEX-SUBJECTINDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 10 Cell Selection by Antigen in the Immune Response

GREGORY W. SISKIND AND B A R U J BENACERRAF Phylogeny of Immunoglobulins

M. GREY HOWARD Slow Reacting Substance of Anaphylaxis

ROBERTP. ORANGE A N D K. FRANK AUSTEN Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response

OSCARD. RATNOFF Antigens of Virus-Induced Tumors

KARLHABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARD AMOS

AUTHOR INDEX-SUBJECT INDEX

xv

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Electron Microscopy of the Immunoglobulins

N. MICHAEL

GREEN

Nafionol Insfifufe for Medical Research, Mill Hill, London, England

I. Intioduction . . . . . . . 11. Electron hlicroscopy at the Molecular Level 111. Electron Microscopy of IgG . . . . A. Results Obtained by Shadowing . . B. Results Obtained by Negative Staining C. The Question of Conformational Change IV. Electron Microscopy of IgM . . . . V. Comments and Corclusions . . . . References . . . . . . .

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1 2 6 6 7 14 17 26 28

Introduction

Chemical techniques have provided extensive information about the structure of the constituent pcptide chains of immunogIobulin molecules and the way in which they are linked to each other (reviewed by Cohen and Porter, 1964; Cohen and Milstein, 1967). They have also shown how the chains interact to give the compact Fab and Fc fragments which are produced by splitting of a few peptide and disulfide bonds. A different approach is required to determine the overall layout of the molecule and the spatial relationships of the fragments to each other. Before electron microscopy and X-ray crystallography had reached their present level of development the only approach to this problem was through hydrodynamics. Sedimentation, diffusion, and viscosity measurements consistently showed that the IgG molecule was either highly hydrated or asymmetric (Neurath, 1939; Oncley et d.,1947). A plausible hydration of 0.2 ml./gm. was usually assumed, from which the axial ratio of about 6: 1 was calculated. IgM has an even higher frictional ratio ( f / f o = 1.9) (Miller and Metzger, 196:3a), and in these terms would have an axial ratio of about 1O:l. The asymmetry of IgG was supported by the early electron micrographs and a rod-shaped or ellipsoidal model was accepted for some time. However, in 1965, Noelken et aL pointed out that there were other possible interpretations of the high fractional ratio and intrinsic viscosity, which were more consistent with the chemical evidence. The Fab and Fc fragments showed normal fractional ratios and viscosities and so were not unusually hydrated or asymmetric, It was suggested that these three fragments were joined in Y formation by a relatively flexible 1

2

N. 'MICHAEL GREEN

region of peptide chain, of which the extensive hydration could explain both the high frictional ratio and the susceptibility to enzymatic attack. The contribution of electron microscopy to the solution of the problem provides the main subject for this review. Brief reviews of the subject have appeared elsewhere (Horne, 1965, 1968; Stanworth and Pardoe, 1967). The use of ferritin-labeled antibody as a specific marker for cellular constituents has been treated elsewhere (Andres et al., 1967) and is also the subject of a forthcoming article (Rifkind, 1969), so it will not be considered here. II.

Electron Microscopy at the Molecular Level

The recent application of electron microscopy to the study of structure of macromolecules followed the exploitation of negative contrast methods for the study of viruses (Brenner and Horne, 1959). The limiting factor both now and in the earlier work was not the resolving power of the microscope (about 5 A.) but the difficulty in obtaining sufficient contrast with specimens of molecular dimensions (Valentine, 1961). Unless the thickness of a protein molecule is greater than 70 A. (mol. wt. 150,000), it will not, if untreated, scatter a sufficient proportion of the incident electrons to render it visible against the usual background of carbon film. The first advance in technique was the use of metal shadowing (Williams and Wyckoff, 1945) which proved very successful with virus particles and was particularly useful for revealing detail of the surface and the height of the particle. The unavoidable granularity (20 A.) of the evaporated metal film limited the effective magnification to about 50,000, which was not quite sufficient to reveal subunit structure in proteins. The method was, however, used to determine the lengths of some highly asymmetric molecules (Hall and Doty, 1958) and provided one of the earliest pictures of unattached antibody molecules (Hall et al., 1959) . Two other general methods have been used for enhancing contrast. Positive staining, although satisfactory for sectioned material, is of little use at the molecular level because it is difficult to combine sufficient stain with the specimen (Valentine, 1961). Negative staining (more accurately, negative contrast), on the other hand, has proved to be both simple and effective for the study of viruses and of a variety of protein molecules. In its simplest form a droplet of the dilute (0.01%)protein solution, mixed with 2% sodium phosphotungstate or other suitable salt, is applied to a carbon (or nitroccllulose-supported carbon) film on the grid. After removing thc exccss fluid the film is allowed to dly. The molecules appear as low-density footprints in the thin layer of surrounding phosphotung-

ELECTRON hfEROSCOPY OF THE IMMUNOGLOBULINS

3

state. Success depends on obtaining a faithful replica of thc molecule in a uniform a~norphouslayer of a stain of high weight density. The properties and uses of various heavy metal salts havc been discussed by Valentine and Horn(. ( 1962) and by IIornc ( 1968). Much of the work with antibodies has employed phosphotungstatc, but recently silicotungstate, introduced by Wilcox et al. (1963) for use with vii-uses, has been found to give a slightly less granular background. It is more stable at neutral pH than phosphotungstate (Baker et al., 1955) and the surface activity of its solutions causes it to spread more evenly at low protein concentration. A variation of the negative staining technique, described by Valentine et al. (1968) for use with enzymes, is worth repeating here with some additional details, in view of the cxcellent results it has given with antibodies. The molecules were picked up on carbon film dcposited on freshly cleaved mica, by dipping the mica, film upward, into the protein solution (30-60 pg. antibodylml.). The solution penetrated between the hydrophilic mica and the floating hydrophobic film and the molecules were adsorbed on the carbon in a few seconds. The film was transferred on the mica to a dish of 2%sodium silicotungstate, where it was left floating for a minute or two. A 400-mesh copper grid, coated with a thin layer of adhesive, was placed on the film, followed by a square of adsorbent paper (e.g., newsprint). The paper was removed together with the adhering grid and film and placed on filter paper to drain. The grid dries in a short time and is ready for examination. Uranyl salts have been used by Hoglund (1967a,b) and give higher contrast and greater pcnetration than the tungstates, but this advantage is offset by the more granular background. Uranyl fonnate, in particular, penetrates further into molecules and between subunits (Leberinan, 1965; Finch and Holmes, 1968), and this has been turned to advantage by Svehag et al. (1969) who were sometimes able to resolve H chains from each other and from L chains in fragments of IgM (see below). Objections that negative staining and drying may disrupt labile protein molecules are difficult to refute and cannot be disregarded. In some cases it has been shown that the biological activities of enzymes (Valentine et al., 1968) and antibodies (Chesebro et al., 1968) are not affected b y drying down in the presence of the stain. A further check for artifacts can be made by comparing the volume estimated from the linear dimensions with the molecular weight of the macromolecule ( Rowe, 1966). The curves in Fig. 1 facilitate such estimations for spherical and cylindrical subunits. Unfortunately this can only be a very rough comparison since there are several possible sources of error in the measurcment of overall linear dimensions. The blurred out-

4

N. MICHAEL GREEN

0

20

40 Diameter

(A)

60

80

FIG. 1. Relation between linear dimensions and molecular weight for protein subunits of various shapes. ( A ) Cylindrical subunit of diameter d A. and height 10 A.; ( B ) cylindrical subunit of diameter d A. and height d A.; ( C ) spherical subunit of diameter d A. The molecular weights were calculated from the volume using (the partial specific the expression M = ( N / v ) * V .If a value of 0.73 is used for volume), then M = 0.82V, where V is the volume of the molecule in A.’. Curve A can be used to calculate the molecular weights of cylindrical subunits of any length.

v

lines of the molecules introduce a subjective element which can be only partially overcome by averaging. The apparent size of a spherical or cylindrical object is likely to decrease as the thickness of the layer of stain increases and this is a factor which is difficult to control. This is vividly illustrated by the appearance of the projecting “fiber antigens” of the adenovirus (Valentine and Pereira, 1965; Valentine, 1969), which appear as uniform rods (20 A. diameter) when separated from the virus, but of which the proximal ends vanish in the thick layer of stain surrounding the virus when they are attached. Similarly, IgG bound to ferritin

ELECTRON MICROSCOPY OF THE IMMUNOGLOBULINS

5

appears much narrower in the thicker stain surrounding the ferritin than it does when separated from it ( Fig. 4; and Feinstein and Rowe, 1965). A further source of error derives from the unknown third dimension of a molecule seen only in profilc in negative stain. Sometimes the strength of the contrast can provide qualitative information (see discussion of Fig. 7 ) but noiinally it is necessary to use shadowing to estimatc the third dimension of a molecule and this is not very accurate for small particles. However, in some favorable cases the presence of different, clearly assignable orientations of the molecule may permit all three dimensions to be determined accurately ( t 5 A.) from a negatively stained preparation (Valentine et al., 1968). Molecular weights of many proteins agree well with those calculated from the linear dimensions (Horne, 1965) which gives confidence in the general validity of the method. Several recent papers support this conclusion (Valentine et al., 1966, 1968; Pcnhoet et al., 1967) and show that it is often more accurate to measure center-to-center distances between subunits than to try to estimate the absolute size of a molecule. Unfortunately this approach has not been applicable to the immunoglobulins because of the variable relationships between positions of subunits. Although many protcins appear to survive the process of negative staining without damage, it must be admitted that some proteins do dissociate into subunits in the negative stain and others, such as serum albumin, may be completely disorganized so that they give no appreciable contrast. These undesirable effects may sometimes be eliminated by pretreatment of the protein with glutaraldehyde to produce stabilizing cross-links (e.g., Valentine et al., 1966, 1968). Broadly speaking, negative-contrast electron micrographs provide information about the number, size, and arrangement of subunits in a protein molecule. Attempts have been made to see structure within subunits, but a careful study (Mellema et al., 1968) suggests that such is largely artifact. Under favorable circumstances (thinly spread stain and homogeneous specimens) the smallest detectable protcin would be about 20 to 25 A. diameter (mol. wt. 5000-10,000), but even with a diameter of 35 A. (20,000-30,000 mol. wt.) the contrast is poor. When contrast is good, it is possible to resolve objects as little as 10 A. apart, since the ions of the dodecatungstate stains are about 10 A. in diameter (Baker et al., 1955) and can penetrate between them. Sometimes subunits or molecules which are actually in contact may appear to be separate because of partial penetration of stain between them. Finally, it is worth remembering that the negatively stained eIectron micrograph is a c01lcction of two-dimensional profiles and that apparent changes in shape

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N. MICHAEL GREEN

under different experimental conditions may reflect changes in preferred orientation of the molecules on the carbon film superimposed upon any change in molecular structure. Ill.

Electron Microscopy of IgG

Early attempts to observe combination between antigen and antibody in the electron microscope (Anderson and Stanley, 1941) were made even before the introduction of shadowing techniques. They succeeded in showing an increase in diameter of particles of tobacco mosaic virus from 150 to 600 A. after combination with antibody. Little further was done until Easty and Mercer (1958) made an estimate of the length of the antibody link from the appearance of thin films or sections of ferritinantiferritin precipitates. They observed a halo of low density material 300-400 A. thick around the iron core of the ferritin, which was consistent with the length of about 300 A. for the antibody molecule suggested by hydrodynamic measurements ( Neurath, 1939; Oncley et al., 1947).

A. RESULTSOBTAINED BY SHADOWING The first pictures in which individual antibody molecules were resolved were obtained with shadowed preparations of rabbit antibody (Hall et al., 1959) unattached to antigen. These showed molecules the width of which was greater than their height and the dimensions of which corrected for the thickness of the shadowing metal, were 250 x 40 x 40 A. The length was calculated as a weight average and was, perhaps, unduly influenced by a prolonged heavy tail to the distribution, The number-average length was only 170 A. which was more consistent with the molecular weight (Fig. 1). It is interesting to note that Hall et al. attempted to polymerize their antibody using a divalent hapten, in order to locate the binding sites, a technique which has met recently with considerable success (Valentine and Green, 1967). Unfortunately the affinity of the antibody for the hapten was not high enough to give significant polymerization. From the few dimers that were present, Hall et al. concluded that the molecules probably aggregated end to end. Shortly before this work was published the advantages of neg at'ive contrast methods (for work on viruses and protein molecules) were emphasized by Brenner and Horne ( 1959). The technique was much simpler than that of shadowing and enabled resolution of much finer details, so that it rapidly displaced the earlier technique at least for the cxamination of small molecules. The improvement in detail is clearly illustrated in the article by Valentine (1961) on methods of contrast enhancement.

ELECTRON MICROSCOPY O F THE IMMUNOGLOBULINS

7

Recent investigations of antibocly molecules have employed negative staining methods almost exclusively, sometimes with shadowing used in parallel, to provide supplemcntary information ( Feinstcin and Rowe, 1965)- The clearest results obtained by shadowing (Hoglund, 1967a, 1968) are consistent with a structure for IgG containing three subunits arranged in a variety of configurations. Shadowing has also been used to reveal the surface structurc: of ferritin-antiferritin precipitates in antibody excess ( Robinson, 1966), since precipitates are in general too bulky to be studied by negative staining methods, but it is difficult to interpret the rcsults in molecular terms. B. RESULTSOBrAINED

BY

NEGATIVESTAINING

Most electron micrographs of free antibody molecules ( Valentine, 1959; Feinstein and Rowe, 1965; Valentine and Green, 1967) have shown a disappointing lack of cliaracteristic structure. Thcy appear usually as irregular globular particles with a maximum dimension of about 120 A., nowhere approaching the length of 250 A. deduced from hydrodynamic evidence. In contrast, other protein molecules of similar inolccular weight have shown charactcristic shape and sometimes a subunit structure ( Horne and Greville, 1963; Penlioct ct nl., 1967). The application of the method to antigcn-bound antibody was more rewarding. Hunimeler et al. (1962) observed threadlike molecules as a fuzz on the surface of poliovirus particles. Clearly resolved single antibody links were first obscrvcd by Almeida et al. (1963) in their study of antibodies to the eicosahcdral wart and polyoma viruses (550 and 450 A. diameters, respcctively ) . In antibody excess they confirmed the presence of a layer of threadlike molecules on the surface of the virus dirccted outward in an approximately radial fashion. In antigen excess, single antibody molecules could be seen as thin straight rods (width 34 A . ) linking the virus particles together. The mean length was 150 A. and the maximum length 270 A. It was argued that the full length of the antibody molecule would rarely be visible because of the curved surface of thc virus and, therefore, that the longest links gave the best approximation to the true length of the molecule. Alineida et nl. concluded that the most probable dimensions were 35-40 x 250-270 A. Although this implied a high molecular weight (240,000 t 40,000), it was consistcnt with the axial ratio obtained from sedimentation results. They also commented on the occasionally beadcd appearance of the molecules suggesting several subunits strung togethcr. But this could have been a consequence of the thick layer of stain which makes it difficult to obtain good contrast with the thin antibody molecule the diameter of which is less than one-tenth

8

N. MICHAEL GREEN

7

of that of the virion. It is likely that many of the attached antibody molecules remained undetected, which might account for the sparse distribution of clearly visible antibody molecules in the preparations. Elek et al. (1964) examined Salmonella flagella agglutinated by rabbit IgG antibody. The small diameter (120 A.) and linear geometry of the flagella provided favorable conditions for revealing the antibody, but few single molecules were observed. In antibody excess the flagella, like the virions of Hummeler et al. (1962), were covered with a continuous layer of radially projecting threadlike molecules about 140 to 160 A. thick. Smaller proportions of antibody led to agglutination, and from the interflagellar distance the length of the molecules linking the flagella was calculated ( 180 A , ) . More recently, Hoglund ( 1967a, 1968) used the small satellite tobacco necrosis virus (diameter 180 A.) as antigen and obtained pictures of rodlike links between virions rather shorter than those of Almeida et al. ( 100-150 A . ) , He also obtained similar results with antibodies to T2 bacteriophage ( Hoglund, 196%). These results all tended to confirm the conventional interpretation of the high frictional coefficient of IgG in terms of a molecule of high axial ratio. In addition, they provided evidence that the antigen binding sites were located at opposite ends of the long axis of such a molecule. This view was embodied in the model proposed by Edelman and Gally (1964) which also incorporated the chemical evidence on the arrangement of peptide chains and the size of the binding sites. There was already, however, some preliminary evidence of a more complex molecule. Lafferty and Oertelis ( 1963) had obtained pictures of influenza virus combined with excess of antibody in which single antibody molecules could be seen to be bent into a loop linking two neighboring surface antigens on the same virion. Similar effects had also been seen by Almeida et al. (1963) but were partially discounted because of the possibility of superimposition of two different antibody molecules and the consequcnt difficulty in arriving at a clear interpretation. At this stage the evidence from electron microscopy was very confusing. The antibody molecule, like the cloud of Polonius, was sometimes globular, sometimes elongated, sometimes looped. This polymorphism was emphasized by Feinstein and Rowe (1965) using antibodies to ferritin, the smaller diameter (105 A . ) of which allowed a clearer picture of the attached IgG to be obtained. They observed a maximum dimension for uncombined IgG (rabbit and human) of 105 A. either in negative stain or after metal shadowing. Combination with the ferritin led to a marked change in appearance of the antibody molecules. Thin rods (15 A. across), frequently bent in the middle, were

ELECTRON iWC13OSCOPY O F THE IbfMUNOGLOBULINS

9

observed joining the ferritin molecules together. The maximum separation of the ferritin molecules was about 140 A., but Feinsteill and Rowe assumed that part of the antibody was obscured by the ferritin and concluded that its fully extcnded length was about 200 A. A central swelling, often seen on the thin strands was rcmovcd by pcpsin and somewhat straighter links wcre them observcd. Reduction of the pepsin product gave pictures of Fab fragments projecting 60-70 A. from the suiface of the ferritin. Feinstein and Rowe stressed the variable angle between the arms of the molecule and proposed a globular model for IgG, which could open up about a hinge in the Fc region when combined with antigen, to give an elongated structure of about double the length of the uncombined molecule. They did not attempt to reconcile the globular shape of the uncombined antibody with the hydrodynamic results. The Fab fragments were also observed by Almeida et al. (1965), when bound to polyoma virus. They appcared as short rods (25 x 70 A. ) projecting radially from the viral surface, and, thus, resembling closely those observed on ferritin by Feinstein and Rowe (1965). Valentine and Green (1967) avoided the problem of visualizing and measuring molecules of antibody bound to a large antigen by employing a small bivalent hapten. Bisdinitrophenyl( DNP)-octamethylenediamine reacted with an equivalent amount of high affinity rabbit anti-DNP antibody to give soluble polymers the shape of which revealed the arrangement of the three fragments of the IgG molecule (Fiq. 2). Polygonal rings containing any number from three to ten or more distinct IgG molecules could be seen. Dimers and linear polymers were also present. [A number of different fields have been published elsewhere (Valentine, 1967, 1969; Cohen and Milstein, 1967; Kabat, 1968), and together these provide an adequate impression of the variety of structures that were observed.] The short projections present at cach corner of the polygons could be removed by digestion with pepsin, leaving the rings intact. Each projection was, therefore, an Fc fragment and each corner of a polygon was the center of a Y-shaped IgG molecule. The edges of the polygons were, therefore, dimers of two rod-shaped Fab fragments linked end to end by a molecule of bivalent hapten. This was confirmed by reduction of the products of peptic digestion, which broke the disulfide bonds and liberated the hapten-linked dimers (35 x 130 A ) of Fab. These results confirmed some of the conclusions of Feinstein and Rowe (1965), whereas the greater resolution provided evidence for a more precise molecular model (Fig. 3 ) , similar in many respects to that put forward by Noelken et al. (1965). The angle between the two Fab

FIG.2. Polymers produced by reaction of rabbit anti-DNP IgG with an equiv-

ELECTRON hIICRObCOPY OF THE IMMUNOGLOBULINS

11

-25

FIG.3. Scale diagram of a molecule of rabbit IgG based on measurements of the dimensions of cyclic trimers and on chemical evidence ( Cohen and Porter, 1964). The lengths of the Fab and Fe fragments are 10% greater than those published previously (Valentine and Green, 1967) following a more extended set of nieasurements. The mean distance between the extremes of F c fragments in twenty cyclic trimers was 245 A. (range 215-270 A ) . The variable orientation of the Fc fragment probably accounts for the rather wide range of the measurements. The molecular weight of each fragment (calculated from Fig. 1A) would be 52,000, assuming a cylindrical cross section and making no allowance for the rounded corners illustrated. The relative positions of the L and H chains in the Fab fragments and the orientation of the cleavage plane between them are unknown and, therefore, arbitrary. This has been emphasized by reversing the positions of the L and H chains relative to that shown in a previous diagram (Valentine and Green, 1967). The arrow between the two halves of the F c fragment indicates the position of the twofold symmetry axis observed both in crystals of Fc (Goldstein et al., 1968) and of human IgG myeloma protein (Terry et al., 1968). The location of the binding site in a cleft between L and H chains is consistent with the available evidence on the roles of the two chains (Cohen and Milstein, 1967) but cannot be regarded as firmly established. The smooth contour of the junction between hapten-linked Fab’s is consistent with the central location of the binding site.

d e n t amount of a divalent haptcn ( his-DNP-octamethylenediamiue ). The antibody molecules are centered at the corners of the polygonal shapes. The Fc fragments project from the corners and the Fab fragments form the edges of the polygons. Single molecules ( M ) and tlimers ( D ) can also be seen. Arrows indicate edgewise profiles of dimers. The electron micrographs sliown in Figs. 2, 4, and 7 were all made by the technique of Valentiile et a/. ( 1968) described in Section 11, using sodirtin silicotriiigstate as the negative stain. hlaguificatiorl: X400,OUO.

12

N. MICHAEL GREEN

arms varied between about 10' in dimers to 180" in some of the large rings and open chain polymers; there was no suggestion of a preferred angle. The Fc arm was not always symmetrically disposed between the Fab's, and its apparently variable size suggested that it was not confined to the plane defined by the two Fab arms and the carbon film. This was confirmed by the characteristic appearance (1) of some of the dimers seen edge on (Fig. 2; Valentine, 1967, Fig. 3a). This flexibility allows a single IgG molecule to link pairs of suitably orientated antigenic sites at any distance between about 40 and 140 A. and provides a simple explanation for the facility with which IgG can form cross-linked aggregates with a variety of antigens. Bridging distances longer than 140 A. can only be explained by postulating a considerable stretching or unravelling of the molecule for which there is no evidence in the electron micrographs of the cyclic polymers or of the anti-DNP antibody bound to DNP-ferritin (Fig. 4; Valentine, 1967). This bridging distance is consistent with recent results of Hoglund (1967a,b, 1968) but is significantly shorter than inany of the earlier estimates discussed above. The wide range of angles observed between the three arms suggests a flexible hinge region with low-energy barriers between the various alternative conformations. It could also be argued that the energy barriers are high and that the IgG exists as a population of stable isomers, but this is not supported by the uniform amino acid sequence of the hinge region of rabbit IgG (Cebra et al., 1968), since a unique sequence would be unlikely to foId into a large number of different stable conformations. The results of fluorescence polarization ( Weltman and Edelman, 1967; Wahl and Weber, 1967) do not provide much assistance on this question of flexibility. They suggest that the IgG molecule rotates as a single unit and that any rotations of the fragments relative to each other are too slow to affect the polarization. However, this requires only that the rotational relaxation time of the fragments relative to each other he increased about 20-fold over that of the free fragment (from about 0.05 psec. to a few microscconds) which implies only a small potential energy barrier ( 1-2 kcal. ) hindering rotation. Rotational relaxation times of the fragments greater than microseconds are not, therefore, excluded by these experiments ( Edclman, 1967). One observation that is difficult to reconcile with a very flexible hinge is the high density of this region in the electron micrographs. It is usually as well contrasted as the Fab and Fc fragments themselves, and there appears to be no systematic penetration of stain, which might have been expected if the region had consisted of rather open polypeptide chains. Occasionally there does appear to be a slight separation of the Fc from

ELECTROX 3lICROSCOPY OF THE IMMUNOGLOBULINS

13

FIG.4. Rabbit anti-DNP IgG coinl>inetl with DNP-ferritin ( 8 DNP groups per molecule ) . (Valentiiic and Grcen, iii~publisliedexperiments; Valcirtine, 1967). Many unbound Y-shaped anti1)ody inoloculcs can be scan as wrll as those linking the ferritin molecules. The apparcirt width of t h r fcrritin-boiuntl antibody is smaller than that of free antibody becniise of the greatrr t h i c h s s of staiti surmunding the fewitin. hfagirification: X400,OOO.

14

N. MICHAEL GREEN

the Fab fragments, and in the pepsin-treated polymer (Valentine and Green, 1967) a clear division can be seen between the two Fab fragments. This observation of a compact hinge conflicts slightly with the hydrodynamic model of Noelken et nl. (1965) which in most respects agrecs well with the model that is shown in Fig. 3. If the axial ratio of IgG is taken from the elcctron micrographs as approximately 3:1, then a frictional ratio of 1.47 implies 0.9 ml./gni. of hydrodynamically trapped solvent (Edsall, 1953). The fractional ratios and dimensions of the fragments show that they are not highly solvated, so that more than half of this solvent would have to be in the hinge region, implying a much more open structure than is seen in the electron micrographs. An alternative analysis of the hydrodynamic data (Charlwood and Utsumi, 1969) by the method of Bloomfield et al. (1967) led to rather similar conclusions. They showed that it was possible to account for the sedimentation constant of IgG in terms of those of its fragments, only if the centcr-to-center distances of the Fc from the Fab fragments was 78 & 10 A., some 20 A. greater than that iiiexured on Fig. 2. These discrepancies are probably significant and may indicate that the hinge region in solution is less compact than it appears to be in the electron micrographs. This would also be consistent with its susceptibility to proteolysis.

C. THEQUESTION OF CONFORMATIONAL CHANGE The electron microscopic evidence for the three-armed modcl was derived from polymers obtained with bivalent hapten. Electron micrographs of single antibody molecules often do not show this structure clearly, but it is not certain whether this is a result of technical difficulties or of a genuine change in structure. The idea that the IgG molecule undergoes a change in conformation when complexecl with polyvalent antigens has received support from many quarters. The main evidence comes from the new immunological activities of antigen-antibody complexes, such as their ability to fix coniplement, to bind to skin receptors, and to induce new antibody response (e.g. Kabat and Mayer, 1961; Gel1 and Kelus, 1967; Henney and Ishizaka, 1968). Complexes with simple haptcns do not show any of these properties, whereas nonspecific aggregates of IgG or of Fc fragments do. The new properties oftcn appear to be more a consequence of aggregation than of specific combination with antigen followed by specific change in Conformation. Elrctron microscopy is a w r y insensitive test for conformational change since it can only dctcct changes in stability or arrangemcnt of

ELECTROS hIICROSCOPY O F THE IMMUNOGLOBULINS

15

whole subunits. It may bc: argued conversely that any change seen h ~ this method is likely to reflect a fairly substantial altcration in the molecule. Feinstein and Rowe (1965) suggested a correlation between the appearance of the new immunological propcrties and the apparent opening up of the molecule suggested by their electron micrographs. The inore detailed picture obtained by Valentine and Gwen (1967) did not support such an extensive change though there was sonic evidence for a more clcarly defined subunit structurc whctn thc IgG molccules were polymerized. ‘The most serious obstacle to reaching a firm conclusion about the changes that take place on combination with antigen has been the difficulty exprienced in obtaining wcll-stained preparations of uncombined IgG molccules. Where uniform, well-contrasted specimens are obtained, thc tripartite nature of thc single molccule becomes clear. For example, occasional single Y-shaped molecules can be seen among thc polymers formed with bivalcnt hapten (Fig. 2 ) . The clearest singlc molecules that have been observed so far were in preparations of anti-DNP antibody combined with DNP-ferritin ( Fig. 3; Valcntine, 1967). Thesc elrctron micrographs were made for direct comparison with the results of Feinstcin and Rowe (1965) and they illustrate two further points of interest. First, the lengths of the antibody molecules linking ferritin molecules togethcr (aliout 140 A ) is not significantly greater than that observed in the cyclic polyincw; second, thc. appearance of the ncgatively contrasted molecule depends on the thickness of the stain. The connecting strands in thc thicker stain surrounding the ferritin appear to be little morc than half thc width of the molecules in the thinner areas away from the ferritin. There are two further factors which lead to enhancement of the regular appearance of polymerized molecules of IgG and, hence, to ; i n impression of a change in conforniation. First, the molecules lic on the grid in the same orientation (apart from variation in the anglc hetween the arms) ;md, sccond, the union of the Fa11 arms end to cncl emphasizcs their length. This sccond factor can hc c,liminatcd b y isolating the corners of the cyclic polymers with an appropriate mask so that constituent molecules are seen in isolation. Profiles traced in this way arc shown in Fig. 5 where t h y are compared with those of sing](. molecnlcs. Taking nll these factors into account. I would conclude that there is no firm electron-microscopic evidence for an extensive change in the molecular conformation accompanying comhination with antigcn. HOWTVCT, tlierc. clearly must he some change to account for the new immiinological properties. Sincc, tlwse are associated mainly with the Fc fragmcnt of the inolcculc, thcy could wcll be ;I rcsiilt of slight chances in the hing(’ rcxgion ( Fcinstcin and Rowc, 1965).

7

N. MICIIAEL GHEEN

FICA5. Tracings of IgG moleciiles at a magnification of X300,OOO. ( a ) Taken from corners of cyclic polymers; (11) single molecules from Fig. 4; ( c ) single molecules from Valentine and Green (1937). The single molecule approximates to an isosccIes triangle with the sides slightly longer than the base, of which the dimensions are 120 X 120 X 102 A. (iiienn of ineasurenients on twmty-five molecules). The corresponding dimensions for molecules taken from the corners of cyclic trimers was 121) x 120 x 115 A.

Although therc are no obvious differences between the populations of antibody molecules shown in Figs. 2 and 4, there could be a differcnce in the fraction of molecules with large inter-Fab angles, which could be critical for the binding of the complex to a third component. If this binding is multivalent as it appears to be for C', ( Muller-Eberhard, 1968j, then this effect could well be amplified, since it would be dependent on the proximity of several molecules in the appropriate open conformation. An inteiprctation of this type would also be consistent with the formation of weak complexes between free TgG or TgM and C , ( Muller-Eberhard and Calcott, 1966j, The suggestion of an increased angle between the Fab's is supported by the results of the measurements of single and polymerized molecules, summarized beneath Fig. 5. Whereas the molecules at the corners of cyclic trimers can be represented by a triangle that is approximately equilateral ( 120 A. x 120 >< 115 A.), the single molecules shown in Fig. 4 have one dinicnsion that is significantly shorter than the other two ( 120 x 120 X 102 A. j. This suggests that the angle between the Fab fragments in the most probable conformations of the nioiioiner is appreciably lcss than GO", so that angles greater than GO" may only be common in complexes with polyvalent antigens. This argument can only he tentative, first, since it assumes the IgG molccules shown in Fig. 4 provide a representative sample of the conformations present in solution and, second, since thr difierence between 102 and 115 A. is barely significant.

ELECTRON hIICROSCOPY OF THE Ih$MUKOGLOBULINS

17

The optical rotatory dispersion ( O R D ) spectrum is a more sensitive indicator of confoimational change than is electron microscopy, and it is worth considering briefly R few relevant observations. Changes in Q, have been observed in soluble complexes of antibody with scrum albumin, with ferritin, and with a synthetic trivalent hapten (Ishizaka and Campbell, 1959; Henney and Stanworth, 1966), but, except for the last example, it cannot be certainly concluded that they are due to changes in the antibody molecule, because of the laige contribution from the antigen to the total rotation. Coinplexcs with simplc haptens that have been examined do not fix complement, nor do they show any changes in ORD ( Steiner and Lowey, 1966; Cathou et nl., 1968). Howevcr, the correlation between complement fixation and change in optical rotation docs not always hold. The complexcs of anti-DNP with biq-DNP-octamcthylenediaminch do fix coinplcmeiit ( N. Hyslop, unpublished evpeiiments ) although their ORD spectrum is indistinguishable from that of the cornplcx with univalent DNP aminocapl-oate ( N. M. Green, unpublished experiments). Clearly more experimental work is rcquircd, preferably with simple antibody-hapten systems and purified components, to obtain a clcar mswcr to the problem. It will probably be more informative to look directly at the binding intcractions in such systems then to make ORD measurements on the antigen-antibody complexes, since the critical hinge region may have a somewhat Ioosc and variable structure which will make no characteristic contribution to the ORD curve. In support of this view, it has been found that proteolytic splitting of this part of the n~oleculeis without effect on the ORD or circular dichroism of antiDNP antibodies (Stc-iner and Lowey, 1966; Cathou et al., 1968) nor docs it affect the ORD of IgM (Dorrington and Tanford, 1968). IV.

Electron Microscopy of IgM

The chemical evidence for the structure of IgM is lccs extensive than for that of IgG. Much of it derives from the study of Waldenstrom macroglobuhs ( Miller and Metzgcr, 1965a,b), hut similar results havc been obtaincd with ralhit IgM antibodies (Lamni and Small, 1966). A fairIy consistent picture of the overall layout of the peptide chains in thc moleculc has bcen provided by this work which has shown many similarities between the 7 S subunit of Ighii and the IgG molecule. Each 19 S molecule (mol. wt. 900,000) contains five 7 S wbunits linked together 13y single disulfitle tionds, possibly nccir the C - t c m k n l ends of the FC fragments (Abel and Gray, 1967). Each 7 S subunit contains two H ( p ) chains and two L ( h or A ) chains joincd b y 21 p'ittein of disulfide bonds differing little froin tllclt in IgG ( hIi1lc.r and \Ictzqcr. 1965b). It has l)c.cn

18

N. MICHAEL GREEN

possible to obtain Fab and Fc fragments homologous with those derived from IgG by slight modification of the proteolytic procedures used so successfully on the smaller molecule. Digestion with pepsin ( Mihaesco and Seligmann, 196813; Kishimoto et al., 1968) or trypsin (Miller and Metzger, 1966) gave Fab p or F(ab), p fragments, depending on the extent of proteolysis. The Fc p fragments (3.2 S), similar to those from IgG, result from the usual digestion with papain in the presence of cysteine ( Mihaesco and Seligmann, 1968a). If the activating thiol was removed before the reaction, a much larger Fc (10.6 S ) was obtained in which five 3.2 S fragments were united by disulfide bonds to give a structure with a molecular weight of 320,000 (Onoue et al., 1968b). The task of the electron microscopist is to assemble these fragments to give a molecule consistent with the rather varied and unusual profiles shown in his pictures. Electron microscopy may also help to answer the question of the number and location of the binding sites, which is still under discussion. Scveral groups of workers (e.g., Onoue et ul., 1965; Frank and Humphrey, 19168) gave evidence for only five sites in the whole molecule (one for each subunit) which implicd that the two Fab p fragments in each subunit differed from each othcr. Recent work (Merler et al., 1968; Stone and Metzger, 1968; Ashman and Metzger, 1969) suggests that there are ten sites, a finding which is more consistent with the other chemical evidence. It has been suggested that these sites belong to two different classes ( Onoue et al., 1968a ) . The earliest electron micrographs of IgM ( Hoglund, 1965; Hoglund and Levin, 1965) were of shadowed preparations. They showed ellipsoidal molecules (300 x 200 A , ) , which appeared to be much too large for a molecular weight of 900,000 (Rowe, 1966). Later studies by Hoglund (1967a,b) using negative staining also showed molecules about 250 A. across, with a smooth approximately circular profile, but no indication of their thickness was obtained. Occasionally the stain (uranyl acetate) penetrated into the molecule and a suggestion of a more open structure of rodlike components was obtained. Other authors have consistently found two different characteristic appearances of IgM molecules, dependent on whether they were free or bound to the surface of an antigen. The bound antibody often appeared as a well contrasted bar (170 x 35 A.) about 80 to 100 A. from the surface of the antigen and linked to it by a number of thin strands which ;ire difficult to define and count. This structure has been seen on fragments of erythrocyte mcmbrane ( Humphrey and Dou~mashkin,1965) and on Salinonella flagella ( Feinstein and Munn, 1966; see, also, Cohen and hlilstein, 1967). When the antigenic sites were prcsent on virus particles rather than on an extended surface, a slightly different picture was

ELECTHOR; 1\IICHOSCOPY O F THE I2r4MUSOGLOBl.JLIh’S

19

FIG.6. Poliovirus aggregated by rabbit Ighl antilmtly. The antibody molecule appears as a well-contrasted bar linked to the viral surface by thin, soiiietiines invisible, strands. Occnsionally a central ring structure can be seen ( a ). Magnification: ( a ) X200,OOO (Chesebro imtl Svehag, 1969); (11) X400,OOO ( Svehag and Bloth, 1967).

obtained (Fig. 6 ) . The central bar was not always straight and arms protruded from it in all directions, sometimes linking virus particles together (Almeida et al., 1967; Svehag and Bloth, 1967). Sometimes the link appeared as a continuous straight bar up to 370 A. long, and it has in fact

20

N. MICHAEL GREEN

FIG.7a FIG.7. R l t anti-DNP IgM. The contrast is weak apart from bright spots, 3040 A. across, seen on the projecting arms or near their bases, but not in the center of the molecule, The mean span of the molecule is about 270 A,, the mean area 31,000 A?, and the arms are not more than 90 A. in length, Magnification: ( a ) X400,OOO; ( I ) ) X1,100,000 (unpublished work of R. C. Valentine and R. Binaghi).

ELECTRON MICROSCOPY O F THE IMMUNOGLOBULINS

21

FIG. 7b

been suggested that IgM may be a long flexible rod 50 x 370 A. ( Almeida et al., 1967). However, such a simple picture is difficult to reconcile both with the chemical evidence that the molecule contains five 7 S subunits and with the electron micrographs of uncombincd IgM. Svehag and Bloth (1967) described the IgM molecules attached to poliovirus as multiple (2-1)looped striicturcs like repeated “in.” The number of l o o p varied, and in antigen excess extended tails could be seen linking two or more virions together (Fig. 6 ) . The maximum length was 360 A. (mean 330 A ) , and the mean distance from the vertex of a loop to the virion was 85 A. It was suggested ( Svehag et ul., 1967b) that each loop (sometimes with a projection from the vcrtex) might represent a 7 S subunit of IgM and, thus, resemble the loops of IgG observed on other virus particles (Lafferty and Oertelis, 1963; Almeida et d.,1963; Chesebro and Svehag, 1969). Uncombined IgM appears usually as a multiarmed ameboid structure spread out on the carbon film. This was first observed for Waldenstrom niacroglobulins, for rabbit antipoliovirus IgM ( Svehag et aZ., 1967a,b), and for whole bovine IgM (Feinstein a i d M u m , 19167). This is illustrated in Fig. 7 which shows rat anti-DNP IgM. The appearance is very different from that of IgM bound to antigen, and this led Feinstein and Munn

22

N. MICHAEL GREEN

(1966) to suggest a marked change in structure when the antibody coinbined with membrane, flagellum, or virus. However, careful consideration of the geometrical factors involved show that the two contrasting appearances are by no means incompatible with each other. For the most part, the uncombined molecule shows very weak contrast suggesting that it is disclike rather than ellipsoidal. The central area of the moleculc appears particularly thin. The mean area of such molecules (taken from Fig. 7 and other similar fields), measured by superimposing tracings on graph paper, was 31,000 A.' (mean of 30, range 27,000-36,000 Az).From this area and from the molecular weight (900,000) and partial specific volume (0.73), an average thickncss of 35 A. was calculated, very close to the thickness of the strands seen when the molecule is bound to antigen. It therefore seems likely that the highly contrasted bar seen with bound antibody is an edgewise view of the central disc and that this is linked by hinged arms to the surface of the antigen. Since the bound molecules are seen most clearly when the antigen surface is perpendicular to the carbon film, thc aspect of the molecule obtained is totally different from that when it is adsorbed directly on the carbon film. The picture becomes slightly more complicated when the binding surface is provided by several virus particles-arms may project in any direction from the central disc (Fig. 6)-but the basic interpretation can still be maintained. If this is correct, then the arms represent Fab fragments attached to a central disc of Fc fragmcnts and the high contrast spots on the ameboid IgM molecules (Fig. 7) are probably Fab arms viewed end on. This interpretation can be supported and elaborated following further extensive work by Svehag and his colleagues. They took into account the chemical evidencc and concluded that the IgM molccule was basically a cyclic pentamer (Svehag et al., 1967a, 1968a,b; Chesebro et al., 1968), the units being held together by disulfide bonds bctwecn Fc p fragments, which would then form a central ring. This suggestion was supported by the characteristic appearance of a small proportion of the IgM molecules, which showed such a ring with a central hole (Fig. 8a), rather than the continuous thin disc suggested by Fig. 7. These were seen in about twenty preparations of IgM from different sources (S.-E. Svehag, personal communication), but only occasionally (e.g., Fig. 6a) when the IgM was bound to antigen, presumably because edgewise views of the molecule are favored in this situation. The arms of the pentamer were about 35 x 125 A., but it was not often that all five were clearly resolved. If there are only five arms and there are ten Fab p fragments to be accounted for, either each arm consists of two Fab's or onc of each pair of Fab's is somehow incorporated into the central disc (Steffen, 1968). The

ELECTHON hfICROSCOPY OF THE IMMUNOGLOBULINS

23

sccond hypothesis involves an improbable asymmetry, which would imply two different ,U chains in the one molecule, and would be difficult to reconcile with the evidence for ten binding sites mentioned above. The presence of two Fab p fragments in cach arm would avoid such difficulties and it coulcl account for the very variable appearance of the arms, sometimes short, sometimes fused with their neighbors, and sometimes more than five. This suggestion is supported by two papers on the submit s and protcolytic fragments of IgM (Svehag et al., 1969; Chesebro and Svehag, 1969) . Preliminary studies of alkylated IgM subunits, produced by reduction with 0.2 hl mercaptoethanol (Chesebro et aZ., 1968) showed very thin (15 A.) strands about 100 A. long with a knob of diameter 50 A. at one end. This would appear to be too sinall to represent a subunit of rnolecular weight 180,000 but would be consistcnt with the size of a half subunit containing one light and one heavy chain. It is known that reduction (0.2 A t iiiercaptocthanol) cleavcs the disulfide bond between the two halves of the subunit (Miller and Metzger, 1965b), and it is likcly that negativc staining in silicotungstate could cause separation of these two halves since they are held togcther only by noncovaleiit bonds. Such dissociation has been observed by Valentine and Green ( unpublished experiments ) with the polymcrs of anti-DNP IgG, discussed above. In silicotungstate, these dissociated after mild reduction ( 0.01 91 mercaptoethanol, pH 7.4) into thin strands which were probal-jly hapten-linked dimers of half molecules. A morc detailed study (Chesebro and Svehag, 1969; Svehag and Bloth, quoted in Svehag et al., 1969) using milder reduction conditions, which should give covalently linked subunits ( Millcr and Metzger, 1965b), showed units of two sizes 90 x 35 A. and 90 x 55 A. The appearance of the larger one, amounting to up to 30%of the total, was more consistent with the expected molecular weight, but it is difficult to see how it coulcl be assembled to give all the variable profilcs of IgM. Reoxidation of the reduced subunits led to regeneration of 30 to 50% of the antibody activity and to IgM molecules of characteristic appearance. Fragments obtained by proteolytic digcstion provided clearer information about the substructure of the IgM molecule ( Svehag et nl., 1969). The large (10.6 S ) Fc ,U fragment was obtained using papain in the absence of cystine (Onoue et al., 1968b) and w a s separated from smaller fragments on Sephadex. Electron microscopy showed two types of structure: ( 1 ) a ring with a central (40 A.) hole, an outer diameter of about 85 A,, and a number (nciirer to ten than to five) of radial projections, 20-25 A. long ( 8b, 8c); ( 2 ) “screwlike” structures, 40 x 200-500 A. long,

24

N. MICHAEL GREEN

FIG.8. IgM and its fragments. All scale lines are 200 A. ( a ) Intact molecule showing central ring structure. The central hole has a diameter of 40 A. and the five arms are 70-100 A. X 35 A. Magnification: X470,OOO (S.-E. Svehag, unpublished observation). ( b ) Fc p fragments from papain digest of human IgM. The outer diameter (taken froiii a number of difFerent niolecules) is 85 A. A number of sIiiall (20 A , ) projections can often be seen. Magnification: X460,000 (S.-E. Svehag, unpublished observations). ( c ) Aggregated Fc ,u fragments from papain digest of human IgM. Magnification: X230,OOO (Svehag et al., 1969). ( d ) F ( a b ) ? ,u from pepsin digest of human IgM. Club-shaped Fab ,u fragments are joined in pairs by their narrow ends, which cannot always be seen. Magnification: X330,OOO (Svehag et al., 1969).

consisting of chains of several subunits, each unit being a double bar orientated at right angles to the screw axis. The ring structures probably originated from the five 3.2 S Fc p fragments which form the center of the IgM molecule. The screwlike structures probably arise froin the rings by breakage of a disulfide bond followed by rotation of the subunits relative to each other and consequent opening up of the ring. The longer (500 A ) polymers could then be produced by a further disulfide interchange with another ring or oxidative dimerization with another chain. The initial ring opening could have been brought about by the -SH of the papain itself since the digestion mixture contained several moles of papain for each mole of IgM. Small (3.2 S ) Fc p fragments were sometimes seen as dimeric structures, the stain having penetrated between the two halves of the molecule.

ELECTRON MICROSCOPY O F T H E IMhfUNOCLOBULINS

25

In the s m i e p a p c ~ Svehag , ct nl., examined Fall’ and F( ah’)? l’c obtaincd by pcpsin digestion ( Mihacsco and Scligmann, l968n; Kishimoto et mZ., 1968). ’The monomcrs wcrc rod-shapcd i d oftell q p g a t c d sido to side. Sometimcs thcy appeared to consist of two parallel strands, one 80 x 15 A. and the other 50 and 15 A., possibly represcmting half the H chain and the L chain, respectively. If this interpretation is correct then in order to account for their niolecrilar wcights the folded H and L chains would appear to have a ribbonlike cross section about 15 x 35 A. Thc F(ab’), p fragments (Fig. 8 d ) consisted of two parallel Fah’ p,’s linked by their narrow ends, though it was not always possible to resolve the, linking region. The electron micrographs which showed the division between H and L chains and betwcen the two sections of H chain in the Fc fraginent were all obtained with uranyl fortnate as thc negative stain, of which the small size facilitates pcnetration. Thcse effects were not observed when the larger silicotungstate ion was used ( S.-E. Svehag, pcrsonal communication), and this may account for thc failure to sec any such divisions in the Fab and Fc fragments of IgG (Valentine and Grcvii, 1967). Most electron micrographs of IgM are consistent with its i‘or~nulation as a cyclic pcntainc~r,provided that due allowance is made for differences in orientation. The question of whethcr there are five or ten arms may still be regarded :is open. Svehag et al. (1969) concluded that the Fa11 fragments could not be seen as separate units in the whole IgM moleculc and that each arm represented a pair of fragments which were not in&pendently hinged. Stone and Mctzger (1968) used a similar model, with steric hindrance betwcen pairs of sites, to explain the inability of thcir anti-Fc, IYaldenstrom IgM ( YR,Lay) to bind morc than five molecules of antigen, whereas tlie separated Fab p fragments could bind tcn. Howevcr, a minority of IgM molecules show a clear five-armed structure, and it is much easier to nccount both for the molccular polymorphism and for the localized areas of high contrast within the molecule (Figs. 7, 8a; and Chesebro et al., 1968) if a model with ten independently hinged Fall arms is assumed. This would imply that each 7 S subunit had thc same basic Y structure as the molccule of IgG. Thcrc must, of course, be considerable differences of detail since the p chain is some 208 longer than the y chain. This extra length may be iiccountcd for by the narrow tail (30 x 15 A , ) of the Fab p fragment and by the 20-25 A. projections from the ring-shaped Fc fragment. The conformational changes in IgM which may occur when it binds to surface antigens are even less well characterizcd than are those in IgG. The complex certainly binds and activates complement very effectively, ,(,

26

N. MICHAEL GREEN

so it is likely that changes parallel to those in IgG occur near the junction region of the two Fab fragments of each subunit. This would provide further support for the idea that the two Fab arms have some freedom to move independently. It is possible that IgM from different sources may differ in this respect, but so far there is no good evidence for this. V.

Comments and Conclusions

It is unlikely that improvements in electron-microscopic technique will show finer details of structure than have already been obtained, although it may eventually be possible to resolve more clearly the individual H and L chains (perhaps better called H and L subunits, since they possess a definite tertiary structure). One of the next developments is likely to be the study of further antibody types and classes. Detailed morphology of IgG has been reportcd only for rabbit antibodies (Feinstein and Rowe, 1965; Valentine and Green, 1967), however, thc chemical and physical similarities between antibodies from different species suggest that there are likely to be few detectable interspecific differences detectable by electron microscopy. A preliminary examination of yl and y2 anti-DNP antibodies from guinea pigs (Valentine, Green, and Binaghi, unpublished experiments) showed some cyclic polymers with bifunction haptens, similar to those seen with rabbit antibody. Polymerization was less extensive and more open chain structures were present, possibly because of the lower affinity of the antibody ( K = 2 x M ) . It will be of interest to examine antibodies with known differcnces in the pattern of disulfide bonding such as the different classes of human IgG ( Frangione et al., 1969) and mouse IgA ( Abel and Grey, 1968) to see if this affects the angular relations between the fragments. IgM from different species shows no distinguishable differences, although the serum a,-macroglobulin, of unknown function, has a quite different shape, resembling the Russian xc (Bloth et al., 1968). Bifunctional haptens may be useful both for clarifying the subunit structure and for the formation of relatively simple antibody complcxes, of which the interaction with complement components may be observable by electron microscopy. It is, therefore, worth making a few general comments about their use. The bis-DNP-polymethylene diamines used by Valentine and Green (1967) were very insoluble in water, so that they had to be dissolved in dimethylformamide and added very slowly to the antibody. If the dissociation constant of antibody for DNP was M the reagent precipitated and did not react quantigreater than tatively. It is possible to apply the technique to lower affinity antibodies only by use of water-soluble bis-DNP compounds (e.g., derived from

ELECTI3OK hIICROSCOPY OF THE IhIhIUNOGLOBULINS

27

diaminosuccinic acid; N. M. Green, unpublished experiments). Further problcws aris? if K > lo-' R I , bccause of the low protein concentration employed for clectron microscopy. For example, N suitable concentration of antibody is about 50 pg./ml. ( 3 x lo-; AZ) which limits the hapten concentrations also to this order of magnitude. Use of higher concentrations of hapten to saturate the sites would give predominantly monofunctional binding, defeating thc purposc of the experiment. Attempts to produce specific polymerization of rat anti-DNP IgM using bis- ( DNPaminocaproy1)diaiiiinosuccinate gave ncgativc results, presumably because the dissociation constant ( 3 x lo-' M ; Binaghi and Oriol, 1968) was too high. I t may be possible to circumvent this limitation by mixing antibody and hapten at higher concentration and fixing with glutaraldehyde before dilution. In the case of IgM it may not even be necessary to fix thc polymers provided that there arc polyvalent links between molecules and thc molecules are taken up on the carbon film ininiediately after dilution. One furthcr application of bifunctional reagents in which the two functional groups are different may be mentioned. For example, a compound containing 110th a DNP group and a reactive group X, specific for say the catalytic center of a multisubunit enzyme, could be used to bind anti-DNP antibody (or a univalent Fab fragment) to this site. This could enable the arrangement of catalytic sites within the molecule to be determined b y electron microscopy. This would be a high resolution modification of the use of ferritin-labeled antibody, with the added advantage that it would be unnecessary to prepare antibody specific for the catalytic site. The specificity would bc provided by the appropriate reactivc group, X. Electron microscopy, like alniosc all physical techniques for the study of protclin structure, provides only a limited view of the protein molecule, so thdt the clc,ctron micrographs can rarely be interpretcd unequivocally without consideratioii of the results from other techniques. The results discusscd in this review do lcad to structures for both IgM and IgG reasoualdy consistent ~ i t hmost of the other physical and chemical evidence, although a few qiicstions remain which cannot yet be resolved. Oiic: of thest. is tlie cxtent of the eonformational changes accompanying tlie binding to polyvalent antigens, which lead to well-defined biological effects. In this revicw, I havc discounted most of the earlier electroniiiicroscopic evidence in favor of cxtensivc, changes since most of it can be sntisfactorily intciprcted without invoking anything more than selection of certain pre-existing conformations b y the antigen. There still reInnins the possi1)ility that the differcnccs olxervcd in cblectron micro-

28

N. MICHAEL GREEN

graphs bctwcen free and bound antibody reflect genuine large changes in molecular structure and are not merely the consequence of variation in orientation or in stain thickness. However, stronger evidence in support of this view is required before it can be accepted, for these differences imply rather extensive deformation of the Fab and Fc fragments, which is nt variance with their bchatior in solution as stable globular proteins. Active stretching of an antibody by a large antigen is an unlikely occurrence in the absence of a specific chemical mechanism. Since the mcan trmslntional kinetic energy of a molecule is independent of its size, large antigens are no more likely to produce such effects than are small ones, unless forces due to liquid flow or surface tension ale involved. Steric interference between large antigens may, however, tend to stabilize 3 more open conformation of an IgC antibody. ACKNOWLEDGMENTS This review was to have been written in collaboration with the late Dr. R. C. Valentine. His contribution can be seen clearly in the electron micrographs used for illustration as well as in the interpretations, which owe mnch to his influence. I would also like to thank Dr. R. R. Dourinashkin for much helpful advice and assistance in evaluating the evidence.

REFERENCES Abel, C . A,, ancl Grey, H. M. (1967). Science 156, 1609. Abel, C. A., and Grey, H . hl. (1968). Biochemistry 7, 2682. Almeicla, J. D., Cinncler, B., and Howatson, A. (1963). J. Exptl. Med. 118, 327. Alnieida, J. D., Cinnder, B., and Naylor, D. (1965). Inimzinochernisliy 2, 169. Almeida, J. D., Brown, F., and Waterson, A. P. (1967). J . Inimunol. 98, 186. Anderson, T. F., and Stanley, W. Ivl. (1941). J. Biol. Chem. 139, 339. Andres, G. A., Hsu, K. C., and Seegal, B. C. (1967). I n “Handbook of ExperimentaP Immunology” ( D . hl. Weir, ed.), p. 527. Blackwell, Oxford. Ashnian, R. F., and Metzger, H. (1969). J. Biol. Chcm. 244, 3405. Baker, M. C., Lyons, P. A., and Singer, S. J. (1955). J . Am. Cheni. Soc. 77, 2011. Binaghi, R., ancl Oriol, H. ( 1968). B d . Soc. Chim. Biol. 50, 1035. Bloomfield, V. A,, Van Holde, K. E., and Dalton, W. 0. (1967). Biopolyrners 5, 149. Bloth, R., Chesebro, B., antl Svehag, S.-E. (1968). J. Ezpfl. Aged. 127, 749. Brenner, S., and Horne, R. W. ( 1 ). Biochint. Biophys. Acfa 34, 103. Cuthou, R. E., Kiilczycki, A,, and Haber, E. (1968). Biochem. J. 7, 3958. Crbra, J. J,, Steiner, L. A., and Porter, R. R. (1968). Biochen~.J. 107, 79. Cliarlwood, P. A,, and Utsnmi, S. (1969). Biockem. J. 112, 357. Chesehro, B., and Svehag, S.-E. (1969). J. lniniunol. 102, 1064. Chcscbro, B., Bloth, B., antl Svehag, S.-E. (1088). J. E x p t f . Med. 127, 399. Cohen, S., and Milstein, C. ( 1967). Atloan. I n i t t i u n d . 7, 1. Cohen, S., antl Portcr, R. R. ( 1964). Adunn. ItnmtmoZ. 4, 287. Dorrington, K. J,, a n d Tanfortl, C. ( 1968). J. BioZ. C h e m 243, 4745. Easty, G . C., ant1 hlet.ccr, E. 13. ( 1958). lmmtmoZog!/ 1, 353.

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Etlelinan, G. hf. (1967). Nobel Symp. 3, 281. Wiley (Interscience), New York. Edelman, G. M . , and Cally, J. A. (1964). Proc. Natl. Acad. Sci. U.S. 51, 846. Edsall, J. T. (1953). In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 1, p. 549. Acudemic Press, New York. Elck, S. D., Kingsley Smith, B. V., and Highman, IV. (1964). lmnirinology 1, 353. Feinstcin, A,, and M u m , E. (1966). J . Phy.sioZ. ( L o n d o n ) 186, 64P. Feinstein, A,, and Xlunn, E. ( 1967). 171 “Handhook of Experiinental Immunology” ( I ) . hl. Weir, etl.), p. 277. Blackwell, Oxford. Fcinstein, A,, and Rowe, A. J. (1965). Natirre 205, 147. Finch, J. T., and Holines, K. (7. (1968). I n “Methods in Virology” ( K . Maramorosch and H. Koprawski, eds.), Vol. 3, p. 352. Academic Press, New York. Frangione, R., Xlilstcin, C., ancl Pink, J. R. L. (1969). Nature 221, 147. Frank, M. %I., and Humphrcy, J. H. (1968). J. ExptZ. Med. 127, ‘367. Cell, P. C,. H., and Kelns, A . S. (1967). Adoan. Inimunol. 6, 461. Goldstein, D. J., Humphrey, R. L., and Poljak, R. J. (1968). J. Mol. B i d . 35, 247. Hall, C. E., and Doty, P. (1958). J. AVL.Chcm. Soc. 80, 1269. Hdl, C. E., Nisonoff, A., and Shyter, H. S. (1959). J. Biochem. Biopkys. Cytol. 6, 407. Henney, C. S., and Isliizaka, R. (1968). J. Inmitinol. 100, 718. Henney, C. S., and Stnnworth, D. R. (1966). Natirre 210, 1071. Hoglurid, S. ( 1965). Proc. European Rcgioiial Conf. Electron Microscopy, Pragtrc, 1964, Vol. B, p. 55. Hiiglund, S . (1967a). Nohel S y m p . 3, 259. Wiley (Interscience), New York. Hiigluiid, S. ( 196,713) . Virology 32, 662. Hoglund, S. (1968). J. Gen. Virol. 2, 427. Hiiglund, S., :ind Levin, 0. (1965). J. Mol. B i d . 12, 866. IIorne, R. W. (1965). I n “Qu;intitdtive Electron Microscopy” ( G . F. Bahr and E. H. Zeitler, eds. ), p. 316. Willianis & Wilkins, Baltiinore, Maryland. Horne, R. W. ( 1968). In “hlethods in Virnlogy” ( K. Maramorosch and H. Koprowski, ecls.), Vol. 3, p. 522. Acadeinic Press, New Yolk. Horne, R. W., and Creville, C . D. (19G3). J. Mol. Biol. 6, 506. Hummeler, K., Anderson, T. F., ancl Brown, R. A. (1962). Virolo,g!g lG, 84. Humphrey, J . H., and Ilourmashkin, R. R. (1965). In “Complement” ( G . E. W. Wolstcnholme and J. Knight, eds. ), p. 175. Churchill, London. Ishizakn, K., and Campl)rll, D. H. (1959). J. Inimunol 83, 318. Kalxit, E. A. ( 1968). “StructitraI Concepts in Imninnohgy and IminuI-lochemistry,” 13. 192. Holt, New York. Kalxtt, E. A., and Mayer, hl. hl. ( 1961) , “Experiinental Immunocliemistr~,”p. 133. Thomas, Springfieltl, Illinois. Kishiinoto, T,, Onoue, K,, and Yaniamura, Y. ( 1968). J. Imniurtol. 100, 1032. Lnfferty, K. J., and Oertelis, S. (1963). Virolog!/21, 91. Lamin, bl. E., and Small, P. A. (1966). Biochenti.ytry 5, 267. Lcberman, R. ( 1963). J. Mol. B i d . 13, 606. Mellenia, J . E., Van Britggen, E. F. J., and Gruber, M. (1968). J . Aid. B i d . 31, 75. hlerler, E., Karlin, L., and hlatsumoto, S. ( 1968). J. Biol. Chcnt. 243, 386. Mihaesca, C . , and Seligmann, M. (1968a). I . Exptl. Med. 127, 431. Mihaesco, C., and Seligmann, hl. ( 196813). Immunoclwmistry 5, 457. Miller, F., ancl Metzger, H. ( 1965a). J. Biol. Chern. 240, 3325. Miller, F., and hfetzgcr, H. ( 19651) ). J. Bid. Chctn. 240, 4740.

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Miller, F., and Metzger, H. (1966). J. Biol. Chem. 241, 1732. Miiller-Eberliard, H. J. (1968). Aduan. I~ninirnol.8, 1. hliiller-Eberhard, H. J., and Calcott, M. A. ( 1966). Immunoclaeniistry 3, 500. Neurath, H. (1939). J. Am. Chem. SOC. 61, 1841. Noelken, hl. E., Nelson, C. A., Bnckley, C. E., and Tanforcl, C. (1965). J. B i d . Chem. 240, 218. Oncley, L., Scatchard, G., and Brown, A. (1947), J. Phys. Chem. 51, 184. Onoae, K., Yagi, Y., Grossman, A., and Pressinan, D. ( 1965). Immunochemistry 2, 401. Onouca, K., Grossberg, A. L., Yagi, Y., and Pressman, D. (196%). Scietice 162, 574. Onouc, K., Kishimoto, T., and Yamamura, Y. ( 19681,) . .1. Inzmrittol. 100, 239. I’enhoet, E., Kochman, M., Valentine, R. C., and Rutter, W. J. (1967). Biocliernistry 6, 2940. Hifkintl, R. ( 1968 ) . I n “Methods in Immunology and I~nmunoclie~~iistry” ( C. A. Williams and M. W. Chase, eds.), Vol. 3. (in pi-ess). Academic Press, New York. Robinson, 1. 1’. ( 1966). J. Mol. B i d . 17, 456. R o w , A. J. (1966). J. M o l . Biol. 16, 553. Stanworth, D. R., and Pardoe, G. ( 1967 ). In “Handbook of Experimental Imniunology” ( D. XI. Weir, ecl.), p. 298. Blackwell, Oxford. Steffen, C. ( 1968). Z. lninirinilatsfnrsch. Allerg. Klim. lmrnunol. 135, 395. Steiner, L. A., and Lowey, S. (1966). J. B i d . Chem. 241, 231. Stone, A I . J., and hletzger, H. (1968). J. Biol. Cheiti. 243, 5977. Svrhag, S.-E., and Bloth, B. ( 1967). Virologil 31, 676. Svrliag, S.-E., Chesebro, B., and Bloth, B. (1967a). Science 158, 933. Svchag, S.-E., Chesebro, B., and Bloth, B. ( 1967b). Nobel S!/mp. 3, 269. Wiley (Interscierice), New York. Svehag, S.-E., Chesebro, B., and Bloth, B. (1968a). J. Exptl. Med. 127, 749. Svchag, S.-E., Chesebro, B., and Bloth, B. ( 1968b). Bull. Soc. Chim. Biol. 50, 1013. Svehag, S.-E., Bloth, B., and Seligmann, M. (1969). J. Exptl. Med. ( i n press). Terry, W. D., hlatthems, B. W., and Davies, D. R. ( 1968). Nutiire 220, 2.39. Valentine, R. C . (1959). Nature 184, 1838. Valentine, R. C. (1961). Arloan. Virirs Res. 8, 287. Valentine, I{, C. (1967). Nohel Symp. 3, 251. Wiley (Interscience), New York. Valentine, R. C. ( 1969). Proc. Europeaii Regional Cotif. Electron Microscopy, Rome, 1968. VOl. 2, p. 3. Valentine, R. C., and Green, N. M. (1967). J. Mol. Biol. 27, 615. Valentine, R. C., and Horne, R. \V. (1962). In “The Interpretation of Ultrzstructnre” ( R. J. C . Harris, ed. ), p. 263. Academic Press, New York. Valentine, R. C., a i d Pereira, H. G. (1965). J. Mol. B i d . 13, 13. Valentine, R. C., LVrigley, N. G., Scrutton, hl. C., Irias, J. J., and Utter, M. F. (1966). Biochcmistq 5, 3111. Valentine, R. C., Shapiro, B., and Stadtinan, E. R. (1968). Biochemistry 7, 2143. Wahl, P., and Weber, G. ( 1967). J. Mol. B i d . 30, 371. Wcltman, J. K., and Edelnian, C . M. (1967). Biochemistry 6, 1437. Wilcox, W. C., Ginsberg, H. S., and Anderson, T. F. ( 1963). 3. Expt!. Med. 118, 307. \\’illianis, R , C., and Wyckoff, R. W. G. (1945). Proc. Soc. Exptl. B i d . hled. 58, 265

Genetic Control of Specific I m m u n e Responses’ HUGH 0.McDEVITT’ A N D BARUJ BENACERRAF Loborafory o f Immunology, Nofional lnsfifufeo f Allergy and Infectious Diseases, Nafionol lnsfitutes o f Health, Bethesda, Maryland and Division of Immunology, Department o f Medicine, Stanford University School o f Medicine, S f a n f o r d , California

I.

Introdaction

.

.

.

.

.

.

.

.

.

.

.

.

.

11. Constitutional Differences in Individual Responses to Con~plrx Multi-

deterininant Antigens

.

.

.

.

.

111. Analysis of the hlechanism of Gene Action

I\’.

. .

. .

. ,

. .

. ,

Genetic Differences iii Immrine Response to Defined Protein Antigcms

V. Grnetic Differences in Immune Rrsponses to Synthetic Polypeptide . . . . . . . . . . . . Antigens .

. .

.

.

The Immune Response to Linear Random Copolymers of L-a-Amino Acids . . . . . . . . . . . . . B. The Iminiinc Rcaponsc~ of Guinea Pigs to Poly-L-Lysine and to . . . . . . . Hapteii-I’ol\.-~-Ly\ine Conjugates C. The Hesponse of Mice to Aranclie:l, hlnltichain Amiiio Acid Copolymers . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .

A.

VI.

I.

Introduction

The immune responsc to a specific antigen is a complex process which must involve genetic control at several levels. Following introduction into the animal, the antigen must interact with one, and probably several, cell types, thereby initiating a complex process of cell division and diffcrentiation which results in the appearance of plasma cells producing specific antibodies against the antigen and in the appearance of sensitized lymphoid cells capable of specific intcmction with the antigen. Not all of the steps in this process are known, but it is clear that genetic control could be exerted at many different points. Experimentally, the genetic control of specific immune responses can be approached in two ways: ( a ) by structural analysis of the products of the immune response-the antibodies; or ( b ) by scarching for and analyzing genetic control of the ability to recognize and respond to specific antigens. Genetic and structural analysis of normal immui~oglobuliiis,inyeloma proteins, and antibodies ( 1 ) has produced a great dcal of information ___ ‘ \Vork supported iii part b y U.S.P.11.S. grallt ilI 07757 Senior Iiivc’stig!;itor,Arthritis Fountlation.

31

32

HUGH 0. h l C DEVITT AND BARUJ BENACERRAF

about the genetic basis of antibody structure but has not yet given us a clear picture of the genetic basis of antibody specificity. The rabbit heavy-chain, allotypic markers controlled by the a locus are found on y , a, and p heavy chains (2, 3 ) . Since the a locus allotypic markers appear to be controlled by allelic genes, whereas the cistrons controlling the y-, a-, and p-immunoglobulin classes are nonallelic and present in all animals, this finding implies that the rabbit H-chain is controlled by at least two different structural genes. The evidence presently available indicates that the structural diff ercnces correlated with individual a allotypic markers are present on the N-terminal or variable half of the rabbit H-chain ( 4 ) . This finding further suggests that the segment of the variable region containing the a markers is coded for by a singlc gcrm line g e m since, if thcre were a large nuinbcr of such genes, crossing over would quickly lead to the loss of the genetic polymorphism represented by allotypes. The rabbit H-chain allotype data is, therefore, compatible with the existence of at least two separate cistrons-one coding for the variable region and one coding for the constant region-which combine in some way to code for the entire H-chain. Structural analysis of myeloma proteins has led to a similar conclusion for the human ( 5 ) (where three K subtypes have been described) and mouse light chain ( 6 ) . Amino acid sequence studies on mouse K-type Bencc-Jones myeloma proteins have shown that there are at least two different K-chain variable regions differing by four amino acid residues in length, both of which havc been found on K-chains with the same constant region amino acid sequence ( 6 ) . The implication of these results is that light chains are also coded for by two different structural genes, but that, in this casc, there are several variable region genes found in association with the same constant region gene. Thus, the results of genetic and structural analyses of immunoglobulins are in accord in indicating that both light and heavy chains have an N-terminal variable region and a C-terminal constant region, and that these regions are coded for by separate structural genes. The genetic control of antibody specificity is less well understood, and two nicchanisms have been postulated for generating the variability seen in the N-terminal regions of both light and heavy chains. One postulated mechanism is that there exist a large number of germ line genes, each of which codes for a particular variable region sequence ( 6 ) . The alternate postulate is that thcrc arc vcry few germ line, variable region genes, with a very high degree of somatic variability introduced by somatic recombination ( 7 ) or somatic mutation ( 8 ) . There are arguments for and against both of these hypothetical mechanisms, but there is no convincing

GENETIC CONTHOI, OF SI’ECIk’IC I \ I l I U S E

RESPOSSES

33

evidence for eithcr one. At the present time it is difficult to reconcilch the allotypic data on the rabbit I-I-cliain ( 2 - 4 ) with the existc>nccof a large number of germ line V genes coding for the entire variable segment of the chain. It is conceivable that enough sequence or allotype data will accumulate to permit a choice betwecm the two postulated mechanisms for the origin of antibody variability, and thus lead to greater understanding of the genctic mcchanisni by which nntibody structure has evolved and is controlled. It is possible, however, that a coinplet-e understanding of the genetic control of specific immune responscs will require genetic studies on the inheritance of the ability to rccognize antigenic determinants and to synthesize specific antibodies. This ~7ouldrequire the detection of individual animals which arc incapaldc of selected specific rcsponscs. Considering the enormous hctcrogeneity of the iinmune response, the likelihood of finding animals unabIe to respond to R particular antigen would be expected to be low, but studirs of this problem have, nevertheless, resulted in the detection of a wide variety of specific nonrespondcrs and have shown that the ability to respond to specific antigens and to specific :intigenic configurations is under direct genetic control. This revicw is concerned with a description of these studies and an evaluation of their implications. It is written in the expectation that the final rcxsults of these experiments will complement gcnetic and stnictural analyses of immunoglobulins to permit a more complete picture of the genetic basis of the imm LIIW response. II.

Constitutional Differences in Individual Responses to Complex Multideterminant Antigens

In an early study, Gorcr and Schutze were able to demonstrate a correlation between antibody formation and resistance to Snlnmrze&z infections in strains of mice which were geneticnlly resistant to this type of infection ( 9 ) . One of the most straightforward demonstrations that genetic factors play a role in the capacity to form specific antiliodics was presented by Scllcil~el( 1 0 ) in 1943. Random-1)red guinea pigs were iinmuiiizcd with diphtheria toxoid and were separated into good and poor responders to this antigen. There thcn followed several gencrations of selective brccding in which good rcq~onderswcrc brcd with good responders and poor respondcrs with poor. Thc results of s e ~ ~ geiiera~ a l tions of such sclectivc breeding are shown in Fig. 1. It is clerir that b y such a process of sclcsction it is possible to producc populations of guincn pigs that are unifonnly p c i d or uniforinly poor responders to diphthcria

34

HUGH 0. M C D E V I T T AND BARUJ BENACERRAF

a0

-

60 40 20 -

CURVE 2 100-

a0 -

-

60 40 20

-

-

51751 F,[1791 FJ2251 511621 51691

FIG.1. Curve 1 s h o w the percentage of good antitoxin pro-icers (dashed line) and poor antitoxin producers (solid line) in successive generations of inatings between good producers. Curve 2 is similar, and shows the results for matings between poor antitoxin producers with progressive selection for good or poor response. ( Froln I. F. Scheibel, Actn Pothol. Illicrobiol. Scnnd. 20, 464484, 1943. )

toxoid, and Scheibel suggests that the small number of generations required for such a selcctivc process indicates that there were relatively few genes segregating betwcen the good and poor responders. By using a similar approach, Biozzi et al. ( I 1 ) were able to select for thc capacity to procluce sheep rcd cell agglutiiiins in random-hred Swiss

GENETIC COSTROL O F SPECIFIC IMMUNE RESPONSES

35

mice. By the ninth generntion, thcre was a thirty-fold difference in mean heinagglutinin titer in the high- and low-responding populations. It is intcresting to notc that selection for response to sheep red cells also led to a similar segregation in the rcsponse to pigcon eiythrocytcs. In addition to these detailed genetic studies, nunierous observations with a wide variety of antigens and in several different species have shown that there is either reproducible strain variation or a heritable influence on ability to respond to particular antigens. Fink and Quinn ( 1 2 ) found marked diff ercnces in the quantitative ability of inbred strains of mice to respond to a battery of five different antigens. Ipsen ( 1 3 ) showed that there were large differences between inbred strains of mice in the dose of tetanus toxoid required to elicit the same antitoxin titer. Dineen ( 1 4 ) found a large quantitative variation in the ability of diffcrcnt inbred strains of mice to produce antisheep red ccll antibody. Carlinfanti ( 1 5 ) demonstrated in man a statistically significant correlation between thc isohcmagglutinin titer of parents and offspring. Stern, Brown, and Davidsohn ( 1 6 ) studied the production of natural antisheep red cell agglutinins in two inbred strains of mice, their F, offspring, and the two reciprocal backcrosses. The results demonstrated a quantitative, dominant genetic effect. Playfair ( 1 7 ) dctcctcd differences in the number of antisheep red cell plaque-forming cells produced in different inbred strains of mice after a single standard dose of s h c q red cells given on the seventh day of life. Analysis of the F, and backcross populations resulting from a cross between strains producing very diff erent numbers of plaque-forming cells indicated a definite genetic effect. Sobey and Adams ( 2 8 ) found a heritable factor controlling the response of mice to thc antigens of Rhixobizim meliloti and to two strains of influenza virus. Sobey, Magrath, and Reisner (19 ) have recently shown that randoin-bred mice can be selectively bred for inahility to respond to bovine serum albumin ( BSA ) . Thcir results are very similar to those of Scheibel ( l o ) ,since within five generations they were ablc to produce offspring which were over 90% unresponsive to BSA. However in a subsequent study, Hardy and Rowlcy ( 20) demonstrated that these genctically unresponsive mice were, in fact, able to respond quite vr7eIl to BSA when they were given n lower dose of antigen. Polak et ab. ( 2 1) studying hyperscmsitivity to reactive cliemicals have demonstrated a striking, antigen-specific, genetic control of contact sensitization in guiiiea pigs. Thcy showed that strain 2 guinea pigs could readily be sensitized to potassium dichromate and beryllium fluoride, but not to mercuric chloride, whereas strain 13 guinea pigs could not bc sensitized to potassium dichromate and beryllium fluoride but were

36

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

rpadily sensitized to mercuric chloride. By using Hartley guinea pigs, these workers showed that crosses bctwccn two strong reactors or two negative reactors gave rcwlts compatible with genctic control of contact sensitivity to these metals. The immune response of about 20 inbred mouse strains to influenza viruses: was: studied by Von Sengbush and Lcnnox (22) following n single injection of virus suspensions. Antibody was assayed by i t 5 inhibition of red cell agglutination. Thc mouse strains could be grouped roughly into good responders and poor responders to all influenza virus strains investigated. Crosses of several of the good respondcrs by one of the poor-responding str‘iins ( DBA/2) yielded in general good responders. However, a most interesting finding was that the mating of two lowresponder strains, DBA/2 N, yielded hybrids that were good rcsponders to FM1 and WSE viruses. (These were the only poor x poor hybrids tested.) These findings are of considerable interest as they indicate a multigenic control of the immune response of mice to influenza virus antigens. The immune responsc of domestic inbred mice to the erythrocyte antigens Ea-1“ and Ea-1” found only in wild type Mus mnsculus has been studied by Gasser (23). Domestic mice arc type Ea-lo. Gasser has shown that several domestic inbred strains differ in their ability to produce agglutinating antibodies against Ea-la and Ea-1”. Breeding studies have shown that this difference is due to a single Mendelian recessive factor linked to the agouti coat color locus in the Vth linkage group. This genetic control, designated Ir-2 by Gasser, maps very close to two minor histocompatibility loci, H-3 and H-6, in preliminary mapping studies. Thc possible significance of linkage between gcnetic controls of the immune response and loci determining histocompatibility antigens will be discussed below (Section V,C). However, it should be noted that the gene controlling the Ea-1 erythrocyte antigens is located in the XVIIIth linkage group ( 2 3 ) , and that anti-Ea-la or anti-Ea-1’’ antibody from a highresponding strain was not absorbcd by erythrocytes from a low-responding strain. Both of these findings suggest that low response to Ea-la or En-1” is not due to a sharing of antigenic determinants bctween the wild type erythrocytes and those of the low-responding domestic strain (see Section 111). All of the studies cited above have established the existence of constitutional differences in immune response to complex, niultideterminant antigens. Breeding experiments have confirmed the genetic origin of these differences, and have indicated that there are often multiple genes involved, as might be expected for the control of responses to complex

x

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

37

antigens. Thc operation of multiple genes is shown by the rcquircmicnt for several generations of selcctive hrecding to obtain uniform population$ of low nnd high responders. While the data do indicatc that thcre arc numerous instances of gcnetic control of specific immune responscs, they do not permit us to draw any conclusions conceiiiing the mechanism of gene action. Ill.

Analysis of the Mechanism of Gene Action

Studies with antigens of known structure or synthetic polypeptides of restricted heterogeneity have shown that, in sevcral systems, there are dominant, autosonial genes controlling specific immune responses. Many of these systems have been extensively investigated in an attempt to deteiniine the mechanism of gene action. Before describing in detail the observations and conclusions made in each of these system5 (Sections IV and V ) , we would like to present an analysis of the different levels at which genetic factors could be expected to affect the immune response to a specific antigen. In studying any genetically controlled immune response to a specific antigen, an attempt should first bc made to determine whether the ability to respond is a dominant or recessive trait. It is a general rule that an animal is immunologically tolerant to his own antigcns, and it is likely that there will be instances of genctic control in which an animal is unable to respond to a particular antigenic determinant because it c r o s reacts with his self-antigens. Since moat self-antigens are under dominant or codominant genetic control, inability to rcspond would be expectcd to be a dominant trait and ability to rcspond a recessive trait. For this rcasoii, genetic controls in which ability to respond is recessive should first be considered to be examples of “cross-tolerance.” This hypothesis can be testccl by showing that antibody to the antigenic determinant in question produced in another strain also reacts with cells or antigens of the strain which is unable to rcspond. This type of gcnctic control of response to cert,un antigens has been extensively discussed by Cinadcr (24). In the analysis of dominant genetic control of the immune response to a specific antigen, three major mechanisms of gene action must be considered. First, a gene may control antigen-specific processes that are completely unrelated to any aspect of the immune response. It is obvjous that this is the first possibility that must bc ruled out before a useful analysis of any experimental system can be carried farther. The second type of gene may control ‘I process which is ‘in integral pait of a specific immune response but is not responsible for the development of specific cells or

38

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

the structure of specific immunoglobulins. For example, a gene conccrncd with the binding of certain antigens to macrophages or macrophage components might express a certain degree of specificity for the antigen. The third and most important type of genetic factor to be considered is onc which would control the antigen receptor of a specific cell and/or the structure of specific immunoglobulins. These three types of gene action may be differentiated by the detailed study of ( i ) the specificity of thc sensitized cells and/or of the antibody produced; (ii) the results of attempts to transfer the capacity to respond with various cell populations; and (iii) the identification in such transfer experiments of the origin (donor versus recipient) of the cells responding and of the antibodies produced. IV.

Genetic Differences in Immune Response to Defined Protein Antigens

Arquilla and Finn presented some of the earliest evidence that the specificity of antibodies produced in different animals in response to the same antigen was under genetic control (25). Thcse authors used an insoluble insulin-cellulose conjugate saturated with antibodies from a reference rabbit antiserum, and then tcsted the ability of anti-insulin antibodies from strain 2 or strain 13 guinea pigs to add onto the antibody-insulin-cellulose complex. In several different experiments, strain 2 antisera contained antibodies which bound to portions of the insulin molecule not already covered by antibody from the reference rabbit antiserum. This was in marked contrast to strain 13 anti-insulin antisera, which were unable to bind to the insulin-cellulose conjugate after saturation with antibody from the reference rabbit antiserum. This result indicates that strain 2 guinea pigs produce antibodies to portions of the insulin moleculc to which strain 13 animals are unable to produce antibodies. Since it was impossible to demonstrate additional binding when either strain 2 or strain 13 antiinsulin antisera were used to saturate the insulin-cellulose conjugate prior to the addition of either strain 13 or strain 2 antisera, it was concluded that the two strains of guinea pigs produce antibodies to antigenic sites on the insulin molecule which are in close proximity. These studies can be criticized on the grounds that the assay system detects only hemolytic antibody and that a quantitative difference in the amounts of antibody of differing specificities might appear to be a qualitative difference. Despite these objections, the evidence supports the concept of genetic control of the ability to produce antibodies of different specificities against the same antigen (26). Further support for this concept was obtained through the use of modified insulin derivatives. Removal

GENETIC CONTROL OF SPECIF'IC IMMUNE RESPONSES

39

of the eight C-terminal ainino acids from the insulin B-chain resulted in a much greater loss of renctivity with strain 13 than with str,iin 2 anti-

insulin antisera. Conversely, reaction of thc N-terminal cu-miiiio groups on the A- and B-chains with fluorescein resulted in a prefercntl'il loss of reactivity with strain 2 anti-insulin antisera. These results indicate that strain 2 guinea pigs produce antibodies that react preferentially with the N-terminus of the insulin A- and R-chains, whereas strain 13 guinea pigs produce antiboclieq that react prefcrentially with the C-terminal portion of the insulin molecule (27). While these results are compatible with some heritable genetic control of the structure of antibody-combining sites, a much more detailed analysis of the mechnnism of gene action will be required before this conclusion can be supported with any force. Evidence supporting the concept that the recognition of an antigen is genetically controlled ( see Section V,B ) was obtained by Armerding and Rajewsky (28) who studied the immune responsc of rats to porcine lactic dchydrogenase ( LDH ) isoenzymes. The LDH isoenzymes are tetrameric molecules composed of two typcs ( A and B ) of iinmunologically non-cross-rencting subunits in all possiblc combinations. Outbred Sprague-Dnw ley m d Wistar rats respond equally well to LDH-B,, but differ markedly in their response to LDH-A,. Sprague-DawIcy rats respond well to LDH-A4,while Wistar rats respond poorly, only at higher doses of antigens, and fail to give a clear-cut secondary response. However, when nonresponder (Wistar) rats were primed and boosted with A-subunits coupled to B-subunits, definite secondai y responses were observed, suggesting that the gcnctic dcfect in Wistar rats is a inilure to recognize the A-subunit as antigenic, in the presence of a normal ability to produce anti-A antibody. The ability to respond to LDH-A, is inherited as a single dominant gene which is not linked to allotype marker\ very probably located on Iight chains of rat immunoglobulins (28) ( see Section V,C ) . V.

Genetic Differences in Immune Responses to Synthetic Polypeptide Antigens

A. THE IMMUNE RESPONSE TO LINEARRANDOM COPOLYMERS OF L-WAAIINO ACIDS When random copolymers of L-@-aminoacids became available to immunologists (29-32), it was soon realized that the immiinogenicity of these materials for experimental animals and man depended primarily upon their degree of complexity. Although the precise amino acid sequence of the random copolymers studied is not known, thc structural

40

HUGH 0. MCDEVITT AND BARUJ BENACERRAF

TABLE I RANDOMLINEARCOPOLYMERS L-PAMINO ACIDS“

IIESPONSE O F VARIOUS SPECIES TO

OF

Coinpositionh

Micec

Rahbit.s

0/58 0/38 0/20 20/20

8/17 60/101

20/20 17/35 10/10 -

5/12 30/4 1 9/12 7/11 4/6 59/59

Ciiinen pigs 68/181

6/21 0/18 3/7 7/24 lO/22 3/5 8/8

Man 0/30 -

0/4 :3/3

2/6 11/12

16/20

From P. Pinchuck and P. H. Maurer, in “ltegdatiori of the Antibody Respoilbe” (B. C i n d e r , ed.), Thomas, Springfield, Illinois, 1968. Sribscripts refer to molar percentage of amino acid in copolymer. Niimher of iespoiicleis/tiiin~l)er immunized.

heterogeneity of these compounds clearly depends upon the number and the relative proportions of the different amino acids which they contain. Thus polymers of a single amino acid or copolymers of two amino acids are considerably less complex than copolymers of three or four amino acids. In the evaluation of the results of immunization with these materials, which are gencrally wcak antigens even in responder animals, it should be stressed that their antigenicity has been evaluated in nearly all instances (except in man) after repeated immunization with complete Freunds adjuvant to ensure maximum responses (30, 3 2 ) . As shown by Pinchuck and Maurer ( 3 3 ) , the immunogeiiicity of thesc synthetic polypeptides for different species increases, with the number of amino acids they contain both with respect to the number of responding animals and the amounts of specific antibody produced. Thus, as shown in Table I, random copolymers of four amino acids-glutamic acid, lysine, alanine, and tyrosine (GLAT)-or of three amino acids-glutamic acid, lysine, and alanine (GLA)-arc immunogenic in the majority of the animals tested. In contrast, homopolymers of single L-amino acids are rarely antigenic. Poly-L-alanine, poly-L-glutamic acid, and poly-L-tyrosine do not induce specific immune responses (30, 32, 3 3 ) . Poly-L-lysine (PLL) and poIy-L-arginine are only immunogenic in guinea pigs possessing the PLL gene, as discussed in Section V,B. The nonantigenic homopolymers may, nevertheless, behave as excellent haptens and induce specific antibody synthesis when bound to immunogenic carriers (34, 3 5 ) . Co-

GENETIC CONTROL O F SPECIFIC IMMUNE RESPONSES

41

polymers of two L-amino acids (or of two L-amino acids with only a vcrv small proportion of a third amino acid), e.g, gliitamyl-alanine ( G A ) , glutamyl-lysine ( GL ) , glutamyl-tyrosinc ( GT), arc most intcresting compounds since they are able to induce a significant immune respoiisc only in certain individuals of a givm spccics and in some inbred strains but not in others (Table I ) (30, 31, 36, 37). Copolymers GA, GL, and GT are recognized as antigens by some but not all random-bred guinea pigs and rabbits (30, 33, 3 6 ) and by none of the random-bred or pure strain mice tested so far ( 3 8 ) . Similar observations were also made by Sinionian et al. ( 3 9 ) on the response of inbred rats to several copolymers of several amino acids. In this species, there also exist largc quantitative strain variations in immune responses to the same polypeptide. The ability of a particular strain to respond well to one antigen did not correlate with its ability to respond to a second sti-ucturally different copolymer. The pattern of response to these homopolymers and copolymers with relatively simple strrrctnrc, suggested that the individual variations observed could be explained by the presence or absence of specific genes controlling the capacity to recognize thcw respective structures as antigenic determinants ( 36, 40). Breeding experiincnts have lwcn performed with two of thesc systems: (1) the immune response of guinea pigs to PLL and to hapten conjugatcs of this homopolymer, which will be discussed in Section V,B; and ( 3 ) Pinchuck and Maurer investigated the immunogenicity for mice of a random copolymer of glutamic acid and lysine with only 5% alanine, Gj7Ll8Aj( 4 1 ) . This polypeptide was found to induce specific antibodies in only 47%of random-bred Swiss mice a s detemiined by passive hemagglutination following several courses of immunization with complete adjuvants. This copolymer was also immunogenic in the following mouse inbred strains: C3H/ HeJ, BALB/cJ, 1391J, but not in C57RL, A / J, or CBA/ J strains. (If, however, the relative content of alanine was raised from 5 to lo%, all mice including the nonresponding strains formed antibodies against GLAIo after standard himunization with this polypcptide. ) The pattern of transmission of the ability to respond to GLA, in the progeny of responder and nonresponder Swiss mice was then investigated (41) . ( 1) The mating of nonresponder parents produced consistently nonrcsponder offspring. ( 2 ) The progeny of the mating of responder mice consisted of 19 responder and 3 nonresponder offspring; when 2 of these nonresponders were, in turn, mated they produced only nonresponder offspring (Table 11). ( 3 ) Furthermore, the specific F, hybrids of responder C3H/HeJ nonresponder C57BL strains were all able to form antibodies to GLAj whcn immunized with

x

42

HUGH

0.

MC DEVITT AND BARU J BENACERRAF

TABLE I1 I M M U N E 1 t . E S P O N S E S TO COPOLYMER O F GLUTAMIC ACID,

(GL&)

BY O F F S P R I N G O F

R.ESPONDERA N D

LYSINE,A N D

~ 0 N R E S I ' O X I ) E R HIVISS

ALANINE

MICE

.4ND I N B R E D h f l C E "

1':trents

1'rogelI)b

Bot,h noiiresponder Swiss Both respoider Swiss C3H/HeJ C67BL/6J c:m x C57HL P I

0/19 19/22 6/6 O/X 29/29

From .'I I'iiichiick stid P. H. & h i r e r , .J. h'.rpL/. Alrrl. 122, 67.5, 1965. Ntimber of iespoiic~eis/ririni~ie~ immiinixed.

this antigen. These results indicate that the ability of mice to form an immune response to GLA, is governed by the presence of an autosomal dominant gene. Unfortunately no attempts have yet been made in this system to transfer the capacity to respond to GLA, from responder to nonresponder mice with cells known to be concerned with the immune response (spleen or bone marrow cells). All these experiments on the immune response to random copolymers of L-a-amino acids have concerned themselves with individual differences in the ability to respond to some of these polymers and with the genetic control of the capacity to respond to antigens with relatively restricted heterogeneity but not with the specificity of thc antibodies produced. However, even in those instances [as in the responses of mice to GLA, (41 ) or to branched copolymers, discussed in Section V,C, and of guinea pigs to hapten-PLL conjugates (35,36, 4 0 ) ] where the ability to form a significant immune response is shown to be controlled by single dominant autosomal genes, the antibodies produced are clearly heterogeneous with respect to antibody class, specificity, and affinity. It would, therefore, be of considerable importance for the interpretation of these findings if the specificity of the antibody populations produced b y individual responder animals or by responder inbred strains to some o€ these antigens could be shown to be characteristic of the individual or of the strain and if these specific properties of the antibody population could be shown to be inheritable. In an attempt to achieve this result, the specificity of the antibodies produced by inbred mice to selected copolymers was investigated by Pinchuck and Maurer in collaboration with Bcnacerraf ( 33, 4 2 ) . Thc patterns of cross-reactivity with related polypeptides displayed by antibodies produced by different mouse strains or diffcrent individual mice

GENETIC COSTROL OF SPECIFIC IhlhlUXE RESPONSES

43

to different copolymer were investigated. For instance, inbred mice were immunized with G,,,A,,T,, or GLA,, and the cross-reaction of the specific 'intibodies with GA, GT, and GL, respectively, were investigated. The pattrm of cross-rcactivity w a s found to be charactcristic of the strain (Table 111) which indicates that genetic factors are concerned with the T IRLTC 111 PRECIPITIV I:F4< T l O \ b OF I \ H R E U .1\TI-C;I.i

T411 IC

hfOLT\E s T R 4 I h IIY P E R IZ IR Il~\E

I( li)-.I1,4\1\ E-TYRO~IYE (( ;,ZT)

sER.\"

St rail1 of mice

I'ci~cciI t age -re:ictioii

C:S11/1 IeJ

(76)

aitli

t :.vr

loo~o'y4'21)'

G so.Ir0 c: ,oT L O

3)''

-1,

121

120/.J ( %)

I 00yoy802)" 40 9,5

n From P. Piiichiick :tiid P. H. ~ I a i i r e r In , "RegiiI:~tioii of the *kirtibo(Iy I ~ e s p o n ~ e " (B. Ciiinder, ctl.), Tliornas, Spiiirgfieltl, Illinois, 1968. h c

Percent of homcilogoris ant igm. hlicrograms A41)S / m l .

capacity to recognizc specific detcrminants on these molecules. These observations are analogous with the findings of Arquilla and Finn (25, 26) concerning the specificity of the antibodies produced by strain 2 and strain 13 guinea pigs to bovine insulin (discussed in Section I V ) which showed that these two strains of guinea pigs produced antibodies directed to differcnt deterniinants on the insulin molecule. If the genetic factors controlling the ability to respond, respectively, to GLA,, haptenPLL, or (T,G)-A--L and ( H,G)-A--L ( scc Section V,C) arc, indeed, concerned with the ability to recognize specific detcrminants on these niolecules, it is not surprising that unifactorial genetics have been observed only with such synthetic polypeptides of limited complexity. With more complex antigens the multiplicity of genes concerned and, therefore, of possible responses no longer permits genetic andysis.

B.

RESPONSE OF GUINEAPIGS TO POLY-I,-LYSINI: AND HAPTEN-POLY-L-LYSINE CONJUGATES

T H E Ih4h.fUNE

TO

1. Ncituie of the Response As originally olwrvcd by Kantor, Ojeda, and Benaccrraf ( 3 6 ) , approxiniately 30% of Hartley strain guinea pigs immunized with 0.001 to 1 mg. of 2,4-dinitrophenyl-pol!'-r.-lysinc ( DNP-PLL ) in complete

-kljuvatit and zaliiie

10-19

3

D S P-PLL

75-85

2

DSP-PLL RS i

2

DXP-(;L

1

"

13

13-19

1 'b (1 39-0 70) 9 1 (10 5-7 7) .5 69 (6 6-4 7) DSP-GL (100 pg./ml.) 7.71 (7 .6.i-6.78)

1 07

-

-

9 .i (20 9-3 4) 3 0,i (4 4-1 731

-

-

( 1 22-0 97)

6 84 (6 84-6 81)

DNP-G L

DSP-GL

( 1 pg.ln11.) 6.74

( 0 . 0 1 pg./ml.) 3.14 (3.72-2.36)

(7.30-3.46)

1 .?8 (1 6 1 .i6)

-

tlinitrophenyl; PLL = p ~ l y - i ~ l y s i BYA ~ ~ e ;= bovine seriim a1t)iimiii; GL = glritxniyl-Iysine. roiitrts in DN.1 f r o m experimental culttires with antigen . Tallies > 1 indicate stimrilatioti of DXA This number is the ratio, c'otiiit9 i l l D S - 1 from roiitrol cultures withorit antigen

DSP

=

ci P

3 6" cl

td

m

z

P 0

GENETIC COSTROI, OF SPECIFIC 1MMUhTE RESPONSES

45

Freund's adjuvant containing 0.5 mg./ml. Mycobacterium butyricurn ( Difco complete adjuvant ) produccd an immune response characterized by the development of delayed hypersensitivity to DNP-PLL and, at 2 to 3 weeks after immunization, b y the synthesis of high serum levels (1-2 mg./ml.) of anti-DNP-PLL antibodies, belonging to both ys- and y ,-immunoglobulin classes. Their lymph nodc cclls are able to respond to DNP-PLL in culture with increased DNA synthesis (43, 44) (Table I V ) . This reaction and delaycd hypcrscnsitivity rewtions to DNP-PLL ( $36) show specificity for the PLL carrier a s well as for the haptcn--a phenomenon which is known to cliaracterizc the response of sensitized cells to hapten-protein conjugates ( 4 5 ) . In contrast, nonresponding guinea pigs fail to bcconie delayed sensitive to DNP-PLL (35, 36) or to display in vitro evidence of cellular immunity (43, 4 4 ) (Table \') and do not produce anti-DNP antibodies detectable by double diffusion in agar gel or anaphylaxis with DNP-proteins (361, or by cquililx-ium dialysis with ,"H D~P-E-arninocaproic acid ( 3 5 ) . The two available inbred strains of guinea pigs also differ markedly in their rclsponse to this antigen; strain 2 guinea pigs show the characteristic rmpoiise to DNP-PLL, displaying both delayed sensitivity and high sei-um concentrations of specific antibodies, whereas strain 13 guinea pigs show no evidence of an immune rcsponse whcn injected with DNP-PLL in Difco compIcte adjuvant ( 4 0 ) (Table V I ) . The same guinea pigs (responders) with the ability to respond to DNP-PLL can also be sensitized by unconjugated PLL ( 3 5 ) and to conjugates of PLL with other haptens, immunologicidly unrelated to DNP, such as the benzylpenicilloyl and the p-toluenesulfonyl haptens ( 4 6 ) . Guinea pigs that do not recognize PLL as an antigen arc not able to form significant immunc responses to any hapten coupled to the homopolymer. Respondcr guinea pigs immunized with PLL in complete adjuvants develop clelayed hypersensitivity to this polymer but no detectable serum antibody ( 3 5 ) . In addition, only the identical randombred Hnrtley strain and strain 2 guinea pigs capablc of responding to PLL and to hapten conjugatcs of this polymer recognize as antigcms a copolymer of 1,-glutamic acid and ~-lysinc,G,,,,L,,,(the subscripts refer to the molar pcrcentage of the amino acids), and DNP conjugates of this copolymer, although no significant immunological cross-reaction can bc detectcd betwecn GL and DNP-PLL eithcr in the specificity of the antibodies produccd or in thc response of thc sciisitized cells i n viva or in oitro to these antigens ( 3 6 ) . Strain 13 guinea pigs do not rcspond to GL or to DNP-GL ( Tal~leVI ). Thc virtual idcntity olwrved in the response to PLL and GL, is pro1xtl)ly clxplaincd l ~ thc y c3xistcnce in CI, of c>xtc.nsivc

T.IBI,F. V E F F E C T OF h T I G E X OK THE i / l T’ifW

LY?,lPII X O n E C E L L S FROM DNp-pLL.B&k OR DKP-GL“B‘~

ISCo R I W R . 4 TlOS OF 3 H - T H Y U I ~ I S E 1.U

G E S E T I C S O S R E S P O S D E R G1’ISE.A PIGS IMMUSIZED \YITH

3

Test. aittigens

0

DSP-PLL.BS.1

so.d a y s

S o . of

animals

Tmmiuiizing aiitigen

after

DSP-PLL

DNP-PLL

or DSP-PI,L.OT’A

OVA

?

imrniiiiizatioii

(10 pg./ml.)

(1 pg./ml.j

( 1 pg./ml.)

( 1 pg./ml.1

5

4

DSP-PLL

-

DKI’-PLL.BSA

7

DTP-PI,L.OV.~

11-21

0.707 (1.47-0.438)

2

DSP-C;I,

1% 19

DSP-GL ( I 0 pg.lml.1 1.13 (1.17-1.09)

)

31

14-13 2”

0.712c (0.91-0.40) -

1.03 (1.4-0.77) 1.24 (1.26-1.22) 1.04 (1.41-0.34) DXP-GL (1 ~g./nil.‘l 1.13 (1.20-1.11)

-

-

7.2

-

4.8 (10.2-1.86)

DNP-GL (0.01 pg./ml.) 0.93 (1.37-0.50)

3 0 (5.0-2.3) -

Fium I. Green, B. B. Levine, If-. E. Paul, and B. Benacerraf, in, “Xiicleic Acids in Immruiology” (0.J. Plescia aiid W. Brawl, eds.), Springer, Berlin, 1968. D S P = diuitropheiij-1; I’LL = poly-I,-lysine; BS.1 = hovine serum a1’r)iimin; GI, = glutamyl-lysine; OVA = O V ~ L ~ ~ ) I I I I ~ ~ I I . coiiiits in DXL1from experimental cidtiires wit.h untigell . Values > 1 iiitlicate stimulntioti of D S . l This niimher is the ratio, counts in D S A from coiitrol ciilt.ures without. antigen synthesis.

cci 4

* 3

m

&C

LI

m

2!

P

n

1 ki

IMMUNE RESPONSES OF STRAINS 2 Guinea Pig strain (No. of animals)

Antigen

AND

TABLE VI 13 GUINEAPIGSTO DNP-PLL

Adjuvant (mg./ml.)

2 (4)

DPS-PLL

0 5 M. butyricirni

(4)

DNP-PLL

(4)

DNP-GL

10 hl. tubercitloszse 0 5

M . bulyrtciini (4)

DIP-GL

10

M. 13 (8)

DIP-PLL

(8)

DXP-PLL

DNP-GL

Stimulation of DNA synthesisb (10 pg. Ag/ml.)

++++ ++++ ++++ ++++

++++ ++++

hTeg.

Seg.

Keg.

Keg.

WITH

DIFFERENTADJUVANTS"

Serum anti-DNP antibodies a t 26 days" (mg./ml.)

Av. % bindingd 3H DNP-EACA

DSP-GL

0 3

8.5

2F

2 3-3 9

89

8

1 4-2 4

87

ATot nieasiirable

7

0-0 1

2
Xeg.

Not mea~rirable

0

Keg.

Sot measurable

3

M . biit?)riclrnl (8)

DNP-GL

10

M . t iiberculoszs a

D N P = dinitrophenyl; PLL = poly-zilysine; GL = glutamyl-lysine. Stimulation of incorporation of 3H-thymidine into DNA by lymph node cells in culture. Precipitin analysis with DSP-bovine fibrinogen. Binding of 0.1 ml. 10-8 M 3H DKP-eaminocaproic acid (EACA) by 0.1 ml. serum. M yobacterzitm tuberciilosis H37Rv strain.

8

0 82-0 94

M. tiibercirloszs (8)

m

88

Oittilrzcirm

10

cl

1 29-2 1

tic brrculosis

0 3

M.

Del. sensitivity to 10 pg. Ag

AND

48

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

PLL sequences. In contrast with the identical behavior of GL and PLL, in PLL responder guinea pigs, immunization of Hartley strain guinea pigs with a copolymer of L-glutamic acid and L-alanine, not containing lysine, and with DNP-PLL shows that these two compounds can be immunogenic in different individual animals ( 33, 43, 4 8 ) . In all the experiments described above, immunization was carried out in complete Freund’s adjuvant with low doses of Mycobacterium butyricum or of Mycobacteriurn tubercdosis H37Ra, 0.5 mg./ml. (commercial Difco adjuvant). Under these conditions the differences between responder guinea pigs (Hartley and strain 2 ) and nonresponder guinea pigs (Hartley and strain 13) immunized with DNP-PLL and especially with DNP-GL are striking, and the responses can be considered to be all or none (49). Nonresponder animals show no evidence of cellular immunity or of synthesis of specific antibodies (Table VI). If, however, the dose of mycobacteria in the adjuvant is increased to 10 mg./ml. and/or a more powerful adjuvant is used ( M . tuberczcZosis H37Rv strain), nonresponder guinea pigs, both Hartley and strain 13, at 3 to 4 weeks after immunization with DNP-PLL, produce very low levels of p anti-DNP antibodies, generally well below 0.1 mg./ml., but still do not show delayed sensitivity to DNP-PLL or other in vitro evidence of cellular hypcrsensitivity ( 50). The response to DNP-GL, a nearly neutral polypeptide, after immunization with the higher dose of mycobacteria is even weaker -strain 13 does not form significant levels of anti-DNP antibodies against DNP-GL, and HartIey strain nonresponder guinea pigs synthesize lower levels of anti-DNP antibodies than after immunization with DNP-PLL. In summary, if higher doses of mycobacteria or more powerful adjuvants are used to immunize with DNP-PLL the differences observed between responder and nonresponder guinea pigs remain qualitative with respect to their capacity to display cellular sensitivity but become quantitative ( although marked, generally over 20-fold) with respect to their ability to form anti-DNP-PLL antibodies ( 50). A study of the response of guinea pigs to the corresponding D-polypeptides with the two types of adjuvant is very informative in this respect. Strain 2 ( 5 1 ) and Hartley strain guinea pigs, irrespective of their PLL responder status, do not become sensitized to DNP-poly-D-lysine (PDL) (35) or to DNP-D-GL and do not form detectable levels of specific antibodies following immunization with 0.5 mg./ ml. M Y C O ~ U C terium butyricum as adjuvant. However, the production of low levels of anti-DNP antibodies, but no cellular immunity, is observed after immunization with adjuvant containing 10 mg./ml. Mycobacterium tuberculosis H37Rv ( 5 2 ) .Thus the response of random-bred guinea pigs to the

GENETIC CONTROL O F SPECIFIC I M M U N E RESPONSES

49

DNP conjugates of the D-polypeptides is analogous to that of the PLL nonresponder guinea pigs to DNP-PLL or DNP-GL. As shown below (Scction V,B,7), increasing the adjuvant may be interpreted as exerting a “schlepper” effect (35, 53) in nonresponder guinea pigs where the DNP-PLL or DNP-GL molecules may behave as haptens. 2. Breeding Experiments Mating experiments with random-bred Hartley strain guinea pigs have permitted an analysis to be made of the genetic control of the response to DNP-PLL. The study of the offspring from the matings of responders, of nonrespondcrs, and of the F, generation from strain 2 and strain 13 guinea pigs shows that the ability to respond to DNP-PLL and, therefore, to GL and DNP-GL is inherited as an autosomal dominant which has been designated as the PLL gene ( 4 7 ) . (These experiments were carried out by immunization with 0.5 mg./ ml. Mycobacterium butyricum as adjuvant, under conditions where nonresponder animals show no evidence of an iinniune response.) This conclusion is based on the following evidence from Levine and Benacerraf (40, 5 4 ) (Tables VII and VIII): (1) the mating of nonresponder parents produced nonresponder offspring; ( 2 ) the progeny from the mating of responder parents consisted of 82%responder guinea pigs; ( 3 ) crosses between nonresponders and guinea pigs heterozygous for the responder trait resulted in a progeny consisting of nearly 50% responder animals ( S 4 ) (in this experiment, heterozygosity was established by the capacity of responder guinea pigs to yield both responder and nonresponder offspring); (4) the F, generation of the mating of strain 2 and strain 13 guinea pigs all TABLE VII A\NTIGENICiTY OF D N P 2 r - P L L , , 6

IN OFFSPRING O F

RESPONUER PARENTS

A N D NONRESPONDER P A R E N T S “ , ‘

50

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

TABLE VIII OF OFFSPRING OF THE MATING(NONRESPONDERS X HETEROZYGOUS PERCENTAGE RESPONDERS) WHO ARE IMMUNERESPONDERS TO HAPTEN-POLYLYSINE CONJIJGATESG.~ OfTspritig Family SO.

I:espoiitlers

Nonresponders

I I1 I11 IV '1' 1'1

VII VIII IX X Total a

14 (45 3%)

17 (54.7%)

From I). B. Leviiie and B. Benacerraf, Science 147,617 (1965).

* The offspring were immiinized with 0.1 mg. of DNPZJ-PLL3la in complete adjuvant.

Itesponders gave positive allergic reactions to the iinmuriiziiig antigen and their serums showed antibodies to DNP. Nonresponders showed no evidence of an immune response t u DNP-PLL. (DSP = clinitrophenyl; PLL = poly-r.-lysine.)

rcspondcd to DNP-PLL as do strain 2 guinea pigs. No evidence of sex linkage was noted in any of these experiments. 3. Studies on the Metabolism of DNP-PLL in Responder and Nonresponder Guinea Pigs

Considering that DNP conjugates of polypeptides of D-lysine are not or only marginally immunogenic, even in guinea pigs capable of responding to DNP-PLL (51, 5 2 ) , and that these polypeptides are poorly degraded ( 5 5 ) , the possibility that nonresponder guinea pigs lack a proteolytic enzyme able to degrade PLL was investigated. Lymph node macrophages from both responder and nonresponder guinea pigs were shown to phagocytize DNP-PLL to the same degree (56). The L polymer, in contrast to DNP-PDL was degraded equally well an vivo into small fragments excreted in the urine of responder and nonresponder guinea pigs. In addition, spleen extracts from both respondcr and nonresponder guinea pigs degraded fluorescein conjugates of PLL in vitro (55).

51

GENETIC CONTROL O F SPECIFIC IMMUNE RESPONSES

4. hlinimum Size of PLL Antigen

Schlossman and associates investigated the ability of DNP conjugates of L-oligolysines of various sizes to induce characteristic immune responses in guinea pigs with the PLL gene (57). Their earlier experiments showed that the smallest molecule consistently immunogenic was a,N-DNP-octalysine. Strain 2 (Table IX) and Hartley responder guinea pigs immunized with a,N-DNP-octalysine or a,N-DNP-nonalysine developed delayed sensitivity to these antigens and synthesized significant levels of anti-DNP antibodies of which the binding energy was almost totally directed to the tetra- or hexalysine conjugate (57, 58). In spite of this, a,N-DNP-hexalysine and lower polymers were not immunogenic and were not able to elicit delayed hypersensitivity reactions in guinea pigs immunized with the higher polymers (59). Guinea pigs sensitized with a,N-DNP-octa- or nonalysine responded to these antigens in vitro with increased DNA synthesis, but the reaction could be neither elicited nor inhibited by larger concentrations of a,N-DNP-hexalysine (60). These experiments demonstrate that a molecule with a minimum of eight lysyl residues is required both to be recognized as an antigen by guinea pigs with the PLL gene and to elicit the response of sensitized TABLE I X RESPONSEOF STRAINS2 AND 13 GUINEAPIGSTO INJECTION OF CY.N-DNP-OLICOLYSINES~ Skin reaction (No. of animals)

Antigens Strain 2 ~~,N-DNP-oligo-r,lysitie (W = 8.4) or,N-DNP-riona-L-lysine CY,N-D N P-oc t a-L-1ysine LY, N-DNP-hept a-L-lysirie CY, N-D NP-hexa-L-lysine a,N-DNP-penta-L-lysirie a,N-DNP-tetrs-L-lysiiie ~~,l\i-DNP-poly-~-lysilie ( A = 80) Strain 13 a,N-DNP-oligo-I,-lysirie (fi = 8.4)

Tested

Iriimediate (3-6 hr. av.)

30 0 9

0 0

10 ) )

Delayed (24 hr. av.)

0 0 0 0 0 0 0 0

0

0 0 0 0

5

o

3

0

Passive cutaneous anaphylaxis

0

0

52

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

cells, in spite of the fact that the serum antibodies produced in response to these antigens bind equally well a,N-DNP-hexalysine. The studies of the immunogenicity of DNP-oligolysines of various sizes, with the DNP determinant on a specific c-amino group, and of the specificity of the immune responses observed could provide considerable information concerning the process controlled by the PLL gene. For this purpose c,N-DNP-oligolysines of various length with the DNP hapten conjugated either to the N-terminal or the C-terminal lysine were synthesized by Paul et al. (61) using the Merryfield technique. The observations of Schlossman et al. were confirmed. Only r-DNP-oligolysines that contained at least eight or nine lysyl residues induced delayed hypersensitivity and were able to elicit delayed reactions and to stimulate increased DNA synthesis by specific lymph node cells in &To. Extensive cross-reactions were observed in these experiments between 1 e,N-DNPnonalysine, 9 c,N-DNP-nonalysine, and also DNP-PLL ( 61 ) . 5. The Response of Guinea Pigs with the PLL Gene to Protamine, DNP-Protamine, and Poly-L-Arginine

The gene in guinea pigs controlling the response to PLL and GL was found to determine also their ability to respond to protarnine, DNPprotamine, and poly-L-arginine. Protamine, a small positively charged protein with a very high arginine content and no lysine, and DNPprotamine were shown by Green, Paul, and Benacerraf ( 6 2 ) to be able to induce specific immune responses only in guinea pigs with the PLL gene. Only strain 2 and Hartley strain guinea pigs whose responder genetic status had been verified by their ability to respond to immunologically unrelated GL were able to develop delayed sensitivity to DNP-protamine and to form anti-DNP antibodies (about 0.3 mg./ml.) following immunization with DNP-protamine in complete Freunds adjuvant containing 0.5 mg./ml. Mycobacterium butyricum. Strain 13 and PLL nonresponder Hartley guinea pigs did not show these responses. Similar results were obtained with unconjugated protarnine. In preliminary experiments strain 2 but not strain 13 developed delayed sensitivity to poly-L-arginine and to DNP-poly-L-arginine following immunization with these antigens in complete adjuvants. 6. Cell Transfer Experiments

As discussed in Section 111, cell transfer studies are essential for an adequate analysis of gene action in these systems. Attempts must therefore be made to transfer to nonresponder guinea pigs the capacity to respond to DNP-PLL or GL with lymphoid cells or macrophages from

GENETIC CONTROL OF SPECIFIC IMMSJNE RESPONSES

53

animals with the PLL gene. Transfer of the capacity to respond to DNPPLL to lethally irradiated nonresponder random-bred Hartley guinea pigs has been successfully carried out by Foerster et al. with bone marrow cells from responder Hartley guinea pigs ( 6 3 ) .The genetic responder status of the animals had been determined by previous immunization with GL. A major di5culty with these experiments was the high mortality (over 70%)from graft versus host reactions in the recipient animals. Nevertheless, of the 15 guinea pigs which survived the transfers and were immunized with DNP-PLL in complete adjuvants, 13 developed delayed sensitivity to DNP-PLL. The lymph node cells of 9 of these animals responded to this antigen in vitro with increased DNA synthesis, and 10 recipient guinea pigs also produced low concentrations of antiDNP antibodies. As control for these experiments, bone marrow cells from 5 nonresponder guinea pigs were transferred to 5 irradiated nonresponder animals. None of these animals developed delayed sensitivity, cellular immunity, or antibodies to DNP-PLL when immunized with this antigen in complete Freund's adjuvant. A more appropriate experimental system involves the transfer of immunocompetent cells from strain 2 X strain 13 F, animals into lethally irradiated stain 13 guinea pigs. Accordingly, the process controlled by the PLL gene has been successfully transferred by lymph node and spleen cells from strain 2 x strain 13 F, animals to lethally irradiated strain 13 animals whose bone marrow had been reconstituted by strain 13 bone marrow. Thirteen such transfer experiments were performed and 10 of the 13 recipient strain 13 guinea pigs gained the capacity to form an immune response to DNP-PLL or to DNP-GL following the transfer of F, lymph node and spleen cells. The cell populations used contained about 95%cells with the appearance of small or large lymphocytes and 5%cells staining vitally with neutral red (Green and Benacerraf, unpublished data). These experiments demonstrate that the capacity to recognize PLL conjugates is expressed in the cells of the bone marrow and of lymph nodes and spleens of responder guinea pigs, but the cell type involved has not yet been identified.

7. The Behavior of DNP-PLL As a Complete Antigen in Guinea Pigs with the PLL Gene and As a Hapten in Nonrespondm Guinea Pigs Nonresponder guinea pigs lacking the PLL gene do not develop cellular immunity (delayed sensitivity) to DNP-PLL and form very low concentrations of specific antibodies only when high levels of mycobacteria are used as adjuvant. However, PLL is a highly charged molecule and can form stable aggregates with negatively charged foreign albumins

54

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

IIESPONSE

TABLE X NORMAL ANI) BSA-TOLERANT NONRESPONUER GI11 N E A DNP-PLL.BSA I N COMPLETE FREUND'S ADJUVANT^^^

OF

Normal

P I G S TO

BSA-ToleraritC

Anti-DNP Coric. (mg./ml. serum)

No.

Anti-DNP Conc. (mg./ml. serum)

2 3 4 5 6 7 8 9 10 11

1.11 0.82 2.46 1.78 1.88 2.23 1.26 1.97 1.67 1.85 1.25

12 13 14 15 16 17 18 19 20 21 22

0.55 0.56 0.02 0.21 0.16 0.25 0.13 0.34 0.16 0.16 0.30

Av.

1.66

Av.

0.26

NO.

1

From G. A. Theis, I. Green, B. Benacerraf, and G. W. Siskind, J . Immunol. 102, 513 (1969). b Guinea pigs immunized with 100 pg. of DNP-PLL.BSA emulsified in complete Freund's adjuvant with 0.5 mg./ml. Myobactwium butyricum. Animals were skin tested a t 7 clays af!.er immunization with 10 pg. DNP-PLL and bled to death at 14 days after immuriixation. Antibody concentration was determined by precipitin reaction with DNP bovine fibrinogen. (DNP = dinitrophenyl; PLL = poly-lilysine; BSA = bovine serum albumin.) c Tolerance to BSA was induced by four injections of 1 mg. BSA dissolved in PBS (phosphate buffered saline) given over a 2-week period ending 7 days before immunization with DNP-PLLsBSA. Q

where the albumin can act as an immunogenic carrier. By this device, nonresponder guinea pigs immunized with complexes of DNP-PLL with ovalbumin or BSA, in complete adjuvants with 0.5 mg./ml. Mycobmterium butyricum, can be induced to form high concentrations of antiDNP-PLL antibodies (1-2 mg./ml., Table X ) in the absence of delayed sensitivity or in vitro evidence of cellular immunity to DNP-PLL ( 3 5 , 4 3 , 6 4 ) (Table V). An immune response to the carrier albumin is essential for the synthesis of high levels of anti-DNP-PLL antibodies in spite of the fact that anti-DNP-PLL antibodies and anticarrier albumin antibodies are synthesized in different plasma cells ( 6 5 ) . Thus, the establishment of tolerance to the carrier albumin markedly decreased the ability of anti-DNP-

GENETIC CONTROL O F SPECIFIC IhIMUNE RESPONSES

!5S

PLL albumin complexes to stimulate the formation of anti-DNP-PLL antibodies (43, 6 4 ) (Tablc X ) . The immunological specificity of the an ti-DNP-PLL antibodies synthesized by these nonresponder nnin~alswas coniparcd with that of the antibodies produced by responder guinea pigs immunized with DNPPLL alone, and was shown to be identical, with most of the binding affinity directed against DNP-lysine and partial specificity (less than 1 kcal./mole) directed against the PLL molecule ( 3 5 ) . The ability of an immunogenic molecule to act as a “carrier” for DNP-PLL in guinea pigs lacking the PLL gene may explain in part why nonresponder guinea pigs immunized with this antigen and high doses of mycobacteria were able to synthesize low serum levels of anti-DNY antibodies ( 5 0 ) . The inycobacteria may have acted as a carrier in a manner similar to the foreign albumin. The much weaker response observed with DNP-GL, a weakly charged molecule, is compatible with this interpretation (49, 50).

8. Analysis of the Process Controlled by the PLL Gene a. The PLL gene is concerned with a process which is an integral part of the immune response, sincc the capacity to respond to DNP-PLL can be transferred by bone marrow cells from responder guinea pigs, that is, by cells or precursors of cells directly involved in the immune response (63). b. The most challenging finding in this and in other genetic systems is the observation that a single dominant genetic factor controls the immune response to a family of antigens which have structural features in common (in this case sequences of positively charged amino acids, lysine or arginines). The induction of the immune response in the PLL system as well as the stimulation of the response of sensitized cells in vim or in vitro requires the identical immunogenic antigen with eight or more lysyl residues (57-60). (Blocking of all the €-amino groups of DNP-PLL renders the molecule nonantigenic in responder animals.) Furthermore, in this system as in other systems controlled by single dominant genes, the immune response observed is heterogcneous both as to immunoglobulin class and as to specificity and affinityof the antibodies produced. This is particularly clear in the case of anti-DNP-PLL antibodies ( 3 5 ) . c. The immunogenicity requirements for the response to DNP-PLL ( a minimum of eight lysyl residues) and the specificity of cellular immunity to this antigen arc similar (59-61) but differ from the specificity of the anti-DNP-PLL antibodies produced ( 5 8 ) , suggesting either that the inimune receptors on the antigen-sensitive cells and specific serum

56

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

antibodies later produced do not have identical specificities or, more probably, that the DNP-PLL antigen undergoes interaction at two levels of specificity, one of which is under the control of the PLL gene. d. In favor of this last interpretation is the observation that immune responses to haptens in hapten-protein systems require previous or simultaneous responses to the carrier molecule and that tolerance to carrier molecules abolishes the responses to haptens they bear (24, 43, 64, 66). According to this view, immune responses to PLL, GL, polyarginine, or to hapten conjugates of these molecules would require an identical interaction of the antigen with a molecule of which the synthesis is controlled by the PLL gene and which is specific for the sequences of charged amino acids that characterize these antigens, before specific antibodies can be synthesized against other determinants they carry. It is conceivable, in addition, that such an interaction can determine which determinants are recognized. An analysis of where such an interaction takes place await the precise determination of the type of cells capable of transferring responder status.

MULTICHAINAMINO ACID C. THE RESPONSEOF MICE TO BRANCHED, COPOLYMERS The ability of inbred strains of mice to make antibodies to branched, multichain, synthetic polypeptide antigens bearing a restricted range of antigenic determinants is a quantitative genetic trait (67). This trait appears to be controlled to a large extent by an autosomal dominant gene, which has been designated immune response-1 ( Ir-1) . A schematic structural diagram of the type of antigen used in these studies is shown in Fig. 2. These antigens are synthesized by polymerizing

j4olylysine

--l-rT-

Po Iy - D, L- a I a n in e

- Poly (tyrosine,

glutornic ocid)

FIG. 2. A schcniatic tliagrarii of the structural pattern of (T,G)-A--L 509. (From H. 0. McDevitt and M. Sela, J. Exptl. hled. 122, 517531, 1965.)

GENETIC CONTROL OF SPECIFIC IM M U N E RESPONSES

57

side chains of poly-D,L-alanine on the E-amino groups of a long backbone of PLL. This results in a branched, multichain polypeptide, polyalanyl-polylysine (A-L), which obeys thc general rulcl for linear amino acid polymers that copolymers of one or two amino acids arc not immunogenic in mice (38). The addition of short, random sequences of tyrosinc and glutamic acid to the amino termini of the poly-o,L-alanine side chains converts A--L into (T,C)-A-L, a good antigen in some strains of mice. It should be noted that the sequences of tyrosine and glutamic acid at the tips of the side chains are random and not identical on every side chain. Substitution of histidine or phenylalanine for tyrosine in the sidechain termini produces (H,G)-A--L or (Phe,G)-A--L, closely related antigens in which the nature of the antigenic determinant is varied on a similar structuraI background. When CBA and C57 mice are immunized with the maximal immunizing dose of (T,G)-A--L. CBA's respond poorly while C57's respond well. Exactly the opposite result is found with (H,G)-A--L: CBA mice respond well, whereas C57 mice respond poorly. The F, hybrid between CBA and C57 responds well to both antigens, and reciprocal backcross progeny segregate as a 1:1 mixture of the F, and the respective homozygous parent animals (67, 68). Figure 3 illustrates the genetic segregation in antibody response to (T,G)-A--L. The horizontal axis plots the antibody response of individual mice in tcrnis of the percent of antigen bound in an antigcn-binding assay, The vertical axis represents the number of animals falling into a given percentile of percent antigcnbound values. Since a wide variety of control experiments have excluded dose-response differences (67) or different responses due to adjuvant, age, or sex, these results indicate that the ability of mice to respond to ( T,G)-A--L is a genetically controlled, quantitative, dominant trait. Since substitution of histidine for tyrosine in the antigenic determinant results in a reversal of the high- and low-responding strains, as noted above, this genetic trait, Ir-1, may be concerned with the recognition of the antigenic determinants. Preliminary analysis of the Ir-1 gene showed that there was no significant quantitative difference in distribution among the immunoglobulin classes of the antibodies produced to (T,G)-A--L in high- and lowresponding strains (69) and that in a segregating backcross population [ (CBA x C57) F, CBA], no linkage was found between Ir-1 and the Tg region coding for the mouse immunoglobulin allotypes. This indicates that the Ir-I gene is not associated with the known structural genes coding for the F, fragments of mouse immunoglobulin heavy chains (69). Further analysis of the mechanism of action of the Ir-1 locus has been

x

58

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

81

8l

0

c 57

CBA

CBA x C57 FI

25

81

50

75

I00

CBA x C 5 7 FI x C 5 7

4

0

25

50

75

100

% ANTIGEN BOUND

FIG. 3. Immune response of mice given 10 pg. (T,G)-A--L 509 in complete Freund's adjuvant, and boosted with 10 p g . of the same antigen in saline. (From H. 0. McDevitt and M. Sela, J. Erptl. Med. 122, 517-53'1, 1965.)

carried out, as indicated in Section 111, by the use of cell transfer studies, linkage studies, and studies of the specificity of the antibodies produced.

1. Cell Transfer Studies Since preliminary analysis (69) failed to show any association of the Ir-1 gene with the level of immunoglobulin class or with immunoglobulin allotype, cell transfer studies were undertaken to find out whether the ability to respond well to a particular synthetic polypeptide antigen was a trait closely related to the process of antibody formation and, therefore, transferable with immunocompetent cells. McDevitt and Tyan ( 7 0 ) found that it is possible to transfer the ability to respond well to (T,G)-A--L from (C57BL/6 x C3H) F, responder animals into lethally irradiated, nonresponder C3H parental recipients by the transfer of 100 to 150 million adult unfractionated

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

59

TABLE XI ADOPTIVE TRZTWER O F i PRIMARY RESPONSETO (T,G)-A--L TO LETHALLY IRRADIATED C3H HOSTSW I T I I IC3H X C37BL/6) PI SPLEENCELLP~

Recipiei it3 (No. of ariimdsj

Primary stimiiliisc (lay, after radiatioii

Thymectomized C3H 9 (7) Normal C3H 0 (7) Normal C3H 61 (9) Thymectomized C3H 9 (5) n’orxnal (C3H X C57BL/6) FI 0 ( 5 ) Sormal (CSII X C37BL/6) 171 0 (4)

0 0 21 21 21 0

Summary:

Titer 10 days after iecoiidary stimiiluid 52, 19, 10, 58, 0, 0, 65 ( 1/50) 13, 60, 41, 51, 50, 8, 54 (1/50) 39, 8, 49, 45, 0, 63, 52, 50, 73 (1/50) 46, 17, 54, 72, 26 ~ 5 0 ) 76, 59, 58, 50, 47 (1/50) 69, 76, 71, 6.5 (1/.50)

F,+ C3H

=

FI C31I + C3II

= =

171 --t

38% (19/2X) 63% (9/9) 8%

From IT. 0. McDevitt and M. L. Tyaii, J. Esptl. dled. 128, 1-11 (1068). &Spleen cell dose in all these transfers wab 100-150 X lo6 (C3H X C57BL/6) F, spleen cells per iecipient. (T = tyrosine; G = glr1tltmlc acid; A = polyalanyl; L = polylysine.) c First stimulus was 10 pg. (T,G)-A-L 309 iri complete Freurd’s adjuvant; second stimulus was 10 pg. (T,G)-.4-L-309 i i i aqueous solution given 3 weeks later. d Titer is average percent antlgeil I,ourid. Figures in parerilheses indicate dilution at which antisera were titered.

spleen cells. These results are summarized in Table XI. This transfer was carried out using normal, nonimmunized spleen cells and was successful in 19 of 28 attempts whether the recipients were immunized immediately after transfer or 3 weeks after transfer and whether or not the recipient animals were thymectomized. These results strongly support, but do not prove, the conclusion that this genetic control (Ir-1) is directly related to the process of antibody formation. The possibility still rcmains that the successful transfer of any type of cell from a responder into a nonresponder strain (e.g., liver cells and skin cells) would result in the transfer of an cnzyme system required for proper metabolism of the antigen. Conclusive support for the hypothesis that this genetic control is an integral part of the process of antibody formation was obtained from another type of cell transfer experiment. In these experiments, thymectomized, lethally irradiated (C57BL/6 x DBA/2) F, mice [who normally respond well to (T,G)A-L] were given fetal liver cells and a thymus implant from CBA (nonresponder) fetuses and then immunized with (T,G)-A--L at 60 and 100

60

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

days after irradiation and cell transfer. All the chimeras failed to respond to (T,G)-A--L, but 12 of 13 subsequently responded well to (H,G)-A--L, to which the CBA strain normally responds well (71 ). This experiment indicates that the genetic control of the ability to respond to (T,G)-A--L and (H,G)-A--L is exerted through a mechanism directly related to the immune response, since thymectomized, irradiated mice which were responders to (T,G)-A--L and nonresponders to (H,G)-A--Lcould be converted into the opposite phenotype by the transfer of fetal liver and thymus from a strain of the opposite phenotype ( 71 ). Preliminary evidence (M. L. Tyan and H. 0. McDevitt, unpublished data) indicates that it is possible to transfer the ability to respond well to (T,G)-A--L by the transfer of partially purified peripheral blood lymphocytes, although efforts to accomplish this transfer with thoracic duct lymphocytes ( 71 ) have been unsuccessful. Further experiments will be required before the site of action of the Ir-1 gene can be definitely localized to a particular cell type. In summary, the evidence indicates that the Ir-1 gene acts in a cell type directly involved in the process of antibody formation, now tentatively identified as a peripheral blood lymphocyte. 2. The Linkage of Ir-1 with the Major Histocompatil~ility ( H - 2 ) Locus This linkage was first suspected when it was found that C3H.SW mice responded well to (T,G)-A--L,although they are genetically identical with C3H/DiSn mice ( a known low-responding strain), except for the H-2 locus-C3H.SW being H-2” and C3H mice being H-2k. Similar anomalous results were found with B1O.BR mice ( H-2k) which are congenic with C57BL/10 ScSn mice (H-2”). The B1O.BR mice respond poorly to (T,G)-A--L, although C57BL/10 mice respond well to this antigen, These anomalous results suggested that the ability to respond well or poorly to (T,G)-A--L had been bred into these strains by the breeding process which put a different H-2 allele on the same genetic background. This suggestion implies that ability to respond (Ir-1) is linked to the H-2 locus. Linkage was established in backcross tests in which it was shown that the ability to respond well to (T,G)-A--L is linked to the H-2h allele, whereas the ability to respond well to (H,G)-A--Lis linked to the H-2“ allele ( 7 0 ) .Although three recombinant animals were tentatively identified in the initial backcross test, only one has been fertile and this animal was not, in fact, a recombinant. Further testing of backcross populations is continuing, but at present there is no conclusive evidence that Ir-1 and H-2 are not identical. Extensive testing has shown that there is a regular correlation be-

TABLE XI1 THERELATIONSHIP BETWEEN H-2 TYPEA N D IMMUNE RESPONSETO

THE

A-L SERIESOF ANTIGENS" ~~

(T,G)-A-L

Strain A/J A.BY c57 D1.LP C3H.SW BALB/c DBA/2 CBAb C3H/HeJ B1O.BR AKR DBA/lC SJL A.SW WB/Re SWR

Antigenbound

(H,G)-A-L

(%)

Range

No. of animals

k

10 78 69 59 79 28 34 12 17 6

-

5-15 62-87 53-82 40-95 52-91 0-55 11-53 0-27 9-26 2-14

-

9 9 10 10 10 8 10 10 10 10

-

9

6

S

5

4-12 3-7

S

0 0 0

8 10 6 10 10

H-2 Type

a b b b

b d d

k k k

W

?

Antigenbound

(Phe,G)-A-L

KO.of

Antigenbound

( %)

Range

animals

(%I

Range

No. of animals

77 0

61-83

<5

0-16 0-9 0-13 15-63 0-27 39-77 61-82 60-83 60-78

8 6 22 10

75 73 69 73 73 72 65

73-76 73-75 67-71 72-73 70-74 68-75 53-74 69-72 72-75 69-74 72-74 69-76 0-39 6-22 37-62 71

10 5 9 6 6 9 6 7 10 10 8 10 10 5 5 2

<5 5 42 11 70 71 68 70 0 5 0 0 0

0-18

8

10 10 9 8 10 10 10 10 6 10 10

71

74 71 73 74 13 15 49 71

From H. 0. McDevitt and A. Chinitz, Science 183, 1207-1208, 1969. Copyright 1969 by the American Association for the Advancement of Science. * These animals were produced from CBA's maintained for many years a t the National Institute for Medical Research, Mill Hill, London. c A special H-2s, tufted (tf), shoretail (T) linkage strain responded in a manner identical to DBA/1 mice.

! 8

0

E

62

HUGH 0 . MC DEVITT AND BARUJ BENACERRAF

tween H-2 type and the pattern of the immune response to a series of three related, branched, synthetic polypeptide antigens. These results are shown in Table XI1 ( 7 2 ) . The variety (at least five) of patterns of response of different H-2 type strains to the three antigens used indicates either that there are multiple loci controlling the ability to respond to each of these antigens or that there are multiple alleles (five or more) at a single locus, Ir-1. The detection of a strain (SJL) unable to respond to poly- ( Phe,Glu) - p ~ l y - ~ , ~ - A l i i - - p ~ l y -[ (~Phe,G - L y ~ ) -A--L] and the detection of a strain ( D B A I l ) which responds only to (Phe.G)-A--L has permitted an independent test, in strains not related to CBA or C57, of the linkage of ability to respond (Ir-1) with the H-2 locus. This test was carried out using the offspring of the cross [ ( DBA/1 X SJL) F, X SJL] and indicated a close linkage between the ability to respond to (Phe,G)A--L and the H-21 allele ( 7 2 ) .This finding is of considerable importance and will be referred to below (Section V,C,3). The close association between the H-2 locus and the Ir-1 locus raises the question of whether therc is any functional relationship between thc two genetic factors or if, in fact, they are identical. Several points must be taken into account, First, it is possible to exclude antigenic crossrcactions between synthetic poIypeptide antigens and H-2 antigens as the cause of the nonresponder state found in some strains. In addition to the fact that ability to respond is a dominant trait (Section 111) the demonstration that fetal liver from a respoiidcr strain can transfer the ability to respond to a nonresponder strain virtually cxcludes “crosstolerance” as a mechanism of gene action. If such antigenic cross-reaction did exist, the fetal liver would be rendered tolerant on injection into the nonresponder strain prior to its maturation ( 7 1 ) . Further genctic studies (73) have permitted a much more precise localization of the Ir-1 locus. Figure 4 gives a schematic diagram of the linear arrangement of the genes controlling several H-2 specificities within the H-2 locus; Ss is a gene coding for a “serum substance” protein ( 7 4 ) which is apparently unrelated to any H-2 specificity but which maps in the middle of the H-2 locus. The arrows above the diagram indicate the approximate position at which crossovers have been detected between H-2“ and H-Zh alleles to produce the recombinant H-2 types, H-2’‘and H-2 ( 7 5 ) .The arrows below the diagram indicate the positions of the crossovers which have been detected between H-2d and H-2“ alleles, giving rise to H-P-Ss’ and H-2”-Sst1alleles, as well as the location of the crossover lietween H-2”-Ss’ iind H-2‘, giving risc to the H-Y-Ss’ allele. Immuiiization of the H-2’‘ and H-21 strains with (T,G)-A--L and (H,G) -A--L indicates that the genetic control of the ability to respond

63

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

Soarre of

Left-hand Ir-2

a

~ j p e

ptrt

Reiponhe to

Right-hand part

I

1

J,

1)

1,

I>

11

1)

a

i

1

I>

(T,(;)-A--L

(H,G)-A--L

LOW High LOW High

High Low High Low

From H. 0. McDevitt, D. C:. Shreffler, aud J. H. Stimpflirig, J . Clin. Irmest. Absli.

48, 57a (1969).

This table is a siimmary of the antibody iespoiise of iiormal arid recombinant H-2 alleles arising from t,he crossovers indicated in t,he upper part of the diagram in Fig. 4. Three H-2h strains were tested, B.1OX (In),B.10A (2R), and B.10A (4R).All of these H-2h recombinant alleles carry the right-hand part, of the H-2%locus and respond as do H-P mice. Two €1-2' st,rains were tested [BlO..A (311) and B1O.A (.5IL)]. Both of these recombinant, H-2' alleles contained the right-li:iiid part, of t,he H - P allele and both respond as normal H-2b mice respond. This indicates t,hat the Ir-1 lociis is either in the right-hand part of the H-2 locus or in the nii1t.h niouse linkage group somewhere to the right of the H-2 lociis.

to these two antigens lies to the right of this crossover, as is shown in Table XIII. Inimunization of the recombinant H-2 types, H-2"-Ss1, H-2OSsh, and H-2"-Ss1, indicates that the genetic control of the ability to respond to (H,G)-A--L lies to the right of these crossovers as well and, therefore, to the right of the Ss locus. However, immunization of H-2'-Ss' mice with (H,G)-A--L gives a uniformly high response, indicating that the genetic control of the ability to respond to (H,G)-A--L lies to the left of this crossover. Figure 5 is a diagram of this crossover. Barring a double crossover event in the derivation of this crossover strain ( a possibility which is not yet ruled out), this result would indicate that the Ir-1 locus lies between the Ss locus and the genes controlling H-2 specificities 11 and 31. Since the Ss locus controls a circulating serum protein which is apparently unrelated to any H-2 specificity, it is quite possible that this region of the ninth mouse Iinkage group controls proteins which are unrelated to H-2 but have other functions. If Ir-1 does, in fact, map in this region, it will first be necessary to determine the cell type in which Ir-1 is expressed (i.e., a particular type of lymphocyte or a macrophage) before studies can be undertaken concerning the difference between responder and nonresponder cells of this type.

64

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

,

I

FIG.4. A schematic representation of the portion of the ninth mouse linkage group containing the H-2 locus. The H-2 locus is a large locus controlling many separate serological specificities of the major histocompatibility antigens in the mouse. The various H-2 alleles are made up of combinations of thirty odd different specificities. They segregate as alleles, but this locus is actually a pseudoallele since it contains many cistrons and since recombination has been detected within it by the crossovers indicated by the arrows (75). The numbers are the code numbers for various scrological specificities. The arrows above the diagram represent the approximate position of crossovers between H - 2 and H-Zb which gave rise to the H-2h and H-2' alleles. The arrows below the line indicate the approximate position of crossovers between H-Zd and €I-2' alleles to give rise to the indicated H-2 recombinant types. The two alleles at the Ss locus, Ss" and Ss', are for high and low levels of the S5 serum protein. L e f t - hand limit of H - 2 .

Right -hand limit of H-2.

FIG.5. A schematic representation of the crossover event which gave rise to the H-2'-Ss' recombinant chromosome indicated by the dotted line. The H-2"Ss' chroniosome carries the right-hand half of H-2' and the left-hand half of H-2d. Animals carrying this recombinant chromosome give a uniformly high response to ( H,G) -A--L, indicating that the Ir-1 gene is localized to the right of the crossover event (indicated in Fig. 4 ) which gave rise to the H-2'-Ss1 allele, since H-2' animals are high responders to (H,G)-A--L. In the crossover shown in this diagram, the crossover occurs between a high responder to (H,G)-A--L (the H-2"-Ss1 allele) and a low responder to (H,G)-A--L (the H-2' allele). Mice carrying the recombinant H-2 allele, designated H-2'-Ss' show a uniformly high response to (H,G)-A--L. This evidence, together with the evidence produced by the other recombinants between H-2d and H-2k indicated in Table XI11 and Fig. 4, localizes the Ir-1 locus between the Ss locus and H-2 specificities, 11 and 31.

GENETIC CONTROL OF SPECIFIC IM M U N E RESPONSES

65

An alternative possibility must be kept in mind, i.e., the Ir-1 locus is, in fact, a locus coding for as yet undetected H-2 specificities and that these specificities on the surface of ceIIs, such as lymphocytes or macrophages, determine whether or not that cell can interact with antigens of a particular chemical structure. In view of the large number of genetic controls of specific immune responses already described, this implies a very high order of complexity and discrimination of cell surface antigens. There is a priori no reason to believe that this is not, in fact, the case. The linkage between susceptibility to some forms of inurine leukemia and the H-2 locus, which has recently been reported by Lilly ( 7 6 ) , as well as the Iinkage between ability to respond to the Ea-laJ’ erythrocytc antigens and the H-3 and H-6 loci described by Gasser ( 2 3 ) , suggest that we may be dealing with a general biological phenomcnon, whatever its mechanism. 3. The Genetic Control of Antibody Specificity

The genetic control of the immune response to (T,G)-A-L, (H,G)A--L, and (Phe,G)-A--L is highly specific for the amino acid composition of thc antigenic determinant (see Table MI). There is a s yet no definite evidence concerning the affinity of anti- ( T,G ) -A--L antisera from highnnd low-responder strains for the antigenic determinant. This experiment will require antigens of the sanac type as (T,G)-A--L which have identical amino mid sequences on each side chain of the molecule. Strong evidence for the genetic control of antibody specificity has, ho\vever, been obtained with branched multichain, synthetic polypeptide antigens in which the side chains are composed of poly-L-proline instead of pOIY-D,Lalanine (77-79). These antigens were synthesized to test the role of the poly-D,L-alanine side chains in the immunogenicity of thc A--L series of antigcns. Two antigens of this type have bcen tcstecl: poly-( Tyr,Glu)Pro--Lys, or ( T,G )-Pro--L, and poly- ( Phe,Glu ) -Pro--Lys, or ( Phe,G) Pro--L. The results are summarized in Table XIV. It is clear that the presence of poly-L-proline side chains has a very marked effect on the immunogenicity of the entire molecule. ( T , G ) Pro--L elicits the same response in CBA and C57 mice, two strains that show a very marked difference in response to (T,G)-A--L. Thc SJL mice arc the best responders to (T,G )-Pro--L, although they arc poor re$ponder\ to ( T,G)-A--L. Ccnctic segregation of antibody response to ( T,G) -Pro--L in the DBA/ 1 x SJL cro5s is shown in Fig. 6. The segrcgation is similar to that seen for anti-( T,G)-A--L rc’sponse in the CBA x C57 cros$ except that ( a ) the F, x SJL offspring are all intermediate or low, although they

66

HUGH 0. MCDEVITT AND BARUJ BENACEFiRAF

ANTIBODYRESPOSSEOF

TABLE XIV MICE TO THE SAME (A-L) AND (PRO-L)

INBRED ON

RANDOM

SEQUENCES

Antigen-bound (Av. %)" Straiii CBA c57 DBA/1 SJL

H-2 type (T,G)-A--L (Phe,G)-A--L k t) 4 S

12 (lo) 69 (hi) 6 (10) __ 5 (lo) __

71 (hi) 69 (hi) 74 (hi) 1 3 (10)

(T,G)-Pro-Lb

(Phe,G)-Pro-Lb

8 (10) 7 (10) 3 (10) 33 (med)

33 (med-hi) 21 (med) 39 (hi) 40 (hi)

Antiserum dilution, 1/500. As can he seen, (T,C)-I'ro--L is a weaLer immunogen than (T,G)-A--L and (Phe,G)A--L. More clear-cut segregation is seen when these antisera are titered a t a dilution of 1/50 as is shown i n Fig. 6. (Phe,G)-Pro--L, on the other hand, is tt very potent antigen. However, it. is tlifficult to titer with this antigen becalm of a high degree of spontaneoiis aggregation. (Phe,G)-Pro--L is approximately 60-70% spontaneously precipitalile, and the antigen remaining in the supernatant after centrifugation is only 64% precipitahle by specific antiserum. The titers given here are, therefore, deceptively low and very much higher titers are obtained when these same antisera are titered with (Phe,G)-&-I, as is shown in Fig. 7. Dilutions on the order of 1/50,000 are required before DBA/l anti-(Phe,G)-Pro-L antisera begin to show a drop in their ability to bind (Phe,G-)A-L. 11

b

would be expected to be intermediate or high-no explanation has yet been found for this anomalous result; and ( b ) there is a complete absence of linkage of high anti-( T,G)-Pro--L response to the H-2" allele in the F, x DBA/1 backcross. If we ignore the anomalous response of the F, X SJL backcross mice, these results indicate that response to (T,G)Pro--L is under a genetic control which is superficially similar to that operating at the Ir-1 locus but qualitatively different in that it is not linked to the H-2 locus. Pending further analysis, this genetic control has been tentatively designated Ir-3. Genetic control of antibody specificity is found when the immune response to (Phe,G)-Pro--L is studied in the DBA/1 x SJL cross. The response of DBA/1, SJL, F,, and the two backcross generations to (Phe,G)-Pro--L when titered with 3H-acetylated ( Phe,G )-Pro--L is striking in that there is a nearly equal response to this antigen in all five groups, However, when the same antisera are tested for their ability to bind (T,G)-Pro--L, a segregation for ability to bind (T,G)-Pro--L is immediately apparent; SJL anti-( Phe,G)-Pro--L binds (T,G)-Pro--L better than DBA/ 1 anti-( Phe,G) -Pro--L. The expected segregation is seen in the F, x DBA/1 and F, x SJL antisera, although the difference between high and low responders is not great. In this situation, as well, there is no linkage of anti-( T,G)-Pro--L binding capacity with the H-2s allele.

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

67

RESPONSE TO (T,G)-Pro--L.

:T

DBA/I

SJL

4

20 D R A / i X SJL

F,

LL

D B A / l X SJL F, X D R A / I

:I-

D B A / l X SJL

4-1

n

25

0

%

5

X SJL

n

50

ANTIGEN

75

100

BOUND

FIG. 6. The antibody response of mice immunized with (T,G)-Pro--L and titered with (T,G)-Pro--L labeled with "'I. The format of this figure is similar to that for Fig. 3. The horizontal axis plots the p e r c a t antigen bound in an antigen-binding assay, whereas the vertical axis plots the number of animals falling into a given percentile for antigen-bound values. A11 antisera were titeied at a 1/50 dilution. In the third graph from the top, the closed bars represent those animals that carry the H-2" allele donated by the SJL grandparent. In the bottom graph the open bars indicate the animals that carry the H-21 allele donated by the DBA/1 grandparent. There is no linkage between immune response and H-2 allele.

Figure 7 illustrates the genetic segregation seen when anti- ( Phe,G)Pro--L antisera are tested for their ability to bind (Phe,G)-A--L. There is a clear-cut genetic segregation; DBA/ 1 mice are the highresponders, and ability to bind (Phe,G)-A--L is clearly linked to the H-2 locus in the F, x SJL backcross. This is virtually identical to the results obtained in this cross when (Phe,G)-A--L is used as the immunogen ( Section V,C,2). The results presented in Figs. 6 and 7 show definite genetic control of the specificity of antibodies produced against the same antigen in two

68

HUGH 0. MC DEVI'IT AND BARUJ BENACERRAF RESPONSE TO (Phe,G)-Pro--L (Titered with (Phe, GI-A--L.) DBA/l

D B A / l X SJL F,

8T

D B A / i X SJL F, X D B A / 1

D E A / l X SJL

F;

X SJL

n

231m

00

25

Yo

50

ANTIGEN

75

100

BOUND

FIG. 7. The immune response of mice immunized with (Phe,G)-Pro--L and titered with tritium-labeled (Phe,G)-A--L at a 1/500 dilution. There is a very marked segregation in the production of antibodies that bind (Phe,G)-A-L, and this is even more apparent when the same sera are titered in this manner at higher dilutions. In the bottom graph the open bars indicate the animals carrying the H-2Rallele donated by the DBA/1 grandparent. There is definite close linkage between the ability to produce antibodies that bind (Phe,G)-A--L and the H-2" allele.

genetically different strains of mice. This genetic control appears to be the result of two loci, the Ir-1 and Ir-3 loci, each affecting the response to (Phe,G)-Pro--L. Thus the ability of F, x DBA/1 mice to produce antibodies capable of binding (T,G)-Pro--L is not linked to the H - 2 allele donated by the high-responding SJL parent, whereas the ability of F, x SJL mice to produce antibodies capable of binding (Phe,G)-A--L

GENETIC CONTROL OF SPECIFIC IMMUNE IiESPONSES

69

is linked to the H-2s allele donated by the high-responding DBA/1 parents. In summary, the evidence presently available indicates that there are different loci controlling the immune response to antigenic determinants of different amino acid composition and that the operation of the highresponding allele at either of these loci markedly alters the specificity of the antisera produced to the same antigen. Recent evidence indicates that the antibodies binding ( T,G)-Pro--L and ( Phe,G) -A--L in response to (Phe,G) -Pro--L are largely non-crossreacting, separate antibody populations, The high degree of specificity of the Ir-1 and Ir-3 loci is consistent with the interpretation that these gene loci control the recognition of the antigenic determinant.

VI.

Conclusions

Several autosomal dominant genes have been identified which govern the ability of guinea pigs and mice to form specific immune responses to synthetic polypeptide antigens with a limited number of amino acids and with relatively simple structure: the PLL gene in the guinea pig and the Ir-1, Ir-3, and GLA, genes in the mouse. The evidence from cell transfer experiments clearly shows that these genes control processes that are an integral part of the specific immune responses. The first question that should be asked is, “Do these specific genetic factors, which operate in different specific immune responses, control an identical process, or do they act at different levels in the immune response?” In other words, does the information obtained with the various systems investigated imply one or several levels of genetic control of specific immune responses? It is reasonabIe to conclude, as thcre is no evidence to the contrary, that these four genetic factors act at an identical level and that the various gene-antigen systems described illustrate the same basic phenomenon and, therefore, what can be learned from one system can be applied to another. Before an attempt is then made to analyze the function controlled by these “immune response genes” the characteristic features common to the various genetic systems discussed should be summarized. 1. The genes concerned with specific immune response control the synthesis of molecules or govern recognition processes which are specific for certain amino acid sequences on the antigen. It is clear that d8erent genetic factors control the responses to different polypeptide antigens. 2. A relatively large number of immune response genes probably exist, since several such dominant genes have been discovered in a rela-

70

HUGH 0. MC DEVITT AND BARUJ BENACERRAF

tively short time (at least three in the mouse) when immune responses to a limited number of synthetic polypeptides with only a few amino acids have been studied. 3. These individrral dominant “immune response genes” may control the response to several antigens with similar structure in spite of the fact that the immunological specificity of the antibodies formed to each of these selected antigens may be different, although cross-reacting. Furthermore, the antibody responses governed by these single dominant genes, to DNP-PLL, to (T,G)-A--L, or to GLA, consist of antibodies which are very heterogeneous as to class, specificity, and a6nity. In fact, there is no evidence that the antibody popuIations synthesized to antigens of which the responses are largely controlled by single dominant genes are less heterogeneous in these various respects than antibodies formed against more complex antigens. 4. Nevertheless, in spite of the heterogeneity of the antibodies produced to antigens of which the responses are governed by immune response genes, these factors appear to determine to a considerable extent the specificity of the antibody population synthesized, as shown by the patterns of cross-reactivity of the antibodies with antigens related to the immunizing antigcn and by the studies of the response of inbred guinea pigs to bovine insulin. This can be interpreted to indicate that the action of the immune response genes may result in the recognition or selection of antigenic determinants against which specific antibody is synthesized. To explain these phenomena two possible sites for gene action may be considered. a. A specific immune response gene may control the recognition of an antigen or of its determinants if it is concerned with the structure of the variable segment of an immunoglobulin chain. If this is the case, the available data would require that such a gene would impose certain restrictions on the specificity of the immunoglobulins and yet pcrmit a heterogeneous population of antibodies of related specificities to be synthesized. This could result if the genetic factors discussed would code for only a portion of the variable segments of the immunoglobulin chains. Alternatively, if they code for the entire variable segments of the chain, a limited degree of diversity must be postulated to be generated by somatic mechanisms. Todd and Mandy (80) have recently shown that the rabbit heavy chain allotype markers located in the N-terminal portion of the heavy chain are linked to new allotype markers ( A l l , A12) located in the constant region of the heavy chain, This suggests that entire heavy chains (and, by analogy, entire light chains) are coded for by closely linked genes. Since Ir-1, and the ability of rats to respond to LDH-A,, are not linked to allotype markers, the genetic factors discussed here

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

71

could not be entire variable region structural genes, tinless such genes are inserted between thc structural gencs coding for the N- and C-terminal portions of heavy and light chains. There is as yet 110 prccedent for such a mechanism. In addition, the observations that PLL nonresponder guinea pigs immunizcd with DNP-PLL albumin complexes produce high levels of anti-DNP-PLL antibodies whose specificity cannot be distinguished from the specificity of the antibodies produced by responder guinea pigs also argue against variable region structural genes as the site of gene action. b. Another possible interpretation of the data postulates that an antigen undergoes specific interactions at at least two levels, one of which would be under the control of the specific immune response genes discussed in this review and would be specific for a configuration determined by certain amino acid sequences on the antigen. The specificity of this interaction would not be identical with that of the antibody populations later produced. The postulatcd molecules controlled by such genes would have to react with the antigen before antibody synthesis against specific determinants on the antigen could be stimulated. It is conceivable that such an interaction could also determine which antigenic determinants on the antigen may stimulate antibody synthesis. Several instances of specific antibody synthesis which depend upon the previous recognition of other determinants on the antigen have been reported in the immune responses of PLL nonresponder guinea pigs to DNP-PLLBSA (35) and of rats (28) and rabbits (81) to lactic dehydrogenase. These can be considered examples of the “specific carrier function” originally described by Landsteiner (53). In this sense the immune response genes discussed in this review could be considered to be responsible for the specific recognition of carrier function. If this is the case, all the available evidence indicates that there must exist a large number of these genes with a high degree of specificity. Their possible relationship to histocompatibility antigens (23, 7 2 ) remains to be elucidated.

REFERENCES 1. Herzenberg, L A., hlcDevitt, €I. O., and Herzenberg, L. A. (1968). Advan. Genet. 2, 209-244. 2. Todd, C. W. (1963). Riochem. Biophys. Res. Comnzun. 11, 170-175. 3. Pernis, B., Torrigiani, G., Amanct, L., Kehis, A. S., and Cebra, 1. J. (1968). Iminunologq 14, 445-451. 4 . Koshland, M. E. (1967). Cold Spring Harbor Symp. Qziant. R i d . 32, 119-127. 5. Milstein, C . ( 1967). Nature 216, 330. 6. Hood, L., Gray, W. R . , Sanders, B. G., and Dleyer, W. J. (1967). Cold Spring Harbor Sump. Qrrairt. B i d . 32, 133-145.

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HUGH 0. MC DEVITT AND BARUJ BENACERRAF

7. Edelman, G. M., and Gally, J. A. (1967). Proc. Natl. Acad. Sci. U.S. 57, 353. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

Lennox, E. S., and Cohn, M. (1967). Ann. Rev. Biochem. 36, 365406. Gorer, P. A., and Schiitze, H. (1938). J. Hyg. 3 4 647-662. Scheibel, I. F. (1943). Acta Pathol. Microbiol Scand. 20, 464-484. Biozzi, G., Stiffel, C., Mouton, D., Bouthillier, Y., and Decreusefond, C. (1968). Aim. Inst. Pasteur 115, 965. Fink, M. A., and Quinn, V. A. (1953). J . Immunol. 70, 61-67. Ipsen, J. (1959). J. Immunol. 83,448-457. Dineen, J. K. ( 19%). Nature 202, 101-102. Carlinfanti, E. J. (1948). Immunology 59, 1-7. Stern, K., Brown, K. S., and Davidsohn, I. (1956). Genetics 41, 517-527. Playfair, J. H. L. (1968). Immunology 15, 35-50. Sobey, W. R., and Adams, K. M., (1961). Australia J . Biol. Sci. 14 588-593. Sobey, W. R., Magrath, J. M., and Reisner, A. H. (1966). Immunology 11, 511513. Hardy, D., and Rowley, D. (1968). Immunology 14,401407. Pol& L., Barnes, J. M., and Turk, J. L. (1968). Immunology 14, 707-711. Von Sengbush, P., and Lennox, E. S. Personal communication. Gasser, D. L. (1969). J. Immunol. 103, 6&70. Cinader, B. (1968). I n “Regulation of the Antibody Response” (B. Cinader, ed. ), pp. 3-53. Thomas, Springfield, Illinois. Arquilla, E. R., and Finn, J. (1963). Science 142, 400-401. Arquilla, E. R., and Finn, J. (1965). J. Exptl. Med. 122, 771-784. Arquilla, E. R., Miles, P., Knapp, S., Hamlin, J., and Bromer, W. (1967). Vox Sang. 13, 32-35. Armerding, D., and Rajewsky, K. (1969). Proc. XVIIth Colloquium: Protides of the Biological Fluids, Bruges, Belgium (in press), Katchalski, E., and Sela, M. (1058). Adcan. Protein Chem. 13, 243. Maurer, P. H. (1964). Progr. Allergy 4 1. Gill, T. J., 111, and Doty, P. ( 1961). J. Biol. Chem. 236, 2677. Sela, M. (1966). Aduan. Immunol. 5, 29. Pinchuck, P., and Maurer, P. H. (1968). In “Regulation of the Antibody Response” (B. Cinader, ed.), pp. 97. Thomas, Springfield, Illinois. Maurer, P. H. ( 1965). Federation Proc. 24, 184. Green, I., Paul, W. E., and Benacerraf, B. (1966). J . Erptl. Med. 123, 859. Kantor, F. S., Ojeda, A., and Benacerraf, B. (1963). J. Exptl. Med. 117, 55. Ben-Efraini, S., and Maurer, P. H. (1966). J . Immunol. 97, 577. Pinchuck, P., and Maurer, P. H. (1965). J . Exptl. Med. 122, 665. Simonian, S. J., Gill, T. J., 111, and Gershoff, W. N. (1968). J . Immunol. 101, 730. Levine, B. B., Ojeda, A., and Benacerraf, B. (1963). J . Exptl. Med. 11% 953. Pinchuck, P., and Maurer, P. H. (1965). J. Exptl. Med. 122, 673. Pinchuck, P., Maurer, P. H., and Benacerraf, B. Unpublished observations. Green, I., Paul, W. E., and Benacerraf, B. (1968). J. Exptl. Med. 127, 43. Green, I., Levine, B. B., Paul, W. E., and Benacerraf, B. (1968). In “Nucleic Acids in Inimunology” (0. Plescia and W. Braun, eds.), p. 288. Springer, New York. Benacerraf, B., Paul, W. E., and Green, I. (1969). Ann. N.Y. Acad. Sci. (in press ) .

GENETIC CONTROL OF SPECIFIC IMMUNE RESPONSES

73

46. Levine, B. B., Ojeda, A,, and Benacerraf, B. (1963). Nature 200, 544. 47. Benacerraf, B., Green, I., and Paul, W. E. (1967). Cold Spring Harbor Symp. Quant. BioZ. 32, 569. 48. Benacerraf, B. ( 1968). 1n “Hegulation of the Antihody Response” (13. Cinder, ed. ), p. 85. Thomas, Springfield, Illinois. 49. Lamelin, J. P., Paul, W. E., and Benacerraf, B. (1968). J. Immunol. 100, 1058. 50. Green, I., Benaccrraf, B., and Stone, S. (1969). Federation Proc. 28, 564. 51. Levine, B. B. (1964). Nature 202, 1009. 52. Paul, W. E., Green, I., Levine, B. B., and Benacerraf, B. Unpublished observations. 53. Landsteiner, K., and Simms, S. (192.3). J. Exptl. Med. 38, 127. 54. Levine, B. B., and Benacerraf, B. (1965). Science 147, 517. 55. Levine, B. B., and Benacerraf, B. (1964). J . Exptl. Med. 120, 955. 56. Vassalli, P. Unpublished observations. 57. Schlossman, S. F., Yoran, S., Ben-Efraim, S., and Sober, H. A. (1965). Biochemistry 4, 1638. 58. Schlossman, S. F., and Levine, H. (1967). J. lmmunol. 98, 211. 59. Schlossinan, S., Ben-Efraim, S., Yoran, S., and Sober, H. A. (1966). J. Exptl. Med. 123, 1083. 60. Stulbarg, M., and Schlossman, S. F. (1968). J. ZmmunoE. 101, 764. 61. Paul, W. E., Siskind, G. W., Stupp, Y., and Benacerraf, B. (1969). Federation Proc. 28, 570. 62. Green, I., Paul, W. E., and Benacerraf, B. (1969’). Proc. Natl. Acad. Sci. (in press ). 63. Foerster, J., Lamelin, J. P., Green, I., and Benacerraf, B. (1969). J. Exptl. Med. (in press). 64. Theis, G. A., Green, I., Benacerraf, B., and Siskind, G. W. (1969). J. lmmunol. 102, 513. 65. Green, I., Vassalli, P., and Benacerraf, B. (1967). J. Exptl. Med. 125, 527. 66. Paul, W. E., Thorbecke, G. J., Siskind, G., and Benacerraf, B. (1969). Immunology 17, 85-92. 67. McDevitt, H. O., and Sela, M. (1965). J . Exptl. Metl. 122, 517-531. 68. McDevitt, H. O., and Sela, M. (1967). J . Exptl. Med. 126, 969-978. 69. McDevitt, H. 0. (1968). J. Immunol. 100,485-492. 70. McDevitt, H. O., and Tyan, M. L. (1968). J. Exptl. Med. 128, 1-11. 71. Tyan, M. L., McDevitt, H. O., and Herzenberg, L. A. (1969). Transphtation PTOC.I, ( 1 ) 548-550. 72. McDevitt, H. O., and Chinitz, A. (1969). Science 163, 1207-1208. 73. McDevitt, H. O., Shreffler, D. C., and Stimpfling, J. H. (1969). J. Clin. Inoest. 48, 57a. 74. Shreffler, D. C . (196s). Wistar Inst. Symp. Monogr. 3, 11-19. 75. Snell, C. D., and Stimpfling, J. H. (1966). In “Biology of the Laboratory Mouse” ( E . L. Green, ed.), pp. 457492. McGraw-Hill, New York. 76. Lilly, F. (1968). J. E x p t l . Med. 127, 465-473. 77. Jaton, J-C., and Sela, M. (1968). J. Biol. Chem. 243, 5616-5626. 78. Mozes, E., McDevitt, H. O., Jaton, J-C., and Sela, M. (1969). J. Exptl. Med. (in press). 79. Mozes, E., Jaton, J-C., Seln, M., and McDevitt, H. 0. (1969). J. Exptl. Merl. ( i n press).

74

HUGH 0. MC DEVIm AND BARUJ BENACERRAF

80. Todd, C. W., and Mandy, W. J. (1969). Proc. XVIIth Colloquium: Protides of the Biological Fluids, Bruges, Belgium (in press ).

81. Rajcwsky, K., Rottlander, E., Peltre, G., and Muller, B. (1967). J. Exptl. Mecl. 126, 581.

The Lesions in Cell Membranes Caused by Complement JOHN H. HUMPHREY AND ROBERT R. DOURMASHKIN Nafional lnsfifutefor Medical Research and Imperial Cancer Research Fund Laboratories,

Mill Hill, London, England

I. Introduction . . . . . . . . . . . . . 11. Description of Holes’ Produced by the Action of C’ . . , , A. Appearance of the C’-Induced Holes . . . . . . B. Size of the Holes . . . . . . . . . . C . Relationship to Electron-Microscopic Lesions Produced by Other . . . . . . . . . . . . Agents . 111. Holes Produced by C’ in Substrates Other than Erythrocyte Memliranes A. Mammalian Nucleated Cells . . . . . . . . B. Bacterial Cell Walls . . . . . . . . . . C. Virus Particles . . . . . . . . . . . D. Bacterial Lipopolysaccharides . . . . . . . . . IV. The Relationship of Holes to Sites of Damage on the Cell Surface V. Occurrence of Multiple Holes (Clusters) at Single Sites of Damage . . VI. The Number of Antibody Molecules Required to Produce a Lesion . . . . VII. The Stage of C‘ Action at Which Holes Are Formed VIII. The Nature of C‘ Holes . . . . . . . . . . A. Qualitative Studies . . . . . . . . . . B. Chemical Studies . . . . . . . . . . . IS. Artificial Membrane Models . . . . . . . . . X. Biological Significance of the Terminal C‘ Lesion . . . . . References . . . . . . . . . . . . .

I.

75 77 77 82 84 8S 85 87 88 88 88 92 95 98 101 101 104 108 110

113

Introduction

Although it is now well recognized that the lysis of cells, to whose surface suitable specific antibody has been attached, is not the only or even the most important action of complement, this Iysis is, nevertheless, a striking phenomenon and its mechanism has becn the subject of much inquiry. The first clear evidence about the nature of the damage to the cell came from Green ct al. (1959a) who studied the cytotoxic action of rabbit antibody and complement on Krebs ascites tumor cells in vitro and observed that, within a few minutes, most of the smaller intracellular ions and molecules such as potassium, free amino acids, and ribonucleotides were lost from the cell together with a large but variable proportion of the intracellular ribonucIeic acid (RNA) and protein. These _

_

~

Since the word “hole” is often used i n a hypothetical sense in this review, it could lxt written in quotation niarks. Scc Scvtion IV. 75

76

JOHN H. HUMPHREY AND ROBERT R. DOURMASHKIN

materials escaped through a cell membrane which appeared to be altered but unruptured, as judged by phase and electron microscopy, and still able to discriminate to some degree against the passage of larger molecules. When the effect of antibody and complement on ascites tumor cells and on erythrocytes was examined in the presence of sufficiently high external concentrations of bovine serum albumin (BSA) in the surrounding medium, the cell membranes lost their capacity to retain potassium and exclude sodium as before, but the outflow of intracellular protein was prevented (Green et aE., 195913). The authors concluded that the action of antibody and complement produces functional “holes” in the cell membrane, large enough to perniit the free exchange of water and ions such as K‘ and Na’ between the interior of the cells and the suspending medium, but too small to allow the passage of protein niolecules of the size of BSA-a sufficient concentration of extracellular BSA can counteract the osmotic pressure of the intracellular proteins and prevent gross swelling of the cell and further stretching of the holes. Mayer (1961a) proposed on the basis of mathematical analysis of the kinetics of immune heniolysis that the production of a single irreparable lesion or “hit” in the cell membrane is sufficient to cause lysis of an erythrocyte. Since cell lysis occurs only at the last stage of the sequence of activation of successive complement components, the terminal lesion would presumably be that responsible for loss of osmotic regulation by the cell membrane, and it seemed likely that the holes postulated by Green and his colleagues might correspond to the lesions required by the one-hit theory. The first direct electron-microscopic evidence for the production by antibody and C’ of lesions resembling holes on erythrocyte membranes was obtained by Dourmashkin and Humphrey (Humphrey, 1963). This chapter is concerned with the nature and causation of these lesions and their relationship to the terminal stage of the lytic action of C’. It is not intended to discuss the whole mechanism of C’ action, especially in its earlier stages, since this has been extensively reviewed by Muller-Eberhard in a previous volume of this series ( Muller-Eberhard, 1968) . When we undertook our assignment we hoped that the problem of the nature of the holes was approaching a solution. This goal has not been reached, although a considerable amount of information has been gained. Not all this information has been published, and some of it only briefly. In order to make the revicw more nearly complete, it has been necessary in Sections IV and VII to draw more extensively than we would have chosen on material which is still unpublished, but which it is hoped will be published fully in due course.

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

II.

77

Description of Holes Produced by the Action of C'

As will become apparent in the course of this review, lesions characteristic of the action of C' have been now observed by electron microscopy in a wide variety of situations in which cell lysis is produced. In each case the method used has been essentially the same, namely, negative staining as developed for the study of virus structure by Rrenner and Horne (1959). For this technique to be applied to cell membranes, it is necessary that these should be thin and flattened, which is difficult to achieve unless the cells have been lysed either before or after thc action of C'. In the case of erythrocyte membranes, a procedure which has been found by the authors to give reliable results is as follows. All particulate matter, apart from the cells or membranes, is first removed from solutions employed by high-speed centrifugation if necessary. After the action of C', any unlysed cells are removed by brief centrifugation at low speed, and the membranes are then sedimented at 50 to 100,000 g for 30 minutes, They are next washed once or twice with 0.01 AI phosphate buffer, pH 6.5-7.0, by high-speed centrifugation under similar conditions and the pellet is suspended in a 2%solution of sodium silicotungstate followed immediately by two drops of 10%aqueous neutral formalin. Preparations for electron-microscopic examination are normally made at once by drying a drop of the suspension on a grid coated with Pailodion and carbon, aIthough good preparations can usually be achieved after storage under these conditions in the cold overnight.

A. APPEARANCE OF THE C'-INL)UCED HOLES The appearance of the holes varies little, whichever type of membrane, antibody, or complement is used. When the surface of the membrane lies flat on the electron microscope grid, a dark central portion of the hole is observed, surrounded by a clear ring. The surrounding ring may be single, double, or it may have tiny spikes radiating from it; sometimes the ring is incomplete. The dark central portion may be irregular in outline (Fig. 1 ) . It appears to be an indentation in the membrane surface, filled with negative stain, and the surrounding ring to be a relatively raised edge. When the folded edgc of the membrane is seen (especially with human C' holes), the clear ring may be seen projecting from the membrane surface, forming a hollow cylinder filled with negative stain (Fig. 2 ) . These forms are not unlike bubbles, the tops of which have been burst as a result of thc negative stain, and resemhlc micellar structures seen in

78

JOHN H. HUMPHREY AND I-IOBERT R. DOURMASHKIN

FIG. l a . Sheep erythrocyte membrane treated with rabbit anti-Forssnian antibody ( IgM ) and guinea pig complement. Magnification: X400,OOO.

THE LESIONS IN CELL MEhfBRANES CAUSED BY COAII'LEhfENT

79

Frc:. Ib. ~ o r i m dhuman rrythrocytcs meniblatie trcnted with atiti-I a n t i h d y arid human complement. Note the difl'ercnce in the sizc of liolcs i i i Fig. l a and b. Magnification: X400,OOO.

80

JOHN H. HUMPI-IREY AND ROBERT R. DOURMASI€KIN

FIG. 2. Escherichia coli cell wall treated with fresh human serum containing natiiral antibody. Note that the lesions along the edge of the wall fragment resemble liollow cylinders, filled with negative stain. The “cylinders” project beyond the edge of the wall fragment. Similar appearances were seen with erythrocyte menilxanes bearing ninny holes. hlagnificntion: X400.000.

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

81

FIG.3a. Thin section of a pellet of hmiian erythrocyte nienibranes, lysed with water, and fixed with OsO,. Pernianganate staining was eniployed. Note the thin, “trani-line” appearance (see text, Section II,A ). hlagnification: :<.700,000. ( From Donrmashkin antl Hosse, 196%.)

saponin-cholestcrol-lecithiii mixtures by Glauert and Lucy ( 1969). The analogy with bubbles is reinforced by the appearance of erythrocyte membranes examined in convcntional thin sections stained with potassium periiianganate. In Fig. 3b, which shows human erythrocyte incinbraiics containing a large number of C’ lesions, it can be seen that the normal “tram-line” appearance is lost and the mciiibrancs look foamy and thickened, as though micclle forination had occurred within then1 (Humphrey et nl., 1968). It must be borne in mind in interpreting the

Frc. .3b. Thin section of a pellet of human erythrocyte membranes pretreated with anti-I antihotly arid hrinian coinplement and Ixwing verv miiny holes. The SYCtions were fired antl stained iix iii Fig, 3a. Note the thickeiird, foamy appearance of the nienibraiies. hlagnification: X200.000. ( F r o m Huinplirey et [il,, 1968. )

82,

JOHN H. I-IUMPI-IREY AND ROBERT R . DOURMASI-IKIN

appearance of cell membranes examined after fixation in osmic acid, dehydration, and embedding in epoxy resins that a substantial loss of lipids occurs during the processing ( Morgan and Huber, 1967). The materials stained by osmic acid do not necessarily represent the lipids as they exist in the ccll membrane (Chapman, 1968). Occasionally, human crythrocyte membranes, when left in hypotonic solution for some time, will show ring-shaped structures with dark centers. These are usually larger than complement holcs, are irregular in size, and will not appear in membranes fixed with fornialin immediately after lysis. They probably are “stromalytic” forms (Baker, 1964) seen cnd on.

B. SIZEOF

THE

HOLES

The sizes and appearance of the holes produced by antibody (either IgM or IgG) from different species on different types of cell membrane and with different sources of C’ are generally similar. Data from the literature and recent remeasurements of our own have been assembled in Table I, which records the mean internal diameter of the holes and takes no account of the outer raised ring. It may be sccn froin this table that there is a consistent small difference bctween the size of the holes made in unfixed cell membranes by human C’ ( 100-110 -4,)(Fig. Zb) and by guinea pig C’ (85-95 A. ) ( Fig. l a ) . This difference has been recorded by more than one group of workers and is certainly significant. In the few instances when measurements of holes made with C’ from other sources have been recorded, they seem to be closer to thosc with guinea pig than with human C’. A further minor difference, which is also significant, has been obscrvcd by ourselves when the substratc has been sheep eiythrocyte membranes prefixed with formaldchyde (which does not damage the Forssinan antigen nor affect the relevant properties of the lipids). The holes in these are some 20 A. smaller than those made with the same C’ in unfixed membranes. On the other hand, when the substrate was a gr‘im-negative bncillus lipopolysaccharide, spread as a thin layer on an electron microscope grid, the holes were slightly larger than in the corrcspondinq bacillary walls or in particles of lipopolysaccharide ( Fig. 7). Evidence is discusscd below that the holes represent micelles in a predominantly lipid outer Iaycr, and it iq possible that the difference in size (with the same source of C’) is due to lateral compression or stretching of the niicellcs according to the stage of the underlying nonlipid matrix.

TABLE I SIZESOF HOLESPRODI-CED R Y ASTIBODYFROM VARIOI:S SPE~IES

Snl,st~l.atc

Sorrnal sheep erythrocyr.es Sornial sheep eryt,hrorytes Formaldehyde-fixed sheep eryt hrucyt~e membranes Sormal human eryt hrucytes Sorrnal hurn:tn erythrocytes S o n n a l human erythrocytes

PSH erythrocyt,es PSH eryt lirocy t es PI13 erythrocytes coated with Shfqdla 0

Satlire of aiitil,otly I~LaIhit19 8 1:al~bit7 8 I t d h i t 19 8 Hiiman 19 S (anti-I) IIuman 19 S (mit,i-hj 1iahhit, ail t i-human erythrocyte Hiimaii 19 S (:inti-I) .lcitlified Iiuniaii seriirn Ralil~it10 S aiiti-S'hiqc~/~~.

: i I L t,igen Gi.arn-neg:itive bacillsry wnlls [Escicerichin. Huninii u r ralhit. 19 S c o / i : Shigel/cc shiquc; l'eillonellu n/ca/esceris) I'eillonella alcalesccns lipopolysaccharide Ralhit (particles) Escherichin coli 1ipopolSsncclisricle Hiimaii 19 S (particles) Eschrrichia coli 1ipopolysacch:~ride(thin Human 19 S layer 011 grid) ____

1Iean internal diameter" of holes (in b.)with diR'ereiit. C'

2TJ

(;iiiiiea pig

c, 5

Hiim:iii

109.4

88 100 ( I ) 104 ( l j 10% 8 (1)

Other

8.i Xi

8 8 . 6 [rnbbit (l)]

66

-

-

-

F

2

zn Fr

zm

~

-

-

-

-

103, 100.2 (11 101 3 (1) 1o:',.x (1)

96, 88 ( I , -

-

-

-

110

9.j, 90 (2)

80-100 [calf (311

-

8.5-90 (2)

-

100..i

-

-

111.3

91

-

'9

2 5a

!2 n

+

g z

8

5r m 5 ____

= The values with 110 figure i n paleiithem are nieaiurenieiits by the present anthurs, thwe marked (1) are from Korsect 01. (1966); (2) from Bladen rt al. (1967); (3) from Bladeii et al. (1966).

5

on G3

84

J O H N H. HUMPHREY AND ROBERT R. DOURMASHKIN

C. RELATIONSHIPTO ELECTRON-MICROSCOPIC LESIONS PRODUCED BY OTHERAGENTS Membrane defects detected by negative staining have been described after treatment of erythrocytes with a number of hemolytic agents. None of these are sufficiently similar to C’ holes to be confused with the latter. 1. Saponin The morphological changes in cell membranes treated with solutions of saponin were first noted during an investigation of Rous sarcoma virus ( Dourmashkin et al., 1962). Hexagonally arranged arrays of holes were seen, each 80-100A. in diameter; a clear ring, made up of a number of small globules, surrounded each hole. Subsequently, Bangham and Horne (1962) and Glauert et nl. (1962) demonstrated that the effect of saponin on cell membranes could be reproduced in a system of artificial membranes made by emulsifying cholesterol and lecithin in aqueous saline solutions. Molecular models were proposed by Lucy and Glauert (1964) suggesting the rearrangement of cholesterol, lecithin, and saponin in an expanded network of micelles. 2. Filipin Filipin is a complex polyene antibiotic that kills fungi by interacting with lipids present in the membrane. There is evidence that sterols, such as cholesterol, are primarily affected, but certain components of the filipin complex also react with phospholipids as well (Sessa and Weissman, 1968). Electron micrographs of membranes from cells lysed with filipin or artificially prepared “polysomes” from cholesterol-phospholipid mixtures treated with filipin show lesions not unlike those seen in immune hemolysis; however, they appear to be somewhat larger than complement holes, measuring approximately 125 A. ( Kinsky et al., 1967). 3. Streptolysin 0

Rabbit or sheep erythrocytes lysed with preparations of streptolysin 0, showed relatively large defects in their membranes on electron microscopy. These defects, measuring about 400 A. in diameter, were approximately round and were edged by double rings. Similar defects were seen in membranes treated with certain preparations of phospholipase C (Dourmashkin and Rosse, 1966). 4. Pliospholipase C

Phospholipase C (sigma) produced large (400-500 A,) circular defects in erythrocyte membranes (Dourmashkin and Rosse, 1966) and

*rmLESIONS

IN CELL MEMBRANES CAUSED BY COMPLEMENT

85

virus coats (Padgett and Levine, 19f35; Kemp and Howatson, 1966). These defects were edged by doubled rings which became detached from the membrane and could be obscrved separately. It was later shown that phospholipase C from the same origin could interact with cholestcrollecithin mixtures to produce similar changes. The presence of cholesterol was necessary for this to occur. Heating the phospholipase to 100°C. prevented its action, which argues against the lesions being due to a nonenzymatic effect ( Simpson and Hauser, 1966).

5. Lysolecithin Lysolecithin or synthetic lysophosphatidyl ethanolamine are wellrecognized hemolytic agents. When they are added to a lecithincholesterol dispcrsion, discs of uniform diameter are produced, each disc consisting of two lamellae annealed around their edges to enclose n water-containing compartment ( Bangham and Horne, 1964). A similar event occurs on treating erythrocyte membranes with these agents ( Dourmashkin and Rosse, 1966). We have never seen holes resembling those produced by C’. 6. Streptolysin S

Partially purified streptolysin S, prepared from RNA broth cultures of streptococcal strains that do not make streptolysin 0, failed to produce lesions identifiable by negative staining or by conventional thin sections on rabbit or sheep erythrocyte membranes, although the membranes had evidently disintegrated during the course of hemolysis ( unpublished observations b y the authors). Ill.

Holes Produced by C) in Substrates Other than Erythrocyte Membranes

A. MAMMALIAN NUCLEATEDCELLS Because of the greater technical difficulty involved in examining the membranes of nucleated mammalian cells and because these have not been used to any great extent in theoretical studies of the mechanism of C’ action, relatively little has been published about the detailed nature of the lesions produced by C’. However, Humphrey and Dourmashkin (1965) showed photographs of the membrane of Krebs mouse ascites tumor cells lysed by the action of rabbit 7 S antibody and rabbit or guinea pig C’, in which multiple holes were present identical with those produced by similar C’ in erythrocytes ( Fig. 4 ) . Humphrey ( 1968), in a discussion of C’-dependent histamine release from rat peritoncnl mast cells hy rabbit antirat Ig and antirat mast cell antibodies, showed electron micrographs taken by Dourinashkin of le-

86

TOHN H. HUMPHREY AND ROBERT €3. 1)OUIIMASHKIN

FIG. 4. Krebs ascites tumor cell membrane after treatment with rabbit IgC antibody and giiiiie.i pig compleinent. Magnification: X240,OOO. (Fiom Humphrey and Doulniashkiii, 1965.)

THE LESIONS I N CELL MEMBRANES CAUSED BY COhfPLEMENT

87

sions produced h y antirat Ig and rabbit C’. Again typical C’ holes were presrwt. In contrast, meinbraw\ of mast cclls from rats with anaphylactic (hornocytotropic) sensitivity to ovalbumin, after contact with the antigen, contained no c’ holes hiit showcd spherical protrusions about 250 A. in diameter. The significance of these protrusions (which werc not unlike the micclles produced by lysophosphatides ) is uncertain. However, it is of interest that histamine release by homocytotropic antibody is riot dependent on added C’ and that the biochemical mechanisms, so far as they are understood, can be distinguished by the use of esterase inhibitors (Bccker and Austen, 1966). B. BACTERIAL CELLWALLS Gram-negative bacteria are killed b y the action of antibody against their 0 somatic antigcm and C’. Electron-microscopic evidence of the formation of typical C’ holes on Eschericlzia coEi cell walls has been published b y Bladen et al. (1966) and Glynn and h4ilne (1967) and on Veillonella alcalescens by Bladen et al. (1967). Humphrey and Dourmashkin ( unpublished) have obscwed similar C’ holes on Shigella shigac: cell walls and on protoplast membranes from Bacillus lichenifomis. The appearance and si7e of the holes (Table I ) are characteristic of the species of C’ employed. Glynn and Millie ( 1967) examined whether lysozyme played any part in the production by human C’ of holes in Escherichia coli. They showed that rcnioval of all detectable lysozymc activity from the antibody and C’ by absorption with bentonite or addition of antibody against human lysozyme did not affect the forination of holes. The prevmce of C’ and of small amounts of antibody ~ v a sessential both for killing and lysis of the bacteria in the prcsencc of lysozyme. When lysozymc was inhibitcd by antilysozyme the rate of killing was diminished and the occurrence of lysis was very greatly delayed; both were restored to normal by addition of egg white lysozyme. However, when lysozyme had been removed by bentonite absorption, both killing and lysis werc stoppcd, and were not returned to normal b y addition of egg whitc lysozyme. They concludcd, in agreement with others (Amano et al., 1954; Muschel et a!., 1959: Wardlaw, 1962), that thc primary damagcx is the result of the action of C’ and antibody but that lysozyme accelerates the killing and especially the lysis of the bacteria. However, the failure of egg white lysozyme fully to restore the activity of bentonite-trcated human serum was taken to indicate the nced for an unidentified bentonitc ahsorbable factor for full bactericidal activity. The holes are certainly large enough to permit the passage of lysozymc (mol. wt. 14,000). It is worth commenting that the

88

JOHN H. HUMPHREY A N D ROBERT R . DOURMASHKIN

presence of lysozyme does not affect the appearance of the holes; this is presumably because the substrate in which the holes are formed is independent of the underlying mucopcptide.

C. VIRUSPARTICLES Berry and Almeida (1968) studied preparations of avian infectious bronchitis virus grown in chickens. When treated with unheated rabbit antibody, typical C’ holes about 100A. across were seen on the viral envelope. Similar holes were produced by unheated rabbit antichick embryo fibroblast antiserum, which also contained antibody against the viral coat. Heated rabbit antisera produced no holes, nor did unheated fowl ( homotypic) antiserum that contained antibodies against the petal shaped projections of the virus but not against the underlying viral coat. These findings indicate that the viral coat contained host antigens and that the coat was also capable of acting as a substrate for the lytic action of C’.

D. BACTERIALLIPOPOLYSACCHARIDES Lipopolysaccharides isolated from gram-negative bacteria in a more or less native state can be “dissolved in aqueous media to give clear or faintly opalescent solutions containing small but variable-sized particles in suspension. These are readily adsorbed directly onto carbon-coated electron microscope grids or onto fine bentonite particles, which can, in turn, be deposited on the grids. Bladen et al. (1967) examined lipopolysaccharide extracted by phenol-water extraction from Veillonella alcalescens, and Humphrey et al. (1968) examined the native lipopolysaccharide complex secreted by a lysine-requiring strain of Escherichia coli 12408 grown in lysine-deficient medium (Taylor et al., 1966). With fresh, normal, guinea pig or human sera, which contain both C’ and “natural” 19 S antibodies ( authors’ unpublished observations), typical C’ holes were observed. When C’ activation was prevented, such holes were not formed. The addition of hyperimmune rabbit serum did not notably increase the number of holes, which were, however, very plentiful with the normal sera alone. IV.

The Relationship of Holes to Sites of Damage on the Cell Surface

It is a crucial question whether the holes described above represent the lesions in the cell membrane actually responsible for loss of osmotic regulation or are only epiphenomena accompanying some other more fundamental kind of damage. Until more is known about the detailed

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

89

arrctngernent of lipid and protein molcculcs in cell membranes which constitute their osmotic barrier, it is difficult to statc with certainty that no other derangement, unclctected b y electron microscopy, underlics the visible holes. However, the correlation between the appearance of holes and C’-induced lesions appears to be complete. Thus, in all systems examined that involvcd lysis of cells by the action of C’ (human, she<,p, rabbit, or guinea pig erythrocytes; Krebs ascites tumor cells; rat mast cells; and various gram-ncgative bacteria ) , typical holes have been observed but not when the cells were lysed by other agents. Furthermorc, under conditions when lysis of erythrocytes by C’ was incomplete the membranes of unlyscd cells, separated by low-speed centrifugation and subsequently lysecl osmotically, never contained holes. Holes are not produced by antibody with or without inactivated C’ nor by C’ alone (provided that any pre-existing antibodies have been absorbed out first). The strongest evidence that the holes arc the actual lesions comes from the expcrinienty of Borsos et al. (1964), using the standard system of rabbit anti-Forsman antibody, sheep erythrocytes, and guinea pig C’. The erythrocytes were treated with antibody and reacted with C’ to yield cells in the statc EAC’14. These were then reacted with purified guinea pig C’2, previously titratcd in the same system, so as to give, under standard conditions, cells with a predicted number of EAC’ 142 sites, that is, sites at each of which thc action of excess of the remaining C’ coniponeiits [supplied as citrate or cthylenediaminetetraacetate ( EDTA ) guinca pig serum] woulcl lead to sufficient damage to the membrane to cause cell lysis. After treatment with citrate guinea pig serum the fragments of lysed cell membranes were washed and examined by negative staining in the electron microscope. The areas of and the number of holes on a sufficient number of fragmcnts werc measured to permit calculation of the number of holes on a complete erythrocyte membrane (26 p’). Agreement betwecn the nunilx~rof sites of damage predicted and the number of holes w a s rcniarkably close. In this system, therefore, it appears that a single hole represents a lesion sufficient to lyse the cell. Confirmation of this conclusion has bcen obtained in hitherto unpublished experiments by Dourmashkin and Humphrey, in which highly purified rabbit IgM anti-Forssman antibody [prepared by elution from formalin-fixed sheep erythrocytes, as described by Frank and Humphrey ( 1968)l was used with sheep erythrocytes and carefully absorbed whole guinea pig C’. The amount of antibody required to cause 50%lysis of the cells with excess of C‘ under standard conditions was measured. It was assumed that each lytic lesion would correspond to a single hole and that the number of lesions per cell would follow a Poisson distribution

90

JOHN H. HUMPHREY AND ROBERT R. DOURMASHKIN

m W

2i 1 a LL

0 K W

m

z 3 z

00206 10 NUMBER

0 24 68

OF 'HOLES'

0 2 6 10 14

PER SQUARE MICRON

OF

MEMBRANE

FIG.5. Production of holes in sheep erythrocyte membranes by rabbit IgM antiForssinan antibody. Evidence that one hole represents a lesion: First, 2, X lo8 sheep erythrocytes were incubated for 1 hour at 37OC. with varying amounts of highly purified rabbit IgM anti-Forssinan antibody (Frank and Humphrey, 1968) and a large excess (1/20) of carefully absorbed guinea pig C'. Then, after incubation, the lysed cell membranes were collected by centrifugation at 100,000 g , washecl with 0.01 M phosphate buffer pII 7 , and examined in the electron microscope by negative staining. A large number of photographic plates were taken of cell membrane fragments selected a t random. The photographs were eniarged, and the areas of meinbrane and the numbers of holes measured. The amount of IgM antibody required to cause 50%hemolysis was measured in the same experiment, and it was assumed (from the one-hit theory) that this amount of antibody was sufficient to cause a mean of 1.4 lesions per lysed cell. The arrows indicate the expected numbers of holes per square micron of membrane if ( u ) a single attached IgM antibody inoleciile is sufficient to activate C' to cause a lesion, and ( b ) one hole represents one lesion. (Hatchecl blocks-plates with no holes; black blocks-plates with stated number of holes per square micron. )

(Mayer, 196lb), so that the amount of antibody sufficient to lyse half the cells would produce an average of 1.4 lesions per cell lysed. The erythrocytes were then treated under the same standard conditions with various multiples of the determined amounts of IgM, and the number of holes per unit area on membrane fragments was measured. Comparison of the

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

91

number of lesions predicted with thc number of holes observed showed excellent agreement ( Fig. S) . These findings indicate that a single hole may represent the hemolytic lesion, at least under certain circumstances. As is discussed below in Section VIII, thc holes are probably bubbles or micelles in the outer lipid layer of the membrane. It may be argued that beneath them lies another laycr which is actually responsible for permeability control. The question arises whether the dimensions of the holes arc compatible with what is known about the permeability changes induced by C’. Several workers (Green et d., 19S9b; Sears et al., 1964; Frank et al., 1965) have shown that C’ hcmolysis, but not the free passage of small ions, is prevented by the presence of BSA in the external medium at a concentration of 22.5%or above. Since hemoglobin does not leak out under such circurnstanccs, the lesions must he small enough to retain hemoglobin. The Stokes radii of HSA ant1 hemoglol~inare 37 and 30.S A., respectively (quoted by Ackers, 1964). Scars et al. (1964) found that dextran 40 was also effective, h i t that dextran 20 was not fully so, and dextran 10 was ineffrctive. The Stokes radii of the dextrans arc. not accurately known but their behavior in gel filtration suggests that their effective sizes are substantially greater than would be expected for globular proteins of similar molecular weights ( Andrews, 1966). Sears et al. (1964) quotc the effective radius of dextran 20 as 32 A. It seems a reasonable assumption, therefore, that the lesions must be such that the limit of exclusion is for molecules with cffective diameters about 60 A. The intcrnal diameters of the holcs, as measured by electron microscopy, is greater than this (Table I ) . Unfortunately we have no means of knowing what were the cffective diameters of the holes in the cell membranes before drying. Hydration or adsorption of ions at the edges and any tendency for the holes to be cup-shaped would diminish their cffective diameters; furthermore, for significant diff usion of macromolecules to occur, the effective diameter of a pore must be greater than that of the molecules ( i.e., greater than 60 A , ) . If a s c ~ o n dpermeability barrier were present beneath the layer in which t h e holes are formed, it would need to have pores so near in size to the holes which are described that we consider the most reasonable, though admittedly unproven, conclusion to be that the holes represent the effective. lcsions produced b y C’. Evidently when, as discussed in Scction V, a site of damage is the result of the formation of a cluster of holes the lesion will, in fact, be multiple, althouSh each individual hole will retain its characteristic permeability properties, unless the holes actually run together. Sears ef al. ( 1964) obscrvcd that ccrtain antiscra (rabbit and human ) against human A erythrocytes, acting with human C’, appeared to cause lesions greater

92

JOHN H. HUMPHREY AND ROBERT R. DOURMASHKIN

than 65 A. in diameter, which permitted the escape of intracellular hemoglobin even in the presence of high external concentrations of BSA or of human hemoglobin. A probable explanation for this is given hy Hosse et nl. (1966). They showed that the diameter of the individual holes produced by the action of human C’ on human erythrocytes [either normal or from subjects with paroxysmal nocturnal hemoglobinuria (PNH)] was 99-104 A. irrespective of whether the antibody used was human anti-I, anti-A, or of the Donath Landsteiner type or rabbit antihuman erythrocyte. However, when very many holes in clusters were formed these were seen to fuse and to result in what amounted to tears in the membrane. V.

Occurrence of Multiple Holes (Clusters) at Single Sites of Damage

In the previous section evidence was discussed that a single hole may represent a site of C’ damage in a system involving sheep erythrocytes, rabbit IgM antibody, and guinea pig C’. However, in certain other systems the number of holes observed in fragments of erythrocyte membranes, adjusted to the area of a whole erythrocyte, may greatly exceed the expected number of lesions. The holes are similar to those seen in the experiments described above, and there is nothing to indicate that they are functionally different, but numerous holes close together tend to occur in clusters on a membrane fragment as well as some which appear singly. One system in which this phenomenon was observed was when sheep erythrocytes were lysed by varying amounts of highly purified rabbit IgG anti-Forssman antibody in the presence of excess guinea pig C’, as in experiments designed to ascertain how many IgG antibody molecules pcr cell are required to initiate a lesion (see Section YI). Figure 6 shows the findings in such an (unpublished) experiment. In order to obtain sufficient membrane fragments with holes for examination in the electron microscope without having to examine a very large number of plates, it was necessary to use conditions in which extensive or complete hemolysis occurred. The quantity of IgG antibody required to cause 50%lysis under standard conditions in the presence of 1/67 C’ was ascertained, and the erythrocytes were treated under similar conditions with multiples of this quantity ranging from 1.33 to 33.0. About 100 photographs of membrane fragments were taken in each case, and the number of holes counted and the membrane areas measured. In the figure are indicated the numbcr of plates with a given number of holes per square micron of membrane when different amounts of antibody were used. (The area of membrane was not constant from plate to platc.) According to the one-hit thcory, at 50%lysis the expected mean number

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

60

50

93

MULTIPLE OF 50% HEMOLYTIC DOSE OF IgG ANTIBODY

40 x 133

30

20

>x 267

IIl
FIG. 6. Formation of mi~ltipleholes (clusters ) on sheep erythrocytes by rabbit IgG anti-Forssinan antibody and guinea pig C'. The experimental design was similar to that in Fig. 5, except that pnrified IgG anti-Forssman antibody was employed, and the final guinea pig C' coccentration was 1/67, Several multiples of the 50%heinolytic amount of antibody were used. This figure illustrates how, at low ratios of anti-

c',

some nienibrane fragments have inany more holes than would be expected body to if one lesion corresponded to one hole. (Hatched Mocks-plates with no holes; black blocks-plates with the stated ntmilier of holes per square micron. )

of lesions per whole eiythrocyte membrane (26 p ? ) would be about 0.7, averagcd ovcr all thc cells (h4aycr, 1961b). It is striking, espccially at the lowcr multipIicitics of antibody, th'it some membrane fragments were seen which contained far more than the expectcd number of holcs. The distribution shown in this figure does not, however, give a true picture of thc average number of holcs per wholc erythrocyte, since the memb r m c fragmcnts with no or few holes werc generally larger than those

94

TOHN 13. HUMPHREY AND ROBERT R . DOURhlASHKIN

with many holes. Nevertheless, the skew distribution of holes indicates that some occurred in clusters, and that clusters were more frequent as the amount of antibody decreased while the concentration of C’ was held constant. It was not clear whether cluster formation with IgG antibody was duc to the W R Y in which C’ was fixed or to local movement of antibody on the cell surfacc. (Although the antibody used was avid, evidence was obtained that transfer from cell to cell could occur to a small extent when erythrocytes to which this IgG antibody was attached, and which wcre then washed, wcre incubated with untreated erythrocytes). In other experiments reported by Rosse et at. (1966), erythrocytes from patients with PNH and from nonnaI human donors were lysed with human TgM (anti-I) antibody and large amounts of human C’. Again, electron-microscopic examination of the membrane fragments revealed a mean number of holes per cell very much higher than that expected from the extent of hemolysis. Since there is no reason to believe that the onehit theory does not apply to human cells and C’, these observations are not compatibIe with the hypothesis that a single lesion is always represented by a single hole. They might, however, be explained in one of two ways: either the terminal stage of human C’ activation at a single site generates a sufficient amount of some surface-active agent to cause extensive local micelle formation in the membrane (see Section VIII) or there is multiple binding of C’3 at a single site, as has been indicated b y Mullcr-Eberhard et al. (1966), and a variable number of the bound C’3 molecules activate the succeeding C’ components. We have attempted to tcst the second posibility by comparing the lysis of human erythrocytes by huinan C’ and human anti-I antibody [prepared as described by Rosse et al. (1966), but from a less hemolytically active serum] under conditions in which one or the other reagent was limiting. It was nece5sary to increase the sensitivity of the erythrocytes to lysis b y human C’ treatment with 2-aminoethylisothiouronium bromide (AET), which renders normal cells as sensitive to C’ as PNH cells (Sirchia and Dacie, 1967). Thc experimental results in Table I1 indicate that when C’ was limiting thcre were few clusters (not exceeding those due to the action 011 these cells of human C’ alone, to which they are abnormally sensitive) and that the proportion of membranes with single holes increased with the degree of hemolysis. However, when antibody was limiting and c’ in excess, hemolysis was accompanied by the presencc of more meinliranes with clusters than with single holes. Frank, Dourmashkin, and Humphrey ( unpublished observations ) havc also txamincd the lesions produced by human C’ on sheep erythrocytes sensitized with rabbit anti-Forssman Igh4 antibody. When antibody WRS limiting and C’ prcsent in cxccss the membmnes contained clustcrs

95

THE LESIONS I N CELL MEMBRANES CAUSED BY COMPLEMENT

IiYhIi

OF

T l R L E I1 dET TREATED HVMAYERYTHROCYTE5 B Y A \ D B Y H ~ U AAYTI-I L ANTI BODY'^

IIUMAN

c’

S O .of membiaiie\ o r fragment\ with Dilutioii of human anti-I 1/ 1/9600 1/4X00 1/2400 l/l200 1/600 1/300 1/1>0 I/ m 1/9600 1/4XOO 1/2400 1/1200 1/600 1/300 l/l>O a

Dilutioiiof

Hemolysih

SO

Siiigle

hiimnn C’

( %)

holes

holes

Clusters

1/YO 1/ : 3 0 1/:10 1/30

6 ;i 20 34 50 64 7;.

77

4

-

-

2 -

1/30 1/30 1/30 1/30 1/60 1/60 1/60 1/60

1/60 1/60 1 /60 1/60

8:) X,.

4.5 3 X 10 3 19 28 .i 44 .54 63

-

-

-

-

-

-

-

-

-

48 59

9

2

17

2

Hiimaii eryt~hrorytesmade seirsitive to C’ by treatment, wit.h ANT were incubated

at, 37°C. with varying amoiuits of purified hiinian IgM antibody at. different, concerit.ratioiis of hiininn C’. After iiicxibat,ioii any intact, cells were lysed osmotically, and the

washed memhraiies and membrane fragments were examined for the presence of holes either singly or iu chisters.

iii

the electroii microscope

of holes, whereas when the degree of lysis was limited by the C’ concentration, clusters were not observed and the nuniber of holes was approximately that expected from the number of lcsions predicted from the onehit theory, Thus the formation of multiple holes in clusters appears ( a ) with human C’ in cxcess, and lysis is limited by the amount of human or rabbit IgM antibody, and ( b ) with guinea pig C’, when this is in excess and lysis is limited by the aiiiount of rabbit IgG, but not IgM antibody. Although these findings might be taken to support the idea that clusters of holes are formed by multiple activation of C’3 at a single site, they certainly do not prove its correctncss. VI.

The Number of Antibody Molecules Required to Produce a Lesion

Humphrey and Dourmashkin ( 1965) used purified rabbit IgG and IgM antibodies against the Forssman antigen to correlate directly the

96

JOHN H. HUMPHREY AND ROBERT R. DOURMASHKIN

number of antibody molecules attached to sheep erythrocytes with the number of holes per cell membrane detected by electron microscopy after lysis in the presence of guinea pig C’. The antibodies were labeled with lZ5I,and known numbers of molecules were added per cell, more than sufficient to lyse all the erythrocytes in 2 hours at 365°C. in the presence of a 1:50 dilution of C’, preabsorbed with sheep erythrocytes. Many membrane fragments were examined; the number of holes seen md the membrane areas were measured; and the number of holes per whole membrane (26 p 2 ) calculated. Using their purest IgM antibody, the ratio of the number of antibody molecuIes to the number of holes was found to be 1.15 to 3.4 (mean 2.1), whereas with their best IgG antibody the ratio varied from 1: 308 to 1:4100. Using the same antibody preparations, they also measured the number of antibody molecules per erythrocyte required for 50%lysis, and calculated on the basis of Mayer’s one-hit theory how many molecules were on average necded to cause 1 lesion per cell. The numbers for IgM antibody were 2.4 and for IsG about 4000. The maximum number of rabbit IgM Forssinan antibody molecules which can attach to a sheep erythrocyte was estimated to be about 100,000, and of IgG molecules about 600,000 (Humphrey and Dourmashkin, 1965). In the case of rabbit IgM antibody and guinea pig C’, it had already been shown that the number of holes corresponded to the number of theoretical hits. They argued that in the case of IgM antibody it was very unlikely that two molecules would arrive by chance at 2 neighboring sites out of 100,000, and that a single molecule would suffice to c a s e a lesion. The fact that about 2 molecules were required in their experiments could be attributed to working in the absence of a sufficient excess of the C’ components. The validity of this explanation was supported by subsequent experiments (see Section IV) in which it was found that, in the presence of higher concentrations of C’ ( 1:12.5), at various multiplicities of antibody molecules to erythrocytes the number of holes per cell was linearly related to the number of antibody molecules per cell. Borsos and Rapp (19651) introduced the technique of C’la fixation and transfer, by which the number of C’1 molecules activated in a system can be measured by transfer of the C’la to EAC’4 cells, which are then lysed by addition of a large excess of the remaining C’ components. They found a linear relationship between the amount of IgM antibody and the amount of C’1 activated over a wide range of antibody concentrations and aIso concluded that 1 molecule of antibody was sufficient to sensitize a shcep erythrocyte to the action of guinea pig C’. Thus this conclusion

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

97

seems to be soundly based, although in those systems where a lesion is represented by a cluster rather than by a single hole (see Section V ) , a one-to-one relationship between the number of holes and of IgM antibody molecules is not to bc expected. Similar measurements have not been reported for other lytic systems, but Rowley and Turner (1966) have examined how many molecules of rabbit IgM antibody against the 035 lipopolysaccharide antigen of Salnioitella oddaide were required on average to opsonize one bacterium, so that it would be ingested and killed by macrophages in the peritoiieal cavity of a living mouse. They found that a minimum of 8 molecules were needed and suggest that C’ activation is a part of the opsonic mechanism, as proposed by Nelson ( 1962). In Humphrey and Dourmashkin’s ( 1965) attempts to correlate directly the number of holes and the number of IgG rabbit anti-Forssman antibody molecules per erythrocyte, their results showed a much wider variation than in the case of IgM. This may have been partly due to the tendency discussed in Section IV for IgG antibody to give rise to clusters of holes, so that a few fragments of membrane with clusters may greatly exaggerate the number of holes estimated per cell. It may also have bccn due to the tendency reported by Coltrn et nl. (1967) for IgG antibodies to fix C’la to a marked but variable extent less well at elcvated temperatures (30°C. ) than at lower temperatures (6°C. ). No account was taken of this in Humphrey and Dourmashkin’s experiments. However, they suggested that their findings could be explained if 2 adjacent IgC, molecules on the erythrocyte surface were required to activate C’ and cause a hole. They calculated that if a sheep erythrocyte has 600,000 combining sites for IgG antibody, about 800 moIecules would be required to attach at random so that there would be an even chance of 2 molecules being adjacent ( assuming that each site has two effective neighboring sites) and concluded that their findings were coinpatible with this cstimated figurc. Humphrey ( 1967) showed that the amount of rabbit IgG anti-Forssman antibody required to lyse sheep erythrocytes in the presence of a given amount of guinca pig C’ was approximately 860 times greater than the amount needed to produce a similar degree of lysis when the crythrocytes, with IgG antibody attached, were also trcated with an excess of guinea pig y - antirabbit IgG. On thc assumption that a solitary molccule of rabbit IgG antibody on the erythrocyte surfacc would not activate C’ but that subsequcnt attachment of 3 or 4 molecules of thc anti-IgG would do so effectively, this finding supports the calculation that roughly 800 nioleculcs of IgG antibody per shcep erythrocyte are reqtlired to activate. C’ at one site,

98

JOHN H. HUMPHREY AND ROBERT R . DOURMASHKIN

Clearer, though still indirect evidence that 2 or more 7s antibody molecules in close proximity on the cell are required to fix 1 molecule of C’la was provided by Borsos and Rapp (1965b) who found that the number of C’la molecules fixed was proportional to a power of the antibody concentration slightly over 2 (2.1-2.6). That this conclusion is also valid for IgG antibody in immune complexes is suggested by the observation of Cohcn (1968) that IgG rabbit antiovalbumin, of which the antibody-combining sites were intact but of which the C’-fixing ability had been destroyed by amidation and benzylation, inhibited C’ fixation by intact antibody in thc presence of ovalbumin to an cxtent predictablc from calculations based on the assumption that 2 intact, adjacent, antibody molecules arc necessary for c’fixation. Most of the experiments discussed in this section were done with rabbit IgM and IgG antibodies and guinea pig C’. Gcneralization of the conclusions to other systems may not be justified; indeed, even among rabbit IgM antibodies, some antibodies that fail to activate C’ have been described ( Hoyer et al., 1968).

V11.

The Stage of C’ Action at

Which Holes

Are Formed

If the holes are, indeed, the lesions that lead to the loss of osmotic control by cell membranes, it should follow that they are formed at the final stage of C’ activation, i.e., by the action of C’9 (MidIer-Ebcrhard, 1968). Jn the case of C’ holes made in isolated bacterial lipopolysaccharides, where no question of osmotic regulation arises, Gewurz et a!. (1968) showed that all the components of guinea pig C’, from C’1 to C’9, were consumed. Interestingly, they found that lipopolysaccharide incubated with fresh normal serum (containing, presumably, natural antibody) consumed relatively little C’l, 4, and 2 but relatively much more of components C’3 to C’9, compared with aggregates composed of rabbit anti-RSA and BSA made at equivalence. In this respect lipopolysaccharide behaved like zymosan. Gewurz et al. found that consumption of thc tcrminal C’ components depended, nevertheless, upon the prior activation of C’l, 4, and 2. Although the extensive activation of the C’3 components onward remains to be explained, it is consistent with the present authors’ observation th‘it remarkably large numbers of holcs are produced by C‘ on lipopolysacchiiride substrates in the presence of quite small amounts of natural antibody. Examination of erythrocyte membranes in thc electron microscope at the stagc EAC’142 by Rorsos et a?. (1962) did not reveal any abnornialities, Mullrr-Ebcrhard ( 1965) stcites that electron microscopy rcvealed

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

99

the characteristic ultrastructural lesioiis following human C’9 action, and quotes a note by Hadding et 01. (1966) reporting that 110 such lesions could be detected with certainty in membranes of cells containing C’8 sites. Increased fragility of erythrocytes at the C’8 stage has been reported by Linscott and Nishioka (1963), by Stolfi (1967), and by Gotze et al. (1968), and it would be interesting to know whether any changes in the membrane are present at this stage. Frank et nl. (1964) showed that 0.09 hl EDTA inhibits the transforination of a cell in the E* state (i.e., which has been acted upon by all the C’ components and has one or more irrtprable lesions) to a ghost which has leakcd its hemoglobin. They also showed (1965) that the conversion of E D to a ghost involved three steps. The first, which was temperature-dependent, took place in the presence of 0.09 M EDTA; the second, which was not temperature-dependcnt occurred only when the EDTA concentration was lowered to 0.01 M ; and the third, which was also indcpendent of temperature, was blocked by the presence of 25% BSA in the external medium. This last is presumably the osmotic swelling which occurs after the holes are formed. The first two steps could be the chemical or enzymatic action of C’9 on the cell membrane, followed by some rearrangement of the constituents to form the holes. It would be of considcrable interest to verify this by examining membranes fixed at each stage, but, unfortunately, in the present author’s experience, the number of lesions per cell which may be expected to be produced by thc methods described is so small that definitc conclusions could not be drawn. Information about the nature either of C’9 or of the holes should cast light on each other. It is suggested by Miiller-Ebcrhard that C’S is bound firmly to the cell surfacc and that C’9 reacts with ceII-bound C’S. In the case of sheep crythrocytes which had reacted with human C’S, Hadding and Muller-Eberhard (1967) found that lysis resembling that causcd by C’9 could be brought about by 1,lO-phenanthrolinc and they postulated that chelation of ferrous ions was involved in the final step. Giitze et al. (1968) using EAC’1-8 prepared with guinea pig C’ were unablc to confirm this suggestion. These workers prepared highly purified C’9 from pig serum and found that its activity was abolished by 0.1 111mercaptoethanol but not by reagents rcactiiig with -SH or with serine hydroxyl groups. It was also inhibited rcyersibly by 10F A1 Cu2+or 10;”M Zn’+, but irreversibly by either lo-” A1 Fez+or 10.’ h l Fe:“ ions. Apart from thc suggestion that heavy metal ions may be involvcd, the modc of action of C’9 is at present unclcar.

100

JOHN H. HUMPHREY AND ROBERT R. DOURMASHKZN

FIG.7. Electron micrograph of Escheiichia cola lipopolysaccharide ( LPS ) applied directly to grid, then treated with human complement ( 1:20) containing

THE LESIONS IN CELL MEMBRANES CAUSED BY COMPLEMENT

VIII.

The Nature of

101

C’ Holes

A. QUALITATIVE STUDES Evidence has been accumulated that C’ holes represent a rearrangement of the predominantly lipid outer layer of the cell membrane, in which small micelles of lipid constituents form circular or spherical structures, the centers of which become filled with stain and can be viewed in the electron microscope. It was found soon after the holes were first observed that their appearance was not affected-except that it was actually sharpened-by treatment of membranes bearing holes with trypsin or with buffer at pH 2.5 to detach antibody and/or C’ components. We have since treated holes in Esclierichia coli lipopolysaccharide on electron microscope grids with pronase, and, again, the only effect is to sharpen but not to alter their appearance (Fig. 7, authors unpublished work). It was also found that typical holes could be pro-

FIG.8a. Formalin-fixed sheep erythrocyte nieml)ranes, bearing very many complement holes (guinea pig), were fixed for 10 minutes in OsOl and negatively stained. Magnification: X400,OOO. ( From Humphrey ct al., 1968.) natural antibody. The grids were subsequently floated on 0.1%pronase for 1 hour. Pronase did not change the appearance of the holes. Coinpleiiient holes are seen both on LPS particles and also on LPS spread oiit on grid; their bizes are different (see Table I ) . Magnification: X225,OOO.

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duced by the action of anti-Forssman antibody and C’ on erythrocyte membranes prepared by osmotic lysis and then hardened by treatment with 2% formaldehyde in thc cold (Humphrey, 1963; Humphrey et a]., 1968). Such treatment, which does not destroy thc Forssman antigen, made it possible to obtain stabilized erythrocyte membranes bearing holes which could be subjected to extraction procedures with organic solvents and yet leave the membrane matrix sufficiently intact for subsequent electron-microscopic examin a t’ion. Extraction of an aqueous suspension of the formalin-fixed membranes with ether did not affect the holes, but alcohol-ether removed them to a large but variable extent, and chloroform-methanol (2: 1 ) removed them completely ( Fig. 8 ) . These observations were interpreted to mean that the holes are present in a predominantly lipid layer on the membrane, some or all of the constituents of which are removed by solvent extraction. After formalin-fixed, sheep erythrocyte membranes bearing C’ holes

FIG.81). A preparation similar to that in Fig. 8a after extraction with chloroform-methanol for 1 hour. After this treatment, coniplemeiit holes could not be found. Magnification: X400,OOO. (From Humphrey et al., 1968.)

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had been extracted with alcohol-cther in the cold and shown to have few or no remaining holes, thcy wcie treated afresh with anti-Forssnian antibody and guinca pig c’ and rc-examined. Holes were again visible, though less numerous than before thc extraction procedure ( Humphrey et al., 1968). Since about 5x of the Forssman antigen on the membranes had survived alcohol-ether extraction and was available for combination with antibody ( unpublished observation ), this finding implies that a fresh substrate in which C’ hole$ could be formed was furnished during the vcond treatment with antiserum and C’. Erythrocyte membranes contain a complex mixture of lipids, which differs mxkedly from species to species (e.g., human and shcep), yet the C’ holes in each arc similar. It seemed probable, thercfore, that further analysis of the nature of the holes would be easier if a simpler substrate werc used. Humphrey et at. (1968) chose the native Iipopolysaccharide complex secreted by Escherichia coli 12408 when grown in a lysinedeficient medium, since this material was a typical rough strain 0 antigen the composition of which had been characterized by Knox et nl. (1967). It contained 60%of Iipopolysaccli~uide,26% of extractable phospholipid (largely phosphatidylethanolamine ), and 11%of peptide. The presence of peptide permitted the material to be tracc labeled with radioactive iodine, and so enabled the amount adsorbed onto carbon-coated electron microscope grids or glass surfaces or onto bentonite particles to be nieasured easily. As already mentioned in Section 111, C’ holes were readily produced by the action of fresh normal human or guinca pig serum on this material, even without the addition of extra antibody from specifically immunized rabbits. Such holes could be completely removed by extraction with chloroform-methanol, but only if acetic acid was also added. The requirement for acetic acid was interpreted as due to the need to overcome tight bonding of the lipids by the layer of activated carbon on the grids; the labeled lipopolysaccharide itself was not removed by the solvent. These findings confirmed that the holey were formed in some lipid substrate on the surface of the grid, supplied by the lipopolysaccharide complex and,/or by the scruni used as the source of C’. In order to tcst the second possibility, freshly prepared carbon-coated grids were floated on solutions of highly purified simple antigens, namely crystallized human serum albumin (HSA) or Type 3 pneumococcus polysaccharide, and washed. The grids with adsorbed antigens were then incubated with various dilutions of specific rabbit antiserum and guinea pig C’. Over a fairly narrow range of antibody dilutions the grids showed scanty but typical C’ holes, controls without antibody or with heated C’ showed none (Humphrey ct al., 1968). The original HSA, even though

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crystallized, was found by thin-layer chromatography to contain about 1%of a mixture of lipids resembling those in normal serum; however, substantially more serum lipids than would be accounted for by the adsorbed HSA became adsorbed onto the grids during the contact with antiserum and/or C’. These findings were interpreted as showing that adsorbed serum lipids are able to provide a substrate for the formation of holes by activated C’, though not necessarily to imply that they are the usual substrate on cell membranes. There is evidence that at least some plasma phospholipids exchange quite rapidly with those on the surface of erythrocytes (Lovelock et aZ., 1960) and that plasma unesterified cholesterol exchanges with that on a variety of biological membranes (Hagerman and Gould, 1951; Graham and Green, 1967). The possibility arises, therefore, that the difference in the size of holes made by human C’ and guinea pig C’, noted in Section 11, is due not to differences in the end product of C’ action but because the membrane lipids are affected by lipids in the serum used as the source of C’. We attempted to test this by examining holes made on Escherichia coli lipopolysaccharide complex by small amounts of fresh guinea pig serum in the presence of a 20-fold excess of heated human serum (1hour at 56°C.) and vice versa. In each case the size of the holes was characteristic of the active C’ and was unaffected by the presence of lipids derived from the heated serum ( authors’ unpublished observations).

B. CHEMICAL STUDIES Humphrey et al. (1968) reported preliminary studies in which Escherichia coli-lipopolysaccharide ( LPS ) complex was firmly adsorbed onto carbon-coated glass cover slips, which were treated with fresh or heated human serum or buffer solution. Controls included cover slips without LPS treated in parallel. Six hundred square centimeters of cover slip surface would adsorb about 1 mg. of LPS and so provide sufficient material for analysis of the lipids by thin-layer chromatography. Similarly treated electron microscope grids were floated on the solutions at each stage, so as to check that many holes were made on the LPS by fresh serum but none in the other preparations. The lipids extracted with chloroform-methanol-acetic acid were examined in several solvent systems. A wide variety of lipids from heated or unheated serum was found to be adsorbed onto the cover slips, whether or not they had LPS on them. The phosphatidylethanolamine of the LPS was not evidently affected by the formation of holes, and no lysophosphatides were present in any of the samples; there appeared, however, to be some small changes in the distribution of cholesterol and its esters and of free fatty acids.

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The use of carbon-coated surfaces had two disadvantages. The first was that the amount of serum lipids adsorbed was so large as to seem likely to ohscure any possi1)le minor cffclcts due to activation of C’; thc second was that such surfaces contained, besides carbon, variable amounts of a mixture of lipidlike materials which were shown to be derived from the carbon arc rods uscd for coating the cover slips. (These survived heating the rods to high tcmperaturcs and are prcsumed. though withoat proof, to be higher paraffins from the bonding material used in making the rods.) In further unpublished experiments, the LPS was, therefore, adsorbed onto bentonite, which could firmly bind the complex up to 40%of its dry weight. The sm‘dler bentonite particles were suitable for electron-microscopic examination, and it was found that abundant holes were produced by C’ on the adsorbed LPS. When experiments similar to those described in the preceding paragraph wcre performcd with LPS on bentonite, the bentonite was found to adsorb serum lipids, though to a lcsser extent than the carbon-coated cover slips. Again, thinlayer chromatography of the extracted lipids failed to show any clear differences betwcen those from LPS trcated with fresh serum with many holes or with heated serum, with none (Fig. 9). Subsequent re-cxtraction of the bentonite with solvents containing 1.0 N HCI or with Na dodecylsulfate and urea gave no indication that any lipid remained after the first extraction. The position at present is that, despite the evidence that C’ holes are formed in a surface lipid layer, no chemical evidcnce of changes in the lipids has been detected. It is possible to add two further pieces of negative evidence. The first relates to the possibility that some altcmtion in cholesterol is involved in the formation of holes. This was tested by treating erythrocyte membranes with saponin, which solvatcs in cholesterol layers to produce the hexagonal micellcs described in Section 11, or with digitonin, which combines with cholesterol to form pseudocrystalline structures on erythrocyte membranes (Dourmashkin and Rosse, 1966). Neither saponin nor digitonin affected the appearancc of preformed C’ holes, nor did pretreatment of the membranes prevent the subsequent formation of holes by antibody and C’ ( Dounnashkin, unpublished observations). Although these findings do not cxclude thc poqsibility that cholesterol is an important constituent of the lipid layer in which hoIes are formed, they make it improbable that any specific effect on cholesterol is involved. The second possibility, advocated by Munder et al. (1965) but disputed by Keller ( 1965), is that c’ acts on cell membranes by the generation of lysolecithin. Humphrey et al. (1968) looked for evidencc that lysophosphatides were present in erythrocyte mcmbranes as a result of

FIG. 9. Effect of C' on surfacc lipids. Thin-laycr chromatogram on silica gel G.

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the action of C’. Although whcn added to crythrocyte suspensions, even in sublytic amounts, lysolecithin or lysophosphatidylethanolarnine could readily be recovered and detected in solvent extracts of the cell membranes, no increase in lysophosphatides in the membranes was found to accompany extensive production of holes by C’. In fact, whcn the sera used a s the sources of antibody and of C’ containcd no detectable lysophosphatides initially, none were detected in the C’-treated cell memhranes; furthermore, no 1ysophosphatidc.s were found in extracts of LPS even when extensive hole formation had occurred. An additional, though Icss compelling argument against the participation of lysophosphatides is that the menibrimes of erythrocytcs lysed by adding lysolecithin to the suspending ineclium did not show any typical C’ lesions. Smith and Becker (1968) examined the changes in total lipids that occurred when 2 x 10’” sheep erythrocytes scnsitized with rabbit Forssman antibody were incubated with guinea pig C’. They detected an increase in titratable acid, in parallel with a dccrease in serinc or choline phosphatides, which obeyed first-order kinetics. Antibody and activc C‘ were both required for thcsc. changes to occur. No such changes wcrc found when cells which had reacted with C’ as far as C’3 or even C’7 wcre incubatcd alone, but thcy did occur on addition of partially purified C’8 and C’9 or of EDTA guinea pig serum. The authors suggest that the lipid alterations were due to the action of one of the terminal components of C’ on the cellular intermediates. Because thc lipids examined were cxtracted from the mixture of erythrocytes and scruni (or scrim fractions), it is difficult to relate them to specific changes in the cell membranes. Calculations made from the authors’ data suggest that 50%lysiis of 2 X of the lipids extracted I,y chloroforiii-iiic.tlianol 2 : 1 ( v / v ) from two systems which h;id I,een treated with antibody and heated C‘ ( n o holes) or active C’ ( m a n y holes). Thc systems were as follows: ( I ) forlnalin-fixed sheep erythrocyte mernbranes ra1,bit anti-Forssnian Ig51 antibody f heated guinea pig C’; ( 3 ) as ( I ), h i t using fresh guinea pig C’; ( 4 ) Escherichin coli LPS ac1sorl)ecl onto Iientonite particles heated human seruin; ( 5 ) as ( 4 ) , but using fresh human ser~iiii (eqiial ainorints of reagents were employed far coinparison in each system); ( 3 ) reference prcparation of rat hrain polar lipids and standard neiitial lipids. I.ipids were identified as n, cholesterol rsters; 17, triglycerides: c, fatty acids; d, cholesterol; c, polar lipids remaining at the origin on neutr;il lipid plates; f , nrutral lipids at the first solvent front ( C :51 :W ) ; g , cerebrosides; h, phosphatidylcthanolaniines; i , sulfatides; i, phosphatidylcholines; k, sphingomyelins; I , phosphatitlylseriiie; m, Iysophospliatidylcholine; n, gangliosides. [Neutral lipids \\‘ere separatcd in petioleiim ct1ier:etlier :glacial acetic acid ( 7 3 :25 : 2) ; polar lipids were sepalatrd i i i chloroforin : methanol :\vater ( 14 : 16:1 ) to 15 ciii a i d then in it-propanol : 12.5%acpeotls h’H,OH (4:1 ) to 8 cm.] T l ~ scliroinato~rupl~y wits rarricatl ont by Sheila N. Payiw.

+ +

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JOHN H. HUM P HR E Y AND ROBERT R . DOURMASHKIN

1O1O cells was accompanied by the release of about 0.025 p M of titratalile acid, i.e., about 1.5 x 10” molecules of acid were releascd for each cell lysed. Since only half the cells were lysed and their experimental conditions were siich that single holes would be expected to accompany each lesion, this would imply a change in the improbably large number of 10” molecules of phosphatide per hole if the substrate were phosphntide in the membrane. Furthermore, when heated guinea pig serum was used, increases in titratable acid were sometimes observed up to half those found with fresh serum, although no hemolysis occurred. For numcrous reasons, especially those advanced in Section VII1,A above, we propose that C’ holes are due to a rearrangement occurring to form micellar structures in a predominantly lipid surface layer. The fact that holes are similar in appearance and size when generated by C’ from a given species on a wide variety of membranes, and even on a pseudomembrane formed by thc adsorption of serum lipids onto a carbon-coated surface, argues in favor of the micelles being formed by the local action of a similar or identical detergentlike agent in each case. Nevertheless, the consistent difference observed between the size of the holes formed by human C’, on the one hand, and by guinea pig C’ (and, from very limited data, rabbit and calf C’), on the other, suggests that the end products of C’ from different sources may differ. The hypothetical agent could be generated from adsorbed C’ components (either C’9 or earlier) or could arise by alteration or removal by C’8 or 9 of substances preexisting in thc membrane. Unfortunately there are at present few clues as to what this hypothetical agent might be. IX.

Artificial Membrane Models

Model lipid membranes have been used increasingly for electronmicroscopic study of the action of surface-active agents, such as lysophosphatides ( Bangham and Home, 1964), saponin (Lucy and Glauert. 1964), or filipin (Kinsky et al., 1967). They have recently been applied to the study of antigcn-antibody reactions in the presence of C’ by Barfort et d. (1968) and by Haxby et d . (1968). Barfort et a2. prepared himolecular membranes from sphingomyelin and a-tocopherol and nieitsiired the decrease of electrical resistance in 0.1 A{ NaCl when solutions of various antigens were on one side and of specific antisera, on the other. Very marked changes of resistance occurred when active C’ was present in the antiserum, but not in its absence. Haxby et al. (1968) prepared . hposonics,” a term describing an aqueous colloidal suspension of sphcrules of lipid mixtures, a certain proportion of which may be completely closrd, thereby tr‘ipping n quantity of aqueous solution. These “

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authors used the lipids obtained by extraction of sheep or becf crythrocytes with chloroform-nieth;~nol, they also prepared liposomes with sphingomyelin ( o r ~~1losphaticlylcholinc), cholesterol, and dicetyl phosphate, into the membranes of which was also incorporated a methanolsoluble extract of the erythrocytes (presumed to contain Forssman antigen). The liposomes wcre formed in an aqueous medium containing glucose, which became trapped within them, so that subsequent damage to the liposomc membranes could be nionitorcd by the release of glucose, assayed by a sensitive spectrophotomctric method. Haxby et al. found that on incubation with anti-Forssman antibody and C’, glucose was released from the liposomes. The requirement for antibody and active C’ was absolute. In the case of liposomes prepaied from sphingomyelin, cholesterol, and dicetyl phosphate, but without Forssinan antigen, no release occurred even when antibody and C’ were present. These authors mention that clectron-microscopic examination of lipo~omeswhich released glucose rapidly, revealed “pits” typic‘il of C’ action. Such experiments would seem to prove that C’ holes are formed in a lipid substratc. They do not, however, enable conclusions to be drawn concerning the detailed nature of the substrate in which the holes are formed, became of the inevitable adsorption of lipids or lipoproteins from the C’ and/ or the antiserum. It would be of considerable interest to test the effect of purified antibody and C’ frce from lipid on a simple meinbranc system or even on isolated bacterial lipopolysaccharides. Unfortunately all the usual methods for removing lipids from serum or plasma cause inactivation of hemolytic C’. Dalmasso and Muller-Eberhard (1966) succeeded in removing nearly all thc lipids from human serum, apart from those associated with serum albumin, by prolonged high-speed centrifugation after adjusting the density of serum to 1.21 with NaBr. This procedure inactivated C’3, 4, and 5, hut hemolytic activity could be restored by adding back the missing coinponmts in a highly purified form containing no detcctable lipids. Dalmasso and Mdller-Eberhard found that if albumin was also removed b y ammonium sulfate fractionation from the lipid-depleted plasma, rendering it practically lipid-frec, hcmolytic activity could still be restored with purified C’3, 4, and 5. Thus, lipid-free C’ can evidently lyse sensiti7cd sheep erythrocytes. However, their membranes already contain a complcx mixture of lipids, and it does not follow that lesions would equally be foimed on a simpler substratum. Wc attemptcd ( Humphrey. Dourinashkin, and Payne, unpublished observations) to test this using LPS on electron microscope grids. The denser fraction of human srrum, subjected to prolonged centrifugation at a density of 1.21, retilined its macroglobulin

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antibodies but lost its hemolytic activity (tested on AET-treated human group A erythrocytes) and its capacity to make holes in LPS. Both hemolytic activity and the capacity to make holes in LPS were restored by adding purified human C’3, 4, and 5 kindly provided by Dr. MullerEberhard. However, our reconstituted human C’ still contained some lipids, about 10% of those in the original serum. When the albumin fraction was also removed, and the serum was lipid-free, it still possessed its macroglobulin antibodies, and about one-tenth of the original hemolytic activity was restored by adding C’3, 4, and 5, but it no longer formed holes when applied to LPS under similar conditions. So far as they go, these observations imply that when the substrate is LPS some essential ingredient for the formation of holes is supplied by the serum lipids and, also, are consistent with the findings discussed in the previous section. There is considerable uncertainty at present about the structural organization of the numerous components of which cell mernbrancs are coniposed and about the way in which their selective permeability properties are maintained (e.g., see Chapman, 1968). The phenomcna discussed in this chapter have not generally been considered in relation to thc more gencrnI probIem of cell membrane structure. However, they may be relevant in two respects: first in emphasizing the readiness with which plasma lipids or lipoproteins can be adsorhed onto a variety of surfaces, so as to provide a layer that resembles a cell membrane at least in its susceptibility to the action of C’; second, in showing that a single hole or bubble about 100A. in diameter, occurring in the outer predominantly lipid Iayer, can cause an irreparable loss of the selective permcability properties of the cell. X.

Biological Significance of the Terminal C’ Lesion

Despite the fact that rabbits without C’6, mice without C’5, and men with little or no C’2 can survive in good health (see Miiller-Eberhards review, 1968), it is difficult to suppose that the complex mechanism involving the terminal C’ components and culminating in cell lysis would have evolved and siirvived in so wide a variety of vertebrate species if it did not have significant biological value. In this chapter, two points relcvant to this have been brought forward. The first is that a single IgM antibody molecule against ccll surface component9 can activate C’ so as to damage the cell lethally. It is probable that this occurs especially, and perhaps only, when the antigenic groups on the cell surface form a closely spaced, rcyeating pattern, allowing multipoint attachment of the pluri-

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valent IgM antibody ( Frank and Humphrey, 1968)-a condition satisfied by antigcms such as thc Forssman antigen on erythrocytes and the 0 antigens of grain-negative bacteria. A similar requirement for closely spaced similar antigenic groups is also likely in the case of IgG antibodies, since otherwise it would not be possible) for 2 molecules to b c ~ o m eattached adjacently, as appears to be necessary for C’ activation by IgG to take place. In tlie case of gram-negative bacteria, the action of C’ is enhanced by the subsequent action of lysnzyme, which not only erodcs the underlying cell wall but renders the inncr protoplasinic inembranc accessible, in turn, to the action of antihody and C’ (Glynn, 1969). The whole mcchanism seems well designed to make the best uscb of weakly avid “natural” IgM antibody, and of the IgM antibodies produced early in a primary response (especially against the surface antigens on particulate materials). The second point is that the lesion produced by activation of C’ at a single site may consist not of one hole but of a cluster. It is conceivable that this may represent a useful amplification of the action of C’; for example, if a cell or a microbe were able to withstand the damage caused by one hole but not that caused by several. However, we have no evidence about this, and thr circumstances in which clusters are produced have not yet been adequately defined. Two important riders need to be attached to any generalizations about the damage to cells by C’. The first is that the lytic action of C’ appear5 only to be effective whcn C’ activation occurs actually at or very close to the susceptible surface of the membrane. For example, attachment of bacterial lipopolysaccliaride 0 antigens to sliecp erythrocytes or to PNH human erythrocytes (Rosse et ul., 1966) rendcrs the cells extremely sensitivc>to the lytic action of C’ and specific antibody against the 0 antigen. However, we have ohserved in unpublished experiments, that when Type 3 pneumococcal capsular polysaccharide ( S3) is attached to erythrocytes by the method of Askonas et al. (1960), lieniolysis with C’ occurs only in the presence of vcry much higher concentrations of anti-S3 than are needed for liemagglutination or for fixation of C’ by the erythrocytes coated with S:3. Electron microscopy of tlie menibrancs of cells treated with anti-S3, with or without C’, revealed that the polysaccharide coating became aggregated by the antibody into iinev(~imasses which were no longcr closely applied to the membranes. Consequently, although C’ was fixed extensivcly, little or none of it was attached to the membrane. Rowley and Turner ( 1968) report cxperinients in which living Salnionella rrtlelnide organisms n ~ r econjugated with RSA, mouse 7-globulin or aggregated human IgG. Thc organisms wcrc thcn treatcd with guinea pig

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C’ and rabbit antisera against the attached proteins or against Salmonella adelaide itself, and the amount of antibody needed to kill them was estimated. Coated organisms retained their sensitivity to the antibacterial antibody but were only killed by lo5 times more anti-BSA or lo6 times more antimouse y-globulin and were not killed by any quantity of antibody against aggregated IgG. Thcse findings were taken to imply that the effectiveness of C’ was inversely related to the distance of the site of activation from the surface. Similar conclusions could perhaps be drawn from the observation that human iso- and autoantibodies are more effective at causing C’-dependent lysis of human erythrocytes pretreated with trypsin, papain, or ficin than of normal ceIIs (Mollison, 1967), although in this case increased binding of antibody after enzyme treatment may be a sufficient explanation. The conclusion that holes are formed only when C’ activation occurs very close to the susceptible membrane implies that one or more of the activated C’ components remains lytically effective in free solution only for a very short duration-as appears to be the case for C’3 ( Muller-Eberhard, 1968). The second rider is that antibodies in some classes of immunoglobulin fail to activate C’ and that not all antibodies in those classes that do activate C’, namely, IgM and IgG, are equally efficient. It has already been mentioned that Hoyer et al. (1968) were able to distinguish a hemolytic and nonhemolytic form of rabbit anti-Forssman IgM antibody. In some species, notably guinea pigs, one kind of antibody ( y , ) can inhibit C’ fixation by another ( y 2 ) by competition for the same antigenic sites (Benacerraf et al., 1963). There is evidence that this may occur in other species also (Humphrey, 1968), and it would be interesting to know whether human antibodies of classes such as IgG4 and IgA which do not fix C’, could inhibit C’ fixation by antibodies of the other IgG subclasses. Among human isoantibodies, it is well recognized that some, such as IgM immune anti-A, are much more frequently capable of causing heinolysis than others, such as IgM natural anti-A, and that anti-Rh antibodies of any class are scarcely ever hemolytic (Mollison, 1967). Furthermore, even among the IgM anti-I antibodies found in cases of macroglobulinemia with cold agglutinins, there is surprisingly little correlation between the hemolytic and agglutinating capacities of sera from different subjects (Dacie, 1962). Some of the differences may be due to the way in which the antigenic groups are disposed at the cell surface, and some, for example, in macroglobulinemia with cold agglutinins, to selective removal of the more avid antibodies by the patient’s own erythrocytes in uiuo. However, it is evident that there is still much

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to learn before the significance of C’-dependent lytic reactions is fuIIy understood. ACKNOWLEDGMENT W e wish to express our thanks to Mrs. Sheila N. Payne for collaboration in some of the experiments discussed above and for reading the manuscript.

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Gotze, O., Haupt, I., and Fischer, H. (1968). Nature 217, 1165. Graham, J. M., and Green, C. (1967). Biochem. J. 103, 16C. Green, H., Fleischer, R. A., Barrow, P., and Goldberg, B. (1959a). J. Exptl. Med. 109, 511. Green, H., Barrow, P., and Goldberg, B. (1959b) J. Exptl. Med. 110, 699. Hadding, U., and Miiller-Eberhard, H. J. (19’67). Science 157, 442. Hadding, U., Miiller-Eberhard, H. J,, and Dalmasso, A. P. (1966). Federation Proc. 25, 485. Hagerman, J. S., and Gould, R. G. (1951). Proc. Soc. Exptl. Biol. Med. 78, 329. Haxby, J. A., Kinsky, C. B,, and Kinsky, S. C. (1968). Proc. NatZ. Acad. Sci. U . S. 61, 300. Hoyer, L. W., Borsos, T., Rapp, H. J., and Vannier, U’. E. (1968). J. Exptl. Med. 127, 589. Humphrey, J. H. (1963). 3rd Intern. Symp. ImmunopathoZ. p. 369. Schwabe, Basel. Humphrey, J. H. (1967). Nature 216, 1295. Humphrey, J. H. (1968). In “Biochemistry of the Acute Allergic Reactions” (K. F. Austen ancl E. L. Becker, eds.), p. 249. Blackwell, Oxford. Humphrey, J. H., and Dourmashkin, R. R. (1965). Ciha Found. Symp. Complenierit p. 175. Churchill, London. Humphrey, J. H., Dourmashkin, R. R., and Payne, S. N. (1968). 5th Intern. Symp. Immunopathol. p. 209. Schwabe, Basel. Keller, R. (1965). Intern. Arch. Allergy Appl. Irnmuno!. 28, 201. Kemp, C. L., and Howatson, A. F. (1966). Virology 30, 147. Kinsky, S. C., Luse, S . A., Zopf, D., van Deenen, L. L. M., and Haxby, J. (1967). Biochim. Bi0phy.y. Acta 135, 844. Knox, K. W., Cullen, J., and Work, E. (1967). Biochem. J . 1803, 192. Linscott, W. D., and Nishioka, K. ( 1 9 6 3 ) . J. Ex&. Med. 118, 795. Lovelock, J. E., James, A. T., and Rowe, C. E. (1960). Biochcm. J. 74, 137. Lucy, J. A,, and Glauert, A. M. (1964). J. Mol. Biol. 8, 727. Mayer, M. M. (1961a). In “Immunochemical Approaches to Problems in Microbiology” ( M . Heidelberger and 0. Plescia, eds.), p. 268. Rutgers Univ. Press, New Brunswick, New Jersey. Mayer, M. M. (196lb). In “Experimental Immunochemistry” ( E . A. Kahat and M. M. Mayer, eds.), 2nd ed., p. 180. Thomas, Springfield, Illinois. Mollison, P. L. (1967). “Blood Transfusion in Clinical Medicine,” 4th ed., p. 250. Blackwell, Oxford. Morgan, T. E., and Huher, G. L. (1967). J. Cell B i d . 32, 757. Muller-Eberhard, H. J. (1968). Adoall. Immunol. 8, 1. Miiller-Eberhard, H. J., Dalmasso, A. P., and Calcott, M. A. (1966). J. Exptl. Med. 123, 33. Mnnder, P. G., Ferber, E., and Fischer, H. (1965). Z . Natirrforsch. 20b, 1049. Muschel, L. H., Carey, W. F., and Baron, L. S. (1959). J. Immrmol. 82, 38. Nelson, R. A . ( 1962). 2nd Intern. Symp. Immunopathol. p. 245. Schwabe, Basel. Padgett, F., nncl Levine, A. S. (1965). Virology 27, 633. Rosse, W. F., Dourmashkin, R. R., and Humphrey, J. H. (1966). J. Erptl. Med. 123, 969. Rowley, D., and Turner, K. J. (1966). Nature 210, 496. Rowlcy, D., and Turner, K. J. (1968). Notcrre 217, 657.

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Sears, D. A., Weed, R. J., Swisher, S. N., and Trabold, N. (1964). j . Clin. lnoest. 43, 975. Sessa, C . , and Weissman, C. ( 1968). J. Biol. Chem. 243,4364. Simpson, R. W., and Hauser, R. E. (1966). Virology 30, 684. Sirchia, G,, and Dacie, J. V. (1967). Nature 215, 747. Smith, J. K., and Becker, E. L. (1968). j . Immvnd. 100, 459. Stolfi, R. (1967). Federation Proc. 26, 362. Taylor, A,, Knox, K. W., and Work, E. ( 1966). Biochem. J. 99, 53. Wardlaw, A. C. (1962). J. Exptl. Med. 115, 1231.

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Cytotoxic Effects of Lymphoid Cells in Vitro PETER PERLMANN AND GORAN HOLM Deportment o f Immunology. The W e n n e r - G r e n Institute. University o f Stockholm. Stockholm. Sweden

I . Introcluction . . . . . . . . . . . . . I1 Methods . . . . . . . . . . . . . . A . Target Cells . . . . . . . . . . . . B. Effector Cells . . . . . . . . . . . . C . Survey of Methods . . . . . . . . . . . D General Considerations . . . . . . . . . . E Concluding Remarks . . . . . . . . . . I11. Different in Vitro Models . . . . . . . . . . A . Cytotoxic Effects on Antigenic Target Cells of Lymphoid Cells from . . . . . . . . . . Sensitized Donors . 3. Induction of Cytotoxicity of Lymphoid Cells from Norma1 Donors . . . . . . by Antibodies to Target Cell Antigens C. Cytotoxic Effects of Lymphoid Cells Triggered by Target CellBound Complement . . . . . . . . . . D . Nonspecific Cytotoxicity of Lymphoid Cells Activated by Phyto. . . . . . . hemagglutinin or Other Stimulants E Target Cell Destruction by Lymphoid Cells from Nornial Donors . . . . . . . . after “in Vitro Sensitization” . . . . . . IV Some in Viuo Implications of the in Vitro Models 4 . Delayed Hypersensitivity . . . . . . . . . B. Autoimmunity . . . . . . . . . . . C. Graft-versus-Host Reactions . . . . . . . . . D. Allograft Rejection . . . . . . . . . . E . Tumor Defense . . . . . . . . . . . V Summary . . . . . . . . . . . . . . References . . . . . . . . . . . . .

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I.

117 119 119 120 121 125 126 127 127

144 153 156 168 172 172 175 177 179 181 183 185

Introduction

Lymphoid cells havc effector functions in certain tissue-damaging immune reactions . Cell-mediated tissue injury is believed to be of prime importance in delayed hypersensitivity. in some of the experimental and human autoimmunities. in the various allograft phenomena. and in somc forms of tumor rejection . The basis for this belief are. in brief. the typical histopathological picture of the tissue Iesions seen in these states. the various graft-versus-host phenomena. and the fact that tissue-destructive immune reactions can often be provoked in nonsensitized recipients by 117

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PETER PERLMANN AND GORAN HOLM

transfer of lymphoid cells from sensitizcd donors rather than by immune serum. During recent ycars, a number of morc direct in vitro models have been designed in order to throw light on the mechanism of these tissuedamaging reactions. It is now well established that sensitized lymphocytes, upon contact with antigen, will inhibit thc in vitrn migration of macrophages and other leukocytes (Rich and Lewis, 1932; David et al., 1964). This inhibition is brought about by soluble mediators released from the reacting lymphocytes (Bloom and Bennett, 1966; David, 1966; Svejcar et al., 1968) and is believed to be of importance for the initiation of the delayed hypersensitivity reaction. In a different in vitro model, it has been shown that lymphoid cells from sensitized donors will destroy tissue culture cells carrying the antigen to which the cell donor is sensitized. This type of cytolytic reactions has been encountered in a great variety of immune situations, comprising all those mentioned in the first paragraph. The reactions are, therefore, believed to be in vitro correlates to corresponding effector activities of lymphoid cells in vivo. The cell that initiates the in vitro cytotoxic reaction has been assumed to be the sensitized lymphocyte, equipped with its own recognition sites for antigen on the cells which are destroyed. Although this may be true in many situations, it now seems clear that “normal” lymphoid cells can become cytotoxic to other cells by a variety of pathways. There is little doubt that this “nonspecific” cytotoxicity in vitro also is of importance in wivo. The study of the various pathways by which lymphoid cells can become cytotoxic has been helpful for our beginning understanding of their effector role in cell-destructive reactions in general. This review will deal with the in vitro cytotoxicity of lymphoid cells. After a brief discussion of some methodological issues, various in vilro models will be described. In a final section, the possible bearing of these models on cell-mediated tissue destruction in vivo will be discussed. Parts of this subject have previously been treated in this series (Dutton, 1967; Wilson and Billingham, 1968). In this article, the cells which are destroyed upon addition of lymphoid cells are called target cells or targets. The cells which achieve this are called effector celk. The designation lymphoid cells rather than lymphocytes has been chosen in order to put some emphasis on the fact that the effector cell populations used in many experiments are relatively crude mixtures, containing not only lymphocytes, heterogeneous by themselves, but monocytes or macrophages as well. Frequently, polymorphonuclear leukocytes are also present. The significance of lymphocytes and other

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cell types for the cytotoxic rcactions t\dI lie discussed in the different sections. The tcrm setrsitizcrl cell or .sen.sitixtl hp1]hOC~/tedcsignate thc antigeIi-reactivc lymphocytc~swith mtiliody-like reccytors believcd to bc produccd by thc cells which carry them. In other words, it applies to those lymphocytes that are thought to bc) instrumental in delaycd hypersensitivity and similar reactions. In contrast, the term cellmediated reaction is used in a hroad sense. It applies here to those reactions in which lymphoid cells function as the effectors, regardless of whether or not sensitized lymphocytes are involved. It draws a border line between cellmediated reactions and those which are brought about hy humoral antibodies and complement but without participation of lymphoid cells. Obviously, this definition does not exclude participation of either humoral antibody or complement acting in conjunction with lymphoid cells. II.

Methods

Cell-mediated cytotoxicity in vitro is the manifestation of complicated cellular interactions. It is tested by adding lymphoid cells in excess and under tissue culture conditions to target cells of various kinds. After n certain time which varies depending on the particular system under study, destruction of the target cells takes place and can bc recorded. The many different methods which have been applied for the assay of cytotoxicity do not necessarily always nicasure the same reactions. Thcy are also subjected to different kinds of artifacts which must be taken into account when the results obtained with different methods are to 1~ compared.

A. TARGET CELLS All kinds of tissue culture cells may serve as target cells. When the stt1dic.s involve lymphoid cells from donors sensitized to target cell antigens which are stable in vitro, the cultures can be propagated for a long time. This is truc for the major transplantation antigens (Rosenau and Moon, 1964) and for some of thc tumor-spccific antigcns. I n contrast, some “tissue-specific” antigcns disappear within 1 week of culture (for references, see Dumonde, 1966). Studics of cytotoxic reactions in autoimmune diseases, therefore, usually requirc cultures of freshly explantcd tissues. Established cell lines of normal tissucs or tumors are frequently uscd (Brunner et a?., 1966; Holm and Perlmann, 1967a). Such cell lines have the advantage of being adapted to tissue culture conditions. They arc easy to culture as monolayers which in most instances can bc>converted

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PETER PERLMANN AND GORAN HOLM

to suspension cultures. Cells grown in nionodisperse suspension are more suitable target cells than cells attached to glass. They are not influenced by enzymes or other factors which may detach the cells which grow in monolayer (Lundgren et al., 1968a). They are probably available for contact with the effector cells from all sides, and this may increase the sensitivity of the method. Furthermore, established cell lines consist of one cell type, in contrast to the mixture of two or more cell types usually encountered in primary cultures. Chicken erythrocytes have also been employed as target cells. When these erythrocytes are passively coated with antigen, they are excellent targets for lymphoid cells from donors sensitized to that antigen or, after reaction with humoral antibody, for lymphoid cells from normal donors ( Perlmann and Holm, 1968). It is a notable finding that macrophages, cultured on glass, may serve as target cells for lymphoid cells from donors sensitized to thcir transplantation antigens ( Brondz, 1964). In other situations, macrophages from sensitized animals are cytotoxic to antigenic target cells (Granger and Weiser, 1964). Antibody-forming target cells have been used by Friedman (1964). The reduction of plaque-forming cells in the Jerne assay after contact with lymphoid cells from sensitized donors was taken as a measure of cell destruction. Different types of target cells may vary in susceptibility to cell-mediated lysis (Holm, 1967a; Brunner et al., 1969b). However, even target cells of one type may sometimes vary in susceptibility when different batches are compared. Comparison of the susceptibility of target cells of different types may, therefore, be difficult to quantitate. B. EFFECTOR CELLS Lymphoid cells are obtained from the lymphoid organs or from the circulation. When prepared from lymph nodes or spleen, the common techniques for preparation of cell suspensions are applied ( for references, see Ling, 1968). Comparison of the cytotoxic activity of cells from different origin must be done with caution. With spleen and lymph node cells it has sometimes been found advantageous to place small pieces of tissue on a grid (Trowell, 1959) from which lymphocytes are allowed to fall on a target cell monolayer underneath (Vainio et al., 1964; Holm, 1966). However, this method is difficult to quantitate. It is essential to start the experiments with cell suspensions in which !N-100% of the lymphoid cells are viable. Cells from different animals may vary highly in their viability in vitro (for references, see Ling, 1968). Mouse cells in particular will die much faster during the course of an

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experiment than human or guinea pig cells. Since the cytotoxicity of lymphoid cells is a fmiction of viable cells, differenccs in viability will affect the results. This has to be takcn into account when cytotoxicity of lymphoid cells from cliff erent specics are comparcd. In most experiments, the lymphoid cells consist of mixtures of lymphocytes of various sizes, monocytes, and macrophagcs. Polymorphonuclear leukocytcs and red cells are also present in many cases. Some of these cells (monocytes, macrophages, poIymorphonucIear leukocytes) may be cytotoxic by themselves (Granger and Weiser, 19M; Lo Buglio et al., 1967) or may die and release cytotoxic factors (Lundgren et al., 1968a). Moreover, these cell types may interact with each other in the course of the cytotoxic rcaction. To ascribe effector functions to a special cell type, it is essential to use homogeneous suspensions with as little admixture of other cell types as possible. Thoracic duct lymph is, therefore, preferred as the source of lymphocytic effector cells. In other cascs, the analysis requires the application of purification procedures. Even then it may be difficult to exclude the participation of small numbcrs of nonlymphocytic cells in the reaction, particularly when the number of lymphoid cells added to target cells is high.

C. SURVEYOF METHODS 1 . Destruction of Manolnyers

In these methods, disruption of target cell monolayers is evaluated after incubation with lymphoid cells. In a common modification, part of the monolayer is selected undcr the microscope and photographed before, during, and after incubation with the effcctor cells (Govaerts, 1960; Koprowski and Fernandes, 1962; Berg and KZllkn, 1963). Thesc methods have considerable drawbacks: the reactions are slow (48 hours or more) and evaluation of cell damage is subjective. Due to variations in target and effector cell densities, the important effector cell/ target cell ratios cannot be controlled from one part of the monolayer to anothcr. In the phque technique by Granger and Weiser (1964, 1966), later applied by Moller and Moller ( 1965), drops of suspensions containing known numbers of lymphoid cells are added to the target monolayer in well-defined areas. A cytotoxic reaction is recorded as local reduction of target cell density ( = plaque formation). The intensity of thr plaques is This allows a semiquantitative evaluusually scored from to ation of cell damage. With the plaque techniquc, the ratio of effector cells to target cells can be controlled to some extent. Small numbers of effcctor cells are

+ ++++.

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PETER PERLMANN AND GORAN HOLM

needed for each plaque and several drops of effector cells can be placed on each monolayer. The method is rapid and the results are easy to read. However, long incubation times are often required for development of the plaques (Moller and Moller, 1965; Moller et al., 1966; Lundgren, 1969) . In all monolayer techniques the detachment of cells from the substrate is taken as reflecting the cytotoxic activity of the effector cells. Detached cells are usually seen to be damaged or dying (Rosenau and Moon, 1961; Biberfeld et al., 1968; Ax et al., 1968). However, various nonspecific factors, such as enzymes released from dying polymorphonuclear leukocytes or from other cells may also be responsible for detachment of undamaged target cells (Lundgren et al., 1968a). Non-specifically affected cells will usually not survive very long either, and their detachment will introduce errors in the evaluation of the results. 2. Cell Counting

Target cells from stock cultures are counted and added to flatbottomed tubes or petri dishes. When the cells have become attached to the glass, the effector cells are added. After incubation, the supernatant is usually discarded. The remaining target cells are removed from the glass mechanically or by trypsinization. Dead cells are stained with vital dye and the number of unstained cells are counted (Taylor and Culling, 1963; Brondz, 1964; Moller, 1965h). Alternatively, target cell nuclei are counted after digestion of the cytoplasm with citric acid (Rosenau and Moon, 1961; Wilson, 1965a). Since the culture medium is usually discarded prior to counting, only target cells attached to the gIass will be enumerated. When this is the case, these methods do not distinguish between cell death and cell dctachment ( see preceding paragraph). Moreover, the number of surviving ceIls is influenced by the number of cell divisions during incubation. Lymphoid cells may have growth-promoting feeder effects on the target cells (Taylor and Culling, 1963; Wilson, 1965a; Roseuau, 1968) and this may tend to give erronous results. Target cell multiplication can be suppressed by X-irradiation before incubation with the effector cells (Wilson, 1965a). Counting methods are relativcly laborious. Their methodical error is rather big and several tubes (usually 3-6) must be run in parallel. They can only be used when target cells and effector cells can be distinguished microscopically by differences in size or shape. However, in spite of these disadvantages, the methods have been used by several authors and form the basis of quantitative studies (Wilson, 1965a; Brunner et nl., 1966; Berke et al., 1969a). Several of the previous disadvantages are avoided

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in some recent micromodifications ( Ulrich and Kieler, 1969; Takasugi and Klein, 1969).

3. Inhibition of Colony Formation These methods can be regarded as variants of the cell counting techniques. Hellstrom and Sjogren (1965) assayed the plating efficiency of tumor target cells after incubation with antibodies and complement. In a later modification (Hellstrom, 1967), thc target cells are allowed to form a noiicoiifluent monolayer overnight before lymphoid cells are added. The number of target cell colonies is counted after 3-4 days of culture. Brunner et al. (1966) described a slightly different technique where the target crlls after contact with effector cells were suspended arid cultured in a semisolid medium. The colonies were counted 1 week later. The colony inhibition method is very sensitive. When the effector cclls are added to nonconfluent nionolayers, the effective lymphocyte/ target ccll ratio may be very low, even when the number of lymphocytes added for each target cell is as high as 5000 (Hellstrom, 1967). Rapidly growing target cclls, adapted to tissue culture conditions, are more suitable than primary cultures. The method has mostly been used for studies of cytotoxic reactions directed against tumor cells. The colony inhihition techniques measure the survival of single cells which are capable of attaching to glass and of forming colonies by niultiplication. The method can, therefore, only be applied to certain types of monolayers. As in other monolayer methods, errors may arise because of en7yinatic or otherwise “nonspecific” detachment of cells from the glass.

4 . Isotope Release from Labeled Target Cells Release of radioactive markers from target cells is frequently used for quantitative evaluation of cell damage. In general, after incubation of isotopically lnbcled target cells with cytotoxic effector cells, the total radioactivity of the sample and that of thc cell-free supernatant (or of the insoluble residue) are determined. Rclcased (or retained) isotope, ~is~ially expressed as perceiitage of total radioactivity, reprcseiits a cuinulative measure of cell darnage. Target cell growth and multiplication will influence the results only marginally. Isotope can be introduced into target cells by making use of their metnbo’fism, or chemically. a. Deox!/ribonticleic Acid (DNA ) . \Vhcn DNA-synthcsizing target cclls are incubated with ”or I ’C-thymidine, thc label is incorporated into DNA. Dnmaged cells clo not release DNA unless completely disin-

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PETER PERLMANN AND GORAN HOLM

tegrated ( Green et al., 1959a; Klein and Perlmann, 1963). Since damaged cells arc susceptible to proteolytic enzymes (Hirata, 1963), isotope rcIease from affected cells is measured after treatment of the target cells with trypsin ( Klein and Perlmann, 1963). Isotope released from damaged cells during the experiment is reutilized b y DNA-synthesizing cells. Horn'ever, reutilization can be blocked by addition of cold thymidine (Holm and Perlmann, 1965). The advantage of the method is the stability of the marker. Dcoxyribonucleic acid-labeled cells can be used for long-term incubations with the effector cclls (Holm et nl., 1964; Vainio et al., 1964; Holm and Perlmann, 1965; Holm, 1966). However, the method is rather insensitive. Thus, with cells, doubly labeled with thymidine-'% and chromate-'j'Cr and exposed to lymphocytes, significant damage was detected with 'ICr within 1 to 3 hours, but with l'C-thyniidine damage was first detected after 18 hours of incubation (Holm and Perlmann, 1967a). 13. Proteins. These can be labeled with radioactive amino acids; protein-bound label is readily released from damaged cells. The spontaneous release of label may vary for different cell types and reutilization may be difficult to control (Bickis et al., 1959; Perlmann and Broberger, 1963). c. 3'P-Pliosphate. This label is incorporated into P-containing constituents of both cytoplasm and cell nucleus. It can be used for determination of cell-niediatcd cytotoxicity in short-term experiments ( Ellem, 1958; Perlmann and Broberger, 1963). Spontaneous release and reutilization may be difficult to prevent in long-term incubat'ions. d. "Cr-Chromate. This has been used as a routine label in studies of the survival of red cells in hemolytic diseases. Sanderson (1964) and Wigzell ( 1965) adapted this label for quantitation of antibody-induced lysis of nucleated cells in vitro. Later, "'Cr-chromate was also found to be an excellent label for detcrmination of cell-mediated lysis of tissue culture cells growing in suspension (Holm and Perlmann, 1967a) or in monolayer ( Holm, 1967c) and of chicken erythrocytes (Perlniann et al., 1968). The method has since been adapted for the same purpose by other authors (Brunner et al., 1968a; MacLennan and Loewi, 1968a; Berke et al., 1969a). Chromium-51 is noncovalently bound to proteins and other cell constitucnts. The chromate is reduced during binding and isotope is not reutilized (Bunting et al., 1963; Holm and Perlmann, 1967a). From 80 to 95%of the radioactivity is released from dead cells (Wigzcll, 1965; Holm and Perlmann, 1967a). Labeling is rather stable; thus, only 1 5 4 0 % is spontancously releascd from tissue culture cells in suspension during 24 hours of incubation at 37°C. Most of this comes probably from spon-

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taneously damaged or dying cells. Under optimal conditions, spontaneous W r relrase from chicken erythrocytes usually does not exceed 5%in 1 to 2, days (Perlmann et al., 1968). Because of a minimum of handling, the error of this method is very small. Therefore, release of “Cr represciits a very sensitive index of cell damage, which sometimes: can bc measured within less than 3 hours (Holm and Perlmann, 1967a: Brunner et al., 1969b). 5. Inhibition of Isotope Incorporation Changes of the nietabolism of tissue culture cells upon contact with lymphoid cells can be assayed as inhibition of incorporation of labeled precursors. Incorporation into protein of radioactive amino acids, added to the incubation mixture, has recently been used for this purpose (Granger and Williams, 1968; Granger and Kolb, 1968). This method has also bcen used to study the effects of 5oluble cytotoxic factors (Williams and Granger, 1969). When measured in this way, cytotoxicity reflects primarily inhibition of growth. The method suffers from the drawback that both the effector cclls and the target cells may synthesize protein. Incorporation of isotope into the cffcctor cells can be avoided by the use of precursors which are not utilized by lymphoid cells. Incorporation of radioactive thymidinc into DNA in a cell mixture during the first 24 hours of incubation will takc place primarily in the target cells (Ming et al., 1967). However, even in this c x e , the results may be difficult to evaluate.

D. GENERAL CONSIDERATIONS I . Nature of Cell Injurg The principal methods discussed above measure in part different types of target cell injuries. This raises the question to what extent results obtainccl with different methods are comparable. It will be noted, that detachment of target cells from the substrate is a component of all methods in which usc is made of target cells growing in monolayer. Dctached cells are usually dead or will rapidly die. AS slready statcd, detachment will often lead to loss of target cells for causes which may be unrelated to the activity under study. In the colony inhibition assay and in ccll counting methods, cxctxpt when nondividing target cells are used, growth inhibition is one manifestation of injury. When supravital staining is used, changes in membrane perme,ibility arc recorded. This is also the casc with all iiicthods in which iso t op rclcase i s mcwxircd. Growth inhibition nnd changes of mcmilirane

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PETER PERLMANN AND GORAN HOLM

permeability, provoked by the effector cells, may be related and result in cytolysis (Briinner et ul., 196th; Berke et al., 1969a). Present data speak in favor of the opinion that membrane permeability increases carly during lysis (Green et al., 1959a,b). Methods that predominantly measure such alterations seem to be the most sensitive ones. In conclusion, there may be doubts about the interpretation of cytotosicity measured by methods based mainly on detachment of cells from glass. Growth inhibition and increased membrane permeability may reflect related phenomena, provided the experiments are performed under otherwise comparable conditions. 2. Choice of Controls

The choice of controls is crucial. Usually, target cell damage in the presencc of effector cells is compared with that in the samples which contain target cells without additions. Alternatively, target cells are incubated with lymphoid cells which are not expected to be cytotoxic. This second type of control would seem to be preferable since it provides culture conditions similar to those prevailing in the test samples. However, the lymphoid cells may have feeder effects on the monolayer, and these controls then grow and multiply faster than the controls free of lymphoid cells (Taylor and Culling, 1963; Wilson, 19651; Rosenau, 1968). In primary cultures and with low plating efficiency, only 10%of the cells may sometimes be found in the controls at the end of an experiment (Moller, 1965a). The results of such experiments must be evaluated with caution since there is no real base line to which the cytotoxic effects of thc effector cells can be referrcd. The release of radioactivity from cells labeled with isotope which is not reutilizcd is independent of most of the factors discussed above. Thc base line for estimation of the cytotoxic effects of lymphoid cells is usually provided by samples in which lymphoid cells are absent.

E. CONCLUDING REMARKS The interaction of lymphoid cells and target cells represents a dynamic situation which continuously changes during incubation. Some of the variables that govern the reactions have been described above. Others, such as differences in target cell susceptibility, state of sensitization of the donor of the lymphoid cells, death of effector cells, or transformation or selection of lymphoid cells during incubation will he discussed in the following sections. Thc discussion in this chapter can be summarized with the prescntntion of some criteria for the ideal method to determine cell-mediatetl

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cytotoxicity: ( 1 ) high sensitivity to enablc detection of cell damage within the shortest possible time of incubation; ( 2 ) low error allowing measurement of small differences; ( 3 ) quantitative and kinetic measurements of cell damage; ( 4 ) low spontaneous cell damage in the controls (“background”); ( 5 ) independence from detachment of target cells from substrate; and ( 6 ) inhibition or promotion of target cell growth b y culture conditions do not affect the results. None of the methods available at present fulfills all these criteria. Monolayer methods and methods measuring the incorporation of radioactive precursors are far from thc ideal. Cell counting techniques and the colony inhibition method are better. Most critclria are fulfilled by the method that measures release of ”Cr. It can be applied in most experimental situations and with all types of target cells. Ill.

Different in Vifro Models

A. CYTOTOXIC EFFECTS ON ANTIGENICTAHGET CELLS CELLS FROhf SENSITIZED DOKORS

OF

LYhrPHOID

The first experimental approach along the lines discussed above was that by Govaerts (1960). This author studied the cytotoxic effect of lymphoid cells from the thoracic duct of dogs, sensitized by a kidney allograft, on donor kidney cells in tissue culture. Recipient lymphoid cells became aggregated to the cultured cells which were destroyed within 24 to 48 hours. Lymphoid cells from normal dogs or dogs who had received a third party graft were not cytotoxic. However, in contrast to what since has been found by many, recipient serum strongly enhanced the weak cytotoxicity exhibited by recipient lymphoid cells in normal serum. Moreover, addition of complcmcnt further potentiated the cellmediated cytotovic effect. A large number of similar inwstigations has since been performed in many laboratories. In Table I, sollie of this work has been compiled. The different papers have bcen ordered according to the immune systems studied. No attempt has been made to present a complete list. Rather, reference has been made to papers considered to be representative for work done in this field. 1. Aggregation of L~jinplioirlCel1.Y to Target Cells.

Whcwever microscopic observation h a s been included in the work, aggregation of lymphoid cellc to target cells has been sccn. This is most c y d y observed when the target c ~ l l sare tissue culture cell5 in monolayer. In these cacc’s, lymphoid cc.11~from normal donors, or from donors sen-

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TABLE I I n Vilro CYTOTOXICITY OF LYMPHOID CELLSIN DIFFERENTIMMUNE SYSTEMS Authors

Target cells

Effector cells

Method

1. Transplantation Antigens

Govaerts (1960)

Dog, kidney cells Peritoneal or thoracic Monolayer dact cells, graft recipients Spleen cells, BALB/c Monolayer, cell Rosenau and Moon Mouse, L-cells (1961, 1964) (CJH), other immiinized with C3I-I counting cell lines or C57B1 tissue Wilson (1963) Mouse or rat Lymph node cells Cell coiinting kidney cells (mouse), thoracic duct cells (rat), sensitization by allogeneic skin grafts Wilson (1965n) Rat, tnmor cell Lymph node cells, 6ho- Cell counting lines racic duct, cells, rats sensitized by allogeneic skin grafts or immunization Vairiio et ol. (1964) Mouse, fibroLymph node and spleen Isotope release blasts, various cells, allogeneic immu(W-thymidine) H-2 genotypes iiization Monse sarcomas, Lymph node cells, mice Cell countiiig Broridz (1964, 1968) macrophages, immunized with alloH-2 genotypes geneic tumor cells Granger and Mouse, A/Jax Peritoneal cells, C57B1 Monolayer (plaqiie Weiser (1964) fibroblasts mice immunized with technique) Sarcoma I (A/Jax) Miiller (1965h) Mouse sarcomas, Spleen and lymph node Cell coiinting cells, mice immuiiized various H-2 genot,ypes with allogeneic cells Brunner el (11. Mouse, mastoSpleen cells, C57B1, Cloiiing assay, cell (1!)66) cyt.oma immiinized with cotinti Iig (DBA/2 urigiri) DBA/2 cells Monse, mashSpleen cells, C57B1, Isohpe release ( 5 0 ) Bruniier et c t l . (196%) cytomn immunized with (DBA/2 origin) I)BA/2 cells Hashimoto and Rat,, sarcoma Peritoneal exudate, frac- Cell couiit,ing Sudo (1968) (Yoshida strain) tionated into different cell t,ypes, Donryu mt,s immunized wiUi t,iimor Lurrdgren (I!KN) H\im:iii skin Blood lymphocytes, skin Munolayer (Plaqiie fil)rol)lnsts transplanted patients technique)

CYTOTOXIC EFFECTS OF LYMPHOID CELLS

.bit

hors

Target cells ~

Effector cells

in Vitro

129

Method

~~~

2. Expe~iinentul$utoimnziine Diseases

Koprowski and Dog, brain tissue E:sperimeitt.nl allergic Sloiiolayer Fernandes (1962) etirephalit,is. Lymph node cell, Lewis ra(s imnirinizecl with giiinea pig spinal cord Rose 6.1 ul. (1969) Rabbit, thyroid .iiitoimmriiie thyroitlit,is. hloiiolaycr Lymph node cells, cells rnk)l)its imniuiiized with thyroglohulin, thyroid esiracts Experimental allergic hloiiolayer Berg and KallBii Eat, glia cells encephalitis. Blood (1963) motlonuclear cells, t.horacic duct cells, rabbits or rats immiinized with porcine spinal rord Aiit,oimmune thyroidit.is. Monolayer Biorklund (1964, R,zt,, thyroid Thoracic duct, and explants 1968) lymph node c,ells, rats immiiiiized with rat t.iiyroid Munolayer Rat, myelinated Ksperiment al allergic fibers of trineriritis. Lymph iiotle geminal ganglia cells, rats imniuiiized with rat or rabhit peripheral nerve Rat, kidney cells I1:xperinieiital “a~itoirn- Isotope release Holm (1966) (14C-thymidine) mrine” nephrosis. B h J d , mononuclear cells, lymph node, spleen cells, rals immruiized with rat, kitliiey l~oc:dmuscle lesions, Monolayer Rat., skeletal Kakulas (1966) muscle nitisclr fiber necrosis. Lymph node cells, rats immiinizetl with rat skeletal muscle I

-

~

3. I‘arious Antzgens Taylor atid Crilling RIorise (L-stlain), Splecn cells, B.\LB/c Cell countiiig ( 1963) guinea pig mice or guinea pigs imniritiized with L-cells fihroltlast s

Contitiired

130

PETER PERLMANN AND GORAN HOLM

TABLE I (Conlinucd Authors

Target cells

Effector cells

Method

Holtzer and Winlrler Human amnion Blood mononuclear cells, Monolayer (1968) (cell line), lymph node o r spleen guinea pig kidcells, guinea. pigs senriey cells sitizetl with human amnion cells MacLennan and Chang cells (hu- Lymph node cells, rats Isotope release ( W r ) immunized with Chang Loewi (1968b) man cell line) cells Perlmann et nl. Chicken erythro- Blood moiioriiiclear cells, Isotope release ("'Cr) spleen cells, guinea (1968, 19691)) cytes coated pigs immunized wit,h with purified protein derivaMyeobucteriu tttberculosis tive (PPD)

4. H umun Diseases Perlmanii and Human colon cells Ulcerat,ive colit,is. Blood Isotope release Broberger (1963) nionoiiuclear cells (W-amino acid, 32P)

Hedberg and KalIBn (1964)

Human fibroblasts R.heuniatoid arthritis, systemic I L I ~ U Serythematosiis, psoriatic arthropat,liy. hlononuclear cells from syiiovial fluid Berg and KallBn Rat, glia cells Miihiple sclerosis. Blood mononuclear cells (1964) Rraunst,einer el uI. Human amtiion R heiimat.oid arthrit,is. (1964) cells Lymph node cells Trayanova et al. Human fibrohlasls Systemic lupus eryt,he(1966) and kidney cells matosus. Blood mononuclear cells Watson el ul. (1966) Human colon cells Irlcerative colitis. Blood mononurlear cells Snkernick el a / . Humail fibrol)lasts Iiheiimat,oitl arthritis. (1968) 131ood monorruclear cells

Monolayer

Monolayer Monolayer hlonolayer

Monolayer h'lonolayer

5 . Anamal Tumors (Tumor-Spccijc fi'euctzons)

Itosenau and Morton (1966)

Mouse, methylSpleen cells, syngeneic cholanthrenemice immunized with induced sarcoma tumor i n C3I-I or C.57Bl

Cell counting

CYTOTOXIC EFFECTS OF LYMPHOID CELLS

in Vitl.0

131

TABLE I (C‘ontinicetl)

Authors Alexander et 01. (1966h) Phillips (1967)

IIellstroni et al. (1968%)

Hellstroni et nl. (1969b) Heppner and Pierce (1969)

Target cells

bIouse letiltemiai Spleeii cells, dlogeneic cells of D13.412 mice ininirinizetl with origiii tiinlor Mouse, Sarcoma I 1,ymph node cells, C57Bl (A/J:ix origin) mice iinmiiriized with tiimor hIoilSe, iIlet.hyl1,yrnpfi node cells, cholaii t.tiretre o r t unior-t)earing alloplastic tlisc-ingeneic, syngeiieic, 01’ ducetl samiinas auit,ologviis mice in C3H or BhLB/c Mouse, Noloiiey Lymph node cells, t umoi-bearing synsarconia in BALB/c or C3H geneic mice Mouse, spoiitaLymph node cells, autolnenw mammary ogoris tiimor virusluniors carrying BAL13/c mice, foster-fed on C3H; or virus-free BX LR /c 6. Himuin Tiimors

Hellstrnrri rt ( 1 96x1))

rtl.

Hellstriiiri et a / . (196%)

Bubenik et al. (1969,)

Effector cells

Seiirolil:tst,oiiiat

(7‘li?tL(JI’-SpC’CL$C

Method Cell counting

Cell c o i i n h g

Coloiiy inhibition

Colony inhibil ioii

Colony inhibition

Keuctions)

Xritdogoiis or allogeiieic Colony irihiljitioii bli~odinononuclear cells Xut,ologr)iisor allogeiieic Coloiiy inhibition M)od mononuclear cells

Wilm’s tiimor, vnrioiis aclenocxrcinoin:ts :tiid sarcomits Bladder cnrciAutologoiis or allogeneic Cell coiinting iioma blood moiioiiiic1e:i.r (microniodificacells tion)

sitized to unrelated target cells, can be washed off. In contrast, lymphoid cells from donors sensitized to target cell antigens cannot easily be removed by washing (Rosenau and Moon, 1961; Koprowski and Fernandcs, 1962; Taylor and Culling, 1963; Wilson, 1963; Brondz, 1964; Vainio et al., 1964). An increased stickiness of lymphoid cells from donors injected with Freund’s complete adjuvant has been described (Koprowski and Fernandes, 1962; Holtzer and Winkler, 1968) but is not a rcgular finding. Aggregation usually precedes morphological changes and death of the target cells ( Rosenau and Moon, 1961; Koprowski and Fernandes, 1962).

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PETER P E R L M A N N A N D GORAN HOLM

2. Target Cells

a. Nature and Properties Affecting tlie Cytotuxic Reaction. By definition, a common feature for the target cells discussed under this heading is their possession of antigens to which the donors of the lymphoid cells are sensitized. In all c a m studied thus far, these antigens arc supposed to be surface components. Thcy can be naturally occurring constituents of the target cell surfacc; antigens, foreign to the cclls and artificially attached, may also be effective (Perlmann and Holm, 1968; Perlmann et al., 1969b). This is of some importance since it simulates conditions which may occur in an organism in connection with delnycd hypersensitivity. Lymphoid cells from donors immunized to serum proteins in tlie incubation medium in which target cells were growing were not cytotoxic: to these cells (Rosenau and Moon, 1964; Taylor and Culling, 1965). (However, in Section III,D,I, it will be shown that antigens unrelated to target cell antigens under difEerent experimental conditions can induce cell-mediated cytotoxicity. ) A sufficiently high dcnsity of antigenic determinants on the cell surface is known to be essential €or the cytolytic reaction induced by humoral antibody and complement (Moller and Moller, 1962; Winn, 1962). Although there is some evidence that the density of antigenic receptors influences the course of cell-mediated cytotoxicity, other properties of the target cells and the experimental conditions are probably also of importance (Brunner et al., 1969b). b. Allogeneic Inhibition. From experiments to be discussed latcr (Moller, 1965a,b; Moller and Moller, 1965) (see Section III,D,2), it was suggested some years ago that in vitru destruction of antigenic target cells by lymphoid cells from sensitized donors constitutes a case of allogeneic inhibition. Thc latter is defined as a nonimmunological phenomenon in which cells may be killed through an unknown (possibly suicidal) mechanism when coming into close contact with cells of different genctic (i.e., allogcneic) origin (Hellstriim and Moller, 1965; K. E. Hellstrom and I. Hellstrom, 1967). Thus, although close contact between lymphoid cells from immune donors and antigenic target cells may be established by immunologically specific reccptors on the cell types, the actual killing would be due to confrontation of the target cells with foreign histocompatibility antigens on the lymphoid cells ( Moller, 1965a,b; Moller and Moller, 1965). However, since the time when this concept was advanced, several papers have been published in which lymphoid cells and target cells were from genetically compatible donors. Rosenau and Morton (1966), I. Hellstrom and K. E. Hellstrom (1967), Hellstrom

CYTOTOXIC EFFECTS OF LYMPHOID CELLS

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133

et al. ( 1968a), and Brunner et al. (1969b) have shown that experimentally induced tumors in micc arc destroyed in tumor-specific reactions by syngeneic lymphoid cells from scnsitized animals. Tumor-specific reactions in autologous combinations have recently also becn dcscribed for human tumors (Hellstrom et al., l W b , c ; Bubenik et al., 1969a). Other examples are organ-specific destruction of kidney or thyroid cells by syngeneic lymphoid cells from rats immunized with kidney or thyroid, respectively (Holm, 1967c; Biorklund, 1968), and of human colon cells by autologous lymphoid cells from patients with ulcerative colitis (Watson et al., 1966). Such findings do not rule out the possibility that differences in histocompatibility or, perhaps, other cell-type-specific differences in surface architecture may influence the course of lymphoid cell-target cell interactions. Most importantly, however, it is obvious &at lymphoid cells may become cytotoxic to other cclls within an organism in a strictly autologous situation. 3. Immunological Specificity and the Significance of Humoral Antibodies

a. Specificity. Different types of controls have been used to prove the immunological specificity of the cytotoxic reaction. Thus, lymphoid cells from tumor-bearing animals, or from animals immunized with X-irradiated tumors, have been shown to destroy tumor cells in a specific reaction. Tumor cells with noncross-reacting tumor antigens were not affected (Rosenau and Morton, 1966; I. Hellstrom and K. E. Hellstrorn, 1967; Hellstrom et al., 1968a). Similar results have recently been obtained with several human tumors. In nitro destruction of certain tumors has been obtained both with the patients’ own lymphoid cells and with those from other patients carrying a tumor of the same type. In contrast, these lymphoid cells did not affect cells from healthy organs of the type from which the tumors originated. Neither did they destroy cells from unrelated tumors (Hellstrbm et al., 1968b,c). Among other things, these findings provide convincing evidence for the occurrence of tumor-specific antigens and 3 corresponding immune response for a variety of human tumors, Unexpectedly, in view of what was heretofore known of animal tumors, these antigens seem to be both tumor and organ specific. For a full discussion of these problems thc recent review by Hellstrijm and Hellstrom (1969) should be consulted. In work with other immune systems (Table I ) , the specificity of the reactions has usually been controlled in a similar manner. A more detailed analysis of immunological specificity has thus far only been possible in those cases in which the target cell$ carried serologically and gcnetically well-defined transplantation antigens. Rosenau and Moon ( 1964) found

134

PETER PERLMANN AND GORAN HOLM

that lymphoid cells from BALB/c mice (genotype H-2d), immunized with spleen cells from C3H mice (H-2k), killed cells from established cell lines of C3H origin but did not affect fibroblasts originating from thc strains DBA-2 (H-2d) or C57B1 ( H-2b). The results were reversed when the lymphoid cells wcre from donor mice immunized with C57B1 spleen. Target cells of rat or human origin were not affected. Lundgrcii (1969) has reported similar results for the human HL-A system. A detailed analysis, within the H-2 locus of the mouse, was recently reported by Brondz ( 1968) who used methylcholanthrene-induced sarcoma cells as immunogen, lymph node cells as effector cells, and peritoneal macruphages as target cells. Serum from the sensitized mice was taken on the same day as the lymph node cells. Several strain combinations within the H-2 system were tested. In all cases, the resulting cell-mediated cytotoxic reaction was specific for the H-2 antigens of the target cells. Moreover, in order to be cytotoxic, the lymph node cells had to be taken from donors sensitized to all or almost all of the foreign H-2 antigens on the target cells, When the donors were sensitized to only half of the antigcns or less, thc targets were not killed, in spite of the fact that hcmagglutinating and cytotoxic antibodies were shown to be present in the serum of the lymph node donors. Rrunner et al. (1969b) have made similar fiiidings. No cytotoxic reaction could be produced by combining lymph node cells from two donors, each sensitized against a different fraction of the H-2 antigens on the target cells (Brondz, 1968). These findings establish a high degree of specificity of the cell-mediated reaction, with exacting requirements exceeding those of the serological reaction. Thcy may possibly indicate certain requirements of carrier specificity, resembling those operating in delayed hypersensitivity (for references, see Turk, 1967). The author assumed that, in order to trigger a cytotoxic reaction, the activc lymphoid cell must carry multivalent receptors, each receptor with specificity for all foreign H-2 antigens on the targct cells. Other more appealing explanations may he found, however. b. Hutnorcil Antibodies. It is often stated that the cytotoxic action of lymphoid cells from sensitized donors 011 antigenic target cells is an in wiiro expression of delayed hypersensitivity or a similar state rather than of a humoral antibody response. It is implied that cytotoxicity reflects an activity of sensitized cells rather than one of humoral antibody. This argument is based on several lines of evidence. Thus, the optimal time for harvesting active cclls from sensitized donors may coincide with the development of allograft rejection rather than with humoral antibody production (Brondz, 1964; Brunner et al., 196%). When humoral antibodies are present they may not he cytotoxic (Broberger and Perlmann,

CYTOTOXIC EFFECTS OF LYMPHOD CELLS

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135

1963; IVilson, 196%; Holm, 1966; Hellstriim et al., 196Sa,b). However, in many cases no correlations liavc, bcen cst,il)lishcd and cytotoxic antibodies have sonietimes hcmi seen in high titcrs (Rosenau, 1963; Berg and Kallch, 1964, Hellstrbm et al., 196Sb,c, 1969b). In an alloimmunc>system (H-2) of the mouse, Biunner et al. (196Sa,b) found an cxccllent correlation between formation of 19 S (but not 7 S ) antibodies and thc appearance of cytotoxic cells. In the guinea pig, Perlmann et al. (1969b) found lymphoid cells cytotoxic to purified protein derivative (PPD) coated target cells cvcn when delayed hypersensitivity in the donors could not he detected by means of skin testing. Attempts to elute nntibody-like activity from lymphoid cells of sensitized donors ( Taylor and Culling, 1963; Wilson, 196%; Brondz and Bartova, 1966) or to confer activity on lymphoid cells fioin normal donors by exposure to antiserum in vitro have mostly been unsuccessful (Rosenau, 1963; Perlmann and Broberger, 1963; Brondz, 1964; Wilson, 1965a; Moller, 1965b); see, however, Section III,B. For cytotoxicity, humoral antibodies require participation of complement. In experiments with cell-mcdiated cytotoxicity, heat inactivated serum is usually included in the incubation mixtures and addition of complemciit has no effect ( Brondz, 1964; Wilson, l965a, Moller, 1965b; Rosenau, 196s). Exceptions to this rule have becn noted ( Govaerts, 1960; Perlmann and Broberger, 1963). There are also cases in which cytotoxic effects of lymphoid cells in uitro have been demonstrated in complete absence of serum (Rosenau and Moon, 1961; Rosenau, 1968). Other arguments for cell-mediated cytotoxicity being the expression of a cellular activity, independent of humoral antibody, are derived from observation of the inhibitory effects of the lattcr. Thus, treatment of mouse target cells with heat-inactivatcd alloantibodies against their H-2 antigens before or during cxposiuc to lymphoid cells from immunized allogeneic mice strongly reduced the cell-mediated cytotoxic effects ( Moller, 1965b; Brunner et al., 1967, 196Sa). The immunological specificity of this inhibition was clearly established. Moreover, only when a major part of the relevant antigens was blocked by antibody, the cellmediated rcaction w'is abolished (Mauel et al., 1969). Both 7 S and 19 S alloantibodies have been found to be protective ( Bruiiner et a]., 1968a). Similar although less clear-cut inhibitions have occasionally been observed in other imniiuie systems (Holm, 1967c; MacLeiirian and Loewi, 1968b). Most recently, an immunologically specific inhibition of cellmediated cytotoxicity in vitro by humoral antibodies has been described for several human and animal tumors ( Hellstriim et al., 1969a). Thesc in vitro results are indicative of an enhancement-like inhibition

136

PETER PERLMANN AND GORAN HOLM

(Kaliss, 1958) by antibodies on the efferent ( =effector) side of the immune response (Brunncr et al., 1968b). An afferent or central inhibition (Billingham et al., 1956) by humoral antibody on the inductive phase of the immune response may also be reflected by a reduction of cellmediated cytotoxicity in uitro. Brunner et al. (1968b, 1969a) found that humoral alloantibody ( mouse), passively administered in connection with immunization, reduced the in vitro cytotoxic effects of lymphoid cells harvested from the treated animals within 1 to 3 weeks after grafting. Both 7 S and 19 S antibodies were suppressive. However, formation of humoral antibodies ( 1 9 s ) in the treated animals was also strongly reduced. Since course and manifestation of the immune response reflect a balance between cellular and humoral factors, it is not really contradictory that inhibition by antibody of in vitro cytotoxicity does not represent a general finding. Thus, Brondz (1965) was not able to achieve inhibition of target cell killing by lymph node cells with alloantibodies within the H-2 system of the mouse. In some of the tumor-specific systems, recently described by Hellstrom et al. (1969a), humoral antibodies did not inhibit in vitro cytotoxicity of lymphoid cells on the tumor cells (colony inhibition). These “inactive” sera were from donors in which the tumors had regressed and which often contained antibodies which were cytotoxic to the cells in the presence of complement (Hellstrom et al., 1969b). In summary, cell-mediated cytotoxicity in vitro may reflect a state of delayed hypersensitivity or a related condition of a sensitized donor. However, from what has been said it is also clear that too far-reaching generalizations in this direction are unwarranted. That humoral antibodies in certain cases can induce in vitro cytotoxicity of normal lymphoid cells will be more fully discussed in Section II1,B. It would be surprising if such reactions would not also take place in some of the cases where the lymphoid cells are obtained from sensitized donors in which antibody formation takes place concomitantly. In this sense, the in vitro reactions are not less complex than are the in vivo manifestation of delayed hypersensitivity, tumor or allograft rejection, or tissue damage in autoimmunity.

4. Kinetics Target cell destruction by lymphoid cells generally proceeds slower than lysis induced by humoral antibody and complement. This does not by itself constitute a valid argument against participation of antibody and Complement in cell-mediated cytotoxicity. The rate of target cell destruction will depend on a variety of factors such as nature of target cells, immune state of the donor of the effector cells, the number of these

CYTOTOXIC EFFECTS

OF LYMPHOID CELLS

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137

cells, and the assay system. The course of the reaction will also be dependent on the experimental conditions that affect the viability of the lymphoid cells. A certain excess of lymphoid cells over target cells seems always to be necessary to obtain measurable target cell destruction. The most complete kinetic analysis has first been reported by Wilson (1965a) who used suspensions of irradiatcd tumor cells (cell lines) from inbred rats. The lymphoid cells were from lymph nodes or thoracic duct of allogeneic rats, sensitized against the histocompatibility antigens of the target cells by means of skin allografts. The lymphoid cells werc added in excess and target cell destruction was evaluated by counting of nuclei (Section II,D,2). Detectable destruction of target cells was first noted after about 20 hours of incubation but was virtually complete at 50 hours. Target cell survival was inversely related to the number of Iymphoid cells added. The exponential relationship found was similar to a “single-hit” inactivation phenomenon, suggesting that one single “sensitized lymphocyte would suffice to affect adversely one target cell. It could also be extrapolated that 142%of the lymphoid cells added in these experiments were immunologically active ( i.e., destructive for target cells), This course of target cell destruction is typical for most of the reports in which cell-mediated cytotoxicity was studied under similar conditions. The influence of the assay system on the reaction rate is brought out by some experiments of Brunner et d. (1966), using mouse mastocytoma cells of the DBA/2 strain as targets and, as effectors, spleen cells of C57B1 mice, sensitized against DBA/Z. The assay system was either microscopic counting of surviving target cells, or measurement of their ability to form colonies in semisolid medium after contact with the effector cells (Section II,D,3). Like Wilson, the authors found an inverse and exponential relationship of target cell survival to number of spleen cells added. However, whereas it took from 24 to 4s hours to demonstrate a reaction by microscopic counting, this was demonstrable, after 3 hours and complete after 12 hours in the cloning assay. In later experiments (Brunner et nl., 196Sa,b) these authors compared the cloning assay with 51Cr-releasefrom labeled mastocytoma cclls. Interestingly, in these different assays, target cell destruction had very similar kinetics. In the same immune system with spleen cells taken at peak response after one immunizing injection, Brunner et al. (196913) have recently been able to achieve almost complete target cell destruction within 1 to 2 hours, when the effector cells were added to the mastocytoma cells at a ratio of 100:1. At lower ratios, a certain time lag was observed and the rate of target cell destruction decreased. However, even at lymphocyte/

138

PETER PERLMANN AND GORAN HOLM

target cell ratios as low as 1:1, up to 20%of the targets were lysed within 6 hours. The data suggested that one “sensitized effector cell killed more than one target cell. It was also shown that the rate of destruction was different for target cells of different types. Thus, at lymphocyte/ target cell ratios of 100: 1, embryonic fibroblasts in suspension or allogeneic spleen cells were destroyed at much lower rate than mastocytoma cells, in spite of the fact that the same H-2 antigens were involved.

5. Effector Celh

a. Origin and Activity of Lymphoid Cells. Spleen cells, lymph node cells, blood lymphocytes, or thoracic duct cells may vary somewhat in cytotoxicity when their effects are compared on the basis of equal numbers of lymphocytes. Moreover, activity levels in cell populations of different origin will also vary at different times after immunization (Wilson, 1965a; Moller, 1965b, Bruiiner et al., 1969b). Thymus cells from immunized donors are only slightly active or inactive ( Brunner et al., 1969b ) . It is assumed that the activity of a cell sample reflects the concentration therein of active cells rather than varying degrees of cytotoxic activity on the level of the single cells. In experimental animals, all authors have found peaks of activity at about 1 to 2 weeks after antigenic challenge (Brondz, 1964; Wilson, 1965; Brunner et al., 196913). Mode and intensity of immunization are decisive for the cytotoxic activity of the effector cells, with cells from heavily immunized donors exhibiting the strongest effects (Brunner et al., 196913; Perlmann et al., 1969b). The nature of the immunogen is also important, with living antigenic cells being more efficient than X-irradiated cells or extracts (Hellstrom et al., 1968a; Brunner et al., 1969b). This may be the reason for the finding of relatively active lymphoid cells in human tumor patients and in some autoimmune diseases. It may be noted that the highly efficient cytotoxicity described by Brunner et al. (1969b) was achieved by immunizing C57B1 mice with living mastocytoma cells of DBA/B origin. When DBA/2 spleen cells were the immunogen, the cytotoxic activity of the lymphoid cells, tested against several DBA/S targets, was much less. b. Lymphocytic Cells. The facts reported in the preceding paragraphs and the common microscopic picture of clustering of lymphocytes to target cells have led to the general assumption that lymphocytes also are cytotoxic effector cells in uitro. From the data published thus far it cannot be concluded which cells within the functionally heterogeneous populations of lymphocytes (Gowans and McGregor, 1965; Miller and Osoba, 1967) participate in in vitro cytotoxicity. From indirect evidence already referred to (Section III,A,4,b) the assumption may be made that

CYTOTOXIC EFFECTS OF L Y m w o m CELLS

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139

the lymphocytes that trigger delayed hypersensitivity and similar conditions in vivo, or some of their descendants, may function as cytotoxic cffector cells in vitro. However, such indirect evidence does not help to resolve the question of the relative role in in vitro cytotoxicity of antigenreactive and thymus-derived lymphocytes as compared to that of bone marrow-derived precursors of antibody-producing cells ( Mitchell and Miller, 1968). It is not known, for instance, whether or not antibodyproducing plasma cells or their prccursors exhibit cytotoxic activities in vitro. Even if not directly cytotoxic by themselves, they may contribute indirectly by releasing small amounts of antibody capable of inducing cytotoxic activity of normal lymphocytes by one of the pathways described in the following paragraphs and in Section II1,B. c. The Contribution of Monocytes, Alacrophages, and Polymorphonuclear Leukocytes. At the present stage of our knowledge, the conclusions that lymphocytes are acting as effector cclls in in vitro cytotoxicity requires many qualifications. Thus, although some cell populations, such as those from thoracic duct, consist of 98 to 99% lymphocytes, most authors have made experiments with ccll populations in which lymphocyte concentrations may have varicd from 75 to 95%. Since the effector cells are added to the target cells in excess, sometimes at very high ratios (1000: 1 or more), a small fraction of active nonlymphocytic ceIls may very significantly contribute to target cell damage. There is evidence that macrophages from sensitized donors also are cytotoxic to target cells in vitro by phagocytic and “nonphagocytic” mechanisms ( Bennett et al., 1963; Old et nl., 1963; Granger and Weiser, 1964, 1966). It is likely that the same applies to monocytes of the pcriphcral blood. Whether or not polymorphonuclear leukocytes from sensitized donors may exhibit specific cytotoxicity in vitro is unknown. When antibody-producing cells are present, and this is the rule rather than the exception, cytotoxic activities of macrophages, monocytes, and polymorphonuclear leukocytes will most likely be enhanced. Interactions between lymphocytes and other cells cannot be disregarded in this context. Cellular interactions of this type have recently attracted growing attention in several relevant situations, such as in connection with the induction of humoral immunity by macrophages ( Fishman and Adler, 1963; Ford et al., 1966; Mitchison, 1968; Unanue and Askonas, 1968). I t has been shown that the reaction of a small number of sensitized lymphocytes with antigen will lead to profound changes in activity of macrophage populations by cmission of a soluble factor (David, 1966; Bloom and Bennett, 1966; Svcjcnr et al., 1968). Conversely, lc~ the reaction of a few niacrophagcs with antigrw sCm1s to be c a p ~ ~ b to

140

PETER PERLMANN AND GORAN HOLM

induce morphological transformation and DNA synthesis in lymphocytes (Cline and Swett, 1968). It is obvious that the relative role of these and similar interactions for the cytotoxicity of lymphoid cells on target cells in vitro requires further studies. 6. Cytotoxic Mechanisms a. Contact. It has already been described that a characteristic feature of cell-mediated cytotoxicity is the antigen-specific aggregation of lymphoid cells to target cells. Instrumental in bringing about contact are antibody-like receptors on some of the lymphoid cells. When contact between lymphoid cells and target cells is prevented, damage of the latter does not take place (Rosenau, 1963; Wilson, 1965a). When two target cell populations, carrying different antigens, are simultaneously exposed to lymphoid cells from a donor immunized against only one of the antigens, only the target cells carrying the relevant antigen are destroyed (Brunner, personal communication). Although these findings indicate that contact is necessary for the cytotoxic reaction they do not reveal its mechanism. Microscopic observations and time-lapse cinematography with target cells in monolayer shows that some lymphoid cells, after random movement, will become temporarily attached to the target cells which finally seem to undergo osmotic lysis (Rosenau, 1963). These studies do not prove whether or not lysis is actually produced by the attached cclls. Treatment of mouse lymphoid cells with alloantibodies against H-2 antigens that distinguished them from the target cells in a transplant a t'ion immune system did not inhibit their cytotoxic effect ( Mauel et al.. 1969). Thus, when the lymphoid cells are from sensitized donors, allogeneic inhibition i.e., contact of the targets with foreign alloantigens on the lymphoid cells, does not lead to target cell death (Section III,A,S,b). On the other hand, addition of heat-inactivated antilymphocyte serum, made in a diffcrent species, efficiently prevented the cytotoxicity of both mouse and guinea pig spleen cells (Mauel et al., 1969; Lundgren, 1969). It remains to be established whether this inhibitory effect was due to prevention of efficient contact bctween the cell types or to physiological inactivation of the lymphoid cells. A possible immunoglobulin nature of receptors on the lymphoid cells is suggested by the rcsults of Winkles and Arnason ( 1966). These authors inhibited the demyelinating activity in vitro of lymph node cells from rats immunized with nerve tissue b y treatment with rabbit anti-rat IgA. b. Phyyiologicnl State of Lymphoid Cells. Many authors have shown that the lymphoid cells have to be alive in order to be cytotoxic. No

CYTOTOXIC EFFECTS OF LYMPHOID CELLS

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141

specific cytotoxic effects have thus far been obtained with dead cells or with extracts or eluates of cells from sensitized donors (Brondz, 1964; Wilson, 1965a; Brondz and Bartova, 1966). Although the lymphoid cells may die during the experiment when the conditions are unfavorable for their survival, availahle evidence suggests that they are not killed in the course of thc cytotoxic reaction (Wilson, 1963). In Fig. 1 it is shown that the cytotoxic potential of a suspension of lymphoid cells from sensitized mice remained unchanged when the cells were added to a fresh batch of target cells after having killed the first one. Figure 1 demonstrates a good similarity of the kinetics of target cell destruction on thc two occasions. This speaks against death of cffector cells as being a major event, at least in short-term experiments. It possibly favors the

I

2

3

4

5

6

HOURS

FIG. 1. Cytotosic effect of lymphoid cells (spleen) from sensitized mice (C57BI anti-DBA/?) on srispensions of DBA/B mastocytoma cells, labeled with ”Cr. Target 1-100: specific isotope release (ordinate) during 2 hours of incubation of targets with an excess of 100 spleen cells per target cell. During this period most target cells were lysed. In parallel tubes, unlal,eled target cells were incubated with lymphoid cells under identical conditions. After 2 hours of incubation, the suspensions were ccntrifuged and the siiiiie niunber of fresh hut “‘Cr-labeled cells was now added. Isotope release from these cells is shown h y ciiii’e cksignated “Target 2-100.” Control-100: cytotoxic effect of a second sample of lymphoid cells from the same batch, first incubated for 2 hours without targets. The cells were then centrifuged and “Cr-labeled target cells were ndded as clcwxibed. Target 1-30 (etc. ) : parallel experiments with cells from the same batches but perforrned a t spleen cell/target cell ration of 30: 1. Tiiiie of experiiiient shown on abscissa. For other cxperimental details, see Brunner rt al. ( l968a). (Froni Brunncr ct nl., 1AliOa. )

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opposite supposition that reaction with antigen may enhance the cytotoxic potential of the effector population. If true, this could be due to either activation of previously "sensitized cells or recruitment. Some authors have succeeded in making normal lymphoid cells cytotoxic by exposing them to ribonucleic acid ( R N A ) or ribosomes from sensitized cells (Gerughty et al., 1966; Wilson and Wecker, 1966; Bondevik and Mannick, 196s). These preliminary results suggest one possible mechnnism for recruitment. Since the fraction of cytotoxically activc cells in a sensitized population is relatively large (Wilson, 1965a; Biunner et al., 1966), recruitment of originally inactive cells may play a role (MacLennan and Harding, 1969). However, more experiments are needed to establish this important point. Lymphoid cells from sensitized donors will aggregate to target cells at low temperatures, but undcr these conditions 110 destruction of the latter ensues (Wilson, 1967a). This reflects the fact that metabolic processes are necessary for the cytotoxic reaction. Experiments with metnbolic inhibitors support this notion. Antimycin A, suppressing electron transport in the respiratory chain (Chance and TVilliams, 1956) also impaired cytotodcity when applied to the lymphoid cclls (pretreatment) at concentrations that inhibit respiration without being toxic during the experimental period (Perlmann et al., 1969b). Inhibitors of RNA and protein synthesis, such as Imuran (Wilson, 1965b), actinomycin D, and cycloheximide (Brunncr et al., 1968a), inhibit cytotoxicity when applied at concentrations that blocked these metabolic activities without being toxic for the cells in the incubation mixture. However, inhibition with cycloheximide was partial and reversible. The residual activity ( approximately 50%) found after cycloheximide treatment was Explained as reflecting the presence in or on the cells of a pool of protein needed for cytotoxicity. When the lymphoid cells werc treated with low concentrations of trypsin, no inhibition was obtained in long-term experinients ( Brondz and Bartova, 1966). However, trypsin treatment abolished the cytotoxicity of lymphoid cells in short-term experiments ( Mauel et al., 1969). Inhibition by trypsin was only temporary and was coniplctely reversible. When trypsin treatment was followed by cycloheximide, cytotoxicity was strongly and irreversibly supprcssed. This suggested that protein, removed by trypsin, could not bc replaced at necessary concentration when protein synthesis was blocked. It may be that thc protein needed for the lytic reaction represents the antibody-like receptors, rcquired for the initiation of thc cytotoxic rcaction. Cytotoxicity of lymphoid cells has also Iwen suppressed by cthylcnc-

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diaminetetracetic acid (EDTA) (Mauel et al., 1969). Inhibition was reversible and activity could be fully restored either by washing or by addition of Ca2+and Mg". It is not known whether this treatment affects contact or the lytic process. In long-term experiments (48 hours), X-ray at high doses ( 15,000 r ) seemed to be inhibitory, probably because it affected the viability of the lymphoid cells under the experimental conditions applied ( Rosenau and Moon, 1966). In short-term experiments, D N A synthesis and cell division are not required for the manifestation of cytotoxicity in uitro ( Maud et al., 1969). Contradictory results have been obtained by hydrocortisoiic treatment, probably because of differences in experimental conditions ( Rosenau and Moon, 1962; Mauel et al., 1969). The mode of action of this drug in this system is open to speculation.

7. Conclusions Cytotoxicity mediated by lymphoid cells from scnsitized donors may be viewed as a two-step phenomenon. The immunological specificity of target cell destruction resides in the first step in which contact between effector cells and targc t cells is established. A prerequisite for specificity is the presence of antibody-likc receptors on some cells in the effector cell population. Available evidence points to lymphocytes as cells possessing such receptors. It is assumed that these receptors also are manufactured by the lymphocytes on which ihey occur but this remains to be proven and is not necessarily a general rule. In any case, small amounts of released antibody may induce aggrcgation of cells, lymphocytes, and others, not originally cquipped with specific receptors synthesized by them (sec Sections III,B,C, and D ) . The results described above strongly support the notion that target cell lysis is brought about in a second step, following the contact established in the first step. There is no evidence that the antibody-like receptors of the first step also participate in the lytic reaction which probably lacks specificity in an immunological sense. It requires the participation of viable and metabolically active effector cells. It is possible that cytotoxic factors which nonspccifically inhibit target cell growth are released from the effector cells upon contact with target cell antigen. However, most of the evidence available to date suggests that cell-to-cell contact or at least close proximity between the ccll types is a principal requirement for the lytic step. This docs not exclude that target cell destruction is brought about by local relcnse of toxic substanccs, liberated from effector cells aggregated to the target cc~lls.Such mediators may be hydrolytic enzymes, components of thc coniplement system (Section III,C), or other factors. It is also possible that early changes in phospholipid metab-

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olism lead to activation of detergent-like agents on thc surface of the effector cells (Fischcr et al., 1968). Entirely different mechanisms may be at play, however. Although phagocytosis does not seem to be a major event, target cell destruction may involve surface processes rclated to pinocytosis or phagocytosis. It remains to be cstablished whether the sensitized lymphocytes that initiate target cell destruction by reacting with antigen in the first step, also are executing the lytic reaction. Some of the microscopic and cinematographic studies wouId seem to suggest this. However, the importance of interactions between lymphocytes or between lymphocytes and other cell types in the effector population has already been pointcd out and cannot be overemphasized. Such interactions may lead to recruitment of originally nonsensitized lymphocytes or of other cell types and thereby actually trigger or amplify the lytic reaction. The question of participation of cells other than lymphocytes in the cytotoxic reaction bccomes particularly pertinent when relatively crude cell mixtures arc used as effector cells. When niacrophages, polymorphonuclear leukocytes, and nntibody-secreting cells are present, it can be safely assumed that target cell destruction may occur along a number of different pathways. Which of these will predominate may depend on the immune state of the cell donor, thc type of target cells used, and the assay system. OF CYTOTOXICITY OF LYMPHOID CELLSFROMNORMAL B. INDUCTION BY ANTIBODIESTO TARGET CELLANTIGENS DONORS

The cytotoxic reactions produced by lymphoid cells from donors sensitized to target cell antigens are immunologically specific and, therefore, require the presence of antibody-like receptors on some cells in the effector population. In spite of some negative results referred to in Section III,A,3,b, recent studies have provided evidence that humoral antibodies may induce cell-mediated cytotoxic reactions in &fro. In brief, antigenic target cells, treated with certain heat-inactivated antisera will be damaged when exposed to a modcrate excess of lymphoid cells from unsensitized donors. The antisera arc effective at dilutions too high to give rise to conventional complement-mediated lysis in the absence of lymphoid cells. 1. Description of Models a. General. Chromium-51-labeled chicken erythrocytes, coated with various protein antigcns, such as PPD or guinea pig thyroglobulin, have been used as target cells. Thc antigens were attached to the red cells by means of tannic acid treatment. When PPD was used (Perlmann and

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Holm, 1968; Perlmann et d., 1969b), active sera were obtained from guinea pigs, immunized with Bxillus Calmette-Guerin ( BCG ) or killed tuberclc bacilli. The c ffector cells were spleen cells or blood lymphocytes from norinal guinea pigs, and cytotoxicity was assayed as isotope release. In the controls, the antisera n7erc replaced by heat-inactivated sera from normal guinea pigs. In othcr controls the lymphoid cells were left out or repIaced by kidney cells or red bIood cells. These as well as guinea pig thymus cells wcrc inactive. There was no species barrier; guinea pig lymphoid cells could be replaced by lymphoid cells from human peripheral blood or even from chicken, autologous to the erythrocytes used as targets. In contrast, blood lymphoid cells from patients with chronic lymphatic leukemia or Burkitt’s lymphoma cells wcre inactive. In thiF model, only sera from hyperimmunized animals wcre active. There was no correlation between their hemagglutinating titers towaid PPD-coated erythrocytcs and their activity in cell-mediated cytotoxicity. The antithyroglobulin sera were also from guinea pigs, immunized with guinea pis thyroglobulin in complete Freund’s adjuvant ( Wasserman and Packal6n. 1965). However, in these cases, one single injection was sufficicnt to produce highly active sera as well as thyroiditis within 3 to 4 weeks after challenge ( Wasserman et nl., 1969). In later work use was made of natural target cell antigens. The cells most widely used in the authors’ laboratory are Chang liver ceIIs (human ccll strain), grown in suspension cultures ( Holm and Perlmann, 1969a,b) and chicken erythrocytcs ( Pcrlinann and Perlmann, 1969). In both systems, antisera are from hyperimmunized rabbits and lymphoid cells from human periphcral blood or thoracic duct. Column-purified blood lymphocytcs are cytotoxic while thymiis cells and leukemic lymphocytes are inactive or only weakly active. Chang cell9 have also been used in a similar system by MacLennan and co-workers ( MacLennm and Loewi, 196%; MacLennan and Harding, 1969; MacLennan et al., 19G9a). These authors used lymphoid cells of blood, lymph node, and spleen origin from humans, rats or rabbits. Antisera were either from rabbits or from rats. Antibody-induced cytotoxicity was seen in some but not all combinations. High concentrations of normal serum or antiseiwn wcw inhibitory. Taylor and Culling (1968) have used mouse fibroblast treated with guinea pig antimouse serum and guinea pig spleen cells. Lundgren et nl. (1968b) used sheep fibroblast monolayers, cuposed to rabbit antishccp erythrocyte serum, and purified human blood lymphocytcs. Cytotoxicity was assayed by the plaque twhnique. In umnbcr of 1ium:un sera, RlncLcnnm et nl. (196911) recently found ‘1 factor \vhich rcndcrs Chang cclls susceptible to lysis by normal human lymphoid cells. The factor had the properties of an antibody (7s)

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specific for Chang cells. Thus, in this system, all components of the reaction were of human origin although the Chang cell antigen involved may have been of viral origin. Bubenik et al. (1969b) have recently obtained similar results in work with inbred ducks. The antibodies were alloantibodies fioni the Campbell strain and were directed against the transplantation antigens of the Peking strain. Purified blood lymphocytes from normal Campbcll ducks lysed Peking targets in the presence of such antibodics. Both eiythrocytes and embryonic fibroblasts were destroyed. Lymphocytes from Campbell ducks tolerant to Peking transplantation antigens were as efficient as normal lymphocytes. b. Kinetics and Morphology. For positive cytotoxic reactions, lymphoid cells are added in numbers exceeding those of the target cells. Optimal ratios vary according to variations in antiserum, lymphoid cells, and target cells, but 25:l-5O:l seems to be optimal in several systems ( Holm and Perlmann, 196913; Perlmann and Perlmann, 1969). Under optimal conditions, isotope release from Chang cells or chicken erythrocytes can be observed within 1 or 2 hours and usually reaches completion ( 100%lysis) within 1 day. Purification of lymphocytes (human blood) affects the time course but not total. lysis. Microscopic observation of mixtures containing normal lymphoid cells, antibodies, and target cells renders results similar to those described for mixtures of lymphoid cells from sensitized donors and antigenic target cells. Thus, when target cells were tissue culture cells in monolayer, lymphocytes were seen to aggregate on their surface. With crude mixtures of blood leukocytes, aggregation was pronounced within a few hours. With purified lymphocytes it was delayed (Holm and Perlmann, 1969b). When both target cells and effector cells were of human origin, the antiserum was first exhaustively absorbed with lymphocytes from the donor providing the effector cells in the cytotoxicity experiment. This treatment neither abolished aggregation nor the cell-mediated cytotoxic effect. With chicken erythrocytes and purified human lymphocytes, erythrophagocytosis by what appears to be lymphocytes has been observed ( < 1%of the Iymphocytcs, with ncver more than one erythrocyte/lymphocyte) (Perlmann and Perlmann, 1969). With Chang livcr cells as target cells, phagocytosis has not been observed. 2. Cytotoxic Mechanisnis

a. Distinction from Complement-Znduced Lysis. Lysis brought about by humoral antibody and complcment is usually completed within less than 1 hour, whereas antibody induced cell-mediated lysis requires several hours even under optimal conditions. However, the validity of

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0 1

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FIG. 2. Cytotoxic effect of lymphoid cells from human blood on suspensions of antibody-treated Chang cells. The target cells were labeled with “Cr and treated for 30 minutes with heat-inactivated rabbit anti-Chang cell serum, diluted as indicated on the abscissa. After washing, 5 X 10’ Chang cells were incubated either with 10% fresh guinea pig sernni in the absence of lymphoid cells (squares), or with 125 X 10‘ lymphoid cells arid 10%heat-inactivated guinea pig serum (open circles). Filled circle: lymphoid cells added together with phytohemagglutinin. Ordinate: isotope release in samples, corrected for spontaneous release from the Chang cells (=26.4%) after 20 hours of inculmtion. (From Holm and Pcrlmann, 196Ha.)

such evidence for a distinction between lytic mechanisms may be debated. More important arguments can be based on quantitative considerations. Thus, extremely high dilutions of antiserum, not lytic with complement in the absence of lymphoid cells, are fully effective in the presence of the latter but in the absence of complement. An example is shown in Fig. 2. In this experiment, the heat-inactivated antiserum was from a rabbit, hyperimmunized with Chang cells (Holm and Perlmann, 1969a,b). Figure 2 illustrates the extreme cytotoxic potency of the serum in the presence of lymphoid cells from human blood. As a rule, strong cell-mediated cytotoxic effects with the lymphoid cells in moderate excess

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can still be seen at serum dilutions from lo-" to lo-'. Similar results have been reported by MacLennan et al. (1969a). The same is found with chicken erythrocytcs pretreated with rabbit antiserum and exposed to highly purified human lymphocytes. In contrast, the complement-dependent titers of these antisera at optimal concentrations of either guinea pig or rabbit complement and measured after 3 or 20 hours of incubation never exceeded 1:500 ( Holm and Perlmann, 1969a,b; Perlmann and Perlmann, 1969; MacLennan et nl., 196%). There is other indirect evidence against common lytic mechanisms. Thus, some antisera known to be highly lytic in the presence of complement alone seem to bc quite inactive when tested with lymphoid cells. Rabbit antisheep hemolysin ( anti-Forssman ) at high dilutions lyses chicken erythrocytes with complement but only at very high concentrations with lymphoid cells (human peripheral blood) (Perlmann et al., 1969a; Perlmann and Perlmann, 1969). Since the antibodies in these sera are about 90%of the 19 S class, it may be suspected that complcmentfixing 19 S antibodies are not involved in the cell-mediated cytotoxic reaction. This emphasizes the similarity of cell-mediated cytotoxicity to the IgG-induced red cell sphering and fragmentation by mononuclear cells, recently clescribed by Lo Buglio et al. (1967). In the anti-Chang cell system, MacLennan et al. (1969a) werc able to separate rat antibodies by fractionation through Sephadex. Thc complement fixing lytic antibodies were predominately of macroglobulin nature but had little effect on Chang cells in the presence of lymphoid cells. Antibodies inducing cell-mediated cytotoxicity were predominately of 7S-type. The results support the notion that different cytotoxic mechanisms are operative in conventional complement dependent lysis and cell-mediated cytotoxicity. More work with immunoglobulins belonging to well-defined subclasses is, however, required to establish this. The data do not exclude a nonconventional participation of the complement system in cell-mediated cytotoxicity ( Section II1,C). b. Mode of Antiserum Action. Heat-inactivated antisera at high dilutions can be incorporated in the incubation mixture. Full effect is also obtained when the target cells are pretreated with the antiserum and are washed before incubation. With guinea pig spleen cells and guinea pig anti-PPD serum, it has also been possible to obtain cytotoxicity by first exposing the lymphoid cells to antiserum and adding them to the target cells after washing (Perlmann and Holm, 1968; Perlmann et al., 1969b); in other systems this did not work (Holm and Perlmann, 1969b). The positive results were obtained with spleen cells from guinea pigs, and it is possible that the cytotoxic cells in these reactions were macro-

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phages, since thcw is good evidence that macrophages (and monocytes ) havc rcceptor sitcs for cytophilic antihoclics ( Beiken and Hcnaccrraf, 1966; Davey and Asherson, 1967). Cytophilic iunti1)otlics with affinities for lymphocytes are not known with certainty. Lo Buglio ct al. (1967) have provided some evidence that “certain lymphocytes” as wc~llas nionocytes specifically bind to the Fc portion of IgG attached to red cells. This binding leads to trapping, deformation, and damage of the red cells. It can be inhibited by IgG in the medium. When lyinphocytes arc the effector cells in the cytotoxic reaction, it is most likely that formation of antigenantibody complexes on the target cell surface provides the sites for induction of cytotoxicity. Available evidence, although limited, suggests that there is no correlation between cytotoxic or hemagglutinating titers of an antiserum and its cytotoxic potency in the cell-mediated test. It is possible, that the antibodies active in this system belong to a special immunoglobulin subclass. This problem has not bcen studied. c. The Zmportance of Contact. In Section III,A,f3, it has bcen shown that contact between lymphoid cells and antigenic target cells is an important feature of the cytotoxic reaction produced by lymphoid cells from sensitized donors. The microscopic appearance of antibody-induced reactions suggests that cell-to-cell contacts are also of importance in this system. When chicken erythrocytes, coated with eithcr PPD or thyroglobulin were mixed in equal proportion and were exposed to lymphoid cells in the presence of antibodies against one of the antigens, only the cells on which the antigen-antibody complexes werc formed became destroyed (Perlmann and Holm, 1969). A similar experiment is shown in Fig. 3. Chicken erythrocytes treated with antibody at high dilution were mixed with untreated chicken erythrocytes and exposed to purified human lymphocytes. When the treated erythrocytes were labeled with 51Cr,isotope release took place rapidly. When thc untreated erythrocytes were labeled, a slowly proceeding lysis first became apparent after several hours of incubation. It could bc assumed that this slow lysis was either due to release of a cytotoxic factor from the lymphocytes, or due to transfcr of antibodies from unlabeled to labeled cells. This latter explanation is strongly supported by thc second diagram in Fig. 3. In this experiment, the target cells were a 1: 1 mixture of chicken and duck erythrocytes. The rabbit anti-chicken erythrocyte antibodies did not cross react with the duck cells and no lysis was seen when the isotopic label was in the duck cells. The results were reversed when rabbit anti-duck antibodies were applied ( Per1m:inn and Perlmann, 1969). Very similar rcsults have been obtained with Chang cells (Holm and Perlmann, 1969b; MacLen-

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FIG. 3. Cytotoxic effect of column purified lymphocytes from human blood on mixtures of antibody treated and untreated erythrocytes from chicken or ducks. Before incubation with lymphocytes, part of the chicken erythrocytes was treated with heat inactivated anti-chicken erythrocyte serum (rabbit), diluted 1:10'. The other part included in the target cell mixture (chicken or duck erythrocytes, respectively) was treated with heat inactivated normal rabbit serum. 2.5 X 10" lymphocytesjlo? erythrocytes were added after washing. Ordinates: percent isotope release. Abrcissus: hours of incubation. Filled circ2es: target cell mixtures consisting of one part antiserum treated "Cr-labeled chicken erythrocytes and, in upper diagram, one part unlabeled chicken erythrocytes, or, in lower diagram, unlabeled duck erythrocytes. Open circles: the same but with the "Cr-label in the erythrocytes which had not been treated with antiserum. Filled and open triangles: aliquots of the same target cell mixtures incubated without lymphocytes. ( From Perlmann and Perlmann, 1969. )

nan and Harding, 1969). Experiments of this type provide good evidence that antibody-induced cytotoxicity requires contact between effector cells and targets. Local participation of cytotoxic factors in the area of contact is, however, not excluded. d. Inhibition of Cytotoxicity. The lymphoid cells have to be alive in order to be cytotoxic. Inhibition of cytotoxicity by pretreatment of the effector cells with antimycin A indicates that the antibody-induced reaction also is dependent on energy-requiring effector processes ( Perlmann et al., 1969b). The antibody-induced cytotoxicity of normal lymphoid cells is as efficiently inhibited by xenogeneic antilymphocytic serum ( ALS) as that of lymphoid cells from sensitized donors (Section III,A,G,b). An example

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is shown in Fig. 4 in which lymphoid cells from human peripheral blood were pretreated with heat-inactivated rabbit ALS and the target cells ( Chang cells) with hcat-inactivated rabbit anti-Chang cell serum ( Holm and Perlmann, 1 9 6 9 ~ ) The . dilution of ALS needed for inhibition is proportional to the dilution of anti-Chang cell serum used. It is of interest that even those ALS conccntrations ( >1:25) that stimulated a major proportion of the lymphocytes in the effector populations to morphological transformation and D N A synthesis (James, 1967), completely inhibited the antibody-induced cytotoxicity (Holm and Perlmann, 1969~). This suggests but does not prove that ALS inhibits the reaction by affecting lymphocyte-target cell contact rather than by a metabolic block. However, it is not known whether the cells that are stimulated by ALS also are involved in the cytotoxic reaction. e. Effector Cekls. CeIl-mediated cytotoxicity, induced by antibody, is brought about by thoracic duct cells and by highly purified lymphocyte

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FIG.4. Inhibition of lymphocyte-mediated cytotoxicity by antilymphocyte serum ( ALS). Chromium-51-labeled Chang cells were treated with rabbit anti-Chang cell serum diluted 1:lo' ( circles) or 1:10' (triangles ). After washing the target cells were incubated with column-purified human blood lymphocytes, pretreated with normal heat-inactivated rabbit serum or with heat-inactivated rabbit ALS, diluted as indicated on the abscissa. Ordinate: isotope release corrected for spontaneous release from Chang cells (27.4%)after 20 hours of incubation. For further explanations, see legend to Fig. 2. (From Holm and Perlmann, 1969c.)

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preparations (e.g., Fig. 3 ) . Presence of monocytes, macrophages, and polymorphonuclear leukocytes will often speed up the cytotoxic reaction (Holm and Perlmann, 196913; Perlmann and Perlmann, 1969). These cell types are phagocytic for antibody-coated cells. They may become cytotoxic by themselves, although different targets will then vary in susceptibility. In addition to exhibiting direct cytotoxic effects, small numbers of nonlymphocytic cells may participate in in vitro cytotoxicity by interaction with lymphocytes as discussed in Scction III,A,5,c. 3. Conclusions

The antibody-induced cytotoxicity of lymphoid cells from nornial donors resembles that of lymphoid cells from sensitized donors. Certain antibodies are capable of inducing this reaction at very high dilutions and with highly purified lymphocytes as effector cells. Antibodies possessing this capacity may occur in xeno-, allo-, and autoimmune situations. Cytotoxicity does not require differences in histocompatibility between lymphoid cells and target cells. The reaction is most easily induced by antibody bound to antigen on the surface of the target cells which are destroyed. Since antigen-antibody complexes are known to stimulate lymphocytes to blast transformation and cell division ( BlochShtacher et al., 1968; Moller, 1969) and since malignant lymphocytes or thymus cells which are difficult to stimulate (for references, see Ling, 1968; Oppenheim, 1968) arc not cytotoxic, it can be inferred that blast transformation and cytotoxicity of lymphocytes are induced by the same basic reaction. This does not imply that blast transformation or cell division are necessary for cytotoxicity. The lytic reaction requires direct contact bctween effector cells and the antigenic target cells. Available data speak against the rclease of cytotoxic factors into the medium as being the cause of target cell destruction. The lytic process exerted by the effector cells is energy-dependent and may involve surface reactions such as increased motility. When the effector cells are purified lymphocytes, cytotoxicity is not due to phagocytosis but may be based on similar surface activities. It is most likely similar to thc recently described action of monocytes and “lymphocytic monocytes” on human red blood cells coated with IgG in the absence of complement (Lo Buglio et al., 1967). Although this lytic reaction is different from the conventional antibody-complement-induced lysis, the participation of complement cannot be ruled out ( Section II1,C). When present in the effector populations, white cells other than lymphocytes will contribute to target cell destruction. Lysis may then follow a course different from that outlined above.

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C. CYTOTOXIC EFFECTSOF LYMPHOIDCELLSTRIGGERED BY TARGET CELL-BOUND COMPLEMENT

1 . General Activation of the complcment system is necessary for cytolysis produced by humoral antibodies. The coinplement-fixing antibody initiates n relatively well-defined sequence of reactions which takc place on the surface of the target cell and which ultimatcly lcad to lysis. Once complemcnt activation has been triggered, antibody is no longer needed and lysis will be produced without its participation. Decisive for the lytic step is the activation of the terminal components C’8 and C’9 of the complement system (for references, see Miiller-Eberhard, 1968). The complement system comprises a series of interacting proteins. It is known that this system, in addition to its cytolytic role, has other biological functions. I t is well established that partial activation of complement, comprising only its first four components, leads to aggregation phenomena known as immune adherence (R. A. Nelson, 1953; D. S. Nelson, 1963). Instrumental in this reaction is cell surface-bound C’3 (Nishioka and Linscott. 1963). Cells with affinity (“receptor sites”) for activated C’3 will aggregate to those cells on which C’3 activation has taken place. Both macrophages and a certain fraction of lymphocytes have been shown to possess such receptor sites (Lay and Nussenzweig, 1968). Immune adherence of monocytes to C3-carrying erythrocytes is an efficient mechanism for erythrophagocytosis, promoted by xenogeneic 19s antibodies which have no opsoiiizing propertics in the absence of cornplemcnt ( Huber et a]., 1968). Other biologically important functions of the complcnient system are generation of anaphylatoxins, leucotactic factors, and histamine release from mast cells (for references, see MullerEberhard, 1968). The cell-mediated cytotoxic reactions described in Sections II1,A and B are always performed in medium containing heat-inactivated serum or, in some cases, no serum at all. Although there are a few exceptions (Section 111,A,3,11),addition of complement to a mixture of antigenic targct cells and lymphoid cclls from sensitized donors does not promote cytotoxicity. This has been takrn to indicate that ccll-mediated cytotoxicity has a different mechanism from that produced by humoral antibody complement. This argument can probably bc acccpted as valid, although the reservation must be madc th‘it small amounts of complement carried or produced by cells, or present in the heat-inactivated serum, inay contributr to cytotovicity wh(w smill amounts of complcment-fixing anti-

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bodies are also present or produced. Howevcr, in view of the complexib of the complement system, the important question is whether C’ participates in cell-mediated lysis by pathways different from those operativc in the conventional lytic reaction. The results described below suggest that this may be the case.

2. Cell-Mediated Lysis of Chicken Red Blood Cells Carrying Partially Activated Coinplenient a. Target Cells Carrying C’3. In these experiments, Wr-labeled chicken red blood cells were used as target cells (Perlmann et al., 1969a) The cells were treated with small amounts of heat-inactivated rabbit antisheep hemolysin, reacting with Forssman antigen common for sheep and chicken eiythrocytes. At this concentration the antibodies, consisting primarily of 19 S immunoglobulin, were not capable to produce cellmediated lysis by themselves ( Section III,B,2,a). The antibody-carrying cells were then treated sequentially with isolated and purified components of human complement and were finally exposed to an excess of lymphoid cells from hunian peripheral blood ( 2 5 lymphocytes per target cell). Target cells treated with antibody alone or with the first three complement components (C’l, C’2, C’4) ( Muller-Eberhard et al., 1966; Polley and Muller-Eberhard, 1968; Cooper and Muller-Eberhard, 1968) were not lysed within 24 hours after addition of the lymphoid cells. The isotope release from such erythrocytes was low and indistinguishable from that seen in the controls to which no effector cells were added. However, whcm C’3 was also bound to the target cells, the lymphoid cells promoted an extensive lysis. This cytotoxic reaction required 10-20 hours to reach completion and was only seen when the effector cells were viable. It is based on energy-dependent effector processes, since pretreatment of the lymphoid cells with antimycin A, which blocks respiration, also inhibited the cytotoxic reaction. About 75%of the effcctor cells used in these experiments were lymphocytes. Fractionation into two fractions-one containing 80% monocytes and approximately 20% lymphocytes (Bennett and Cohn, 1966) and, a second, consisting of glass-bead purified lymphocytes ( Rabinowitz. 1964)-indicated that the monocyte-enriched fraction was highly cytotoxic. In contrast, the purified lymphocytes wcre not cytotoxic ( MiillcrEbcrhard et al., 1969). This suggested that the effector cells in these cxperiments were monocytes. However, since about 20% of the effector cells were lymphocytes, R lynq~hocyte-monocyte interaction in the cytotoxic reaction cannot be cntirely excluded. Cytotoxicity seemed to he due to a nonphagocytic process ( Miiller-Eberhard et al., 1969).

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b. Turget Cells Carrying C’7. Isotope-labeled chicken erythrocytes treated with antibody and the first four complement components, were brought in the C’7 stage by addition of isolated C 5 (Nilsson arid MullerEberhard, 1965) and functionally purified C’6 and C’7 (Nilsson and Miiller-Eberhard, 1967). In these experiments the fetal calf serum ( 5 % ) in the medium was fractionated chromatographically in order to remove small amounts of C’8 and C’9 activities, normally present even after heat inactivation. The C’7-carrying erythrocytes were completely lysed within a few hours when exposed to the lymphoid cells (Perlmann et al., 1969a; Miiller-Eberhard et al., 1969). No lysis occurred in the controls even when the C’7-carrying cells were artifically aggregated by means of phytohemagglutinin ( P H A ) . Lysis was not due to small amounts of C’8 bound to human erythrocytes in the effector population. It required viable effector cells and was also inhibitcd by antimycin A. In contrast, inhibition of protein synthesis in the effcctor cells by means of puromycin had no blocking effect. In this system both glass-bead-purified

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Frc. 5. Damage of complement-target cell coinplexes by human blood lymphocytes, piirified on glass-bead columns. Target cells consisted of “Cr-labeled chicken erythrocytes, first treated with diluted, rabbit, 19 S antibody to boiled sheep erytlirocyte stroiiiata and, subsequently, with purified Iirtinan complement components. Inc~ilmtion inistiires contained 8 X 10’‘ lymphocytes and 1 X 105 target cells. Ordiii;rte: percent isotope release. Abscissa: time of incubation. EAC‘1-4, EAC’1-3, EAC’1-7: target cell~omplement complexes Imild up by sequential addition of components up to C’4, C’3, ant1 C’7, respectively. Black symbols: incubation mixture containing Iymphocytcs. Open symbols: inculxition mixtiires containing 1 X 10’ unlabeled and untreated chicken erythrocytes instead of lyinphocytes. ( From MullerEberhard et al., 1969. )

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lymphocytes and the monocyte-enriched fractions were cytotoxic (Fig. 5 ) . The velocity of this cytotoxic reaction resembles that produced by addition of C’8 to red cells in the C’7 stage. It is, however, considerably slower than lysis produced by adding both C’8 and C’9. The results can be interpreted to mean that some cells in the effector population carry (or produce) C’8, instrumental as the lytic factor in this cell-mediated cytotoxic reaction. Some preliminary experiments suggest that a thermolabile substance, functionally similar to C’8, can be extracted from human blood lymphocytes and lyse red cells in the C’7 stage (Miiller-Eberhard et al., 1969). 3. Conclusions

These results are limited to a single type of target cells, and presently it is not known if other cell types would behave in a similar way. However, the data indicate that complement in some situations is of importance for induction of cell-mediated cytotoxicity. The two reactions described above probably represent two entirely indcpendent modes of complement action. The cell-mediated destruction of C’3-carrying target cells is related to phagocytosis, following a step of immune adherence. This partial activation of the complement system may be an important mechanism for cell-mediated cytotoxicity in uiuo. The lymphocyte- and monocyte-mediated destruction of target cells carrying C’7 is believed to reflect a different phenomenon. If it is correct that C’8 is the lytic factor in this model, cytotoxicity will be triggered by target cell-bound C’7 (for references, see Miiller-Eberhard, 1968). It may be asked whether C’8, provided by the effector cells, could also act as a cytolytic factor in the cytotoxicity models described in other sections of this chapter. This would either require a buildup of C’8-susceptible sites on the taget cells by an unknown pathway or a direct activation of effector cell-bound C’8. In both cases, the conventional sequence of reactions initiated by antibody would have to bc by-passed. In preliminary experiments, it has been seen that both the antibody and the PHAinduced cytotoxicity of human lymphoid cells (Section III,B,D) may be inhibited by rabbit antiserum to human C’8 and C’5 but not by antiserum to C’lq, C’2, C’4, and C’3 (data to be published). If confirmed by further experiments, activation of C’8 could actually be assumed to be of some general importance for cell-mediated cytotoxicity.

D. NONSPECIFICCYTOTOXICITY OF LYMPHOID CELLSACTIVATED BY PHYTOHEMAGGLUTININ OR OTHERSTIMULANTS It was found some years ago (Holm et al., 1964) that nonspecific aggrcJgation by PHA of human or aninial lymphoid cells to target cells in

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tissue culture led to destruction of the latter. Later studies showed that cytotoxic effects of lymphoid cells from normal donors can be brought about by a variety of nonagglutinating agents, known to stimulate lymphocytes to blast transformation and DNA synthesis. Therefore, “activation” of some cells in the effector population can be considered as the most important basis for cytotoxicity in this model. Lymphoid cells activated by these stimulating agents are destructive to many kinds of target cells including cells originating from the donor.

1 . Diferent Stiniulants a. Reactions Induced by PHA. When lymphoid cells are added to tissue culture cells in monolayer in the presence of PHA, they become aggregated and cannot be removed by washing. The microscopic picture is indistinguishable from that seen after addition of lymphoid cells from sensitized donors to antigenic target cells (Holm et al., 1964). Aggregation is later followed by destruction of the monolayers. Similar reactions have been observed with tissue culture cells from rats and mice (Holm and Perlmann, 1965; Moller and Moller, 1965; Rosenau, 1968; Malchow et al., 1969), tumor cells from mice (Moller, 1965a,b), human fibroblasts (Moller et al., 1966; Lundgren and Moller, 1969) and human tumor cells (Chu et al., 1967), established human cell lines in suspension ( Holm and Perlmann, 1967a,b; Mac Lennan and Loewi, 1968a,b), and with chicken red blood cells (Perlmann et aZ., 1968). Human red blood cells, target lymphocytes, and some human lymphoma cells seem to be relatively resistant to PHA-induced cytotoxicity (Holm, 1967a; Perlmann et al., 1968). The cytotoxic activity of lymphoid cells can also be induced by pretreatment with PHA before adding them to the target cells. The PHA which is added in these experiments is a mixture, consisting of proteins with both hemagglutinating, leukoagglutinating and mitogenic properties (for references, see Naspitz and Richter, 1968). Absorption of the hemagglutinating substances does not abolish mitogenicity (Nordman et al., 1964; Robbins, 1964) and, likewise, does not remove the principle inducing cytotoxicity. The latter is destroyed by heating to 100°C. for 5 minutes (Perlmann et al., 1968). Phytohemagglutinin induces blast transformation and DNA synthesis in a large fraction of the lymphocytes from the peripheral blood of humans and animals. Under optimal conditions, DNA synthesis in human lymphocytes reaches a first peak within 2 to 3 days of culture. The response to PHA is dose dependent and decreases sharply at too high concentrations of the drug (for references, see Ling, 1968). When cytotoxicity of human or chicken lymphoid cells was studied as a function of PHA concentration, there was a good correlation between the dose de-

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pendency of the cytotoxic reaction and that of DNA synthesis (Holm and Perlmann, 1967b; Perlmann et al., 1968). However, there was no correlation in time, since cytotoxicity was close to its optimum at a time when DNA synthesis had hardly started. This indicates that cytotoxicity and DNA synthcsis are independent expressions of lymphocyte stimulation, Lymphoid cells from some animals such as mice do not respond as well to PHA as do those of humans, guinea pigs, and other animals (Ling, 1968). When special precautions are taken, blast transformation and DNA synthesis may be achieved but the number of responding cells is lower than that found in humans. This is probably one major reason for some of the conflicting results which have been reported for PHAinduced cytotoxicity of lymphoid cells in mice (see below, Section III,D,2). b. Other “Nonspecific” Mitogens. Stimulation of lymphocytes by PHA is assumed to be mediated by immunologically nonspecific cell receptors (for references, see Ling, 1968).Another lymphocyte-stimulating agent, believed to act via similar pathways as PHA, is present in culture filtrate from staphylococci (Ling et al., 19%). This agent induces cytotoxicity of lymphoid cells from human blood to tissue culture cells in monolayer and in suspension ( Holm and Perlmann, 196%).The staphylococcal filtrate induces blast transformation and DNA synthesis in a large fraction of the human circulating lymphocytes but is nonagglutinating; its action is slightly slower than that of PHA. Pretreatment of human lymphocytes with this agent for several hours or longer (2 days) produced strongly cytotoxic cells ( Holm and Perlmann, 1967b). Cytotoxicity was well correlated to the degree of blast transformation induced by the staphylococcal filtrate. It was also noted that the staphylococcal filtrate and PHA had additive effects on cytotoxicity. c. Antigens Unrelated to Target Cell Antigens. Lymphocyte stimulation in vitro to blast transformation and DNA synthesis is induced by antigens to which the donor of the lymphocytes is sensitized (for refer-. ences, see Ling, 1968). By definition, this stimulation requires presence of antibody-like recognition factors on some cells in the lymphocyte population. Antigen-induced stimulation of human peripheral blood lymphocytes proceeds at a slower rate than that induced by PHA and compriscs a smaller fraction of the cells (Ling and Holt, 1967). After pretreatment with PPD, human lymphoid cells from blood of tuberculin-positive donors became cytotoxic to allogeneic tissue culture cells. Cytotoxicity was correlated to the degree of stimulation achieved by the antigen in parallel incubations (Holm and Perlmann, 1967b).

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Since there was no reason to believe that an immunological relationship existed between PPD and the target cells, it was concluded that cytotoxicity was iiii1iiui7ologically nonspecific. Similar results by Ruddle and Waksmxn (1967, 1968a,b,c) confirm and extend these findings. These authors used lymph node cells from rats sensitized to various antigens such as PPD, bovine ./-globulin, or ovalbumin. Treatment of the lymph node cells with antigen induced a cytotoxic activity when they were added to monolayers of rat fibroblasts. Destruction of the latter, recorded by the plaque technique or by cell counting, was first seen after 48 hours and was maximal at 72 hours. The destructive activity was specific for the sensitizing antigen and both allogeneic and syngeneic monolayers were destroyed. Short preincubation of the lymph node cells with antigen was sufficient to produce cytotoxicity. Similar results have also been reported by Lundgren et al. (1968b) who exposed to Salmonella vaccine, purified human lymphocytes from donors sensitized to Salmonella. Lundgren and Moller (1969) also obtained cytotoxicity with human lymphocytes and Streptolysin 0, an antigen against which most humans are sensitized (Ling, 1968). These authors measured plaque formation on autologous or allogeneic fibroblast monolayers. d. Mixed Leukocyte Culture. Mixed culture of lymphoid cells from humans (Bain et al., 1964) or experimental animals (for references, see Dutton, 1967; Wilson and Billingham, 1968) will lead to blast transformation and DNA synthesis. The degree of this stimulation correlates well with differences in the major histocompatibility antigens of the cell donors (Dutton, 1965; Wilson, 1967b; Wilson et al., 1967; Albertini and Bach, 1968). Preincubation of allogeneic lymphoid cells from human peripheral blood in mixed culture for up to 1 week and subsequent transfer of the mixtures to unrelated human tissue culture cells led to lysis of the latter. Lysis was correlated to the degree of blast transformation ( Holm and Perlmann, 1967b). 2. Allogeneic Inhibition

From experiments with target cells and lymphoid cells from normal mice, some authors concluded that the PHA-induced cytotoxicity was an in vitro manifestation of allogeneic inhibition, assumed to be a nonimmunological surveillance mechanism in uiuo (K. E. Hellstrom and I. Hellstrom, 1967). As already stated (Section III,A,2,b), allogeneic inhibition was originally defined as the passive destruction of cells exposed to foreign histocompatibility antigens. Allogeneic inhibition probably

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represents a special case of the phenomenon known as contact inhibition of growth (Eagle, 1965; Eagle and Levine, 1967). This means that it is probably not limited to differences in histocompatibility antigens, although such differences may give rise to more pronounced effects than other smaller differences in cell surface structure or cell surface physiology. In brief, the following evidence has mostly been given in support of this hypothesis; 1. Mouse tumor cells or fibroblasts in tissue culture were destroyed when exposed to lymphoid cells or lymphoma cells from nonimmune donors of allogeneic or F, hybrid origin. Exposure to syngeneic cells seemed to have no effect (Moller, 1965a; Hellstrom et al., 1965, 1967). In order to obtain destruction, the cells were treated with PHA or rabbit antimouse serum. This was done to bring the cell types into close contact. Since F, hybrid lymphoid cells or some of the tumor cells were expected not to react “immunologically” against parental strain antigens, allogeneic inhibition was implicated. 2. Similar cell destructive effects were obtained by exposing mice tumor cells in tissue culture to X-irradiated lymphoid cells from F, hybrids or to extracts of allogeneic or F, hybrid origin (Hellstrom et aZ., 1964, 1967; Moller and Moller, 1965). 3. Cytotoxicity was reduced when the experiments were made in the presence of alloantibodies to the H-2 antigens on the lymphoid cells ( Moller, 1967). Experiments with other material have since shown that allogeneic inhibition and PHA-induced cytotoxicity are separate phenomena. Thus, in the presence of PHA, chicken red blood cells were destroyed by autologous and allogeneic lymphoid cells. No quantitative differenccs in target cell lysis were seen over a wide range of experimental conditions Perlmann et al., 1968). Rapid destruction of human fibroblast monolayers was also obtained with PHA-treated and purified human blood lymphocytes from both autologous and allogeneic donors (Lundgren and Moller, 1969). However, in some cases, in which the effector cells were relatively crude mixtures of human blood cells, allogeneic cells seemed to be more efficient than autologous cells (Mdler et al., 1966; Chu et al., 1967). Malchow et al. (1969) have recently made similar observations with cells from rats and mice. The destructive action of antigen-activated lymph node cells from rats affected both syngeneic and allogeneic monolayers ( Ruddle and Waksman, 1967,1968a). There may be a number of explanations for the differences in results obtained with different materials. The most obvious reasons for the ap-

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pearance of allogeneic inhibition effects in experiments with mice were> ( 1 ) rcduced viability of both target cells and lymphoid cells during incubation, ( 2 ) marginal response of the latter to PHA ( Albller, 1965a,b), and ( 3 ) choice of syngeiieic lymphoid cells as the base line for the determination of the allogeneic or F, effects (Hcllstrom et al., 1967). Target cell destruction due to allogencic inhibition was weak and most likely due to reactions different from those obtained with lymphoid cells which are easy to stimulate with PIIA. Recent quantitative experiments by Malchow et al. (1969) may serve to illustrate this point. These authors found that addition of syngeneic lymphoid cells to rat or mice targets gave clear-cut cytotoxic effects after PHA treatment. Effector cells from F, hybrids had slightly stronger effects in mice but not in rats. The cytotoxicity of allogeiieic lymphoid cells was slightly stronger than that of syngeneic cells in both cases. Thcse results suggest that differences in cell surface architecture may affect the interaction between lymphoid cells and targets in a quantitative rather than in a qualitative manner.

3. Effector Cells a. Lymphocytic Cells. In the model involving PHA, cytotoxicity is obtained with those populations of lymphoid cells that are easily stimulated to blast transformation and DNA synthesis. Cells which are d a c u l t to stimulate (for references, sec Ling, 1968) were only weakly cytotoxic. This was seen with both human and rat thymus cells (Holm and Perlmann, 1965; Holm, 1967d; Perlmann et al., 1968). Blood lymphocytes from patients with chronic lymphatic leukemia were not cytotoxic under conditions where normal blood lymphocytes had strong effects. Lymphoid cells from blood of patients with Hodgkin’s disease had a reduced cytotoxicity, paralleling a reduced reactivity in skin tests with PPD (Holm et al., 1967). Burkitt lymphoma cells, which have the morphological appearance of transformed lymphoid cells, were not cytotoxic (Perlmann et at., 1968). It is not definitely established which of the functionally heterogeneous lymphocytes from the circulation or the lymphoid organs respond by transformation and proliferation when exposed to PHA or antigen. There is some evidence that the lymphocytes responding to PHA are thymus dependent (Meuwissen et al., 1968; Greaves et d., 1968). Recently, Tursi et a!. (1969) collected lymphoid cells from mice treated with ALS in vivo. These lymphocytes were not cytotoxic when added to target cells in tissue culture in the presence of PHA. This parallels the loss of graft-versus-host reactivity of ALS-treated mice as seen in similar experiments (Boak et al., 1967; Naysmith and James 1968).

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Thus, available evidence suggests that the PHA- and thymus-dependent lymphocytes also trigger in vitro cytotoxicity. This does not imply that they also are the effectors of the lytic reaction. Stimulation by antigen of lymphocytes from sensitized donors often reflects a state of delayed hypersensitivity and is thus believed to be typical for those cells that participate in its induction (Mills, 1966; Oppenheim et al., 1967; Oppenheim, 1968). Antigen-induced cytotoxicity to fibroblasts of lymph node cells from rats sensitized to various proteins has been shown to be correlated to delayed hypersensitivity rather than to Arthus reactivity (Ruddle and Waksman, 1968b). Moreover, in experiments with hapten-conjugated proteins, skin reactivity as well as cytotoxicity in vitro could only be elicited with the hapten bound to the carrier protein used for sensitization. Although these results suggest a relationship between delayed hypersensitivity in vivo and antigen-induced cytotoxicity in vitro, it is not clear whether this is a general rule. Antigeninduced transformation has in some cases been seen to be correlated to antibody formation rather than to delayed hypersensitivity ( Oppenheim, 1968). It is not known whether or not antibody-forming cells or their precursors also are cytotoxic in vitro when exposed to antigen or other stimulants. b. Monocytes, Macrophages, Polymorphonuclear Leukocytes. In the controls of the cytotoxicity experiments, red blood cells or tissue culture cells were sometimes included as “effector cells.” The results have always been negative (MiilIer and Moller, 1965; Holm, 1967a; Perlmann et at., 1968). In PHA-induced cytotoxicity, thoracic duct cells and purified lymphocytes from the peripheral blood were cytotoxic for tissue culture cells ( Holm et al., 1964; Lundgren et al., 1968a; Holm and Perlmann, 196913). Admixture of polymorphonuclear leukocytes, monocytes, or macrophages will affect the course of the interactions in different ways. Human Chang liver cells in suspension seemed to be relatively resistant to polymorphonuclear Ieukocytes (Holm and Perlmann, 1967a, 196%). When human skin fibroblasts were exposed to human peripheral blood lymphocytes contaminated with a few percent of polymorphonuclear leukocytes and monocytes, monolayer destruction took place rapidly regardless of whether or not PHA was present (Lundgren et al., 1968b). Release of toxic substances (hydrolytic enzymes) from adversely affected polymorphonuclear leukocytes was assumed to contribute to monolayer destruction (see Section 11,CJ). Chicken red blood cells, exposed to effector cells consisting of 80-9m polymorphonuclear leukocytes and 1020% mononuclear cells (monocytes and lymphocytes), were rapidly lysed by these cells in the presence but not in the absence of PHA. Kinetically,

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this lysis was quitc different from that obtained with highly purified lymphocytes and crythrophagocytosis was pronounced ( Perlmann and Perlmann, 1969). How PHA produced these effects is unknown. Although PHA does not induce DNA synthesis or mitosis in polymorphonuclear leukocytes or monocytes, it has been shown to activate metabolic processes in their nuclci (Killander and Rigler, 1965; Darzynkievicz et d., 1969). Whcn agents other than PHA are used for activation, the importance of contaminating cells has not as vet been established. Addition of antigen to the cells from a sensitized donor can be expected to release various cytotoxic activities of both polymorphonuclear leukocytes and macrophages. The question of interaction between lymphocytes and other white cells must not be overlooked. Recent evidence suggests that stimulation of lymphocytes to transformation and mitosis in many situations may be enhanced by other leukocytes (Gordon, 1968: Oppenheim et al., 1968; Hersh and Harris, 1968). Interaction of allogeneic lymphocytes in mixed culture in citro in heat-inactivated serum has bcen shown to lead to the production of leukotactic ( Ramseier, 1967) and macrophage inactivating factors (David, 1966; Bloom and Bennett, 1966; Svejcar et al., 1968). Such interactions may also be of significance for the cytotoxic reactions discussed in this section.

4 . Kinetics The rate of target ccll destruction by activated lymphoid cells is roughly similar to that described for the previous models. It varies with the mode of assay and the naturc of the target cells. With tissue culture targets in monolayers, visible effects may appear within a day, but optimal effects are mostly not scen until after several days (Lundgren and Moller, 1969). An excess of lymphoid cells over target cells is required. Quantitative studies have so far only been performed with PHA-stimulated lymphoid cells froin human peripheral blood and "Cr-labeled Chang liver cells in suspension. Under optimal conditions, significant isotope release could be observed after a few hours and lysis was usually completed within 24 hours. Howevcr, lymphoid cells from different donors may show considerable individual variations ( Holm and Perlmann, 1967a). Lysis at a fixed initial concentration of Chang cells increased signioidally with the log of lymphoid cell concentration-50% lysis at 24 hours required lymphocyte/target cell ratios from 5 : l to 25:l.The results seemed to suggest that a considerable proportion of the effector cells was active and capable of killing target cells within the experimrntal

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pcriods. However, these data do not exclude that target cell damage is brought about by release of toxic agents or lymphocyte-activating factors from a small number of originally active cells.

5. Cytotoxic Mechanisms a. Contact. When contact between lymphoid cells and target cells was prevented by Millipore membranes, target cell damage did not take place (Holm et al., 1964). On the other hand, mixed aggregation of lymphoid cells and target cells is not by itself detrimental to the latter. No cytotoxic effects were seen when aggregation was brought about by polylysine (Rosenau, 1963; Holm and Perlmann, 1965) which docs not stimulate lymphocytes. Light- and electron-microscopic observations ( Biberfeld P L al., 1968'I and time-lapse cinematography ( A x et al., 1968) of the PHA-induced interaction of lymphoid cells with tissuc culture cells indicated that the aggregated lymphocytes were highly mobile. Adhesion of lymphocytes to target cells by their uropod (McFarland and Heilman, 1965; MacFarland et al., 1966) was relatively rare. Emperipolesis (Pulvertaft, 1960) was not seen. Detachment of target cells from monolayer and/or lysis was observed before lymphocyte transforniation took place. z?. Physiological State of Efector Cells and lnhibitor Studies. Lymphoid cells from human (Holm, 1967a) or chicken blood (Perlmann et al., 1968) do not die in the course of the PHA-induced cytotoxic reaction. As in the previous models, lymphoid cells must be viable in order to produce cytotoxic effects. Cells killed by heat or extracts of normal lymphoid cclls were not cytotoxic (Holm et al., 1964; Holm ant1 Perlmann, 1965; Holm, 1967b; Lundgren and Moller, 1969) . X-irradiation and treatment with hydrocortisone (Moller et al., 1966) or treatment with inhibitors suppressing nucleic acid or protein synthesis did not affect PHA-induced cytotoxicity ( Holm, 196717; Lundgren and Miiller, 1969). In contrast, treatment of the lymphoid cells with antimetabolites that block glycolysis or respiration did inhibit cytotoxicity ( Holm, 19671~; Holm and Perlmann, 1968). It can be concluded that the PHA-induced cytotoxicity is a function of encrgy-requiring processes. Since nucleic acid synthesis or protein synthesis is not necessary, cytotoxicity is obviously not bound to blast transformation or mitosis. There is actually no direct proof that the transforming cells also arc' the executors of the cytotoxic reaction. Stimulation of lymphocytcs by PHA is known to lead to early changes in phoqpholipid mctabolism (Fisher and Mueller, 196s; Kay, 1968) and to early nuclear changes (Killander and Riglcr, 1965; Pogo et al., 1966; Darzynkiewicz et al., 1969; Riiigeitz et al., 1969).

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Particularly the first-mentioned changes are indicative of alterations in cell surface activity and should be of importance for cytotoxicity. It could be expected that activated lymphoid celIs, because of enhanced metabolic requirements, become cytotoxic for target cells by competing with them for nutrients in the medium. Howel-er, when purified human blood lymphocytes were treated with the antibody-like plant protein Concanavalin A (Sumner and O’Kane, 1948; Goldstein and Iyer, 1966) they were strongly stimulated to blast transformation and DNA synthesis but were not cytotoxic for chicken rcd blood cells. Moreover, Concanavalin A potentiated PHA-induced stimulation but completely blocked cytotoxicity. It did not inhibit the PHA induced mixed aggregation of lymphocytes and target cells. The inhibition was completely reversible when Concanavalin A was removed from the lymphocytes by treatment with n-methyl-D-niannoside after one hour but not after 20 lirs of interaction. This is furthcr evidence for the notion that blast transformation and cytotoxicity are separate phenomcnn ( Perlmann ct nl., 1970). Similar results were obtained when human lymphocytes were treated with hc,it-inactivatcd antiserum (rabbit) to human immunoglobulin ( Fig. 6). Under the evperimeiital conditions applied, the antibodies completely suppressed PHA-induced cytotoxicity but did not block PHAinduced transformation and DNA synthesis. Antiserum to human serum albumin had no inhibitory effects. On the contrary, this antiserum induced a slight cytotoxicity of the lymphocytes even in the absence of PHA. The results point to the interesting possibility that lymphocyteassociated y-globulin also may havc rcccptor functions: in PHA-induced cytotoxicity. However, other cxplanations cannot be excluded ( see following paragraph). Rabbit ALS stimulate human lymphocytes to blast transformation and DNA synthesis to almost the same extent as PHA (for references, see James, 1967). Human lymphocytes tieated with heat-inactivated ALS absorbed with Chang cells were not cytotoxic for jlCr-labeled Chang cells ( Holm and Perlmmn, 1969a,c), even \vhen thc antiserum was applied at stimuhting concentrations. Antilymphocyte s c ~ aalso inhibited PHAinduced cytotoxicity. I t is not known whcJther this inhibition of cytotoxicity in vitro is related to the immunosuppressive action of ALS in vivo. Light-microscopic and electron-microscopic observations suggested that the impairment of cytotoxicity was at least partly due to changes in pattern of lymphocyte target cell aggregation and inhibition of peripolesis ( Biberfeld et nl., 1969). Similar inhibitory effects:, recently noted by Lundgrm ( 1,undgren c’t a/., 1968a; Lundgren, 1969) , werc also interpreted in this way.

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i

I6

FIG.6. Inhibition of phytoheniagglutinin ( PHA )-induced cytotoxicity of human blood lymphocytes by rabbit antiserum to human immunoglobulin G (ICG). Ordinate: percent isotope release after 15 hours of incubation of “Cr-labeled chicken erythrocytes with lymphocytes. Abscissa: lymphocyte/erythrocyte ratio. Each tube contained 1 X lo5 erythrocytes. Squares: lymphocytes pretreated with 25% heatinactivated normal rabbit serum for 30 minutes and then washed. Circles: lymphocytes pretreated with 25%rabbit antihuman-ICG. Solid lines: incubation in presence of PHA. Dashed line: incubation without PHA, or without lymphocytes, respectively. (From Perlmann and Holm, 1969.)

c. Cytotoxic Factors. The evidence presented in the preceding paragraphs suggests that cytotoxicity of activated lymphoid cells requires contact between effector cells and targets. In the initial work by Holm et al. ( 1964), no cytotoxic factors could be recovered from the incubation medium of PHA-treated lymphoid cells. However, Ruddle and Waksman ( 1968c) recently presented preliminary evidence for the occurrence of factors inhibiting target cell growth after incubation of rat fibroblasts with PPD-treated lymph node cells from rats sensitized to tubercle bacilli. Similar findings have been reported by Granger and Kolb (1968) and Granger et al. (1969) in various immune and nonimmune cytotoxicity models in mice, guinea pigs, and man. Granger and Williams (1968), Williams and Granger (1968,1969), and Kolb and Granger (1969) found that culture media, obtained from lymphocytes stimulated by PHA, antigen, or mixed culture inhibited L-cell fibroblasts and other target cells. These factors were called nonspecific “cytotoxins” and were assumcd to be effectors of cell-mediated cytotoxicity in general. Their generation would depend on preceding lymphocyte activation. This adds to the already impressive list of factors, such as interferon (Grecn et aZ., 1969),

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lcukotactic factor ( Raniseier, 1967; Ward et d., lS69), specific and nonspecific blastogenic factors, permc&ility factors, and low- and high molecular transfer factors, nll assumrd to he given off from activated lymphoid cells [for other references, see Lawrence ( 1969) 1. The relation of the “cytotoxins” to these factors is prcscntly the matter of some confusion. These findings do not necessarily contradict the notion that the lytic reaction requires close contact between eff cctor cclls and target cells, since cytotoxic factors may primarily exhibit local effects. Much of the evidence referrcd to in this and previous sections points to the fact that remote bystander cells in a target cell culture are not destroyed during incubation under proper conditions. When target cell destruction requires long incubations, thc situation probably becomes more complex. However, even if soluble cytotoxic mediators were involved, the factors referred to above iiiay be of minor significance. In these reports, cytotoxicity was primarily measured as monolayer destruction or a s growth inhibition, both known to be susceptible to changes of thc tissue? culture medium. In PHA-activated lymphocytes, lysosome formation is nn early event (Hirschhoni et al., 1967). When the lymphoid cc.11~die, as in the experiments of Granger and Kolb ( 1968), or when supernatants from blast cell cultures are used, toxic lysosomal enzymcs may become enriched in the medium. Such factors are also released to the mcdiuiii when lymphocyte suspensions are contaminated with other leukocytes (Lundgren et a)., 196%). This has becn the casc in almost all reports referrcd to above. Lysosomal enzymes may cither act on the targct cells directly, or indirectly b y changing the niediuin. Cytolysis may- or inay not follow secondarily to growth inhibition. Possible cytotoxic inediators produced by living and mctabolically active lymphocytes may be of a different nature>.They may be related to the complement system ( Section III,C) or may be ccll-bound surfaceactive agents generated by changes in phospholipid metabolism of the activated cells (Fisher and M~leller, 1968; Kay, 1968; Fischer et d., 1968). Much more work is needed to permit distinction between tissue culture artifacts and possible cytotoxic mediators of biological inqiortaiicc,.

6. Conclusions The PHA-induced cytotoxicity of lymphoid cells from normal donors serves as a model for thc similu cytotoxicity exhibitcd by cells activated by othcxr, morcl physiologic,il stiiniilant\. Common for 2111 models i u the rcquiremcnt of living and mctalmlic~tllyactive effector cc4s, susceptible

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to stimulation. Cytotoxicity is an energy-rcquiring process. It is set in motion by activation of lymphocytcns by antigen or other stimulants. H 0 ~ 7 ever, although the smie lymphocyte-stimulating reactions are probably involvcd, blast transformation and DNA synthesis per se are not required for cytotoxicity. There is no relationship in immunological specificity between the stimulating agents and antigens on the target cells. In this sense, cytotoxicity is nonspecific. Both autologous, syngeneic. and d o geneic target cells are affected. Contact between effector cells and target cells is not needed for activation of the lymphoid cells. However, contact seems to be necessary for target cell destruction at least during carly phases of interaction. Surfacch bound effector sites on the lymphoid cells may be needed for target crll destruction but the nature of such hypothetical sites is unknown. The strongly enhanced surface motility and peripolesis of activated lyniphocytes is considered to be of importance for cytotoxicity. The concept of contactual target cell destruction does not exclude local release of cytotoxic mediators. Mechanisms for target cell destruction, discussed in previous sections ( I I I , A , B , and C ) are also applicable to this model. This is also true for the cytotoxicity of polymorphonuclear leukocytes or niacrophages and for the possible importance of interactions between these cell types and lymphocytes.

E.

CELLDESTRUCTION BY LYMPHOID CELLS FROM NORMAL DONORS AFTER “in Vitro SENSITIZATION” TARGET

1. Cytotoxicity of “Normal” Lyinphoid Cell-s

When lymphoid cells are from donors sensitized to target cell antigens, the cytotoxic reaction is initiated by immunologically spccific reactions. In all the other models, receptor units or lymphocyte-activatiiig agents are introduced experimentally in order to produce cytotoxic effects. Lymphoid cclls froin normal donors are usually not cytotoxic without addition\. On the contrary, growth-promoting fecder effects of normal lymphoid cells ha\^ been o h \ e i i d ( Section II,D,2). Howcver, exceptions to this rule have also been notcd. Thus, Stuart (1962) found that spleen cells from norin,il mice destroycd human tissue culture cells in monolayer within 48 hours. Spleen cells from immunized mice produced thr same effects at a higher rat?. Hcat-killed spleen cells or extracts were inactive. Similar obscrv
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This cytotoxicity of normal lymphoid cc~llsuiay have various causes. It inay be due to preceding scwiitizntion of the donor b y aiitigcm crossrcacting with target cell antigciis or it may be nonspecific after stimulation of lymphoid cells by components in thc ciilturc medium. I t is often difficult to distinguish such cause's from cqierimimtal artifacts.

2. “ I n Vitro Setasitizntioii” of Lymphoid Cells Hirschhorii et af. ( 1965) ol)servcd that human lymphoid cells dcstroycd allogencic fibroblast without addition of PHA after 7 to 8 days of culture. \l’hcw the lyrnphoid cells were traiirfcwd to ;I new culture of the same origin, it \viis rapidly d(~stroycd.Fil~rohlnstcultures of diffcrcmt origin were not affected to the same dcgrec. Phytohemagglutinin was said to enhance this effect. The interprc~tationwas that an in uitro scnsitization had taken place. However, the basis for these conclusions was not very substantial. Ginsbtirg and Sachs ( 1965) and Ginsburg ( 1965) iiindca siinilar observations with lyiiiph node or thoracic duct cells of rats. Thc cells bverc, grown 011 1nonol;tyers of rat or inice fibroblasts for scveml days. Dcstruction of the monolnycrs wiis observed after 6 days a i d w a s preceded by the appearance of extensive numbcw of large py~-oninophiliccells ( LPC ) . Transfer of these cells to fresh monolayers of the saine type was followed l)y accelerated dchstruction. This was not the case with moiiolaycrs of other o r gin. Thyinus cells were inactive. Lalieling with “H-thyinidine suggested tliat tlw LPC which iippcm~don the sccond and third day were dcrived froin sinall lynpliocytes ( Ginsburg e t d., 1967). The immunological specificity of cell destruction after transfer of thc lyniphoid cells in thcsc experinwnts w a s not establishcd with certainty. \\%en working with rats, Cins1,urg ( 1965) aiid Ginsburg and L ~ i g ~ i ~ i o f f ( 1968) fouiid that lysis of allogencic monolnycrs w a s not very pronounced or did not occur at all, i n spite of blast traiisfonnation ;uld aggregation of the lynq3hoid cells to the monolayers. Strong lytic effects werc. o111y s w n in cqx~rirnentsin which rat lymph nodc cells were scc,dcd 011 inousc filirol,lasts ( Ginsbiirg, 1968) . The culture conditions necc’ssary to produce lytic effects seem to be critical (Ginsburg, 1965, 1968; I k d w et ul., 1969ii). In general, rnt lymph node cells ;ire first grown im iiious(~filirolilast monolayers (originator monolayrm) for 5 days, with i\ chmgc of medium on clay 4. Thcl cells :ire thcw trmsfvrrcd to fresh inonolnyers ( ttBst nionol;iyc-rs ) wherc t1ic.y rc~iclia p c a k of iiiaturiition and growtlr. Thcy iiiust lie traiisfcwed to a third moiiolnycr \vithin 48 hours in o r d c ~to avoid dctcrioration. FoIIowing niaturation, lirolifrration slolvs tlo\vn anid disiii tcgration starts even

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in case of renewed plating on fresh monol'iyers. The lytic power of thc cell populations is correlated to this growth pattern. In order to ascertain the immunological specificity of the lytic reaction, the experiments were performed with fibroblasts from inbred mice of different H-2 type. Lysis was estimated by observation of the plates or by cell counting ( Ginsburg, 1968). Although test monolaycrs of the same H-2 origin a s the originator monolayers were rapidly lysed, there was no or only moderate destruction when the test layers were of different H-2 origin. It was concluded that the rat lymph node cells had become specifically sensitized to 13-2 antigens of the mouse. The specificity of the cytotoxic reactions was not absolute. The cross-reactions were assumed to reflect an activity of lymphoid cells sensitized to those individual H-2 antigens that were common for various strains. Weak histocompatibility antigens of loci other than H-2 did not seem to give rise to sensitization. In a later report (Berke et al., 1969a), the specificity of the lytic reaction to mouse fibroblasts of C57B1 and C3H origin was assayed by the quantitative "Cr-release technique ( Section II,C,4,d). Evaluation of the results is difficult since the response of the rat lymphoid cells to C3H monolayers was much weaker than that to C57B1. The release of "Cr was also used for a kinetic study of the cytotoxic reaction (Berke et al., 1969a). Lysis plotted as a function of the numbers of LPC added had the kinetics of a first-order reaction. This suggested a relationship of one LPC for each target cell lysed. The lytic power of these precultivated lymph node cclls was very strong. On the basis of quantitative considerations, it was felt that the proportion of active cells in the total population of LPC approached 100%. It was concluded that the H-2 antigens on the target cells trigger transformation and proliferation of small lymphocytes. This results in the formation of a homogenous population of rapidly dividing cells, specifically sensitized to the inducing antigens. Since no cytolytic factors were recovered from homogenates of LPC ( Ginsburg, 1968), cytotoxicity was assumed to require contact between effector cells and targets. This was also supported by microscopic observations and b y time-lapse cineniatogrqhy (Ax et al., 1968). In the latter study, waves of almost syiichronous mitoses were observed during the fourth and fifth days. Large numbers of the lymphoid cells differentiated into LPC. These highly mobile cells clustered around the targct cells. Many had a typical uropod ( McFarland and Heilman, 1965; McFarland et d.,1966) which seeincd to be of importance for attachment to the target cells. Target cell destruction (detachment from moiiolaycr and lysis) followed at days 5 to 7. In an additional report, Rcrkc et al. (1969b) attempted to compare

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the cytotoxicity of in oitro scnsitizecl LPC with that induced by treatment of lymphoid cells with PHA. It was stated that the pyroninophilic cc)lls, formed aftcr prtincubation of rat lymph nodc cc~llson mouse monolaycrs, were morphologically distinct from those produced b y PHA. 'The PHAinduced blast cells gave rise to a population of medium-sized lyniphocytes. Moreover, PHA sccmed to inhibit in oitm sensitization. In thesc experiments PHA did not produce cytotoxic cells at all.

3. Comments The data reported above raise a number of important questions. The most puzzling result is the apparent immunological specificity of the lytic reaction. It is implied that the rat is capable to mount in citro responses specific for different H-2 antigens of the mouse. Even if recent findings of an immunological relationship betwcen major trnnsplantation antigens in different animal species would be in favor of this possibility (Abeyounis and Milgrom, 1969), it is obvious that confirmation of these results is needed. Regardless of specificity, it must be asked in what way the cytotoxic cells described hy Ginsburg and colleagues are rclatcd to those described in the preccdiiig sections. It has been seen that exposure of rat lymph node cells to antigen induces nonspecific cytotoxicity to both allogeneic and syngeneic fibrohlasts ( Ruddle and Waksman, 196Sa). Pretreatment of human lymphoid cells with PPD or mixed culture with allogeneic lymphoid cells for 5 to 6 days gave rise to blast cell-containing populations which were highly toxic for tissue culture cells (Holm and Perlmann, 196711). In contrast, the effectox cells described by Ginsburg and collcagues do not seem to exhibit nonspecific cytotoxicity at all. If correct, at least two explanations may bc found for thcse discrepancies : ( 1 ) the cytotoxic LPC produced by in oitro sensitization are differentiated cells which have lost their original capacity to induce nonspecific cytotoxic reactions-thc appearancc of antigen-specific receptors on their surface or other factors could account for this; ( 2 ) the LPC found by Ginsburg and colleagues are descendants of lymphocytes different from those responding to stimulation with nonspecific cytotoxicity. It may be of some importance to note that blast transformation and proliferation in miwd culture, reflecting an immunologically specific response of certain lymphocytes to foreign transplantation antigens, cannot easily be induced by cells other than lymphoid cells (Hardy and Ling, 1969). In contrast, the population of LPC formed after in 2jitro sensitization seems to reflect stimulation of some cells by transplantation antigens on fibroblasts. Onc wonders, therefore, whether they represent prccursors of an antibody-

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forming cell typc ( Mishell and Dritton, 1967; Dutton and Mishc,ll, 1967). Further eupcrimc>nts are needed to decide bet~7cen thcw or othcr alternatives. IV.

Some in Vivo Implications of the in Vitro Models

In the preccding sections it has been shown that cell-mediated cytotoxic reactions in witro can be induced in a variety of ways. It is not only sensitized lymphocytes which specifically damage antigenic target cells. After activation by unrelated antigen, sensitized lymphocytes niay also become cytotoxic to target cells of any origin. Lymphoid cells from nonsensitized donors will exhibit a similar type of apparently “nonspecific” cytotoxicity when exposed to alloantigens on foreign cells or to other stimulating agents. Most importantly, minute amounts of certain humoral antibodies in the dbsence of coinplement may also induce cell-mediated cytotoxicity. Finally, antibody in conjunction with partially activated complement may give rise to similar cell-mediated activities. Cytolytic mechanisms of the same kind as those seen in witro will be at play as major or minor links in thc chain of evcnts which lead to cellmediated tissue d‘image in wiuo. In the following sections, an ‘ittempt has been made to define some in wizjo situations whcrc the diffcrent in wilro inodcls would seem to fit in. On the basis of some well-known facts and a number of rather nrbitrarily chosen examples, possible applications of the various in uitro models have been pointed out. The purpose of this section is not to explain what takes place in siwo, but rather to outline sonic of the directions for further studie5 of tissue-damaging mechani~~ns.

A.

DELAYED HYPERSENSTTIVITY

1. The Tuberculin Reaction and Similar Phenomena The histological picture of delayed hypersensitivity ( DLH ) as described for the tuberculin skin reaction is in most species charxterized by an early accumulation of polymorphonuclear leukocytes. Later, at thc peak of the macroscopical infiltration after 24 to 48 hours, mononuclear cells predominate around small vessel5 in dermis, subcutanous fat, and connective tissue ( Waksman, 1960; Turk, 1967). Focal necrosis of mu$cle fibrils and fat has been described (Kolin et al., 1965). Thc composition of the cellular infiltrate in DLH varics from species to species and from antigen to antigen within a species. Delayed hypersensitivity to tubrrculin and other antigens can he transferred to nonsensitizcd recipients with lymphoid cells but not with scwim from ininiune donors (for refercnccs, see Bloom and Chase, 1867).

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In recipients d q ~ l o t c dof lymphoid cclls by hcavy X-irradiation, DLII could not be elicited after trmsfcr of lymphoid cells from sc.nsitizec1 donors ( Cunrmings et a/., 1955). The number of laheled cells at thct tchst site in transfer c~xprimrmtswith isotope-labc~lcdcclls sc'cnis to 1)e small ( 1-2% of thc infiltrating cells) m c l can wcll be explained oil the 1)asis of random distribution. The majority of the infiltrating niono1iuclear cells are of host origin (Feldman and Najarian, 1963; h3cCluskey ct d., 1963; Lubaroff and Waksnian, 1967). It is generally assunird that nonsensitized cells activcly participate in the manifc~stationsof DLH, although it is initiated by the reaction of stmitized lymphocytes with antigen. The mechanism of activation of noiiscnsitized cclls is not known. It is thought that thc relcmse of factors from a few sensitized lyrnphocytcs ~ h i c hinhibit macrophage migration in vitro ( Bloom and Bennett, 1966; David, 1966; Svejcar et al., 1968) also may be of importance for retainmcnt of macrophages and perhaps other leukocytes at the DLH test site in tjiuo. The same and/or othcxr soluble factors may activate nonsciisitized lymphocytes and provoke iriflammatioii by increasing vascular permeability (Inderbitzin et al., 1966; Spector and Willoughby, 1968). Although DLH can be elicited when circulating antibodics cannot be demonstrated (Uhr et al., 1957; Gcll, 1961; Holtzcr and Winkler, 1967), it has been explained by some authors (Karush and Eiscm, 1962) as a manifestation of the action of minute amounts of high-affinity antibodics. A common argument against this assumption is that elicitation of DLH differs from humoral antibody by its requirements of carricr-specificity (for refcrences, see Turk, 1967) and immunogenicity (for references, sec Schlossniaii, 1967). Antigen-induced stimulation of sensitizcd lymphocytes to blast transformation ( Mills, 1966; Oppenheim et ol., 1967; Stulbarg and Schlossman, 1968 ) and releasc of macrophage migration inhihitor (David and Schlossman, 1968) has specificity requirements siinilar to those ncmled for elicitation of DLH. However, synergism between humoral antibodies and lymphoid cells in DLH has been shown to occur (Asherson and Loemi, 1966). In a passive transfer model in guinea pigs, 24 hours skin reactions to bovine serum albumin, bovine 7-globulin, and other antigvns coulcl be induced in nonscnsitized recipients after trunsfcr of either serum or peritoneal exudatc cclls from seiisitizcd donors. Siinultaneous transfer of iminunc cells and serum produced strong cutancous reactions. The histology of the cutancous lesions after transfer of serum alone was characterized by infiltration of polymorphonuclear leukocytes. These were absent after 48 hours. When both serum and cells from sensitized donors were trmwferred, polymorphonuclear leukocytcs predomiiiatcd during the first hours. and liistiocytes and lymphocytes at 48 hours.

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It was thought that potentiation of DLH by immune seruni was in part due to provocation of a local inflammatory reaction and in part due to local retention of antigen on thc site of the skin rcaction ( Asherson, 1967). In the preyent state of knowledge, attempts to find in vivo correlates to the in viiro models discussed in this article must by necessity be vague. If it is assumed that in DLH the antigen is retained at the test site where it is injected, the chain of events will be started by some sensitized lymphocytes, entering the site by chance and reacting with antigen. At this point one or more of the following cytotoxic mechanisms, all with in vitro counterparts, may come into play: ( 1) Sensitized lymphocytes may exert a direct cytotoxic action on antigen-coated tissue cells ( Sections III,A,E), ( 2 ) Sensitized lymphocytes may become activated by interaction with antigen-this will lead to nondiscriminatory injury of surrounding tissue cells, as described in Section III,D, ( 3 ) Free antigen-antibody complexes or complexes on the surface of macrophages may be formed if humoral antibodies are present or locally produced in some delayed reactions. These complexes may then stimulate ( Bloch-Shtacher et al., 1968; Cline and Swett, 1968; Moller, 1969) and, thereby, induce a cytotoxic action of a certain fraction of nonsensitized lymphocytes. Antigen-antibody complexes adsorbed to tissue cells may also induce direct destruction of the latter by activation of nonsensitizcd lymphoid cells (Section 111,B). ( 4 ) Immune-adherence-like phenomena may lead to tissue destruction. These may be induced by C’3 on cells on which antigen-antibody reactions have taken placc. Although the role of complement in DLH is not established, it has been found that decomplementation of rats with antigenantibody complexes reduced the intensity of DLH (Ncveu and Biozzi, 1965). Willoughby et al. (1968) have also shown that treatment of sensitized guinea pigs with antiserum to C’3 abolishcd their ability to dcvelop skin reactions in DLH. 2. Certain Infectious Diseases Delayed hypersensitivity may participate in some pathological changes associated with active tuberculosis (for references, see Turk, 1967). Intense cell-mediated immunity to mycobacterial antigens seems to be responsible for the lesions in tuberculoid leprosy. In contrast, in lepron-tatous leprosy, cell-mediated immunity is deficient ( Waldorf et al., 1966; Turk and Waters, 1968). In this variant of leprosy, the bacteria proliferate throughout the body-humoral antibodies to the bacteria are formed but granulomatous Iesions are not seen. Cellular immunity connected with increased nonspecific resistance is an important part in the defense against certain bacteria, parasites ( e.g.,

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Leislzmcinia ), and fungi ( Suter and Ramseier, 1964; Mackaness and Blanden, 1967). However, dcxstruction of microorganisms hy contact with lymphoid cells from sensitized donors hiis not yet been dcscribed. Recently, Dineeii et al. (1968~1,l))\verc' able to trnnsfcr immunity to a hellnintic infection ( Trichostro,ig!/lrrs colzrbriforvnis ) into syngeneic recipients with lympliocytcs from mesentcrial lymph nodes or Peyer's plaques of immune guinea pigs. Iinmune serum had no effect. Transferred lymphocytes, labelcd with "Cr, were shown to localize at the site of the infection in close vicinity to thv microorganisms. These were eliniinated provided they wcre hit in a suscclptihle stage of their development. Transferred lymphocytes from nonimmune donors were not found in the small intestine. The experiments demonstrate the role of cell-mediated immunity in host resistancc to helmints and suggest that parasites may somrtinics be destroyed by dircct cytotoxic action of lymphoid cells. B. AUTOIMMUNITY Autoimmunity is usually recognized by the presence of humoral autoantibodies. Although autoantibodies may be responsible for tissue lesions in some diseases such as the hemolytic anemias (Dacie and Worlledge, 196S), their pathogenic role in most disease processes is unknown. On the basis of the histopathological picture of the tissue lesions and of transfer experiments, cell-mediated immune reactions are considered to be instrumental in the production of tissue lesions in certain autoimmune diseases. Cell-mediated destruction of tissue culture cells has bccn reportcd for several human arid experimental autoimmunities (Table I; Section II1,A). Coexisting humoral antibodies may theii act synergistically or antngonistically to the cellular mediators. The pathogencsis of the brain lesions in experimental allergic encephalitis ( E A E ) may serve a s c~xample.This discwe can be produced in rats, guinea pigs, and other sprcies by injection of allogeneic or xenogeneic brain extracts ( for references, see Paterson, 1966). Clinical signs of EAE usually develop within 2 wwks after the injection of antigen. The lesions are characterized by heavy infiltration of lymphoid cells in the white ii1attc.r of the central ncrvous system. Delayed hypersensitivity can be elicited by injection of brnin itntigen ( Shaw et al., 1965). Similarly, evidence for cellular sensitivity was obtnined in guinea pigs by antigeninduced inhibjtion of macrophage migration ( David and Paterson, 1965). It has also been shown that thymectoiny of newly hatched chickens suppressed their ability to manifest EAE (Jankovie and Ihaneski, 1960). In contrast, chickcm made agammaglobulinemic by bursectomy and total-

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hody irradiation developcd EAE uni~np~iired when injcctcd with bovine spinal cord (Hlaw et ul., 1967). Experimental allergic encephalitis can be successfully tranxferrcd to tolerant or syngencic recipient rats by lymphoid cells but not with serum ( Patcrson, 1960, 1966). IIowcvcr, local administration of antibrain serum into the later'il vcntricle of normal animals induced histological lesions characteristic for EAE (Jankovi; e t uZ., 1965). Lymph node cells from diseased animals 1 week after immunization damaged glial cells in tissue culture. In these experiments, immune serum in the presence of complcment was not cytotoxic ( Koprowski and Fcrnandes, 1962). However, other authors have described complement-dependent cytotoxicity to glia cells or demyelination of brain tissue cultures by huinoral antibodies from animals with EAE (Berg and Kallkn, 1962, 1965; Appel and Bornstein, 1964). Thus, at certain stages of EAE there is evidence for the coexistence of lymphoid cells and antibodies with the potential to destroy brain cells. Antibodies may participate in the production of EAE either by exerting conventional cytolytic reactions in conjunction with complement or by interacting with lymphoid cells as described for the in &ro model of Section II1,B. Experimental cvidence for the last-mentioned effects: iq lacking. In Lewis rats, EAE is fatal in 50%of the animals. In contrast, EAE in Wistar rats runs a benign course. Complement-fixing antibodies to brain antigen were found from the fourteenth day after immunization in most Wistar rats but not in Lewis rats. If Lewis rats were intensely treated with serum containing complement-fixing antibodies, the severity of EAE and the number of diseased animals was reduced in comparison with control groups (for referenccs, sce Paterson, 1966). This is evidence for antagonistic effects of antibodics during certain stages of the disease. Tissue culture experiments similar to those described in Section II1,A (Brunner et al., 1967, 1968a,b) would seem to be able to throw light on the mechanism of this inhibitory action. Support for the interaction of autoantibodies and lymphoid cells in the production of autoimmune tissuc lesions was provided by Brown et al. (1967) who studied the induction of experimcntal orchitis in guinea pigs. Iminuiiizatiou schedules which produced either circulating antibodies or DLH to testicular antigens did not provoke orchitis. Only when DLH and antibodies were present at thc same time were testicular lcsions observed. Antibodies bound to the antigen in testes and DLH to testicular antigens appeared simultaneously, whereas circulating antibodies were not detected until later after immunization. Although other mechanisms: are probably involved, it would be of interest to establish whether or not

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the tissue-hound antibodies concerned also were capable of inducing cell-mediated cytotoxicity a s w c ~ nin 2;itro (Section III,B),

C. CRAFT-VERSUS-HOST REACTIONS I. Graft-r;ersus-Host Reactions after S!/stemic Tramsfer of Allogeneic Cells The systemic transfc,r of imniunocompetcnt lymphocytes into an allogeneic host may produce graft-versus-host ( GvH ) reactions (for references, see Sinionsen, 1962; McBride, 1966). Snidl lymphocytes injected intravenously into a foreisn host proliferate mainly in tlic white pulp of the spleen and in the cortex of thc lympli nodcs. Transferred lymphocytes, labeled with isotope or equipped with chromosome markers, have been found to differentiate into large pyroninophilic cells, from which sinall lymphocytes e i i i c y y aftcr several divisions ( for refercwces, scc Go\vans nnd McGregor, 1965). Since this is not seen in syngeneic recipients, thc imrnunologic~ilbasis of the renctions is established. Griift-\~ersus-liost reactions induced in immunologically maturc recipients a r usunlly ~ ~ self-limiting and tlic transferred cclls are probably eliminated by host-versus-yraft rcactions ( for references, see McBride, 1966). In recipients \vho arc unablr to niakc an inimunc response to the transferred cells, these cc~llsmay survive and produce allogcwc~icdisease. The acute type of allogeneic disc.ase is rapidly lcdial. It is cliarxterized by runting, skin rashes, and diarrhea. Ll’lirn runting aniinals were transplanted with skin froni animals of thck strain donating the lymphocytcs, the transplmts \vcw acceptccl and did not show the smie changes as the original rrcipient skin. In contritst, aiitotransplilnted skin was rapidly rejected (Billinghum et al., 1962; Stastny et ul., 1963). Massive infiltration of histiocytes, lymphocytcs. and plasma cells was seen in tlw areas of skin injury. Intcractions betwecm donor m d host lymphoid cells during early stagcs of systemic GvH may produce nctivated lymphoid cells. T h e may provoke tissue lesions b y mech;inisms similar to those dcscrihed for tissuc’ culturc damnge by lymphoid cclls stimulated in mixed culture (Section I I l ,D J,d). In later phases of this form of GvH, scmitized lymphocytes or their dcsccndants ( Section IV,D ) and/or mitibodic,s may damage host tissue in a specific way.

2. Graft-verws-Ho,st Renction.s

(if lcr

Loctil lransfer of Cells

Brcwt i u i d kledan~nr ( 1963, 1966) d(wri1)cd ii tii~wrculiii-likc cutnrc*action in guinea pigs, induccd b y intracutanuous inoculation of

11(’o11s

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immunocompetent lymphocytes from normal donors to allogeneic, irradiated hosts [normal lymphocyte transfer ( N L T ) ] . The first phase of NLT starts 6 hours after inoculation and is maximal after 48 hours. The flare-up phase attains maximal intensity after about 5 days. I t is followed by a fade-out, ascribed to immunological recovery of the host. The first episode is regarded as a manifestation of recognition of foreign host antigens by the donor cells. No reaction is obtained when the donor cells are syngeneic to the recipient or are of F, hybrid origin. Exposure of the host guinea pig to 500 to 1500r abolishes their immunological capacity. However, it does not impair the early phase of NLT, induced by donor lymphocytes injected 3 days later. The first phase of NLT in guinea pigs is not affected by treatincnt of donor cells with imniunosuppressive drugs or antimetabolites which inhibit protein or RNA synthesis. In contrast, the flare-up phase is abolished by treatment with antimitotic agents or X-ray. The flare-up is regarded as a manifestation of sensitized donor cells which have arisen after contact with host antigen. The participation of host leukocytes in the first phase of NLT may vary from species to species. Ramseier and Billingham ( 1966) obtained R NLT-like reaction in hamster skin by injection of allogeneic lymphoid cells. No reaction was seen when the donor cells were introduced into the skin of irradiated hosts. On the other hand, inoculation into such hosts of a mixture of donor cells and allogeneic lymphoid cells from a different strain provoked a reaction (Ramseier and Streilein, 1965). This suggested that the injected lymphocytes were unable to react with the antigens of host skin. In vitro, lymphocytes are usually better stimulated by allogeneic lymphoid cells than by nonlymphoid cells ( Hardy and Ling, 1969). These observations shcd light on the requirement of host lymphoid cells in NLT. In Brent and Medawar’s experiments, thc recipient guinea pigs were irradiated with 6 0 r. However, the apparent iminunologicnl rccovery of the recipients during the fade-out phase indicated that the animals were not completely deprived of immunocompetent cells. Heavily irradiated lymphocytes which are unable to proliferatc are known to stimulate allogcneic. lymphocytes in mixed culture in oitro ( for rcferences, see Ling, 196s). Stimulation of donor cells in NLT in irradiated hosts may be brought about in a similar way. In the early phases of NLT, tissue lesions may be induced by direct cytotoxic effect5 of activated and proliferating donor cells. This would be in analogy to thc nonspecific cytotoxicity in titro of lymphoid cell.; activated by contact with allogcneic cells (Section III,DJ,d), In n011-

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irradiated animals, lymphoid cells of the recipient may become destructive to autologous tissue after stimulation by allogeneic donor cells. In the later phases of NLT, sensitized lymphocytes may arise and participate in the specific destruction of antigenic tissue cells (Section 111,E). At this stage, cell-mediated tissue dcstruction may be either impaired ( Section III,A,3,b) or enhanced ( Section II1,B) by small amounts of humoral antihodics. Elkins ( 1964, 1966) described the destruction of kidney parenchyma after subcapsular injection of allogeiicic lymphocytes. This reaction was also regardcd as local GvH. Treatment of the recipient with increasing doses of X-irradiation progressivcly impaired the kidney destmction. Transplantation antigens of the kidney were apparently not involved; kidney destruction was also brought about by lymphoid cells from parental strain donors, injected into a pxental strain kiclncy that had been transplanted to a F, hybrid host (Elkins and Guttmm, 1968). In this situation, the F, hybrid lymphocytes will stimulate the donor cells which, then, m a y become nonspccifically cytotoxic to the autologous kidncy tissue (Section III,DJ,d).

D. ALLOGRAFTREJECTION After transplantation of solid tissue to a nonsc.nsitized allogencic recipient, the first morphological c1iangc.s take place within the paracortical ( thymus-depciideiit ) iireas of regional lymph nodes. In these, large pyroninophilic cclls develop, prcbsuniably from small lymphocytes, induced to differentiation and proliferation by contact with foreign transplmtation antigens from the graft. ( for references, see Gowans and R/lcGregor, 1965). In rats and mice, sensitized lymphocytes appear in the circulation 6 days after grafting and can be demoiistrated by adoptive transfer experiments (Billinghmn et oZ., 1963). In such experiments, only a sinall proportion of the inononuclcar cclls that infiltrate the graft arc' of donor origin ( Najarian and Fc~ldman,1962; Rilliiigham ct al., 1963). AS in DLH, most infiltrating iiiononuclriir cc~lls:ire of host origin. By :'H-thyinidine labeling of regional lymph node cells draining a skin allograft in rabbits, Prendergast ( 1964) ~ h o w ~that c l labcblc,d cells infiltrated the graft. Howc>vcr, infiltration was not specific; labclcd cells also appeared in an unrelated allograft as w ~ l LLS l in delayed cutaneous skin rcwtions induced by unrelatcd antigcn. Hall ( 1967) studicd cc~llsin the lymph drainiug local nodes from homografted shwp. On the cighth day after transplantation when thr graft already showed signs of rejection, il considerable uuniber of liasophilic cells app;ired in the lymph leaving the draining

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node. No such cells wcre seen in the lymph afferent to the node. Experiments with 3H-thyniidine-labeled basophilic cclls suggested that such cells in the graft were randomly selected froiii the blood. It is assumed that the small nuniber of sensitized lymphocytes that enter the graft initiates its rejection. The in uitro experiments discussed in Sections III,A and II1,E suggest that these cells may destroy graft cells by direct cytotoxic action. It has recently been shown that the lymphborne blast cclls, which are discharged from a node draining an allograft, also exhibit in witro cytotoxicity to allogencic cells of graft type ( Denhani et al., 1969). Howcver, the inajority of the mononuclear cells in the graft arc primarily lionsensitized cells. Recent findings (Stcinmuller, 1969) suggest that lymphoid cells, present in the graft, may serve to stiinulate lymphoid cells of the recipient to participate in the rejcction process. This could bck by a “mixed culture”-type induction of localized cytotoxic responses, as indicated in Section III,DJ,d. It is also possible that recruitment of lionsensitized lymphocytes could be provoked by direct transfer of specific information from sensitized cells ( Gerughty et ul., 1966; Wilson and Wecker, 1966; Alexander et nl., 1967; Boiidevik and Rilannick, 1968). Finally, sensitized cells that react with antigen will also release nonspecific soluble factors which may confer cytotoxicity onto nonsensitized cells, lymphocytes or others. HaSek et uf. ( 1968) recently achieved allograft rejection in tolerant ducks by transfer of large volumes of irnmunc~ serum or isolated 7-globulin. Microscopic examination of the grafted tissue revealed two patterns of rejection-onr of acute rejection with vascular damage, necrosis, and predominately granulocytic infiltration nnd one of a firstset rejection, where mononuclear cells were predominating. As already discussed, some of these sera have now also been found to contain graftspecific alloantibodics capable of inducing cell-mediated cytotoxicity in uitro ( Rubenik et al., 196%) ( Section III,B,I ). Somewhat similar results have rcwntly also been reported by Spong et a / . (1968). These authors worked with antibodies from rats undergoing a first-set renal allograft. Parenteral injection of such IgG fractions into norriial rats produced perivascular mononuclear infiltratc>sin the kidney. The lcsions were indistinguishable from those accompanying early rejection. Cochrutn et d.( 1969) i n d e similar findings in goats after local perfitsion of the autologous kidney with immune serum. A further illustration of thc rolc of humoral antibodies for rejection of solid grafts has becn providcd by Clark et (11. ( 1968). A dog kidney was placed for a few hours into an ;illoqc‘ncic recipicwt who was heavily irradiattd after presensitization with donor tissue. The kidney was thcw rctransplanted in to

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the donor, where it was rapidly rejcctcd. Thc rejected kidneys were heavily infiltratcd with polyinolphoiiricl~~~r granulocytes and mononuclear cells. These rcports suggest that a syncq$stic intcmction between humoral antibodies and lymphoid cells, at least i n crrtain situations, may be of importance for allograft rejection. In Scction III,B it has becm shown that minutc :unounts of certain antibodies to targct cell antigens are sufficient to provoke cell-mediated lytic reactions in uitro. In analogy, cellmediated graft rcjcbction may be induced by small amounts of antibodies to transplantation antigens. Antibody may be produced locally in the graft or elsewhcrc and may interact with lymphoid cells infiltrating the graft. It should also be noted that induction of ccll-mcdiated cytotoxicity b y antibody does not necessarily require cytophilic properties ( Holm and Perlmarin, 196921). From what has been scen in zjitro, the titers of such antibodies may be too low to be detected with common techniques. When produced within the graft, the antibodies may be :illsorbed locnlly. Synergism between antibodies and lymphoid cclls will probddy gain relatively more importance during later stages of a first-set rejection or in a second-set situation when production of 7 S antibodic3s may be more pronounced ( Hutchin et al., 1967).

E. TUMOR DEFENSE Tumor defense by immunological means has the characteristics of tissue rejection. The mechanisms which apply to allograft rejection are, therclfore, also of importancc~for defense against neoplasm. The situation will be coinplicated by the inherent growth potential of thc tumor, its capacity to metastasize, and its possible inhibitory effects on thc immune apparatus of tlic host. It is assumed that imnwnological defense against tumors is most important in prcwwting thc establishment of a tumor. Once a tumor has been established thck capacity of the immune 2IppafiIh1s to achieve rejection may be more in doubt. Prerequisite for an immune response against autochthonous neoplastic tissue is the existence of tumor-specific antigens. From work with experimental animals it has been known for some time that such antigens exist in a variety of tumors, induced cither by viruses or by chcmical or physical means (for references, see Prehn, 1965; Klchin, 1966). More recently, tumor-specific antigens havc also been dctected in human tumors such as the Rurkitt lymphonia (Klein et a]., 1966, 1967), malignant melanoma (Morton et nl., 1968) a i d carcinoma of the colon (Gold and Freedman, 1965a,b), In these cases, evidence for tumor-specific antigens was provided by means of humoral antibodies. I-Io\vever, most tumor-specific

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antigens have the characteristics of transplantation antigens, and cellmediated immune mechanisms have been considered to b e essential for tumor destruction. Evidence for occurrence of cell-mcdiated immunc rcactions and their significaiicc for tumor defense has recently 11een reviewed by Hellstrhm and Hellstrom ( 1969). Therefore, this discussion will be restricted to a fcw aspects related to the in oitro models treated in this articlc. In vitro neutralization of tumor cells and adoptive transfer of tumor immunity can be achieved with lymphoid cells from sensitized animals whereas immune serum usually has no effect (for references, see Hellstriim and Hellstriim, 1969). Alexander et nl. (1966a) inoculated rat sarcoma cells into sheep. Three days later, lymphoid cells were harvested from the efferent lymph from the draining node and were injected into tumor-bearing rats. By this procedure, specific retardation of tumor growth in the rats was obtained, and no GvH reactions were seen. It was postulated that a transfer of “sensitizing information” from the donor cells to cells in the recipient was responsible for this. Later experiments with RNA extracts supported this view (Alexander et nl., 1967). The in vitro cytotoxicity of lymphoid cells from tumor-bearing animals has also been taken as evidence for the importance of cell-mediated immunity. Typical examples have been given in Table I (Section II1,A). In the human, striking rcsults have recently been published for neuroblastoma (Hellstrom et al., 196Sb). Other neoplasms in which the sensitive colony inhibition assay has given positive evidence for cell-mediated cytotoxicity in human cancer are adenocarciiioma of lung and of colon, malignant rnclanoma, thyroid carcinoma, squamous cell carcinoma, mammary carcinoma (Hellstr6ni et nl., 196Sc), and carcinoma of the bladder ( Bubcwik et al., 1969b). The mechanisms discussed above for allograft rejection may all be involved in tumor destruction. There is no doubt that tumor drfense as a rule reflects a complicated balance of both cellular and humoral fuctors. Enhancement of tumor growth by inhibition of cellular responses is well known from animal studies in transplantatioii-imniuiie systems ( sers Section III,A,3,b ) . There is also evidence for enhancement of tumor growth by antibody in situations where experimentally induced tumors were transplanted into syngeneic recipients ( Moller, 1964; Bubenik and Koldovsky, 1965, 1966; Bubenik et al., 1965). However, antibodies, which are capable of destroying tumor cells by complement-dependent cytolysis are also frequently found and may coexist with cytotoxic cells. Recent examples are human iieuroblastoma and colon carcinoma mentioned above (for other references, see Hellstrom and HellstrGm, 1969). Such

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183

cytotoxic antibodies may be an important part of the defense system. Howcver, from what has h w n said in Section IJI,R, it is rcasona1)le to c’xpect that humoral iiiitibodic,s also may inducc> cell-iiiediated tumor destruction b y mc~hanisms diffcreiit from conventionnl complementdependent lysis. Small amounts of locally relcased antibodies may be candidates for this type of synergistic action. This hypothesis can be subjected to experimental test. V.

Summary

In thc left column of Table 11, ten cell-mcdiated cytotoxic reactions, all described in this review, have been tabulated. Operationally, three TABLE I1 POSSIBI.E in Viuo CORRELATES O F i / ~V i ( m CYTOTOXIC REACTIONS Cytotoxic reaction (Cx)

iii

Possil)le it, v i ~ ocorrelates

tissiie ciiltiire

1. Cx of lymphoid cells sellxitized t o t.arget cell air t igeiis 2. Cx of lymphoid cells sensitized to mivruljial o r other aiit~igeiis:ttlsorbetl to target cells 3. Cx IJf lymphoid (.ells after in, uitro seiisi(izrttiori

Allograft.. Tumor defense. Some aiitoinimiltie diseases. IAat,ephases of graft.-vs.host reaction Delayed hypersetisitivit y

Sensitized (*ellsin allograft. situat,ions arid perhaps ot.her coiidit,ioris?

4. Cx of iioitseiisit,izetl lymphoid cells irt- Allograft, reject ion. Timior defense. Sonie aiitoirnimiiie diseases diiced by ant,il)odies 1.0 target. cell :itit,igerrs I>el:iyrcl Iiyperstwsit ivit y? ,r). Cx of nonsensitized lymphoid cells iiitluced by niit~ihotliest,o niiwol)id o r ot.her aiit.igens atlsorl)ecl t.o 1,argel. cells 6 . Cx o f nioiiocyt.es iiitliiced I)y artivitt.ed tZll coiirlitioiis iii which C’-fixing :tnti\)otlie-: aiitl c~omplenientare preheiit C‘3 on t,arget. cells

7. Phytohemagglut iriiii-itiducetl cx of

Nolle

rwnseiisit.izetl lymphoid cells 8. Cx of troirseiisitizeti lyniplloitl cells itct.ivat.ed “iicitispecifcally” b y Imct.erial prodriots (st,apliyloc.orc:tl filt,rat,e, st,rept.olysiti S) 9. Cx of notiseiisit izetl lymphoitl cells Kormal lymphocyte transfer a i d other act,ivat.ed t)y :tlloant.igeiis (“mixed graft-vs.-host, reac,tiotis. Some allograft, sit iiat,ioiis? criltrire”) 10. Cx of lymphoid cells seiisiiized to Delayed hyperseiisilivity. Some chronic infect i o i i s ? ant,igeii i i o t assori;ttetl with t):trge( cells

184

PETER PERLMANN AR‘D GORAN HOLM

groups of reactions can be distinguished. In the first group, cytotoxicity is specific; this nicnns, thc destroyed target cells carry surface antigens to which the donor of the lymphoid cells is sensitized. l’he second and third group comprise reactions which may be called “nonspecific.” Nonspecific implies only that there is no apparent relationship betwccm antigens on the target cells and the specificity of the agcnts that trigger cytotoxicity. In thc second group, nonspecific reactions are induced by humoral antibodies to target cell antigens. These reactions do not involve conventional-type complement activation. Antibody-mediated induction of cytotoxicity seems to be most efficient when the antibodies are complexed to antigens on the target cclls. Exceedingly small amounts of certain antibodies are sufficient to start a reaction. In the third group, cell-mediated cytotoxicity is started by a variety of agents, known to qtimulate lymphocytes to blast transformation and DNA synthesis. Available evidence suggests that all nonspecific cytotoxic reactions of both the second and the third group (except the monocytemediated reaction, No. 6, Table 11) are triggered by activation of those lymphocytes (probably thymus-dependent) that are susceptible to stimulation. However, activation to cytotoxicity is an early event which may or may not be followed by transformation and proliferation of the activated lymphocytes. When the lymphoid cells are from donors which arc sciisitized to target cell-associated antigens, cytotoxicity will be specific (reactions 1 and 2, Table 11). When they are from donors sensitized to antigens not associated with target cells, contact with antigen will trigger nonspecific reactions (rcaction 10, Table 11). This and other evidence suggest that the lytic mechanisms are similar in all cases. Both specific and nonspecific cytotoxicity has been obtained with purified lymphocyte suspensions. In all these cases, contact between lymphocytes and target cells seems to be necessary for target cell Zysis. Lysis is a manifestation of energy-dependent processes in the effector cells. It may include surface activitics related to pinocytosis or phagocytosis. The concept of contactual lysis does not excludc. participation of solublc mediators during later phases of the reaction. There is some evidence that activation of cell-borne C’8, one of the terminal components of the complement system, could also be of significance for cell-mediated lysis. Stimulation of lyniphocytcs is known to lead to release of pharmacologically active substances. Such factors may amplify the reaction by activation of originally inactive lymphocytes, and, when present, of monocytes, macrophages, or polymorphonuclear leukocytes. Conversely, some of these ccll types may also affect the reactivity of lymphocytes.

CYTOTOXIC EFFECTS OF LYhIPHOID CELLS

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185

The biologicul signific;uicc>of lyniphocytr stiiiiulation. \vhich Lilso co11stitutcs the basis of thr+ cptotoxicity, is not c k ~ It. h a s rc,ccntIy bc~c.11 reported ( Scsction 111,E; rcxtion :3, Tahlc. 11 ) that in &it/()stirnulatioii of rat 1yinpliocytc.s under spcdic.tl conditions gave rise to homogcncous populations of large pyroiiinophilic cr.11~.Most of th \\’ere stlid to be specifically sensitized to the antigens which gave rise to prolifer.ition. These cells were assinncd to lie identical with the sciisitizcd cells arising in allograft reactions and similar situations in c;ioo. In these. exprriments nonspecific cytotoxicity was not observed. If correct, this would mean cither that stirnulntcd lymphocytes may diffcrtxntiatc into cells which have. lost their nonspecific cytotoxic potential or that the cell typcs which give rise to sc~nsitizcd populations arc’ diffcrcmt from thosr involvc~din nonspecific cytotoxicity. The cytotoxicity of lymphoid cclls from sensitized donors has l~ccw assumed to be an in Gitro manifestation of DLH or of similar states. Although this inay bc true in many situations, it is not a gencml rule. Most of [he i n Gitro models described in this article also Iiavc their counterparts in viuo. This implies that even in thcx organism, tissuc. damage may &her be induced by sensitized lymphocytes or by nonsensitized lymphocytes, p r h a p s activated b y small amounts of locally produced an tibody, hy contact with alloantigeii on foreign lymphocytes, or otherwise. 1n uiuo, the various specific and nonspecific reactions will occur simultaneously or in sequence. In the right column of Table IT, possible in uiuo correlates of the different in uitro models have been compiled. Some of the hackground for this has been given in Section \’. Thr relative role of diffcwnt cytotoxic rcactions for tissuc darnagc~in cico remains to cqlored. 1 x 3

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Transfer Factor’ H. S. LAWRENCE Infectious Disease and Immunology Division, Depurtment of Medicine, N e w York University School of Medicine, N e w York, N e w York

I. Introduction . . . . . . . . , . . . 11. Definitions and General Principles . . . . . , . A. Selection of Donors . . . . . . . . . B. Selection of Recipients . . . . , . , . C. Protocol . . . . . . . . . , . D. Method of Systemic Transfer . . , , . , . E. Methods of Local Transfer . . . . . . . 111. Transfer of Delayed Hypersensitivity with Viable Blood Leukocytes . . . . . . . . A. Quantitative Variables B. Transfer with “Lymphocytes” . . . . . . . C. Transfer of Contact Sensitivity-A Special Case? . . . D. Transfer of Tuberculin Sensitivity via Blood Transfusions . E. Transfer of Delayed Reactivity to Kveim Antigen . . F. Transfer via Renal Transplants . . , . , . C:. Summary and Conclosions . . . . . . . and hlechanism of Action , IV. Transfer Factor-Characterization A. Introduction of Leukocyte Extracts . . . . . . B. Confirmation of Transfer with Lenkocyte Extracts in hlan . . . . V. Nature and Properties of Dialyzable Transfer Factor A. Prepared from DNase-Treated Leukocyte Extracts . , B. Prepared from Sonically Disrupted Leukocyte Extracts . . VI. Transfer Factor and in Vitro Correlates of Cellular Imtnunity A. Inhibition of Xlacrophage Migration . . . . . B. Lymphocyte Transformation . . . . . . . C. Target-Cell Destruction . . . . . . . . . VII. Mechanism of Action of Transfer Factor itz V i m and in Vitro VIII. Transfer Factor and Mechanisms of Cellular Irnmune Deficiency . . . . . . . . . . . . Diseases A. Agammaglobulinemia . . . . . . . . . B. Boeck‘s Sarcoid . . . . . . . . . , C. Hodgkin’s Disease . . . . . . . . . D. Systemic Neoplastic Disease . . . . . . . IX. Transfer Factor and Reconstitution of Cellular Immune Deficiency A. Generalized Vaccinia . . . . . . . . . B. Disseminated Moniliasis . . . . . . . .

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196 199 199 200 201 201 201 202 202 209 210 212 212 215 216 217 217 226 229 229 231 234 234 238 244 245 248 248 249 251 252 252 253 254

’Work from the author’s laboratories has been supported by the U. S. Public Health Service Research Grant AI-01254-14 and Training Grant AI-0005-11 and in part by the Stieptococcal and Staphylococcal Commi~sion of the Armed Forces Epidemiological Board. 195

196

H. S. LAWRENCE

C. Leproinatous Leprosy . . . . . . . . . D. Hazards of Reconstitution with Intact Immunocompetent Cells E. Possible Applications of Dialyzable Transfer Factor . . X. Transfer Factor, Immunological Surveillance, and Tumor Innnunity XI. Conclusion . . . . . . . . . . . . References . . . . . . . . . . . .

I.

. .

. .

.

.

255 256 256 258 259 261

Introduction

Delayed-type hypersensitivity or cellular immunity, as this category of immune responses is now designated, has become the recent object of intensive scrutiny in the laboratory and in the clinic. This concentrated attention has resulted in the cataloguing of meaningful experimental data at an exponential rate. Cellular immunity is thus rapidly emerging from the obscurity and confusion which had for so long been its particular fate. Progress in rescuing this class of reactions from the ambiguous position into which they had fallcn, was hampered by the critical lack of an immune reagent, akin to an immunoglobulin, to measure. This hard reality resulted in difficulties in the conceptual as well as in the practical approaches to the problem. Compounding this impediment, and perhaps a more serious defect, was the failure of delayed hypersensitivity to conform to any of the successive theories of antibody formation or central dogma which captured popular attention at any given moment. The ambiguity which dogged this area of investigation is understandable in view of its slender qualifications as an immune response-namely, the requirement for prior specific sensitization. The added inadequacies posed by scoring an indolent, inflammatory, cutaneous reaction as the final arbiter of the event, had also contributed to this dilemma (Chase, 1965a; Eisen, 1967; Lawrence, 1956; Pappenheimer, 1956; Uhr, 1965, 1966; Turk, 1967; Humphrcy, 1967). Thus matters stood until the discovery of cellular transfer by Landsteiner and Chase (1940, 1942) and, subsequently, pioneered and extended by Chase ( 1945, 1953, 1965a,b, 1967). This critical observation marked a turning point in an otherwise bleak future. It provided at once the nearest approximation to an immunological reagent for analysis of mechanisms of delayed reactions and clearly separated the latter from these responses mediated by serum immunoglobulins (Chase, 1965a; Lawrcnce 1956) . Our work on transfer factor bcgan with the dcrnonstration that cellular transfer of tuberculin hypcrscnsitivity can be accomplishcd in humans using viable leukocytes obtaincd from sensitive donors ( Lawrence, 1949) . Thcse observations led to the finding that leukocyte extracts are as effective as viable cells in the transfer of delayed hypersensitivity in humans

TRANSFER FACTOR

197

(Lawrence, 1954b, 1955) which allowed the search for the identity of the material( s ) responsible for transfer factor activity to begin. It resulted in the discovery that transfer factor is a dialyzable moiety of <10,000 mol. wt. which is not immunogenic nor immunoglobulin-like in character (Lawrence et d., 1963) and has biological properties suggesting a replicating informational molecule. The latter intcrpretation may be a consequence of transfcr factor functioning as a derepressor of normal lymphocytcs. These cells when exposed to specific antigen then undergo repeated division and clonal proliferation (Marshall et al., 1!368b; Valentine and Lawrence, 1968a). Along the way our laboratory has evaluated certain in oitro correlates of cellular immunity in relation to the in vioo events mediated by transfer factor (David et al., 1964a; Al-Askari and Lawrence, 1969; Marshall et al., 1969; Valentine and Lawrence, 1969; Lebowitz and Lawrence, 1969). We have used the term “transfer factor” to designate thc specific factor or factors in leukocytes responsible for thc transfer of delayed hypersensitivity (Lawrence and Pappenheimer, 1956; Pappenheimer, 1956). The latter is a convenient descriptive term employed with the appreciation that more than one factor may be operative in the reaction. Transfer factor is used to describe either the active moiety residing in specifically sensitive, viable blood leukocytes or is applied to the equivalent activity liberated from living cells mechanically ( lysis, freeze-thawing ) or immunologically (interaction with specific antigen), as well as to the more highly purified low-molecular-weight preparation separated from subcellular constituents by dialysis and concentrated by lyophilization. The purpose of this present revicw is threefold: ( I ) to review the growing literature and evaluate the present status of transfer factor 20 years after the original observation in man; ( 2 ) to come to some understanding of thc molecular basis for and the mechanisms involved in cellular immunity, in general, and the role of transfer factor, in particular; and ( 3 ) to evaluatc the practical application of these experimental observations to the investigation and treatment of diseases charactcrized by cellular immune deficiencies. It will be helpful for purposes of orientation to outline our current view of transfer factor and its mcchanism of action at this junctuie. With this preview. the reader will be bctter equipped to interprct the meaning of the experimental data collectcd irrespective of whether viable leukocytes, leukocyte c>xtracts,or dialyzable transfcr factor are used to transfer sensitivity. Our work has raised a nunibcr of siniplc questions concerning transfer factor; namely, is it antigen, is it antibody, is it an informational mole-

198

H. S. LAWRENCE SENSITIVE LYMPHOCYTES

REAGENT

RESULT

DELAYED CUTANEOUS SENSITIVITY

AG

IN VlTRO

t

TRANSFORMATION

HEAT-LABILE

[NONSENSITIVE

1

LYMPHOCYTES

CLONAL PROLIFERATION

@'

-[ ]

INCUBATE IG

HEAT-LABILE NONDIALYZABLE SUPERNATANT MATERIAL

DELAYED CUTANEOUS SENSITIVITY

SUPERNATANT

FIG.1. Diagrammatic comparison of in wiwo and in witro activities of dialyzable transfer factor, antigen-liberated transfer factor, and lymphocytc-activating material. See also Fig. 6.

cule, or is it a derepressor of normal lymphocytes? The experimental results gathered to datc have narrowed the mechanism of action of transfer factor to two major alternative possibilities. Transfer factor may function either to convey immunological information to and/or act as a derepressor of normal lymphocytes. In either case, a select, small population of normal circulating lymphocytes following exposure to transfer factor become antigen responsive, transform, and undergo clonal proliferation. This view is derived from correlative in vivo and in vitro data which will be developed chronologically in detail below, The experimental results upon which this conclusion is based are diagrammatically detailed in Fig. 1. In this current view, attention is paid to transfer factor as a dialyzable low-molecular ( <10,00O)-weight moiety that is not antigenic and is not an immunoglobulin or a protein (Lawrence et al., 1963). By exclusion, the main candidates for this activity are polynucleotide and/ or polypeptide chains in the dialyzate. The biological activity of dialyzable transfer factor has suggested properties that are usually associated with informational molecules. Nevertheless, it has begun to appear more likely from recent data, that transfer factor may function as a derepressor of

199

TRANSFER FACTOR

TABLE I SOME I’ROPERTrES OF T R A N S F E R

Biochemical (TE’ imaffected I)y)

Biological Endows recipient with specific sensitivity of donor Sensitivity is systemic Onset early (hours) ; duration long (mos.-year) Minute dosage WBC effective: as little as 0.01 ml. local transfet ; as little as 0.1 ml. systemic transfer

Capacity for transfer depends on degree donor sensitivity and dosage WBC used Negative donors incapalde ICxtracts or cell-free supernatants as effective as viable cells Does not cross species barrier in vivo (1

b

FACTOR"^'

2-5’ oi 3 7 O C - 6

Inimunological

hr.

Interacts with b u t IS not iietitralized by antigen Distilled water lysi, WBC desensitized by antigeii Neg. WBC antigen + no transfer Freeze-thaw 10 cycles No detectable AB in donor WBC extract Deep freeze--5 months No detectable AB in skin or serum of recipient a t time of maximum transferred sensitivity DNase Not active sensitizatinn early onset

+

R.Nase

DNase

+ trypsin

Not passive sensitization long diiration Repeated test with antigen may increase ititeusity and duration of t,ransferred sensitivity-yet is not necessarily it,s cause

Ileprodiicetl from Lawrence (19601,). Tic, transfei factor; IYBC, white 1)lood cell\; AB, antibody.

normal lymphocytes which, upon subsequent exposure to antigen, undergo transformation and clonal proliferation. In order to understand how this current view has evolved a detailed survey is required of the experimental data upon which it is based. This is summarized in Table I, reproduced from an earlier summary which is still applicable (Lawrence, 1960) and brought up to date by tabulating additional properties of dialyzable transfer factor in Table I1 (Lawrence et al., 1963). II.

Definitions a n d General Principles

A. SELECTION OF DONORS

I have discussed elsewhere the need to choosc leukocyte donors from among noiinal indivicluals who have donated blood for clinical use and have not transmitted hepatitis (professional donors). This obvious hazard of cell transfer in humans can be eliminated by this rigorous selective

200

H. S. LAWRENCE

T..IBLE I1 SOME I’ROPERTTES O F n I A L Y Z h B L E ‘rR.\NSFER

Biological Same properties as extract: I’rornpt onset (I1orus) I A J I duration I~ ( >1 yr.) Eqiial intenshy

Biochemical

Small qirmtifies 4 magnified effects

Immiinologicitl

Soliible, tiialyzihle, l yophilizalh

Not immunogenic

< 10,000 mol. wt. No prtrtein, albiimiii, U - or 7-globiiliii Orcinol-positive

Dihsociable from t raiispln I itation nntigens

ACTOR"

Pol ypep t itle/polynurleotide composition

Imrmu:ologically spevific Converts normal lympliocytes in vttro and in vico to aritigen-responsive state Transformation and clonal proliferation of cowerted lymphocytes exposed to antigen

Iunctivatetl at 56T30 min. Iiesists pancreatic Inforrnat.iona1molecule/derepressor/receptor site? 1XNase Retains potency-5 yr.

a Data from Lawrenre P.! (11. (1963), Lawrerice a i d Zweiman (1968); Rapaport r l ul. (196.5a); Marhhall ct nl. (1969); arid Valentine aiid LtLwietice (1968a, 1969).

dcvice. The next criterion for an effective donor is possession of intense delayed skin reactivity to the specific antigen under consideration. Naturally, the donor should not be afflicted with the disease relevant to the specific antigen ( e.g., tuberculosis and coccidioidomycosis) or with other diseases.

B. SELECTIONOF RECIPIENTS The recipient should ideally be absolutely rather than relatively negative to the test antigen. We have arbitrarily excluded as recipients individuals with any trace of erythema or induration, no matter how trivial or evanescent. Most recipients were deliberately tested on more than one occasion bcfore transfer, particularly when tuberculin or coccidioidin was used but not with more potent antigens such as diphtheria toxoid. There has been criticism of testing recipients before transfer with the implication that this somehow induces active sensitization or prepares thc recipient to respond to transfer factor (Bloom and Chase, 1967; Turk, 1967). This intcrpretation may arise from the expcricnce with guinea pigs, where repeated testing with tuberculin [purified protein derivative ( PPD ) ] does, indeed, induce delayed-type skin reactions-frequently

TRANSFER FACTOR

201

as early as at the application of a second test. However, in humans, it has been deliberatcly deinoiistrated that although repcated testing with tuberculin may elevate a latent sensitivity, it does not ordinarily by itself actively inducc a statc of delayed hypersensitivity (Furcolow et al., 1941). W e went out of our way to demonstrate that this principle applics for coccidioidin as well ( Rapaport et nl., 1960b).

C. PROTOCOL The experimental principle utilizes concentrates of leukocytes obtained from heparinized venous blood by sedimentation. The leukocyte preparations may then be washed and treated in a variety of ways. Thc leukocytes may be frozen or frozen and thawed and treated with deoxyribonuclease (DNase) and stored in the deep freeze without loss of transfer factor potency for at least 4 to 5 years and, therefore, presumably indefinitely. Alternatively, such extracts may be dialyzed and the lyophilized dialyzate stored for 5 years at refrigerator temperatures without loss of potency upon subsequent transfer. This stability of the material, aside from the operational advantages, allows aliquots of transfer factor to be prepared from the same donor on repeated occasions and stored until ready for use.

TRANSFER D. METHODOF SYSTEMIC The technique of systemic transfer has been the method most generally employed by ourselves as well as by others. In this technique, leukocytes, leukocyte extracts, or dialyzates containing transfer factor may be injected either intradermally, subcutaneously, or intramuscularly into the delfoid region; the route of injection is of no consequence. The test with antigen is then performed intradermally over the volar surface of the opposite forearm, usually 18 hours later, and the delayed reaction evolves exactly as observed in naturally sensitive individuals.

E. METHODSOF LOCAL TRANSFER The injection of antigcn into a prepared site is a modification of the Prausnitz-Kiistner technique ( Metaxas and Metaxas-Buhler, 1948) : leukocytes, leukocyte extracts, or dialyzable transfer factor are injected intradermally into the volar aspect of one forearm; 24-48 hours later, antigen in one test is injected intradermally atop the leukocyte depot and another simultaneous injection of antigen is placed in an unprepared skin site on the volar surface of the opposite forearm; then the delaycd reaction evolving in each site is recorded in the usual fashion. We have only scored the reaction in the remote site when local transfer is used,

202

13. S. LA-NCE

because of thc conb-ibution of the leukocytes to the inflammatory response locally (i.e., normal lymphocyte transfer reaction). This contribution is eliminated when dialyzable transfer factor is used to prcpare local skin sites (Lawrence and Zweiman, 1968). The preincubation of sensitive leukocyte extracts with specific antigen is a technique which provides instant transfer of delayed sensitivity (Rapaport et al., 1960a). A remote site is also tested simultaneously with antigen intmdermally, usually the volar surface of the opposite forearm, to detect systemic sensitization. Again, in our studies we have scorcd only the reaction in the remote site as the end point. Unless otherwise stated the literature reviewed below has generally employed the method of systemic transfer detailed above. Ill.

Transfer of Delayed Hypersensitivity with Viable Blood Leukocytes

There is now an extensive literature on the transfer of delayed hypersensitivity using viable blood leukocyte preparations. It will be instructive to review selected aspects of these accumulated data while keeping in mind the fact that the cells used are vehicles of transfer factor, from which transfer factor is dissociable as a dialyzable, low-molecular-weight moiety which is not antigenic and is not antibody. Evidence will be discussed below to demonstrate that cells bearing transfer factor may be rejected via a homograft response, but transfer factor itself is not rejected. A.

QUANTITATIVE VARIABLES

The earliest cell transfers in man underlined certain principles which are still operative and of importance to the success of the undertaking (Lawrence, 1949, 1952). From the outset it has been evident that transfer of delayed hypersensitivity is an all-or-none response-either a delayed reaction is transferred or the recipient remains negative to the antigen. This principle is most evident when cells from a nonsensitive donor are used; with rare exceptions no transfer of sensitivity occurs, and no amount of repeated testing with antigen can make up for this deficit. The two most critical variables governing successful transfer of sensitivity are ( 1 ) the intensity of the cutaneous reaction expressed by the leukocyte donor and ( 2 ) the dosage of leukocytes (transfer factor) employed, If one deliberately chooses a donor with minimal or moderate degree of skin sensitivity to the specific antigen under study, the recipient either may not respond at all or, if sensitivity is transferred, it is likely to be feeble in intensity and of short duration. To achieve a substantial degree of sensitivity which is enduring in the recipient, it is necessary to

TRANSFER FACTOR

203

choose a donor with an intense degree of cutaneous sensitivity and to cmploy an adcquatc> volunic of cclls. Of coiirsc, there is evidence for variations in the rccipicwt or “rc~sponder”using cqual volumes of cells from the same donor, but this is a retrospective impression rather than an cxtensivcly documcntcd, prcclictable fact ( Lawrcncc, 1959a; Wanviek et al., 1960; Jeiisen et a]., 1962). The role of cell dosage‘ is illustratcd by the finding that 0.1 nil. (85 x 10‘’) cells were sufficient to transfer tuberculin sensitivity to each of two recipients, whereas 0.05 ml. (4.2 x 10:) cells obtained from the same donor and given to each of the two same recipients had previously failed to transfer sensitivity. Thus, although 0.05 ml. packed cells from a very sensitive donor are insufficitwt to transfer tuberculin sensitivity by the technique of systemic transfer, double this amount (0.1 ml.) of cells is effective. It is of interest that prior tissue sensitization using leukocytes from the same donor to the same recipicnt did not block the transfer despite the homograft response to the cells transferred on the second occasion ( Lawrenccx, 1919). The critical role of cell dosagc and dilutional factors when small numbers of cells are used is underlined by our experience with the technique of local transfer (i.e., where antigen is injected atop leukocyte depots in the forearm rather than in a remote site). In this situation as little as 0.01-0.02 ml. (8.5-17 x loG)leukocytes when challenged with antigen result in transfer of intcwsc local as well as enduring systemic sensitivity. The engagement of the few antigcn-responsive cells, in the entire leukocyte population, by antigen results in an amplification of the total response. By this local tcchnique, one-tenth the voluinc of cells usually required for transfer of sensitivity when the systemic inethod is used, are now effective. The juxtaposition of antigen and sensitive cells (transfer factor) thus magiiifics the expression of sensitivity with this system tenfold. This finding is a general one and has been documented in the transfer of coccidioidin scnsitivity and skin homograft sensitivity as well ( Rapaport et al., 1960a; Lawrence et d., 1960). In addition to the demonstration that an adequate dose of cells from a sensitive donor is obligatory, it was also shown that cells obtained from negative tuberculin reactors were incapable of transferring sensitivity as was serum obtained from a positive reactor. The latent period between ‘Cell dosages in our reports have been given in milliliters of packed leukocytes, where each 0.1 ml. is equivalent to ca. 85 X lo6 cells. With the fibrinogen method of sedimentation, the concentrated cell pellet generally contains 7070 lymphocytes and other mononuclear cells. Where cell dosage is given in iniililiters, a conversion value of 85 X 10“ cells per 0.1 ml. will also be given for uniformity.

204

H. S . LAWRENCE

the injection of transfer factor and appcarance of remote cutaneous sensitivity is gencrally 1&24 hours. However, in one recipient, a negative screening tuberculin test site bccame positive within 8 hours of injection of transfer factor in the shoulder. A similar short latent period ( 4 hours) was also observed in a screening test site when diphtheria toxoid sensitivity was transferred by means of leukocyte extracts injected into the shoulder. (Lawrence and Pappenheimer, 1956). Following the transfer of tuberculin sensitivity in humans, a similar transfer of delaycd sensitivity to streptococcal proteins ( bacterial cells: SK-SD; M-substance) was undertaken, and the rcsults followcd the pat tern of those achieved with tuberculin as an antigen (Lawrence 1952, 1954a). In addition, a rcverse local transfer effect was observed whereby the quiescent intradermal depots of leukocytes in the shoulder flared up with tuberculin-type reactions 5 days after transfer and coincident with an intense reaction to a test with streptococcal hl-substance in the forearm. Since these initial reports on the cellular tr,ansfer of delayed hypersensitivity in man, the general applicability and unifoiin reproducibility of the observations reported have been subjected to extensive confirmation. The ease with which delayed hypersensitivity has been transferred in humans to a variety of bacterial, fungal, viral, protein, and transplantation antigens emphasizes the unusual responsiveness of man to transfer factor. This conclusion is reinforced by our own cumulat.we experience over the past two decades, notable for the regularity of successful transfer accomplished in 143 out of 152 consecutive attempts. This represents an incidence of 94%successful transfers. In our initial work we had turned our attention to the evaluation of leukocyte extracts in the transfer of delayed hypersensitivity ( Lawrencc, 1954b, 1955). However, the literature detailed below continued to use viable leukocytes for transfer and will be reviewed from this aspect, even though the results are the same whether intact cells, leukocyte extracts, or dialyzable transfer factor are uscd. Using the technique of local transfer, Urbach et nZ. (1952) confirmed the occurrence of prompt local as well as cnduring systemic tuberculin sensitivity in normal individuals. However, when this tcchnique was applied to patients with sarcoidosis, there resulted only the transfer of local, but not systemic, sensitivity and the sensitivity transferrcd was evanescent. Extension of this approach using dialyzable transfer factor is discussed below (Lawrence and Zweiman, 1968). Good et nl. (1957) in a series of studies in agammaglobulinernic patients, transferred delayed hypersensitivity to streptococcal proteins I

TRANSFER FACTOR

205

( SK-SD) . In addition to clarifying the immunological defect in agammaglobulineinic subjects, thcy found, as we had reported earlier (Lawrence 1949, 1952), that the transferred delayed reaction appeared promptly in the recipient (48 hours after transfer) and persisted for a long time thereafter ( 4 months to <1 year). Parallel skin tests with SK-SD repeatedly applied to control subjects did not result in the acquisition of delayed rcactions to this antigen. This study also oonfirmed the lack of detectable Serum antibody in rccipients of blood leukocytes described carlier ( Lawrence and Pappenheimer, 1956) despite vigorous priming of the leukocyte donors with typhoid-paratyphoid A and B (TAB) vaccine. In addition, Good et nl. (1957) showed that contact sensitivity to DNFB could be transferred from either actively sensitized, nornial subjects or from againmaglobulineinic patients to other normal individuals. Large volumes of leukocytes (5-8.4x los) were injected subcutaneously to accomplish transfer. Good (1959) also indicated that neither 150 ml. nor 200 ml. of plasma from donors with severe delayed reactions to DNFB, when given to normal individuals, was capable of transferring contact sensitivity. He also reported a flare-up of quiescent leukocyte sites in the shoulder at the time of a positive reaction to DNFB in the forearm of the recipicnt. IVanvick et al. ( 1956) transferred delayed sensitivity to “cat scratch antigen” in normal individuals with blood leukocytes (260-1000 X 1 W ) obtained from patients recovered from this disease. A 150-cc. plasma injection from a sensitive donor was ineffective for transfer. For a variety of reasons, which need not be dctailed here, the disease is presumed to be of viral etiology. The preparations of the antigen and the delayed reaction evoked have many similarities to the Frei test for lymphogranuloma venereum, and to the Kveim test for sarcoidosis. Portcr (1957) also reported on the development of tuberculin sensitivity in an ag~~mmaglobulineniic patient following inoculation with Bacillus Calmctte-Guerin ( BCG ) vaccine. Blood leukocytes obtained from this child subsequent to vaccination, transferred tuberculin sensitivity to ii normal infant. hIoreover, despite transfer of t u b e r d i n sensitivity, no antibody to the poIysaccharide antigen of the tubercle bacillus could be detected. Braunsteiner et al. (1958) transferred to normal individuals delayed sensitivity to tularemia antigen ( tularin ) and to tuberculin. Good ( 1$159),in further studies on agammaglobulinemic and on normnl cliildrcn, actively sensitized each group with diphtheria tosoicl-ant;to\;in-sp~cific proeipitates, prepared according to thc method of Uhr et (11. (1957). Following the appearance of delayed reactions to

2of3

H. S . LAWRENCE

diphtheria toxoid, blood leukocytes obtained from cither agammaglobulinemic or normal donors were shown to transfer delayed hypersensitivity to toxoid. Delayed reactions to horse 7-globulin, following the injection of similarly prepared specific precipitates, were also actively induced in agammaglobulinemic and in normal children. Blood leukocytes obtained from both latter groups of donors transferred to normal individuals delayed hypersensitivity to horse y-globulin. When lymph node slices are empIoyed instead of blood leukocytes, both Good et a l (1957) and Martin et al. (1957a) were able to transfer to agammaglobulinemics the capacity to respond to antigen with the development of circulating antibodies. In addition, Martin et a2. ( 1957a) found that tuberculin hypersensitivity was also transferred by the same lymph node cell population that transferred the capacity for antibody response. Gitlin et al. (1959) reported on the transfer of delayed hypersensitivity to heat-killed vaccinia virus to agammaglobulinemics, using blood leukocytes obtained from normal individuals. Schlange ( 1954) had reported the transfer of tuberculin sensitivity by means of exchange blood transfusions (750-850 cc.) given to infants suffering from erythroblastossis fetalis in the first or second day of life. The blood donors were normal adults, only one of which was tested and known to be tuberculin positive. Wanvick et al. (1960) devised a carefully detailed protocol to investigate this question in depth, which resulted in several interesting and significant observations. Since infants and young children served as recipients, it was possible to establish on a large scale ( a ) the specificity of transfer and ( b ) the unlikelihood that the explanation of transfer factor activity is elevation of latent sensitivity, The 23 donors chosen exhibited various combinations of positive and negative reactions to a battery of delayed allergens such as tuberculin, SK-SD, mumps, diphtheria toxoid or tuberculin, histoplasmin, trichophytin, and SK-SD. The 28 recipients wcrc uniformly negative to these antigens. A most important conclusion established by this carefully controlled study is that newborn infants and neonates are incapable of responding to transfer factor or of expressing delaycd sensitivity following transfer during the first month of life. This result was controlled by employing split aliquots of leukocytes from the same donor and showing transfcr was effective when given to a child of 9 months, or to children 2, 3, 8, or 11 years of age, or to three adult controls. It should also be noted that the dose of leukocytes administered (2.5 x lo” to 6.4 x lo9) was in great e w e s and more than adequate to insure sncces4ful transfer, as was the combination of routes of administration employed ( e.g., exchange trans-

TRANSFER FACTOR

207

fusion alone or combined with subcutaneous and intramuscular injection of leukocytes from bone marrow and from blood). This result provided additional evidence far the conclusion (Lawrence, 1959a) that the transfer of delayed hypersensitivity is clearly not a “passive transfer” but requires a specific contribution by the host. The nature of the host’s contribution to the reaction will be discussed in detail below. Of further interest was the reinforcement on a larger scale of earlier findings (Lawrence, 1959a) that donors with negative skin tests do not transfer sensitivity, the degree of sensitivity transferred parallels the degree of sensitivity of the donor, the transfer of sensitivity is immunologically specific and the recipient acquires the exact profile and approximate intensity of delayed reactivities expressed by the donor. In addition to transferring delayed sensitivity to tuberculin, SK-SD, and diphtheria toxoid, the authors also transferred sensitivity to histoplasmin, trichophytin, and mumps antigens for the first time. In other studies, Kelly et nl. ( 1960) and Good et al. (1962) were unable to transfer delayed sensitivity to any of 13 patients with Hodgkin’s disease, whereas 22 of 23 control subjects injected with blood leukocytes developed delayed reactions to one or another of the antigens to which the donor \vas sensitive. Six of the 22 normal recipients who responded to transfer factor had received an equal aliquot of leukocytes obtained from the same donor as did 6 of the Hodgkin’s disease patients who failed to respond. Becker et nl. (1961) noted that 5 of 6 normal subjects developed delayed sensitivity to ragweed extract following injection of this antigen in adjuvant ( Arlacel A-Drake01 emulsions). Employing blood leukocytes obtained from 3 of the sensitized indilliduals, these authors demonstrated local as wcll as systemic transfer of delayed sensitivity to ragweed antigen in 4 individuals. Extending these observations, Slavin et nl. (1963) studied patients with natural ragweed allergy who uniformly failed to develop delayed sensitivity to ragwced after injection of this material in emulsion. They found that blood leukocytes, obtained from 4 of 10 such naturally sensitive subjects in this category, transferred delayed ragweed sensitivity to normal individuals. Again 24-hour skin biopsies of transferred delayed reaction sites revealed marked perivascular infiltration with mononuclear cells, Moreover, when blood leukocytes were obtained from 20 patients with immediate allergy to ragweed, 14 of whom had prolonged treatment with aqueous ragweed extract, no transfer of sensitivity resulted. Fazio and Calciati (1962) in their attempt to transfer tuberculin sensitivity to patients with Hodgkin’s disease used as leukocyte donors pa-

208

H. S. LAWRENCE

tients with inactive tuberculosis who were exquisitely sensitive to tuberculin. They injected 0.5 ml. packed leukocytes (ca. 425 X loGcells) intradermally and subcutaneously into each of 10 normal recipients and into each of 7 patients with Hodgkin’s disease. Each of the normal recipients developcd systemic tuberculin sensitivity ( 15-30 mm. ), whereas each of the Hodgkin’s disease patients failed to develop sensitivity. Of particular significance was the failure to detect any evidence of local sensitivity when tuberculin was injected atop intradermal depots of leukocytes in 4 of the 7 patients. They record only one questionable reaction ( 5 mm.) in one patient of the latter group. These results, particularly those obtained by challenging the local leukocyte site with tuberculin, confirm the earlier report of Kelly et 02. (1960) and suggest that the anergy in Hodgkin’s disease is complete. More recent attempts by Muftuoglu and Balkuv (1967) to transfer delayed hypersensitivity to patients with Hodgkin’s disease have been equally unsuccessful. Their results supply additional confirmation of those of Kelly et al. (1960) and of Fazio and Calciati (1962) in that 21 of the 22 patients with Hodgkin’s disease failed to become reactive to tuberculin following transfer. Only one Hodgkin’s disease patient exhibited a questionable degree of sensitivity (6 mm.), whereas 9 of 10 normal recipients developed tuberculin sensitivity following transfer. Moreover, 3 of 4 patients with acute leukcmia acquired tuberculin sensitivity following transfer as did the 2 patients with chronic myeloid leukemia. Additionally, 3 of 4 patients with lymphosarcoma and 2 patients with carcinoma also developed tuberculin sensitivity following transfer, but the single patient with reticulum-cell sarcoma failed to do so. Of interest, however, is the observation that each of the individuals in this latter group who failed to develop tuberculin sensitivity following transfer (1normal, 1 acute leukemia, 1 lymphosarcoma, and 1 reticulum-cell sarcoma) were on maintenance doses of cortisone, whereas 6-mercaptopurine (6 MP), nitrogen, mustard, busulfan, or X-ray therapy was given to the patients who did develop sensitivity. From the minimal size of the reactions transferred to this latter group of recipients, these dmgs may have depressed the intensity of the response, Steroid administration has bcen shown to inhibit the expression of delayed sensitivity whether acquired activcly or following cell transfer both in guinea pig (Cummings and Hudgins, 1952) and in humans (Kirkpatrick et nl., 1964). These results clearly separate the patients with Hodgkin’s disease, characterized by absolute anergy, from the other lymphomatous diseases by virtue of a responsc in the latter, howcvcr feeble, to transfer factor (cf. Editorial, 1967).

TRANSFER FACTOR

209

13. TRANSFER WITH “LYMPHOCYTES”

Slavin and Garvin (1964) extended their earlier observations on the transfer of delayed hypersensitivity to pollen antigens (ragweed, timothy) from actively sensitized donors to normal recipicnts. These authors separated lymphocytcs from granulocytes and plateIets by passing blood through a siliconized glass bead column. The final cell suspensions separated from erythrocytes by polyvinylpyrrolidone (PVP) and washed 3 times, contained 94-98% lymphocytes. When such “pure” lymphocyte suspensions were injected into negative recipients, transfer of the respective delaycd reaction resulted in 8 of 12 attempts. The dosage of lymphocytes employed ranged from 65 to 150 x loGcells. A most important finding of this study was the demonstration that lymphocytes or mononuclear cc~11salone are sufficient for the transfer of delayed hypersensitivity and granulocytes or glass-adherent cells are not obligatory for this effect. The results achieved also emphasize again the immunological specificity of cellular transfer and the criticaI role played by the dcgree of donor sensitivity as well as the dosage of cells used in determining a positive result. In a study evaluating the ‘hormal lymphocyte transfer” reaction in patients with advanced malignancy, Hattler and Amos (1965) studied the response to the cellular transfer of delayed hypersensitivity in 18 patients. They used 90-98% pure blood lymphocyte suspensions for transfer, prepared by a nylon column technique, in dosages ranging from 2.5 x 10” to 6 x loGcells. The donors possessed varying patterns of positive or negative reactions to mumps, trichophytin, candida antigen, tuberculin, and histoplasmin. The recipicnts were unifoi-mly unreactive to this battery of antigens. The authors observed that only the sensitivities possessed by the donor wcre transferred to the recipient in every instance. Their results confirm once again the finding of a direct correlation between the intensity of the reaction to a specific antigen expressed by the donor and the acquisition of, as well as the intcnsity of, the reaction expressed by the recipient. Moreover, the immunological specificity of transfer factor was again rc-emphasized by the finding that in 17 of 18 instances the specificity was complctte and the recipients reacted or remained unreactive in thc exact pattern dictated by thc multiple antigenic spccificities encountcwtl in thcb donor ( e.g., donor mumps positive, trichophytin positivc, tu1,erculin ncgativc-recipient mumps positive, trichophytin positive, tuberculin nrgativc) . Four attcmpts at local transfer with frozen and thawed lymphocytcs wcrc reported to be unsucces5ful in these patients, whereas viable lymphocytes from the same donor tested with the

210

H. S. LAWRENCE

same antigen were reactive. Since normal individuals do develop local as well as systemic sensitivity when sites of leukocyte extracts are injected with the specific antigen (Rapaport et al., 1960a) the latter finding suggests that the cancer patients studied merely loaned their skin to reveal an interaction between viabIe cells bearing transfer factor and antigen. This conclusion is reinforced by the uniform failure of these patients to develop systemic sensitivity. The meaning of this type of result will be discussed in detail below (see Section VIII).

c.

TRANSFER OF CONTACT

SENSITIVITY-A

SPECIAL

CASE?

The variable results achieved in attempts to transfer contact sensitivity stands in contrast to the repetitive ease with which transfer of bacterial, fungal, and viral types of delayed hypersensitivity is accomplished. This is not a new development, since the same vicissitudes characterized earlier reports of successful transfer of contact sensitivity in humans, when antigen-induced blister fluids were used as the source of “cells” in the Urbach-Koenigstein technique (reviewed in Urbach and Gottlieb, 1946; Lawrence, 1959a; Bloom and Chase, 1967). Following the report of successful transfer of tuberculin sensitivity with blood lcukocytes in humans (Lawrence, 1949), Baer and Sulzberger (1952) attempted transfer of contact sensitivity by this means with variable results. Of 27 recipients, 21 remained negative, 4 became positive, and 2 exhibited doubtful reactions. In a subsequent similar attempt, Baer et al. (1952) reported only 3 positive results in 66 recipients of sensitive leukocytes. Haxthausen (1947, 1952, 1953) in a series of attempts at transfer of contact sensitivity in humans also achieved consistently negative results. In one study, Haxthausen (1952) reported only 1 positive result in 66 recipients of leukocytes from contact-sensitive donors. The validity of these negative results is questionable, since he was also unable to transfer tuberculin sensitivity using the same protocols in twelve consecutive attempts. We have discusscd previously Good’s transfer (Good et al., 1957) of sensitivity to DNFB from both sensitized agammaglobulinemic and normal donors to negative recipients. Epstein and Kligman (1957) reported on the successful leukocyte transfer of contact sensitivity to three chemicals: pentadecylcatechol ( PDC ) , p-nitrosodimethylaniline (NDMA), and dinitrochlorobenzene (DNCB). Contact sensitivity to PDC seemed easier to transfer than that to NDMA or DNCB. In their study, transfer of sensitivity to PDC was reported successful in 17 of 19 attempts; the sensitivity was systemic in distribution and persisted from 5 to > I 4 months in 5 recipients. The authors achieved transfer with

TRANSFER FACTOR

211

blood lcukocytes; blood transfusions; leukocytes in blister fluids; and blister fluids from which the Ieukocytcs had been removed by centrifugation. More recently, Harber and Baer (1961) again attempted to transfer contact sensitivity to DNCB in humans via blood transfusions without success (see Mohr et al., 1969, for transfer of tuberculin sensitivity by this means). Freedman and Fish (1962) have reported on the transfer of delayed sensitivity to procaine by means of blood leukocytes. Supernatants of leukocyte extracts prepared by sonic, vibration were only effective in the transfer of procaine sensitivity in one instance where the same donor’s viable cells were also effective. Leukocyte extracts obtained from two additional donors whose viable cells failed to transfer were also found ineffective. One questionable transfer ( 5 x 8 mm.) occurred using supernatants prepared from frozen and thawed leukocytc extracts obtained from a donor whose viable cells had failed to transfer sensitivity. Brandriss ( 1968) using preparations of dialyzable transfer factor obtained from donors naturally sensitive to tuberculin and actively sensitized to DNCB obtained similar results for contact sensitivity. He failed to transfer DNCB sensitivity to each of 8 recipients of dialyzate from the doubly sensitive donors, whereas 4 of the 8 recipients of this group developed tuberculin sensitivity following transfer. The method of transfer was systemic and the DNCB sensitivity was tested by patch test. Thus, the cumulative experience with repeated attempts to transfer delayed hypersensitivity of the contact type is quite variable. It is evident that the transfer of contact-type sensitivity is difficult or may be impossible in humans and yet so easy and predictable in the guinea pig (Chase, 1954). This spccies difference is ironic in view of the ease with which blood transfusion, leukocytes, leukocyte extracts, supernatants, or dialyzates transfer bacteria1, protein, fungal, or viral types of delayed sensitivity in man as compared in the guinea pig (Lawrence, 1959b). There are certain differences in contact vs. bacterial type of delayed hypersensitivity, however, which may play a role in this regard. The nccd to sensitize actively the prospective leukocytc donor to the simple chemical under study prior to transfer of sensitivity, represents a departure from the situation encountered in delayed hypersensitivity to bacterial, fungal, arid viral antigens. In the latter instances the leukocytc donor is taken as he is without further sensitization. Moreover, the sensitivity being transferred has resulted from an earlier infection with a replicating intracellular microbe contrasted to a finite quantity of antigen applied to the skin. The importance of these or other unknown circumstances in controlling the outcome of transfer of contact sensitivity can only be

812

H. S. LAWRENCE

postulated at this time and the cxact role of each cannot be designated with certainty.

D. TRANSFEI< OF TuuEn<:ULIN SENSITIVITY VIA BLOODTRANSFUSIONS It will be recallrd that Schlange (1954) claimed to have transferred tuberculin sensitivity to erythroblastotic infants in the first few clays of lifc following exchange blood transfusions ( 750-850 cc. ) . However, Warwick et d. (1960)were unable to transfer delayed sensitivity to a battery of antigens up through the first month of life despite use of a large volume of cells (2.S x lob to 6.4 log) and a variety of routes of injection including exchange blood transfusions alone or in combination with subcutaneous and intramuscular injections of packed leukocytes. This result stands in contrast to their successful transfers in recipients 9 months of age or older. More recently, Mohr et al. (1969) in a carefully controlled study demonstrated the transfer of tuberculin sensitivity to 5 tubcrculin-negative surgical patients who had each received a whole blood transfusion ( 500 ml. ) from tuberculin-positive donors. The authors also report the transfer of tuberculin sensitivity by mcans of plasma alone to a tuberculin-negative patient with chronic lymphocytic leukemia. The transferred reaction became negative 1 month aftcr the plasma transfusion, when the patient was treated with chlorambucil and prednisone. Since transferrcd tuberculin reactions are indistinguishable from those acquired following natural infection, the authors sensibly suggest that candidates for prophylactic isoniazid therapy on the basis of conversion to tuberculin positivity, be queried about receiving blood within the preceding year or two. We would strongly endorse this advice on the basis of the following properties of transfer factor alone: ( 1 ) stability during storage (8 hours at 37"C., 5 years frozen, 5 years lyophilized), ( 2 ) its resistance to exogenous [DNase, ribonuclease ( RNase) , trypsin] and endogenous ( lysosoma1 hydrolases) enzymes, ( 3 ) liberation from sensitive cells in the presence of antigen or when cells are slightly damaged physically (the latter property suggests an explanation of the capacity of plasma alone to transfer scnsitivity as noted in this study), and ( 4 ) the regularity with which the transferred scnsitivity is found to persist for 1 to 2 years ( Lawrence 1959a, 1960b).

x

OF DELAYED REACTIVITYTO KVEIM ANTIGEN E. TRANSFER

The Kveini rcaction has many of the temporal and histological characteristics of a uniquely indolcnt, delayed reaction and is usually only

TRANSFER FACTOR

213

dctccted in paticnts with sarcoidosis in an activc stage of the disease. Normal individunls do not icact to Kvrim antigen and thc latter thus derives its vidiic as a cIi‘~gno\tictest specific for sarcoidosis (Cliaw, 1966; Isracl, 1965). Lelxicq and Vcihaegcn ( 1963) have repoitecl on thc transfer of positivc, Kvcim reactions from 5 sarcoid paticnts to 20 normal individuals b y nicans of blood leukocytes. The 5 leukocytc donors were Kveim-positive patients with clinical manifestations of active sarcoidosiy. Each of the 20 recipients developed a small nodule at the Kveim tcst site following tranqfer. A biopsy of each nodule, which devcloped at the site of the tiansferred Kveim reaction, was done 1 month after injection. Histological excmination of the biopsied nodules revealed an epithelioid granuloma in each; 15 nodules reproduced thc exact picture seen in Kveim-positive sarcoid patients, and 5 nodules were described as “sarcoidlike” with scattered epithelioid cells and occasional giant cells, lacking the true aspect of a sarcoid granuloma. Attempts to transfer Kveim I C actions with scrum are reported negative. The samc Kveim antigen was given to a control group of 104 normal subjects who had not received leukocytes. A sarcoid nodule was detected in 3 of these individuals. Thus of 20 normal recipients, 15 developed typical and 5 atypical sarcoid nodules following transfer with Kveim-positive leukocytes. In the above experiments, an unspecified number of recipients were tested with Kveim antigen alone and some with Kveim antigen plus aluminum hyclroxide suspension as adjuvant. Since the aluminum hydroxide could be questioncd as contributing to the granulomatous reaction, Lebacq and Verliaegen ( 1961) repeated the above experiments using only Kveim antigen alone to test the recipients. In this experimental protocol, 6 Kveim-positive patients with active sarcoidosis served as lcukocyte donors and 10 noimal subjects wcre selected as recipients. The dosage of leukocytes obtained was 0.5 ml. (ca. 425 x 10‘) prepared from 100 ml. heparinized blood donated by each of the 6 patients. Two preparations of Kveim antigen were employed for testing; a gauze filtered suspension of ground sarcoid lymph nodes and a sonicated suspension cleared by centrifugation and clefatted with ether. Both preparations gave strongly positive Kvcim reactions in 3 patients with sarcoidosis. Only 1 of the controls showed evidence of granulomatous reaction on biopsy. In this repetition of their original observations, using Kveim antigen without adjuvant, 4 of 8 recipients of Kveim-positive leukocytes dcveloped typical histological evidence of a positive Kveim test, and 1 recipient developed an atypical Kveim reaction. Tuberculin-positive, Kveim-negative leukocytes did not transfer Kveim reactions on two occasions. Thus,

214

1% S. LAWRENCE

the second study of Lebacq and Verhaegen (1964) appears to confirm the initial observations without the nagging doubts raised by the possible contribution of aluminum hydroxide to the granulomatous nodules observed in the recipients of the first study. Recently, Behrend et al. (1968) have reassessed this question and report confirmation of the results reported above. These authors employed purified lymphocyte ( 95%) suspensions obtained from Kveimpositive patients with active sarcoidosis. They used the technique of local transfer injecting 0.1 ml. Kveim antigen intradermally into the volar surface of the left and the right forearm of 11 normal recipients. They used small dosages of lymphocytes (6-8 x lo6 equivalent to ca. 0.01 ml. packed cells) and injected viable cells intradermally adjacent to the Kveim antigen in the left forearm. The Kveim implant on the right forearm was similarly infiltrated with Hank's solution alone. Five control subjects were injected with lymphocytes alone. The authors describe the evolution of a normal lymphocyte transfer (NLT) reaction in recipients at 24 to 72 hours. However, this reaction disappeared, and 42 days later small nodules developed (1-4 mm.) in the 11 recipients only at the Kveim antigen site where sensitive lymphocytes had been juxtaposed. Histological examination of the biopsied nodules revealed that 6 of 11 recipients exhibited classic Kveim reactions described as typical epithelioid granuloma with giant cells without necrosis. The histology in 2 recipients was cquivocal or atypical and, in the remaining 3 recipients, was read as negative, with only lymphoid perivascular infiltrates. Transfer was attempted in 4 additional recipients using sonically disrupted lymphocytes and the same experimental protocol without success. When biopsied, histological examination of nodules in 3 of these recipients was read as negative. The fourth recipient had developed no reaction. Thus, of 11 attempts with viable lymphocytes, an unequivocal Kveim reaction was transferred to 6 recipients and an equivocal reaction developed in 2 recipients. Four attempts to transfer Kveim reactivity using lymphocyte extracts were unsuccessful. The authors interpret the 48-hour reactions as normal lymphocyte transfer reactions. Since the transferred lymphocytes may be rejected b y just such a homograft response long before the Kveim nodule appears, 42 days later, they correctly conclude that the host cells have been sensitized by donor lymphocytes. Taken together the results of the two studies of Lebacq and Verhaegen (1963, 1964) and the above results of Behrend et aZ. (1968) give cumulative weight to the conclusion that cellular transfer of the Kveini reaction from sarcoid patients to normal individuals is, indeed, possible.

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215

These observations tell much with regard to the pathogenesis of the lesions in sarcoidosis and thc origin of the cellular immune dcficiencies to commonly encountered antigens that characterize this diseasc. This topic will be developed in greater detail below ( scc Section VIII).

F. TRANSFER VIA RENAL TRANSPLANTS In an extensive and well-controlled study of the immunological status of donors and recipients of renal transplants, Kirkpatrick et al. (1964) reported the transfer of delayed hypersensitivity to 18 consecutive recipients of renal allografts. Each recipient acquired the delayed responsiveness with the identical specificity possessed by the kidney donor. Donors and recipients were skin tested preoperatively with a battery of six antigens (tuberculin, histoplasmin, coccidioidin, mumps, Candida, and trichophytin) . The 18 recipients of renal allografts who were not receiving steroids were retested with the same battery of antigens postoperatively. The majority werc retested within 24 hours of surgery and all by the twelfth postoperative day. The authors found that each of the 18 recipicnts reacted to at least one antigen to which he had been previously nonreactive, and the transferred reaction corresponded to the specific reaction detected in the donor in each instance. Of the total of 49 positive reactions detected in the donor group, 40 were transferred to the recipients. In the donor group, the mean diameter of the positive reactions detected was greatest to mumps, trichophytin, and Candida and the frequency of successful transfer was greatest to these three antigens. The authors thus confirmed the correlation between the size of the donor’s reaction and the frequency of transfer. This seems to apply to all of thc antigens studied with the exception of histoplasmin. Histoplasmin, Candida, and trichophytin donor reactions of 1.7 cm. induration resulted in less than 20%incidence of transferred histoplasmin sensitivity compared to an 80%incidence for the transfer of trichophytin sensitivity and a 90% incidence for the transfer of Candida sensitivity. However, the reactivities to mumps and Candida which caused the most intense reactions in the donors wcre transferred most frequently. The mean diameter of histoplasmin (1.7 cm.) was twice as large as that of coccidioidin (0.8 cm.) and of tuberculin ( 0.8 cm. ), yet histoplasmin reactivity was transferred less frequently. The authors attribute the transfer of delayed hypersensitivity to at least two sources of immunocompetent cells: donor blood in the kidney vasculature, which is transfused into the recipient, and lymphatic tissue surrounding the kidney and ureter. Since the majority (13/20) of the

216

€1. S. LAWRENCE

recipients manifested the transferred delayed sensitivity on the first day after renal transplantation, the authors conclude that donor cells had rapid and easy access to the recipients circulation. The remainder of the recipients (7/20) developed delayed sensitivity 4-11 days after transplantation. Rejection of the renal transplant had no effect on established transferred delayed reactivity. The latter finding as well as the prolonged duration of transferred sensitivity observed in humans become understandable in the light of our subsequent demonstration ( Rapaport et &'., 1965a) that transfer factor is separable from the histocompatability antigens residing in leukocytes.

G. SUMMARYAND CONCLUSIONS In this section we have reviewed the extensive data collected using viable leukocytes and/or lymphocytes for transfer of delayed hypersensitivity. The potent biological activities of transfer factor have been delineated and the conditions governing the initiation and reproducibility of the phenomenon in normal and diseased individuals have been analyzed. In searching for an understanding of the mechanism of action o€ transfer factor, certain principles have emerged. Moreover, in consequence of the general uniformity of results secured in a variety of laboratories under diverse experimental conditions, certain generalizations are justified. Of prime importance in understanding the mechanism of action of transfer factor, has been the clear evidence that the phenomenon is not a passive transfer in view of the long-lived effects observed in most recipients. It is also most unlikely that active sensitization causes the recipient to acquire the delayed responses of the donor in view of the prompt appearance of transferred reactivity. This has occurred as early as 4 hours and with infrequent exceptions is usually prcsent by 24 to 48 hours after transfer. Other evidence against the interpretation of active sensitization or elevation of latent sensitivity is the failure to detect specific serum antibody in recipients of transferred leukocytes or leukocyte extracts. The consistent inability of negative reactors to transfer sensitivity and the correlation between the intensity of donor reactions and the occurrence, frequency, and/or intensity of transferred sensitivity are quantitative variables which clearly point to the donor's immunological experience as thc critical determinant of this response, rather than that of the recipient. This conclusion is re-emphasized by the exact confirmation of the specificity of the reactions in the recipicnt to the reactivity expressed by the donor, when multiple sensitivities are transferred. These consistent

TRANSFER FACTOR

217

obscrvations as well as their meaning are at variancc with the conclusion that thc entirc response is due to elevation of a Intcnt sensitivity possessed by the recipient (Turk, 1967). IV.

Transfer Factor-Characterization

and Mechanism of Action

A. INTRODUCTION OF LEUKOCYTE EXTRACTS We have seen how in most respects the results of viable cell transfer in man paralleled and confirmed thc results attained by Chase (1959, 1967) and others in the guinea pig-in particular, the obligatory need for a donor with specific sensitivity and the dependence of the intensity of the recipient’s reactivity upon both the degree of donor sensitivity and the quantity of cells transferrcd. However, it soon became apparent that the phenomenon in humans (Lawrence, 1949, 1952) was of a different order of magnitude from that observed in the guinea pig (Lawrence, 1959b; Bloom and Chase, 1967). The differences encountered in humans relate to the small numbers of cells required to transfer sensitivity to outbred recipients and the prolonged duration of sensitivity following transfer. These observations led to the discovery of another major difference from experimcntal animal species, whereby extracts of leukocytes prepared by water lysis or freeze-thawing were shown to be as effective as viable cells in the transfer of delayed hypersensitivity (Lawrence, 1954b, 1955). 1. Transfer of Bacterial Sensitivity In this work, delayed sensitivity to streptococcal M-substance was transferred using the systemic method with leukocyte extracts prepared by distilled water lysis. Of the strcptococcal proteins, M-substance ( Type 1 ) was chosen because of its poor antigenicity and relative purity (Lancefield and Perlmann, 1952; Lawrence, 1952). Delayed sensitivity to M-substance was readily transferred by lysed leukocyte extracts and followed the pattern established when viable cells were used. For rxample, leukocyte extracts prepared from 0.5 ml. (ca. 425 x loGcells) obtained from a moderately sensitive donor ( PeB) failed to transfer sensitivity. The same recipient was, subsequently, dcliberately given extract prepared from a small volume of cells (0.08 ml., ca. 65 x lo6 cells) obtained from an exquisitely sensitive donor ( F I T ) and the recipient still remained negative to M-substance. Finally, the same recipient, when subsequently given extract prepared from an adequate dosage of cells (0.4 ml., ca. 340 x l o G ) obtained from the same exquisitely sensitive donor (FIT), promptly developed marked ( 3 f ) sensitivity to M-substance. The re-

218

H. S . LAWRENCE

peated testing with M-substance alone did not cause this recipient to develop sensitivity until lie received thc required dose of transfer factor from a markedly sensitive donor. We have also taken pains in each oi' our other studies to demonstrate the temporal relationship between the acquisition of sensitivity and internally controlled, deliberate administration of an adequate quantity of the specific transfer factor (Lawrence, 1949, 1952; Lawrence et al., 1960; Rapaport et al., 1960a). Leukocyte extracts prepared by freezing and thawing through 7 to 10 cycles were found equally effective in the transfer of delayed sensitivity to streptococcal M-substance, and the results were identical with those achieved using viable cells or their lysates. Similarly, these results were confirmed when tuberculin sensitivity was transferred with frozen and thawed leukocyte extracts prepared from tuberculin-sensitive donors as a vehicle for transfer factor. Leukocyte extracts prepared from 2 tuberculin-negative donors failed to transfer sensitivity, despite repeated tuberculin testing of the recipients. Thus, results achieved with leukocyte extracts when two different methods of preparation were employed and two different delayed hypersensitivities evaluated were the same as when viable cells had been used, particularly in terms of the requirements for adequate donor sensitivity and adequate dosage of cells as well as in the prompt appcarance of transferred sensitivity (18 hours) and in its prolonged duration ( >10 months). These findings represented a major departure from prior experience and also allowed the first attempts at identification and characterization of the active principle, transfer factor, to begin. Here, then, was a nonliving preparation which when injected into an adult human endowed him with the specific delayed hypersensitivity of the donor promptly, systemically, and for prolonged periods. The dilutional aspects of this effect alone are impressive considering the minute quantity of extract effective in relation to the total surface area of the recipient plus an intraand extravascular fluid compartment of 35 liters. The magnification of the biological cff ect prompted the idea that transfer factor may be a self-replicating entity or by some other means induced the recipient's cells to make more transfer factor. To test this idea, we attempted serial transfer of sensitivity from individual A to €3 to C (Lawrcnce, 1955). The results are summarized in Table 111. As may be seen delayed sensitivity to two different antigens, using two d8erent preparations of cell extracts, is amenable to transfer from a natively sensitive donor ( A ) to a primary recipient ( B ) and, from there, to a secondary recipient ( C ). Of particular significance are (1) the prompt

TRANSFER FACTOR

219

~

(1

6

Adapted from published d:tt:t (IAWience, 195.5). \\'13C, white blood cell<.

appearancc of transfer factor in the M-positive recipient's cells, coinciding with the positive cutaneous reaction, as early as day 3 after transfer and (2) the continued presence of transfer factor in the tuberculin-positive recipient's blood cells as late as the third week after transfer. This simple experiment excludes the possibility of carryover of antigen or antigcn-RNA complexes, as well as the existence of a unique type of antibody in leukocyte extracts as explanations of the transferred sensitivity. The results also suggest more than mere random redistribution of transfer factor among the recipient's total cell populations. Operationally, it appears that the recipient's circulating cells have acquired the same characteristics of the donor cells in relation to delayed sensitivity, namely, mediation of skin reactivity and capacity to transfer such skin reactivity to another individual. From this experience, one may conclude that the recipient's cells bear transfer factor, as do the donor's cells, for at least as long as the recipient's cutaneous reaction remains positive. Rather than exclude the question of replication of transfer factor, the positive results of serial transfer favored this interpretation and fostered the conclusion that the recipient's circulating leukocytes are intimateIy involved in the process. Whether the exact locus of the initial transfer occurred in central or peripheral lymphoreticular cells could not be derived from these results. However, as will be developed below, current in uitro studies coupled with in vivo transfers offer evidence that the injected transfer factor engages a discrete population of small circulating lymphocytes in the recipient's blood (Valentine and Lawrence, 1968a). These earlier experiments (Lawrence, 1955) led to the treatment of leukocyte extracts with the specific enzymes, DNasc and RNase, in an attempt to inactivate transfer factor, should it be associated with or de-

220

H. S. LAWRENCE

pendent upon molecules known to have self-replicating potential. For this purpose tuberculin-positive leukocyte extracts were prepared from each of two donors, with a documented capacity to transfer marked degrees of tuberculin sensitivity, for subsequent transfer to 16 consecutive tuberculin-negative recipients. Eight recipients received untreated leukocyte extracts and served as controls, while 4 alternate recipients received DNase-treated extracts, and another 4 alternate recipients received RNase-treated extracts. Tuberculin sensitivity of an equivalent degree and duration was transferred to each of the 16 recipients, and it was concluded that treatment with DNase or with RNase had no effect on transfer factor. In subsequent attempts to inactivate transfer factor, leukocyte extracts were treated sequentially with DNase and then subject to tryptic digestion with crystalline trypsin; this treatment also was without effect on the transfer of tuberculin sensitivity (Lawrence, 1959a). Thus transfer factor began to emerge as a hardy material not only resistant to endogenously liberated lysosomal enzymes at 37OC., but also to the exogenous addition of DNase, RNase, and DNase plus trypsin. These results are so uniform and reproducible that all leukocyte extracts prepared for transfer in this laboratory since 1953 have been treated routinely with pancreatic DNase to facilitate handling and injection of the material. Although these data were all very interesting and suggested exciting possible mechanisms of action for transfer factor's effects, they only excluded certain possibilities for transfer factor ( e.g., a superantigen, polymerized DNA or RNA, or a tryptic susceptible protein) and suggested that continued attempts at identification and characterization would be a protracted and arduous undertaking. We turned next, therefore, to an immunochemical approach to the problem and sought to establish a relationship between transfer factor, delayed sensitivity, and circulating antibody (Lawrence and Pappenheimer, 1956).The selection of the diphtheria toxin-antitoxin system arose from extensive earlier studies of a large series of naturally sensitive individuals, which showed that minute quantities of highly purified toxin or toxoid elicited intense delayed reactions. Although delayed reactivity to toxoid was always accompanied by a high titer of circulating antitoxin, no direct correlation between the size of the delayed reaction and the titer of antibody could be made (Pappenheimer and Lawrence, 1948). In addition to employing a reasonably well-characterized and highly purified protein antigen (Pappenheimer, 1956), this system afforded a quantitative, scnsitive biological neutralization test for the cle-

TRANSFER FACTOR

221

tection of antitoxin, even when present in very low concentration ( <0.01 pg./ml.). Transfer of delayed sensitivity both to diphtheria toxoid and to tuberculin was readily achieved using DNase-treated leukocyte extracts obtained from Schick-negative donors with pronounced delayed reactions to toxoid and only moderate sensitivity to tuberculin. Nevertheless, circulating antibody could not be detected in recipients expressing delayed rcactions to toxoid nor in the skin site of the transferred delayed reaction nor in the leukocyte extracts used to transfer sensitivity. The test with tuberculin was employed as an indicator that transfer had occurred and allowed testing of recipients with toxoid to be selective and specific. The suggestion that prior tests with antigen prcparcs or conditions negative rccipients of transfer factor to develop a delayed reaction has been repcatedly raised (Bloom and Chase, 1967; Turk, 1967) and was critically examined in thir early study. It was documented that the testing of recipients before transfer had no effect on transfer of sensitivity. This was clearly the case whether the rccipient had been tested as long as 2 years or as recently as 3 days before transfer. Moreover, it was also demonstrated that delay in testing the recipient with specific antigen did not affect the transferred reaction. For example, the test with toxoid was deliberately withheld in one recipient for 19 days following transfer. The reaction transferred was maximal for that recipient and comparable to reactions elicited 19 days after transfer in two other recipients who had each been previously tested at 4 and 11 days after transfer. It was also shown that incubation of specifically sensitive blood cell populations with antigen (toxoid or PPD at 37°C. for 1 hour) failed to block transfer of sensitivity. The interaction of the appropriate antigen with specifically sensitive cells did result, however, in the libcration of the related transfer factor into the cell-free supernatant solution. Mere incubation of sensitive cells without antigen at 37°C. for 1 hour was sufficient to result in the liberation of some transfer factor into the cellfree supernatant, as shown by the transfer of sensitivity to toxoid or to tuberculin, respectively, if cells are damaged. This study indicated that delayed sensitivity occurred in recipients of transfer factor without concomitant circulating or cell-bound antibody in the leukocytes or the skin site. The failure to detect antibody following transfer with blood leukocytes was confirnied by Good et al. (1957) in agammaglobulinemic patients. The failure of blood leukocyte extracts to transfer the capacity for antibody synthesis or a secondary response has

222

H. S . LAWRENCE

also been observed by Maurer (1961) and by Rapaport et al. ( 1960a). In this regard the finding of Good et al. (1957) and Martin et al. (1957a) that the capacity for immunoglobulin synthesis can be transferred by lymph node cells to agammaglobulinemic recipients is of considerable interest. In the study by Martin et al. (1956, 1957a,b) lymph node sliccs transplanted to a patient with acquired agammaglobulinemia, transferred both the capacity to synthesize antibody and at the same time tuberculin sensitivity, Moreover, antibody synthesis persisted for only 110 days until the lymph node slices were rejected via a homograft response, whereas the transferred tuberculin sensitivity persisted considerably beyond this time and was still present when last tested at >300 days. The preceding series of observations taken together clearly indicate that either circulating blood leukocytes are entirely devoid of or scantly populated with cells that can initiate antibody formation following transfer; yet antibodyforming cells are present and functional in adequate quantities in lymph node cell populations, which also contain cells bearing transfer factor for delayed sensitivity. A further separation is indicated by the rejection of the antibody-forming cells via a homograft response from which transfer factor is exempt. The latter separation becomes understandable when viewed from our subsequent observations (Rapaport et al., 1965a),which demonstrated transfer factor for delayed sensitivity is separable from leukocytic transplantation antigens and, although the cellular vehicles of: transfer factor may be rejected via a homograft response, transfer factor itself is not. Of additional interest are other observations secured with the diphthcria toxoid system ( Lawrence and Pappenheimer, 1956) which demonstrated that interaction of specifically sensitive leukocytes with antigen (tuberculin, toxoid) not only failed to block the transfer of sensitivity but also resulted in the liberation of transfer factor from the cells to the supernatant solution. These observations suggested that transfer factor interacted with antigen, but unlike known antibody or even postulated “cell-bound antibody, transfer factor was not neutralized by antigen. Further studies with Pappenheimer on this question attempted to evaluate the specificity of the interaction of transfer factor and antigen (Lawrence and Pappcnheimer, 1957). The results of one of these experiments are diagrammatically shown in Fig. 2. Of interest is the selective release by PPD of the tuberculin transfer factor and not the toxoid transfer factor from a mixed population containing toxoid-sensitive as well as tuberculin-sensitive cells. The observations secured in these experiments indicate that cell-bound transfer

223

TRANSFER FACTOR LIBERATION OR TRANSFER FACTOR FROM SENSITIVE CELLS INCUBATED WITH ANTIGEN" NEGATIVE RECIPIENTS REACTION POST-TRANSFER TUBERCULIN

0.5 ml WBC 2 t 5 y P

P

D CELL-FREE / ~ l

3t

DIP. TOXOID

0

SPIN

f

I t

TBC TOX + +

++++

37',

I HOUR 0

EXTRACT

It

0 5 mlWBC ALONE DATA FROM LAWRENCE AND PAPPENHEIMER (1957), EACH (t)= lOxl0 m m INDURATION AND ERYTHEMA

FIG.2. In vioo activity of transfer factor liberated from sensitive cell populations by antigen i n Gitro. See also Fig. 6.

factor very likely interacts with specific antigen, and this interaction liberates the transfer factor from the cells with which it is associated. These results were confirmed when the principle derived from the in vitro experiments was applied in vivo by Oliveira-Lima (1958). He showed that leukocytes obtained from 6 donors who were tuberculin sensitive and streptococcal sensitive could transfer both streptococcal and tuberculin sensitivity to 6 recipients negative to both antigens. He next desensitized the same 6 donors to tuberculin, to the point of negative skin reactions, and showed their leukocytes could now only transfer streptococcal but not tuberculin sensitivity to 6 additional recipients negative to both antigens. 2. Transfer of Fungal Sensitivity

Since the question of elcvation of latent sensitivity inevitabIy ariscs with the use of common, although not necessarily ubiquitous antigens, such as tuberculin, streptococcal proteins, and diphtheria toxoid, we next trrrncd to coccidioidin as a test system. Coccidioidin sensitivity was selected for transfer in view of the natural

224

H. S. LAWRENCE

restriction of this fungus to isolated areas of the western portion of this country (Salvin, 1963). As a prelude to the transfer of coccidioidin sensitivity, it was demonstrated that although repeated intradermal testing with coccidioidin may elevate a latent state of sensitivity, this type of antigenic exposure does not actively induce delayed reactions to coccidioidin in negative reactors (Rapaport e t al., 1960b). Lcukocytes were collected from hcalthy coccidioidin-sensitive donors in the endemic area in California, frozen and flown to New York. Dcoxyribonuclease-treated leukocyte extracts were prepared and injected into negative recipients who were residents of the eastern seaboard (Rapaport et al., 1960a). The transfer of coccidioidin sensitivity was readily accomplished, and it persisted for as long as 1to 1% years (i.e., was still present at the end of that time, which was as long as the recipients could be followed). This study provided a unique opportunity to observe the time course of transferred sensitivity under controlled conditions for the absence of exposure to antigen in the natural environment or in the form of a skin test. For this purpose 12 consecutive coccidioidin-negative recipients were restricted to one skin test with coccidioidin ( a ) 1 week before transfer, ( b ) 1 week after transfer, and ( c ) either 1 or 1%years after transfer. It was observed that each of the 12 individuals acquired coccidioidin sensitivity following transfer. Six of this group of recipients had reverted to a negative reaction when retested at 1 or 1% years after transfer. These 6 recipients had expressed weak ( I f , 2-1) reactions immediately following transfer. The other 6 recipients who remained coccidioidin sensitive after this long interval, had expressed strong (3+, 4+ ) reactions immediately following transfer. This result clearly shows that the duration of transferred sensitivity in the absence of repeated exposure to antigen is correlated with the intensity of the reaction initially transferred. We have already indicated the latter is, in turn, a function of the intensity of the donor's sensitivity as well as the dosage of cells employed. To test the postulated presence and role of antigen carried over in leukocyte extracts used for transfer, we also incubated leukocyte extracts prepared from negative donors with coccidioidin before injection of the mixture. This treatment failed to result in the transfer of coccidioidin sensitivity. It was concluded that transfer of sensitivity to a fungal antigen had been regularly accomplished, that a good case for de novo transfer had been made, and that, although repeated exposure of transfer factor recipients to antigen may play a role, it is not in itself the cause of the transferred sensitivity. Moreover, to effect transfer of sensitivity, a specific pre-existing nioiety (and more than just a mixture of indifferent enzyme-

TRANSFER FACTOR

225

digested leukocyte extracts and antigen) is obligatory. This specific moiety must be supplied by the leukocyte populations of an individual expressing the appropriate delayed reaction or transfer will not occur. 3. Transfer of Homograft Sensitivity

We next turned to the adaptation of transfer factor to an analysis of mechanisms of skin homograft rejection in humans (Lawrence, 1957, 1965b,c, 1967; Lawrence et al., 1960). For the first time it became necessary to sensitize actively leukocyte donors with a particular individual's tissues to produce a state of deIayed hyperscnritivity (expressed as accelerated graft rejection ) . This was accomplished by actively sensitizing individual B to A's skin by four sequential exposures in the form of a homograft. The anti-A transfer factor prepared from B's DNase-treated lcukocyte extracts was then injected into a third individual C.Individual C would either already have skin homografts obtained from individuals A and D, E, or F, in residcnce, or in other protocols the grafts would be applied 2 or 3 wecks after transfer had occurred. In either case thc recipient of anti-A transfer factor reacted as if sensitized to A's tissues only (i.e., accelerated rejection of graft within 18 hours of transfer or after surviving 4 days) but not to the tissues of D, E, or F (i.e., giving first-set reactions-8-10 day survival ) . Thus anti-A transfer factor was selective in causing the host to respond as if he had met only A's skin before. Nonsensitive leukocyte extracts, leukocyte extracts from a desensitized donor, and scrum from scnsiti7ed donors, all failed to transfer accelerated homograft rejection. These studies established that ( a ) de novo sensitivity is, indeed, transferred; ( b ) transfer factor is highly spccific in promptly ( 18 hours) initiating the rejection of only the A-graft and not grafts from other individuals; ( c ) testing of recipients with antigen just before or just after transfcr is not an indispensible rcquirement for initiating the enduring effects observed; ( d ) as demonstrated for bacterial and fungal hypersensitivity, the function transferred by leukocyte extracts consists of a prompt initiator of tissue damage and a mechanism for reduplication in the recipient as in the donor; ( e ) although serum immunoglobulins are produced following activc sensitization to homografts and play a subsidiary role following organ transplantation, transfer factor alone causes rejection of homografts. Thus at least onc mcchanism of homograft rejection has been defined and analyzed in human suhjccts (Lawrence et al., 1960, 1962). These data also demonstrate that a specific transfw factor is claboratcd by the host exposcd to another individual's tissues or organs, with speci-

226

H. S. LAWRENCE

ficity directed only against that particular donor. Hence, we believe the immunosuppressive effects achieved with drugs (6 MP, steroids) and, more particularly, with antilymphocyte serum, are due to the neutralization or engagement of those few small, circulating lymphocytes which bear the transfer factor elaborated against the antigens of the individual donating thc organ (Lawrence, 1968a). Subsequent studies ( Rapaport et al., 1965a,b) designed to identify and characterize human transplantation antigens in subcellular leukocyte fractions, revealed that the material which actively induces homograft sensitivity is associated with the endoplasmic reticulum and is nondialyzable. However, the leukocyte extract dialyzate, which contains transfer factor does not have such antigenic activity. In the homograft system of delayed hypersensitivity, the antigens which actively induce sensitization are separable from the moiety (transfer factor) which can only transfer sensitivity but not actively induce it. Thus, although the cellular vehicle of transfer factor may be rejected via a homograft response, transfer factor itself is not rejected. This finding may explain the prolonged duration of transferred sensitivity in humans compared to the short duration observed in outbred experimental animals, where the transferred cell populations are rejected and their function terminated (Harris and Harris, 1960; Warwick et al., 1962).

B. CONFIRMATION OF TRANSFER WITH LEUKOCYTE EXTRACTS IN MAN As we have detailed above, our report on the transfer of tuberculin sensitivity using viable cells was accepted until the results in man began to depart from experience with experimental animals ( Lawrence, 1955, 1959b). The publication of our findings using leukocyte extracts was generally received with reservations. This guarded reception is understandable when viewed against a background of the acknowledged failure to transfer sensitivity with leukocyte extracts in animals and the bleak fate of scattered reports claiming to have done so. The checquered history and current status of the problems surrounding attempts to transfer with leukocyte cxtracts in animals is the subject of an excellent critical review by Bloom and Chase (1967) and this topic, therefore, need not be further elaborated. Nevertheless, the experience in animals notwithstanding, the claim of transference of delayed hypersensitivity with leukocyte extracts in man was confirmed in explicit detail and the observations extended in several laboratories. Freedman et al. (1957), using the technique of local transfer, compared the efficacy of viable cells with frozen and thawed leukocyte extracts and with mechanically disrupted leukocyte extracts in the transfer

TRANSFER FACTOR

227

of tuberculin sensitivity. The Ieukocyte donors were tuberculin sensitive and, in addition, had urticaria1 responses to ragweed or penicillin. Tuberculin sensitivity was transferred to four recipients who developed sensitivity at the leukocyte extract site and at remote sites as well. A singlc recipient remained tuberculin sensitive for 4 months before reversion to a negative state. Leukocytc extracts obtained from a tuberculin-negative donor failed to transfer sensitivity. Leukocyte extracts obtained from patients sensitive to tuberculin and ragweed or to tuberculin and penicillin transferred delayed sensitivity to tuberculin but not immediate sensitivity to ragweed or penicillin. Leukocyte extracts obtained from patients with anaphylactic sensitivity to horse serum failed to transfer this type of sensitivity, and precipitable or anaphylactic antibody to horse serum could not be detectcd in the leukocyte extract. The supernatants of the leukocyte extract preparations were discarded and only the sediment resuspended in saline was used. Such supernatants will contain a variable quantity of transfer factor Iiberated from the disrupted cells (Lawrence and Pappenheimer, 1956). The possible contribution of viable sensitive leukocytes, injected intradermally at control sites and not tested with antigen, cannot be assessed from the data given. Maurer's studies (1961) on delayed reactivity to ethylene oxidetreated human serum provide the most clearly documented confirmation of the capacity of leukocyte extracts to transfer delayed sensitivity. The alteration of human serum proteins by this treatment created new antigens to which neither donor nor recipient had been exposed. Thus the recipicnt was not skin tested prior to transfer. Prospective leukocyte donors were actively immunized with the antigen and developed marked delayed sensitivity but no detectable serum antibody even after repeated immunizations by intradermal test over 1?1:years. Using the systemic method of transfer and frozen and thawed leukocyte extracts obtained from sensitive donors, strong delayed reactions (20-35 mm.) were regularly transferred to 5 consecutive recipients. In one instance, delayed sensitivity to tuberculin as well as to denatured human serum albumin (HSA) was transferred. Three recipients who served as controls were given viable cells obtained from the same donors and each developed sensitivity comparable to that achieved with leukocyte extracts. Biopsy of the skin site at 24 hours in a recipient of leukocyte extracts revealed perivascular lymphocytic infiltration compatible with a delayed reaction. The absence of detectable serum antibody in the leukocyte donor did not appear to affect the transfer of delayed hypersensitivity. Additionally, it was observed that all of the recipients had retained the delayed sensi-

228

H. S. LAWRENCE

tivity transferred when tested 1 year after the initial transfer and skin test. Like the transferred cocciclioidin scnsitivity described by Rapaport et al. (1960a), transfcrrcd dclaycd reactivity to denatured HSA also persisted for a year in the absence of rcpeated cxposure to antigen in the cnviroiiment or via skin tcst. Maurer’s results are esthetically more satisfying than ours in view of the additional advantage of not having to screen the recipient with a skin test before transfer. Maurer’s (1961) study would seem to answer clearly the criticism repeatedly voiced that recipients of transfer factor may be sensitized by the pretransfer skin test. An entirely similar result had been obtained earlier in the transfer of skin homograft rejection-another system that doesn’t require testing before transfer (Lawrence et al., 1960). Jensen et al. (1962) undertook an elaborate study of the specificity of multiple transfers of tuberculin sensitivities to human, Battey, and avian strains of tuberculin, using frozen and thawed cells. Twenty-six leukocyte donors were selected with varying patterns of reactivity to PPD-S (human tuberculin), PPD-B (Battey tuberculin), and PPD-A (avian tuberculin), The leukocytes used for transfer were collected and stored frozen (-20°C.) until prior to their use (the dose vaned from 0.1 to 0.65 ml. packed leukocytes). Of 26 recipients, 18 became positive to one or more of the three tuberculins to which the donor was sensitive, and 8 recipients remained negative to all three tuberculins. In control studies of 6 recipients of frozen cells from negative reactors, 2 developed positive reactions. Upon retesting one of the “negative” donors had a weak but definite reaction to PPD-B. Histoplasinin sensitivity was transferred in only 1out of 6 attempts. Barain and Mosko ( 1962) fractionated leukocyte extracts prepared by sonication and assessed the activity of various fractions. The leukocyte extract was centrifuged at 40,000 r.p.m., the sediment discarded, and the supernatant concentrated by dialysis, preparatory to chromatography on a diethylaminoethyl ( DEAE ) cellulose column. Four fractions were separated, and each transferred a moderate degree of tuberculin sensitivity in a random manner to 11 of 22 recipients studied. The sensitivity transferred was transient, lasting as long as 2 months in only 3 recipients. Four recipients of similarly derived leukocyte fractions prepared from tuberculin-negative donors remained negative despite repeated skin tests with tuberculin. The authors conclude that repeated testing of recipients with tuberculin did not cause the maintenance of the transferred sensitive state nor induce it in recipients of negative leukocyte fractions. The moderate intensity of the reactions transferred and their short duration is a likely reflection of the mild to moderate degree of sensitivity of the

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donors selected (15 mm. or greater reaction to 0.0002 mg. PPD) and the chance distribution of variable quantities of transfer factor in each fraction prepared. Brown and Katz (1967) using frozen and thawed leukocyte extracts transferred tuberculin sensitivity from a single tuberculin-sensitive adult donor to each of 17 tuberculin-negative children. The recipients comprised a group of 5 normal children, 4 children with marasmus, and 8 children suffering from kwashiorkor. The authors concluded that malnutrition had no affect on the host’s response to transfer factor, despitc the poor response of similarly malnourished children to active sensitization by BCG vaccination or active infection with the tubercle baccillus. V.

N a t u r e a n d Properties of D i a l y z a b l e Transfer Factor

A. PREPARED FROM DNASE-TREATED LEUKOCYTE EXTRACTS Progress in the efforts to isolate and identify transfer factor was greatly facilitated by the finding that it is a dialyzable moiety of low molecular weight (Lawrence et uZ., 1963). With this simple procedurc, transfer factor was separated from all nondialyzable cell constituents of >10,000 to 40,000 mol. wt. The protocol in this study consisted of placing DNase-treated extracts of frozen and thawed leukocytes (0.3 to 0.9 ml., 255-605 loGcells), obtained from donors with the appropriate delayed sensitivity, in a Visking cellulose sac and dialyzing in a (1:1) ratio against distilled water, overnight in the cold room. The dialyzate was then filtered and injected into the shoulder of the tuberculinnegative or coccidioidin-negative recipient and the respective skin tests made in the forearm. It was demonstrated that tuberculin sensitivity was transferred to 11 recipients of dialyzable transfer factor, of which 7 developed marked ( 4f ) reactions and 4 developed moderate (2+ ) reactions to tuberculin. Treatment with 50 pg. RNase had no effect on thc capacity to transfer tuberculin sensitivity and no inhibitors to RNase activity were demonstrable in the dialyzate. The findings obtained with tuberculin were confirmed when coccidioidin was uscd as a test material. Of 11 recipients, 7 developed marked ( 4 + ) reactions and 4 devcloped moderate ( 2 + ) reactions to coccidioidin following transfer with dialyzates prepared from coccidioidin-sensitive donors. It was found that transfer factor could be lyophilized without impairing its activity. This facilitated dialysis with the result that greatcr amounts of transfer factor appeared in the dialyzate as determined by the increased intensity of coccidioidin sensitivity transferred.

x

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H. S. LAWRENCE

DIALYZATE -ABSORPTION SPECTRA AFTER SEPHADEX G-25

c 9 cc W BC EXTRACT

+

DNASE CIALYLATE

-

A SEPHADEX COLUMN

0 8 8 __

TUBE NO

RECIPIENT 1

1

SKIN REACTICNS-l WEEK PCST-TRANSFER

*I[

-

3+

[3

FIG. 3. Transfer of coccidioidin sensitivity with active fraction (peak 11,
The dialyzable transfer factor exhibited the same properties as the parent leukocyte extract-namely, prompt reactions in most recipients which persisted for prolonged periods (e.g., 9-15 months). Occasional failures occurred early in the course of the study when smaller volumes of dialyzate were prepared. It is of interest that repeated testing with either tuberculin or coccidioidin in those recipients in whom transfer failed to occur, did not by itself cause any delayed reactions to these antigens. Bence-Jones protein or papain-digested y-globulin fragments added to the inside contents of the dialysis sac as markers did not appear in the dialyzate (see Table 11). Material prepared from DNase-treated leukocyte extracts was dialyzed against 200 ml. distilled water for 18 hours, lyophilized, and passed through a Sephadex G-25column. The total eluate was pooled and, following a second lyophilization, redissolved and injected into negative recipients. Marked degrees (4+ ) of coccidioidin sensitivity was transferred on three consecutive attempts. These results are illustrated in Fig. 3 where a more precise separation into two peaks is shown. The fractions collected under peak I ( <40,000 and > 10,000mol. wt. ) failed to transfer delayed sensitivity to coccidioidin to 1 recipient, whereas the fraction collected under peak I1 ( <10,000 mol. wt. ) transferred a marked ( 3 f ) degree of coccidioidin sensitivity. Later testing showed that the recipient of the fraction under peak I de-

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veloped minimal by clefinitc sensitivity ( I f ) probably due to slight con tamination. Attempts to preparc an antibody to transfer factor by injcction of dialyzatc in Frcund’s adjuvant into rabbits werc unsuccessful ( Lawrence et al., 1963, unpublished obscrvations ) . Moreover repeated injection of dialyzable transfer factor in high dosage weekly, over a year’s interval, in man has failed to elicit antibody production. I t thus appears that dialyzable transfer factor is not immunogenic in animals or man (Valentine and Lawrence, unpublished observations ) . Subsequent studies, undertaken as part of a series to identify and characterize human transplantation antigens in subcellular leukocyte fractions, revealed that the dialyzate of leukocyte extracts was incapable of actively immunizing recipients to reject a skin homograft from the leukocyte donor. Thus dialysis separates transplantation antigens from transfer factor ( Rapaport et al., 1965a).

B. PREPARED FROM SONICALLY DISRUPTED LEUKOCYTE EXTRACTS

The dialyzable nature of transfer factor, its low molecular weight, and polypeptide-polynucleotide composition have each been confirmed by several investigators. Baram and Mosko (1965) confirmed the capacity of dialyzates of leukocyte extracts to transfer tuberculin sensitivity to 6 of 7 negative recipients. In addition, activity was detected on one occasion in a nondialyzable fraction as well. They prepared supcmatants from frozen and thawed leukocyte extracts obtained from tuberculinsensitive donors. Dialyzates prepared from two tuberculin-negative reactors failed to transfer tuberculin sensitivity. They found, as we did, transferred sensitivity was present promptly ( 3 days) and persisted in the 2 recipients who were tested 6 months after transfer. Mycobacterial antigens in leukocyte extract supernatants and in dialyzates were sought and not detected. Absorption spectra performed on dialyzate passed through Sephadex G-25 gave a similar pattern to the one described earlier (see Fig. 3; Lawrence et al., 196:3). The dialyzate was found to contain pentose, hexose, lipid phosphorous, protein-nitrogen, and other unidentified substances. Baram et al. (1966) continued studies on the dialyzable and nondialyzable materials capable of transferring tuberculin sensitivity and partially characterized each. One partially purified active fraction was further concentrated by filtration through Sephadex G-25. Although multiple peaks were detected on elution, activity was present under only one peak, and was found to be of low molecular weight and contain a

232

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high concentration of ribose but no protein or lipid. This peak was analyzed and reported to be composed of a polynucleotide lacking uracil. No precipitin bands could be found on agar-gel dif€usion nor by immunoelectrophoresis, when this peak was tested with antisera against human serum, human leukocytes, or purified human yG-globulin. These data exactly fit the low-molecular-weight dialyzable transfer factor previously described. Transfer activity was also associated with a larger-molecularweight fraction found associated with +globulin. Polynucleotide fractions and A1 fractions prepared from the negative donor pool failed to transfer tuberculin sensitivity yet exhibited identical chromatographic and absorption spectra patterns as those prepared from tuberculin-sensitive donors. Shifts of transfer factor activity from fraction to fraction were detected by Baram et al. (1966) which is consistent with our observation that increased activity appears in the dialyzate as the volume of dialyzate is increased (Lawrence et al., 1963). Arala-Chaves et al. (1967) have also isolated a dialyzable transfer factor of low (5,000-15,000) molecular weight. They prepared leukocyte dialyzates by sonic vibration of the cells, followed by ultrafiltration of the extracts. Such dialyzates from three different donors readily transferred tuberculin sensitivity, whereas the residue failed to transfer sensitivity, Separation on Sephadex G-25 revealed two peaks. Material collected under peak I transferred sensitivity and that collected under the smaller peak I1 failed to do so. They comment on the polynucleotidepolypeptide composition of the dialyzate. They also found no obvious differences between dialyzates from positive or negative donors and also failed to detect immunoglobulins in the dialyzate. Fireman et nl. (1967, 1968) obtained similar dialyzates which they fractionated on Sephadex G-25. An active fraction was isolated from the dialyzate eflueiit which transferred tuberculin sensitivity. This active fraction was further purified on Sephadex G-10, and the activity was eluted in the region for materials with a molecular weight in the range of 700 to 4000. Similar fractions, prepared from a tuberculin-negative donor, failed to transfer tuberculin sensitivity. These in vivo studies were a prelude to an in vitro transfer of tuberculin sensitivity which will be discussed below (see section VI) . In the preceding studies, the leukocyte extracts were prepared by either mechanical disruption or freezing and thawing and the resultant whole extract or a supernatant fraction dialyzed. Brandriss (1968) treated the frozen and thawed leukocyte extracts with pancreatic DNase before dialysis, exactly as outlined in the original report (Lawrence et al., 1963). He investigated the capacity of dialyzable transfer factor to trans-

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fer contact sensitivity to DNCB, using leukocytes obtained from tuberculin-sensitive donors who wcrc actively scmsitized to DNCB. Employing dialyzatcs prepared from such doubly-sensitive leukocytes, transfctr of tulmculin scnsitivity w'ts accomplished in 4 of 7 recipients, whereas 110 transfer of DNCB scnsitivity was achicved in 8 recipients of the same dial yzate. The ultraviolet absorption spectrum of the dialyzates all had a peak at 252-255 mu. The shape of the curves were similar to that found by Baram and Mosko (1965) although thcir peak was at 245 mp. When optical density was plotted versus tubc iiuinber after passage of two dialyzates through Sephadex G-35, two peaks were noted at 260 and 280 nip arid their patterns were similar to that obtained by Lawrence et al. ( 1963), (see Fig. 3 ) . The nature of the host's contribution to the in vivo mechanisms of delayed hypersensitivity and the mode of action of transfer factor have emerged from the use of dialyzable transfer factor in the study of the cellular immune deficiency in patients with Boeck's sarcoid (Lawrence and Zweiman, 1968). Advantage was taken of the inability of sarcoid patients to respond to transfer factor with systemic sensitization. Equivalent aliquots (0.05 ml. packed cclls, ca. 40 x 10O cells) of various transfer factor preparations obtained from the same tuberculin-sensitive donor were assayed for activity in an internally controlled situation provided individually by each of 7 recipients. Since dialyzable transfer factor is freed of transplantation antigens and othcr cell constituents and since it is not iinmunogenic, preparation of local skin sites with intradermal dialyzate alone causcs 110 inflammatory reactions of any kind. Using such highly purified preparations of dialyzable transfcr factor, it was found that local transfer of tuberculin sensitivity was accomplished in 5 of 7 patients, wcak systemic sensitization occurred in 2 of 7 patients, and neither local nor systemic sensitization occurred in 2 of 7 patients. Transfer with dialyzable fractions was again unaffected by treatment with 5, 50, or 500 p g . pancreatic RNase. However, local transfer was abolished by heating dialyzate at either 56" or 100°C. in a water bath for 30 minutes. Dialymtcs were similarly prepared from another tuberculin-positivc donor and similarly injected into each of 5 normal recipients, and each developed enduring systcmic as well as local sensitivity to tuberculin. Like Baram and hlosko ( 1965) we found that the residue of the leukocyte extract remaining within the dialysis sac possessed variable activity. From this study it was concluded that dialyzable transfer factor is capable of causing prompt local transfer in normal individuals and in patients with sarcoidosis, and the feeble systemic transfers (10 mm.) observed in 2

234

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patients may reflect an increased dosage of transfer factor or increased responsiveness of the lymphocytes of the 2 patients so responding. Again, the finding that pancreatic RNase has no effect on transfer factor takes the latter out of the category of RNA-antigen preparations of Fishman and Adler (1963) and of Bondevik and Mannick (1968) which are inactivated by pancreatic RNase. The insensitivity of transfer factor to pancreatic RNase takes on new meaning, however, in conjunction with the inactivation of transfer factor by heat (56" or 100OC.). Pancreatic RNase insensitivity and heat lability are two unique properties of doubled-stranded RNA which serve to distinguish it from singlestranded RNA. This could be of importance if the activity of transfer factor is ultimately demonstrated to reside in the RNA polynucleotides present in the dialyzate ( Billeter and Weissmann, 1966). Utilizing an identical protocol, we have also evaluated the capacity of patients with advanced cancer to respond to local injections of dialyzable transfer factor ( Solowey et al., 1967). Dialysates were obtained from donors sensitive to streptococcal proteins ( SK-SD) . The recipients were 10 cancer patients anergic to a battery of antigens (SK-SD, PPD, mumps, diphtheria toxoid, histoplasmin, and coccidioidin ) . Employing the dialyzates, the local technique of transfer was undertaken, and 10 of the 10 recipients developed both local and systemic SK-SD sensitivity. Each recipient exhibited intense reactions to SK-SD at the local site of dialyzate antigen injection. However, the systemic reactions transferred were feeble ( 10 mm. ), and the duration of transferred sensitivity was short compared to that usually seen in normal individuals. In addition to transferring another modality of delayed sensitivity with dialyzable transfer factor, these observations suggest that patients with extensive malignancy express a cellular immune deficiency state in their handling of transfer factor.

+

VI.

Transfer Factor and in Vitro Correlates of Cellular Immunity

A. INHIBITION OF MACROPHAGE MIGRATION We next turned to the development of in vitro techniques for further clues to the identity and mechanism of action of transfer factor. The capillary macrophage migration technique described by George and Vaughan (1962) and extensively studied in our laboratory in collaboration with David, Al-Askari, and Thomas, provided leads toward understanding in vivo mechanisms of delayed hypersensitivity responses (David et al., 1964a,b,c,d). It was soon apparent that this reproducible technique is a sensitive indicator of delayed reactions induced by bac-

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teria, proteins, and simple chemical compounds. The technique was, therefore, readily and successfully adapted to the in vitro analysis of the immunological events encountered in other states of altered tissue reactivity believed to be expressions of delayed hypersensitivity, namely, homograft reactions ( Al-Askari et al., 19651; Al-Askari and Lawrence, 1969) and experimental autoimmune disease (David and Paterson, 196Fj). The subsequent findings by Bloom and Bennett (1966) and, independently, by David (1966) that an interaction between sensitive lymphocytes and specific antigen induces the synthesis of a protein which causes normal macrophages to stick to each other, has brought the analysis of this system to the critical point of identifying and characterizing this heat-stable, nondialyzable protein which migrates in the region of albumin molecules and does not appear io be an immunoglobulin (David, 1967, 1968; Bloom and Bennett, 1968). Our initial results using this technique demonstrated an effect on normal cell populations when sensitive cells were added in the presence of antigen (David et al., 1964b ) . This observation suggested that transfer of immunological information from cell to cell might occur. The search for a material akin to transfer factor in the guinea pig, therefore, became a icasible venture with this sensitive system. Moreover, the prospect of adopting this technique to study transfer factor in humans was a constant preoccupation. However, Dr. David and I were unable to find evidence for the operation of a transfer factor in the guinea pig system, and we encountered difficulties in adapting human blood leukocyte populations to the capillary mcthod. Successful results were obtained by Thor and his colleagues (Thor, 1967, 1968; Thor and Dray, 1968a,b, Jureziz et d., 1968). In these studies, lymph nodes were obtained from tuberculin- or histoplasmin-sensitive donors at operation, minced, and the dissociated cell populations placed in culture at 37OC. for 3 days. The cells, consisting mainly of lymphocytes and phagocytic cells, were then harvested, placed in capillary tubes, and cultured in the presence of tubcrculin (PPD) or histoplasmin. The results obtained in humans paralleled those obtained in the guinea pig. In thc absence of antigen, positive cells migrated out of the capillaries and negative cells were not affected by tuberculin (10 pg./ml. ) or histoplasmin ( 30 pg./ml. ) . Tuberculin-sensitive, histoplasminnegative cells were inhibited by tuberculin but not by histoplasmin, and, conversely, histoplasmin-sensitive, tuberculin-negative cells were inhibited by histoplasmin but not by tuberculin. Specific and complete correlation of inhibition of cell migration and skin tcst reactivity was obtained in 27 patients sensitive to either tuberculin or histoplasmin, to both antigens, or to neither.

236

H. S. LAWRENCE

Thor and Dray (1968b) extended the above observations and transferred to nonsensitive lymph node cells the capacity to be inhibited. This was done with a phenolized RNA extract prepared from lymph nodes cells of specifically sensitive donors. Nonsensitive cell populations were shown to migrate freely in the presence of tuberculin and histoplasmin before addition of the RNA extract, Similarly prepared nonsensitive cell populations were inhibited by tuberculin but not by histoplasmin, after preincubation with an RNA extract prepared from the ceIIs of a tuberculin-sensitive donor. Conversely, nonsensitive cell populations were inhibitcd by histoplasmin but not by tuberculin, after preincubation with a RNA extract prepared from the cells of a histoplasmin-sensitive donor. Finally, nonsensitive cell populations were inhibited by either tuberculin or histoplasmin, after incubation with an RNA extract prepared from the cells of a tuberculin- and histoplasmin-sensitive donor. Serum obtained from sensitive donors had no effect on the migration of sensitive cells in the presence of antigen. The capacity of specifically sensitive RNA extracts to transfer sensitivity in vitro was abolished by treatment with pancreatic RNase; however, treatment with DNase or with trypsin were without effect. The authors point to thc similarity of their RNA extract to other preparations studied ( Fishman and Adler, 1963; Bondevik and Mannick, 1968) and the possibility that an RNase-sensitive, RNA-antigen complex cannot be excluded. The differences between their human RNA extract and transfcr factor are discussed (e.g., transfer factor unaffected by RNase) along with work done in their laboratory which indicated the active component of the RNA extract is nondialyzable and has a sedimentation value >4 S but <28 S. The authors suggest tcsting the RNA extract for in vivo activity and transfer factor for in vitro activity in that system, as an cxtension of their findings. Thor et al. (1968) have recently reported on the inhibition of migration of normal guinea pig macrophages with migration inhibitory factor ( MIF) produced by incubating sensitive human leukocytes with specific antigen (tuberculin, histoplasmin, coccidioidin) . Apparently, the MIF produced by human lymphocytes exerts the same specific effects on guinea pig macrophages as that described for guinea pig MIF (Bloom and Bennett, 1966; David, 1966). This finding has been rccently applied to test for human transfer factor activity in the guinea pig macrophage migration system. Paque et al. (1969) report on the transfer of histoplasmin sensitivity in vitro to nonsensitive human leukocyte cultures by mcans of 1yzatc.s containing transfer factor prepared from histoplasmin-sensitive leukocytes. Sensitive human ccll lyzates, plus nonsensitive human leukocytes, plus histoplasmin,

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yield on culture a supernatant material ( M I F ) which inhibits migration of normal guinea pig macrophages in the presence of histoplasmin but not when exposed to coccidioidin. The observations on the transfer of sensitivity to human cell populations with an RNA extract were recently extended to guinea pigs with equal success (Jureziz et al., 1968). In the guinea pig system, peritoneal exudate cells were incubated with phenolized RNA extract, prepared from lymph nodes and spleens of tuberculin- or coccidioidin-sensitive animals. Migration of exudate cells was specifically inhibited by the antigen to which the RNA extract donor was sensitive. In control studies, RNA extracts prepared from nonscnsitive animals were without effect, as were nonsensitive cells preincubated with antigen alone. The demonstration in humans of the transfer of the capacity to synthesize MIF with either an immunologically specific RNA extract or leukocyte lyzates promises to clarify the relationship of transfer factor ( a n informational or derepressor molecule) to MIF (an effector molecule) in humans. Hopefully, studies of this type with parallel demonstrations extended to the guinea pig system, may help to resolve the vexed question concerning how or why the guinea pig differs from man in respect of transfer factor in vivo (Lawrence, 195%; Bloom and Chase, 1967; Turk, 1967). In this connection the recent transfer by Dupuy et al. ( 1969) of tuberculin sensitivity to guinea pigs by means of plasma obtained from sensitive donors after X-irradiation is pertinent. These authors have achieved transfer in two-thirds of the animals so treated. In control studies, plasma obtained from sensitive donors unexposed to X-irradiation, as well as plasma from nonsensitive X-irradiated donors, failed to transfer. Additionally, nonsensitive guinea pig spleen cells incubated with plasma from X-irradiated sensitive donors also transferred tuberculin sensitivity. The activity conferred on normal spleen cells could not be removed by repeated washing. The authors postulate the factor or factors involved in transfer may represent the thymus-dependent antibody or “IgX” of Mitchison ( 1969). They report that the active material is nondialyzable and is stable to lyophilization; and they conclude that its relation to dialyzable human transfer factor remains to be elucidated. There is a growing literature on the in vitro transfer of specific reactivity by cell fractions in experimental animals. This new approach is exemplified by the in vitro transfer of 5pecific reactivity with an RNA extract from scnsitive to nonsensitivc guinea pig lymphocyte populations (Jureziz et ul., 1968); as ell as the transfer of reactivity from sensitive to nonscnsitive rhesus monkey lymphocyte populations by leukocyte

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lyzates containing transfer factor (Baram and Condoulis, 1969); also the transfer of specific reactivity by dialyzable transfer factor, obtained from sensitive human leukocytes, to mouse lymphocyte populations ( Adler and Smith, 1969). These in vitro transfers, coupled with the in vivo transfer of tuberculin sensitivity to guinea pigs with plasma containing cell-liberated activity just discussed (Dupuy et al., 1969), suggest that differences between the behavior of transfer factor in man and in experimental animals are being resolved gradually as more similarities are finally beginning to emerge. B. LYMPHOCYTE TRANSFORMATION The initial reservations concerning the accuracy and meaning of the lymphocyte transformation response as a reproducible in vitro correlate of delayed hypersensitivity reactions ( Robbins, 1964) have been largely resolved (Dutton, 1967; Wilson and Billingham, 1967). This has been due in large part to the objective quantitation afforded by the application of radioistopic and radioautographic techniques. The result has been the perfection of an exquisitely sensitive indicator of delayed hypersensitivity which generally parallels but is more discriminating than the skin test, in that latent states of delayed sensitivity are readily detectable (Lawrence, 1968b). Fireman et al. (1967) have adapted the lymphocyte transformation reaction to a study of the effects of dialyzable transfer factor with promising results. They prepared blood lymphocyte cultures from tuberculinnegative and from two types of tuberculin-positive individuals: one group exhibited native tuberculin sensitivity; and the other group had been tuberculin negative and acquired tuberculin sensitivity following the in vivo injection of an isolated active fraction (Sephadex G-10) of dialyzable transfer factor. They found the addition of transfer factor alone had no effect on cultures of negative cells or on either type of positive cell population. Negative cell cultures to which specific transfer factor plus tuberculin were added responded with transformation to lymphoblasts 3 times background level. Tuberculin-negative dialyzate, with or without added tuberculin, had no effect on cultures of negative cells but seemed to inhibit transformation of both typcs of positive cells. Finally, transformation in the presence of tuberculin was observed in cell cultures taken from individuals who had become tuberculin positive following an injection of transfer factor in uiuo, 1-4 weeks earlier. Of additional interest was the suggcstion of rccruitinent, when transfer factor plus tuberculin was added to cell populations obtained from natively

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sensitive donors or sensitized recipients of in vivo transfer factor. The additional increment of transformed cells caused by transfer factor plus PPD on natively sensitive lymphocytes was 6%above the figure obtained with PPD alone; the increment of transformed cells caused by transfer factor plus PPD on lymphocytes from recipients of in vivo transfer factor was 4%above the figure obtained with PPD alone. This observation suggests recruitment of nonreactive lymphocytes by transfer factor in vitro over and above the pool of committed cells available in nature or following transfer factor’s original recruitment in vivo. Valentine and I (unpublished observations) had embarked independently on a similar approach to the same problem as a consequence of our earlier findings that dialyzable transfer factor is not antigenic and that dialysis separates transfer factor from Ieukocytic transplantation antigens. We also found that incubating dialyzates of tuberculin transfer factor alone with nonsensitive lymphocyte cultures exerts no detectable effect until tuberculin is added. A small but consistent percentage of nonsensitive lymphocytes (ca. 3%) were transformed by the seventh day when cultured in the presence of transfer factor plus PPD-a result confirmed by measurements of thymidine uptake. Of interest is the failure of dialyzable transfer factor alone to cause transformation of sensitive cells as well as nonsensitive lymphocyte populations. Since sensitive lymphocytes in this system respond to minute quantities of antigen, this lack of transformation of positive cells suggests that the dialyzate does not contain antigen-polynucleotide complexes. This conclusion is also supported by the failure to induce antibody to repeated administration of dialyzable transfer factor in vim. In this connection, the insensitivity of transfer factor to pancreatic RNase also argues against the possibility of an antigen-RNA complex contributing to the biological effects observed. The effects of most of the antigen-RNA complexes studied are abolished by RNase (see, also, Thor and Dray, 1968b). In another type of experiment, we reassessed the immunological specificity of transfer factor as a small molecule and also compared in vitro transformation of normal lymphocytes by transfer factor plus antigen with the results of in vivo transfer. Here, dialyzable transfer factor prepared from leukocyte extracts of a tuberculin-positive, diphtheria toxoid-negative donor was cultured with blood lymphocytes obtained from an individual with negative reactions to tuberculin and to toxoid. This individual would later serve as the in vivo recipient of transfer factor. Transformation of the cultured nonsensitivc lymphocytes occurred only in those tubes containing dialyzable transfer factor plus tuberculin but not in the respective control lymphocyte cultures to which

240

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had been added transfer factor plus toxoid, or toxoid alone or tuberculin alone, or transfer factor alone. The same tuberculin-positive, toxoid-negative dialyzate was then injected into the tuberculin-negative, toxoid-negative individual, from whom the lymphocytes had been obtained for the in vilro studies just described, and resulted in the in vivo transfer of marked (3f ) tuberculin sensitivity while the recipient’s reaction to diphtheria toxoid remained negative. Following the in vivo transfer, blood lymphocytes obtained from the same individual, which had been unaffected by tuberculin or toxoid before transfer, now underwent transformation when cultured in the presence of tuberculin alone but not in the presence of toxoid alone. This result, although derived only from one series of experiments, suggests that the in vitro transfer with dialyzate engages the small, circulating lymphocyte and induces it to respond only to the appropriate antigen; and that this same event occurs following an interaction of transfer factor and circulating small lymphocytes in vivo resulting in the transfer of cutaneous reactivity, as well as the capacity of the recipients lymphocytes to respond only to the appropriate antigen; and that despite the small molecular size of transfer factor, the transfer achievcd both in vitro and in vivo is immunologically specific (Lawrence and Valentine, 1969). Since the in vitro effects of dialyzable transfer factor were not as intense as we had anticipated from its potent in vivo activity, we adapted earlier experiments to the lymphocyte transformation reaction. It will be recalled that incubation of blood cell populations sensitive both to tuberculin and diphtheria toxoid for only 1 hour in the presence of tuberculin (PPD) resulted in the liberation only of the tuberculin transfer factor into the cell-free supernatant, which now transferred tuberculin but not toxoid sensitivity in vivo. The cell sediment had lost the capacity to transfer tuberculin sensitivity, but retained the capacity to transfer toxoid sensitivity. Control cell populations incubated without tuberculin retained the capacity to transfer both tuberculin and toxoid sensitivity and no in vioo activity was detected in their supernatants (see Fig. 2; Lawrence and Pappenheimer, 1957). Valentine and Lawrence ( 1968a, 1969), applying this principle, find that incubation of tuberculin-sensitive blood lymphocytes with tuberculin for 24 to 36 hours produces in the cell-free supernatant, a material which when added to nonsensitive lymphocytes causes the latter to undergo transformation, repeated cell-division, and clonal proliferation ( i.e., lymphocyte-activating material ) . This in vitro transfer of cellular immunity is antigen dosc-dependent. Thc activity in such supernatants is abolished by heating at 56°C. for 30 minutes, a property shared by dialyzable transfer factor (Lawrence and Zweiman, 1968)

TRANSFER FACTOR

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and one which distinguishes this material from the heat-stable MIF of Bloom and Bennett (1966) and of David (1966). The lymphocyte-activating material in such supernatants is also distinct from transplantation antigens, since the activity is not scdimcntcd at 100,OOOg. However, it differs from dialyzable transfer factor in that the supernatant activity is nondialyzable. We continue to study this antigen-liberated lymphocyte-activating material and its relationship to dialyzable transfer factor, as well as to other products resulting from an interaction of sensitive lymphocytes and antigen. Thus, the in vitro observations on dialyzable transfcr factor reveal the transformation of relatively few (ca. 3%)of the normal lymphocytes in vitro and of the recipient’s cells in viuo, despite transfer of marked skin sensitivity; whereas, the antigen-liberated supernatant material has consistently resulted in larger (15-208) population of transformed cells at the end of the samc culture period ( 7 days). The question as to how this effect is amplified remains. Does antigen liberate transfer factor from a few sensitive lymphocytes to recruit a large population of nonsensitive cells as originally postulated by Pappenheimer ( 1955, 1956) or is transfer factor a vehicle for immunological information passed from cell to cell? We examined this question by studying cultures of lymphocyte populations under the continuous observations of time-lapse cinematography for periods of 1 week (Marshall et al., 1968). For this purpose a plastic ring was cmployed which held all the lymphocytes captive under a single microscopic field and cells could neither migrate into or out of the field of vision. This device had been successfully employed to study the effects of phytohemagglutinin ( PHA) on lymphocyte cultures by Marshall and Roberts (1965). Our initial efforts were directcd toward the behavior of tuberculinscnsitive lymphocyte populations incubated with tuberculin ( Marshall et al., 1968a). These rcsults revealed that 2% of the total lymphocyte population was antigen rcsponsive and underwent transformation, repeated cell division, and clonal proliferation. Thus, at least after 48-72 hours of tube incubation, we found no evidcnce for recruitment, and the large numbers of transforined lymphocytes ( 2 0 4 0 % )observed at the end of 7-day cultures arose from the very few antigen-responsive cells by a process of repeated cell division resulting in the proliferation of a clone 1969). of antigcn-rcsponsivc. cclls (sec Figs. 4 and 5; Marshall et a?., W e next applied thc cincrnatography technique to look for recruitment in cu1turc.s of nonsensitive lymphocyte populations to which had Iwen added active supcrnatants ( i.e., supernatants containing inateria1 produced by tuberculin-scmsitive lympliocytcs incubated with tuberculin (Valentine and Lawrence, 1968a,b, 1969). Surprisingly, an equally small

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H. S. LAWRENCE

FIG.4. An individual microchamber demonstrating tuberculin-stimulated human lymphocyte proliferation on ( a ) day 3; ( b ) day 4; ( c ) day 5 ; and ( d ) day 6 of culture, The proliferating Iymplioblasts are the large, light colored, refractile cells. (Reproduced from Marshall et al., 1969.)

percentage of nonscnsitive lymphocytes were initially engaged by the lymphocyte-activating material ( ca. 2%). This small percentage of cells in the presence of tuberculin underwent transformation, repeated cell division, and clonal proliferation, which amplified into 15 to 20%of transformed cells by the seventh day of culture. This whole sequence was quantitatively, qualitatively, and temporally exactly as had been observed when naturally tuberculin-sensitive lymphocytes were cuItured in the presence of tuberculin. Again, no evidence of recruitment was observed under the conditions of these experiments. Nevertheless, those few nonsensitive cells that were induced to proliferate by the active material present in the supernatant, must by definition have been recruited-since their counterparts in control cultures gave no evidence of transformation or clonal proliferation in the presence of antigen alone, This type of result especially when dialyzable transfer factor is used, supplies direct experimental evidence in favor of Burnet’s (1959) clonal selection theory, particularly the finding of an immunologically specific engagement of a highly restricted number of cells in the total lymphocyte population. The implication of this finding is that the few normal cells

FIG. 5. Reconstruction of the fate of a single lyniphoblast through the 64 cell stage after exposure of tuberculin-sensitive human lymphocytes to tiiberculin. Generation times shown in hours over appropriate lines; those marked with ( * ) are average times for a group of cells where it was impassible to follow the fate of a single cell but where the mitoses within the group were clearly seen. (Reproduced from Marshall et al., 1969.)

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€1. S . LAWRENCE

engaged may be a clone that is predestincd to become tuberculin sensitive should that particular tuberculin-negativc lymphocyte donor ever have occasion to meet the tubercule bacilhs. The time-lapse cinematography approach to the in vitro study of captive populations of lymphocytes providcs a powerful tool for the investigation of problcnis of exactly how many sensitive or antigen-responsive circulating lymphocytes exist and whether and under what precise conditions recruitment of initially nonresponsive cells occurs. In this connection it is of interest that Barani and Condoulis (1969) have recently reported evidence for the presence of a transfer factor in rhesus monkeys. They sensitized monkeys to keyhole limpet hemocyanin ( KLH ) and prepared blood leukocyte lyzate-containing transfer factor. The KLH-sensitive leukocyte lyzate when cultured with nonsensitive monkey lymphocytes caused the cells to transform in the presence of KLH antigen as measured by thymidinc uptake. Nonsensitive leukocyte lyzatcs were without effect on control populations of nonsensitive lymphocytes plus KLH. The activity was found nondialyzable and stable upon incubation at 37°C. Increasing the concentration of sensitive leukocyte lyzate does not increase the number of cells engaged. The authors comment on the small number of cells susceptible to sensitization and the possibility of specific clones being involved. In relation to intraspccies transfer in vitro, it is of interest that Adler and Smith ( 1969) havc reported the transfer of tuberculin responsiveness to nonsensitivc mouse lymphoid cells in culture, following incubation with tuberculin-sensitive, dialyzable human transfer factor. The converted mouse lymphoid cells respond with transformation to addition of tuberculin following transfer, as measured by increased thymidine uptake.

C. TARGET-CELL DESTRUCTION Since Rosenau and Moon’s (1961) demonstration of target-cell destruction by lymphocytes sensitized to the tissues used as targets in culture, there have been many refinements and extensions of this observation ( cf. Dutton, 1967; Wilson and Billingham, 1967). Recent work on human “lymphotoxin,” particularly by Granger and Kolb ( 1968) and Kolb and Granger (1968), has shown that the lymphocyte is not an indispensable participant in the reaction, and a variety of stimuli which derepress or initiate lymphocyte transformation (e.g., PHA) result in the production of a soluble, nondialyzable supernatant material responsible for the activity. Holm and Perlmann (1967) and Ruddle and Waksman ( 1967, 1968) have shown that tuberculin-scnsitive lymphocytes are stimulated by tuberculin to cause target-cell death of indifferent tissues in

TRANSFER FACTOR

245

culture. Moreover, Rnddle and Waksman ( 1968) havcx nlso shown that preiiicubation of tuberculin-seiisitivc. rcit lymphocytes results in elaboration of the cytotoxic activity into the supernatant. We liave also observed n similar targct-cell effect on the cloning ability of HcLa cclls incubatcd with tul,crcul~i-sensitivehuman lymphocyte populations triggered by prior incubation for 36 hours with tuberculin (Lebowitz and Lawrence, 1969). Of interest to this discussion of in vitro corrc1atc.s of ccllular immunity is our finding that cell-free supernatants, prepared by incubating tubcrculin-sensitive lymphocytes for 36 hours with tuberculin, are as effective as the lymphocytes themselves in reducing the number of clones of HeLa cells. When sensitive lymphocytes plus tiiberculin or their supernatants were tested, a consistant 93%reduction in HeLa cell clones was observed. Tuberculin-negative control lymphocytes plus tuberculin or thtlir supernatants had no effect, nor did histoplasmin-negative lymphocytes cultured with histoplasmin. The lymphocytotoxin released into such supernatants is nondialyzablc and resists frcezing and thawing but is inactivated by hcat at 56°C. for 30 minutes. Preliminary studies of the cffects of lymphocytotoxin on HeLa cclls by continuous time-lapse cinematography have been undertaken. The effects observed havc consisted of markedly disturbed duration of intcrphase and prophase intervals, with an associated marked reduction or complete cessation of HeLa cell division after 1 or 2 mitoses. The remaining crlls acquire abnoiinal shapes and asciume disordered spatial arrangement, yet no cytolysis was observed. VII.

Mechanism of Action of Transfer Factor in Vivo a n d in Vitro

In vitro studies such a3 the type described in this section have revealed a broad repertoire of latent responscs residing in sensitive lymphocyte populations, one or another of which are set in motion following interaction with specific antigcn. The growing number of activities cataloged for human blood Iyniphocyte populations to date, arc schematic.ally listed in Fig. 6. Among the many u n a a s ~ e r c dqucstions, the relation of dialyzable transfer factor to antigcm-liberated transfer factor is prominent. Since both have the same in vivo propcrties, do both share the same in vitro properties? What is the contribution of transfer factor liberated from sensitivc cells after only 1 hour incubation with antigen to the nondialyzable, lymphocytc-activating material which requircs 24 hours incubation with antigen for its production in the same supernatant solution? Do both these materials compete with cach other for the same few normal lymphocytcs that arc susceptible to activation or do the materials act in

H. S. LAWRENCE

246 WhL

X 1I V I TIE5 LI i i t R D 1 r U

iwro

I’RUPt H TI t S

tlJNC I IONS

SUPERNATANT IN VlVCl

NOT STIJUIEU IN 5UPtHNATANT

TRANSFER CUTANEOUS SENSITIVITY

LYMPHOCYTE ACTIVATING MATEHIAI.

NONDIALYZABLE INACTIVATED 56’ C

NOT DONE SHOULD CONTAIN TRANSFER FACTOR

MIGRATION INHIBITORY FACTOR

NONDIALYZABLE HEAT STABLE

INCITES LOCAL INTRADERMAL REACTION

/ @

IAG

\

\

ANTIGCN LIBEfiAILD TRANSFEH FACIVR

LYMPHOCYTOiOXlC FACiOR

NONDIALYZABLE INACTIVATED

56-C

UNKNOWN

IN V l l R O -~

NUT SIUUIEU IN SUPtRNATANT

ACTIVATES NORMAL LYMPHOCYTES ( 7 DEHEPRESSOIII

AGGLlITINATF9 NDHMAI. MACROPliAGES It f F FCTOHi ~~

TARGET-CELL DEATH IFFFECTORI

FIG. 6. Activities produced by or liberated from antigen-responsive human blood lympliocytcs following interaction with specific antigen. Transfer factorliberated after only 1 hour incubation with antigen. (Lawrence and Pappenheimer, 1956, 1957. ) Lymphocyte-activating material-produced after 24 hours incubation with antigen. ( Valentine and Lawrence, 1969. ) Migration inhibitory factor-produced after 24 hours incubation with antigen. Although MIF is produced by human lymphocytes (Thor and Dray, 1968a,b), properties listed in this figure were obtained in the guinea pig. (Bloom and Bennett, 1966; David, 1966. ) Lymphocytotoxic factor -produced after 36 hours incubation with antigen. (Lebowitz and Lawrence, 1969.)

concert? Is transfer factor in this situation nondialyzable because it has become complexed with antigen during incubation? These questions as well as those relating to the identification and similarity or differences between lymphocyte-activating material, MIF, and lymphocytotoxin represent areas for continued interesting investigation. The critical step in the in vitro and in vivo event is the moment of contact with the particular “lymphocyte” and its specific antigen and the nature of the elusive receptor site, which having been engaged by antigen, initiates the elaboration of the family of molecules with such diverse functions. This primary recognition event may then be adapted to reveal a broad category of secondary effects which are limited only by the ingenuity of the experimental design created to reveal them. Each secondary effect delineated to date has arisen from an in vivo event which prompted the original experimental design. Of the products of sensitive lymphocyte-antigen interaction, only antigen-liberated and dialyzable transfer factor as well as MIF have been demonstrated to mediate in vivo effects. The relevance to in vivo reality of lymphototoxin may be soon forthcoming ( Gowans and McGregor, 1965; Lawrence 1963, 196%).

TRANSFER FACL'OR

247

Thc nature of the r~iitiRCri-r~""ptc)rsite residing in or on sensitive lymphoeytcs m d whethcr it is a cell-nssociatcd iiiimuiio~lohuli~~ of the type postulatcd I)y Kanish and Eiscn ( 1962) or the I g S postulated by Mitchison ( 1969), or yvt another typc of molrcular specics with stcareospcxcific configuration, is the focus of miich current interest. Since the effect of dialyzable transfer factor in converting the very few normal lymphocytes to the sensitive state is expressed only following addition of appropriate antigen, a specific rcceptor site must, therefore, have been conferred on such sensitized cells. These results raise the question whether transfer factor itself is the spccific receptor site as postulated by Pappenheimer ( 1956), since it is known to interact with antigen (Lawrence and Pappenheimer, 1956, 1957), or does transfer factor induce the synthesis of the receptor site? In the conversion of so few nonnal cells to an antigen-responsive state, transfer factor itself is accomplishing a highly specific interaction with a particular reccptor site. The in uitro techniques currently avnilable and the separation and characterization of the diverse specific and nonspecific irnmunologicnl reagents delineated have brought the problem to the point where an approach to further elucidation of this complicated question is possible. The whole question of mediators of delayed-type hypersensitivity and thc delineation of ii molecular basis for cellular immune responses is an extremely active and rapidly moving area of investigation. Thc preceding discussion is, therefore, in the nature of a progress report. The approaches we have discussed are considered in greater detail in the proceedings of two recent symposia ( s e e In viiro correlates of delayed hypersensitivity, 1968, and Lawrence and Landy, 1969). Wc have revicwcd selectcd in h o and in vitro properties of transfer factor a s an initiator of cellular immune responses at a molecular level. Evidence has been presented that transfer factor is neither immunogenic nor immunoglobulin and thc response transferred is neither passive nor active sensitization, in the usual sense in which these terms arc' used. Transfer factor bchaves in some respects like an informational niolccule in its endowment of the entire negative recipient in vim or a select portion of his isolated circulating lymphocyte populations in vitro with the specific cellular immunological mtwiories of the donor. Of thc materials present in thc dialyzatca, the two main candidates for transfer factor activity are polypcptidcs and/or polynucleotides. Serial transfer from individual A to B to C has also diminished the possibility that transfer factor acts as a superantigen, as has our inability to make an antibody to transfer factor in man or rabbit. These biological properties of transfcr factor, coupled with its demonstrated in zjitro and

248

11. S. LAWRENCE

in uiuo effects on nonsensitive lymphocyte populations, emphasize the rcpeated cell division and clonal proliferation of lymphocytes bearing transfer factor when cxposcd to antigen as an explanation for the replication of transfer factor which had been postulated on the basis of carlier data (e.g., serial transfer). Since the polynucleotides present in the dialyzate are rather small to code for the amount of information required, the informational aspects of transfer factor remain to be clarified. From in vitro studies and from the concrete visual evidence supplied by time-lapse cinematography, it is suggested that transfer factor may act as a derepressor of a select population of small lynlphocytes which may have been predestined to become antigen-sensitive under other circumstances, such as natural infection with the tubercle bacillus. VIII.

Transfer Factor and Mechanisms of Cellular Immune Deficiency Diseases

With the introduction of cellular transfer in animals ( Landsteiner and Chase, 1942) and extension of the principle to man (Lawrence, 1949), n technique became available which elucidated mechanisms of dclayed-type hypersensitivity in normal individuals, and thus allowed its confident adaptation to analysis of immunological derangements caused by or associated with disease (Good et aZ., 1962; Chase, 1966).

A. AGAMMAGLOBULINEMIA The most extensive and systematic application of cellular transfer to the study of disease has been that accomplished in agammaglobulinemia by Good and his colleagues (1957, 1962, 1966,1968). These patients were shown to respond to transfer factor with the acquisition of long-lived ( > 1 year), systemic, delayed hypersensitivity despite their documented deficiency in immunoglobulin synthesis. Moreover, transfer with blood leukocytes was found incapable of conferring on such recipients the capacity for antibody synthesis. Furthermore, agammaglobulinamic patients were shown capable of making their own transfer factor, in turn, as indicated by the prolonged duration of the transferred sensitivity. This conclusion was later confirmed by the demonstration that active sensitization ( BCG, horse serum, DNFB ) of agammaglobulinemics resulted in the development of the respective delayed sensitivity which was then shown to be transferred by means of cells to normal individuals ( Good et al., 1957; Porter, 1957). Studies by Gitlin et al. (1959) also demonstrated the cellular transfer of delayed reactivity to vaccinia virus from normals to agammaglobu-

TRAXSFER FACTOR

249

linemic patients. hlartin ct al. ( 1957a) demonstrated that lymph node cell populations, unlike 1)lood cells, wcrcl capable of transferring thc capacity for seruin iinmunoglobulin synthesis. The same cell population^ also transfcwcd ttilierculin sensitivity possessed l)y the donor. Of intcrest is the finding that antibody production in tho recipient was tenniiiated coincident with rejection of the transplanted lymph node after 110 days, yet the transferred tuberculin sensitivity persisted for as long as observations could be niadcb ( >300 days). These observations clearly separated the cell populations concerned with the transfer of serum antibody synthesis from the cell populations mediating thcl transfer of delayed hypersensitivity. In view of the fact that transfer factor for dclayed sensitivity is not rejected via a hoinograft response, these results also suggest that transfer of antibody formation in humans may depcmd upon intact functioning cells.

B. BOECK’SS.41icom In some way related to thc mystery surrounding its pathogenesis, sarcoidosis results in a puzzling immunological dichotomy characterized by a selective loss of delayed sensitivity in the face of normal seruin antibody production. The ancrgy detected was originally thought to be an exclusive impairment of tuberculin hypersensitivity, until later studies revealed a widespread irnpairmcnt of commonly encountcred bacterial, fungal, and viral antigens. This atypical response also extended to active sensitization with BCCr vaccinc and to simple chemicals. This impediment is more of R suspension rather than complete obliteration of delayed sensitivity responses, since cutaneous reactivity re-emergcs during quiescent periods of thc disease (Good et nl., 1962; Israel, 1965; Chase, 1966). It will be recalled that earlicr studies (Lawrence, 1949) had showed that intradermal depots of sensitive leukocytes placed in tubercnlinnegative normal subjects when challenged with tuberculin resulted in development of intense sensitivity at the local cell site and also in remote unprepared skin sites in which simultaneous tuberculin tests were placed. Thus, normal subjects respond with local plus systemic sensitivity whcm this method of transfcr is employed. The latter observations were confirmcd in normal individuals using viablc: leukocytes and thc technique extended to patients with Boeck‘s sarcoid by Urbach et al. (1952). They found sarcoid patients differed from normnls in that only local tuberculin scnsitivity was transferred and systemic sensitivity did not appear. These observations indicated an incapacity of tht, patient to respond to transfer factor and excluded “anticutines” or othcr postulated inhibitors a s causing tuberculin ancrgy. Since

250

13. S. LAWRENCE

intact viable cells were usclcl it appeared as if the sarcoid patient had merely loaned his skin to reveal a local interaction of cells bearing transfer factor 2nd tubcmulin. W e have recently re-examined this problcm in 7 wcll-documented, Kveim-positive, tuberculin-negative patients with sarcoidosis (Lawrenccb and Zweiman, 1968). For this purpose equal aliquots of highly purified dialyzable transfer factor from 1 tuberculin-sensitive donor were used. Intraderrnal injection of dialyzable transfer factor alone affords the decided advantage that it causes no cutaneous reaction immediate or delayed. This study confiimed the observations of Urbach et al. (1952) in that local but not systemic tuberculin sensitivity was transferred in 5 of 7 patients. Two patients, however, did develop feeble (10 mm.) tuberculin reaction in test sites remote from the injection of transfer factor. These exceptions could result either from increased dosage of transfer factor or an increased responsiveness of host cells. Five normal recipients scrved as controls and each developed enduring systcrnic tuberculin sensitivity as wcll as local sensitivity following injections of dialyzable transfer factor. Thesc results indicate an impaired production or function of the patients’ own specific transfer factors for common delayed sensitivities:, rcsulting in the observed anergy. The systemic unresponsiveness to transfer factor suggcsts impairment of host cells (presumably sniall lymphocytes) processing such information. The occasional transfer of systemic! sensitivity and, more particularly, the transfer of local sensitivity itself, suggests that transfer factor may engage a few competent lymphocytes and the immunological deficit is not absolute (cf. differences in Hodgkin’s disease below). The origin of the anergy encountered can be interpreted best in view of recent reports, which wc have discussed above, on the transfer of thc granulomatons clclayed reaction to Kveim antigen from patients with active sarcoidosis to normal individuals ( Lebacq and Verhaegen, 1963,, 1964; Behrend et al., 1968). This observation at the very least suggests: that the Kveim reaction fulfills a critical criterion of delayed hypersen-. sitivity, namely the capacity to be transferred by cells of the leukocyte series. These results become more significant in view of the failure of patients with sarcoidosis to respond as normal individuals to tuberculin transfer factor. Patients with sarcoidosis are, therefore, producing a transfer factor to Kveirn antigen at a time when their production of specific trans-. fer factors to other antigens is suspended. This suggests antigenic corn-. petition with Kveim antigen may precmpt thc natural production or function of transfer factors for other delayed reactions, as well as impair

TRASSFER FACTOR

25 1

the responsiveness of the patient’s cells to injections of transfer factor produced by another individual. Thus the patient’s constant preoccupation with Kveini antigen may be expressed by his amnesia for prior immunological cxpericnccs and by his feeble recognition of the inimunological memories of the clonor conveycd b y transfer factor. These results demonstrate the presence and indicate the extent of a specific immunological deficiency in sarcoidosis and suggest that it arises as a consequence of a selective aberration of those iminunocompetent memory cells which initiate and sustain the production of transfer factor and, thereforc, delayed hypersensitivity responses. We suggcst, therefore, that the anergy of sarcoidosis may represent an expression of malfunction of the particular lymphocytes engaged in the synthesis or rcxcognition of transfer factor and/or the transmission of the message it conveys. Whether this cellular illness results from overcrowding, displacement, antigenic competition, or other more subtle misadventures is conjectural at this time.

C. HODGKIN’S DISEASE Good and his collcagucs (Kclly et al., 1960, Good ct nl., 1962) have also applied cellular transfer to studies of the anergy encountered in Hodgkin’s diseasc, which like the anergy of sarcoidosis occurs in patients with normal inimunoglobulin production. They were unable to transfer tuberculin sensitivity, using the systemic method of transfer, to any of 13 patients with Hodgkin’s disease, whereas 22 of 23 normal recipients developed delayed sensitivity following transfer. This experience was confirmecl by Fazio and Calciati (1962), who, using the local method of transfer, failed to transfer systemic sensitivity. Of greater interest is their failure to inducc even local sensitivity in Hodgkin’s disease patients. Similar findings havc more recently been confirmcd by Muftuoglu and Balkuv ( 1967). This clifferencc from tho response of sarcoid paticnts to transfer factor, suggests a paucity of cnough iiiiinunocompetent cells to even initiate a local reaction in patients with Hodgkin’s disease (cf. Editorial, 1967). Muftuoglu and Ralkuv’s ( 1967) study, howcver, did demonstrate the successfiil transfer of delayed sensitivity to patients with acute and chronic myelogenous leukemia, to patients with lymphosarcoma, and to patients with carcinoinatous disease. The positive results secured in these categories of lymphomatous or malignant disease, separate them from the patients with Hodgkin’s cliscase and suggcst a different qualitative and qmntitativc, involvcrncnt of cc~llularimmunity b y thc Hodgkin’s disease process.

252

H. S. LAWRENCE

D. SYSTEMICNEOPLASTIC DISEASE Hattler and Amos (1965) studied the response of patients with advanced malignancy to transferred lymphocyte suspensions using the local technique of transfer. Their findings demonstrated that only local sensitivity was transferred without evidence of systemic sensitivity in each of 18 recipicnts. This result coincides with the experience accumulated with sarcoid patients (Urbach et al., 1952; Lawrence and Zweiman, 1968) and suggests, perhaps, a similarity of derangement in cellular immune responses. We have evaluated a comparable group of patients with advanced malignancy using dialyzable transfer factor and the method of local transfer (Solowey et “I., 1967). In these studies each of 10 recipients developed intense local delayed reactions ( SK-SD ) and each developed definite, but weak ( 10 mm.), systemic sensitivity that was not enduring. The results of studies assessing the responsiveness of patients with neoplastic disease to transfer factor indicate at least local engagement of immunocompetent host cells with uniformly positive results following local transfer. However, the presence, degree, and duration of transferred systemic sensitivity is variable and probably a reflection of the varying degrees and types of involvement expected in a mixed group of disorders (e.g., type and location of tumor, site, and extent of metastases). IX.

Transfer Factor and Reconstitution of Cellular Immune Deficiency

There is increasing awareness of the central role of cellular immune responses in the resistance to and recovery from infectious diseases of man where the infecting microbe exhibits a preference for residence within mononuclear phagocytic cells, I n all viral infections the intracellular residence is an obligatory prelude to the initiation and progress of the disease. Bacterial infections characterized by preferential intracellular parasitism are exemplified by Mycobacteria ( tuberculosis, leprosy), Salmonella, and Brucelk. Most systemic fungal infections also fall in this category, examples of which are coccidioidomycosis, histoplasmosis, moniliasis. This type of host-parasite relationship exhibits certain features in common, namely, ability of the microbe to resist intracellular destruction, intracellular multiplication of thc microbe, and prolonged intracellular residence without necessarily cell destruction. Much effort and intensive investigation has been directed toward elucidating the contribution of cellular immunity to recovery from infections of this type (reviewed by Suter and Rarnseicr, 1964; Macknness and Blanden, 1967).

TRANSFER FACIOR

853

Thc detection and characterization of naturally occurring primary cellular immune deficiency diseases in man (Good et al., 1962, 1968) and the production of similar secondary immunological derangements by associated disease (e.g., sarcoidosis, hodgkin’s, cancer, leprosy) or by immunosuppressive therapy ( steroids, 8 M P ) , has given new insights into this problcm (Lawrence, 1965n, 1968a). Both the natural absence and the acquircd depression of cellular immunity have resulted in disseminated infection by indigcmous viruses, fungi, and bacteria with fatal consequences to thc afflicted host, often despite appropriate antimicrobial therapy, and despite adequate immunoglobulin levels. A. GENERALIZED VACCINIA In progressive generalized vaccinia following smallpox vaccination, the primary vaccination site fails to undcrgo its usual evolution and regression and instead sprt)ads and becomes necrotic. Lesions of vaccinia appear at distant cutanrous and visceral sites and most cases terminate fatally. Transfer factor has been empIoyed successfully as a therapeutic immune reagcnt in one such infectious disease, gcncralized vaccinia. Kempe (1960) has reported a case ‘of progressivc generalized vaccinia in a 1year-old infant with high-titer serum antibody directed against vaccinia virus. The progressive disscniination of vaccinia virus was not inhibited by administration of human hyperimmune vaccinia1 y-globulin, by debridement, or by amputation of the affected area. However, the local and systemic transfcr of specifically sensitivc blood and lymph node leukocytes resulted in prompt and dramatic regression of the vaccinia lesions with recovery of the child from an otherwise fatal infection. The patient’s recovery w a s coincident with the appearance of transferred local, but not systemic, delayed sensitivity to killed vaccinia vinis. This successful therapeutic application of transfer factor has been confirnmcd in a similar casc of progressive generalized vaccinia occurring in an eldcrly adult ( O’Connell et d., 1964). This patient, like the child described above, also had normal levels of 7-globulin and an adequatc antibody response to the infection. In this instance also administration of vaccinia immune globulin (25 ml. ) did not halt the appearance or progress of vaccinia lesions and a scratch test with inactivated vaccinia virus was neg at’ive. Cellular transfer w a s accomplished using washed sensitive leukocytes obtaincd from 250 nil. blood which were injected intramuscularly. The donor was strongly tubwculin positivc, had reacted to a reccnt vaccination, and gavv a pnpulatc~ddclaycd reaction to inactivated virus. Twenty-

254

11. S . LAT?rREh'C:E

four hours after transfer the patient developed a moderate positive scratch test to vaccinia virus and the site of an earlier, mildly positive tuberculin test enlarged and became painful. Erythema appeared about the edges of the vaccinia1 lesions and increascd local pain was noted. Thc patient's course was one of progressive healing following transfer without residual lesions. Four months after transfer the patient relapsed coincident with reversion to a negative cutaneous reaction to vaccinia virus. Systemic transfer of an equivalent volume of leukocytes from the same sensitive donor, resulted in a local inflammatory reaction around the site of the recurrent vaccinia lesion in the foot immediately posttransfer which was followed by progressive healing of the lesion. Hathaway et al. (1965) have also referred to another patient with vaccinia necrosum who was successfully treated with huffy coat-rich plasma transfusions. This is a passing reference and details are not given. These cnses are instructive for students of infectious disease as well as for students of immunology. The failure of naturally produced or passively transferred serum antibody to eradicate this intracellular infection and terminate the disease provides a critical control of the efficacy of cellular transfer in accomplishing that goal. The coincidencc3 of recovery concomitant with the appearance of delayed sensitivity tci vaccinia virus, as revealed by inflammatory reactions around the lesions or b y dermal test, suggests again a strong causal relationship between delayed hypcrscnsitivity, cellular immunity, and recovery from intracelM a r infection.

B. DISSEMINATED MONILIASIS Buckley et al. (1968) have approached the problem of immunological reconstitution with regard to histocompatibility matching of donor and recipient and using tramfused allogeneic ( parental) marrow cells for transfer. Thc recipient was an ll-year-old patient with chronic, generalized cutaneous moniliasis since infancy which was resistant to repeated courses of y-globulin, amphotcricin, and Mycostatin therapy. No improvement followed administration of specific high-titer human antiserum Immunological investigation revealed normal immunoglobulin levels and noimal antibody responses to diphthcria and tetanus toxoids. Extensive impairment of delayed hypersensitivity was documented by the presence of anergy to a battery of antigens, depressed lymphocytc response to PHA, and prolonged survival of a skin homograft. The patient w x given an intravenous infusion of (342 x lo9) marrow cclls obtained from hcr fathcr without incident. Six weeks aftcr transfer she remained fwe of cutaneous moniliasis, except at several nailbeds,

TRANSFER FACTOH

255

and thc lesions of oral thrush were minimal. Whcn tcstcd 6 months after transfer the patient had acquired those delayed reactivities to which the donor gavc the strongest reactions ( candida and mumps) but no transfer of trichophytin, monilia, or staphylococcal sensitivity was detected. The transferrcd reaction to Canrlidu ( 2 + ) was present at 11 months posttransfer, a s were newly detccted reactions to diphtheria toxoid ( 3 + ) and staphylococcal antigens ( 1+ ). Coincident with the transfer of specific cutaneous reactivity, the responsiveness of thc patient’s lymphocytes to PHA returned toward nornial. The patient’s clinical condition con tinuecl to improve as evidenced by gain in weight and growth and no recurrence of inoniliasis when last observed 11 months after transfer. In spite of repeated attempts, no evidence of patcriial cells was detected either bj7 leukagglutination or karyotyping. The authors consider the possibility that transfer factor liberated from fragments of parcntal lymphocytes that had undcLrgonc hoinograft rejection, may have activated the patient’s own lymphocyte populntion.

C. LEPHOMATOUS LEPROSY We havc suggested clsewherc~that transfer factor may be adapted as a therapeutic immune reagent to convcrt leproniatous lcprosy to the tuberculoid type (Editorial, 1968). The lepromatous type of disease goes virtually imcheckcd by the host-despitc a high-titer of serum antibody bathing his tissues and his inacrophagcs ladcn with bacilli, there is no inflammatory reaction to leprosy baccilli or their products in the paticnt’s skin or rlsewhc~e.hforcovcr, there is ;I loss of prvviously established immunological memories of thc delaycd type as well as a diminished capacity for activc scnsitization. In contrast, the tubcwuloid type of leprosy is charactcrized by a benign, self-limited course-few leprosy bacilli are detectable>. little or no serum antibody response is present, and a markedly positive delayed hypersensitivity to lrpromin persists ( Rees, 1964). Coincident with the above suggestion, it h a s been demonstratt‘d that the mcrgic state of 5 of 13 patients with lepromatous Leprosy was converted to :i positivc lepromin reaction by transfer of leukocytes obtained from positive donors ( d e Bonaparte et al., 1968). This situation is not unique for lepromatous leprosy but is frequently scen in other disseminated infcctions particularly miliary tuherculosis and disseininated coccidioidomycosis. In the latter infections, impaired or absent dcxlaycd hypersciisitivity to the invading microbe has hccn associatcd with ldoodstream invnsion and widespread disscniination. In this situation there is a poor prognosis for rccovery in thc. face of normal serum antibody titcrs nnd often tlcspitc appropriate antiniicrohial therapy.

256

H. S. LAWRENCE

In cach instance, acquisition of the specific delayed hypersensitivity is associated with a favorable prognosis and followed by ultimate recovery for most patients. Whether the serum antibody detected has an “enhancing” effect on the infection as it does for tumor or tissue homografts is not clear at present. In any event, antigenic determinants of bacterial cclls that are camouflaged by antibody may blunt or dissipate a sustaincd delayed type of response and perpetuate the anergy observed (Lawrence, 1965a). OF RECONSTITUTION WITH INTACT D. HAZARDS IMMUNOCOMPETENT CELLS

The adaptation of cellular transfcr to treat patients with disseminated infcction, as a consequence of a natural or acquired cellular immune deficiency state, can be the only life-saving measure available for a desperate and otherwise fatal illness. However, when viable immunocompetent blood cells, particularly lymphocytes, are transferred to a host incapable of rejecting them via a homograft response, a graft-versus-host syndrome may develop resembling the runt disease originally described in experimental animals ( Billingham and Brent, 1959). This untoward complication has been reported by Hathaway et nl. (1965) in 2 infants suffering from progressive vaccinia necrosum, who had been treated with multiple transfusions containing viable leukocytes. Hong et al. ( 1968) also warn against this possibility and advise histocompatibility matching to avoid it if transfusion or cellular transfer is an unavoidable risk in otherwise fatal disseminated infections (see, also, Graft treatment of immunity deficiency, 1969).

E. POSSIBLE APPLICATIONSOF DIALYZABLE TRANSFER FACTOR In such potentially fatal clinical situations, we would suggest the possibility that dialyzable transfer factor may be a safer and more effective immune reagent to attempt rcconstitution of cellular immune deficiency disease. The advantages of dialyzable transfer factor for this purpose are ( I ) it is not antigenic in animals or man (Lawrence et al., 1963 unpublished observations; Valentine and Lawrence, unpublished observations) and ( 2 ) it is freed of all macromolecular cellular constituents including the transplantation antigens and, therefore, cannot cause a homograft response (Rapnport et a]., 1965a). The critical question whether dialyzable transfer factor can reconstitute cellular immunity to a lymphocyte population that is diminished in absolute numbers or otherwisc deranged in function has not been

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257

evaluated. The clues which suggest that this may be possible are slender and as yet sparse. Our own experience with sarcoidosis and neoplastic disease suggests that systemic as well a s local sensitivity may be on occasion transfcrred with dialyzates of leukocytes ( Lawrcnce and Zweiman, 1968; Solowey et al., 1967). Additionally, the child reported by Kempe ( 1960) cured of generalized vaccinia following cell transfer developed only local but not detectable systemic scnsitization to vaccinia virus. Finally, the patient reported by Buckley et nl. (1968) although transfused with parental marrow leukocytes recovered from the disease, developed cutaneous sensitivity, and acquired PHA responsiveness to her lymphocytes, despite a failure to detect evidence of viable parental cells, suggesting engagement of the patient’s cells by transfer factor liberated from parental leukocytes. In this connection, it is of interest that dialyzable transfer factor may be given repeatedly from the same donor or from pooled donors to the same recipient without immunological reaction of any kind to the dialyzate. The only detectable result is a boost in the intensity and duration of transferred tuberculin sensitivity, for example ( Lawrcnce, unpublished results). W e would also point out that the dosages of transfer factor that WE or others have used to date are miniscule and adapted to study the mechanism of delayed-type responses. For treatment, howevcr, much higher dosages given over a prolonged period would be desirable. The means for increasing the dose of transfer factor (e.g., concentration and storage in lyophilized state) and its safety on repeated adniinistration have been demonstrated. Thc application of dialyzahle transfer factor should, therefore, provc of value at least in the reconstitution of those cellular immune dcficiency states wlwre the Iymphocytic defect is not absolute or central. Whethcr transfer factor can favorably influence the outcome of disseminated infection in the extreme categories of cellular immune deficiency cannot bc predicted at this time. In this regard, attempts at transfer of delayed sensitivity with viable lymphocytes have failed in some types of cellular immune dcficiency. In this latter category are ataxia telangiectasia (Peterson et al., 196S, 1966) and, perhaps, the patient with thymic alyniphoplasia who acquired delayed sensitivity to Candidci following a thymic transplant ( Gitliil et nl., 1964). An indication of the potential of transfer factor in a particular deficiency syndrome may, howc~vcr,be gained from in vitro assays of the typc we have considerccl earlier ( c.g., transformation of patients’ Iymphocytcs following incubation u7ith dia1yzal)le transfcr factor, aftcr the appropriatc antigen has 1,ccn addcd) .

258 X.

1-1. S. LAWRENCE

Transfer Factor, Immunological Surveillance, a n d Tumor Immunity

Although an immunological approach to understanding and eflectively dealing with neoplasia is an old idea; it received new impetus with the demonstration of the imniunological principles underlying homograft rejection ( Medawar, 1959) and the demonstration of cellular transfer of rejection of normal tissues (Billingham et al., 1954; Lawrence et al., 1960, 1962) a s well as tumor grafts (Mitchison, 1955). Thomas (1959) postulated that delayed sensitivity ( a n inefficient and sometimes dcstructive attempt at eradication of bacterial cells) and homograft sensitivity ( a defense against the invasion of foreign tissues, unlikely to have developed in an evolutionary sense with this iatrogenic possibility in mind) may cach employ a mechanism that was evolved in multicelluIar organisms with the express purpose of recognizing mutant cells as foreign and rejecting them. Hence, in the face of likely exposure to an unknown number of mutant cells experienced over a lifetime, this dcfense against neoplasia protects many, but not all individuals. This postulated mechanism of defense against neoplastic mutant cells has since been termed “immunological surveillance” by Burnet ( 1967). This suggestion has also received additional indirect support in the increased incidence of tumors observed in animals with dcpresscd cc~llularimmunity following thymectomy (Miller and Osaha, 1967) or prolonged administration of antilymphocyte serum in thymectomized mice ( Gaugas et nl., 1969). It is also evident that some similar mechanism is probably depresscd in recipients of renal transplants who acquire tumor cells possessed by the donor. Such recipients, while on immiinosuppressivc therapy, have a normally functioning renal transplant and develop tumor metastases. Following cessation of immunosuppressive therapy, the patient rejects the donor’s kidney and the donor’s tumor, metastases, and all (Wilson et al., 1968). The simultaneous concurrence of inimunological deficiency syndromes and lymphoreticular malignancies cited by Good and his colleagues (Good et nl., 1962; Peterson et a!., 1966) may afford another example of this association, although whether the immunological deficiency is causal is uncertain. Early attempts to apply the principlc of cellular transfer to this problem in patients with neoplasia have been interesting but not conclusive ( cf. Alexander and Fairlcy, 1967). Woodruff and Nolan ( 1963) treated S patients with advanced carcinoiixitous disease with spleen cells from another individual. The recipients were given an imniunosiippressi~~e drug to prevent rejection of the transferrcd cells and slight improvcmcmt

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259

was notvd in all. Nadlcr and hfoorc ( 1965) took pairs of patients with advanec.d mdignimcy and sensitized each with the other's tumor implant 10-12 d:iys bcfore securing leukocytes from each individual and transferring them back into thc tumor graft donor. Five of the 14 recipients improvcd and 1 had a complctc rcmission without recurrence 1 year later. Thc principle appears a SOU^^ one, but a s yet its cffectivcness is more readily demonstrable in controlled experimental animal models ( cf. .4lcxandc~and Fairlie, 1967). The patient's tumor may, indeed, be regarded iis a homograft by virtue of the acquisition of new antigens a s suggested by Medawar ( 1967). The new appearance of transplantation nntigens differing from the host and distinct for the tumor has been den~onstratedfor methylchol~~nthrc.nc-induccdtumors ( Prehn and Main, 1957) as ivell as for tumors induced by oncogenic viruses (T-antigens) by Rowe ( 1967). Moreover, in human cancer tissue Krupcy el al. ( 1968) have described the prcscncc of a tumor-spccific antigen that is common to all adenocarcinoruatas derived from the digestive tract. It ill lw recalled from the cxperience in the transfer of homograft rejection in humans ( Lawrencc ct ol., 1960) that it w a s obligatory for anothcr individual to be actively sensitized via a homograft before the nppearance of a ncw transfer factor for that graft \wis dcmonstrablc. Moreover, the transfer factor produced in this fashion was highly individual specific, with specificity directed only against the transplantation antigens of the individual providing the homograft for sensitization and not against iintigcns of other individuals. This specificity of transfer factor poses certain operational problems if the tumor bornc by each patient is compriscd of antigens unique to it, since potential donors will not naturally possess a transfer factor specific for that antigenic configuration. However, in uitro techniques of lymphocyte sensitization may yet h e devised to circumvent this barrier. XI.

Conclusion

From the broad field of delayed hypersensitivity and cellular immunity we have r ~ v i e the ~ d cumulativc cxpcrience gainccl from cellular transfer in man over the past two decades. How this data led to the conccpt nnd, hence, to the reality of transfer factor is considcred. Thc central rolc of transfer factor as a common, although ypecific, mcdiator of this type of imniune rcsponse to a variety of bacterial, fungal, viral, protein, and tissue homograft antigcns has been documented. In addition to exploring and extending the biological properties of this ncw immunological principle, investigations have centcared around identification and charactcri7ation of the activc matcrial. This type of

260

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analysis became possible with the demonstration of the efficacy of leukocyte extracts in man. After an initial lag period, thcsc observations werc confirmcd and cnxtended and their validity is no longer in question. The sulmquent discovery that transfer factor is ii nonantigcnic, nonantibody, dialyzable moicty of < 10,000 mol. wt. has greatly facilitated theoretical as well as practical experimental approachcs to the problem. This finding has led to the beginnings of a molecular basis for cellular immune responses. The purification and concentration of transfer factor has also led to its adaptation to a variety of newly perfected in vitro correlates of cellular immunity. In these systems, transfer factor has taken its place as an informational or derepressor molecule among a family of effector molecules produced by antigcn-responsive lymphocytes and possessed of diversc functions. The thrust of current experimental efforts is directcd toward isolation and identification of each of these molecules, with attempts at establishing which may be identical and which different, despite their diverse functions. Time-lapse cinematography of captive sensitive lymphocyte cultures has given concrete visual evidence of how a select few (2%)cells of the total population are antigen-rcsponsive and the magnification of the effects observed results from repeated cell division arid clonal proliferation of these few cells. In vitro studies with dialyzable preparations or antigen-liberated supernatant preparations suggest that transfer factor acts as a derepressor of normal lymphocytes. Yet here again only a small population of cells ( 2 4 % ) is initially recruited or derepressed by transfer factor, which when cxposed to antigen transform and undergo repeated cell division and clonal proliferation. Thus, clonal proliferation of a small population of sensitive cells exposed to antigen suggests replication of lymphocytcs bearing transfer factor rather than replication of transfer factor alone. Recruitment by transfer factor of onIy a restricted number of nonsensitive cells out of the entire population exposed to it suggests that these few cells may be predestined to respond to that particular antigen (e.g., tubercle bacillus) and are held in reserve should the occasion arise. Thc principles evolved from the study of transfcr factor in normal individuals have found extensive application to the elucidation of immunological derangements encountered in diseases characterized by primary or secondary cellular immune deficiency states. Transfer factor has also been applied successfully as a therapeutic immunological reagent in the reconstitution of such cellular immune deficiency disease complicated by disseminated intracellular microbial infections. An increasing awareness of, and some indirect evidence for, the operation of a ccll-

TRANSFER FACTOR

26 1

mediated immunological survcillance mechanism for the disposal of mutant cell$, has resultcd in preliminary attempts at imniuiiological reconstitution of patients siiffering from neoplastic discnse with an appropriatc trmsfer factor. Thus ‘1 study initiated to elucidate mcchanisms of delayed-type hyperwnsitivity at a molecular Ievcl finds itself at the very core of understanding and effectively dealing with a much broader area which encompasses cellular iminunc deficiency disease, honiograit immunity, tumor surveillance. mechanisms, autoimmune disorders, and diverse host-parasite relationships.

ACKNOWLEDGMENTS It is a pleasure to acknowledge the helpfiil suggestions and critical comments of Sir Peter Medawar; as well as discussions with Dr. F. T. Valentine, in colla1,oration with whom the recent itt oitro studies on transfer factor have been done.

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hlaiirer, P. H. (1961 ) . J. Exptl. Med. 113, 1029. hledawar, P. B. ( I967 ). Qricitetl in Annotations, Iininiiiiology and Cancer. Laiicet ii, 1191. Medawar, 1’. B. ( 1 1 ). 1tc “Cclhilar and H i i i i i o r a l Aspcvts of the IIypersensitive States” ( H . S. Lawrence, rtl. ), p. 504. H;iiper (Hoel)eI), New York. Metaxas, RI. N., and Metaxas-Biililer, M. ( 1948). Proc. Soc. E x ) ) t / . Bit)/. M c t l . 69, 163. Miller, J. F. A. P., and 0solx1, D. ( 1967). P h ! / s i o f .Rc~o.47, 497. Mitchison, N . A. (1955). J. E x p h . Med. 102, 157. Mitchison, N. A. ( 1969). 111 “Imn~unologicalTolerance ( M . L a d y and W. Braiin, eds. ), pp. 114-124, Acadcinic Press, New Yo&. Mohr, J. A,, Killcbrew, L., Muchmorc, H. G., Felton, F. G., and Rhoades, E. R. (1969). J. Am. hled. Arsoc. 207, 517. Muftuoglu, A. U., and Balkuv, S. (1967). N . Etigl. J. Med. 277, 126. Nadler, S. H.,antl Moore, G . E. (1965). J. Am. Med. Assoc. 191, 105. O’Connell, C. J., Karzon, D. T., Barron, A. L., Plant, M. E., and Ali, V. hl. ( 1964). Ann. Ititernul M e d . 60, 282. Oliveira-Lima, 0. ( 1958). A m . Rev. Ttiberc. 78, 346. Pappenheinier, A. hl., Jr. ( 1955). J. ~ l ~ I t f L l L 7 1 075, ~ . “59. Pappenheimer, A. hl., Jr. (1956). Harvey Lccttires Scr. 52, 100. Pappenheimer, A. M., Jr., and Lawrence, H. S. (1948). A I I I J. . Hyg. 47, 233. Paque, R. E., Kniskern, P. J., Dray, S., and Baram, P. ( 1969). Fcrleratioti Proc. 28, 629. Peterson, R. D. A., Cooper, hl. D., antl Good, R. A. (1965). A m . J. M e d . 38, 579. Peterson, R. D. A., Cooper, hl. D., mtl Good, R. A. (1966). Am. J. M e d . 41, 342. Porter, H. (1957). Ann. N. Y. Acntl. Sci. 64, 932. Prehn, R. T., and Main, J. hl. (1957). J. Nntl. Cariccr Inst. 18, 769. H;cpaport, I;. T., Liiwrence, l l . S., Millar, J. Li’., l’appagianis, D., and Smith, C. E. ( 19801). J. Imniirtiol. 84, 358. Rapaport, F. T., Lawrence, €1. S., Millar, J. W., Pappagianis, D., and Smith, C. E. (1960b). J. Inimrciiol. 84, 368. Rapaport, F. T., Dausset, J., Converse, J. hl., and Lawrence, H. S. (1965a). Transplnntaiion 3, 490. Rapaport, F. T., Dausset, J., Converse, J. hf., ant1 Lawrence, H. S. ( 19Bq5b).Nutl. Acnd. Sci. Natl. Res. Council. Pirbl. 1229, 97. Rees, R. J. W. ( 1964). Progr. Allergy 8, 224. Robbins, J. €1. (1964). Science 146, 1648. Rosenan, W., and Moon, H. D. ( 1961). J. Nut/. Caficer 1 ~ t27, . 471. Rowe, W. ( 1967). I n “Cross-Rc-acting Antigens and Neoantigens” ( J . J. Trrntin, ed. ), p. 74. Williains & Wilkins, Baltiiiiow, h4aryl;ind. Ruddle, N. H., and Waksinaii, B . H, ( 1967). Scicvicc 157, 1060. R d d i e , N. €I., and Waksman, €3. H. (1968). J. E q d . Mecl. 128, 1237, 1255, 1267. Salvin, S. B. (1963). Progr. Alk!rg~y7, 21:3. Schlange, H. ( 1954 ). Arch. Kinderheilk. 148, 12. Slavin, R. G.: Tennenbaum, J. l., Becker, R. J., Feinberp, A. R., and Feinberg, S. M. (1963). J. Allergy 34, 368. Slavin, R. G., and Garvin, J. E. (1964). Science 145, 52. Solowey, A. C., Rapaport, F. T., and Lawrence, H. S . (1967). In “Histocompatibility Testing,” p. 75. Karger, Base!. Suter, E., and Ramseier, H. ( 1964). Atlr;a?i. Immtitrol. 4, 117.

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Thomas, L. ( 1959). In “Cellrilar and H ~ ~ n i o r aAspects l of the Hypersensitive States” (H.S. Lawrence, etl.), p. 529. Harper ( Hoeber), Ncw York. Thor, D. E. (1967). Science 157, 1567. Tlior, D. E. (1968). Fcderatiou Proc. 27, 16. Thor, D. E., and Dray, S. (196th). J. In~niunol.101, 51. Thor, D. E., and Dray, S. (1968b). J. Imrniinol. 101, 469. Tlwr, D. E., Jureziz, R. E., Veach, S. R., Miller, E., and D r ~ y S. , ( 1968). Nutitre 219, 755. Turk, J. L. ( 1967). “Delayed Hypersensitivity,” p. 105. LViley, New York. Uhr, J. W. ( 1965). Zti “Immunological Diseases” ( h l . Saniter ancl H.L. Alexander, eds. ), p. 305. Little, Brown, Boston, hlassachusetts. Uhr, J . W. (1966). Physiol. Rev. 46, 359. Ulir, J. W., Snlvin, S. B., and Pappenheimer, A. M., Jr. (1957). J. Exptl. Med. 105, 11. Urbach, E., and Gottlieb, P. M. (1946). “Allergy,” 2nd Ed., p. 150. Grime & Stratton, New York. Urbach, F., Sones, M., and Israel, H. L. (1952). N . Engl. J. Med. 247, 794. Valentine, F. T., and Lawrence, H. S. (196%). J. C h .Inn&. 47,9%. Valentine, F. T., and Lawrence, H. S. (196%). Federutiori Proc. 27, 265. Valentine, F. T., and Lawrence, H. S. (1969). Science 165, 1014. Warwick, W. J,, Page, A., ancl Cootl, R. A. (1956). Proc. Soc. Ercptl. B i d . hlecl. 93, 253. Warwick, W. J., Good, R. A., and Smith, R. T. (1960). J. Lab. Clin. Aled. 56, 139. Warwick, W. J., Archer, D. K., ancl Good, R. A. (1962). Ama. N . 1’. Acad. Sci. 99, 620. Wilson, D. B., and Billingham, H. E. ( 1967). Aduan. Inimrinol. 7, 189. Iliilson, R. E., Hnger, E. B., Hampers, C. L., Corson, J. M., Merrill, J. P., and Murray, J. E. (1968). N . E n g l . J. hlcd. 278, 479. Woodruff, M. F. A,, and Nolan, B. (1963). Lancet ii, 426.

Immunological Aspects of Malaria Infection IVOR N. BROWN Division o f Parasitology. Notional Institute for Medical Research. London. England

I. Introduction . 11.

I11.

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IV .

V. VI .

VII . VIII .

IX .

X. XI .

XI1. SIII .

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A . Definitions . . . . . . . . . B . Scope of Review . . . . . . . . T h e Life Cycle of hlalai+~ Parasites . . . . A . Daration of P1asrnodi;il Infection . . . . B . Symptoms and Pathological Efl’ects . . . Innate a r i d Nonspecific Immunity to Malaria . . A . Host Specificity . . . . . . . . B Genetic Constitution . . . . . . . C. Age of IIost . . . . . . . . . D . Environmental Contlitions . . . . . . E . Simu1t;ineou .: Infcctiun . . . . . . Iiiimriiiity Acqllired through Inlection . . . . A . Popiilation Stridies . . . . . . . B . Studies of Experinientally Inducetl Infections . C . T h e Plasniodial Life Cycle and Acquired Immunity Relapses and Antigenic Variation . . . . . Cellular Factors in Mslaria Infection . . . . A . Phagocytosis . . . . . . . . . B . T h e Spleen . . . . . . . . . C . Adoptive Transfer of Rlal~irialIinmiiiiity . . . D. Skin Reactions i n h4ah’ia . . . . . . Antigens of hlalaria Parasites . . . . . . A . Collection of Parasite Material . . . . R . hlalarial Antigens . . . . . . . . Iliimoral Fxctors in Malarial Iininunity . . . . A. hleasurcment of hlalarial Antibocly . . . B . The Protective Effect of hlalarial Antihidy . . Active Immiinizntion to Malaria . . . . . A . Immiinity by Infection . . . . . . B . Living Vaccines . . . . . . . . C . Killed Vaccines . . . . . . . . Experimeiitnl hfodification of I ~ n ~ ~ i [ i n i t v . . . Iiiiniunopatliolo~y . . . . . . . . A . Blood . . . . . . . . . . B . Arito~intibodics and hlalaria . . . . . . . . C . Nephrosis Associated with hlalaria . . . . . . . . . . . Discussion Summary . . . . . . . . . . Refcrcnces . . . . . . . . . . 267

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268 268 269 269 271 274 275 275 276 277 277 277 278 278 279 280 284 288 288 289 293 295 296 296 298 303 303 318 923 323 324

325 329 331 332 :334 :335

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339 340

26s

IVOR N. BROWN

I.

Introduction

Malaria in man is caused by infection with four species of the protozoon Plasmodium ( P . falciparum, P. vivax, P . malariae, and P. ovule). The disease is transmitted in nature by mosquitoes (it can also bc transmitted by the transfusion of infected blood) and is characterized by periodic paroxysms of fever (which are typically intermittent) and associated particularly with anemia and splenomegaly. Malaria is especially the cause of considerable infant mortality since infants suffer the acute form of the disease. In adults, it sometimes occurs as epidemics but more often it is insidious in its effect; it reduces the vigor of communities and by its continued presence causes a retardation of social and economic growth. The effect of malaria on a community is accentuated by the occurrence of other endemic diseases such as schistosomiasis and hookworm. Since Lavaran recognized the causative organism in 1880, knowledge of the biology of plasmodia has rapidly accumulated. Vulnerable links in the parasite cycle have become apparent, and major advances have been made in the development of synthetic antimalarials and in the development of insecticides. The World Health Organization has been committed to malaria eradication as one of its aims from 1955. It has been estimated that in the early 1950’s approximately 250 millioii persons had clinical attacks of malaria and possibly 2.5 million died of the disease in a year (Pampana and Russell, 1955). The impact of the WHO program has been such that of the 1576 million people living in the originally malarious areas of the world (this figure does not include China), 1224 million (77%) have been estimated to live in areas where malaria has been eradicated or eradication programs are now in progress (World Health Organization, 1966). The bulk of the residual exposed population resides in tropical Africa. Mass eradication programs have heightened, however, associated problems of parasite resistance to drug and insect resistance to insecticide. Potentially more dangerous, because malaria may be reintroduced by the reappearance of the vector, there may also be a drop in the immune status of communities through absence of infection ( Bruce-Chwatt, 1963a; Pringle and Avery-Jones, 1966). A recent outbreak in Ceylon in 1967 where over 1,000,OOO cases were recorded perhaps illustrates this ( Rruce-Chwatt, 1968). A.

DEFINITIONS

Hosts show n partial or complete resistance to plasmodia1 infectioii either because they possess innate immunity or because they posseqs acqiiired immiinity. These terms are used as defined below.

Ihf;\lUNOLOC.ICAI~ ASPECTS O F hfALAHIA INFEC‘I‘ION

269

Iiiiiate imniunitty is displnyed by hosts with no previous experience of malarial infection. Such immunity includes the effect of natural antibodies, the normal scavenging function of phagocytes, or the unsuitability of the host for growth of the Plasnioclizrm (for example, because the parasite is not able to utilizc the host’s hemoglobin). Acquired imiiiunity is a state of partial or completc rcsistance existing in a previously susceptible host. Such immunity may be nonspecific (for example, infection of the host with another organism may induce hyperreactivity of the reticuloendothelial system) or specific. Specific acquired immunity derives from the recognition of and the response to plasmodia1 antigen. It may be natiirally acquired by the passive transfer of antibody from inother to fetus or as a result of infection, or be art-ificinlly induced by immunization.

B. SCOPEOF REVIEW This review is p:irticularly concerned with specific acquired immunity to inalaria and includes a brief discussion oidy of innate and nonspecific resistance. Innate immunity has lieen discussed more fully by Taliaferro ( 1949), G a r n h m ~( 1963), and Zuckernian ( 1968). Reviews of acquired immunity include those by Culbertson ( 19-11), Coggeshall ( 1943), and Taliaferro ( 1949). More reccntly the subject has been discussed by Garnham et a!. ( 1963), Sadun et al. ( 1964, 1966), Garnham ( 1967), and Zuckcrman and Ristic (1968). A useful summary can also be found in the World Health Organization ( 1968) publication, “Immunology of Malaria.” II.

The Life Cycle of Malaria Parasites

Suggestions havc been inade in recent years to revise the nomenclature of nialaria parasites and to institute several new genera. These suggestions have not yet received universal a ptance. For the purposes of this review the words “Plasmoclium and plasmodia” will be used in their traditional broad sensc’. A detailed discussion of the morphology, taxonomy, and e\dutioii of plasmodia can be found in Garnhani’s book on malaria parasites ( G : u d i m ~ ,1966a ) . Plasmodia infect not only man but also other mammals (particularly primates), birds, and rcptilcs. The life cycle of a Plasmorlizim infecting primates is illustrated in Fig. 1. Sporozoites (the stage infective for thc vertebrate host) accmnulatc- in largc numbcm in thc salivary glands of an infected rnosquito and, wlicn it takes a blood meal, pass into the tissues of the primate. They do not infect erythrocytes directly but pass into parencliyin~itous cc~llsof thc 1ivc.r. Thif prelimillary stagc is called

270

IVOR N. BROWN

FIG. 1. The life cycle of a primate-infecting Plnsn~odiun~.( From Garnhan~, 1966a.) ( 1 ) Sporozoites from salivary glands of mosquito enter liver cells; ( 2 ) liver cell containing early stages of primary exoerythrocytic parasite; ( 3 and 4 ) stages in development of the primary exoerythrocytic schizont in liver cells; ( 5 ) fully clevelopecl primary exoerythrocytic schizont rupturing and releasing merozoites; ( 6 ) liver cell containing inerozoite of a secondary exoerythrocytic cycle of schizogony; ( 7-9) remaining stages in exoerythrocytic schizogony ending in a second generation of merozoites, ( 1 0 ) 1-ecl cell of circulating blood; (11-14) stages in erythrocytic schizogony in circulating bloocl; ( 1 5 ) fully cleveloped erythrocytic schizont rupturing and releasing erythrocytic merozoites; ( 16-20) repetition of erythrocytic schizogony; ( 21 and 2 2 ) development of male gamctocyte or microgametocyte in circulating bloocl; ( 2 3 and 2 4 ) development of female gamctocyte or macroganietocyte in circulating Iilootl; ( 25) wall of stomach of mosquito; ( 26) exflagellating microgametoeyte producing microgametes in stomach of mosquito; ( 27) macrogamete; ( 2 8 ) microgamete free in stomach of mosquito and seeking macrogamete; (29) zygote, formed by fertilization of macrogamete by a single microgamete; ( 30 ) ookinete formed by elong'ltion of zygote-it is al)out to penetrate cpithelial lining of stomach; ( 3 1 ) oocyst, formed b y ookinete after penetration of stomach wall of mosquito-it lies under elastic membrane on outer surface of stomach; ( 3 2 and 3 3 ) stages in development of oocyst with production of sporozoites; ( 3 4 ) rupture of mature oocyst with dispersion of sporozoites most of which enter salivary g l a ~ ~ dofs mosquito; ( 3 5 ) salivary gland of mosqiiito coiitaiiiing mature sporozoites.

IhfhflUNOLOGICAL ASPECTS OF hI4LARIA 1SFEC"I'IO.V

271

the primary ( i.c., derived from sporozoite) exoerythrocytic or tissue stage. I n the liver parenchyma cell the parasite nuclc~us divides many times and the cell containing the dividing parasite or exoerythrocytic schizont becomes considerably enlnrged. After about 8 days several thousand mcrozoites are liberated. In Plu~sniocliumvivux and PZcr.smor?itrm muluriae the exoerythrocytic stage is thought to persist by rc~infectionof other liver ccJlls by merozoites to procluce secondary exoerythrocytic stages. More usually nierozoites penetrate erythrocytes. Inside the erythrocyte the parasite digcsts thc red cell contcmts and grows rapidly to occupy much of the host cc.11. Malarial pigment is formed as an end product of thc digestion of host red ccll hemoglobin. Ultimately the parasite nucleus dividcs and 8 to 16 claughtcr nierozoites are formed in each schizont. At schizosony thc red cell ruptures and nierozoites are released into the bloodstrc~amto infect more erythrocytes. On penetrating thc red ccll, sonic merozoites develop into male and female gamete-producing cells (micro- and macroganietocytes ) . If these cclIs are taken into the gut of a mosquito a s part of a blood meal, release of the gamctes from the enclosing red cells and fertilization occur. The zygote (ookinete) passes throiigh thc wall of thc gut a n d encysts on its outer surface. I t then undergoes a rcduction division followed by rcpeated mitotic clivisions. The cyst eventiially bursts to release uninucleatc, elongate bodies (sporozoites) into the hemococl of thc host insect from whew they reach the salivary glands. The criteria for the current classification of subgenera of the genus PZusmodirrin refer first to the vcrtcbratc, host and then to various characteristic features of the life cyclc such as size of the erythrocytic schizont, the shape of the gunictocyte, and the site and rate of devclopmeiit of thc exoerythrocytic schizont. This review concerns primarily the mammalian subgcnera and to this piirpose the characteristics of the t h r w inaiiimalian-infectinbr subgcwera are given in Table I. The characteristics of the four human plasmodia are suinmarized in Tablc 11. Reference will, howcver, be made wherc necessary to avian parasites and, apart from their host specificity, these parasites differ from mammalian parasites because ( I ) the exoerythrocytic stage may occur in tissucs other than thc liver (whereas in mammals it is probably restricted to this organ) and ( 2 ) crytlirocytic merozoitc,s c:m givc rise to cxoerythrocytic forms (in mammals, blood-induced infcctions do not initiate tissue stages ) .

A. DIJRATION OF PLASAIOIIIAI. INFECTION After sporozoitc inoculation the duratioir i ~ i dtype of infcction varies amon2 individuals ant1 also accorcling to the spceies of puasite. Relativc

TdBLE I DIFFERESTIAL CHARACTERISTICS OF THE SUBGEXERA OF MAMMALIAN SPECIES OF

Subgenus

Blood schizont

Nerozoites

Shape of garnetoryte

Pigment

Plasmodium

Large

8 o r more

Spherical

Kidespread

Laveranzu

Large

8 or more

Crescentic

Perinuclear

VZ,lckezu

Small

Usually 8

Spherical

\Videspread

or fewer

a

,Ifter Garnham, 1966a.

Prepatent period 5 days or more

5 days or more 3 days or less

~~~?nOdifi?If"

Exoerythrocytic Vertebrate stages host

Examples

Primary

Primates

P . malarioe P. z+rlax P. ovule P. li?towlcsi P. cynomolgi P. faleiparum

Primary and secondary

Lemurs and lower mammals

P. berghci P. oinckei P. chahaiidi

Primary and secondary

Primates

2 0 %J

?: m

z

THELIFE CYCLE

TABLE I1 O F TIIE HTMASPLAShIODIA”

Developmeu t

Exorryitwocytic sta{p Minimal time for development Xo. of merozoites per schizont Secondary exoergtlirocytic st ages Erythrocytic stnqc Timing of cycle “Withdrawal” of segmeiiting forms S o . of nicrozoites per schizotit Parasitemiti-(1)

Usual

(2) Maximal

a

3-7 days 3040,000 Not denlolistrated

48 hours Yes 8-16, double iiifectioiinot iiiico~nnion Extremely variable, high denzities (500,000/mm3) not uncommon in children Can reach 40-50%

From Boyd, 1!)49; Gartiham, 1966a.

8-13 days

.ll>Ollt

Tes

18-17 days 3000 Tes

48 hor1rs so 16

7 2 hours A-0 8-10

48

About 60,000/1nm3

2.5,000/mm3 rarely reaches 30,00O/mm3

Can reach 100,000/n1n13

10-1,5,000

9 days

15,000 \-es

s0

I1Olll.h

x

274

IVOR N. BROWN

to the lifetime of the host, an infection of inan with Plasinoclium falciparurn or Plasmoclium vivax is of short duration; P. falcipciruni does not usually persist for more than a year, nor P. vivax for more than 2 years. In contrast, latent Plasmodircm nialarine infection may persist for over 20 years. A first clinical attack of inalaria is often followed by others which in the abscnce of reinfection are termed relapses (see Section V ) . Aftcr P. fulciparum infection, relapses normally occur soon after the primary parasitemia attack. This Plasmodium is thought to have no persistent exoerythrocytic stage and relapses appear to originate from latent blood infection. On thc other hand, after P. vivux and P. malnriae infection, relapses may also occur long after a primary attack. These plasmodia arc thought to have a persistent exoerythrocytic stage which can initiate crythrocytic attacks in addition to those attacks derived from latent blood forms.

B.

SYhlI’TOMS AND PATHOLOGICAL

EFFECTS

Thc characteristic symptoms and pathology of plasmodia1 infection is caused by the blood stage of the Plasmodium. Paroxysms of fever, accompanied by chills and sweating, arc associated with erythrocytic schizogony. The timing of these paroxysms varies according to the species of Plasmodium. For cxample, Plasmodizini vivax undergoes schizogony eveiy 48 hours and paroxysms occiir every third day, whereas Plasmoclium mnlariae segments every 72 hours and paroxysms occiir every fourth day (see Table 11). The pathological eff ccts of infection are numerous, but the principal effects can bc summarized as follows: 1. The production of a moderate to severe anemia often in excess of that due to parasite destruction. This effect may be due to the invasion of erythropoietic stem cells (as occurs in some avian plasmodial infections) or possibly to an immune reaction to normal erythrocytes (see Section X I ) . 2. The disturbancc of tissue function by toxic products released from the parxite. 3. The disturbance of blood circulation because of blockage of fine capillaries by parasiti7cd cells and parasitized cell debris (such as rcd cell stroma and malarial pigment ) lcading to necrosis and hcinorrhage. The fixed tissucs most noticeably affected b y plasmodia1 infection are the splcen, liver, and bone marrow. All become full of infected crythrocytes, ccll debris, and pigmcnt; the splccn and liver often become enI m g d and their tissucs hyperplastic. In the brain, thc acctimiilation of

IMMUNOLOGICAL ASPECTS OF hfALARIA INFECTION

275

large numbers of parasitjzed cells m i i y lead to local necrosis and cerebral malaria (Edington, 1967). This complication is most often seen in young children with Plc~.smodiziinfalcipumm intcxction. The kidney is also particularly affected by malaria (sec Section X I ) . Blackwater fever is nssociated with acute P. fnlcipurum infection and is characterized by a sudden clinical attack followed by hemoglobinuria. Plnsniocliim m a h i e infection which is, in gcneral, low grade and of long duration, is thought to be responsibk for the nephrotic syndrome obscrvcd in children in parts of Africa and the Far East. Ill.

I n n a t e a n d Nonspecific Immunity to M a l a r i a

Innate resistance of mosquitoes will not be discussed here but has been reviewed by Garnham (1964), Huff (1965), and Zuckerman (1968). Vertebrates show varying degrees of innatc immunity ranging from a complete resistance, throuzh a phasic resistance (in which, for example, apparently normal development of exoerythrocytie stages occurs but no erythrocytes are infected) (World Health Organization, 1968), to an incomplete resistance (in which development of all stages of infection occurs but parasite multiplication is restricted). The mechanisms of innate immunity are ill-defined, but among the factors that can affect observed infection are the species of host, its genetic constitution, its age, and its environment. Immunity may also be nonspecifically acquired by infection of the host with another organism. These factors arc briefly discussed below in order that thcy may be borne in mind during subsequent discussion of specific acquired immunity. They are more fully considered in the articles cited in Section 1,B.

A. HOSTSPECIFICITY In general plasmodia show a marked host specificity. The more closely related the prospective host is to the natura1 host the more likely infection is to occur. Unfortunately for laboratory workers, the human plasmodia are probably thc most restricted of all the mammalian subgenera in thcir ability to infect hosts other than man. Human plasmodia] infections can bc produced in some monkeys and in higher apes but development is often rcstricted to the exoerythrocytic stage with no or only subpatent erythrocytic development. Such infections are usually enhanced by splenectomy (Bray, 1958; Garnham et al., 1963; Gould et al., 1966). The owl monkey, Aottrs iriuirgatus, will support infections of Plasmoclium falcipurum (Geiman and Meagher, 1967), and recent studies (Voller et al., 1969) have indicated that these monkeys show a similar pattern of infection to man. Owl monkeys are difficult to keep in captivity but if

976

IVOR N. BROWN

this problcm can be overcome they could provide, for the first time, a model of human plasmodia1 infection for clinical and immunological study. Simian plasmodia, on the other hand, seem more readily to infect man. Plasmoclium knowlesi of macaque monkeys has been used for malaria therapy in neurosyphilitics. Recent reports of natural transmis5ion of monkey malaria to man (Chin et al., 1965; Deane et al., 1966) have revived interest in the zoonotic potential of the siinian parasitrs ( Garnhain, 1967; Bray, 1968). B. GENETICCONSTITUTION Within a susceptible population there are differences in incidence and severity of infection determined by differences in genetic constitution among the individuals of that population. The variation in susceptibility to Plasmodium berghei shown by inbred mouse strains and their hybrids illustrates this ( Greenberg et nl., 1954; Greenberg and Kendrick, 1957). In human malaria there is evidence that some genetic resistance is conferred by both the sickle-cell gene and the glucose-6-phosphate dehydrogenase ( G-6-PD ) -deficiency gene. The sickle-cell gene, which is responsible for the production of an abnormal hemoglobin, is common in several human populations and in particular, in those of Central Africa. The effect of the trait is most noticeable in young children (1-4 years) who lack significant acquired immunity to malarial infection. The proportion of infants showing parasites, as well as the parasite counts, is lower in trait carriers (Allison, 1957, 1961; Edington and WatsonWilliams, 1965; Gilles et nl., 1967). The reason for this conferred resistance is uncertain, but indirect evidence that other abnormal hemoglobin types also protect against malaria suggests that the mechanical effect of sickling is not responsible. Malaria parasites metabolize hemoglobin, and abnormal hemoglobin may prevent normal development of the parasite. The selective advantage conferred by this gene in malarious areas could possibly account for its survival in their human populations “Abnormal” hemoglobin types may, in addition, determine the effect5 observed by Greenberg and his colleagues and, probably, contribute to the observed host specificity of the malaria parasite. The geographic distribution of the G-6-PD-deficiency trait is remarkably similar to that of Plasmodium fakiparum, but evidence is conflicting as to whether possession of the trait protects against malaria. The studies of Allison and Clyde ( 1961) and Gilles et al. (1967) demonstrated that enzyme-deficient children suffer less from infection than do “normal” children. Most of the conflicting evidence comes from studies of older children (where differences were somewhat obscured by acquired im-

IMMUNOLOGICAL ASPECTS OF hIAL4nI.4 INFECTION

277

tnunity ) or from studies on adtilt white voliintccrs (whcrc. only low parasitemias were used). Little is known of other gtmcltic factors involved in natural inimunity to malaria. Amcrican Negrocs show a marked rcxsistancc to infection with Plasmodiiini vivux ( Boyd and Stratn~aii-Thonias,1933), whereas American Cuucasians do not. A similar resistance can bc observed in many West African Negro populations.

C. AGEOF HOST Young persons and young animals tcnd to be more susceptible than adults to a first infection; after apparent cure they are more likely to rekipse. For exmiple, a Plusnzorliuni berghei infection in suckling and weanling albino rats was almost 100%fatal, whereas in adult rats, weighing nearly 200 gin., only 33%of the animals died (Zuckerinan and Yoeli, 1954). The peak parasitcmia was niuch higher (90%compared with 6%) and occurrcd later (15 days coinpared with 8 clays) in the young rats compartd with thc old rats. On the other hand, the adult rats who died of infection died earlier than the young rats (10.3 days compared with 17.0 days).

D. EXVIHONMENTAL CONDITIONS Variation in environmental conditions can affect susceptibility to malaria. Dietary deficiencies, e.g., p-aminobenzoic acid ( Hawking, 1954; Kretschmar, 1965; Jerusalem and Kretschmar, 1967) and ascorbic acid ( McKec and G c h a n , 1946) limit the in vioo multiplication of malaria parasites, and strcss can acccntuate infection ( Kretschmar, 1964).

E. SIhlULTANEOUS INFECTION Evidence is conflicting as to what effect simultaneous infection with another organism has on malaria infection. Typhoid fever has little influencc on malaria ( Giglioli, 1933) altliough typhoid symptoms inay bc morc: severe in malarious subjccts ( Nazario, 1929). Tuberculosis and malaria inny 11v mutually antagonistic ( Yoeli, 1966; Voller and Rossan, 1969d). Also, ;I concurrent Eperythrozootx coccoides infection protects mice against Plastnoclizim berghei and Plasmodium chabaticli infection (Peters, 1965; Voller and Ridwcll, 1968). The reason for this protection is unknown, but it has been suggested that there may be competition for an unknown substrate or that there may be a nonspecific heightening of phagocytosis. On the other hand, if mice are made anemic by infecting with Haemo1)nrtonelln nitiris and then infected with P. berghei the malaria infcction is potentiated (Hsu and Geiman, 1952). This effcct is

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IVOR N. BROWN

probably duc to known prefcwncc of this specics for infecting thc immature erythrocyte of thc mouse. This p r c h e n c e can also be used to block intection. In normnl mice made polycythcmic by hypertransfusion, ‘1 P. b e r g h i intcvtioii is partidly blocked cven though a large population of mature red cells is availablc for inf-cction. Idcction is coinpletcly blockcd in polycythemic mice by irradiating them 3 days before infection to remove rr~sicliialcrythropoiesis ( Ladda and Lalli, 1966) . Chloroquineresistant P. berghei parasites show a particularly markcd preference for rcticulocytes, and irradiation of normal mice before infection with resistant par‘isites produccs a great diminution in parasitcmia; in addition a high proportion of mice do not become infectcd ( D . C. Warhurst, personal communication) . IV.

A.

POPULATION

immunity Acquired through Infection

STUDIES

How malaria appears in a community depends largely on the inanncr of its transmission. Where transmission is low, the position of the parasite is prccarious. Under these conditions the community does not develop a high level of resistance and, consequently, may suffer infections of epidemic proportions, for example, due to a sudden increase in numbcrs of an effective vector. The parasite most often associated with such conditions of transmission is Plnsmocliuni wivax because of its capacity for late relapses, although Plasmodiiim f a k i p a r u m may appear also. In contrast, in some areas of the world, notably Africa where P. fnlcipariiin predominates, malaria transmission can be continuous and at a high level ( stable nialaria ) although subject to seasonal fluctuation. Studies of hunian populations living in such areas have revealed a remarkably consistent pattern of infection. The incidence (see Fig. 2 ) and density of parasitemia is maximal in young children and declines progressively in the oldcr age groups (Christophcrs, 1924; Taliaferro, 1949; Rrucc-Chwatt, 1963a). During the first few years of life, infections are sevcw and, without treatment, may cause the death of infants. Young children who survive this critical period still suffer heavy parasiteniiar but seem better able to tolerate the infection than infants. This type of immunity is called “clinical” or “antitoxic” immunity. Through older childhood and adolescence, parasite densities decrease until in adult lifc only low levels of parasiteinia are encountered. During this period, when parasite densities are falling, a true antiplasmodial imniunity is thought to be ncquired. MacDonald considers young children as being largely rcsponsihle for continued malarial transmission but knowledgc of the

279

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

1 2 5

10

16

30

40

50

60

Age in years

FIG.2. The incidence by age of malaria parasitemia in Keneba villagers (1950). Keneba is situated in Gambia, West Africa, an area endemic for malaria. Plasmodium fulciparum is the dominant parasite. (From McCregor, 1964.)

natural history of malaria is still incomplete ( MacDonald, 1957; Miller, 1958; McCregor, 1965; Rruce-Chwatt, 1965). B.

EXPERIMENTALLY INDUCED INFECTIONS The use of the experimentally induced infection has the advantage that the source and type (infected blood or sporozoites) of infective material and the timing of infection or reinfection can be controlled. Most studies in this respect concern laboratory animals and birds, but man has also been experimentally infected, either as a volunteer or as a paretic undergoing malaria therapy. The effectiveness of an acquired antiplasmodia1 immunity depends largely on the degree of previous infection [this is illustrated by the expcriments of Maier and Coggeshall (1944) described in Section 1x1. Such immunity niay be antitoxic rather than antiparasitic and show as a suppression of symptoms normally associated with erythrocytic attack. The nature of this immunity is not known. Antiparasitic immunity is most evident during the erythrocytic stage of infection and may show as a lengthened prepatent period after parasite inoculation, a shortening of the primary erythrocytic attack, reduced parasitemia, or a complete clearance of parasites from the host tissues. These expressions of antiparasitic immunity niay act independently of one another. Antiplnsmodial immunity is restricted in its specificity and may be STUDIES OF

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IVOR N. BROWN

effective only against a strain or strains of a given species of Plasmodium (James, 1931; Sinton et al., 1939; Boyd and Kitchen, 1945). This species specificity has been demonstrated in human, simian, and avian malarias ( Gingrich, 1932; Mulligan and Sinton, 1933; Taliaferro and Taliaferro, 1934; Boyd et al., 1936; Manwell, 1938; Taliaferro, 1949; Jeffery, 1966; Voller and Rossan, 1969b,d) and in the rodent malarias (F. E. G. Cox, 1966; Cox and Voller, 1966). After the natural elimination of infection, resistance to further homologous infection lessens although complete susceptibility may never rcturn in some host-parasite combinations. That a true immunity in malaria infection may in reality be an immunity to superinfection has led to much controversy. Sergent et al. (1924) introduced the term “premunition” to describe a nonsterile or coinfectious immunity and compared premunition with sterile or residual immunity. Although useful descriptively, the validity of such a distinction on immunological grounds is questionable. The increased efficacy of immunity to superinfection may be explicable on the basis of an adjuvanted immune response and the waning of such immunity after elimination of the parasite.

C . THEPLASMODIAL LIFECYCLE AND ACQUIRED IMMUNITY Apart from the strain and species specificity of acquired antiplasmodial immunity, there is evidence that those stages of the life cycle that are immunogenic stimulate an immune response specific for themselves. This further specificity is discussed below with reference to the sporozoite, the exoerythrocytic stage, and the blood stage.

1. Sporozoites Whether immunity to sporozoites contributes to immunity acquired in endemic areas is not known. Under natural conditions of transmission they do not appear to provoke an effective immune response. Sporozoites that do not develop within liver parenchyma cells may represent an insufficient antigenic stimulus or a degree of immunity may be developed which is ineffective because injected sporozoites are not in tissue fluids for a long enough period. It could be similarly argued that their brief extracellular life (an hour or less) may not allow for an anamiiestic response. Garnham (1966b) found no evidence of an immunity to Plasmodium cynomolgi bastianelli sporozoites (judging from the number and appearance of liver stages after sporozoite inoculation ) in rhesus monkeys sensitized either by sporozoite-induced infection alone or by sporozoiteinduced infection followed by the injection of large numbers of formalinkilled sporozoites in complete Freund’s adjuvant. However, if sufficient

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

281

killed sporozoites of thc avian Plasmoclium, P. gallinaceurn, are injected into fowls, an apparent immunity to sporozoitcs can be induced (Mulligan et al., 1941; Russell et al., 1942). Fowls vaccinated with sporozoites inactivated by ultraviolet light or by grinding, drying, and subsequent reconstitution, gave varying serrim agglutinin titers against the honiologous sporozoites. Those fowls having titers less than 1: 16000 ( this would includr normal birds ) were susceptible to mosquito-borne infection (mortality 51.4%), whereas most fowls having titers greater than 1:32000 were totally resistant or developed mild infections only (mortality 7.7%).Fowls resistant to sporozoite challenge were, however, compIctely susceptible to intravenous challenge with blood containing P. gallinacerim trophozoites. Recent findings with a similar experimental system ( Richards, 1966) have confirmed these results. Sporozoites werc inactivated by the above methods and also by formalin treatment and by freeze-thawing. Young chicks immunized by any of these preparations developed good serum sporozoite agglutinin titers and were partially resistant to sporozoite challenge but were completely susceptible to challenge with erythrocytic parasites. The birds were protected against death rather than infection, for rcsistant birds were found to be carrying latent infections. Also Nussenzweig et al. (1967, 1969) found that mice sensitized by repeated injection of irradiated sporozoites of Plasmodium berghei did not show blood infections if infected with P. berghei sporozoites, whereas mice sensitized with noninfected mosquito salivary gland tissue developed and died of blood infections, In addition, mice similarly sensitized with P . berghei sporozoites did not develop blood infection after the inoculation of Plasmodium vinckei sporozoites, but nevertheless were completely susceptible to a blood-induced infection with either P. berghei or P. vinckei. Possibly the sporozoites of these two species of rodent malaria show more antigenic similarity than do their blood forms. Mice immune to the blood forms of P. berghei are susceptible to infection with the blood forms of P. vinckei. 2. Exoerythrocytic Stages

Not only sporozoites but also exoerythrocytic stages are apparently unaffected by spccific acquired immunity. Immune chickens and pigeons, when inoculated with sporozoites of Plasmodium gallinaceum and Plasmodiuna relicttim, respectively, showed pre-erythrocytic tissuc stages but no significant parasitemia ( Huff and Coulston, 1946). Similarly, if homologous sporozoites were inoculated into a person immune to

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Plasmodium uivax the development of pre-erythrocytic schizonts in the liver occurred but in the absence of patency ( Shortt and Garnham, 1948). Also, rats recovered from (and resistant to) trophozoite (or sporozoite)induced Plasmodium berghei infeetion developed pre-erythrocytic schizonts in liver parenchymal cells on reinoculation with P. berghei sporozoites (Yoeli, 1966). Liver biopsies taken 48 hours after inoculation of sporozoites showed mature and apparently normal exoerythrocytic schizonts, yet erythrocytes did not become infected. This immunity to the blood stage need not be acquired but may be innate. For example, birds vary in their susceptibility to P. gallinaceum (Huff and Coulston, 1946). Chickens are susceptible to both tissue and blood stages, whereas canaries show a complete resistance to the parasite. In intermediate range geese, ducks, and guinea fowl all show apparcntly normal pre-erythrocytic development but the resulting parasitemia is transient in the goose, only subpatent in the duck, and probably nonexistent in the guinea fowl. Similarly the tissue forms of some mammalian parasites may develop in abnormal hosts which show innate resistance to the erythrocytic stage. The nature of this resistance is not known but can often be lowered by splenectomy, when the hosts suffer transient to moderate parasitemias. The red cells of such animals are, therefore, capable of supporting parasites. I n macaque monkeys infected with Plasmodium cynomolgi sporozoites there is a considerable variation in the number of sporozoites that develop into pre-erythrocytic schizonts, but the number of liver stages produced by a given sporozoite inoculum cannot be correlated with any existing state of immunity (Garnham and Bray, 1956). Also the morphology of the liver stages in “immune” animals is identical to that of liver stages in “normal” animals (see also Yoeli, 1966). It has been observed that liver stages become surrounded by phagocytic cells, but whether this signifies a specific immune response is unknown. Within the liver, development of the parasite is largely intracellular and this may preclude an immune response. However, in the brief period when the exoerythrocytic schizont matures and merozoites are released there may possibly be an antigenic stimulus. 3. Blood Stage

The blood stage of the Plasmodium is the most susceptible to thc effects of immunity. Thus, hosts possessing innate immunity often show normal development of exoerythrocytic stages but no or Iittle erythrocytic infection. Similarly, human subjects previously infected wit11 Plasmodium falciparum, Plasmodium uivax, or Plasmodium ovule become

IMMUNOLOGICAL ASPECTS O F MALARIA INFECTION

283

infected after the injection of homologous sporozoites but the remitting parasitemia is transient only (Boyd et al., 1936, Boyd and Kitchen, 1936; Sinton, 1939a,b, 1940) . Studies of antimalarial antibodies, the cellular reactions to infection, and the effect of existing immunity on bloodinduccd mammalian and avian infections have largely confirmed that the blood stage is susceptible to immune attack. Much of the work rcferred to below pertains to immunity and erythrocytic infection. The asexual erythrocytic Plasmodium seems most susceptible just before and at schizogony (Coggeshall, 1943; Taliaferro and Bloom, 1945; Cohen and McGregor, 1963; K. N. Brown and I. N. Brown, 1965) but, in addition, merozoites may be affected by immunity. The engulfment of free P . fulciparuin merozoites by polymorphonuclear leukocytes has been observed in a warm stage preparation of infected human blood drawn from a relapsing patient ( Trubowitz and Masek, 1968), and presumably similar and more specific mechanisms operate in vivo. In his extensive studies of immunity to plasmodia1 blood forms, Taliaferro ( 1948, 1949, 1967) differentiatcd parasiticidal mechanisms from reproduction-inhibiting factors, making the assumption that specific acquired immunity as it developed was superimposed upon existing innate immunity. Antibody levels were not correlated with the observed effects. The effects of acquired immunity were most noticeable during the period of parasite decline, i.e., just after peak acute parasitemia and crisis. These effects were a decrease in the number of merozoites produced per schizont (which may have been an apparent effect due to more e&cient removal of mature schizonts from the blood) and an increase in the number of parasites that died. In the host-parasite combinations studied when the acute parasitemia subsided and only low parasite levels were detectable, the parasite reproductive rate regained its precrisis level, but the number of merozoites that survived to reinfect new erythrocytes remained at low level only. In some simian infections, well marked degeneration is apparent in erythrocytic parasites in semi-immune hosts (these degencrate forms occur mostly just after peak parasitemia and are called “crisis” forms ) . Gametocyte levels fall subsequent to depression of asexual parasite levels, but it is not known whether gametocytes are directly affected by immunity-there is evidence suggesting that they may be. Plasmodium cynoinolgi ganietocytemia persisted at a high level through the crisis of asexual forms in rhesus monkeys, but infectivity for mosquitoes was nevertheless, markedly reduced (Hawking et al., 1966). Similarly, in experiments involving thv transmission of rodent malaria parasites through mosquitoes, a coininon observation ( Wery, 1968) is that transmission is

284

NOR N. BROWN

more easily obtained if mosquitoes are fed on rodents at an early stage of acute infection. One interpretation of these observations (but not the only one) is that immunity affects gametocytogenesis or gametocytes directly. That Plasmodium falciparum gametocyte levels rise after depression of asexual parasitemia levels with passively administered immune 7-globulin (Cohen et al., 1961) would not be inconsistent with this interpretation for the infectivity of gametocytes was not tested in these experiments. Also P . falciparum may be an unusual parasite because its gametocytes are thought to take 8-9 days to develop. On the other hand, high levels of malarial antibody in rodent serum has no apparent effect on the development of rodent malaria infection in mosquitoes up to the oocyst stage ( Killick-Kendrick, personal communication). In addition, adults living in endemic areas may show low gametocyte levels, but these gametocytes are highly infective for mosquitoes. Possibly gametocytes are antigenically labile and can avoid the host’s immune response (Hawking et al., 1966; Section V ) or a developing immune response may stimulate gametocytogenesis. V.

Relapses and Antigenic Variation

Relapses by definition occur in the absence of reinfection and show as a reappearance of patent blood infection (often accompanied by clinical symptoms) after the primary parasitemic attack has subsided. Of the human plasmodia, Plasmodium vivax and Plasmodium mulariae show the greatest tendency to relapse, Plasmodium falciparum the least. There are three main theories of the origin of relapses. The first theory supposes the persistence of small numbers of erythrocytic parasites that escape the action of drugs or immunity. The second theory presumes the existence of persistent exoerythrocytic stages insusceptible to the effects of immunity developed to the blood phase. The third theory implicates a latent stage of the primary exoerythrocytic schizont (or, perhaps, of the sporozoite but there is no evidence for this). On theoretical grounds, relapses are, therefore, to be classified (World Health organization, 1963) as ( a ) recrudescences which derive from persistent blood stages or ( b ) true relapses (or recurrences) which derive from some form of persistent tissue stage. In practice neither of these two mechanisms alone can account for the origin of relapses (see Bray, 1957a, 1963; Garnham, 1966b for discussion), and it is generally accepted that both may occur after sporozoite-induced infections except where no persistent liver stage has been recorded ( e.g., P . falciparum). After blood-induced infection only recrudescences can occur as there is no infection of the liver.

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

285

Relapses are one of the puzzling features of malaria infections. Their occurrence indicates the survival of the parasite for often extraordinary lengths of time within the body of the vertebrate host. The reason for relapses has provided a subject for much discussion. Explanations of the chronicity of malaria infections include that the host becomes immunologically tolerant to some malaria antigens, that the parasite is poorly immunogenic, or that the parasite, by virtue of its intracellular habitat, is largely insusceptible to the action of immunity. One of the most common cited reasons for relapses is a waning of protective immunity and this receives some support from observations of the depressed antibody levels just before relapses (Coggeshall, 1943) and the enhancing effect of spIenectomy on malarial infections ( Corradctti, 1963). A neglected factor in many discussions of relapses has been the possibility of antigenic lability of the malaria parasite. It is known that immunologically distinct strains, judging from challenge experiments, may exist within a malaria parasite species, and as already indicated the various stages of the life cycle vary in their susceptibility to the effects of immunity. Microorganisms can, however, show considerable diversity of antigenic structure and some species can change rapidly from one antigenic state to another ( Bcale and Wilkinson, 1961) . An infection may be prolonged if a new variant appears when the original antigenic type is removed by the host’s immune response. Among parasitic protozoa, this type of antigenic change has long been recognized in African trypanosomiasis ( K . N. Brown, 1963). The occurrence of antibody-resistant generations in malarial infections was postulated many years ago (see, for example, Schilling, 1934), and H. W. Cox (1959) put forward indirect evidence for antigenic differences between initial and relapse parasitc populations. Mice harboring chronic infections of Plusmodium berghei were more susceptible to challenge with relapse than with parent strain parasites. Repeated antigenic changes of an order that might account for recrudescing simian and human malaria have only recently been demonstrated in the simian Phasmodium P . knozulesi ( K . N. Brown and I. N. Brown, 1965; I. N. Brown et al., 1968a). A schizont-infected cell agglutination test was used (see Section VII1,A). In a rhcsus monkey suffering a recrudescing P. knowksi infection, the surface antigenic structure of erythrocytes infected with mature asexual parasites isolated from a particular relapse population, differed from that of erythrocytes infected with parasites of other relapses isolated from the same recrudcscing infection. Each popuIntion of parasites (“variant”) stimulatcd specific agglutinating antibodies in the host monkey (Table 111). A low level of nonvariant-specific ag-

TABLE I11 TITERS OF

SCHIZOST-ISFECTED CELL A%GGLUTISINS 1N SERUM S,4MI'LES FRO3f A R H E S U S hfONKEY

SUFFE RI SG A RECRIJDESCISG

Parasit.e stabilates

A

Days afterinitialinfection Parasitesin Moodsmear

B

A

1

1 0

7

-

+

22 -

PfaSmod$izr9?lknowksi

T 73 85 92 94 97

C

D

E

T

T

T

34

49

52 59

65

f

-

+

+ + -+ -+ - +

t-

132 167

196

202

209

218

279

295

328

351

-

-

-

-

_

_

Splen. -

-

+ +

i-

Blood inoculation test

INFECTIOS"

+

1

1

1

1

1

1

1

1

1

1

1

1

Antigen A

<10

NT NT NT NT

>1250 10 250 <50

NT

B C D E

1250 <10 10 <SO 110

31250 31250 1250 250 1250

NT NT

NT NT

NT NT

NT NT

NT NT

NT NT

NT NT

Serum

NT

6250 250 250

NT

>781250 >781250 >781250 781250 156250 250 31250 31250 6250 1250 1250 1250 1250 31250 NT

156350 156250 250 250

NT

NT

The monkey was initially infected with parasites of stabilate A, radically cured of the resulting acute infection and then reinfected with A. Parasites were seen in blood smears taken up to day 167 but blood was infective for nmimmUne monkeys until at least day 209. Splenectomy on day 328 did not result in a recrudescence of infection. Stabilates of parasitized cells were isolated either from the host monkey 03 f C) or from recipients of host monkey blood (D E). Schizont-infected cells (antigen) for agglutination tests were collected from nonimnone monkeys inoculated with parasites of the relevant stabiktes. NT = not tested.

+

3

z E0 <

2

IAfMUNOLOGICAL ASPECTS OF MALARIA INFECTION

287

glutinin was dctectable after some weeks of infection. Variation also took place when the infection was subpatent and variants occurring latc in the infection were fully virulent in nonimmune animals. By comparing the agglutinin response to drug-cured infection with onc variant with the response to variants occurring when the infection was subpatent, it was shown that, as a result of prolonged infection, monkeys were able to respond more rapidly and with a higher titer of antibody to new variants. Challenge experiments have shown that the variant-specific response has implications for studies of protective immunity. Monkeys infected with a known variant were radically cured and then rechallenged with the same variant. Such monkeys were susceptible to, and usually died of, infection, but the parasites appearing in the blood were of a new variant type. Antigenic lability could explain the persistence of parasites in the bloodstream but does not explain why recrudescences occur. Some variants may be more virulent, or, alternatively, may differ more markedly in surface antigenic structure from previously experienced variants. The possible effects of other factors such as stress or intercurrent infection cannot be overlooked. That P. knmolesi can vary its antigens has been confirmed by Voller and Rossan ( 1 9 6 9 ~ )who, in addition, have found evidence of such variation in Plasmodium cynomolgi bastianelli (Voller and Rossan, 1969a). Antibody-resistant parasite populations have been isolated from P. berghei infections (Briggs et al., 1968), and from challenge experiments indications of a specific protective response to P. berghei relapse populations has been obtained (I. N. Brown, 1968). By implication, the blood stage of other plasmodia may show a similar antigenic lability. The findings of Wilson et al. (1969), discussed in Section VII, may be indicative of such variation in a human malaria parasite. Whether or not exoerythrocytic stages also show antigenic variation is not known; secondary exoerythrocytic forms may differ antigenically from primary forms, and those plasmodia not showing secondary exoerythrocytic development, such as Plasmodium fakiparum, may lack the capacity for variation in this stage. Likewise the effect of mosquito transmission on the potential variability of plasmodia has not becn investigated. From challenge experiments with P. cynomolgi bastianelli, Voller and Rossan (1969a) found eviclcnce suggestive of a change in antigcnic type after mosquito transmission, but this change was not tested by serological methods. Further investigation is needed to clarify the effect of mosquito transmission on both thc antigenic typc of initial parasitc,mia after sporozoitc inoculation a n d thc spectrum of antigcnic types that can be shown 1)y onc infection. The possibility of antigenic changes occurring within

288

IVOR N. BROWN

one or more phases of the life cycle of malaria parasites is a factor that must be taken into account in the design of immunological experiments. VI.

Cellular Factors in Malaria Infection

Malaria infection is accompanied by pronounced changes in the tissues of the host, which are primarily associated with the erythrocytic stage of infection. Circulatory disturbances, along with an often marked reduction in erythrocyte numbers, contribute to the production of lesions and consequent disturbance of tissue function ( Maegraith, 1948; Fulton and Maegraith, 1949; Fletcher and Maegraith, 1966). The most evident change in the blood itself is a moderate to severe anemia; considerable fluctuation in leukocyte levels occur also particularly at paroxysms (Taliaferro and Kliiver, 1940); in chronic malaria the characteristic picture is a moderate leucopenia with an absolute increase in monocytes. The anemia often exceeds that to be expected from rupture of infected cells alone (Zuckerman, 1964a; Schroeder and Ristic, 1968). After crisis or treatment, reticulocyte counts are high in all forms of malaria; in Plusrnodium berghei infections of mice, this may enhance the infection because the parasite preferentially invades immature red cells. Infection of the red cell causes changes in its physical characteristics. The cell becomes osmotically more fragile (Fogel et al., 1966a), enlarges in some species, and shows changes in its surface properties (Findlay and Brown, 1934; Brown and Broom, 1935). These changes along with vasoconstriction (Fletcher and Maegraith, 1966) may result in marked sludging of infected blood in terminal infections (Knisely et al., 1964). The level of platelets and clotting factors is also depleted during infection which would predispose to hemorrhagic conditions. With some parasites, e.g., Plasmodium falciparum and P . berghei, maturation of blood stages does not occur in the peripheral blood. The infected cells accumulate in the fine capillaries of internal organs. When parasitemia is high this accumulation affects the dynamics of the circulation and may lead to necrosis and hemorrhage of the affected areas. A. PHACOCYTOSIS

Much of the evidence for the involvement of macrophages in the elimination of malarial infection comes from morphological observations of the phagocytosis of parasites, parasitized cells, nonparasitized cells, erythrocytic debris, and malarial pigment. Taliaferro and his colleagues (see Cannon and Taliaferro, 1931; Taliaferro and Cannon, 1936; Taliaferro and Mulligan, 1937; Taliaferro, 1949, 1967) made systematic

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

289

histological studies of the tissues of monkeys and birds killed at intervals during acute infections and after superinfection. Significant phagocytosis was restricted to the spleen, liver, and bone marrow where, it was argued, blood flow was particularly sluggish through networks of phagocytic cells. Phagocytosis was slow during the early stages of infection but at crisis and after superinfcction was greatly enhanced. Similar findings have been reported for Plasmodium berghei infections of rodents (Jerusalem, 1964; Zuckerman et al., 1964; H. W. Cox et a!!., 1966; Zuckerman, 1966). That the spleen, liver, and bone marrow form the primary sites of phagocytosis in human infections also was stressed by Clark and Tomlinson ( 1949) . Surprisingly, in vitro opsonic tests have received little attention. Opsonic factors are detectable in the sera of chickens after superinfection, but not acute infection with Plasmodium gallinaceum and Plasmodium lophurae (Zuckerman, 1945), and opsonins specific for the Plasmodium knotclesi parasitized cell have been detected in the sera of monkeys semi-immune and immune to P . knowlesi malaria (K. N. Brown et al., 1969a) (Fig. 3 ) . It would seem that further research in this field would be profitable.

B. % SPLEEN The precise function of the splecn in immune responses is not understood but it is known to be an important site of phagocytosis and of antibody production. After the intravenous injection of foreign erythrocytes, the initial serum antibody response compared with that found in intact controls is depressed in splenectomized human subjects ( Rowley, 1950b; McFadzean and Tsang, 1956a), monkeys (Saslaw and Carlisle, 1964), rabbits (Taliaferro and Taliaferro, 1950; Draper and Sussdorf, 1!357), and rats (Rowley, 1950a; Wissler et al., 1953). The spleen forms the bulk of the initial antibody in response to intravenous sheep red cells ( Taliaferro and Taliaferro, 1950), but nonsplenic sources are ultimately niorc important as a continuous source of antibody, and the importance of these sites is increased by giving multiple injections of antigen (Taliaferro and Taliaferro, 1951) . Splenectomy has little effect on the antibody response to antigens given by routes other than the intravcnous route; for example, if particulate antigen is given subcutaneously no difference in the antibody response between splenectomized and nonsplenectomized subjects can be found ( Rowlcy, 195011; McFacbcm~ and Tsang, 1956h). Similarly, if a soluble antigen, such as ov:ilbumin, is aclministered to rabbits intramuscularly in complete Freund's adjuvant, only traces of antibody are

290

IVOR N. BROWN

FIG. 3. Photographs of Ciemsa solution-stained mouse macrophnges. Macrophages were allowed to settle on cover slips and then incubated for 2 hours at 37OC.

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made by the spleen. Howcver, upon subsequent intravcmous injection of alum-precipitated ovalbumin the spleen has a higher synthetic ability than other tissues except for the bronchial lymph node (Askonas and Humphrey, 1958). The extent to which the spleen participates in the removal of particles (such as red cells) from the circulation varics. Autologous erythrocytes mildly damaged by chemical or heat treatment or minimally opsoniscd by antibody are removed almost entirely by the splecn (Wagner et nl., 1962; Azen and Schilling, 1964; Crome and Mollison, 1964; Ultmann ancl Gordon, 1965; Marsh et al., 1966). On the other hand, excessively damaged or opsoniscd cells are removed in large part by the liver. Uptake of antigen is an essential prelude to antibody formation and the liver, as is the spleen, is capable of removing large amounts of antigen from the circulation (Ingraham, 1955, Halpern, 1959; Thorbecke et al., 1960) yet does not make significant amounts of antibody (Miller and Bale, 1954; Askonas et al., 1956). Antigen docs, however, persist in Kupffer cells (Garvey and Campbell, 1957), and mice implantcd intraperitoneally with such primed cells givc an anamnestic response upon subsequent injection of spccific antigen (Vredevoe and Nelson, 1963). By analogy, splenic macrophages behave similarly, and the spleen bccomes important in the response to systemic antigen since, within the spleen, macrophages are in intimate contact with lymphoid elements among which are competent cells. A consequence of stimulation with particulate antigen or; in fact, of nonspecific stimulation, such as the injection of carbon particles, is an increase in the size and weight of the spleen. In response to antigen this is seen histologically as a proliferation of lymphoid and macrophage elements. Splenomegaly is one of the characteristic features of plasmodia1 infection. An indication of the prevalence of malaria in a given area is given by the spleen rate, which may be defined as the percentage of the population with a palpably enlargcd spleen; for example, it is possible to differentiate among degrees of endemicity according to the following classification (World Health Organization, 1963) : rate in children 2 to 9 years, 0 to 10% 1. Hypoendemic-spleen 2. Mesoendemic-spleen rate in children 2 to 9 years, 11 to 50% 3. Hyperendemic-spleen rate in children 2 to 9 years, constantly over 50%,adult spleen rate also high with Plasmodium knowlesi schizont-infected cells with added ( a ) normal rhesus monkey serum or ( b ) serum from a rhesus monkey suffering a recrudescing P. knowlesi infection. The final serum concentration in the culture was 1:5. Magnification: X1036.

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4. Holoendemic-spleen rate in children 2 to 9 years, constantly over 75%,adult spleen rate low, but tolerance of the infection high. Within a population, groups of children of age between 2 and 9 years usually have the highest spleen rate. In the age group 10-14 years and in adults, splenic enIargeinent is less. Removal of thc spleen directly, or blocking of its phagocytic activity before infection with erythrocytic stages of malaria parasites, is generally followed by a more acute initial parasitemia than in the intact host. In young animals, which are more susceptible to malaria infections than adults, the accentuation may not be marked. On the other hand, in older animals the effect is often striking and a fataI infection may result. Similarly, if an animal carrying a latent infection is splenectomized, the infection may recrudesce. After a latent infection of short duration the recrudcscent parasitemia is often high and may be fatal. In contrast, splenectomy after a latent infection of long duration may have no effect on that infection or may result in a low-grade recrudescence (Garnham et al., 1968).Splenectomy may affect not only acquired immunity but also innate immunity to the blood stage. Sporozoite-induced infections with human plasmodia in chimpanzees are normally restricted to the liver stage of development but splenectomized chimpanzees can support blood infections of these plasmodia (Bray, 1957b, 1958). Possibly the liver stage is enhanced in such subjects and the effective blood challenge upon maturation of the exoerythrocytic schizonts increased (how sporozoite inoculum can affect initial blood parasitemia is discussed by McGregor, 1965), but in contradiction to this explanation is the observation that primary development of exoerythrocytic stages in rhesus monkeys inoculated with Plasmodium cynomolgi sporozoites is not influenced by removal of the spleen (Garnham and Bray, 1956; see, also, review by Fabiani, 1966). The possible role of the spleen in malarial immunity can be explained by considering the nature of the parasitized erythrocyte and the significance of antigenic variation in relation to splenectomy. As stated above, the surface antigens of erythrocytes containing schizonts of Plasmodium knowlesi differ from those of uninfected erythrocytes. During parasite development the parasitized ceIl becomes modified. A maIarial infection thus results in the appearance at intervals of slightly modified erythrocytes in the circulation. From the studies described above the spleen would be particularly active in the removal of such cells from the circulation and opsonization, whether local, as envisaged by Taliaferro and his colleagues (see Taliaferro, 1949 for review), or systemic would enhance the clearance rate of parasitized cells. Parasitized cells are agglutinated in vizjo (Cropper, 1908; Cannon, 1941; Taliaferro and Bloom,

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1945). The particular susceptibility of splenectomized hosts to iiialaria could be explained, in part, by the high phagocytic ability of the liver which, in the absence of a spleen, possibly removes the bulk of potentially antigenic material from the circulation thus preventing its immediate access to sites of antibody production. Also, after splenectomy the antibody response to a new antigenic type (see Section V ) would presumably be weaker than in an intact host and would result in a resurgence of infection. In a healthy spleen the blood circulation is rapid and intrasinusoidal. In an enlarged spleen, there is also a much slower circulation through the pulp cords. Red blood cells accumulate within this pulp cord circulation and remain in the spleen for longer than they would in a normal spleen. Entry and exit from this “microcirculation” is regulated not only by the mechanical obstruction by an altered splenic architecture but also by the physical properties of the red cells (Richards and Toghill, 1967). Trapping of large numbers of normal red cells in the enlarged spleen probably contributes to the severe anemia characteristic of tropical splenomegaly (Pryor, 1967) and, by inference, to the anemia found in malaria infections. Enlarged spleens also exert a pooling effect on platelets (Gabriele and Penington, 1967) which effect could contributc to thc thrombocytopenia observed in plasmodia1 infections. The contribution of malaria to the etiology of tropical splenomegaly syndrome (big spleen disease) has been discussed recently by Marsden et al. ( 1967) and Pitney ( 1968). Both the spleen and liver show abnormal histological features and a combination of hepatic lymphocytic infiltration ( Fig. 4 ) with hepatic and splenic reticuloendothelial cell hyperplasia is usual. Patients are commonly anemic and their serum shows high IgM levels and a high incidence of rheumatoid factor and cold agglutinins. The precise role of malaria as a causative agent in the development of this syndrome is not known. OF MALARIAL IMMUNITY C. ADOPTIVETRANSFER Phillips ( 1969 ) prepared suspensions of spleen cells, peritoneal cells, and a mixture of lymph node, thymus, and bone marrow cells from rats immune to Plasmodium berghei. These suspensions when injected into normal rats of the same inbrcd strain conferred protection on the recipient rats to an injection of P . berghei infected blood. Similar cell suspensions prepared from nonimmune rats did not confer protection. Not only did the recipients of spleen and lymph node cells from immune donors (1 donor = 1 recipient) show less blood infection than control< but they also cleared infection in 2 to 3 weeks. The immune cell donors

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FIG. 4. Stained section of liver biopsy showing heavy lympliocytic infiltration. Magnification: X265. (From Marsden et al., 1987; by courtesy of Armed Forces Institute of Pathology, Washington; Photograph No. 6G. 2352. )

had themselves previously eliminated infections of 3-6 months duration. The conferred immunity was less if the number of cells transferred was reduced (e.g., if one donor was used to prepare immune spleen cells for two recipients, the patent parasiteniia after challenge was reduced but the animals were less able to eliminate their infections), quickly waned if the transfer of cells was from immune male to nonimmune female rats, and was nonexistent if the cells were disintegrated before injection. Splenectomy of the recipients before injection prevented their clearancc of the challenge infection. It is not known how the cells conferred protection. High levels of protective activity (in a passive serum transfer test) were detected in the serum of immune cell recipients taken 13 days after infection (when parasites had apparently been eliminated). Presumably therefore the protection is to some extent antibody mediated. This does not exclude the possibility of a cell-mediated immunity ( Turk, 1967).

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D. SKINREACTIONS I N MALARIA Intradermal injcctions of malarial antigen have been used in attempts to develop a diagnostic test for malaria ( Herrinann and Lifschitz, 1930; Sinton and Mulligan, 1932; Stratniari-Thomas and Dulniiey, 1940; R/Iakari, 1946) and also to givc information on the immune processes involved in combating malarial infections ( Taliaferro and Bloom, 1945; Dubin, 1948). Taliafcrro and Bloom ( 1945) described the histological appearance of the skin reactions of normal and immune birds and monkeys to the injection of viable parasitized erythrocytes. There was no significant difference between the reactions of normal canaries to Plusmorlium cathemerium or normal Cebus or spider monkeys to Plasmodium brnzilinnum and those reactions given by their immune counterparts. There was an inflaminatorp response in both the normal and the immune. In contrast, there was a marked difference in reactivity between immune and normal rhesiis monkeys to intradermally injected PZusmodiurn knozdesi parasitized cells. In the immune animals the parasitized cells were rapidly agglutinated (see further Section VIII,A,S) and avidly phagocytosed, whereas in the normal animals the parasitized cells were not agglutinated and phagocytosis progressed at a slower rate. Also, inff ammation was more pronounced in die immune monkeys. These studies were not complemented by a description of the macroscopic appearance of the reactions, but these have been described by Phillips et nl. (1969). Monkeys chronically infected with P . knocclesi gave a marked immediate reaction (erythcma) maximal 4 hours after the injection of P . knozulesi schizont infected cells. Normal rnonkcys did not give a reaction. The chronically infected nionkcys showed in addition an inflammatory reaction of the injection site maximal at 24 hours or later. Prcsumably the immediate reactions are a consequence of antibody activity, but it is not known whether the delayed reactions signify a classical hypersensitivity reaction or are an indirect manifestation of the initial antibody-mediated reaction. Support for the first suggestion is provided by the fact that monkeys sensitized with dead P . knotcksi parasitized cells in Freund’s complete adjuvant show stronger delayed reactions than do monkeys sensitized with incomplete adjuvant or chronically infected monkeys, but this does not exclude the possibility of the sccond mechanism. Monkeys sensitized with parasitized cells in complete adjuvant make mtibody to a wide range of malarial antigens. Diagnostic skin reactions for human malaria were described by Herrmann and Lifschitz (1930), who used an aqricous extract of human malaria infected blood, and by Makari (1946), who used an aqueous ex-

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tract of desiccated Plasmodium gallinaceurn avian infected blood, but the underlying mechanism of these reactions is not known. VII.

Antigens of Malaria Parasites

Studies of malarial antigens are complicated by the intracellular habitat of the parasite; to what extent the host recognizes the parasitized cell rather than the parasite alone has still to be determined. It is not known which antigens are immunogenic. Various techniques have been used to detect and, to some extent, to characterize malarial antigens, and these techniques have shown that the localization of parasite antigen is not confined to the parasite itself. Antigen is detectable ( I ) on the surface of parasitized cells at certain stages of development of the parasite, ( 2 ) circulating in the plasma of infected individuals, and ( 3 ) in association with various organ tissues (Ward and Conran, 1966). There are undoubtedly antigens common to many stages of the life cycle, as fluorescent antibody techniques have shown, but little is known of the variation in antigenic composition shown among these stages. Studies of malarial antigens fall into two broad but interrelated categories-those that seek to determine the antigenic composition of and relationships among species and strains and those that seek antigens capable of inducing effective immunity. The approach is perhaps different in each case but there are problems common to both. Not the least of these problems is the collection of sufficient parasite material, especially with human malaria parasites, only limited amounts of which can be conveniently harvested from infected blood. Infected placentas provide a good but erratic supply of Plasmodium fulciparum antigen, but a more regular source is urgently needed. Malaria parasites show only limited growth in vitro and parasite material must, therefore, to a large extent, be collected from infected hosts and then separated from contaminating host material. A. COLLECTION OF PARASITE MATEFUAL 1. Sporozoites By dissecting out the salivary glands of infected mosquitoes, large numbers of sporozoites can be collected. Sporozoites have been used in agglutination and fluorescent antibody tests and for immunization of the vertebrate host, but no detailed antigenic analysis has yet been made. 2. Exoerythrocytic Stages Preliminary attempts (J. Williamson, R. S. Bray, and R. KillickKendrick, personal communication) have been made to separate pre-

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erythrocytic schizonts froin heavily infected mouse liver. An examination of various methods of olitaining suspcnsions of isolated liver parenchymal cells has shown that maximal recovery was possible using thc reagent tetraphenylboron. Liver parenchymal cells can be cultured in uitro mid show Inultipliciition, and this maintcnnncc tc~chniquc~could allow observation of the dcvclopment of preerythrocytic schizonts in uitro and allow attempts to infect suspended cells by addition of sporozoitcs and further attempts at schizont separation and concentration for biochemical and immunological study. 3. Erythrocytic Stages

Infected blood remains the most convenient source of malarial antigen and most studies are concerned with the blood stage. Laboratory strains of malaria parasite, in general, show high parasitemias particularly after splenectomy and this has been used to advantage. The situation is, however, more complicated in human malarias, as has already been mentioned, but may be partially resolved by the use of primates as a source of antigen. Infected blood is collected into anticoagulant and, after washing of the cells, has been used as a source of antigen in, for example, hemagglutination tests (Stein and Desowitz, 1964). For most studies, however, a prerequisite has been further separation of parasite material froin uninfected cells, leukocytes, and platelets. In addition, depending on the synchrony of infection, parasitized cells may vary in maturity and gametocytes may also be present. a. Selective Separation of Infected Cells. Infected erylhrocytes tend to be lighter than uninfected erythrocytes on centrifugation. Pigmented infected cells lie just under the buffy coat. This is a convenient method of obtaining schizont-infected cells for agglutination tests (Eaton, 1938; K. N. Brown and I. N. Brown, 1965) and for immunization (Targett and Fulton, 1965). For more selective separation a variety of techniques has been used. The bulk of leukocytes and platelets from infected pcripheral, heart and placental blood can be discarded as buffy coat after the initial centrifugation step and more complete removal, with loss of parasite material, has been obtained by, for example, dextran sedimentation ( Spira and Zuckerman, 1966), Iow-speed centrifugation in sucrose gradients (Williamson and Cover, 1966; Williamson, 1967), and Millipore filtration (Corradetti et al., 1964; I. N. Brown et al., 1966). By using albumin ( Rowley et nl., 1967) or sucrose gradients (Williamson, 1967), relatively pure isolates of erythrocytes infected with schizonts, trophozoites, or rings can be obtained. Plasmodium falciparum-infected

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cclls from the placenta can be collected using essentially similar procedures ( McGregor et d.,1966). b. Release of Parasites from the Erythrocyte. Several methods of disrupting erythrocytes are available and all show inherent disadvantages. The method of choice depends largely on the reasons for which the parasite cell is required; for metabolic studies the choice may be critical. Hypotonic lysis with distilled water can adversely affect the parasite in a number of ways. Saponin lysis is recommended by Zuckerman (Zuckerman and Ristic, 1968) for its simplicity but, as Williamson (1967) notes, because of its ability to liberate nucleic acids is particularly unsuitable for avian plasmodia, reticulocyte-preferring rodent parasites, or suspensions containing a significant proportion of leukocytes. Immune lysis with antierythrocyte serum involves the introduction of additional proteins into the parasite suspension, not all of which may be removed by repeated washing. Infected cells can also be selectively disrupted in a pressure cell ( DAntonio et al., 1966a) but the process must be carefully controlled to avoid parasite damage. After disruption, parasite cells are deposited by centrifugation and purified by further washing or layering on density gradients. No method yet devised gives completely host-free parasite material.

B. MALARIALANTIGENS Parasite-antigen-rich extracts can readily be obtained from whole or lyophilized infected erythrocytes and “free” parasite cells. Cells have been disintegrated by freezing and thawing, by homogenizing, by sonicating, or by applying pressure (Sodeman and Meuwissen, 1966; Chavin, 1966; Diggs, 1966; Ward and Conran, 1966; D’Antonio et al., 1966a,b; Williamson, 1967; Zuckerman and Ristic, 1968). Lyophilized cells have been extracted by reconstitution followed by sonication or homogenization (Zuckerman 1964b; McGregor et al., 1966; Turner, 1967; Turner and McGregor, 1969la,b; Wilson et al., 1969). Both the supernatant and sedimentable fractions of such extracts have been used in subsequent analyses (see, for example, Mahoney et al., 1966) but a defined subcellular fractionation of antigens has not been reported. Antigens of malarial origin are also obtainable from the serum or plasma of acutely infected monkeys, rats and birds (Eaton, 1939; Chavin, 1966; H. W. Cox, 1966; Williamson, 1967; Todorovic et al., 1968b,c,d) and from patients with heavy Plasmodium falciparum infections (Turner, 1967; McGregor et al., 1968; Turner and McGregor, 1969a; Wilson et al., 1969) . Extracts of plasmodia1 cells consist of a complex mixture of potentially

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antigenic components. For example, after acrylamide electrophoresis of Plasmodium berghei parasite cell extract, a minimum of 15 parasite proteins (Soderman and h4cuwissen, 1966), and 6 proteins of host origin have been idcntified (see, also, Spira and Zuckerman, 1966; Chavin, 1966). Some of these extractable parasite proteins have proteolytic activity (Cook et al., 1961; Sherman et al., 1965). By immunoelectrophoretic analysis with antisera produced by immunization of rabbits or goats, up to 11 precipitinogens of parasite origin have been dcmonstrated in extracts of various malaria parasites, some of which appear to be species specific (Banki and Bucci, 1964; Spira and Zuckerman, 1964; 1966; Chavin, 1966; Diggs, 1966; Williamson, 1967). Strain- or intrastrain (variant)-specificantigens have not been shown yet in this way. Williamson (1967), in a study of Plasmodizinz. knowlesi “cell sap,” found at least 6 niain groups of soluble proteins in the free parasite. The major protein group and the major prccipitinogen group, which contained at least three antigens judging from immunodiffusion tests against immunized goat or monkey antiserum, had a molecular weight of 30 to 33,000 which is similar to the molecular weight of a half molecule of hemoglobin. A product of hemoglobin digestion by plasmodia is malarial pigment which consists of hemin and a protein derived from the breakdown of the globin portion of the hemoglobin molecule (Deegan and Maegraith, 1956; Sherman et nl., 1965). Pigment prepared from the avian Plasmodium, P . lophurne, is antigenic in rabbits, and antisera from immunized rabbits cross-react with host hemoglobin (Sherman et nl., 1968). Whether such pigment is antigenic in birds is not known. Possibly other antigenic components of malaria parasites are also derived from hemoglobin. The most extensive studies of the antigens of human malaria parasites concern Plasmodium falciparum in West Africa. These studies have paralleled an investigation of the precipitating malarial antibody response of individuals resident in an area hyperendemic for malaria ( McGregor et al., 1966, 1968; Turner, 1967; Turner and McGregor, 1969a,b; Wilson et al., 1969) and concern the identity and properties of P. falciparum antigcns, either extractable from heavily infected placental blood or circulating in the serum or plasma of subjects, mostly children, suffering sevcre infc.ction. Turner and McGregor (1969a) identified two main groups of antigens ( a - and P-antigen) in extracts of P. falciparum infected placentas and distinguished between these groups on the basis of their elution position after gel filtration of extracts on Sephadex G-200 (Fig. 5 ) and also on the position of the precipitin lines produced by these antigens in gel-diffusion tests. In subsequent studies, however, a distinction was made on the basis of their heat susceptibility. Wilson et al.

Fraction number

FIG. 5. Gel filtration of antigens extracted by X-press from lyc Plusmodium fulciparum-infected placenta. Three milliliters of sample was applied in 3%sucrose to a column of Sephadex G-200 (2.4 X 77 cm. ). Elution was performed with a buffer of 0.2 M tris-HCl+ 0.2 M NaCl (+O.lX NaN,) at a flow rate of 20 ml./hour and at a temperature of 27OC. Fractions were collected at 10-minute intervals, pooled, and concentrated as indicated, and antigen activity in each pool is expressed as specific activity (titer X concentration factor of pool). Titers were established by gel diffusion using a doubling dilution system, and the antiserum was serum from an immune adiilt Gambian. (From Turner and McCregor, 196%)

(1969) designated P . fakiparum parasite cell antigens stable to heating at 100°C. for 5 minutes as S (stable) antigens, and those antigens of which the activity in precipitation tests was destroyed by heating at 56°C. for 30 minutes at La or Lb (labile) antigens. In addition they detected antigen ( R ) of intermediate heat-susceptibility (Fig. 6 ) . The S antigens probably largely correspond to the a-antigen (associated with the macromolecular peak after Sephadex G-200 chromatography) , and La antigens to the ,@-antigencomplex (associated with the hemoglobin peak after Sephadex G-200 chromatography) of Turner’s classification (Turner, 1967; Turner and McGregor, 1969a). Both La antigens and S antigens were detectablc in placental extracts, but S antigen alone was detected circulating in the serum of heavily infected children. The pattern of precipitin response to these two

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FIG. 6. Heat test to differentiate malarial antigens of Plasmodium fahparzrm. Six sera from immune Gambian adults were tested against an extract of infected placental blood which had been treated as follows: ( 1 ) unheated, ( 2 ) heated in a water bat11 a t 56°C. for 30 iniiiutes, ( 3 ) immersed in a boiling water bath at 100°C. for 5 minutes. Gel diffusion was carried out for 48 hours at 4OC. in 1.5%Noble agar dissolved in 0.05 p Verona1 buBer. The progressive loss of L and R antigens with heating is to be noted. Magnification X3. (From Wilson et al., 1969.)

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types of antigen differed among age groups (McGregor et al., 1968; Wilson et al., 1969). At least three La antigens were identified and seemed to be common to most infected placental extracts. Anti-La-antigen antibodies were detectable in the serum of most individuals from an early age (4-6 years) and wcre also passively transferred before birth. In contrast, there were many S antigens and only few were detectable in one placental extract. Antibodies to S antigens were found mostly in the serum of adults aged 30 years or more; in children the response was weak and transient only (see Fig. 8). During a given parasite attack, only one, or a limited number, of circulating S antigens was detectable even if a range of adult human antisera were used. Possible individual parasite populations produce a single S antigen and mixed infections occur or parasite populations produce more than one S antigen, but the amount of a given S antigen produced varies among populations. Alternatively, Plasmodium falciparum may show antigenic variation. The origin of circulating malarial antigens has still to be determined. They could derive from a modified host erythrocyte, but present evidence is more suggcstive of a parasite cell origin. Wilson et al. (1969) found a complete correspondence between the antigenic specificities of circulating and placental S antigens associated with Plasmodium falciparum infection. Also, in women heavily infected at parturition, serologically identical S antigens were recovered from their serum and their placentas. At least two antigens have been identified in the plasma of chicks acutely infected with Plasmodium gallinaceurn (Todorovic et al., 1968c,d). One of these antigens gave a reaction of partial identity in geldiffusion tests with an antigen present in extracts of P. gallinaceurn parasite cells. These antigens showed genus specificity in tube latex agglutination tests (see Section VIII,A,5) and evidence was also found of their involvement in the agglutination, opsonization, and lysis of P . gallinaceurn-parasitized cells. These antigens differed in their physical properties from those described by Wilson et al. (1969). Why this is so is not known. Factors that must be considered are (1)species differences among plasmodia, ( 2 ) the density of parasitemia at the time of plasma collcction, ( 3 ) whether or not the infected hosts had suffered previous infection and, perhaps, developed an antibody response to some plasmodial antigens (for example, the presence of antibody to L antigens of Plmmodiuin falciparum in plasma of infected children would enhance the clearance rate of the aiitigcn and in gel-diffusion tests could preclude its detection), and ( 4 ) differences in “solubility” among plasmodia1 antigens; Turner and McGregor (196921) remarked on the relative ease of extraction of a-antigens compared with p-antigens from lyophilized

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P. falciparunz-infected placentas; thcy suggested a-antigens might bc associated with the more soluble components of parasite (or parasitized) cells, whereas ,&antigens were possibly of a less soluble, structura
Humoral Factors in Malarial Immunity

Although phagocytosis is undoubtedly important to the control of malaria infection, there are certain features of infection, such as the occurrence of crisis forms, which suggest a direct humoral effect on the parasite is also involved. The composition of the plasma ( o r serum) of an infected host changes during and after plasmodial infection (Maegraith, 1948; Fulton and Maegraith, 1949; Sadun, 1966; Gail et nl., 1967). Among the changes that occur is an increase in the level of immunoglobulin and is particularly apparent in malaria endemic areas (Turner and Voller, 1966; Rowe et al., 1968). The most pronounced change is in IgG and IgM levels but IgA levels also increase. All these three classes of immunoglobulin have been shown to possess specific malarial antibody activity, although it is likely that considerable nonspecific immunoglobulin increase also occurs. Not all malarial antibody is associated with protective immunity but such antibody is useful for diagnosis as is discussed below. A. MEASUREMENT OF MALARIAL ANTIBODY Many methods have been used to demonstrate antiplasmodial antibody. The difficulty throughout has been to correlate the appearance or presence of such antibody with a functional immunity. This may reflect the marked specificity of acquired antipIasmodia1 immunity.

1. Serum Complement and Complement Fixation Tests Early investigators had great difficulty in preparing an antigen to use in this technique (see review by Taliaferro, 1930). Taking advantage of the high parasitemias produced by Plasmadizrm knotol& in rhesus monkeys, Coggeshall and Eaton (1938a) were able to prepare a satisfactory antigen by salinc extraction of ground, dried, parasitized blood. Complement-fixing antibody appeared early in the course of a recrudcscing infection of P . knotoksi, but there was no apparent relation between the number of circulating parasites and thc titer of such antibody, although just before a parasite recrudescence there was a temporary drop in titer. Monkeys and rabbits injected with P . knowlesi antigen preparations had

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high serum complement-fixing antibody titers, but their sera were not protective in passive transfer experiments ( Eaton and Coggeshall, 1939b,c). The test was not specific for P . knowlesi because it would also detect antibody in human serum from individuals who had suffered Plasmodium falciparum or Plasmodium vivax infections (Eaton and Coggeshall, 1939a). This lack of specificity was confirmed by Kligler and Yoeli (1941) who also found that antigen prepared from Plasmodium gallinaceum gave positive complement fixation reactions with human sera that had reacted with P. knowlesi antigen. If an acute P . knowlesi infection was treated at an early stage, no complement-fixing antibody was produced (see also Dulaney et al., 1942). Subsequent studies in the 1940's (Dulaney et al., 1942; Lippincott et al., 1945; Rein et al., 1949; Sutliff et al., 1950) concentrated mainly on the diagnostic value and epidemiological application of the technique. Sutliff and his colleagues found that the test gave a fairly reliable indication of current infection or of past infection within the year preceding the test. Rein's studies on induced P. vivax infections revealed that complement-fixing antibody was detectable about a week after the appearance of parasites in the primary attack, None of these reports gave a full study of antibody production. Essentially similar findings have been reported for Plasmodium berghei infections in rats and mice. Complement-fixing antibody appeared about 8 days after the first appearance of parasites in the blood, and persisted as long as the infection was maintained. As with P. knozulesi, no correlation with protective immunity could be found (Varques et nl., 1951; Schindler and Mehlitz, 196.5). Attempts to increase the specificity of the test for human malarial antibody, by preparing antigen from human Plasmodium, material, were partially successful ( Mayer and Heidelberger, 1946). Difficulty was experienced in preparing sufficiently pure antigens, and some cross-reactions occurred with normal red cell stromata. Mayer and Heidelberger noted that their best P. vioax antigen was probably a fine particulate suspension. Recent work by Mahoney et al. (1966) has supported this view. Soluble extracts of disintegrated P. krwzolesi-parasitized cells contained very littlc complement-fixing antigen. On the other hand, activity was present in the original parasitized cell disintegrate and was retained in the material sedimented by centrifugation at 125,OOOg for 90 minutes. In contradistinction, DAntonio et al. ( 1966a,b) have reported good complementfixing activity in soluble extracts fractionated on Sephadex G-200. By this method, activity was disassociated from normal red cell contamination. The product was stable on storage at 4°C. or minus 70"C., had

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a high molecular weight, and no anticomplementary activity. The specificity of this preparation has yet to be tested. There is considerable evidence that complement is utilized during a malarial infection. In human malaria, titers drop at paroxysm, and in induced infections, serum complement levels are also reduced. At late stages of acute P . knowlesi infections in rhesus monkeys, there was also a sharp drop in titer (Roy and Mukerjee, 1942). These findings have been confirmed recently by Fogel and co-workers ( 1966b). Complement levels in monkeys, hamsters, and birds with acute infections of P . knowlesi, P. berghei, and P . gallinaceurn, respectively, declined steadily as the parasitemia rose. Superimposed on this decline in the monkeys there was a daily fluctuation dependent on the release of merozoites. No evidence could be found that factors such as diet or temperatures were affecting complement levels. The fluctuations in the first, second, and third components of complement paralleled the A uctuations in total complement activity (Cooper and Fogel, 1966). The authors concluded that an immune fixation was occurring but were not able to demonstrate that a malarial antibody was responsible. The results described above would be compatible with the hypothesis that complement-fixing antibody combines with merozoites and with antigen( s ) released at schizogony, either when these are free in the plasma or after their attachment to erythrocytes. However, P. krwwlesi-parasitized cells after hypotonic lysis had a high anticomplementary activity ( D’Antonio et al., 1966b), and it is, therefore, possible that the observed fluctuations in serum complement in acute infections are produced in part by a release of anticomplementary substances at schizogony rather than by immune fixation.

2. Fluorescent Antibody Tests The fluorescent antibody technique (for details, see Voller, 1964) has been used to study the structure of, and antigenic relationship among, stages and species of malaria parasites, but perhaps its main application in this field has been in the study of the antibody response to plasmodia1 infection. In this respect the test has a particular advantage over complement fixation because antigen, in the form of air-dried smears of infected blood, is readily available. A degree of species spccificity can be obtained. Antisera, in general give a better reaction, i.e., react at higher dilution, with homologous, rather than heterologous antigen (Tobie et a!., 1962). By using this test, these authors were unable to diffcrcntiate between the response to two strains of Plusmoclium vivcix, but in a study of induced Plmrnodium falciparurn infections in semi-immune volunteers, Collins et al. (1964) de-

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tected a slight difference in the response to two strains. In spite of these encouraging findings, the fact remains that antiserum used a t low dilution shows considerable cross-reaction with other plasmodia1 species, thus placing limits to the diagnostic value of the technique (see, also, Ingram and Carver, 1963). It is, howevcr, possible to classify malaria parasites on the basis of this test into broad immunospecific groups (Voller and Bray, 1962). The antibody response to infection has been studied both in artificial blood- and sporozoite-induced infections and in natural infections. In primary induced malaria, antibody is not detectable until after parasites have been seen in the blood, even after extended prepatent periods (up to 107 days) (Kuvin et al., 1962a; Tobie and Coatney, 1964). Both IgM and IgG malarial antibody is formed ( Abele et al., 1965), peak titers are reached quickly, and high levels of antibody persist for about 2 months. The titer of antibody then drops but persists at a low level (Kuvin et al., 1962a; Lunn et al., 1966). The increase in the amount of fluorescent antibody contributes to, but is not totally responsible for, the increase in total immunoglobulin which is also detectable soon after infection ( Kuvin et al., 1962b). Later in the course of such infections there is little correlation between immunoglobulin and antibody levels (Lunn et al., 1966). Semi-immune volunteers, if reinfected, respond with much higher levels of antibody than nonimmune volunteers. Little clinical involvement is seen in these patients but parasitemias persist even in the presence of these high antibody levels (Collins et al., 1964). In hyperendemic areas, fluorescent antibody levels are high in the newborn, decline over the first few months of life, and then gradually rise throughout childhood to adult levels (Voller and Bray, 1962; McGregor et al., 1965; Fig. 7 ) . Newborn infants protected by regular drug therapy do not develop antibody and adults similarly protected have lower levels of antibody than unprotected adults (Voller and Wilson, 1964). In view of the known pattern of infection in these populations, the observations would suggest that ( 1 ) the presence of antibody indicates reccnt past infection (this is in agreement with studies on induced malaria) and ( 2 ) thcre is a possible correlation between fluorescent antibody titer and the degree of functional immunity. This would be at variance with the findings of Collins et al. (1964) who found persistent parasitemias in spite of high levels of fluorescent antibody. A more direct approach to the problem of the relation of fluorescent antibody to protection has been made by Targett and Voller (1965) although the situation may be different to that occurring in natural infections. Rhesus monkeys wcre immunized with two injections of killed

IMMUNOLOGICAL ASPECTS O F MALARIA INFECTION

-

I

I,

I

307

IT----------

(Mean age = 33 years)

I

2

3 4

5 6 7 8 9 10 I I 12 13 14 15 16 17

Adults

Age in years

FIG.7. Mean logarithmic titer by age group of malarial antibody in the serum of 446 Gambian subjects. Antibody was titrated using an indirect fluorescent antibody technique. Thin blood films of patients showing more than 30,000 Plasinodiuni fdciparum tropliozoites per cubic millimeter were used as antigen. ( Froin McGregor et d.,1965.)

Plasmodium knorulesi parasites in complete Freunds adjuvant and then challenged. To quote the authors, “There was in most cases an increase in gamma-gobulin of 30-50% (range 8 to 128%)from the beginning to the end of the vaccination period. There was no correlation between the increase in gamma-globulin and the level of protective immunity. The increase was as great in monkeys which showed little or no resistance to challenge as in those which were successfully immunized. The fluorescent antibody titers were mostly very high and, again, the levels were as high in animals which remained susceptible to challenge as in those which were protected.” The level of antibody measured by the fluorescent antibody technique may not neccssarily reflect the level of functional immunity but, nevertheless, does give n fairly reliable indication of past exposure to malarial antigen. 3. Direct Agglutination Tests

The agglutination of sporozoites has been mentioned elsewhere. The discussion below will be confined to agglutination of erythrocytic parasites. The clumping of parasitized cells in vim was noticed as early as 1908 by Cropper but the first significant demonstration of specific in vitro

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agglutination was reported by Eaton (1938). Serum taken from rhesus monkeys at a late stage of a recrudescing infection of Phsmodium knowlesi protected normal monkeys against this parasite ( Coggeshall and Kumni, 1937), and Eaton ( 1938) made the following observations: 1. The same and similarly prepared sera agglutinated monkey red cells infected with mature P. knoujbsi parasties. The sera did not agglutinate uninfected red cells or red cells infected with immature parasites. 2. No agglutinins could be detected in sera from normal monkeys but agglutinins appeared in the serum of infected monkeys between 15 and 45 days after the onset of infection. At an early stage of infection the titers were low but became higher as the infection progressed. 3. Monkeys that had been superinfected had, in general, higher serum agglutinin titers than monkeys that had not been superinfected. (These same sera also possessed stronger protective properties in Coggeshall and Kumm’s experiments. ) 4. In chronically infected monkeys, agglutinins persisted for a year or longer; it is not clear if these monkeys had latent infections or had eliminated their infections. 5. “Free” parasites agglutinated just as well as cells infected with mature parasites but gave no increase in sensitivity. This observation was taken to suggest that the parasite damaged the red cell membrane as it matured, thereby making it more permeable and the parasite more accessible to antibody. Schizont-infected cell suspensions were left to stand overnight to free parasites. Free parasites in this context means a partially lysed suspension of infected cells. Most of the parasites were surrounded by red cell membrane. 6. Serum taken from monkeys with Plasmodium inui infections of 2 to 13 months duration did not agglutinate P. knowbsi-parasitized cells. Plasmodium knowlesi agglutinins were contained in the globulin fraction of antisera (Somogyi, 1939), and the titer of agglutinin depended inore on the duration of infection than on the number of superinfections (Singh and Singh, 1940). The latter authors stressed that only freshly prepared parasitized cells were suitable as antigen for these tests, although Somogyi had claimed that formalin-preserved antigen would keep for several weeks. In some cases, superinfection of monkeys caused a rise in serum agglutinins (Ray et al., 1941), and one monkey injected with a total of 36 ml. of a 102%suspension of killed free parasites over 3%months produced agglutinin to a titer of 1:64. Two other monkeys given similar amounts of freeze-thawed or formalin-treated infected cells did not produce agglutinins. These findings were not related to protection.

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Eaton ( 1938) examined plasmodial antisera repeatedly for the presence of monkey erythrocyte agglutinins. Only occasional slight clumping was visible under the microscope and was not regarded as a serious complication. In studies of bird malarias, however, agglutination of normal erythrocytes has been reported. Manwell and Goldstein (1940) in their passive immunity studies were able to detect a microscopic agglutination of Plasmodium circumflexum-parasitized cells in antiserum but did not mention agglutination of uninfected erythrocytes. On the other hand, Coffin ( 1951b), in similar passive transfer experiments with Plasmodium lophurae, found that antisera collected from large ducks after several infections regularly agglutinated normal duck erythrocytes. The protective effect of an antiserum was mostly removed by contact with Iysed normal duck erythrocytes but not with lysed human erythrocytes (A, rhesus positive). These results would be in agreement with those of Zuckerman (1945) who found antibody in P. lophurae and Plasmodium gallinaceum antisera which opsonized both normal and parasitized cells and which could be absorbed out by normal and parasitized cells. Agglutinating antibody for parasitized cells in rabbit antiP. knowlesi-parasitized cell sera was absorbed out by normal monkey red cells (Eaton and Coggeshall, 1939b). Any parasiticidal effect of such antisera may be due to antibody to the red cell but residual complementfixing antibody to plasmodial antigen was detectable in absorbed sera. Antibody may be made which reacts with host antigens during malarial infection; this has been discussed by Zuckerman (1964a) (see Section XI). Such antibody, if pathogenic, could explain some puzzling features of these infections. After absorption with normal host red cells, rabbit and duck antisera to various avian malaria parasites freed from erythrocytes by saponin lysis still contained agglutinins for free parasites but at low titer (Stauber et al., 1951) . Both group- and species-specific agglutinins were demonstrated. Similar antiparasitic agglutinins were found in the protective sera of Coffin after absorption with normal duck erythrocytes, This procedure absorbed out protective activity which would suggest that antiparasitic agglutinin had little protective effect in his experiments (Coffin, 1951b; Stauber, 1963). Stauber and his colleagues (Stauber et ul., 1951) emphasized that high concentrations of homologous antibody were probably necessary to effect agglutination of erythrocytes infected with avian plasmodia, but Eaton (1938) thought it probable that as little as 1 ml. of a potent monkey anti-P. knozdesi serum injected into an infected monkey could produce sensitization of all the parasitized erythrocytes in the blood-

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stream. Plasmodium knowlesi-parasitized cells injected intradermally into immune monkeys were rapidly agglutinated and taken up by macrophages (Taliaferro and Bloom, 1945). This finding, along with the correlation found between agglutinin titer and protective effect of antisera in Coggeshall and Eaton’s experiments (Coggeshall and Eaton, 1938b) would suggest that in vivo sensitization by antibodies detectable by agglutination tests could play a part in protective immunity. Recent studies (see Section V ) have demonstrated that P. knotolesi parasite agglutination tests may show not only species specificity but also intraspecies specificity (K. N. Brown and I. N. Brown, 1965). Such tests are capable of detecting antigenic changes on the surface of parasitized cells within one infection and some of the implications of such changes have already been discussed. That the variant-specific immune response is probably an essential component of an effective functional immunity to P. knotolesi was shown by challenge experiments. Sensitization of parasitized cells in vivo with agglutinating antibody was demonstrated by transfusing variant-specific antiserum into nonimmune but infected monkeys and then examining the blood of the recipients (I. N. Brown et al., 1968b). Schizont-infected cells were agglutinated in stained blood smears and also in warm stage live preparations, but, in contrast to chronic antiserum (Coggeshall and Kumm, 1937), despite this observed sensitization, variant-specific serum had no observable effect on parasitemia levels. It is not known how the variant-specific immune response results in the elimination of a variant. When parasites were grown in vitro (Trigg, 1967) in the presence of antiserum and complement, agglutination did not occur until the trophozoite stage but then became marked. A reduction of 50%in the multiplication rate was observed compared with controls but the parasites developing after reinvasion looked normal. This reduction may possibly have been due to the physical effect of agglutination restricting escape of merozoites rather than a direct antiparasitic action; also preliminary experiments (K. N. Brown et al., 1969a) have indicated that an opsonin is present in the antiserum and this may have sensitized parasites to phagocytosis by blood monocytes present in the culture suspension.

4. Lytic Tests Despite the extensive studies of complement levels in malaria infection (see Section VI1,AJ ), little consideration seems to have been given to direct lysis of parasitized cells, although lysis of noninfected cells is thought to contribute, perhaps, to the anemia that may accompany ma-

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laria infection ( w e Scction XI,A ) . Possibly schiyont-infected rcd cells, by virtuc of thcir dcgcncratc ‘ippc‘irance, may ahcady be in osmotic cquilibrium with sin ioiindiiig tiswe fluid\. If thi5 were the ca\c, lysi5 of the ccll might not oecur ‘1ftc.r nicni1,ranc~disruption h y antibody and complement. Howt~vcr,such cell\ <11c readily ly\,iblc by hcterologouq antierythrocytc scrum a i d complcment. K. N. Brown et al. (1969‘1) weie not able to detect release of ”Cr from 51Cr-lalielcd, Plasmotliim knozdesi schizont-infected cells incubated with or without added guinca pig complement with fresh antiserum collected from monkeys that had suffercd recrudescing P. knou lesi infection and kno\i n to contain high levels of parasitized ccll agglutinin. 011the other hand, Todorovic et al. (1968d) rcported lysis of Plasmodiurri galliiiaceum schizont-infected cells after in vitro incubation with antisernn to P. gaZliiiaccum “cxplasmodial” antigcn ( 5ec Section V I I ) . Parasites releawd in this way wcw still infectivt, for chickens. Whether iinniunological lysis of parasitized cells occurs in ljiuo is not known.

5. fndirect Agglutination Tests The agglutination by antiserum of particles (inert, e.g., latex, and biological, e.g., crythrocytcs ) on to which antigen has been adsorbed, provides a sensitive technique for measuring small amounts of antibody and likewise, small amounts of antigen, which a t suitable antibody dilution, inhibit this reaction. a. Inert. Collodion particles coated with an alkaline extract of dricd Plnsnzodiziin knozclcsi parasites, using the technique of Cannon and hlarshall ( 1940), agglutinated when mixed with serum from malarious patients at dilutions up to 1: 128 (Dulaney and House, 1941 ). At low serum dillitions ( 1: 8 to 1: 16) some sera from nonmalarious patients also gave a positive agglutination reaction, hiit the difference between these two groups was statistically significant. A tube latcx agglutination test has recently been dcwribed by Todorovic et ([I. ( 196th). Latex particles scnsitized with antigen cxxtractcd from Plasniocliun, fcillincrcercl77-infectecl chicken cells were agglutinated by s c ~ afrom chicks infected with P. gnblimceum. Latex particles sensitized with “c.xplasmodia1” antigen ( sec: Section VII ) from infected chick plasma were agglutinated by sera of humans, monkeys, and rats suffering infcctions with othcr malaria parasites a s WCII as serum from infected chicks. This test, using “explasmodial” antigen, may have applications for diagnosis but its specificity is still ill-defined (see Todorovic et al., 1068b,c,d). b. Biological. The tanned ccll hemagglutination test ( Boyden, 19Fjl)

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has been used to detect antibody developed against a number of parasites, e.g., Toxoplanna, Schktosomu, Amoeba; and Stein and Desowitz (1964) used this test to measure malarial antibody levels. Their results were summarized in the paper by Desowitz et al. ( 19.66). The epidemiological picture appeared similar to that revealed by ffuorescent antibody tests. The serum of individuals living in areas where malaria is prevalent showed high titers of hemagglutinating antibody, but those in areas where antimalarial measures have begun showed progressively waning titers. Similarly, the antibody response of protected children was less than that of unprotected children (Desowitz et al., 1966). Antigen was obtained by making an aqueous extract of parasitized blood and was labile unless stored at -7O”C., although potency was retained for 2 to 3 months at -20°C. Centrifugation at 20,OOOg for 30 minutes resulted in a considerable loss of potency of a Plasmodium berghei antigen prepared in this way; electrophoresis of the antigen revealed at least 4 major protein components. Tanned sheep red cells were sensitized with such antigen and added to dilutions of the antisera under test. Sensitized cells showed a wide spectrum of reaction, for example, P . berghei antigen ( a rodent-infecting Plasmodium) reacted with antisera from subjects suffering monkey and human malaria. Agglutination reactions to higher dilution were obtained in general with homologous antigens. Twenty-eight sera taken from Plasmodium vivax and Plasmodium falciparum patients agglutinated cells sensitized with P. uivax, Plasmodium cynomolgi, or P . berghei antigens (Stein and Desowitz, 1964). The best reactions were obtained with P. vivax antigen ( 1:4000-1 :25,600) ; reactions with P . cynomolgi ( <1: 100-1:6400) and P. berglaei antigen ( <1: 100-1:3200) were of a lower order. However, of 37 control sera taken from apparently nonmalarious patients, 24 gave positive reactions (1:200-1:6400) in the same tests with P. viuux antigen. It was argued that as previous infection couId not be definitely ruled out in these persons, such “false positives” were to be expected. Previously, however, Desowitz and Stein (1962), using this technique to detect antibody produced as a result of P. berghei infection in rats, had found that uninfected rat sera often gave similar false positive agglutination reactions. These side reactions can be eliminated (Bray, 1964). Hemagglutinating antibody appeared a day or two after the onset of parasitemia in a primary P. berghei infection in the rat, reached peak titer soon after the peak parasitemia, and then dropped in titer but was still detectable 12 days after parasites had last been seen in the blood. The serum of rats immune to P . berghei gave the better reaction with P . berghei rather than with Plasmodium vinckei antigen-sensitized cells. Similarly, the serum of rats

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313

immune to P . v i m k e i rcactcd to Iiighcr titer with P. vinckei- than with P. berglzei-sei.lritize~1cells. Possibly the two species of parasite had antigens in common hut also some distinct antigens. There is no demonstrable cross-immunity between the blood forms ot P . cinckei and P . berghei (F. E. G. Cox, 1966). This technique has been applied to the study of antibody levels after Plasnzodiunt knowlesi infection in rhesus monkeys ( Mahoncy et al., 1966). Other methods, such as coniplement fixation and double diffusion in gel were used as a simultaneous measurement of antibody response. Plasmodiunz krwwlesi-parasitized cells in 0.44 A2 sucrose were disintegrated in a French pressure cell, and this material centrifuged at 8OOOg for 10 minutes at 4°C. The resulting supernatant ( S , ) was recentrifuged at 125,OOOg for 90 minutes. Hemagglutination tests using S, were positive but no significant amounts of complement-fixing antigen were prescnt. Hemagglutination tests using Sd-sensitized tanned cells wcre used to study the antibody response of monkeys to priniaiy and to superinfections with P . knozclesi. Antibody appeared 2-12 days after the onset of parasitemia, and titers remained high throughout the period of observation ( u p to 210 days). The antibody levels were not markedly affected by superinfection. Sera from uninfected monkeys or man did not react in these tests but occasional positive rcactions at low titer were found with sera taken from persons suffering from other parasitic diseases. The ser,i of 6 out of 32, patients with Plmniotliuin fulciparzm gave positive reactions at low titer (1:40-1:80) but, on the other hand, a11 16 sera taken from P . uivax patients reacted with titers between 1:lOO and 1:6400. Antibody was more readily detectable using an homologous antigen. In 3 patients with primary P. falcipniw7t malaria, the antibody curves were found to be similar using P . falciparum and P. knozclesi S,-antigen-sensitized cells. The maximiim titers were, however, considcrably higher with P. falcipnruiii antigen ( 1:6400-1 :25800) than with P . knozdesi antigen ( about 1 :400). 6 . Precipitin Tests Although earlier attempts to detect precipitins were largely unsuccessful, Pewny (1918) obtained precipitates within 24 hours when serum from patients with malaria was reacted with an antigen prepared from the blood of a malarious patient. To prepare antigen, clotted blood was digested for several days at 37°C. in twice its volume of distilled water. The supernatant, after centrifugation of this digest, was diluted in salinc and used as antigen. No reaction was obtained using a similar antigen prepared from an uninfected blood. Taliaferro and his colleagues (Taliaferro et al., 1927; Taliaferro and

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Taliaferro, 1928; Taliaferro, 1930) carried out extensive studies of this test in both Honduras and Puerto Rico. In their preliminary experiments, bcst results were obtained with an antigen prepared from a Plasmodium falciparum-infected placenta. The placenta was minced and extracted with an equal volume of ether for several weeks. The resulting residur was then further extracted with Coca’s solution (aqueous solution of 0.5%Nacl, 0.05% NaHCO,, 0.4%phenol) for about a week. This matcrial was filtered and the clear filtrate used as antigen. For the test itself antigen was overlaid on the serum. After incubation at 37°C. for 1 hour followed by several hours in an icebox, the intensity of precipitation at the junction of antigen and serum was noted. Of the sera from 54 malarious patients, 83%gave positive reactions, whereas only 22%of the sera from 32 presumed nonnialarial patients (i.e., negative by thick blood film) rcacted. Antigen prepared in this way was improved by adjusting the pH of the final extract to 7.8 (Taliaferro and Taliaferro, 1928). Of 32 sera from malarial patients, 94%gave a positive reaction, whercas only 1 serum reacted of 32 (3%)collected from persons not having parasites in their blood. The main drawback of such antigen was that it quickly became inactive. Activity could, however, be preserved by drying concentrated P. faZcciparum schizonts in vacuu over sulfuric acid. The resulting powder could then be extracted at will with Coca’s solution. In spite of these encouraging results in Honduras, Taliaferro did not have much success with this technique in Puerto Rico (Taliaferro, 1930). A suitable antigen for precipitation tests can be prepared from cultures of human plasmodia derived from peripheral blood (Row, 1931). Cultures of mature parasites were laked in distilled water and the resulting woolly suspension dried and then extracted with saline. A suitable volume of this extract was overlaid on the serum under test. A positive reaction was taken as the appearance of an opalescent ring at the junction of the two reagents after incubation for 3 to 4 hours at 37°C. As Taliaferro had also found, antisera to Plasmodium vivax as well as to P . falciparum reacted with P . falciparum antigen, and antisera to P. falciparum and P. vivax reacted with P. vivax antigen. Row claimed, however, a degree of species specificity on the basis of intensity of reaction. Also the test was specific for malaria. Malarial antisera gave no reaction with antigens prepared from, for example, Leishmania, various bacteria, or normal red blood cells, and control antisera from persons known not to have suffered from malaria did not react with malarial antigen. The specific reaction of malarial antisera was most evident in antisera collected soon after a paroxysm. Reactivity did not persist; antisera collected when gametocytes had appeared did not give good reactions. Row’s antigen prcparations

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were not suitable for agglutination tests and were not used in coiiiplement fixation tests. Dulaney and Housc (1941) were unable to demonstrate a precipitativc reaction by overlaying sera from malarious patients with antigens preparccl from parasitized human or monkey blood, although these antigens were useful in complement fixation tests. However, positive “precipitative” reactions were obtained using thcse sera and antigens if Goodner’s collodion fixation technique was used (Goodner, 1941). Collodion particlcs were added at the time of mixing serum and antigen, and thc adsorption of these particles b y antigen-antibody complexes produced a visible agglutination. A saline extract of dried Plasmodium lcnowlesi parasites was the most satisfactory antigen. Seventeen of 19 sera taken from persons with positive blood films gave positive collodion fixation reactions. The 2 sera that did not react were taken from persons on quinine therapy. On the other hand, 11 sera taken from persons with negative blood films and no evidence of clinical malaria all gave negative reactions in this test. In spite of the encouraging rcsults obtaincd in the work described above, interrst in this trchnique as a method of measuring malarial antibody languished, and it has revived only within the last few ycars with the introduction of double diffusion in gel techniques. Precipitation in gel techniques have been used to detect both malarial antibodies and malarial antigen (see Section VII). Of the rodent malarias, Plasmodium berghei and Plasmodium vinckei have been most extensively studied. Precipitating antibody has been dctected in the scrum of rats and mice after single or repeated bloodinduced infections, after immunization by infection with attentuated strains, and after vaccination with ground, red cell-free parasite cells (Zuckennan et nl., 1965a; Guberman and Zuckcrman, 1966; Weiss and Zuckerman, 1968). The best response was detected in vaccinated rodents, hut these animals possessed least resistance. Mice infected with attentunted strains, which were highly immunogenic, produced a rty3onsc that differcd in part from both that of rcpeatcdly infected animals and that of vaccinated animals, and furthcr analysis of this different response may lead to the identification of an immunogen. Mahoncy et al. (1966) werc unable to detect precipitin in the serum of rhews monkeys repeatedly inoculated with P. knozalesi-infected blood. Thrce monkeys were reinfccted 4 times at monthly i n t e n d s beginning 68 days after initial infection, but none of the serum samples collected throughout this period prccipitated antigen from P. 1inozc;lesiparasitc cells in double diffusion in gcl tests. This result may reflect the duration of in-

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fection in these monkeys because similarly infected monkeys that had been infected for 18 months or more did have precipitin to parasite cell antigen in their serum (K. N. Brown et al., 1969a). Whether this was because of an increase in the amount of antibody during infection or because of the synthesis of antibody having this property only during late infection is not known. Serum from rhesus monkeys that had recovered from recrudescing P . knozolesi infection also showcd precipitin reactions with a circulating antigen isolated from the plasma of monkeys acutely infected with P . knowlesi ( H . W. Cox, 1966), but the relation of this response to that to parasite cell antigens is not known. In marked contrast to infected monkeys, rhesus monkeys sensitized with killed P . knowlesi parasite cells or parasitized cells in complete Freund's adjuvant, show strong precipitin responses (Mahoney et al., 1966; Williamson, 1967; K. N. Brown et al., 196%). At least eleven P. knozolesi parasite cell antigens have been identified with such antisera. Most monkeys sensitized with P. knowlesi schizont-infected erythrocytes in complete Freund's adjuvant show resistance to subsequent infection with P . knowlesi (Freund et al., 1948; Targett and Fulton, 1965; K. N. Brown et al., 1968, 1969ib)) but whether the precipitating antibody response is concerned with protection is not known. In man, the most extensive studies of the precipitating antibody response to malaria infection concern P. faleiparum in West Africa (McGregor et al., 1966, 1968; Turner, 1967; Turner and McGregor, 1969a)b; Wilson e t al., 1969). Two types of antigen were used-(1) an aqueous extract of infected placental blood and (2) serum or plasma from heavily infected children. The complexity of these antigens has been discussed in Section VII. Antisera were collected from individuals living in the Gambia and the incidence of precipitin lines vaned not only among age groups but also among individuals of one age group. In double diffusion in gel precipitin tests with infected placental extracts, it was found that antibody was passively transferred from mother to child before birth. This reactivity persisted for several months in infants after birth but then declined. The incidence of reactors then increased after about 5 years of age until in adult life most individuals had detectable precipitin present in their serum ( McGregor, 1966). Subsequently this response was analyzed with respect to both heat-labilc and heat-stable parasite cell antigens (McGregor et al., 1968; Wilson et al., 1969; Figs. 6 and 8), and it was found that in children the response to heat-stable antigen was weak but that most adults over 30 years of age reacted to one or more of these antigens. In contrast, the responsc to heat-labile antigens of the

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

S-antigen c a r r i e r s

0 Anti

La-antigen

-

A n t i S-antigen

317

,---.

Age in years

FIG. 8. The age distribution of serum antigen carriers and also of individuals producing antibodies to various malarial antigens. S antigens were collected at Fajara, Gambia, from patients who came from the surrounding Kombo districts during August, 1967, to February, 1968 (161 positive out of 356 heavily infected children). Sera collected in November, 1967, and March, 1968, from the rural village of Mandanr (355 persons) in West Kiang province, Gambia, were tested for antibody to S- and La- antigens, respectively. The sera were screened using a gel diffusion test against three S antigen-containing sera and against an extract of infected placental blood both before and after it had been heated a t 5G°C. for 30 minutes. (From Wilson et d.,1969.)

parasite cell was detectable from early childhood and most individuals over 6 years of age reacted (Wilson et al., 1969, see, also, Section VII), Precipitins have been detected mostly in the IgG fraction of antisera but also in Ighl (Turner, 1967; Turner and McGregor, 1969b). Whether or not the differences in response to P. falciparum antigens described above reflect differences in sensitivity among individuals or reflect differences among strains or variants of the parasite is not known.

7 . Comment Hosts possessing specific acquired immunity to malaria usually have malarial antibody in their serum, and this antibody is detectable by a variety of techniques such as those described above. The converse does not hold true; hosts with detectable serum antibody may not show resistance to infection. Among explanations for this are that the Plasmodium

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is capable of antigenic variation or that humoral factors per se are not responsible for protection. Defined fractionation of plasmodial antigens is required to increase the specificity of serological tests and such fractionations must take into account the biochemical and physical characteristics of plasmodial antigens, their subcellular distribution, and their distribution among the different stages of the life cycle. In addition these properties and distributions must be related io the biology of infection, e.g., fractionated antigens can be used to absorb out antibody from sera known to be protective in passive transfer experiments (see Section VII1,B) or to sensitize susceptible hosts prior to infection.

EFFECTOF MALARIAL ANTIBODY B. THEPROTECTIVE 1 . Congenital Transfer of Immunity First indications of the possible importance of humoral factors came from observations that infants, born of mothers living in areas endemic for malaria, did not suffer unduly from the disease for the first 6 to 12 months of life. Blacklock and Gordon (1925) suggested that the low incidence of infection in these children was the result of an immunity passively acquired from the mother. Experimental confirmation of this view was provided by studies on the congenital transfer of immunity to Plasmodium bergliei by immune rats (for review, see Bruce-Chwatt, 1963b). Immunity was passed from mother to young mostly in the first inilk but to a smaller degree before birth. The protection lasted about 4 weeks. Young suckled on nonimmune mothers wcre similarly protected if antiserum from immune adults was given orally within the first 3 weeks of life. After this time, they were not protected because of a change in the transfer properties of the gut epithelium, but if antiserum was given by another route, it was protective ( Fabiani and Fulchiron, 1953; Briggs et al., 1966). Essentially similar results have been reported for Plusmocliimi vinckei in mice ( Adler and Foner, 1965), although the passively transferred immunity in this case was antitoxic rather than antiparasitic, i.e., it suppressed symptoms rather than parasitemia. In man, the transfer of immunity occurs mostly across the placenta before birth. In endemic areas, adult females had high levels of malarial antibody detcctable by, for cxamplc, fluorescent antibody tests ( Voller and Bray, 1962; McGregor et a ] . , 1965). The infants born in such areas had similar high levels of antibody in their umbilical cord blood. In the infants themselves, antibody levels dropped over the first few months of life, remained at a low lcvel for about 3 years, and then rose again over later childhood to adult levels. This initial lack of response in infants

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could possibly reflect a tolerance induced in fctal life by passage of malarial antigen from the mother. A high level of malarial antibody in cord blood may not necessarily measure the level of functional immunity, but the findings of Edozien et al. (1962) suggest that some is protective. Immunoglobulin G, prepared from umbilical cord bloods of infants born of immune Nigerian mothers, was found (if given in sufficient amounts to suppress parasitemia in local children suffering from severe infections of Plasmodium falciparum. Also, as mentioned previously, infants born of immune mothers do not suffer blood infections for the first a3-6 months of lifc.

2. Experimental Passive Transfer The bulk of evidence that humoral factors are important in the control of malaria infection comes from studies of the effect of passively transferred serum from immune subjects (human, animal, and avian) on infections in nonimmune subjects. The experiments have, in general, taken one of three forms: ( I ) antiserum, or a component of antiserum, has been administered when the recipient was suffering a parasitemia or just after initial infection; ( 2 ) recipients have been challenged with parasites prcincubated with antiserum and the resulting infection compared with that in controls given similar parasite inocula incubated with normal serum; ( 3 ) a combination of ( I ) and ( 2 ) , i.e., preincubation with antiserum and also adniinistration of antiserum to the inoculum recipient. Although earlier experiments of, for cxample, Sotirindhs ( 1936), Kaunders ( 1927), and Neumann ( 1933) strongly suggcsted an active humoral component in malarial immunity, the first convincing demonstration was reported by Coggeshdl and Kurnm (1937). Serum was collected from rhesus monkcys at a late stage of a recrudescing infection of Plasmodium knozdesi produccd by Chemotherapy of the normally fatal first parasitemia. This serum, if given to normal monkeys that had just been infected with P. knotolesi, either markedly prolonged the acute parasitemia or produced a recrudescing infection without the need for additional chemotherapy. To control the acute infection, however, a large amount of serum had to be administered over several days. The protective effect of a particular serimi pool could he titrated by prciincubnting standard niinimal inocnla of 10+ parasitized red cells with varying amounts of sc’rwn (Coggeshall and Eaton, 193811). Prcincubation ( % hour at 37°C. ) w a s morc cffective than mixing parasites and scrum just before inoculation. This suggc-sts th it antibody acts in some way clircdy on the parasite rather than in combination with cellu-

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lar factors (Coggeshall, 1943) or that the level of protective antibody is low and needs time for adsorption. Throughout their studies, Coggeshall and Eaton found that the stage most susceptible to antiserum was the near-mature schizont. In protection tests, if ring-infected erythrocytes were incubated with antiserum, the protective effect was always less than that found in tests where a similar number of schizont-infected cells was used. Possibly the developing parasite was protected until, by its own growth it damaged the erythrocyte and thus exposed itself to the action of antiserum (Coggeshall, 1943). The finding of Eaton (1938) that only cells containing dividing forms of P . knowlesi agglutinated in antiserum, whereas cells containing ring forms or uninfected cells did not agglutinate, would support such a hypothesis. That antiserum which protected monkeys against P . knowlesi had no effect on Plasmodium inui infections and that monkeys immune to P . knowlesi were completely susceptible to P . inui infection would suggest that the ultimate specificity of malarial immunity is, in fact, humoral. Nevertheless, although monkeys immune to Plasmodium cynomolgi were completely susceptible to infection with P . knotdesi, the protective effect of a given anti- P . knowlesi serum pool was enhanced in such monkeys compared with the effect seen in normal monkeys (Mulligan ef al., 1940). Antiserum can thus potentiate existing hyperreactive cellular elements, even though this reactivity is stimulated by another species of Plasniodium. Mulligan et al., also found that passive serum transfer was ineffective in splenectomized monkeys, which finding applies also to rats and Plasmodium berghei infection ( I. N. Brown, unpublished observations ) and re-affirms the importance of the spleen to malarial immunity. The demonstration of passive transfer described above has since been confirmed by other workers, both in animals and birds (see Mosna, 1938; hiianwell and Goldstein, 1940; Taliaferro and Taliaferro, 1940; Fabiani and Fulchiron, 1953; Briggs et al., 1966). The demonstration of such passive transfer in man has come more recently. Plasmodium knowlesi produces a mild infection in man (Knowles and Das Gupta, 1932), and Coggeshall ( 1940) demonstrated that convalescent sera, taken from human volunteers who had been infected with P. knotulesi, protected rhesus monkeys against P . knowlesi infection, whereas serum taken from the same volunteers before infection did not. These studies were, however, complicated by the presence of agglutinins to monkey erythrocytes in some of the human sera. In Plasmodium vivax infections of man the administration of even 500 ml. quantities of whole blood from “hyperimmune” patients did not prevent infection or modify the course of infection in susceptible persons (Boyd and Kitchen, 1943). Cohen and McGregor ( 1963) calculated that

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

321

to administer doses of serum equivalent in human subjects to those used in monkeys, would mean the infusion of about 1500 ml. of antiserum. The need for such large amounts of serum was at first taken to indicate the inaccessibility of the parasite (Coggeshall, 1943) but may reflect antigenic differences among parasite populations, or a low content of protective antibody. A protective effect of monkey antiserum to Plasmodium cynomolgi bastianelli was localized in IgG (Garnham and Cohen, unpublished results quoted in Cohen and McGregor, 1963). Such IgC, given intravenously to monkeys at 150 mg./kg. body weight at an early stage of infection cffectively suppressed parasitemia. In thc two examples quoted, the infection recrudesced about 8 days and 20 days after the administration of IgG possibly because the immunoglobulin was (I) degraded and/or ( 2 ) inhibited the development of an antibody response or of a cellular hyperactivity or because (3) a new variant not experienccd by the antiserum donors may have developed.

T I

1

I

I

I

2

4

6

8

1111

Days

10

y-Globulin therapy

Ftc:. 9. Tvapho~.oitecounts of PI<(stdIuttL\okiparitm nucl P l a s ? t d i t c m trwlariac in a child with a mixed infection treated with y-glol)ulin prepared f r o m immune adults. Arrows show times of administration of y-globulin by intra~nuxularinjection. (From Cohen et al., 1961. )

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23000

Garnetocytes

I

r;r

I

I

I

I

4

6

0

10

Days y-Globulin (intro-mus. i n i )

FIG.10. Traphozoite and gametocyte counts in a child with acute Ptusmodiutn futciparum malaria treated with y-globulin prepared from immune adults. Arrows show time of administration of y-globulin by intramnscolar injection. ( From Cohen et al., 1961.)

A similar protective effect of antibody of the IgG class was demonstrated in man (Cohen et al., 1961). Immunoglobulin was prepared by chromatography on diethylamino cellulose of serum collected from adults living in a hyperendemic area of West Africa; about 95%of such a preparation had a sedirncnt coefficient of 6.78 S. From 1.2 to 2.5 gm. of this IgG was administered over several days to children suffering infections of Plasmodium falciparum or Plasmodium malariae, or both. Doses of over 1.8 gm. suppressed parasitemia (Fig. 9 ) , and the symptoms of infection were also alleviated. Four days after the start of treatment, parasitemia was less than 1%of the initial values. At 9 days, in 8 of the children treated, no trophozoites were detectable, but there was no observable effect on gametocyte counts which tended to rise (Fig. 10). As was found in the experiments of Garnhani and Cohen, the conferred immunity was not long-lasting. y-Globulin-free serum and likewise y-globulin from Europeans did not afford significant protcction, but West African IgC

IMMUNOLOGICAL ASPECTS OF M A L A R I A INFECTION

323

was therapcwtically effective in 9 ehildrcn in Tanzania ( McGregor et al., 1963), indicating an antigenic similarity between the parasites of those areas. The occiirrence of serologically distinct strains was not excluded ( McGregor, 1964) as most of the treated children subsequently showed recrudcwences of infection. The protective effect of maIaiia1 IgG was confirmed by Edozien et nl. ( 1962) in Nigeria (who further demonstrated, as mentioned previously, the suppressive effect of IgG from umbilical cord blood on parasitemia ) and also by Sadun and co-workers. Immunoglobulin G prepared from the serum of West African adults suppressed acute parasiteniia in splenectomized chimpanzees infected with P. faleiparurn isolated from the same area of West Africa but not in chimpanzees infected with P . falciparunz isolated from South East Asia ( Sadun et al., 1966). It is not known whether IgM antibody exerts a similar suppressive effect on parasitemia or can potentiate the effect of IgG antibody. ?-Globulin levels in subjects protected from malaria by chemotherapy ‘ire much lower than in unprotected controls (Cohen et al., 1961) suggesting that malaria infection is n contributing cause of the hypergammaglobulinemia, typical of areas endemic for malaria ( see also, Curtain et al., 1964, 1965a). The rate of y-globulin synthesis in protected Gambians was about half that in the unprotected, but still higher (about 4 times) than that in Europeans. There may also be a genetic factor involved for West Africans resident in Britain for 3 to 10 years still made ?/-globulin at about twice the rate of Europeans. IX.

Active Immunization to M a l a r i a

A. IMMUNITY BY INFECTION Resistance to malaria infection is restricted in its specificity and may show in a number of ways (see Section IV,B). It is most in evidence in those hosts that have controlled the initial infection to the extent that is latent but declines after elimination of infection either naturally or by radical cure (Jeffcry 1966; Voller and Rossan, 1969,a-d) , After radical cure the level and duration of residual immunity depends largely on the degree of previous exposure to infection. For example, rhesus monkeys radically cured of an acute Plasmodium knozclesi infection die if subsequcntly infected with even the homologous strain. On the other hand, in P . knowlesi-infected monkeys which had survived the acute infection with the aid of quinine or antiserum and were suffering recrudescing infections, there was a persistcnce of partial immunity u p to about a year after sterilization of the infection with sulfathiazole ( Maier and Cogge-

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shall, 1944). Complete immunity, i.e., absence of apparent infection judging by blood smear was observed in 20 of 30 monkeys reinfected up to 14 weeks after radical cure of infections that had lasted 2 months or more. The remaining 10 monkeys of this group and all similarly treated monkeys, when inoculatcd aftcr 14 weeks, bccame infected, but no deaths were recorded until the interval between radical cure and reinfection was 52 weeks or greater. Monkeys rechallenged with a strain of P . knowlesi different from that used for the initial infection showed little immunity (see, also, Mulligan and Sinton, 1933). Three of 4 monkeys reinfected within 7 weeks of radical cure of their original infections were partially immune, but the remaining 9 monkeys challenged between 9 and 58 weeks after sterilization were completely susceptible. Reports concerning residual immunity after infection of man are contradictory but, in this respect, may reflect a difficulty inherent in working with man, namely that whether or not he is carrying a latent infection is difficult to ascertain. Some workers claim an effective homologous immunity after one infection (Cove11 and Nicol, 1951), but others report that the intensity of a second, or even third, homologous infection may be only modified in comparison to an initial infection. Also gametocytes produced during second Plasmodium falciparum or Plasmodium vivax infections were still capable of infecting mosquitoes and of transmitting infection (Jeffery, 1966). The implications of such findings as described above to the epidemiology of malaria have been discussed elsewhere.

B. LIVINGVACCINES The possible number of strains or of species of malaria parasite is still not known but may be very large. Possibly for this reason, and perhaps also in view of the known high specificity of acquired malarial immunity, little work has been conducted on the production of a living malarial vaccine. Strains of a given species of malaria parasite may differ in virulence ( e.g., chloroquine-resistant Plasmodium berghei is less virulent than normal P. berghei). The parasitemia produced by a given strain can also be reduced by, for example, feeding the host on a p-amino benzoic aciddeficient diet (Kretschmar, 1966) or by repeated culture of the parasite in a suitable medium (Weiss and Di Giusti, 1964). Strains may also become avirulent (Freund et al., 1948). A strain of P . berghei that had become avirulent in mice, nevertheless conferred an active immunity on mice against the parent P . berghei strain (Weiss, 1965; Weiss and Zuckerman, 1968). Similarly, multiple injections of Plasmodium berghei parasites previ-

IhlhlLJNOLOGICAL ASPECTS O F MALARIA INFECTION

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ously irradiated at levels of 20,000 r. ( 19,000 rad.) or greater conferrccl resistance on recipient rats and mice to subsequent challenge with nonirradiated parasites ( Wellde and Sadun, 1967). Yoeli and his colleagues (Yoeli, 1966; Nussenzweig et al., 1966) and Cox and Voller (1966) found mice infected with Plasmodium chabaudi (mild, chronic) were extremely resistant to Plasmodium oinckei (virulent, fatal) challenge for a period of 9 to 10 weeks after the initial infection with P . chabaucli. This immunity was not effective against P . berghei. In view of these findings and of recent improvements in culture techniques for malarial parasites, which should make it easier to attenuate these organisms, an attenuated living vaccine for primate malarias may be possible.

C. KILLED VACCINES Immunization against the erythrocytic forms of malaria parasites is difficult. Konstansoff (1930) prepared autovaccines from blood of patients by withdrawing infected blood into anticoagulant, lysing the erythrocytes with distilled water, and treating the parasite cells with 0.2 to 0.3%phenol. These killed parasites were then injected into the original patients, and a gradual acquisition of immunity was claimed. A similar approach was made by Boyd and Kitchen (1946) but the parasites were killed in vivo. Large numbers of Plasmodium vivax parasitized cells were injected into the bloodstream of nonimmune patients with high blood levels of quinacrine. The injected parasites quickly disappeared from the circulation. As a result of this procedure, the patients acquired an immunity to subsequent challenge with homologous P. uivax. They became infected but not as severely as would a fully susceptible person. More extensive studies of immunization against P. vivax were reported by Heidelberger and his colleagues in the same year. Two hundred patients suffering from relapsing P. vioax malaria were divided into three groups. The first group was given routine drug therapy, the second was given injections of formolized P. oivax parasites (Heidelberger et al., 1946a)) and the third was given injections of formolized normal human red cell stromata. No difference in relapse rates was found among the three groups ( Heidelberger et al., 1946b). In further experiments, three paretics were given a total of 3.5 to 4 loDformolized P. vivalc parasites by 7 to 9 intracutaneous, subcutaneous, and intravenous injections. On challenge with trophozoites 3 weeks after

x

326

IVOR N. BROWN

the last injcction, there was no cvidencc of protection ( Hcidelbcrger et d., 1 9 4 6 ~ )Similarly, . paticmts sensitized with even 10 x loq formolized parasitcs were completely susceptible to sporozoite challenge and developed normal infection$. There was some evidence in this case that the patients becamc smsitive to vaccinc injection but no innlarial antibody could be dcmonstrated in their sera in the complement fixation test ( Heidelberger et al., 1946~1).Heidelberger suggested that these negative results may have been due to the elimination of a soluble, but essential nntigen(s) during the preparation of the vaccine. The vaccines were prepared by water lysis of infected blood cells and subsequent repeated washing in ice-cold saline of the sedinientable residue. In spite of these unsatisfactory results with human malaria, somc success has been obtained with other species of Plasmodium. Canaries can be protected against bird malaria by giving injections of “killed parasitizcd cells bcfore challenge with infected blood ( Redmond, 1939). Heavily infected blood was drawn into a citrate-urea solution and then stored at -3” to 4°C. for 73 to 96 hours. Birds were given eight injections with 2 days between each injection, the blood from one donor being used to immunize two recipients. After challenge, seven birds proved to be extremely resistant, they showed no patent parasitemia, and another three suffered only transient patent infections. It is not clear from Redmond’s report if the vaccine used was a true killed vaccine. for all cxccpt three birds were found to be carrying latent infections ( b y blood subinoculation tests) before challenge. However, later work by Cingrich (1941) showed that canaries could be protected against Plasinocliuni cathemerium by immunization with large numbers of asexual blood parasites killed by minimal formalin or heat treatment. Birds, with no previous experience of malaria, were given twelve intravenous injections of 1 ml. of a 50% suspension of red blood cells which were 40-60% parasitized. The effectiveness of the immunity produced was demonstrated by an unusually low grade parasitemia of short duration which followed intravenous injection of large numbers of viable parasitcs. Since the number of parasites increased in the challenged birds over the first few days, it was concluded that the immunity elicited by this procedure was not as effectivc as that resulting from infection. Gingrich also found that smaller doses of vaccine (S doses x 0.15 ml.) or the injection of foreign red cells (12 doses x 1 ml.), would similarly protect birds but in these groups only a reduction in the number of deaths compared with the number seen in control birds was observed. These procedures did not influence the height of infection. Injection of hcatkilled parasites (12 doses x 1 ml.) into birds carrying latent infections did not eliminate the parasite.

IhfMUNOLOGICAL ASPECTS OF MALARIA INFECHOX

Ducks vaccinatcd subcutaiwously with killed, frec

327 P~CL.S?~lOt~iZl?~l

lophiime parasitcs acquired a small amount of resistancc to the acute stage of the infection (Jacobs, 1943). Such protection was elicited only by the water-insolul~lcrcsidur3s of thc parasite material. Thc antigcnicity of both whole free p m s i t c s a d insoluble rcsidues wns, however, increased by the addition of siiiall amounts of Staphylococcus toxoid to the vaccine before injcction. On the basis of these results, Jacobs concluded that the effective plasmodia1 antigen was only wcakly immunogenic and was probably an insoluble hapteii. The effectiveness of formalin-killed P . lophrirae vaccine was markedly increased if the plasmodia1 material was incorporated in Freunds-type adjuvant ( Freund et al., 194511) . Ducks were vaccinated three times, with a month between vaccinations, by giving multiple subcutaneous and intramuscular injections of such a vaccine. Upon challenge a month after the last injection with 10" P . lophurae parasites, 7 of 8 birds showed considerable resistance to the acute infection. A11 the birds became infected, but parasitemias were much lower than those seen in control birds, and also of much shorter duration. It is not known whether these birds still carried latent infections. Ducks were similarly protectcd against Plasmodium cathenierium by Thomson et al. (1947). Some protection was afforded by the injection of formolizd parasites alone but did not compare with that afforded by the use of killed parasites in adjuvant. Birds immunized with adjuvant vaccine retaincd the acquired resistance for at least 6 months. Coffin (1951a) confinned that ducks could be immunized against P . lophurae using Freund's technique, but was unable to immunize effectively chickens against Plasmodium gallitiacetcnr. On the other hand, a degree of resistance w7as elicited in chickens immunized with normal chicken red blood cells in Freunds adjuvant. Also 2 chickens immunized with killed P. loplzurae and adjuvant and, subsequently, twice infectcd with P . lophurae, acquired a high level of resistance to P . gallinaceuin. Control birds that had only been twice infected with P . lophurae were only a little more resistant than normal chickens. Richards ( 1966) protected a proportion of young chickens against P . gallinaceurn by giving three intravenous injections of killed, free P. gallinacewn parasites ( = 3 dosrs of 10:' parasitized cells). Fourteen days after the last injection, 23 chickens of the original 80 were partially resistant to a challenge of 2.5 x 10.' P . gallinaceurn-parasitized cells. All the resistant birds developed nonfatal infections which subsequently became latent, The use of saponin as adjuvant did not enhance resistance to erythrocytic forms, although it had some bcneficial effect in sporozoitc immunization experiments.

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Young rats can be immunized with a cell-free extract of PZasmocliurn berghei obtained by saponin lysis of parasitized cclls and subsequent disintegration of the parasite residue in a Hughes press (Zuckerman et al., 1965b). Onc to five inoculations of the resulting material was given to each rat. Partial immunity to challenge compared with control rats was obtained as judged by lower peak parasitemias, lengthened prepatent period, and lower mortality rate. With improvement in the technique of immunization and the availability of mosquito-passaged rodent malaria parasites, this system could perhaps provide a model for investigating the efficiency of vaccination procedures in eliciting protective immunity in a mammalian host to trophozoite and sporozoite challenge, although its use may be sonicwhat restricted by the natural resistance shown by adult rats to P . berglzei. Early attempts to immunize monkeys against Plasmodium knowlesi without the use of adjuvants were unsuccessful (Eaton and Coggeshall, 1939c; Shortt and Menon, 1940) although such procedures induced malarial antibody (Eaton and Coggeshall, 1939c; Ray et ul., 1941). However, most monkeys immunized subcutaneously with two doses of formolized P. knotolesi-parasitized blood (optimally about 15 x 10’ schizonts ) emulsified with complete Freund’s adjuvant, were extremely resistant to a normally lethal challenge (Freund et al., 1945a, 1948). Immunized monkeys, if they became infected, often showed n low grade parasitemia of short duration only and did not subsequently relapse during an observation period of at least 6 months, i.e., they probably eliminated the parasite ( Freund et al., 1948). Although malarial antibody detectable by complement fixation, (Davis, 1948) fluorescent antibody tests (Targett and Voller, 1965) and by precipitin tests (Mahoney et al., 1966; K. N. Brown et ul., 1968) was produced as a result of vaccination, there was no apparent relation between antibody levels and protective immunity. The route of immunization was important ( Targctt and Fulton, 1965). Variable results were obtained using Frcund’s technique of subcutaneous injection, but strong protection was conferred by intramuscular injection of the killed parasites in adjuvant. Vaccination with free parasite aiitigcn material alone or adjuvant alone did not significantly protect, but vaccination with free parasites in adjuvant was protective. Immunization with normal monkey erythrocytes in adjuvant was ineffective (Freund et al., 1948). It is not known if such immunization procedures are equally effective also against sporozoite challenge. The protocol and results of Freund’s experiments (Freund et al., 1948) suggested that antigenic variation by P. knowlesi ( K . N. Brown and I. N. Brown, 1965) does not exclude the possibility of effective vaccination.

IMMUNOLOGICAL ASPECTS OF MALARIA INFECTION

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Consequently his experiments have recently been extended ( K. N. Brown, 1968; K. N. Brown et al., 1968, 1969a,b). Monkeys immunizcd with dead P. knowlesi schizont-infected cclls in “incomplete” Freund’s adjuvant showed only a variant-specific immune rc’sponcc’. If challcnged with the saine variant a5 used for scnsitizution, such monkeys suffcrcd fatal infections by a breakthrough variant population. 111 contrast, inost inoiikcys inimunized with parasites in “complete” adjuvant show a prolonged latent period after challenge, and, in addition, they eliminated their infection after a brief breakthrough parasitemia. This elimination was testcd by blood subinoculation into clean monkeys, which did not produce infection, and also by splenectomy, which did not cause a recrudescence of parasitemia. Levels of parasite agglutinins as detected by schizontinfected cell agglutination tests were esscntially similar in both incomplete and complete Freund’s adjuvaiit-immunized monkeys. Wlietlier the monkeys immunized with complete Freund’s adjuvant showed an otherwise modified antibody response is not known. The possiblc rolc of a nonspecific heightening of phagocytosis analogous to that found by Mulligan et al. (1940) or, indeed, of a ccll-mcdiated immunity cannot be excluded (Phillips et al., 1969). Owl monkeys similarly immunized to Plasmodium falciparum ( Voller and Richards, 1968) developed infections more slowly than controls. Parasitemia was delayed in vaccinatcd animals after inoculation with infected blood although once infection became patent it developed as rapidly as in controls. The effect of such vaccination on sporozoite challenge is not known. X.

Experimental Modification of Immunity

Most of the pertinent early literature has been reviewed by Goble and Singer (1960) who discussed ways by which the reticuloendothelial system could be modified and so affect malarial parasitemia. Procedures that depress reticuloendothelial function or antibody formation, such as splenectomy ( Corradetti, 1963), blockadc with erythrocytes or colloids (Goble and Singer, 1960), or irradiation (Taliaferro and Simmons, 1945), tend to accentuate infection and induce relapses, whereas procedures that stimulate reticuloendothelial function or antibody formation, such as endotoxin administration (Martin et nl., 1967) or injection of Alycobacterium ( Yoeli, 1966) or Corynebacteriunz parutcm ( Nussenzweig, 1967), tend to have an adverse effect on infection. Results have, however, often been contradictory; one cannot generalize as to the cffect of immunosuppressive or adjuvanting procedures among plasmodia1 infections. An infection is a dynamic process spanning days or weeks and its out-

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come is delicately balanced. It may be affected by many factors (see, for example, Section 111); procedures such as the above-mentioned may often affect the host in a number of ways, and may also affect the parasite. Thus, splenectomy in mice removes an important crythropoietic site; a lack of immature cells of erythrocyte series inhibits some rodent malaria infections. Treatments such as irradiation or injection of immunosuppressive drugs, in addition to affecting antibody- and cell-mediated immune response?, may also affect erythropoiesis at certain dose levels. In some cases, parasite replication is inhibited ( Taliaferro and Taliaferro, 1948) though not in all (Walker, 1968). Similarly, treatments such as blockade are often transitory in effect and may be followed by a state of hyperfunction of phagocytic cells. The need for a cautious interpretation of such experiments is illustrated by the effects of corticosteroid administration and of neonatal thymectomy on malaria infection. In rhesus monkeys infected with Plnsmodiurn cynomolgi, cortisone administration over the period of acute parasitemia has little effect on parasitemia (Schmidt and Squires, 1951). Also acute Plasmodium berghei or Plasmodium vinckei infections in mice are not markedly affected by similar treatment (Fulton, 1954, F. E. G. Cox, 1968). However, the ability to resist further infection is lessened. Schmidt and Squires (1951) found that postcrisis P. c!ynomoZgi parasitemias in cortisone-treated monkeys were higher than in nontreated controls. In addition, the administration of high doses of cortisone during latency induced recrudescences. Similarly, F. E. G. Cox (1968) observed that p-methazone-treated mice radically cured of acute P. vinckei infection died if subsequently reinfected, whereas nontreated controls showed strong resistancc to infection after radical cure. These results would seem conclusive yet other workers have published contradictory reports. For example, Findlay and Howard (1952) found that cortisone enhanced acute P. berghei infections in mice, and Singer (1954) reported that cortisone depressed infection. An interpretation of the above results in favor of the suppression of specific acquired immunity may be correct, but that corticosteroids produce these effects nonspccifically cannot be excluded. These experiments are also open to the criticism that cortisone was administered over a long period and that such a dosage schedulc might be expected to accentuate nonspecific side effects if they occurred. Adequate variation of the dosage, timing, and number of cortisone injections would localize a specific effect more precisely. Neonatal thymcctomy may have similar paradoxical effects on malaria infection. As with the corticosteroids, thymectomy can affect specific acquired immunity (Law, 1966; Metcalf, 1966; Miller and Osoba, 1967).

IMMUNOLOGICAL ASPECTS OF hf.\L 4RIA ISFECTION

331

The reaction most consistently depressed by neonatal thymcctomy is cellmediated immunity as manifested by homograft rejection or delayed hypersensitivity. Humoral immune responses especially those to particulate antigens, may also be depressed but many are not. I n rats the ability to resist P . berghei infection is thymus-dependent (I. N. Brown e t al., 1 9 6 8 ~ )In . these experiments only 2 of 21 nonthymectomized 13week-old rats died of infection, whereas 12 of 23 neonatally thymectomized rats died. Higher parasitemias in the thymectomized group were accompanied by severe anemia. Among survivors the duration of acute parasitemia was longer in thymectomized animals of both sexes than in nonthymectomized animals. In contrast, Wright ( 1968) reported that nonthymectomized hamsters died 1-2 weeks after P . berghei infection but that neonatally thymectomized hamstcrs survived for 3 to 4 weeks after infection. At death, the nonthymectomized animals showed low parasitemias only but marked cerebral hemorrhage; thymectomized hamsters, on the other hand, showed high parasitemias at death, were severely anemic but had no marked cerebra1 hemorrhage. Judgin,o from mortality, these rcsults would appear contradictory, and yet both can be interprcted as an effect on the development of immunity. The nature of the impairment in rats is not known, but Wright (1968) argues from the incidence of cerebral complications that thymectomized hamsters wcre deficient in antibody production and that, consequently, no parasitized cell emboli were formed to initiate brain damage. Thymectoniized rats may show a similar antibody-forming deficiency, but the possibility that cell-mediated immunity plays a part in the resolution of infection cannot be overlooked in either case. On the other hand, removal of the thymus from chickens, where it has been shown to affect primarily cellmediated responses, has little effect on Plasmodiztm lophirrae infection, whereas bmsectomy results in higher parasitcmia ( Longenecker et al.. 1966). Assuming the mechanisms of malarial immunity are essentially similar in rodents and birds this would suggest an antibody deficiency in thymectomizrd rodcnts. As in the case of corticostcroids discussed above, a nonspecific effect of thymectomy in the immunological sense cannot be entirely disregarded. These examples emphasizc the nccd for a cautious interpretation of experiments involving manipulation of the immune mechanisms in malaria infcction. XI.

lmmunopathology

Malaria p:irasites live within :ind digest host cells for the greater part of thcir life cycle. Thcy are antigcnically coinplcx and large amounts of

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IVOR N. BROWN

rnalarial antigen is released during the course of infection. Some of this antigen is detectable in serum and can persist in the circulation (Eaton, 1939; McGregor et al., 1968; Todorovic et al., 1968c,d). As a result of infection, immunoglobulin levels, particularly those of IgG and IgM, are elevated though only some of this immunoglobulin is demonstrably malarial antibody. hialarial infections, which tend to chronicity, would thus seem to provide an almost ideal situation for the development of immunopathological complications. Asherson (1968) has discussed some of the ways in which microorganisms may cause such complications. To quote the author, these include: “ ( a ) Liberation of normal or damaged tissue components which then cause autoantibody production; ( b ) modification of normal tissue components by enzyme; ( c ) combination of normal tissue components with products of the microorganism; ( d ) microorganism antigen resembIing mammalian tissue component; ( e ) adjuvant action of the microorganism which may favour an autoimmune response by any of these mechanisms; ( f ) production of rheumatoid factor and conglutinin by the binding of antibody and complement in immune complexes.”

All these situations would seem to apply to malaria parasites, yet few iininunopathological effects have been established.

A. BLOOD The anemia that accompanies malarial infection is often in excess of that to be expected from obscwed parasitemia levels (see Zuckerman, 1964a for review) but is largely unexplained. Enhanced phagocytosis of noninfected cells has been reported by many workers (Zuckerman 1964a, 1966; McGhee, 1965; H. W. Cox et ul., 1966) and is particularly marked at, or just after, crisis, but may persist for some time after the bulk of parasitized cells has been removed from the blood (Fig. 11). An immune process is suggcsted by this close timing to the presumed onset of acquired immunity. A cold agglutinin ( mercaptoethanol sensitive) for trypsinized erythrocytes has bcen demonstrated in the serum of rats during Plasmodium bergliei infection (Kreicr et ul., 1966), and similarly Maycr and Hcidelberger (1946) found an autoantibody in the serum of chronic, relapsing, Plnsmoclium vivax malaria cases associated with patcnt parasitemia. This antibody could be detected in complement fixation tests

333

IhlMlJNOLOC,ICAL ASPECTS O F hfALARTA INFECTION

.

160.

150 140 I30

Number of r e d cells phogocytosed by 100 macrophages . U n i n f e c t e d rbc 0 : Infected rbc

120, I10

100 90

80 70 60

50

.

40

Normal control ratsL*

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with normal human crythrocytcs. Expcrimental evidence of the role of such antibodies in malarial anemia iy lacking. Another possibility is that ancmia rcsults indirectly from the releasc of malarial antigens into the pla.;ma. Antibodies to circulating antigens have been demonstrated (H. W. Cox, 1966; Todorovic et nl., 196th; McGregor et nl., 1968) and anemia can be induced in the absence of parasites by injecting malarious plasma (Corwin and McGhee, 1966; Todorovic et nl., 196%). Malarial antigen does not appear to be directly toxic to erythrocytes, and it has been suggested that an immune process is involved. Whether crythrocyte destruction results from coating of erythrocytes with inal'irial antigen and subsequent opsonisation or lysiy by ma1:trial antibody or, as proposed by Dixon (1966) from coating of erythrocytes with malarid antigen-antibody complexes, is not known. Similar considerations apply to the etiology of blackwatcr fever ( Salisbury, 1949), an hemolytic anemia associated particularly with Plasmodium fakiparum and quinine administration. Despite thv evidence for an immune mechanism, antibodies may not be involved. Enhmcecl phagocytosis coiild result from a nonspecific

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hyper-reactivity of the reticuloendothelial system. The spleen and liver are often considerably enlarged as a result of infection and their tissues hyperplastic. Fluctuations in the level of serum proteins prabably contribute to alterations in the fragility and membrane structure of circulating erythrocytes. Another factor to be taken into account is the marked reticulocytosis consequent to intense parasitemia. Immature red cells are larger than their mature counterparts and differ from them in their physical properties. They tend to agglutinate spontaneously and are readily agglutinated by some Coombs reagents (Jandl, 1960). They behave as if they are mature red cells coated with antibody. All these factors could contribute to an increased sequestration of uninfected cells. Also erythrocytes may be lysed by an hemolytic factor released from the parasite, parasitized cell, or organ tissues affected by the acute pathological disturbances.

B. AUTOANTIBODIES AND MALARIA Malaria has been both directly and indirectly associated with the formation of autoantibodies. The direct association comes from studies of experimental malaria infections which have been shown to induce the formation of Wassermann antibodies (Kitchen et aZ., 1939; Mayer and Heidelberger, 1M6), of antibodies to “normal” erythrocytic components (Mayer and Heidelberger, 1946; Kreier et al., 1966), and of rheumatoidfactorlike antibodies ( Houba and Allison, 1966). Whether such antibodies are pathogenic has not been demonstrated. The indirect association comes from studies of the serum of individuals resident in areas known to be endemic for malaria. Curtain et al. (196%) found an unusually high incidence of cold hemagglutinins and of autoimmune complement fixation with saline extracts of human kidney and liver (Curtain et al., 1965c) in sera of Melanesians of New Guinea. Shaper et a!. (1967) reported a high frequency of circulating heart antibodies in Africans with and without heart disease. Similarly, the incidence of heterophile agglutinins ( Adeniyi-Jones, 1967), of rheumatoid factor ( Shaper et al., 1968), and of rheumatoid-factorlike globulins ( Houba and Allison, 1966) is often high in Africans. These antibodies were in general associated with elevated 7-globulin levels in the individuals concerned. In some cases an association with high malarial antibody titer was demonstrated ( Shaper et nl., 1968). Shaper and co-workcrs d c h e a t e d an immunological syndroinc consisting of high titers of malarial antibody and the prescncc of high IgM levels and circulating autoantibodies to heart, thyroid, and gastric parietal celIs, associatcd particularly with immigrant hut also with indigenous peoples of Uganda.

Ih4MUNOLOGICAL ASPECTS OF MALARIA INFECTION

335

Interpretation of the results of such surveys is difficult. Repeated malarial infection is known to result in high 7-globulin levels, and residents in malaria endemic areas show increased synthesis and turnover of y-globulin (Cohen and McGregor, 1963). On the other hand, other parasitic diseases commonly occur also in malaria endemic areas, and the contribution of malaria to raised 7-globulin levels is thonght to vary markcdly. Malaria is probably inore important in Africa than, for example, in New Guinea ( McGregor and Gillcs, 1960; Curtain et al., 1964). The occurrence of endomyocardial fibrosis, and possibly of some other diseases, in tropical Africa may be related to the inimunological disturbance produced by parasitic diseases such as malaria (Shaper et at., 1968), and similar considerations apply to areas such as New Guinea. On the other hand, Greenwood (1968) pointed out, after a study of the pattern of admissions to a hospital in Nigeria, that diseases in which autoimmune processes are thought to be involved are relatively uncommon in tropical Africa, when compared with their incidence in temperate regions of the world. This might be a reflection of the age distribution of African populations which tends morc to youth than, for example, European populations, but, alternatively, there may be an inverse rclationship between parasitic infection and autoimmune disease.

c.

NEPI-IROSIS

ASSOCIATEDWlTII MALARIA

Both acute and chronic malaria infection can produce kidney damagc ( Fulton and Maegraith, 1949); in acute PZa.smodiuni fakiparum and blackwater fever the most pronounced changes are in the tubules; in contrast. in prolonged malarial infection the tubular lesions are often secondary to glomerular damage ( Giglioli, 1932; Gilles and Hendrickse, 1963). The cause of the damage in cither case is ill-defined. Children in tropical areas show a high incidence of nephiotic syndrome, which is unusual because its peak onset is in late childhood (5-7 years) and because it is, in general, poorly responsive to corticosteroicl therapy. Epidemiological evidence suggests a relation to prolonged Plasmocliunz. malariae infection ( Giglioli, 1968; Gillcs and Hendrickse, 1963, Kibukamusoke et al., 1967). The most extensive studies of this syndrome have h e n in Nigeria (Hendrickse and Gilles, 1963), and the syndrome has been wcll characterised both clinically and pathologically. Despite the observation that the syndrome is in general poorly responsive to antimalarial trcatnient ( Gillcs and Hendrickse, 1963; Kibukamusoke et nl., 1967), the epidemiologicnl rclationship to malaria is so striking w a y implicated. W'1ys of producing that malaria is thought to he in glomerubr damcigc cxpc,rinicntally have becn discussccl by, for cx:lmplv, wmcb

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Unanue and Dixon (1967) and Cochrane (1968), and the most likely explanation of malarial nephrosis is thought to be that glomerular damage is mediated by malarial antigen-antibody complexes, which are trapped in the glomeruli and thus initiate a series of immunopathological processes. Damage could conceivably occur also in other tissues in this way. With respect to the kidney, experimental evidence in support of this view is accumulating. Immunofluorescence studies of the renal tissue of Nigerian children with the nephrotic syndrome have shown that the renal glomeruli contain bound immunoglobulins and the PIc-globulin component of complement; IgM, IgG, and IgA have been detected, but IgM predominates (Dixon, 1966; Allison et al., 1969; Ward and Kibukamusoke, 1969). Similar findings have been reported for Plasmodium cynornolgi-infected rhesus monkeys (Ward and Conran, 1966). By using a direct fluorescent antibody technique, Ward and Conran found that immunoglobulin, P,,-globulin, and, in addition, malarial antigen, were associated with the renal glomeruli of infected monkeys. This association was not detectable in acutely infected animals but was present after 2 or 3 weeks of infection. At this time no or few parasitised cells were seen in the kidney tissues. In the kidneys of nephrotic children a similar association of malarial antigen with the glomerular membranes has also been shown (Ward and Kibukamusoke, 1969) but only in 3 of 13 cases studied. The incidence of other protein deposits was more common in these cases. On the other hand, Allison et al. (1969) were able to elute probable malarial antigen from a basement membrane preparation of the kidney of a nephrotic child and demonstrated its identity by gel precipitation with antigen eluted from P. malariae-infected splenic tissue. In P. falciparum infections, circulating malarial antigens have been detected in association with heavy infection, most often in children ( McGregor et al., 1968; Wilson et al., 1969). These antigens may persist in the circulation and, in children, the antibody response to this type of antigen is transient only. A similar situation may exist, but has not been fully demonstrated, in P. malariae infections. Low levels of antibody, particularly of the precipitating type, in conjunction with circulating malarial antigen could predispose to the formation of soluble complexes in antigen excess. There is some evidence that complexes are formed in the plasma of nephrotic children although their malarial identity has not been established. p,,-Globulin is detectable in the first peak of Sephadex G-200 separations of the serum of nephrotic children as well as in the more usual middle peak which would suggest its incorporation into a macromolecular form, perhaps into an imnlunc complex ( Soothill and Hendrickse, 1967).

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FIG. 12. Pattern of immunoglobulin deposition, judging froin fluorescence, in section of kidney froin an African child with nephrotic syndrome. A granular type of pattern is observed. (From Allison et al., 1969.)

The association of this nephrotic syndrome with P. mzlmiae rather than with P. falcipnruin or P. vivnx is puzzling, particularly in areas such as Nigeria where P. falciparuni is the dominant parasite. Contributing factors may be the longer generation time and, in general, lower and more persistent parasitemia seen in P . mulariae infections. Nigerian children with nephrotic syndrome are in general poorly responsive to steroid therapy; in fact, it may have adverse effects, but some do respond. In addition, in some children the pattern of protein deposition in the glomeruli differs from that normally seen; it is linear rather than granular (Fig. 12). In these cases an autoimmune reaction to kidney tissue may have occurred or is perhaps superimposed on a complexmediated reaction. Malaria may not be the only cause of nephrosis in these children. B. Greenwood and A. Voller (personal communication) have been studying malaria infections in NZR mice as a potential experimcntal model of this system. Plcliminary rcsrdts have been encouraging; young NZB mice show, on blood infection with rodent malaria, pronounced

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kidney damage, increased urine output, and proteinuria. Whether or not this is a consequence of immunological damage has yet to be established. XII.

Discussion

Resistance to malaria can be either innate or acquired. The contribution of innate resistance varies among host-parasite combinations and alone can account for complete susceptibility on the one hand, or, on thc other hand, for complete resistance to all stages of the parasite. Acquired resistance may have nonspecific and specific components. Nonspecific responses may be enhanced without a specific protective response developing. Thus, enhanced resistance resulting from previous exposure to another organism may not necessarily indicate the presence of common antigens but only the activation of nonspecific factors. Specific acquired immunity to malaria is directed primarily to the blood stage of infcction and is evidenced by both immunity to symptoms and immunity to parasites. The nature of antitoxic immunity is unknown; it may be due to antitoxic antibody ( Adler and Foner, 1965), to a host tolerance of toxic products of the Plasmodium, or to a developing antiparasitic immunity, Also the nature of antiparasitic immunity is uncertain. The possible role of antigenic variation in prolonging infections must be remembered (Section V ) . Depending on the host-parasite combination, acquired resistance may result from an immune response to species-, strain-, or, on a lower level, variant-specific antigens. Analysis of the host response into its component parts is difficult. For instance, the lack of general protection afforded by a Plasmodium knotolesi variantspecific immune response in rhesus monkeys may indicate that a cumulative variant-specific response is necessary for protection, but a response to non- or less-variable antigens may be crucial to the eventual resolution of the infection. The wider response may require the prior appearance of variant-specific reactions either for its development or for its action. Malaria infections induce considerable antibody production but this is not always accompanied by increased resistance. Protcction certainly involves huinoral antibody acting directly on the parasite or indirectly as an opsonin. Another possibility is that cell-mediated immunity, or some parallel process such as the synthesis of cytophilic antibody, plays a part in the control of infection. The effect of neonatal thyniectomy on malaria infection in rodents and of complete Freund’s adjuvant, parasite antigen emulsion in stimulating protection to malaria in monkeys could be interpreted in such a way. That immune guinea pig lymphocytes can destroy Leis7~nmnia-infcctedmacrophages in witro has been demonstrated by Bray and Bryceson (1968) and, in malaria, lymphocytcs may similarly

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339

dcstroy macrophagcs that have ingested plasmodia or, perhaps, parasitized erythrocytes directly in organs such as the spleen where during infection blood flow is sluggish. Malarial antibody could iiossibly impedc such reactions and such a masking effect ( a parallel situation may be tumor enhancemcnt ) could contribute to the duration of mnlarial blood infection at subpatent levels. Other contributing factors may be that hosts show immunc tolerance to, or become paralyzed by, some malarial antigens-ciI.culating antigens would he p;irticuliirly relevant in this respect. A host’s ability to eliminate malaria parasites from its tissues is indicative of complete immunity. Criteria such as the lengthening of prepatent or latent period after infcction, the shortening of the duration of patency, and the depression of peak parasitemia, although useful ways of assessing resistance, are expressions of a partial immunity to the blood stage; latency indicates an inability to resolve infection. The concept of premunition, an immunity dependent on latency, has been referred to in Section IV. Though useful descriptively, it would seem best to avoid this term as it implies a distinct imiiiunological mechanism for which there is so far no evidencc. The chief charactcristic of immunity to superinfection is its specificity; evcn morphologically idcntical “strains” of a plasmodia1 species may show no reciprocal cross-protection upon supcrinfection. Why this is so is not known. Possibly different isolations of parasites vary in their genetic constitution. A more precise definition of a “strain” would thus include not only its morphological characteristics but also its spectrum of biochemical and antigenic compositions. Little is known of the genetics of malaria parasites but gcme recombination during the sexual stage of the life cycle in thc mosquito could contribute to the antigenic diversity of parasite isolates. Chromosomal aggregation and diploid or polyploid states may nlso occur in other stages of the life cycle. Developing immunity in a population would tend to select out rarer genetic types, and in areas endemic for malaria selection could be continuous. Aside from other immunological considcrations, the fact that individuals in cndemic areas become infected evcn in aclult life may reflect in part thesct antigenic shifts. XIII.

Summary

Thc resolution of plasmodia1 infection presents a complex problem not only to the infected host but also to the immunologist. M7e do not know what factors are essential to suppression of parasitcmia and eliniination of infection. Both antibodies and phagocytes are thought to be in-

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volved but, despite recent improvements in serological technique, it is not possible to forecast with any degree of accuracy the outcome of an induced infection in a semi-immune host. That plasmodia may be antigenially labile, that plasmodia multiply rapidly, and that they are predominantly intracc~llularin habitat are among factors that must be considered. The amount of fundamental research that is needed is perhaps indicated by thc list of research recommendations drawn up by the WHO Scientific Group on “Imniunology of Malaria” (World Health Organization, 1968). There are now numerous laboratory strains of Plasmodium available for study in rodents, birds, and monkeys. In addition, owl monkeys will support infections with the human Plasmodium P . falciparum and the infection is similar to that observed in man. Plasmodia show limited growth in uitro, but culture systems can now provide, on the one hand, a source of plasmodia1 antigen and, on the other, an isolated system for studying the effects of antibodies and of cells on malaria parasites. Variables inherent in in vivo experiments can be eliminated. It is, however, in nature that the problem lies and fundamental research on the endemic disease is essential. Whether active immuniz a t’ion of infants is possible needs extensive investigation. Also it is not clear what effect mass chemotherapy has on the susceptibility of exposed populations or what is the precise role of malaria as a causative or aggravating agent in tropical splenomegaly, nephrosis, and various other immunopathological syndromes found in malarious regions. In view of the importance of malaria as one of the greatest world causes of mortality ancl morbidity, it deserves greater attention than it has received in the past. ACKNOWLEDGMENTS I thank Dr. K. N. Brown and Dr. F. Hawking for their advice and criticism during the preparation of this manuscript.

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VolIer, A., and Rossan, R. N. (1969b). Trans. Roy. Soc. Trop. Med. Hyg. 63, 57. Voller, A., and Rossan, R. N. ( 1 9 6 9 ~ )Truns. . Roy. Soc. Trop. Med. H y g . 63, 507. Voller, A,, and Rossan, R. N. (1969~1).Trans. Roy. SOC. Trop. Med. Hyg. ( i n press), Voller, A., and Wilson, H. (1964). Brit. Med. J. ii, 551. Voller, A., Richards, W. H. G., Hawkey, C . M., and Ridley, D. S. (1969). J. Trop. Med. Hyg. 72, 153. Vredevoe, D. L., and Nelson, E. L. (1963). Biochem. Biophys. Res. Commrcn. 10, 221. Wagner, H. N., Razzak, M. A., Gaertner, R. A., Caine, W. P., and Feagin, 0. T. (1962). A. M . A. Arch. Intern. Med. 110, 128. Walker, P. J. (1968). J. Protozool. 15, August suppl., 33, Abstr. 121. Ward, P. A., and Conran, P. (1966). Military Med. 131, Suppl., 1225. Ward, P. A., and Kibukamusoke, J. W. (1969). Lancet i, 283. Weiss, M. L. (1965). Proc. 2nd Intern. Conf. Protozool. p. 168. Weiss, M. L., and Di Giusti, D. L. (1964). J. Protozool. 11, 224. Weiss, M. L., and Zuckerman, A. (1968). 1.waeZ J. Med. Sci. 4, 1265. Wellde, B. T., and Sadun, E. €1. (1967). Exptl. Parasitol. 21, 310. Wery, M. (1968). Ann. SOC.Belge Med. Trop. 48, 1. Williamson, J. (1967). Protozoohgy 11, 85. Williamson, J., and Cover, B. (1966). Trans. Roy. Soc. Trop. Med. Hyg. SO, 425. Wilson, R. J. M., McGregor, I. A., Hall, P. J., Williams, K., and Bartholomew, R. (1969). Lancet ii, 201. Wissler, R. W., Robson, M. J., Fitch, F., Nelson, W., and Jacohson, L. 0. (1953). J. Immunol. 70, 379. World Health Organisation ( 1963). “Terminology of Malaria and of Malaria Eradication,” Drnfting Committee Report. World Health Organ., Geneva, Switzerland. \Vorlcl Health Organisation. (1966). World Health Organ. Chron. 20, 286. World Health Organisation. (1968). “Immunology of Malaria,” Report of a WHO Scientific Group. WHO, Geneva, Switzerland. Wright, D. H. (1968). Brit. J. Exptl. Pathol. 49, 379. Yoeli, M. (1966). Bull. SOC. Pathul. Exotique 89, 593. Zuckerman, A. ( 1945). 1. Infect. Diseuses 77, 28. Zuckerman, A. (1964a). Ezptl. Parasitol. 15, 138. Zuckerman, A. (1964b). Am. J. Trop. Med. H y g . 13, 209. Zuckerman, A. (1966). Military Med. 131, Suppl. 1201. Zuckerman, A. (1968). In “Infectious Blood Diseases of Man and Animals” ( D . Weinman and M. Ristic, eds.), Vol. 1, p. 23. Academic Press, New York. Zuckerman, A., and Ristic, M. (1968). In “Infectious Blood Diseases of Man and Animals” (D. Weinman and M. Ristic, eds.), Vol. 1, p. 80. Academic Press, New York. Ziickerman, A,, and Yoeli, M. (1954). J. Infect. Diseases 94, 225. Zuckerman, A., Spira, D., and Schulman, N. (1964). Proc. 1.r.t Intern. Congr. Parasitol., Rome. Zuckerman, A., Ron, N., and Spira, D. (1965a). Proc. 2nd Intern. Congr. Protozool. p. 167. Zuckerman, A., I&mbnrger, Y., and Spira, D. (1965b). Proc. 2nd Intern. Congr. Protozool. p. 50.

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AUTHOR INDEX Numbers in parentheses are reference nimbers and indicate that iin mthor’s work is referred to, although his namc is not cited in the text. Numbers in italics show the page on which the coinplete iefcrence is listed.

A Ahel, C. A., 17, 26, 28 Abele, D. C., 306, 340 Abeyounis, C. J., 171, 185 Ackers, G . K., 91, 113 Adanis, K. M., 35, 72 Adeniyi-Jones, C., 334, 340 Adeniyi, A., 3.36, 337, 340 Adler, F. L., 139, 187, 234, 236, 262 Adler, S., 318, 338, 340 Acller, W. H., 238, 244, 261 Al-Askari, S., 118, 187, 197, 198, 199, 200, 229, 230, 231, 232, 233, 234, 235, 256, 261. 262,264 Alhertini, R. J., 159, 185 Alexander, P., 131, 180, 182, 185, 187, 258, 259, 261 Ali, V. M., 253, 265 Allan, N., 276, 344 Allfrey, V. G., 164, 191 Mison, A. C., 231, 232, 276, 334, 336, 337, 340, 341, 344 Almeida, J. D., 7, 8, 9, 19, 21, 28, 88, 113 Alving, A. S., 326, 344 AIvord, E. C., Jr., 175, 192 Amano, T., 87, 113 Amante, L., 32(3), 33(3), 71 Amos, D. B., 181, 189, 209, 252, 254, 257, 262, 263 Anderson, T. F., 3, 6, 7, 8, 28, 29, 30 Andres, G . A,, 2, 28 Andrews, P., 91, 113 Appel, S. H., 176, 185 Apt, L., 2006, 248, 263 Ada-Chavcs, M. P., 232, 261 Archer, D. K., 2206, 266 Armerding, D., 39, 71(28), 72 Arnason, B. G., 140, 193

Arquilla, E. R., 38, 39(27), 43, 72, 108, 113

Asherson, G. L., 149, 173, 174, 185, 187, 332, 340

Ashman, R. F., 18, 28 Askonas, B. A., 111, 113, 139, 192, 291, 341

Aust, J. B., 204, 205, 206, 210, B1, 222, 248, 263

Austen, K. F., 87, 113 Avery-Jones, S., 2G8, 346 Ax, W., 122, 124, 126, 157, 160, 161, 164, 169, 170, 185, 186, 190

h e n , E. A., 299, 341

B Bach, F. H., 159, 161, 169, 185, 188, 190 Baer, R. L., 210, 211, 261, 263 Bain, B., 159, 185 Baker, M. C.,3,5,28 Baker, R. F., 81, 113 Bale, W. F., 291, 346 Balkuv, S., 208, 251, 265 Bangham, A. D., 84, 85, 108, 113 Banki, G., 299, 341 Baram, P., 228, 231, 232, 233, 236, 238, 244, 261, 265

Barfort, P., 108, 113 Barnes, J. M., 35(21), 72 Baron, L. S., 87, 114 Barren, A. L., 253, 26Fi Barrow, P., 75, 76, 91, 114, 124, 126, 188 I3artholomew, R., 287, 298, 299, 300, 301, 302, 316, 317, 336, 349

Bartova, L. M., 135, 141, 142, 186 Baumgarten, A,, 334, 342 Beale, C . H., 28.5, 341 Becker, E. L., 87, 107, l l , 3 , 175 Bccker, R. J., 207, 261, 265 351

352

AUTHOR INDEX

Beckman, V., 122, 157, 160, 164, 190 Behrend, H., 214, 250, 261 Benacerraf, B., 40(35), 41, 42, 43, 45 (35, 36, 37, 40, 43,44, 45, 46), 46, 48135, 43, 47, 48, 49, 50, 521, 49, 50, 52, 53(63), 54, 55(35, 43, 49, 50, 61, 63, 64), 56(43, 64, 66), 71 ( 3 5 ) , 72, 73, 112, 113, 149, 173, 186, 190, 291, 348 Ben-Efraim, S., 41( 37), 51, 55(57, 59), 72, 73 Bennett, B., 118, 139, 163, 173, 185, 186, 191, 235, 236, 241, 246, 261 Bennett, W. E., 154, 185 Berg, O., 121, 129, 130, 135, 176, 185, 186 Bergheden, C., 160, 161, 188 Berke, G., 122, 124, 126, 169, 170, 186 Berken, A., 149, 186 Berry, D. M., 88,113 Bersack, S. R., 173, 187 Biberfeld, P., 122, 164, 165, 186 Bickis, I. J., 124, 186 Bidwell, D. E., 277, 348 Bill, A. H., 131, 133, 135, 182, 188 Billeter, M. A., 234, 261 Billewicz, W. Z., 306, 307, 318, 345 Billingham, R. E., 118, 136, 159, 177, 178, 179, 186, 191, 193, 238, 244, 256, 258, 261, 266 Binaghi, R., 27, 28 Biorklund, A,, 129, 133, 186 Biozzi, G., 34, 72, 174, 191 Blackburn, W. R., 254, 256, 263 Blacklock, B., 318, 341 Bladen, H. A., 83, 87, 88, 113 Blanden, R. V., 175, 190, 252, 264 Blaw, M. E., 176,186 Bloch, E. H., 288,345 Block-Shtacher, N., 152, 174, 186 Block, J. B., 174, 192 Block, K. J., 112 113 Bloom, B. R., 118, 139, 163, 172, 173, 186, 200, 210, 217, 221, 226, 235, 236, 237, 241, 246, 261 Bloom, W., 283, 292, 295, 310, 348 Bloomfield, V. A., 14, 28 Bloth, B., 3, 19, 21, 22, 23, 24, 25, 26, 28, 30

Blumental, G., 173, 189 Boak, J. L., 161,186 Boesman, M., 232, 238, 262 Bolt, R. J., 130, 133, 193 Boluncl, L., 163, 164, 187, 191 Bondevik, H., 142, 180, 186, 234, 236, 261 Bornstein, M. B., 176, 185 Borsos, T., 89, 91, 96, 97, 98, 99, 112, 113, 114 Bouthillier, Y., 34( l l ) , 72 Boyd, M. F., 277, 280, 283, 320, 325, 341 Boyden, S. V., 311, 341 Boyse, E. A., 139,185,191 Brandriss, M. W., 211, 232, 261 Braunsteiner, H., 130, 186, 205, 261 Bray, R. S . , 275, 276, 282, 284, 292, 306, 312, 318, 338, 341, 344, 348 Breitenbnch, R. P., 331, 345 Brenner, J., 77, 113 Brenner, S., 2, 6, 28 Brent, L., 136, 177, 186, ZSG, 258, 261 Briggs, N. T., 287, 318, 320, 341 Broberger, O., 124, 130, 135, 186, 191 Bromer, W., 39( 27), 7 2 Brondz, B. D., 120, 122, 128, 131, 134, 135, 136, 138, 141, 142, 186 Broom, J. C. 288,341 Brown, A., 1, 6, 30 Brown, F., 19, 21, 28 Brown, H. C., 288, 341, 343 Brown, I. N., 283, 285, 287, 289, 295, 297, 310, 311, 316, 328, 329, 331, 341, 346 Brown, K. N., 283, 285, 289, 295, 297, 310, 311, 316, 328, 329, 341, 346 Brown, K. S., 35, 7 2 Brown, P. C., 129, 176, 186, 191 Brown, R. A., 7, 8, 29 Brown, R. E., 229, 262 Bruce-Chwatt, L. J., 268, 278, 279, 318, 341 Bninner, K. T., 119, 120, 122, 123, 124, 125, 126, 128, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 176, 186, 190 Bryceson, A. D. M., 338, 341

AUTHOR INDEX

Bulienik, J,, 131, 133, 146, 180, 182, 186, 188 Bucci, A., 299, 341 Buckley, C. E., 1, 9, 14, 30 Buckley, R. H., 254, 257, 262 Bukantz, S. C., 304, 346 Bunting, W. L., 124, 187 Burchenal, J. H., 181, 189 Burnet, F. M., 242, 258, 262

C Cacligan, F. C., 275, 344 Caine, W. P., 291, 349 Calciati, A., 207, 208, 2.51, 262 Calcott, M. A., 16, 30, 94, 114, 154, 190 Campbell, D. H., 17, 29, 291, 344 Cannon, P. R., 288, 292, 311, 341, 348 Carey, W . F., 87, 114 Carlinfnnti, E. J., 35, 72 Carlisle, H. N., 289, 347 Carrington, S. P., 284, 321, 322, 323, 342, 345 Carver, R. K., 306, 344 Cathou, R. E., 17, 28 Cebm, J. J., 12, 28, 3 2 ( 3 ) , 3 3 ( 3 ) , 71 Cerottini, J. C., 124, 126, 128, 134, 135, 136, 137, 141, 142, 176, 186 Champness, D. L., 323, 334, 335, 342 Chance, B., 142, 187 Chapman, D., 82, 110, 113 Chapuis, B., 120, 124, 125, 126, 128, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 176, 186, 190 Charlwood, P. A., 14, 28 Chase, M. W., 172, 186, 196, 200, 210, 211, 213, 217, 221, 226, 237, 248, 249, 261, 262, 263 Chavin, S. I., 298, 299, 342 Chesebro, B., 3, 19, 21, 22, 2.3, 25, 26, 28, 30 Chesterman, F. C., 258, 262 Chin, W., 276, 306, 342, 345 Chinitz, A,, 61, 62(72), 71(72), 73 Christophers, S. R., 278, 342 Chn, E. H . Y., 157, 160, 187 ChutnL, J., 180, 188

353

C i n d er , B., 7, 8, 9, 21, 28, 37, 56(24), 72 Clark, D. S., 180, 187 Clark, H. C., 289, 342 Clifford, P., 157, 160, 181, 187, I89 Cline, M . J., 140, 174, 187 Clyde, D. F., 276, 340 Coates, W. A., 325, 344 Coatney, G. R., 276, 304, 305, 306, 342, 344, 345, 346, 348 Cochrane, C. G., 336, 342 Cochrum, K. C., 180,187 Coffin, G. S., 309,325,342 Coggeshall, L. T., 279, 283, 285, 303, 304, 308, 309, 310, 319, 320, 321, 324, 328, 342, 343, 345 Cohen, S., 1, 9, 11, 18, 28, 98, 113, 283, 284, 320, 321, 322, 323, 335, 342, 345 Cohn, M., 32(8), 72 Cohn, Z. A., 154, 185 Collins, W. E., 305, 306, 342 Collste, L., 145, 159, 162, 167, 190 CoIten, H. R., 97, 113 Condoulis, W. V., 238, 244, 261 Connell, D. I., 131, 185 Connor, D. H., 293, 294, 346 Conran, P., 296, 298, 336, 349 Contacos, P. G., 276, 305, 306, 340, 342, 345, 348 Converse, J. M., 200, 203, 216, 218, 222, 225, 226, 228, 231, 256, 258, 259, 264, 265 Cook, L., 299, 342 Cooke, R. A,, 226, 262 Cooper, M. D., 176, 186, 248, 257, 258, 263, 265 Cooper, N. R., 154, 187, 305, 342, 343 Cooper, W . C., 304, 346 Cooperband, S. R., 166, 188 Corradetti, A., 285, 297, 329, 342 Corson, J. M., 258, 266 Corwin, R. M., 333, 342 Cotran, R. S., 121, 148, 149, 152, 189 Coiilston, F., 281, 282, 344 Covell, G., 324, 342 Cover, B., 297, 349

354

AUTHOR INDEX

Cox, F. E. G., 280, 313, 316, 325, 330, 332, 342 Cox, H. W., 285, 289, 298, 333, 342 Craig, J. M., 206, 248, 263 Craige, B., 326, 344 Crome, P.,291, 342 Cropper, J., 292, 307, 342 Culbertson, J. T., 269, 342 Cullen, J., 103, 114 Culling, C. F. A., 122, 126, 129, 131, 132, 135, 145, 192 Cummings, M. M., 173, 187, 203, 208, 228, 262,263 Curtain, C. C., 323, 334, 335, 342

D Dacie, J. V., 94, 112, 113, 115, 175, 187 Dalmasso, A. P., 94, 99, 109, 113, 114, 154, 190 Dalton, W. O., 14, 28 DAntonio, L. E., 298, 304, 305, 342 Darzynkiewicz, Z., 163, 164, 187, 191 Das Gupta, B. M., 320, 345 Dausset, J,, 200, 216, 222, 226, 231, 256, 265 Davey, M. J., 149,187 David, J. R., 118, 139, 163, 167, 173, 175, 187, 192, 197, 198, 199, 200, 229, 230, 231, 232, 233, 234, 235, 236, 241, 246, 256, 261, 262, 264 Davidsohn, I., 35, 72 Davies, D. R., 11, 30 Davis, B. D., 328, 343 Davis, W. C., 180, 187 Davis, W. L., 304, 347 Deane, L. M., 276,343 Deane, M. P., 276, 343 DeBonaparte, Y.,255, 262 Decreusefond, C., 34( l l ) , 72 Deegan, T., 299, 343 Defendi, V., 177, 186 de la Chapelle, A., 157, 191 Delorme, E. J., 180, 182, 185 Demarest, C. R., 325, 344 Denhain, S., 180, 187 De Petris, S., 336, 337, 340 Desowitz, R. S., 297, 312, 343, 347 Dienatl, F., 130, 186 Diggs, C. L., 298, 299, 343

Di Giusti, D. L., 324, 349 Dilley, D., 332, 334, 345 Dineen, J. K., 35, 72, 175, 187 Dingle, J. T., 84, 113 Dixon, F. J., 333, 336, 343, 348 Doebbler, T. K., 129, 191 Donatien, A, L., 280, 347 Dorrington, K. J., 17, 28 Doty, P., 2, 29, 39(31), 41(31), 72 Dougherty, R. M., 84, 113 Dourmashkin, R. R., 18, 29, 81, 83, 84, 85, 86, 88, 89, 92, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 111, 113, 114 Draper, L. R., 289, 343 Draskoci, M., 176, 189 Dray, S., 235, 236, 237, 239, 246, 263, 265, 266 Dreyer, W. J., 32( 6 ) , 71 buhin, I. N., 295,343 Dulaney, A. D., 295, 304, 311, 315, 343, 347 Dunionde, D. C., 119, 187, 295, 329, 346 Dupuy, J. M., 237,238,262 Dutton, R. W., 118, 159, 172, 187, 190, 238, 244, 262

E Eagle, H., 160, 187 Easty, G. C., 6, 28 Eaton, M. D., 297, 298, 303, 304, 308, 309, 310, 319, 320, 328, 332, 342, 343 Edelman, G. M., 8, 12, 29, 30, 32(7), 72 Edington, G. M., 275, 276, 336, 337, 340, 343 Edozien, J. C., 319, 323, 343 Edsall, J. T., 14, 29 Eibl, M., 130, 186 Einheber, A., 329, 346 Eisen, H. N., 173, 188, 196, 247, 262, 263 Elek, S. D., 8, 29 Eliot, T. S., 288, 345 Elkins, W. L., 179, 187 Ellein, K. A. O., 124, 187 Epstein, W. L., 210, 262 Evans, C. A,, 135, 13G, 188

355

AUTHOR INDEX

Evans, C. B., 305, 306, 340, 345, 348 Evans, R. T., 83, 87, 113 Everett, N. B., 169, 188

F Fabiani, G., 292, 304, 318, 320, 343, 348 Fairley, G. H., 258, 2.59, 261 Farmer, J. N., 331, 345 Farthing, C. P.,111, 113 Fazio, M., 207, 208, 251, 262 Feagin, 0. T., 291, 319 Fefer, A., 131, 135, 136, 188 Feinberg, A. R., 207, 261, 265 Feinberg, S. M., 207, 261, 265 Feinstein, A., 5, 7, 8, 9, 15, 18, 21, 22, 29 Feldnian, J. D., 17.3, 179, 180, 187, 190, 192 Feldnian, M . , 122, 124, 126, 169, 170, 186 Felton, F. G., 211, 212, 265 Felts, W. R., 222, 246 Ferber, E., 105, 114, 144, 167, 187 Fernandes, hl. V., 121, 129, 131, 176, 189 Ferreira-Neto, J., 276, 343 Ferris, D. H., 298, 302, 311, 332, 333, 348 Fife, E. H., 298, 304, 305, 342, 343 Finch, J . T., 3, 29 Findlay, G. M., 288, 330, 343 Fink, M. A., 35, 72 Finn, J., 38, 43, 72 Finstacl, J., 248, 253, 263 Fireman, P., 232, 238, 262 Firschein, I. L., 169, 188 Fischer, H., 99, 105, 114, 122, 144, 157, 160, 161, 164, 167, 170, 185, 187, 190 Fish, A. J., 211, 262 Fisher, A. B., 313, 348 Fisher, D. B., 164, 167, 187 Fisher, J. P., 226, 262 Fishman, M., 139, 187, 234, 236, 262 Fitch, F., 289, 349 Fleischer, R. A., 75, 114, 124, 126, 188 Fletcher, K. A., 276, 288, 343, 344 Foerster, J., 53, 55(63), 73

Fogel, B. J., 288, 305, 342, 343 Foker, J . E., 180, 187 Foner, A., 318, 338, 340 Ford, W . L., 139, 187 Foster, W . D., 334, 347 Fox, M., 161, 186 Frangione, B., 26, 29 Frank, M. M., 18, 29, 89, 90, 91, 99, 111, 113 Franklin, E. C., 112, 113, 198, 199, 2008, 229, 230, 231, 232, 233, 256, 264 F r c d n a n , S. O., 181, 188, 211, 226, 259, 262, 263 Freund, J., 316, 324, 327, 328, 343, 348 Friedman, H., 120, 187 Fudcnberg, 11. H., 153, 180, 187, 189 Fujiknwa, K., 87, 113 Fulchiron, G., 304, 318, 320, 343, 348 Fulginiti, V., 254, 256, 263 Fulton, J. D., 288, 297, 303, 316, 328, 330, 335,343,348 Furcolow, M. L., 201, 262

G Gabriele, G. de, 293, 343 Gabrielsen, A. R., 248, 263 Gaertner, R. A., 291, 349 Gail, K., 303, 343 Gajdusek, D. C . , 323, 334, 342 Gally, J. A,, 8, 29, 3 2 ( 7 ) , 72 Canimage, K., 283, 284, 344 Garnharn, P. C. C., 269, 270, 272, 273, 275, 276, 280, 282, 284, 292, 343, 344, 347 Gaivey, J. S., 291, 344 Carvin, J. E., 209, 265 Gasser, D. L., 36, 65, 71(23), 72 Garigns, J. M., 258, 262 Griman, Q. M., 275, 277, 297, 344, 345, 346 Cell, P. G. H., 14, 29, 162, 173, 187, 191 George M., 234, 263 Gershoff, W . N., 41(39), 72 Gerughty, R. M., 142, 180, 187 Gewurz, H., 83, 87, 88, 98, 113 Giglioli, G., 277, 335, 344 Gilchrist, C., 258, 262 Gill, T. J., 111, 39(31), 41(31, 3 9 ) , 72

356

AUTHOR INDEX

Gills, H. M., 276, 319, 323, 335, 343, 344, 345 Gingrich, W. D., 280, 326, 344 Ginsberg, H. S., 3, 30 Ginsburg, H., 122, 124, 126, 169, 170, 186, 187, 188 Githens, J. N., 254, 256, 263 Gitlin, D., 206, 232, 238, 248, 257, 262, 263 Glauert, A. M., 81, 84, 108, 113, 114 Glynn, A. A., 87, 111, 113 Glynn, L. E., 176, 186 Goble, F. C., 329, 344 Goddard, P., 283, 284, 344 a t z e , O., 99,114 Gold, P., 181, 188, 259, 263 Goldberg, B., 75, 76, 91, 114, 124, 126, 188 Goldstein, D. J., 11, 29 Goldstein, F., 309, 320, 346 Goldstein, I. J., 165, 188 Good, R. A., 161, 176, 180, 186, 187, 190, 203, 204, 205, 206, 207, 208, 210, 212, 221, 222, 226, 237, 238, 248, 249, 251, 253, 257, 258, 262, 263, 265, 266 Goodner, K., 315, 344 Gordon, C. S., 291, 348 Gordon, H. H., 304,345 Gordon, J., 163, 188 Gordon, R. M., 318, 341 Gordon, R. S., 222, 264 Corer, P. A., 33, 72 Gorman, J. G., 323, 334, 335, 342 Gottlieb, P. M., 210, 266 Gould, D. J., 275, 344 Gould, R. G., 104, 114 Govaerts, A., 121, 127, 128, 135, 188 Gowans, J. L., 138, 139, 177, 179, 187, 188, 246,263 Grasbeck, R., 157, 191 Graham, J. M., 104,114 Granger, G. A., 120, 121, 125, 128, 139, 166, 167, 177, 179, 188, 189, 193, 244, 263 Grant, P. T., 299, 342 Gray, W. R., 32( 6 ) , 71 Greaves, M . F., 161, 188, 192 Green, C., 104, 114 Green, H., 75, 76, 91, 114, 124, 126, 188

Green, I., 40(35), 42(35), 45(35, 43, 44, 45), 46, 48(35, 43, 47, 50, 52), 49(35, 47), 50(52), 52, 53(63), 54, 55(35, 43, 50, 63, 64),56(43, 64), 71(35), 72, 73 Green, J. A,, 166, 188 Green, N. M., 6, 7, 9, 11, 14, 15, 16, 25, 26, 30 Greenberg, J., 276, 344 Greenwood, B. M . , 335, 344 Greville, G. D., 7, 29 Grey, H. M . , 17, 26, 28 Grossberg, A. L., 18, 30 Grossman, A., 18, 30 Gruber, M., 5, 29 Guberman, V., 315, 344 Guttman, R. D., 179,187

H Haber E., 17, 28 Haddad, Z. H., 232, 238, 262 Hadding, U., 99, 114 Hager, E. B., 258, 266 Hagerman, J. S., 104, 114 Hall, C. E., 2, 29 Hall, J. G., 179, 180, 182, 185, 187, 188 Hall, P. J., 287, 298, 299, 300, 301, 302, 303, 316, 317, 332, 333, 336, 345, 346, 349 Halpern, B. N., 291, 344 Hamburger, Y., 328, 349 Hamilton, L. D. G., 180, 182, 185 Hamlin, J., 39( 27), 72 Hampers, C. L., 258, 266 Hanin, A,, 130, 192 Harber, L. C., 211, 263 Harding, B., 142, 145, 148, 150, 190 Hardy, C. L. S., 298, 299, 316, 345 Hardy, D., 35, 72 Hardy, D. A., 171, 178,188 Harris, J. E., 163, 188 Harris, R. J. C., 84, 113 Harris, S., 226, 263 Harris, T. N., 226, 263 Harvey, J. J., 258, 262 Habek, M., 146, 180, 182, 186, 188 Hashimoto, Y.,128, 188 Hathaway, W. E., 254, 256, 263 Hattler, B. G., Jr., 209, 252, 254, 257, 262, 263

357

AUTHOR INDEX

Haughton, G., 160, 188 Haiipt, I., 99, 114, 144, 167, 187 Hauser, R. E., 85, 11t5 Havemann, K., 214, 250, 261 Hawkey, C . M., 275, 349 Hawking, F., 277, 283, 284, 344 Haxby, J. A., 84, 108, 114 Haxthausen, H., 210, 263 Hedberg, H., 130, 188 Heidelberger, hI., 304, 325, 326, 332, 334, 344, 346 Heilmnn, D. H., 164, 170, 190 Hcllstroni, I., 123, 131, 132, 133, 135, 136, 138, 159, lG0, 161, 182, 188 Hellstram, K. E., 131, 132, 133, 135, 136, 138, 159, 160, 161, 182, 188 Helmstein, K., 131, 133, 186 Hendrickse, R. G., 276, 335, 336, 337, 340, 344, 347 Henncy, C. S., 14, 17, 29 Heppncr, C.. H., 131, 135, 136, 188 Heremans, J. F., 232, 261 Herrmann, O., 295, 344 Hersh, E. M., 163, 188, 191 Herzenberg, L. A., 31(1), 60(71), 62 (71), 71, 73 Hesselbrock, W. B., 304, 345 Hewel, B., 201, 262 Hickman, R. L., 269, 323, 347 Higlnnan, W., 8 , 29 Hill, G. J., 306, 340 Hills, L. A,, 285, 289, 297, 310, 311, 316, 329, 342 Hindle, J. A., 326, 344 Hirata, A. A., 124, 188 Hirsch, M. S., 258, 262 Hirschhorn, K., 152, 167, 169, 174, 186, I88 Hirschhorn, R., 167, 188 Hijglund, S., 3, 7, 8, 12, 18, 29 Holboi-ow, E. J., 176, 186 Holm, G., 119, 120, 122, 124, 125, 129, 130, 132, 133, 135, 145, 146, 147, 148, 149, 151, 152, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 171, 181, 186, 189, 191, 244, 26.3 Holmes, E. C., 181, 190 Holmes, K. C., 3, 29

Holt, P. J. L., 158, IS9 Holtzei, J. D., 130, 131, 173, 189 Hong, R., 256, 263 Hood, L., 32(6), 71 Home, R. W., 2, 3, 5, 6, 7, 28, 29, 30, 77, 84, 85, 108, 113 Hoiiba, V., 334, 336, 337, 340, 344 House, V., 311, 315, 343 Howard, A., 145, 190 Howard, E. M., 330, 343 Howatson, A. F., 7, 8, 21, 28, 85, 114 Hoyer, L. W., 98, 112, 114 Hsn, D. Y. hl., 277, 344 H5U, K. C . , 2, 28 Huber, C. L., 82, 114 Huber, II., 153, 189 Hii(lgins, P. C . , 173, 187, 208, 262 Fluff, C . G., 275, 281, 282, 344 IImniieler, K., 7, 8, 29 IIiimphrey, J. H., 18, 29, 76, 81, 83, 85, 86, 88, 89, 90, 92, 94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 111, 112, 113, 114, 196, 263, 291, 341 Humphrey, R. L., 11, 29 Hotcliin, P., 181, 189 Hutt, M. S. R., 293, 294, 335, 344, 346 Hutton, E. L., 280, 347

I Ilndi, A,, 297, 342 Inai, S., 87, 113 Intlerbitzin, T., 173, 189 Ingrahain, J. S., 291, 344 Ingrain, R. L., 306, 344 Ipsen, J., 35, 72 Irins, J. J., 5, 30 Tshizaka, K., 14, 17, 29 Israel, H. L., 204, 213, 249, 250, 252, 263, 266 Ihaneski, M., 175, 189 Ivnnyi, J,, 182, 186 Iyer, R. N., 165, 188

J Jacobs, H. R., 327, 344 Jncol~son,L. O., 289, 349 James, A. T., 104, 114 James, K., 151, 158, 161, 165, 189, 190

358

AUTHOR INDEX

James, S. P., 280, 344 Jandl, J. H., 121, 148, 149, 152, 189, 334, 344 Janeway, C. A., 206, 248, 257, 263 Janjic, M., 17G, 189 JankoviE, B. I)., 175, 176, 189 Jaton, J-C., 65(77, 78, 79), 73 Jeffery, G. hL, 280, 305, 306, 323, 324, 342, 344 Jensen, K., 203, 228, 263 Jerusalem, C., 277, 280, 344 Johanovskf, J., 118, 139, 163, 172, 173, 189, 192 Johansson, B., 161, 189 JonQk, J., 180, 188 Jones, R., 326, 344 Jureziz, R. E., 235, 236, 237, 263, 266

K Kabat, E. A., 9, 14, 29 Kall=&n,B., 121, 129, 130, 135, 176, 185, 186, 188 Kaku, J., 175, 192 Kakulas, B. A., 129, 189 Kaliss, N., 136, 189 Kantor, F. S., 41(36), 42(36), 43, 4 5 (361, 72 Kaplan, M. H., 334, 335, 347 Karakoz, I., 146, 180, 182, 186, 188 Karlin, L., 18, 29 Karush, F., 173, 189, 247, 263 Karzon, D. T., 253, 265 Kashiba, S., 87, 113 Katclialski, E., 39( 29), 72 Katz, M., 229, 262 Kaunders, O., 319, 344 Kay, J. E., 164, 167,189 Keel, A., 181, 189 Keller, R., 105, 114 Kellumn, M. J., 248, 263 Kelly, A,, 293, 294, 346 Kelly, W. D., 207, 208, 248, 249, 251, 253, 258, 263 Kelus, A. S., 14, 29, 32 ( 3 ) , 33( 3 ) , 71 Kemp, C. L., 85, 114 Kempe, C. H., 253, 254, 256, 257, 263 Kendrick, L. P., 276, 344 Kent, J. F., 304, 346

Kermack, W. O., 299, 342 Ketcham, A. S., 181, 190 Kibukamusoke, J. W., 335, 33G, 343, 349 Kidson, C., 323, 334, 335, 342 Kieler, J., 123, 192 Kiely, J. M., 124, 187 Kies, M. W., 175, 192 Killander, D., 181, 189 Killebrew, L., 211, 212, 265 Kimball, H. R., 278, 342 Kingsley Smith, B. V., 8, 29 Kinsky, C. B., 108, 114 Kinsky, S. C., 84, 108, 114 Kirkpatrick, C. H., 208, 215, 263 Kirman, D. J., 210, 261 Kirrick, S., 166, 188 Kishimoto, T., 18, 23, 25, 29, 30 Kitchen, S. F., 280, 283, 320, 325, 334, 341, 345 Kite, H., Jr., 129, 191 Klein, E., 123, 125, 181, 189, 190, 192 Klein, G., 12.0, 124, 125, 128, 131, 157, 160, 181, 187, 189, 190, 192 Kligler, I. J., 304, 345 Kligman, A. M., 210, 261 Kliiver, C., 288, 348 Knapp, S., 39(27), 72 Knisely, M. €I., 288, 345 Kniskern, P. J., 236, 265 Knowles, R., 320, 345 Knox, K. W., 88, 10.3, 114, 115 Kochnian, M., 7, 30 Kohlschiitter, A., 144, 167, 187 Kolb, W. P., 125, 166, 167, 188, 189, 244, 263 Koldovsky, P., 182, 186 Kolin, A., 172, 189 Konstansoff, S. W., 325, 345 Koprowski, H., 121, 129, 131, 176, 189 Koshlnnd, M. E., 32(4), 33(4), 71 Koskimies, O., 120, 124, 128, 131, 192 Koiintz, S., 180, 187 Kourilsky, F. M., 181, 189 Kretschmnr, W., 277, 303, 324, 343, 344, 345 Krier, J., 332, 334, 345 Krupey, J., 259, 263 Kiilczycki, A., 17, 28

359

AUTHOR INDEX

Kumm, H.W., 308,310,319,,342 Knpper, W. H., 3.34, 345 Kurvin, S. F., 305,306,345,318

Ling, N. R., 120,152,157,158,159,1G1, 171,178,188, 189 r,inscott, w.D., 951, 114, 153, 189, 191 304,345 Lippincott, S. W., Lo lhidio, A. F., 121,148,149, 152,189 L Loewi. G., 124, L30, 135,145,148, 157, Ladda, R., 278,345 173, 185, 190 Lafferty, K.J., 8,21,29 Longenecker, B. M., 331,345 Lagunoff, D., 169,187 Lovelock, J. E., 104,114 LaIli, F., 278,345 Lowcnstein, L., 159,185 Lamb, D.L., 207,208, 251,26,3 Lowcy, S., 17,30 Lamelin, J. P., 48(49), 53(63), 55(49, Liibnroff, D.M., 173,189 63).,. 73 Lucas, Z.J . , 254,257,262 Lamin, M.E.,17,29 Lucy, J. A., 81,84,108,113, 114 Lancefield, R. C., 217,263 Lrindgren, G., 120, 121, 122, 128, 134, Landsteiner, K., 49(53), 71, 73, 196, 140, 145, 157, 159, 160, 162, 163, 248,263 164, 165,167,190 Law, L. w., 330,345 Lrinn, J. S., 306,345 Lawrence, H. S., 118,167.187, 189, 196, Lrise, S. A., 84, 108,114 197, 198, 199, 200, 201, 202, 203, L ~ I I SP., A., 3,5,28

204, 205, 207, 210, 211, 212,216, 217, 218, 219, 220, 222, 223, 224, 225, 226, 228, 229, 230, 231, 232, 233, 234, 235, 237, 238, 240, 241, 242, 243, 245, 246, 247, 248, 249, 250, 252, 253, 256, 257, 258, 259,

261, 262, 263, 264, 265, 266 Lay, W. H., 153,18.9 Lebacq, E.,213,214,250,264 Lebacq, E.G . , 232,261 Leberman, R., 3,29 Lebowitz, A., 197,245,246,264 Lee, S., 180, 192 Lehner, W., 303,343 Lennox, E. S., 32(8), 36, 72 Leon, M. A,, 165,191 Leventhal, B.G., 163,191 Levin, O., 18,29 Levine, A. S., 85,114 I,evine, B. B., 41(40), 42(40), 45(40, 44,46),46, 48(51,52),49, SO, 72, 73 Levine, E. M., 160, 187 Levine, H., 51(58), 55(58), 73 Lewis, M.R., 118,191 Lewis, S. M., 291,346 Lifschitz, M.,295,344 Lilly, F.,65, 73, 139,191 Lindner, R., 276,344

M McBride, R. A,, 177,190 McCIuskey, J. W., 173,190 McCluskey, R. T., 173,190 hlcCullagh, P. J., 139,187 hlcCullorigh, N.B.,206,222,249,264 AlcDevitt, €I. O., 31(1), 56, 57(67, 68, 69), 58, 59, GO(70, 71), 61, 62(71, 72,73),63, 65(78,79), 71, 73 MncDonald, G., 279,345 hlcFadzean, A. J. S., 289,345 McFnrlnnd, W.,1G4, 170,190 hlcGhee, R. B., 332,333, 342, 345 MeGregor, D. D.,138, 177, 179, 188, 246, 263 McGregor, I . A., 279,283,284,287,292,

298, 299, 300, 301, 302, 303, 306, 307, 316, 317, 318, 320, 321, 322, 323, 332, 33.3,335, 336, 342, 34.5, 346, 348, 349 Macintosh, D. M., 334,347 McIntyre, P. A., 3'34, 335,347 Mackaness, G . B., 175,190, 252,264 McKee, R. W., 277,345 MacLennan, I. C . M., 124,130,135,142, 145,148,150,157,190 hlaegraith, B . G., 288, 299, 30.3,335, 343, 345

360

AUTHOR INDEX

Magratlr, J. M., 35, 72 Mahoney, D. F., 298, 304, 313, 315, 316, 328, 345 Maier, J., 279, 324, 345 Main, J. M., 259, 265 Makari, J. G., 295, 345 Malchow, H., 122, 157, 160, 161, 164, 170, 185, 190 Malmgren, R. A., 181, 190 Mandy, W. J., 70, 74 Manni, J. A., 148, 154, 155, 156, 190, 191 Mannick, J. A., 142, 180, 186, 234, 236, 261 Manwell, R. D., 280, 309, 320, 346 Marble, A., 304, 345 Marsden, P. D., 293, 294, 346 Marsh, G. W., 291,346 Marshall, C. E., 311, 341 Marshall, W. H., 197, 200, 241, 242, 243, 264 Martin, C. M., 206, 222, 249, 264 Martin, L. K., 329, 346 Masek, B., 283, 348 Matsumoto, S., 18, 29 Matthews, B. W., 11, 30 Mauel, J., 119, 120, 122, 123, 124, 125, 126, 128, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 176, 186, 190 Maurer, P. H., 39(30), 40, 41, 42, 43, 48(33), 57(38), 72, 222, 227, 228, 265, 291, 348 Mayer, M. M., 14, 29, 76, 90, 93, 114, 304, 325, 326, 332, 334, 344, 346 Meagher, M. J., 275, 344 Medawar, P., 136, 177, 186, 258, 259, 261, 265 Mehlitz, D., 304, 347 Mellema, J. E., 5, 29 Menon, K. P., 328, 347 Mercer, E. H., 6, 28 Mergenhagen, S. E., 83, 87, 88, 98, 113 Merler, E., 18, 29 Merrill, J. P., 258, 266 Metaxas, M. N., 201, 265 Metaxas-Biihler, M., 201, 26.5 Metcalf, D., 330, 346

Metzger, H., 1, 17, 18, 23, 25, 28, 29, 30 Meuwissen, 13. J., 161, 190 Meuwissen, J. H. E. T., 298, 299, 347 Miescher, P. A., 248, 253, 263 Mihaesco, C., 18, 25, 29 Mikulska, Z. B., 131, 185 Miles, P., 39(27), 72 Milgroin, F., 171, 185 Millar, J. W., 201, 202, 203, 210, 218, 222, 224, 228,265 Miller, E., 236, 266 Miller, F., 1, 17, 23, 29, 30 Miller, J. F. A. P., 138, 139, 190, 258, 2&5, 330, 346 Miller, L. L., 291, 346 Miller, M., 279, 346 Mills, J. A., 162, 173, 190 Milne, C. M., 87, 113 Milstein, C., 1, 9, 11, 18, 26, 28, 29, 32 (51, 71 Ming, S. C., 125, 190 Minowada, J., 181, 189 Mirsky, A. E., 164, 191 Mishell, R. I., 125, 172, 187, 190 Mitchell, G. F., 139, 190 Mitchison, N. A., 139, 190, 237, 2.47, 258, 265 Moberger, G., 131, 133, 186 Modolell, M., 144, 167, 187 Mody, N. J., 334, 335, 347 Moller, E., 121, 122, 126, 128, 132. 135, 138, 157, 160, 161, 162, 190 Moller, G., 120, 121, 122, 132, 145, 152, 157, 159, 160, 162, 163, 164, 165, 187, 174, 182,188, 190 Mohan, B. N., 281, 346, 347 Mohr, J. A., 211, 212, 265 Mollison, P. L., 112, 114, 291, 342 Moon, A. P., 269, 323, 347 Moon, H. D., 119, 122, 128, 131, 132, 133, 135, 142, 143, 180, 187, 192, 244, 265 Moore, G. E., 259, 265 Moorhead, J. F., 164, 170, 190 Morgan, T. E., 82, 114 Morgenfeld, M. C., 255, 262

361

AUTHOR INDEX

Morton, D. L., 130, 132, 133, 181, 190, 192 Mosko, M. M., 228, 231, 232, 233, 261 Mosna, E., 320, 346 Mosolov, A,, 130, 192 Most, II., 281, 325, 346 Mouton, D., 34( I l ) , 72 Mozes, E., 65(78, 79), 73 Muchmore, H. G., 211, 212, 265 Mudd, J. B., 299, 347 hliiller, B., 7 1 ( 8 1 ) , 74 Mueller, G. C., 164, 167, 187 MiilIer-Eberhard, H. J., 16, 30, 76, 94, 98, 99, 109, 110, 112, 113. 114, 148, 153, 154, 155, 156, 187, 189, 190, 191 Muftuoglu, A. U., 208, 251, 265 Mukeijee, S., 305, 308, 328, 346 Mnlligan, H . W., 280, 281, 288, 295, 320, 324, 329, 346, 347, 348 Munder, P. G., 105, 114, 144, 167, 187 Munn, E., 18, 21, 22, 29 Murray, J. E., 258, 266 Muschel, L. H., 87, 114

N Nadel, E. M., 276, 344 Nadler, S . H., 259, 265 Najarian, J. S., 173, 179, 187, 190 Naspitz, C. K., 157, 190 Naylor, D., 9, 28 Naysmith, J. D., 161, 190 Nazario, R. C. R., 277, 346 Nelson, C. A., 1, 9, 14, 30 Nelson, D. S., 153, 190 Nelson, E. L., 291, 349 Nelson, R. A., 97, 114, L53, 191 Nelson, W., 289, 349 Nelson, W. E., 201, 262 NBmec, M., 180, 188 Neumann, H., 319, 346 Neurath, H., 1, 6, 30 Neveii, T., 174, 191 Nicol, W. D., 324, 342 Nilsson, H., 165, 191 Nilsson, U.R., 155, 191 Nishimura, S . , 87, 113 Nishioka, K., 99, 114, 153, 191

Nisonoff, A., 2, 6, 29 Noelken, M. E., 1, 9, 14, 30 Nolan, B., 258, 266 Nordman, C. T., 157, 191 Nouza, K., 180, 188 Nowcll, 1’. C., 159, 193 Nussenzweig, R. S., 281, 325, 329, 346 Nussenzweig, V., 153, 189

0 O’Connell, C. J., 253, 265 Oertelis, S., 8, 21, 29 Ojecla, A., 41(36, 40), 42(36, 40), 43, 45( 36, 40, 46), 49(40), 72, 73 O’Kane, D. J., 165, 192 Old, L. J., 139, 185, 191 Oliveira-Lima, O., 223, 265 Oncley, L., 1, 6, 30 Onoue, K., 18, 25, 29, 30 Oppenheim, J . J., 152, 162, 163, 173, 191 Oriol, R., 27, 28 Orton, C., 281, 346 Osoba, D., 138, 190, 258, 265, 330, 346 Ovary, Z., 112, 113 Owen, C. A., 124, 187 P

Packalkn, Th., 130, 132, 135, 138, 142, 145, 148, 150, 191, 192, 193 Padgett, F., 85, 114 Paertan, J., 205, 261 Page, A., 205, 266 Palmer, C . E., 201, 262 Pampana, E. J., 268, 346 Pappagianis, D., 201, 202, 203, 210, 218, 222, 224, 228,265 Pappenheimer, A. M., Jr., 173, 192, 19G, 197, 204, 205, 220, 222, 223, 227, 240, 241, 246, 247, 264, 26<5,266 Paque, R. E., 236, 265 I’aradisi, E. R., 255, 262 Pardoe, G., 2, 30 Parkinson, D., 334, 342 Parrot, F. C., 280, 347 Paterson, P. Y., 175, 176, 187, 191, 235, 262 Patnode, R. A., 173, 187, 203, 228, 263

362

AUTHOR INDEX

Polley, M. J., 153, 154, 189, 191 Patterson, R., 207, 261 Paul, W. E., 40( 35), 42( 35), 45( 35, 43, Porter, H., 205, 865 44, 45), 46, 48( 35, 43, 47, 49, 52), Porter, R. R., 1, 11, 12, 28, 291, 341 49( 35, 47), 50(52), 52, 54( 35, 43), Prehn, R. T., 181, 191, 259, 265 55(35, 43, 49, e l ) , 56(43, G G ) , 71 Prendergast, R. A,, 179, 191 Pressman, D., 18, 30 (35), 72, 73 Payne, S. N., 81, 88, 101, 102, 103, lM, Pringle, G., 2G8, 346 Prioleau, W. H., 181, 189 105, 114 Pekkek, J., 118, 139, 163, 172, 173, 189, Prout, C., 326, 344 Pruzansky, J. J., 207,261 192 Pryor, D. S., 293, 346 Peltre, G, 71(81), 74 Pullman, T. N., 326, 344 Penlioet, E., 7 , 30 Pulvertaft, R. J. V., 164, 191 Penington, D. G., 293, 343 Purba, S., 303, 343 Pereira, H. G., 4, 30 Perey, D. Y. E., 237, 238, 262 PerImann, G. E., 217, 263 Q Perlmann, H., 120, 124, 125, 128, 130, 131, 132, 135, 138, 142, 145, 146, Quastel, J. H., 124, 186 148, 149, 150, 152, 154, 155, 156, Quigley, A., 130, 133, 193 157, 158, 160, 161, 162, 163, 164, Quinn, V. A., 35, 7 2 190, 191, 192, 193 Perlniann,,.'l 119, 120, 122, 124, 125, R 128, 130, 131, 132, 133, 135, 138, 142, 145, 146, 147, 148, 149, 150, Rabinowitz, Y., 154, 191 151, 152, 154, 155, 156, 157, 158, Rajewsky, K., 39, 71(28, 81), 72, 74 159, 160, 161, 162, 163, 164, 165, Raniseier, H., 163, 167, 175, 178, 191, 166, 171, 180, 181, 182, 186, 189, 192, 252, 265 190, 191, 192, 193, 244, 263 Rapaport, F. T., 200, 201, 202, 203, 205, Pernis, B.,32( 3), 33(3), 71 210, 216, 218, 222, 224, 225, 226, Peters, W., 277, 346 228, 231, 234, 252, 256, 257, 258, Peterson, R. D. A,, 248, 257, 258, 263, 259, 264, 265 265 Rapp, H. J., 91, 96, 97, 98, 99, 112, 113, Pewny, W., 313, 346 114 Phillips, M. E., 131, 191 Ray, J. C . , 308, 328, 346 Phillips, R. S., 289, 293, 295, 310, 311, Razzak, M. A., 291, 349 316, 329, 341, 346 Reddy, S., 276, 344 Pierce, A. E., 269, 275, 292, 344 Redington, B. C . , 298, 304, 313, 315, Pierce, G. E., 131, 133, 135, 136, 138, 316, 328, 345 182, 188 Redmond, W. B., 326, 346 Pinchuck, P., 40, 41, 42, 43, 48( 33), 57 Rees, R. J. W., 255, 258, 262, 265 Rein, C . R., 304, 346 (38), 72 Pink, J. R. L., 26, 29 Reisner, A. H., 35, 72 Pisani, T. H., 316, 324, 328, 343 Remold, H. G., 167, 192 Pitney, W. R., 293, 346 Rhoades, E. R., 211, 212, 265 Plaut, M. E., 253, 265 Rich, A. R., 118, 191 Playfair, J. H. L., 35, 72, 161, 192 Richards, J. D. M., 293, 346 Pogo, B. G . T., 164, 191 Richards, W. H. G., 275, 281, 327, 329, PolBk, L., 35, 72, 174, 193 346,348, 349 Poljak, R. J., 11, 29 Richardson, P., 309, 347

363

AUTHOR INDEX

Richter, M., 157, 190 Ridley, D. S., 275, 349 Riflcind, R., 2, 30 Rigler, R., 163, 164, 189 Ringertz, N . R., 163, 164, 187, 191 Ristic, M . , 269, 288, 289, 298, 302, 311, 332, 333, 334, 342, 345, 347, 348, 349 Rohbins, J. H., 157, 191, 238, 265 Roberts, K. B., 241, 264 Robinson, J. P., 7, 30 Robson, M. J., 289, 349 Rodrigue, R., 323, 3.34, 342 Roitt, I. M., 161, 188, 192, 269, 275, 292, 344 Ron, N., 315, 349 Ronni, P. M., 175, 187 Rose, A. S., 326, 344 Rose, M. E., 161, 188 Rose, N. R., 129, 191 Rosen, F. S., 257, 263 Rosenau, W., 119, 122, 126, 128, 130, 131, 132, 133, 135, 140, 142, 143, 157, 164, 180, 187, 191, 192, 244, 265 Rossnn, R. N., 277, 280, 287, 323, 348, 349 Rosse, W. F., 81, 83, 84, 85, 92, 94, 105, 111, 113,114 Rotstein, J., 207, 248, 249, 251, 253, 258, 263 Rottlknder, E., 71(81), 74 Row, R., 314, 346 Rowe, A. J., 3, 5, 7, 8, 9, 15, 18, 26, 29, 30 Rowe, C. E., 104, 114 Rowe, D. S.. 303, 346 Rowe, W., 259, 26.5 Rowley, D., 35, 72, 07, 111, 114, 289, 346 Rowley, P. T., 297, 346 Roy, A. N., 305, 308, 328, 346 Ruble, J. A., 299, 347 Ruddle, N. €I., 159, 1G0, 162, 166, 171, 192, 244, 245, 265 Rudolf, H., 120, 125, 128, 132, 133, 134, 1.35, Is%, 137, 138, 140, 141, 142, 143, 176, 186, 190 Riihe, D. S., 304, 346

Rupec, M., 214, 250, 261 Russell, P. F., 268, 281, 346, 347 Rutter, W . J., 7, 30

S Saave, J. J., 312, 343 Sachs, L., 169, 188 Sadun, E. H., 269, 287, 303, 318, 320, 323, 325, 329, 341, 346, 347, 349 Salisbury, E . I., 333, 347 Salvin, S. B., 173, 192, 205, 224, 265,

266 Sanders, B. G., 32(G), 71 Sanderson, A. R., 124, 192 Saslaw, S., 289, 347 Scatcliard, G., 1, 6, 30 Scheibel, I. F., 33, 34, 35, 72 Schenkein, E. L., 328, 343 Schilling, C., 285, 347 Schilling, R. F., 299, 341 Schindler, R., 119, 122, 123, 128, 135, 137, 142, 176, 186, 304, 347 SchIange, H., 206, 212, 265 Schlosman, S. F., 51, 55(57, 58, 59, 64 ), 73, 173, 187, 192 Schmidt, L. H . , 330, 347 Schoenbecbler, M. J., 298, 304, 313, 315, 316, 328, 345 Schofield, F. D., 293, 294, 346 Schroecler, W. F., 288, 289, 342, 347 Schutze, H., 33, 72 Schulmnn, N., 289, 349 Scrutton, M. C., 5, 30 Scars, D. A., 911, 115 Seegal, B . C., 2, 28 Srki, T., 87, 11,3 SeIa, M., 39( 29, 32), 40(3 2 ) , 56, 57( 67, 68), 58, 65(77, 78, 79), 72, 73 Seligmann, M., 3, 18, “3, 24, 25, 29, 30 Sergent, E., 280, 347 Serri, F., 210, 261 Sewa, G., 84, 115 Shacks, S. J., 166, 188 Shnper, A. G . , 3.34, 335, 347 Shapiro, B., 3, 5 , 11, 30 Shnpiro, H., 3\32., 334, 345 Shnw, C. M . , 175, 192 Sheagren, J. N., 174, 192

364

AUTHOR INDEX

Sherman, I. W., 299, 347 Shields, C. E., 288, 343 Shin, H. S., 98, 113 Shortt, H. E., 282, 328, 347 Shreffler, D. C., 62(73, 74). 63, 73 Shute, P. G., 280, 347 Siddiqui, W. A., 297, 346 Silvers, W. K., 159, 177, 179, 186, 193 Simmons, E . L., 329, 348 Simnis, S., 49(53), 71, 73 Simonian, S. J., 41, 72 Simonsen, M., 177, 192 Simpson, R. W., 85, 115 Singer, I., 329, 330, 344, 347 Singer, S. J., 3, 5, 28 Singh, H., 308, 347 Singh, J., 308, 347 Sinton, J. A., 280, 283, 295, 308, 314, 346, 347 Sirchia, G., 94, 115 Siskincl, G. W., 52(61), 54(64), 55(61, 64), 56( 64,66), 73 Sjogren, H . O., 123, 188 Skamene, E., 180, 188 Skinner, J. C., 305, 306, 342 Slavin, R. G., 207, 209, 265 Slayter, H. S., 2, 6, 29 Small, P. A,, 17, 29 Smith, C . E., 201, 202, 203, 210, 218, 222, 224, 228, 265 Smith, J. K., 107, 115 Smith, R. T., 181, 189, 203, 206, 212, 238, 244, 248, 253, 261, 263, 266 Smith, S. J., 303, 346 Snell, C. D., 62(75), 64(75), 73 Sober, H. A., 51, 55(57, 59), 73 Sobey, W. R., 35, 72 Sodeman, W. A., 298, 299, 347 Solowey, A. C., 234, 252, 257, 265 Sommer, H. E., 316, 324, 327, 328, 343, 348 Soinmerville, T., 320, 329, 346 Somogyi, J. C., 308, 347 Sonak, R., 144, 167, 187 Sones, M., 204, 249, 250, 252, 266 Soothill, J. F., 336, 347 Sotirind&, D., 319, 347 Sovovii, V., 180, 188 Sparks, D. B., 207, 261

Spector, W. G., 173, 192 Spicer, E., 158, 189 Spira, D., 289, 297, 299, 315, 328, 347, 349 Spong, F. W., 180,192 Squires, W. L., 330, 347 Stadtman, E. R., 3, 5, 11, 30 Stanley, W. M., 6, 28 Stanworth, D. R., 2, 17, 29, 30 Stastny, P., 177, 192 Stauber, L. A., 309, 347 Steffen, C., 22, 30 Stein, B., 297, 312, 343, 347 Steiner, L. A., 12, 17, 28, 30 Steinmuller, D., 177, 180, 186, 192 Stembridge, U . A., 177, 192 Stern, K., 35, 72 Stiffel, C., 34( l l ) , 72 Stimpfling, J. H., 62(73, 75), 63, 64 ( 7 5 ) , 73 Stjernswiird, J., 157, 160, 181, 187, 189 Stolfi, R., 99, 115 Stone, M. J., 18, 25, 30 Stone, S., 48( 50), 55(50), 73 Stratman-Thomas, W. K., 277, 280, 283. 288, 295, 304, 341, 343, 345, 347 Streilein, J. W., 178, 191 Stuart, A. E., 168, 192 Stulbarg, M., 51(60), 55( 60), 73, 173, 192 Stupp, Y.,52(61), 55(61), 73 Sudo, H., 128, 188 Siissdorf, D. H., 289, 343 Sukernick, R., 130, 192 Sulzberger, M. B., 210, 261 Sumner, J. B., 165, 192 Sura, V . V., 130, 192 Suter, E., 175, 192, 252, 265 Sutherland, D. E. R., 248, 263 Sutliff, W. D., 304, 347 Svehag, S.-E., 3, 19, 21, 22, 23, 24, 25, 26, 28, 30 Svejcar, J., 118, 139, 163, 173, 192 Svet-Moldavsky, G. J., 130, 192 Swaininath, C. S., 320, 329, 346 Swett, V. C., 140, 174, 187 Swisher, S. N., 91, 115 Szenberg, A,, 157, 160, 161, 190

365

AUTHOR INDEX

Szilvassy, I. P., 332, 334, 345 Szur, L., 291, 346

T Takasugi, M., 123, 192 Taliaferro, L. G., 280, 289, 313, 320, 348 Taliaferro, W. H., 269, 278, 280, 283, 288, 289, 292, 295, 303, 310, 313, 314, 320, 329, 330, 341, 347, 348 Talmage, D. W., 208, 215, 263 Tanford, C. 1, 9, 14, 17, 28, 30 Targett, C. A. T., 297, 306, 316, 328, 348 Taylor, A., 88, 115 Taylor, H. E., 122, 126, 129, 131, 132, 135, 145, 192 Taylor, R. B., 331, 341 Tennenbauin, J. I., 207, 265 Terry, W. D., 11, 30 Tlieis, G. A., 54, 55(a ) ,56( 64), 73 Thomas, L., 118, 187, 197, 234, 235, 258, 261, 262, 266 Thoinson, K. J., 316, 324, 327, 328, 343, 348 Thor, D. E., 235, 236, 237, 239, 246, 263, 266 Thorbecke, C . J., 5 6 ( 6 6 ) , 73, 291, 348 Thumb, N., 205, 261 Tillett, W. S . , 203, 218, 225, 228, 258, 259, 264 Ting, I. P., 299, 347 Tobie, J. E., 305, 306, 340, 345, 348 Todd, C . W., 3 2 ( 2 ) , 3 3 ( 2 ) , 70, 71, 74 Todorovic, R., 298, 302, 311, 332, 333, 348 Toghill, P. J., 293, 346 Toinlinson, W . J., 289, 342 Torrigiani, G., 3 2 ( 3 ) , 3 3 ( 3 ) , 71, 161, 192 Townsley, H. C., 203, 228, 263 Trabold, N., 91, 115 Trager, W., 299, 347 Trautman, J. R., 174, 192 Trayanova, T., 130, 192 Trigg, P. I., 289, 310, 311, 316, 328, 329, 342, 338 Trowell, 0. A,, 120, 192 Trubowitz, S., 283, 348 Tsang, K. C . , 289, 345

Turk, J. L., 35(21), 72, 134, 172, 173, 174, 192, 193, 196, 200, 217, 221, 237, 266, 294, 348 Turner, K. J., 97, 111, 114 Turner, M. W., 298, 299, 300, 302, 303, 316, 317, 332, 333, 336, 345, 348 Tursi, A,, 161, 192 Tyan, M. L., 58, 59, 60 (70, 71), 62( 71 ), 73 Tyler, R. W., 169, 188

U Udeozo, I. 0. K., 269, 319, 323, 343, 347 Uhr, J. W., 152, 173, 174, 186, 192, 196, 205, 266 Ulrich, K., 123, 192 Ultmann, J. E., 291, 348 Unanue, E. R., 139, 192, 336, 348 Urbach, E., 210, 266 Urbach, F., 204, 249, 250, 252, 266 Utscinii, S., 14, 28 Utter, M. F., 5, 30

V Vainio, T., 120, 124, 128, 131, 192 Valentine, F. T., 197, 200, 219, 240, 241, 242, 243, 246, 264, 266 Vrtlentine, R. C.,2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14, 15, 16, 25, 26, 30 VanAlten, P., 161, 190 Van Bruggen, E. F. J., 5, 29 van Deenen, L. L. M., 84, 108, 114 Van Holde, K. E., 14, 28 Vannier, W. E., 98, 112, 114 Varco, R. L., 180, 187, 204, 205, 206, 207, 208, 210, 221, 222, 248, 249, 251, 253, 258, 263 Vargues, R., 304, 348 Vns, M., 159, 185 Vas, S. I., 124, 186 Vasdli, P., 5 0 ( 5 6 ) , 54(65), 73 Vaughan, J. H., 234, 263 Veach, S. R., 236, 266 Verliaegen, H., 213, 214, 250, 264 Verolini, F., 297, 342 Vogelhut, P. O.,108, 113 Voller, A., 275, 277, 280, 287, 293, 294,

366

AUTHOR INDEX

303, 305, 306, 307, 318, 323, 325, 328, 329, 342, 345, 346, 348, 349 Von Doenhoff, A. E., 288, 298, 304, 305, 342, 343 Von Sengbush, P., 36, 72 Vredevoe, D. L., 291, 349

Wilkinson, J. F., 285, 341 Wilks, N. E., 334, 335, 344, 347 Williams, G. R., 142, 187 Williams, K., 287, 298, 299, 300, 301,

302, 303, 306, 307, 316, 317, 318, 332, 333, 336, 345, 346, 349 Williams, R. C.,2, 30 W Williams, T. W., 125, 166, 188, 193 Williamson, J., 297, 298, 299, 316, 349 Wagland, B. M., 175, 187 Williamson, N., 158, 189 Wagner, H. N., 291, 349 Willoughby, D. A., 173, 174, 192, 193 Wahl, P., 12, 30 Wilson, D. B., 118, 122, 126, 128, 131, Waite, J. B., 206, 222, 249, 264 135, 138, 140, 141, 142, 159, 179, Waksman, B. H., 159, 160, 162, 166, 180, 186, 193, 238, 244, 266 171, 172, 173, 189, 192, 244, 245, Wilson, H., 306, 349 265 Wilson, R. E., 161, 186, 258, 266 Waldorf, D. S., 174, 192 Wilson, R. J. M., 287, 298, 299, 300, 301, Walker, H. A., 309, 347 302, 316, 317, 336, 349 Walker, P. J., 330, 349 Walter, A. W., 316, 324, 327, 328, 343, Wilson, W. E. C., 208, 215, 263 Winkler, G. F., 129, 140, 193 348 Ward, P. A., 167, 192, 296, 298, 336, 349 Winkler, K. C., 130, 131, 173, 189 Winn, H. J., 132, 193 Ward, R. A., 275, 344 Wissler, R. W., 289, 349 Wardlaw, A, C., 87, 115 Wolf, A,, 180, 187 Warr, 0. S., 304, 343 Warwick, W. J., 203, 205, 206, 212, 22G, Wolstencroft, R. A., 162, 173, 191, 295, 266 329, 346 Wasserman, J., 130, 132, 135, 138, 142, Woodruff, M. F. A., 258, 266 Work, E., 88, 10.3, 114, 115 145, 148, 150, 191, 192, 193 Worlledge, S . M., 175, 187 Waters, M. F. R., 174, 192 Worms, M. J., 283, 284, 344 Waterson. A. P., 19, 21, 28 Wren, R. E., 329, 346 Watson, D. W., 130, 133, 193 Wright, D. H., 331, 349 Watson-Williams, E. J., 276, 343 Wrigley, N. G., 5, 30 Webb, E. L., 334,345 Wyckoff, R. W. C., 2, 30 Weber, C., 12, 30 Wecker, E. E., 142, 180, 193 Weed, R. J., 91, 115 Y Weiser, R. S., 120, 121, 128, 139, 188 Yagi, Y., 18, 30 Weiss, M. L., 315, 324, 349 Yamaninra, Y., 18, 23, 25, 29, 30 Weissman, G., 84, 115, 167, 188 Yanp, J. P. S., 131, 133, 135, 136, 182, Weissmann, C., 834, 261 188 Welldc, B. T., 269, 287, 318, 320, 323, Yoeli, M., 277, 282, 304, 325, 329, 345, 325, 347, 347, 349 346, 349 Weltman, J. K., 12, 30 IVerner, B., 124, 156, 157, 162, 164, 166, Yoran, S., 51, 55(57, 59), 73 Yuan, L., 231, 232, 233, 261 189 Wpry, M., 283, 340 Z Wliorton, M., 326, 344 Zak, S. J., 204, 205, 106, 210, 221. 222, Wigzell, €I., 124, 193 248, 263 Wilcox, W. C., 3, 30

AUTHOR INDEX

Zeias, I., 122, 157, 160, 161, 1G4, 170,

185, 190 Ziff, M., 177, 192 Zmijewski, C. M., 254, 257, 2/32 Zopf, D. 84, 108,114 Zuckerman, A., 269, 275, 277, 288, 089,

367

297, 298, 299, 309, 315, 324, 328, 332, 333, 344, 347, 349 Zukoski, Ch., F., 120, 121, 162, 165, 190 Zweiman, B., 198, 199, 200, 202, 204, 229, 230, 231, 232, 233, 240, 250, 252, 256, 257, 264

SUBJECT INDEX A

C

Agammaglobulinemia, transfer factor and,

Cells, nucleated mammalian, complement produced holes in, 85-87 Cell surface, relationship of holes to site of damage, 88-92 Cellular immune deficiency, reconstitution, transfer factor and, 252-

248-249 Age, host, malaria and, 277 Allograft rejection, lymphoid cell cytotoxicity and, 179-181 Antibody, malarial, measurement of, 303-318 protective effect of, 318-323 molecules required to produce a lesion,

95-98 Antigenic variations, malaria and, 284-

288 Antigens, branched, multichain amino acid copolymers, immune response to,

56-69

257 transfer factor and, 248-252 Cellular immunity, transfer factor and, 234-245 Complement, artificial membranes and, 108-110 bacterial cell walls and, 87-88 bacterial lipopolysaccharides and, 88 holes produced in erythrocytes, appearance of, 77-82 lesions produced by other agents,

complex multideterminant, constitutional differences in individual responses, 33-37 defined protein, genetic differences in immune response, 38-39 linear random copolymers of L-a-amino acids, immune response to, 39-43 malarial parasites, 296-303 poly-L-lysine and hapten-poly-L-lysine conjugates, immune response to,

84-85 size of, 82-83 holes produced in mammalian nucleated cells, 85-87 nature of holes formed, chemical studies, 104-108 qualitative studies, 101-104 relationship of holes to site of damage,

88-92 site of damage, multiple holes and,

43-56

92-95

Autoantibodies, malaria and, 334-335 Autoimmunity, lymphoid cell cytotoxicity and, 175-177

stage of action forming holes, 98-99 target cell-bound, lymphoid cell cytotoxicity triggered by, 153-156 terminal lesion, biological significance,

110~113

B Bacteria, cell walls, complement produced holes in, 87-88 Blood, pathology, malaria and, 332-334 Blood transfusion, tuberciilin sensitivity transfer by, 212 Boeck's sarcoid, transfei factor and, 249-

virus particles and, 88 Contact sensitivity, transfer of, 210-212

D Delayed hypersensitivity, lymphoid cell cytotoxicity and, 172-

251

368

17s transfer with leitkocytes, contact sensitivity and, 210-212 Kveim antigen and, 2 1 2 2 1 5

369

SUBJECT INDEX lymphocytes and, 209-210 quantitative variables, 202-208 t enaI transplants and, 215-218 tuberculin sensitivity and, 212 Dcoxyribonuclease, transfer factor and,

229-23 1

linear random copolymers of L-a-amino acids and, 39-43 poly-L-lysine and hapten poly-~-lysine and, 43-56 I minunity, adoptive transfer, malaria and, 293-

294

E Effector cell^, cytotoxicitv of lymphoid cells and, 120-121 Electron microscopy, erythrocyte lesions and, 84-85 immunoglobulin G, negative staining, 7-14 question of conformational change,

14-17 shadowing, 6-7 immunoglobulin M, 17-26 molecular level, 2-6 Environment, malaria and, 277 Erythrocytes, complement produced holes, appearance of, 77-82 size of, 82-83 lesions produced by other agents, 84-

malarial, experimental modification of, 329-

331 humoral factors in, 303-323 Immunocompetent cells, intact, hazards of reconstitution with,

256 Immunoglobulin G, electron microscopy, negative staining, 7-14 question of conformational change,

14-17 shadowing, 6-7 Immunoglobulin M, electron microscopy,

17-26 Inmunological surveillance, transfer factor and, 258-259

K

85

Kveim antigen, transfer of delayed reactivity to, 212-215

G Gene action, analysis of mechanism, 37-

1

38 Genetic constitution, malaria and, 276-

Lepromatous leprosy, transfer factor and,

255-256

277

Graft-versus-host reaction, lymphoid cell Leukocytes, delayed hypersensitivity transfer with, cytotoxicity and, 177-179 Guinea pig, immune response to p o l y - ~ 202-217 lysine and hapten-poly-L-lysine conextracts, confirmation of transfer with, 226jugates, 43-56

229

H Hodgkin’s disease, transfer factor and.

251 Host specificity, malaria and, 275-276

I Immune response, branched, multichairi amino acid polymers and, 56-60 constitntional differences in, 3.3-37 genetic differences in, 38-39

CO-

introduction of, 217-226 transfer factor, deoxyrihonuclease and, 229-231 sonication and, 231-234 Lipopolysaccharides, bacterial, complement and, 88 Lymphocytes, transformation, transfer factor and,

238-244 tlelayed hypersensitivity transfer with,

209-210

370

SUBJECT INDEX

Lymphoid cells, cytotoxic effects, different in uitro models, 127-172 in uioo implications of models, 172183 methods, 119-127 summary, 183-185 triggered by target cell-bound complement, 153-158 nonspecific cytotoxicity, phytohemagglutinin or other stimulants and, 156-168 normal donors, destruction of target cells after in uitro sensitization, 168-172 induction of cytotoxicity by antibodies to target cell antigens, 144-152 sensitized donor, cytotoxic effects on antigenic target cells of, 127-144

simultaneous infections, 277-278 skin reactions in, 295-29G Malarial infections, duration of, 271-274 phagocytosis and, 288-289 spleen and, 289-293 symptoms and pathological effects,

274275 Malarial parasites, antigens of, 29C303 collection of, 296-298 life cycle, 269-275 immunity and, 280-284 Membranes, artificial, complement and, 108-110 Mice, immune response to branched, multichain amino acid copolymers,

5W9 Moniliasis, disseminated, transfer factor and, 254-

255

M Macrophages, migration, transfer factor and, 234-238 Malaria, active immunization, infection and, 323-324 killed vaccines and, 325-329 living vaccines and, 324-325 autoantibodies and, 334-335 environmental conditions and, 277 genetic constitution and, 276-277 host age and, 277 host specificity of, 275-276 immunity, 268-269 adoptive transfer of, 293-294 experimental modification of, 329331 humoral factors in, 303-323 immunity acquired through infection experimental infections, 2 7 S 2 8 0 plasmodia1 life cycle and, 280-284 population studies and, 278-279 immunopathology, 331-338 innate and nonspecific inimnnity to,

275-278 relapses and antigenic variation, 284288

N Neoplastic disease, systemic, transfer factor and, 252 Nephrosis, malaria and, 335-338

P Phagocytosis, malarial infection and, 288289 Phytohemagglutinin, nonspecific lymphoid cell cytotoxicity and, 156-168 Polypeptides, synthetic, immune response to, 39-69

R Relapses, malaria, 284-288 Renal transplants, delayed hypersensitivity transfer by, 215-216

S Skin reactions, malaria and, 295-296 Spleen, malarial infection and, 289-293

T Target cells, antihodies to antigens, induction of cytotoxicity of lymphoid cells of normal donors, 144-152

371

SUBJECT INDEX

antigenic, cytotoxic cffects of lymphoid cells from sensitizrd donors on, 127-144 complement, cytotoxicity of Iymphoid cells triggered by, 153-156 cytotoxicity of lymphoid cclls and, 11%

120 destruction by lymphoid cclls from normal donors after in t>ifr.osensitization, 168-172 destruction, transfer factor and, 244-

245 Transfer factor, characterization and niechanism of action, confirmation of transfer in man, 22&229 introduction of leukocyte extracts, 217-226 conclusions, 259-261 correlates of cellular immunity and, 234-245 definition and principles, donor selection, 199-200 local transfer, 201-202 protocol, 201

recipient selection, 200-201 systemic transfer, 201 dialyzable, nature and propertic,s of, 229-234 possible applications of, 2,56257 liistorical, 196199, immunological surveillance and tumor immunity, 258-259 mechanisms of cellular iinmune cleficiency diseases and, 248-252 inechanism of action i n vioo and in uitro, 245-248 reconstitution of cellular immune tleficiency and, 252-257 Tuhercnlin sensitivity, transfer of, 212 Tumor defense, lymphoid cell cytotoxicity and, 181-183 Tumor imninnity, transfer factor and, 258-259

V Vaccines, malarial immunity and, 324329 Viiccinia, generalized, transfer factor and, 253-

254 Virus particles, complement and, 88

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