Advances in Immunology Volume 21

ADVANCES I N

Immunology

VOLUME

21

CONTRIBUTORS TO THIS VOLUME

ANTONIO COUTINHO J. KINDT THOMAS

GORANMOLLER

ROBERTOJ. POLJAK WILLIAM0. WEIGLE

ADVANCES IN

Immunology EDITED B Y F. J. DIXON

HENRY G. KUNKEL

Division of Experiment01 Pathology Scrippa Clinic and Reaearch Foundation La Jolla, California

The Rockefeller University New York, New York

VOLUME

21

1975

ACADEMIC PRESS

New York

Sun Francisco

A Subsidiary of Horcourf Brace Jovonovich, Publishers

London

COPYRIGHT 0 1975, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. N O PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED I N ANY FORM OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM T HE PUBLISHER.

ACADEMIC PRESS, INC.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

LIBRARYO F

CONGRESS CATALOG CARD

NUMBER:61-17057

ISBN 0-12-022421-6 PRINTED IN TH E UNITED STATES O F AMERICA

CONTENTS LIST OF CONTRIBUTORS .

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vii

PREFACE.

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ix

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1 2 2 4 7 29 30

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X-Ray Diffraction Studies of Immunoglobulins

ROBERTO J . POLJAK

I . Introduction . . . . . . . . . . . . I1 . Polypeptide Chain Structure of Immunoglobulins and Antibodies .

. . 111. Techniques of Crystallographic Analysis . IV . Results of Low-Resolution X-Ray Diffraction Studies V . High-Resolution Studies . . . . . . VI . Summary and Conclusions . . . . . . References . . . . . . . . .

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Rabbit immunoglobulin Allotypes: Structure. Immunology. a n d Genetics

THOMAS J .

KINDT

I . Introduction . . . . . . . . I1 . Structural Correlates of Allotypic Determinants . 111. Antigenic Determinants of Rabbit Allotypes . IV . Genetic Relationships among Allotypes . V . Allotypes and the Immune Response . . . VI . Allotype Suppression . . . . . . . . . . . VII . Idiotypes and Allotypes VIII . Conclusion . . . . . . . . . . . References .

35 48 61 65 70 73 75 78 81

Cyclical Production of Antibody as a Regulatory Mechanism in the immune Response

WILLIAM0. WEICLE

I. I1 . 111. IV .

Introduction . . . . . . . . . . . . . Cycling in the Immune Response Synchrony of' Appearance of Antibody-Producing Cells Conclusions . . . . . . . . . References . . . . . . . . .

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87 90 100 107 109

vi

CONTENTS

Thymus-Independent B-Cell Induction and Paralysis ANTONIO COUTINHO AND GORAN MOLLER

I . Introduction . . . . . . . I1 . Hypotheses for Immune B-Cell Activation

111. Some Important Technical Considerations

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IV. Critical Evidence Supporting the One Nonspecific Signal Hypothesis . V . Basis of Thymus Independence (Direct, Specific B-Cell Activation): Competing Concepts . . . . . . . . . . . VI . Molecular Basis of B-Cell Activation . . . . . . . . VII B-Cell Paralysis in Thymus-Independent Responses . . . . VIII . B-Cell Induction and Paralysis by Nonpolyclonal Activator Molecules . . . . . . . . (Thymus-Dependent Antigens) . IX . Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .

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SUBJECT INDEX CONTENTS

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OF PHEVIOUS VOLUMES

114 119 126 145 167 191 207 221 226 227

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

ANTONIOCOUTINHO,Division of Zmmunobiology, Wallenberg Laboratory, Karolinska Institute, Stockholm, Sweden (113)

THOMAS J. KINDT, The Rockefeller University, New York, New York (35) GORAN MOLLER, Division of Zmmunobiology, Wallenberg Laboratoy, Karolinska Institute, Stockholm, Sweden (113)

ROBERTO J. POLJAK, Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, M a y land (1) WILLIAM 0. WEIGLE, Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California (87)

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PREFACE

It is always difficult in any branch of science to assess the relative significance of individual advances. It is a task best left to historians who have the advantage of a retrospective purview. Current developments in immunology are a good case in point. Key information in diverse areas is accumulating at such a rapid pace that there is scarcely time to take stock of the situation and obtain a true evaluation of significance. The papers in Volume 21 relate directly to four of the forefronts that have developed particularly rapidly. In contrast to other areas, there can be little doubt about their long-range significance. Dr. Poljak’s review certainly represents a landmark study. His work on the X-ray diffraction analysis of the Fab fragment of a human myeloma protein has provided the first real insight into the three-dimensional structure of the Ig molecule. Four globular units sharing a basic pattern of polypeptide folding make up the two units from the light chain and the two from the heavy chain. These are connected by linear stretches of polypeptide chains. It is of special interest that the hypervariable portions of the two regions are both positioned at an exposed end of the molecule offering the greatest opportunity for variability at the combining site. Dr. Kindt reviews the long series of interesting studies of rabbit allotypes including some of the exciting recent developments. He is a real leader in the field and his vast knowledge of the intricacies of the system is readily apparent. The rabbit system has held particular interest for a number of years because of the presence of genetic markers in the V regions; similar readily detected antigens have not been found in other species. He discusses his new and very rational theory that multiple structural genes control each V region and that an insertional mechanism into a relatively constant C region is involved. The article by Dr. Weigle dwells on the details of control mechanisms involved in antibody production. The phenomenon of cyclical production of antibody after a single injection of antigen is striking and apparently considerably more common than is usually thought. It is clear that antibody itself plays a primary role in this regulation, but other added factors, such as suppressor T cells and secondary idiotypic antibodies, also may play a role under specific circumstances. The intriguing concept is presented that the cycling phenomenon ix

X

PREFACE

plays a key role in the conservation of memory cells and thus assures continued persistence of antibody. The paper by Drs. Coutinho and Moller represents a very complete review of the mechanism of B-cell activation for the production of antibody. The work with mitogens particularly by these investigators has changed very considerably the earlier concepts. It now appears that the mitogenic effect, which they term polyclonal B-cell activation, is an integral part of antigen activation, particularly where the antigen itself is mitogenic as well. The authors assemble considerable evidence that this is the case with T-cell independent antigens. These questions with respect to T-cell dependent antigens remain unresolved and obviously must await further knowledge concerning the mechanism of action of the various T cell factors influencing B cells.

FRANKJ. DIXON HENRYG. KUNKEL

ADVANCES I N

Immunology

VOLUME

21

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X-Ray Diffraction Studies of Immunoglobulins’ ROBERTO J. POLJAK2 Department o f Biophysics, Johns Hopkins University School of Medicine, Baltimore, Maryland

I. Introduction . . . . . . . . . . . . Polypeptide Chain Structure of Immunoglobulins and Antibodies . Techniques of Crystallographic Analysis . . . . . . Results of Low-Resolution X-Ray Diffraction Studies . . , . High-Resolution Studies. . . . . . . . . . A. Shape, Dimensions, and Symmetry of Fab’ . . . . . B. Amino Acid Sequence of the L and H Chains from IgG New . C. Structure of the Homology Subunits: The “Immunoglobulin Fold” D. Interchain and Intrachain Disulfide Bonds . . . . . E. Location of Isotypic and Allotypic Markers . . . . . F. Structure of the Hypervariable Regions and the Active Site . , G . Structure of a Ligand-Fab’ Complex . . . . . . . . . . . . . . . H. Patterns of Change . I. Changes in Conformation. . . . . . . . . VI. Summary and Conclusions . . . . . . . . . References . . . . . . . . . . . .

1 2 2 4 7

11. 111. IV. V.

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9 11 15 17 18 23 26 27 29 30

I. Introduction

The most direct approach to the study of the detailed threedimensional structure of macromolecules is the method of singlecrystal X-ray diffraction. The determination of the structure of several enzymes and nucleic acids to atomic resolution achieved within the last 10-15 years is an outstanding demonstration of the potential of this approach. Recent developments in methods and techniques have facilitated the determination of more complex structures such as those of multimeric protein molecules. Once the overall multichain structure of immunoglobulins became established, about 12 years ago, it was hoped that amino acid sequence determinations, and eventually X-ray diffraction studies, would provide useful models for the correlation of the function and structure and the genetic mechanisms that control the variability and specificity of antibodies. However, the fact that immunoglobulins were demonstrably heterogeneous seemed to present an insurmount-

’ Supported by Grants AI-08202 from the National Institute of Allergy and Infectious Diseases and E-638, NP 141A from the American Cancer Society. * Recipient of Research Career Development Award AI-70091 from the National Institute of Allergy and Infectious Diseases. 1

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ROBERTO J. POLJAK

able obstacle for X-ray diffraction studies since crystallization, a necessary first step, had always been associated with the presence of a homogeneous preparation of a single molecular species. Just as in the case of sequencing studies, this problem was resolved by the use of myeloma immunoglobulins which provided homogeneous material in sufficient quantities to permit crystallographic studies to atomic resolution. This review treats almost exclusively results obtained in the investigation of the three-dimensional structure of a human Fab' fragment currently under study in the author's laboratory. Results of X-ray diffraction studies which are under way in several other laboratories will be briefly mentioned or described in much less detail. II. Polypeptide Chain Structure of Immunoglobulins and Antibodies

The reader is referred to previous extensive reviews of this subject such as those of Edelman and Gall (1969), Milstein and Pink (1970), Krause (1970), Hood and Prahl (1971), Natvig and Kunkel (1973). Figure 1 shows the diagrammatic structure of a human IgGl molecule and introduces some of the nomenclature that will be used in this review. The variable homology regions, indicated as VH and V Lin Fig. 1 occur toward the N-terminal of the heavy (H) and light (L) polypeptide chains and each include about 110 amino acid residues. The constant homology regions CL, CH1, CH2, and C,.J also include about 110 amino acid residues each. The amino acid sequences of VH and V L are highly homologous and so are those of CL,CHI, CH2,and C& The homology between the V and C regions is weaker, although some features such as intrachain disulfide bonds, illustrated in Fig. 1, are common to all of them. It is well known that the V regions display a variability of sequence that is not observed in C regions. Comparison of V, sequences indicates that this variability is more pronounced in certain regions which are hypervariable in sequence (Wu and Kabat, 1970; Kabat and Wu, 1971). Similar hypervariable regions have been detected in V, sequences (Kabat and Wu, 1971; Capra and Kehoe, 1974). Ill. Techniques of Crystallographic Analysis

Several extensive reviews on this subject are available (e.g., Dickerson, 1964; Holmes and Blow, 1966; Matthews, 1974). A shorter review including preliminary results of X-ray diffraction studies of immunoglobulins has also been published (Poljak, 1973). Briefly stated, X-ray diffraction analysis of single crystals can provide a high-resolution model of a molecule by a Fourier series

X-RAY STUDIES OF IMMUNOGLOBULINS

3

FIG. 1. Diagram of a human immunoglobulin (IgG1) molecule. The light (L) chains (mol. wt. about 25,OOO) are divided into two homology regions, V,, and C,. The heavy (H) chains (mol. wt. about 50,000) are divided into four homology regions, V H , CHI, cH2, and cH3. The CHI and cH2 are joined by a “hinge” region indicated by a thicker line. Cleavage of the IgGl molecule by papain generates Fab fragments (mol. wt. about 50,OOO) consisting of an L and an Fd polypeptide chains, and Fc fragments (rnol. wt. about S0,OOO). Cleavage by pepsin followed by reduction of inter-H-chain disulfide bonds generates an Fab’ fragment consisting of an L and an Fd’ polypeptide chains. Interchain and intrachain disulfide bonds and the N-termini of the L and H chains are indicated. (Reproduced from Poljak, 1973, with kind permission of Plenum Press.)

representation of the electron density, calculated from experimental diffraction data and plotted as a contour map. The conformation obtained from X-ray diffraction studies has been shown to be very close to that inferred to exist in solution for several proteins (see review by Matthews, 1974). Since X-ray measurements are made during prolonged periods of time (hours), only stable conformations can be analyzed, for example, at the beginning and at the end of a conformational change, or at both stages if they can be obtained under controlled conditions (for example, oxy- and deoxyhemoglobin crystals). The development of data-gathering instruments, such as computercontrolled diffractometers and large memory high-speed computers and their peripheral equipment, have made the study of larger, more complex macromolecules easier than it would have been only a few years ago. Even with these technical developments it should be con-

4

ROBERTO J. POLJAK

sidered that since immunoglobulin crystals do not produce the quality of X-ray diffraction data which is observed in smaller proteins (myoglobin and lysozyme, for example) and in direct proportion to their molecular weight produce a much larger quantity of data, the strategy and practice of data collection requires careful attention. The number of reflections that must be measured increases with increasing resolution and is of the order of 104-105reflections for a protein of mol. wt. about 50,000 studied to “atomic” (2-81.)resolution using the multiple isomorphous heavy atom replacement technique. Even at this resolution, knowledge of the amino acid sequence is essential for a complete interpretation of the Fourier map of the electron density. IV. Results of Low-Resolution X-Ray Diffraction Studies

Following the work of Kendrew et aZ. (1958) and of Perutz et al. (1960) the preliminary structural analysis of a protein by X-ray diffraction methods is first attempted at low (usually 6-81.) resolution. This approach is followed because low-resolution studies are capable of providing a general model of the quaternary and tertiary structure of the protein under study and can be completed in a much shorter time than is required for higher-resolution analyses. In addition, low-resolution studies provide a critical test of the experimental techniques and the mathematical and computational procedures which will be used in subsequent (longer) high-resolution studies. Fragments Fab and Fab’ from human myeloma proteins were crystallized by Rossi and Nisonoff (1968) and by Rossi, Choi, and Nisonoff (1969).X-Ray diffraction patterns of the crystalline Fab and Fab’ fragments (Avey et al., 1968; Humphrey et al., 1969) from IgG New gave identical intensity patterns indicating that they have the same structure. It would normally be expected that, since Fab’ is about 10 amino acid residues longer than Fab (in its Fd’ chain), its X-ray intensity patterns should reflect this difference. Subsequent high-resolution studies indicated the C-termini of the Fd’ and Fd chains do not make any significant contribution to diffracted X-ray intensities, probably due to random motion of the ends of the polypeptide chains. From this it follows that Fab’ New and Fab New are equivalent for crystallographic studies. Another crystalline fragment, Fab Hi1 (Rossi and Nisonoff, 1968; Humphrey et uZ., 1969) also proved suitable for X-ray diffraction studies. However, since it contains two Fab molecules per asymmetric unit of the crystal lattice, its study requires about twice the number of intensity measurements than are required for the study of Fab New. Both fragments, New and Hi1 were obtained from human

X-RAY STUDIES OF IMMUNOGLOBULINS

5

IgGl (A) myeloma proteins of allotype Gm(l+, 3-, 4-, 5-) (Rossi and Nisonoff, 1968). The 6-A. resolution study of Fab' New (Poljak et al,, 1972) showed that the Fab' fragment consists of two discrete globular domains, V and C , , containing the V, and VH and the C , and CHI homology regions, respectively. Despite the limitations of a lowresolution study, the two polypeptide chains, L and Fd', were found to be two, continuous, independent stretches of electron density, which originate in one of the globular subunits and, after a region of globular folding in that subunit, extend to the other subunit through a narrow outer bridge of polypeptide chain (Fig. 2). The overall scheme is that of a tetrahedral arrangement of four globular subunits, two of which (V, and C,) are formed by the L chain and the other two (V, and CHI) by the Fd' chain. This type of arrangement is

FIG.2. Four different views (A-D) at 90" from each other, of the 6-A. resolution model of Fab' New. The two continuous chains of electron density described in the text are shown in white and gray. Two globular domains, V and C,, separated by a cleft and joined by two easily accessible connections are clearly seen. Photographs C and D clearly show the approximate tetrahedral symmetry of the molecule. (Reproduced from Poljak et al., 1972, with kind permission of Nature.)

6

ROBERTO J. POLJAK

roughly similar to that observed in hemoglobin (Perutz et al., 1960) where four globular subunits are arranged in tetrahedral configuration, although in hemoglobin there are four independent (two a,two p) polypeptide chains. The presence of discrete globular domains V and C1of Fig. 2, had already been demonstrated by Green et al. (1971) in a study by electron microscopy of the dinitrophenyl (DNP)-binding murine IgA, myeloma protein, MOPC 315. Furthermore, Green and colleagues inferred that each globular domain contained a homology region of both the L chain and the H chain. As expected, the detailed symmetry of the arrangement is more clearly indicated in the 6-A. resolution X-ray diffraction model of Fab’ New. However, the assignment of the V (V, V,) and C, (C, +CH1)domains from the 6-A. resolution study, made on the basis of the occurrence of a cavity-shaped region at one end of the molecule, was not confirmed by correlation with the high-resolution model to be described below. From this correlation, the dark-colored chain of Fig. 2 corresponds to the Fd’ polypeptide chain and the light-colored chain to the L chain. The V domain is the one shown at the left of the model in Fig. 2. Several properties of immunoglobulins and the results of various immunochemical experiments designed to probe their structure and function were explained by the low-resolution model of Fab’ New. For example, cleavage of human myeloma L chains by controlled enzymatic digestion had been shown to generate V L and C ,fragments (Solomon and McLaughlin, 1969; Karlsonn et al., 1969) which have a more compact shape as inferred from their radius of gyration and frictional coefficient, than the intact L chain. Also, Givol and co-workers (Inbar et al., 1972) have demonstrated that cleavage of an Fab’ fragment by pepsin can generate a fragment which he called FV,consisting of the V,, and the V, regions. Figure 2 shows that both the Fd‘ chain and the L chain have an easily accessible linear region around the middle of their length which joins one globular subunit of the chain to the other. These midpoints of both chains are accessible and should be more readily cleaved by enzymatic action than other points of the chains. The average value of 16 A. obtained for the radius of gyration of the C, and V, fragments of human myeloma L chains (Karlsonn et al., 1969) is very close to the value of 15 to 20 A. that can be measured directly in the low-resolution model of Fab’ New. Close inspection of the low-resolution structure appeared to indicate that the L and the Fd chains share a common overall structure such that they could pair with each other as in Fab, or form a dimer with a homologous chain (an L-chain or an H-chain dimer).

+

X-RAY STUDIES OF IMMUNOGLOBULINS

7

FIG. 3. Schematic representation of the structure of an IgG molecule incorporating structural features determined for the Fab and Fc fragments (see text). (Reproduced from Poljak et al., 1972, with kind permission of Nature.)

Amino acid sequence homologies first demonstrated by Hill et al. (1966) led to speculation that the homology regions of immunoglobulins would fold into basic globular subunits of similar three-dimensional structure (the structure specified by a primordial gene) which, suitably repeated, could account for the whole immunoglobulin structure. This speculation is clearly consistent with the low-resolution model of Fab’ New. On the basis of this model and on the symmetry determined for the Fc fragment (Goldstein et al., 1968) and for a whole IgG molecule (Sarma et al., 1971), a schematic picture of an IgG molecule can be represented as shown in Fig. 3. V. High-Resolution Studies

A 2.8-A. resolution study of the Fab’ New fragment (Poljak et al., 1973) led to an atomic resolution model that correlated amino acid sequence data with tertiary structure. Subsequently, a 2.0-A. resolution Fourier map has been calculated and an atomic resolution model was built based on this map and on the amino acid sequence (Poljak et al., 1974). Models of an L (A) dimer at 3.5-A. resolution (Schiffer et al., 1973) and of a V, dimer at 2.8-A. resolution (Epp et al., 1974) have been presented. A 4.5-A. resolution Fourier map of the Fab’ fragment from the phosphorylcholine binding protein McPC 603 has also been published (Padlan et al., 1973). Complexes between Fab McPC 603 and a phosphorylcholine ligand (Padlan et al., 1973) and Fab’ New and several ligands (Amzel et al., 1974) have been studied by X-ray diffraction methods. Some of the results obtained from these studies will be discussed below. A. SHAPE, DIMENSIONS, AND SYMMETRY

FAB’ The 2.8-A. resolution Fourier map of Fab‘ New clearly indicated the molecular shape of Fab’ and the arrangement of the L and Fd’ polypeptide chains, which are essentially as described in the 6-A. OF

8

ROBERTO J. POLJAK

resolution study (Figs. 4 and 5). The overall dimensions of the Fab’ molecule are 80 x 50 x 40 A. and those of the homology subunits are 40 x 25 x 25 A. (“homology subunit” is used here to denote the globular unit of three-dimensional structure that contains the amino acid sequence of a homology region, V,, V H ,C,, or CH1,etc.). A centrally located cleft, openly accessible to solvent divides the molecule into two globular structural domains, V and C, which are linked by two “switch” region sequences. The structural subunits C, and CH1 in the C1 domain interact more closely and are more tightly packed than the V, and V Hsubunits in the V domain. The angle between the

FIG. 4. Model of Fab’ New based.on a 2.8-A. resolution Fourier map. The V (left) and C , (right) domains are separated by a central cleft. Labels show the “switch” region at the midpoints of the L (upper) and Fd‘ (lower) chains. Approximate local twofold axes of symmetry are indicated by two white rods. The four intrachain disulfide bonds and the interchain disulfide bond are marked by white spheres. White tapes connect the a-carbon positions of residues that form disulfide bonds in other immunoglobulin molecules (see text). The tags at the left end of the model indicate the hypervariable positions of the L chain (round tags) and the H chain (rectangular tags). Arrows at the right end of‘ the model point to Ser 154 and Lys 191 (Kern and Oz serological markers, respectively). This view of the molecule closely corresponds to photograph A of the low-resolution model in Fig. 2.

X-RAY STUDIES OF IMMUNOGLOBULINS

9

FIG. 5. Stereo pair drawing of the a-carbon backbone of the Fab' structure oriented as shown in Fig. 4. The thicker trace corresponds to the Fd' polypeptide chain. (Inexpensive stereo viewers to help achieve a three-dimensional effect from this figure can be obtained from the Taylor-Merchant Corporation, New York, N. Y. 10036.)

major axes of the CL and V,, homology subunits is greater than 90" (100"-110"), whereas the angle between the axes of the CHI and VH subunits is smaller than 90"(80"-85"), see Figs. 4 and 5 . As a result, the Fd' chain is more folded over itself, displaying a closer association between the VHand CH1 subunits than is the case for the VL and C Lsubunits of the L chain. It is interesting to observe here that the L (A)-chain dimer Mcg (Schiffer et al., 1973) does not crystallize as a dimer of conformationally identical chains. One of the L chains of the dimer assumes a conformation that appears very close to that of the L chain from IgG New. The second L chain from the Mcg dimer, although identical in sequence to the first, assumes a conformation closer to that of an Fd' chain, with an angle smaller than 90" between the major axes of its V and C subunits. As indicated in Fig. 4, approximate twofold axes of symmetry relate CLto CHI and VLto VH.

B. AMINO ACID SEQUENCE FROM IGG NEW

OF T H E

L

AND

H

CHAINS

High-resolution Fourier maps of protein structures are usually interpreted by a correlation of the electron-density features with the amino acid sequence of the protein under study. Extensive sequence work which has been carried out with different im-

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ROBERTO J. POLJAK 10

20

PCA-SER-VAL-LEU-THR-GLN-PRO-PRD-SER-VAL-SER-GLY 21 27a 27b 270

-RLA-Pw-GLY

-GLN-AFS-VAL-THR-I

SER-~YS-THR-GLY-SER-SER-SER-ASN-ILE-GLY-ALA-GLY -ASN-HIS-VAL-LY 40

50

LeZI-PW-GLY-THR-~-PRO-LYS-LeU-LEU-ILE-PHE-HIS-ASN-ASN-ALA 60

- - - A=-PHE-SER-VAL-SER-LY

S-TRP-TYR-GLN4LN

-

-

70 S-SER-GLY -SER-SER-ALA-THR-LEU-ALA-ILE~

80

90

GLY -LeU-GLN-ALA-GLU-ASP-GLU-ALA-~P-TYR~R-cYS

--

LE

30

100

-GLN-SER-TYR-ASP-AFS-SER-LEU-~

110

VAL-PHE-GLY-GLY -GLY-THR-LYS-LEU-THR-VAL-LEU-AFS-GLN-PRO-LYS-ALA-ALA

120 130 PI(D-SER-VAL-THR-LEU-PHE-PRO-PRO-SER-SER-GLX-GLX-LEU-GLN-ALA-ASN-LYS-ALA-THR-LEU 140 VAL-CYS-LEU-I

LE-SER-ASP-PHE-TYR-PW-GLY

160

150 -ALA-VAL-THR-VAL-ALA-T~-LYS-ALA-ASP-SER

170

SER-PRO-VAL-LYS-ALA-GLY-VAL~LU-THR-THR-THR-PRO-SER-LYS-GLN-SER-ASN-~N-LYS-TYR

180 190 A L A - A ~ - S E R - S E R ~ R - L E U - S E R - L E U - T H R - P R O -SER-HIS S -LYS-SER-TYR-SER 200 210 CIS-GLX-VAL-THR-HIS-GLU-GLY-SER-THR-VAL-GLU-LYS-THR-VAL-ALA-PRO-THLU-CIS-SER

FIG.6. The amino acid sequence of the L chain from Fab’ New. Numbers 27a, 27b, and 27c and gaps (53-59 and 96-97) are introduced to maximize homology with other human A chains. (Data from Chen and Poljak, 1974.)

munoglobulin molecules (see Dayhoff, 1972, for a comprehensive compilation of these sequences) provides data that can be used to interpret a Fourier map of the Fab fragment. The constant character of sequences of the C L and CH1regions has been firmly established in several laboratories. Even in the V regions the patterns of extreme variation in sequence are circumscribed to segments of the polypeptide chain called the hypervariable regions (Kabat and Wu, 1971). However, when the sequence of the L chain from IgG New was determined by chemical methods (Chen and Poljak, 1974) (Fig. 6) and by a study of the 2.8-A. resolution Fourier map, a deletion of seven amino acids was found at positions 53-59. It remains to be seen whether this deletion is peculiar to the structure of the L chain from IgG New or whether it is a more general feature to be found in other immunoglobulin L chains. Other features of the L-chain sequence from IgG New will be discussed in the following paragraph. Residues 27a, 27b, and 27c, which are deleted in many human hchain sequences, do not conform to the sequences observed for A “subgroups” I and I1 (Baczko et al., 1970). Position 31, which is most

X-RAY STUDIES O F IMMUNOGLOBULINS

11

frequently occupied by tyrosine in human, pig, and mice A-chains and which is frequently tagged by affinity labeling reagents (see below), is replaced by histidine. A deletion of two amino acids is introduced at positions 96 and 97 to align the L-chain sequence from IgG New with that of other human A chains. Deletions of one or two amino acids at these positions have been reported before in A chains (Dayhoff, 1972). Sequence determination of the CL region of IgG New verified that it conforms to that observed in other human A chains. Positions 154 and 191 are occupied by serine and lysine, which correlate with the serological (isotypic) markers Kern- (Hess and Hilschmann, 1970) and Oz+ (Appella and Ein, 1967), respectively. Work on the determination of the amino acid sequence of the H chain of IgG New is under way at the time of this writing. Part of the sequence has already been determined and is shown in Fig. 9 (Section V,C).

c. STRUCTURE OF THE HOMOLOGY SUBUNITS: FOLD” THE “IMMUNOGLOBULIN After tracing the path of the polypeptide chains and assigning some of the amino acid residues of the IgG New sequence to features of the electron-density map, it became evident that the homology regions of Fab’ share the common pattern of three-dimensional structure illustrated diagrammatically in Fig. 7. This structure is called the “immunoglobulin fold” to indicate that it is the basic pattern of the homology subunits of Fab and, by extension, of the homology subunits CH2 and C,3 in the Fc fragment. Immunoglobulins can thus be visualized as multimeric proteins made up of repeated subunits, such as that illustrated in Fig. 7. Although the variable homology regions VL and V H share the basic immunoglobulin fold, they differ from the C regions in at least two significant respects. First, they include an extra length of polypeptide chain that does not occur in the C regions (see Figs. 7 and 9); second, the V and C subunits interact with each other ( C , with CH1 and V, with V,) with contacts by residues which are located in different regions of their common three-dimensional structure. In addition to the striking similarity in the folding of the polypeptide chain in each homology subunit, certain amino acid residues such as V LCys 22, Trp 34, Tyr 85, and Cys 87 and the homologous residues in V,, CL, and CH1(see Fig. 9) are a common feature of all the subunits. A similar scheme of basic three-dimensional structure as that of

12

ROBERTO J. POLJAK

FIG.7. Diagram of the polypeptide chain folding in the C,. subunit illustrating the basic immunoglobulin fold. Solid trace shows the folding of the polypeptide chain in the constant subunits, CL and C,1. Numbers indicate C , residues, beginning at NH,+ which corresponds to residue 110 for the L chain. Dotted lines indicate the additional loop of polypeptide chain characteristic of the V, and V, subunits. (Reproduced from Poljak et al., 1973, with permission.)

Fig. 7 has been presented by Schiffer et al. (1973) and b y Epp et al. (1974) as a result of their studies of the structure of a human L (A)chain dimer and a human V, dimer, respectively. Since it is generally accepted that the primary structure of a protein determines its tertiary structure (Epstein et al., 1963), it is logical to expect that homologous sequences will lead to similar patterns of three-dimensional folding. This expectation has been verified for the serine proteases chymotrypsin, trypsin, and elastase where homologous

X-RAY STUDIES O F IMMUNOGLOBULINS

13

sequences (Hartley, 1970) correspond to a similar three-dimensional structure (Matthews et al., 1967; Watson et al., 1970; Stroud et al., 1971). In other families of proteins, such as myoglobinshemoglobins (Perutz et al., 1965) and dehydrogenase enzymes (Buehner et aZ., 1973), which are believed to be derived from a common ancestral gene, a distinct pattern of three-dimensional structure is preserved even when homologies are difficult to establish by comparison of amino acid sequences. The existence of a common structure for the homology regions of immunoglobulins can be taken as supporting the mechanism of gene duplication postulated by Hill et al. (1966). Compared to the families of proteins mentioned above, immunoglobulins retain striking homologies of amino acid sequence, reflecting a relatively recent evolutionary appearance and, possibly, a strong selective pressure resulting in slow divergence. From structural analyses of L chains and of Fab’ New, it is reasonable to assume that all immunoglobulins possess a similar subunit structure in their L ( K or A) chains and H (y, a, p, etc.) chains. The strong homology between the amino acid sequence of p,-microglobulin and the constant homology regions of immunoglobulins (Smithies and Poulik, 1972; Peterson et al., 1972) implies that the structure of µglobulins will also be similar to that illustrated in Fig 7. Turning now to the detailed structure of the homology subunits, they can be described as consisting of several strands (seven in C L , CH1)of polypeptide chain running parallel to the length of each subunit and tightly packed against each other. About 50 to 60% of the amino acid residues are included in the framework of hydrogen bonds, which is characteristic of p-pleated sheet structures formed by antiparallel chains. Two irregular, roughly parallel p sheets surround a tightly packed interior of hydrophobic side chains including the intrachain disulfide bond that links the two sheets in a direction approximately perpendicular to their planes. In the C L subunit, for example, four hydrogen-bonded antiparallel chains (residues 116-120, 132-140, 160-169, 173-182) are included in one of the ppleated sheets. The other p sheet contains three antiparallel chains (residues 147-151, 193-199, 202-208). This structural feature is shown schematically for each of the homology subunits in Fig. 8. The V L subunit consists of a p sheet made up by four antiparallel chains including residues 1-5, 16-25, 53-66, 68-75 and another p sheet consisting of the hydrogen-bonded antiparallel chains that include residues 7-12,31-38, 41-46,83-91, and 94-108 (Fig. 8). The following hydrophobic residues occur in the center of the structure, between the two p sheets: Leu 4, Gln 6, Val 10, Val 18, Ile 20, Cys 22, Val 32, Trp 34, Leu 46, Phe 61, Val 63, Ala 70, Leu 72, Ile 74,

14

ROBERTO J. POLJAK

Leu 77, Ala 83, Tyr 85, Cys 87, Ser 89, Val 98, Thr 103, Leu 105, and Val 107. Very little helical structure can be seen in the subunits. The V, residues 26, 27, 27a, 27b, and 27c (Fig. 4) form one turn of a wtype helix. Since not all A chains have residues corresponding to 27a, 27b, and 27c, not all of them will have this one turn of helical conformation. The V, residues 78-82 and V, residues 87-91 also describe one turn of a helical conformation close to that of a 7~ helix or an a helix. This helical turn has also been reported for the homologous residues of a human K chain (Epp et al., 1974). Since the polypeptide chain folding of the homology regions is very similar, their sequences can be compared by matching residues that occur at the same position in their three-dimensional structure, 141 142 143 144 145 146

cL 171 170 172 169---173

-

52 51 50 49 48 47

VH

I

I

14

I.

118

1

121

2,j8-sg3------

122

209'

192

%]

183 184 185 186 187 188 189 190 191

159 158 157 156

7

I

45

44 41 42 43

I

201 200

I51 152 153 154 30 31 32

113 9311139 11411192 40

I

114

1

129 128 121

A

102 103 I01 105104---100 53 54 55 106 99 1z33 52 56 34'1151 57 107.-. 98

t

if:

161-- 180111134 164 181 133

76 77 78 79 80 81 82

7 2b 27 28 29

I

177 176 178 1501Z121 175'11179 149 122 174 180..-148 123 172173Z1181 147111124 171 182111146 125

167 18611342 166---187 141 140

I

981'

129 130

131

136 135 190 191 49;1~::1

195 196 197

J

FIG.8. Diagram of the hydrogen bonds between main chain atoms for V,, CL,V H , and C H l . Amino acid residues are grouped in two hydrogen-bonded clusters corresponding to the two p-sheets of each subunit (see text). Cysteine residues that participate in the intrachain disulfide bonds linking the two p-sheets are enclosed in squares; C,. residue 213 and CH1 residue 220 from the interchain disulfide bond are also enclosed in squares. Numbers refer to residues of the L-chain sequence given in Fig. 6 and to V Hand CH1residues as given in Fig. 9. (Reproduced from Poljak et al., 1974, with permission,)

J

15

X-RAY STUDIES O F IMMUNOGLOBULINS VL

1 LO 20 21 a b c 30 40 50 - - - - Z S V L T ~ P P S V S G A P - C ~ R V T l S C T G S S S N l G ~ G N H V K U Y ~ ~ L P G T A P K - L L l F H N N A - - - - - -

VH

1 10 20 30 40 50 60 - - - - Z V q L P E S G P E L V S P - G Z T L S L T C T G S T V S T F A V - Y l V W V R q P P G R G L E W l G Y V Q Y H G T S D T D T

CL

120 130 140 150 q P K * A P S V T L P P P S S E E L 9 A N K A T L V C L l S D F Y P G A V - T V A W K - - A D S S - - - - - - - ~ ~ - - ~ ~ - ~ - ~ ~

C"1

120 I30 140 150 160 A S T K G P S V P P L A P S S K S T S G G T A A L G C L V K D Y F P E P V - T V S U N - - - S G - - - - - - - - - ~ - - - - - - - - -

VL

_ _ _ _ -60R F S V S K S G - - - - - - - - - - S S A T L A l T G L q A E D E A D Y Y C q S Y D R S - ~ L R ~ V F G G G T K L T V L R

v,,

70 80 90 100 I10 118 - P L R S R V T W L V N T - S - - - - - - - K N ~ P S L R L S S V T A A ~ T A ~ ~ Y ~ A R B L ~ A G - ~ ~ B ~

CL

I60 170 180 190 200 210 214 - P V K A - - G V E T T T P S K q S N N K ~ A A S S Y L S L T P E q U K S H K S Y S C q V T H - - E G S T - V E K T - V A P T E C S

CHI

170 180 190 200 210 220 - A L T S - - G V H T P P A V L 9 S S G L Y S L S S V V T V P S S S L G T - ~ T ~ l C N V N H K P S N T K - V D K R - V E P K S C

110

70

80

90

100

FIG.9. Alignment of the amino acid sequences of the V,,, V,, CJ,, and CJ,l homology regions of Fab' New obtained by comparison of their three-dimensional structures. Parts of the tentative V, sequence given in this figure were obtained by sequence is as given by Edelman et al. (1969). The Nakashima et al. (1975). The one-letter code for amino acids is taken from Dayhoff (1972). (Reproduced from Poljak et al., 1974, with permission.)

In general, this method gives the same alignments that can be obtained by matching of identical or homologous amino acid residues except for some regions where such relations of identity or homology are not very clear by direct comparison of the sequences. Thus, this procedure is especially useful for the alignment of V and C homology regions (see Fig, 9). As mentioned above, the homology between the V and C regions is obscured by the presence of an extra length of polypeptide chain in the V regions (see Fig. 7) and by the fact that the interchain contacts between C Land CH1and between V L and V, are made by hydrophobic residues occupying different positions along the linear sequences of the V and C regions. AND INTRACHAIN DISULFIDEBONDS D. INTERCHAIN

The L chains of immunoglobulins are covalently linked to the H chains by a disulfide bond [with the exception of some IgA molecules where two L chains are linked to each other by a disulfide bond (Grey et al., 1968)l. The L-chain cysteine residue that contributes to this bond is at the C terminus of the chain (as in human K chains) or adjacent to it (as in human h chains) (see Fig. 8). In different isotypes of H chain, and in different animal species, the cysteine residues that completes the S-S bond is either at position 214 or at about position 131 (Fig. 10). The bonding scheme illustrated in Fig. 10A applies to human IgGl (Steiner and Porter, 1967) immunoglobulins such as IgG New. The interchain disulfide bond illustrated in Fig. 10B, in which the H-chain cysteinyl residue occurs at position 131 is found in human IgG2, IgG3, and IgG4 (De Preval

109

16

ROBERTO J. POLJAK 213

L

s

A

I

213

L B

H

s/s 131

FIG.10. Diagram of two different patterns of interchain disulfide bonds of immunoglobulins (see text).

et al., 1970), in human IgM (Pink and Milstein, 1967; Putnam et al., 1971), in rabbit IgG (O’Donnel et al., 1970), in guinea pig IgG2a (Birshtein et al., 1971),and in mouse IgG2a and IgG2b (De Preval et al., 1970). In the three-dimensional model of Fab’ New the H-chain Cys 214 is at a distance of about 6 A. from L-chain Cys 213 to which it is linked by a disulfide bond. However, position 131 in CHI also occurs at a distance of 6 8, from L-chain Cys 213, so that its replacement by a cysteinyl residue could lead to an alternative interchain disulfide bond as is found in the immunoglobulin molecules listed above. Unusual intrachain disulfide bonds that have been observed in several immunoglobulins can be explained on the basis of the model of Fab New (Poljak et al., 1973). Among this is the intra-H-chain disulfide bond observed in rabbit IgG, linking the polypeptide chain at positions 131-221 (O’Donnel et aZ., 1970). An unusual disulfide bond observed in the V Hregion of a human y l chain from IgG Daw (Press and Hogg, 1970) can also be explained by the close spatial proximity (about 6 A.) of the homologous residues in Fab New. Perhaps the most interesting interchain disulfide bond that has been reported is the one that links VL position 80 to C L position 171 in rabbit antibodies of restricted heterogeneity (Poulsen et al., 1972; Appella, 1973). A comparison of the sequences of these rabbit K chains and the human h chain from IgG New indicates that Cys 80 and Cys 171 in rabbit K chains correspond to Ala 79 and Asn 172, respectively, in IgG New. The side chains of V,, Ala 79 and CI, Asn 172 face each other, and the distance between their a-carbon atoms is about 5.5 A., compatible with the presence of a disulfide bond linking the two homology subunits as observed in rabbit K chains. Thus, the Fab’ model provides an adequate structural framework for the various patterns of interchain and intrachain disulfide bonds that have been established by sequence analyses and gives further support to the postulate that K and h L chains have the same overall three-dimensional structure and that the V, and C H regions of dif-

X-RAY STUDIES OF IMMUNOGLOBULINS

17

ferent classes of H chains (a, y, p, 6, etc.) also have the same overall pattern of polypeptide chain folding.

E. LOCATION O F ISOTYPIC AND ALLOTYPIC MARKERS Human X chains have been classified by serological criteria into two types, namely Oz+ and Oz-. These two serological types correlate with the presence of a Lys or an Arg residue, respectively, at position 191 in the C h sequence (Apella and Ein, 1967). Amino acid replacements have also been detected at position 154, where a Ser residue (Kern- marker) is sometimes replaced by a Gly residue [Kern+ (Hess and Hilschmann, 1970)l. These differences in sequence and serological type are under the control of nonallelic structural genes and are usually called isotypic markers. The positions of the Kern and Oz markers in the structure of C Lare shown in Figs. 4 and 7. They occur in an exposed region of the molecule, accessible to solvent and reagents and separated by a distance of 8 A. Their location is consistent with the fact that they constitute surface determinant groups that can be detected serologically. Human K chains have been classified into three serological types, namely InV(l), InV(2) and InV(3), which are under the control of three alleles, InV',*,InV', and InV3 of a C , structural gene (Ropartz et al., 1964). The InV(1,2) and InV(3) allotypes have been correlated with a Leu/Val substitution, respectively, at position 191 (Milstein, 1966; Baglioni et aZ., 1966), Study of the K Bence Jones protein Cro of allotype InV(1,2-,3-) indicates that the ZnV' allele specifies a valine residue at position 153, which is most frequently occupied by alanine in K chains [InV (1,2,3-)] (Milstein et al., 1974). The antiInV(2) antiserum cannot react with the InV(1)antigen which is taken to indicate (Milstein et d.,1974) that it recognizes Leu 191 encompassing Ala 153. By comparison with the C h structure and in agreement with this interpretation, Leu 191 and Val 153 are exposed antigenic determinants separated by a distance of about 10 A., so that they could be recognized by a single antiallotypic antibody molecule. The a l , a2, and a3 allotypic markers of rabbit H chains are of great interest because they occur in the V Hregions of different classes of H chains ( y , a, p, etc.) (Todd, 1963) and, thus, are frequently interpreted as indirect evidence in support of the existence of separate V, genes, which together with fewer C,, genes (responsible for y, a, p isotypes) specify a complete polypeptide chain. Location of the allotypic markers or of the allotype-associated sequences in a threedimensional model of the V H structure depends primarily on the identification of amino acid alterations which are under the control of

18

ROBERTO J. POLJAK

the a’, a2,and a3 alleles. At the present time there is no clear consensus that this aim has been unequivocally achieved. Following the report of Jaton et al. (1973), it appears that the a’, a2, and a:
F.

STRUCTURE OF THE HYPERVARIABLE REGIONS AND THE ACTIVESITE

A comparison of the primary structure of L chains has revealed that residues around positions 30, 50, and 95 are hypervariable (Wu and Kabat, 1970; Kabat and Wu, 1971) so that the sequences of amino acid residues that occur around those positions are unique for a given L chain. A similar conclusion was obtained by comparison of H chains, although since fewer H-chain sequences have been studied the location of hypervariable regions was less definitive (Kabat and Wu, 1971). In adJition, comparison of the sequences of human subgroup I11 H-chain sequences (Kehoe and Capra, 1971) indicated the presence of a hypervariable region around residues 86-91 which had not been detected before. Amino acid sequence determination and affinity-labeling studies have led to the recognition of three hypervariable regions in guinea pig H chains (Cebra et al., 1974) that occur around positions 25-30, 55-65, and 100-110. The hypervariable regions of immunoglobulins were postulated to specify the conformation of the antigen-binding sites. Support for this postulate was obtained by affinity-labeling studies (Singer et al., 1971; Goetzl and Metzger, 1970; Franek, 1971; Ray and Cebra, 1972; Givol et al., 1971; Fleet et al., 1972) which, in general, labeled residues of V, and V, sequences coincident with, or adjacent to, hypervariable positions. Cross-linking of H and L chains by affinity labels (Givol et al., 1971; Hadler and Metzger, 1971) clearly indicated that H and L chains occur in close spatial proximity at the antigen-binding site or near it. Independently of the results outlined above, the study of human myeloma proteins (Slater et al., 1955) and of induced antibodies (Kunkel et al., 1963; Oudin and Michel, 1963; Oudin, 1966) revealed the existence of “idiotypic” antigenic determinants that are unique and characteristic of every molecule studied. Quantitative investiga-

X-RAY STUDIES OF IMMUNOGLOBULINS

19

tions of the reaction between rabbit anti-p-azobenzoate antibodies and their anti-idiotypic antisera showed that the reaction could be inhibited by the specific benzoate hapten and some of its derivatives (Brient and Nisonoff, 1970). A main conclusion derived from this finding was that the location of the hapten-binding site partially or totally overlapped with that of the idiotypic determinants. Furthermore, the work of Potter and colleagues (Barstad et al., 1974) has shown that mice myeloma proteins that share idiotypic (and hapten binding) specificities also share a common amino acid sequence through the first hypervariable region of both the L and H chains. In a study of mice myeloma proteins with antidextran activity, Carson and Weigert (1973) showed that idiotypic specificity resides in both L and H chains. Reconstituted immunoglobulin molecules in which the L chain differed from the original L chain by substitution of three amino acid residues (at positions 25, 52, and 97) had a recognizably altered idiotypic constitution. The results outlined above can be correlated with the highresolution model of Fab’ New. In the following paragraphs, we briefly discuss this attempted correlation in terms of the location of the regions of hypervariable sequences, the possible structural basis for the recognition of idiotypic determinants by an anti-idiotypic antibody, the conformation of the active site, and the spatial location of amino acid residues that have been marked by affinity labeling of antibodies and myeloma proteins. In the model of Fab’ New, the regions of hypervariable sequences of both H and L chains are fully exposed to the solvent, at one end of the molecule (see Fig. 4).These regions occur at adjacent, exposed bends of tightly packed polypeptide chains that are part of the @pleated sheets of V, and V L .The amino acid side chains projecting into the solvent at this end of the molecule constitute a unique pattern for each immunoglobulin. The hypervariable positions in the L chain are related to the hypervariable positions of the H chain by an approximate twofold rotation symmetry, with the exception of H-chain residues 86-91 which are unrelated to the other hypervariable positions (see Fig. 4). This region is relatively flat, with protruding side chains and with a central channel or pocket 15 A. long, 6 d;. wide at its center, and with a relatively shallow depth, about 6 A. Light chain residues 27-30 delineate the “upper” limit of this channel. Its sides are made of L-chain residues 90-95 to the left (see Figs. 11 and 13) and H-chain residues 102-107 to the right. The lower end of this site is made up by H-chain residues 55-65 and

30-33.

20

ROBERTO J. POLJAK

FIG. 11. A view of the active site region of IgC New. Residue numbers for V,, and V,. correspond to those of Fig. 9.

The area occupied by the positions of hypervariable sequence is about 20 X 25 A. The geometry of this area (see Figs. 11 and 13) will be modified by the presence of different side chains and, more markedly, by deletion (or addition) of amino acid residues to the hypervariable regions of V, and V,. For example, in human A chains, positions 27a, 27b, and 27c (Fig. 6) are frequently deleted. Since these residues are part of a single helical turn, their deletion can be easily accommodated without a major change in the path of the polypeptide chain. However, contacts with antigens will be appreciably modified by this structural change. A similar situation arises in human and mouse K chains in which additional residues are present in the first hypervariable region (Dayhoff, 1972; Barstad et al., 1974) and to deletions or additions that occur at positions 96-97 in human A chains. Deletions (or additions) of amino acid residues are a widespread features of the hypervariable sequences of H chains and they must play an important role in determining the dimensions and the nature of the active site and in defining different antigen-binding

X-RAY STUDIES OF IMMUNOGLOBULINS

21

specificities. The V Hhypervariable regions, extending from positions 50 to 60 and 100 to 110, are longer than the homologous regions of L chains. In particular, this last hypervariable region in VH sequences has been found to consist of a variable number of amino acid residues, ranging from 13 to 20 when counted from (and including) Cys 96 to Trp 108. The homologous loop (Cys 87-Phe 99) contains 11-13 residues in V, and 11 residues in V,. As indicated in Figs. 11 and 13, this hypervariable loop of VHdoes not conform to the approximate local twofold axis of symmetry that relates V, to V H Instead, . it bends toward the L chain making the active site narrower than the corresponding region of L-chain dimers (Schiffer et al., 1973; Epp et al., 1974). In the model of Fab’ New the H chain appears to make a larger contribution to the definition of the active site pocket than does the L chain, and by virtue of a variable length in the region of residues 100-110 different H chains will alter the width and the depth of the active site pocket. Thus, different H chains that pair with the same L chain in induced antibodies (Friedenson et al., 1973) could modulate specificity and affinity not only by a change in the kind of amino acids present in this sequence but also by alterations in the length of this segment of the polypeptide chain. Evidently, L chains will also modulate specificity and affinity and they also exhibit patterns of variation in the length of residues near (or at) the first hypervariable region (Barstad et al., 1974; McKean et al., 1973; Putnam et al., 1973). An additional hypervariable region of H chains extending from position 86 to 91 (Capra and Kehoe, 1974) is not close to the other V Hor V, hypervariable sequences discussed above although it occurs at an exposed bend in an “outside” region of the H chain. It is perhaps interesting that these residues occur at the positions occupied by some of the rabbit allotypic markers (or allotype-related sequences). As mentioned above, affinity-labeling studies (Singer, 1967; see Givol, 1973, for a comprehensive review of these studies) of specific antibodies and myeloma proteins have labeled residues of V, and VH in the hypervariable regions or adjacent to them. For example, the often labeled Tyr 32 of L chains (Goetzl and Metzger, 1970; Franek, 1971; Haimovich et al., 1972) is next to the structural pocket discussed above and openly accessible to solvent and reagents. Lightchain Tyr 93 is inside the pocket at the combining site but less accessible to solvent and reagents as was inferred from affinity-labeling experiments (Franek, 1971). Heavy-chain residues Lys 54 (Haimovich et al., 1972), Tyr 33, Lys 59, Tyr 60, and some residues at positions 99-119 (Cebra et al., 1971, 1974) are also accessible to solvent

22

ROBERTO J. POLJAK

and immediately adjacent to the combining site. Other residues that have been labeled, such as Tyr 85, Tyr 86, and Cys 87 in the L chain and Tyr 96, Tyr 97, Cys 98, and Ala 99 in the H chain (Singer et al., 1971; Press et al., 1971) are at the core of their structural subunits and are not accessible to solvent, It is encouraging to see that, in general, predictions about the residues that should be located at the active site, based on analyses of amino acid sequence data and on affinity-labeling experiments, have been confirmed and correlated with features of three-dimensional structure by X-ray diffraction studies. Since the model of the active site discussed above was obtained from the structural analysis of only one immunoglobulin, how generally applicable can one expect this model to be? The answer to this question clearly resides in the sequence characteristics of the hypervariable regions discussed above. The general size of the area where the hypervariable residues occur can be expected to remain roughly constant. As outlined above, the dimensions of the structural pocket (the active site) will be determined by the sequences of both the L and H chains and by the particular combination of an L and H chain that pair to make an antibody molecule. The length, and especially the width and depth of the active site, will be determined by the variation in amino acid sequences and in particular by the deletions (or additions) that may be present in the hypervariable regions. On the basis of amino acid sequences currently available, it can be concluded that the “bottom” of the active site will be formed by the side chains of residues, such as Tyr (or Trp)90 in L(X) chains and Trp 47 and residue at position 99 in H chains, and other residues which occur in this area. The proximity and the nature of the residues that occur in some sequences of L and H chains suggest that not all combinations of L and H chains may be structurally possible (at least without some conformational change) because of steric hindrance. From this point of view, it is clear that not enough information is currently available about the amino acid sequence requirements for the association of L and H chains, By using the active site of Fab‘ New as a basic structural model, it is possible to attempt a correlation of structure and function in the well-characterized MOPC 315 anti-2,4-dinitrophenyl (DNP) murine myeloma protein (Eisen et al., 1968; Goetzl and Metzger, 1970; Green et al., 1971; Haimovich et al., 1972; Francis et al., 1974). The L(X) chains of MOPC 315 and of IgG New are highly homologous and contain the same number of residues in the first and third hypervariable regions. A comparison of the tentative sequence of V, New

X-RAY STUDIES OF IMMUNOGLOBULINS

23

(Fig. 9) and that of MOPC 315 (Francis et al., 1974) shows that the third hypervariable region of V Halso includes the same number of amino acids in both chains. It is, therefore, feasible to fit the MOPC 315 sequence to the structural model of V Land V HNew. The model of the MOPC 315 binding site that can be built from this comparison includes several distinctive features. Light-chain Tyr 34 is at the upper limit of a narrow, shallow pocket at the active site, L-chain Trp 98 and Phe 103 and H-chain Phe 105 are located at the 6-10 A. deep “bottom,” H-chain Trp 47 and Phe 50 constitute the lower limit, and other aromatic residues, such as L-chain Phe 99, H-chain Tyr 104, and Phe 34, contribute to the “sides” of the binding site (to follow this description and obtain a picture of the site, see Figs. 11 and 13). The high density of adjacent aromatic side chains present at this site is striking and correlates with the observed specificity of MOPC 315 IgA for DNP and other haptens including benzene and naphthalene aromatic ring moieties. The shallow depth of the active site (6-10 A.) is in agreement with the electron-microscopic study of a complex between MOPC 315 IgA and a bifunctional bis(DNP-@-alanyl)diaminosuccinate hapten (Green et al., 1971) in which the DNP groups occur at a short distance (15 A.) from each other and join the Fab arms of two different molecules of IgA through their active centers. On the basis of the model of Fab’ described here, antigenic idiotypic specificity should reside in the exposed, unique stereochemical conformation provided by the combination of hypervariables sequences of a given pair of L and H chains. The individually specific conformational variations affecting V Hand V, should easily be recognized by a molecule of complementary structure, i.e., an antiidiotypic antibody. Since haptens are bound to this region of idiotypic specificity (see Section V,G) and cannot be deeply buried in it, the binding of a specific hapten will compete with that of an antiidiotypic antibody and will block recognition of the immunodeterminant groups by the anti-idiotypic antibody (Brient and Nisonoff, 1970; Carson and Weigert, 1973).

G. STRUCTURE OF A LIGAND-FAB’ COMPLEX Human myeloma immunoglobulins have been shown to react with a variety of haptens and antigens in a way that closely resembles the behavior of induced antibodies (Eisen et al., 1967; also, see reviews by Metzger, 1969; Krause, 1970). Several immunoglobulins secreted by induced plasma cell tumors in Balb/c mice (Potter and Boyce, 1962; Potter, 1968, 1970) have also been

24

HOBERTO J. POLJAK

FIG. 12. Chemical structure of vitamin K,OH [3-(3’-hydroxy-3’, 7’, ll‘, 15’-tetramethyl hexadecyl) 2-methyl-1,4-naphthoquinone], a ligand of IgG New. (From Amzel et d.,1974.)

found to react selectively with haptens and to exhibit properties similar to those of induced antibodies, including common idiotypic specificities (Cosenza and Kohler, 1972; Lieberman et al., 1974). The general picture that has resulted from these various studies is that myeloma immunoglobulins share structural and functional properties with induced antibody molecules and that it is reasonable to assume that extensive screening of a myeloma protein may eventually clarify its antigen (hapten)-binding specificity. A key point in defining the specificity for a myeloma protein is that the affinity constant for the myeloma immunoglobulin-hapten complex should be comparable to that of induced antibodies, i.e., higher than 104-105 literslmol. Lower-affinity constants, which are frequently observed for several ligand-protein complexes, will not satisfy this condition and would not be assumed to be of physiological significance (Eisen et al.,

1970). A screening assay to test the possible ligand-binding activity of IgG New (Varga et al., 1974) led to the identification of several compounds such as uridine, orceine, menadione, and others that bind with a low-affinity constant ( K 1 x lo3 liters/mol.) and a y-hydroxy derivative of vitamin K,, namely vitamin K,OH (Fig. 12) which binds with a higher-affinity constant ( K = 1.7 X lo5 liters/mol.) Crystallographic analysis of the vitamin K,OH-Fab’ New complex to 3.5-A;.resolution (Amzel et al., 1974) indicated that vitamin K,OH is bound to the active site region of Fab’ New. The menadione moiety of vitamin KIOH is bound to the upper part of the pocket at the center of the active site and surrounded by hypervariable residues of both the L and H chains (Fig. 13). It makes close contacts with the side groups of Tyr 90 and the peptide chain of Gly 29 and Asn 30 in V, and with residues 100-102, 47 (a constant Trp residue in human V Hsequences), 50, and 58 in V,. Crystallographic studies of the Fab’ New complexes with the low-affinity ligands menadione, orceine, and uridine indicated that these ligands bind to the same part of the active site to which the methyl naphthoquinone rings of vitamin K,OH are bound. Since the affinity constants for these compounds

-

X-RAY STUDIES OF IMMUNOGLOBULINS

25

are substantially lower than that observed with the Fab’ New-vitamin K,OH complex it was concluded that the total binding energy of the Fab’ New-vitamin KIOH compIex is derived from the contacts made by the quinone rings and by the phytyl chain with the Fab’ fragment (Amzel et al., 1974). No major conformational change was observed after binding of vitamin K,OH to Fab’ New. These results are similar to those obtained by Padlan et al. (1973), who examined the phosphorylcholine-IgA McPC 603 (mouse myeloma protein)

FIG. 13. Drawing of vitamin K,OH bound to the active site of IgG New, based on a model built from a 3.5 A. resolution difference Fourier map. The HI,H,, and H , designate hypervariable regions of the H chain, and L,and L,,the first and third hypervariable segments of the L chain, respectively. Light-chain Tyr 90 and Arg 95 are at the bottom of the active site, between the L and H chains, Heavy-chain residues 35 and 54 (a constant Trp vesidue in human H chains) are in close contact with the phytyl chain of vitamin K,OH. See text for a more complete description of this complex. (Reproduced from Amzel et al., 1974, with permission.)

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complex by X-ray diffraction techniques to 4.5-A. resolution and concluded that the phosphorylcholine hapten binds to a cleft located between the VL and V, homology subunits, without inducing major conformational changes in the Fab' fragment of IgA McPC 603. Crystallographic studies of the hapten-Fab' complexes provide a model for interactions between antigens (haptens) and antibody molecules. These interactions occur as close contacts with a large number of amino acids at the hypervariable positions of both L and H chains, involving mostly van der Waals interactions. The interactions with haptens of variable length is consistent with an active site that is divided into subsites (Schechter, 1971; Givol, 1973). Even a relatively long hapten molecule such as vitamin KIOH will not occupy the entire length of the active site. Finally, the size of the active site estimated by the experiments of Kabat (1966) and of Weigert et al. (1974) is in agreement with the model obtained by study of the Fab' New-vitamin KIOH complex.

H . PATTERNSOF CHANGE Some of the variability observed in amino acid sequences of immunoglobulins can be discussed in terms of three-dimensional structure. Since L chains have been more extensively sequenced than H chains, it is easier to discuss variable and constant features of V,, sequences in relation to the three-dimensional structure of Vh New. A number of Gly residues appear as constant features of human h-chain sequences. Glycine residues are known to occur with high frequency in p bends or hairpin turns, in which a polypeptide chain is reversed by about 180" (Crawford et al., 1973). Nearly all of the invariant Gly residues of Vh occur at hairpin bends around positions 14-15, 27-30, 39-40, 67-68, and in a bend (close to 90" around residues 75-76 in V;, New. Another bend, at positions 92-93, includes residues of the third hypervariable region. Most of these bends involve a constant or nearly constant Pro-Gly or Ser(Thr)-Gly sequence. A similar conclusion has been derived from the crystallographic study of a V, fragment (Epp et al., 1974). The constant sequence Phe-Gly-Gly-Gly at positions 99-102, which is not part of a bend, can be explained by close intrachain contacts of the Gly residues with other invariant residues of VL or with the main polypeptide chain, with the exception of Gly 101 which could be replaced by a bulkier residue as is observed in K chains. Phenylalanine 99 is part of an interchain contact and on this basis it can be considered important for VL-VH assembly. Similar considerations apply to the homologous sequence Trp-Gly-X-Gly, positions

X-RAY STUDIES OF IMMUNOGLOBULINS

27

101-111 of VH(related to V Lpositions 99-102 by a local twofold axis of symmetry). Other residues such as Tyr 35, Gln 37, Ala 42, Pro 43, and Asp 84 which are constant or nearly constant in V h are involved in close contacts with the V Hsubunit. Constant residues, such as Asp 81 and Tyr 85, Glu 82 and Tyr 142 (in C,) make intrachain hydrogen bonds. The hydrophobic amino acid side chains that occur in the center of the structure, between the two p sheets, are invariant or are replaced by side chains of similar hydrophobic residues. All of these constant or nearly constant residues occurring at bends in the internal hydrophobic core or contributing to intersubunit and intrasubunit bonds must be important for the preservation of structure. They represent more than 50% of the residues of the V h sequence. Mutations that alter these residues and result in different combinations which are compatible with the requirements of a constant three-dimensional structure can best be explained by a process of evolutionary germ-line gene divergence, rather than by somatic mutation. In marked contrast to these requirements, the nature of the amino acid side chains at hypervariable positions is not affected by any visible structural limitations. IN CONFORMATION I. CHANGES

Green (1969) has recently reviewed the evidence obtained by electron microscopy concerning possible major conformational changes in the immunoglobulin molecule following the binding of antigen. Many papers dealing with this subject have appeared since then, and this section is not intended to review this subject in detail. Among others, papers by Pilz et al. (1970, 1973), Warner and Schumaker (1970), Ohta et al. (1970), Padlan et al. (1973),and Amzel et al. (1974),deal with subjects that are directly related to problems of conformation and conformational changes. As discussed in a previous section, no major conformational changes were observed in Fab’ fragments after binding of ligands (Padlan et al., 1973; Amzel et al., 1974). However, as discussed by these authors, the occurrence of such changes cannot be ruled out because ( I ) these experiments were carried out in the crystalline state, in which the Fab structure may already be present in a conformationally altered state, and (2) the monovalent haptenic groups, phosphorylcholine and vitamin KIOH, can only interact with some of the side chains of the active center and, thus, they may not be capable of triggering a conformational change. The results of the crystallographic studies that are reviewed above suggest that L chains and Fd chains can exist in more than

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I

FIG.14. A view of the *carbon backbone of Fab‘ New. The V and C, domains, the L chain (open trace), the Fd‘ chain (thick trace), and the local, approximate twofold axes (dashed line) relating the V Lto the V,, subunit and the CI, to the C,,1 subunit are shown. The switch regions of both chains are indicated by short arrows. A possible motion of the V and C, domains is indicated by the long arrow (see text). (Reproduced from Poljak et al., 1974, with permission.)

one conformation. In the Mcg L-chain dimers (Schiffer et al., 1973), two identical L chains assume different conformations such that one of them makes an angle larger than 90”between the major axes of its V , and CLsubunits, whereas in the other L chain the corresponding angle is smaller than 90”. As discussed in a preceding section, the Fab structure can approximately be described as a tetrahedral arrangement of homologous subunits covalently linked (V, to C Land V, to CH1) by linear accessible regions of the polypeptide chains (“switch” regions) in which the Fd chain is bent to a larger extent than the homologous L chain. On the basis of these observations, which indicate flexibility at the switch regions, and assuming that a conformational change takes place after antigen binding, it is possible to visualize that the Fd chain could undergo a “closingopening” movement as illustrated in Fig. 14. Since a disulfide bond joining V, to CLhas been established for some rabbit antibody molecules (Poulsen et al., 1972; Apella, 1973) the flexibility of the L chain can be assumed to be more limited than that of the H chain where such a bond has not been demonstrated. In summary, the Fab structure suggests a potential for a confor-

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mational change resulting in a relative movement of structural subunits (as has been demonstrated for hemoglobin molecules) (Perutz, 1970). However, the existence of such a change remains to be demonstrated in a convincing way. VI. Summary and Conclusions

The three-dimensional structure of the Fab‘ fragment of a human myeloma immunoglobulin (IgG1 (A)) has been elucidated to atomic resolution by X-ray diffraction analysis. Fragment Fab’ consists of two globular domains, V and C1, separated by an interdomain space accessible to solvent (see Figs. 2 and 4). The V and C1domains each consist of two globular subunits corresponding to the VLand V, and to the CLand CHI homology regions, respectively. These four homology subunits are arranged in a tetrahedral configuration and are linked (VLto CL and V Hto CH1)by linear stretches of polypeptide chain which correspond to the switch regions. The four homology subunits of Fab’ closely resemble each other, sharing a basic pattern of polypeptide chain folding or immunoglobulin fold (see Fig. 7). The existence of this underlying structural pattern further supports the postulate that a gene-duplication mechanism gave rise to the subunit structure of immunoglobulins. Comparison of the three-dimensional structures of the homology subunits allows alignment of their amino acid sequences (see Fig. 9) and indicates that the V, and VH subunits include an additional loop of polypeptide chain (see Fig. 7). A prominent feature of secondary structure in each homology subunit is the presence of two irregular p sheets traversed by strands of antiparallel polypeptide chains and including -60% of the amino acid residues. These roughly parallel p sheets surround a hydrophobic interior of invariant or semi-invariant amino acid side chains and are joined at the center of each subunit by the intrachain disulfide bond. Different patterns of interchain disulfide bonds linking H and L chains, and unusual intrachain disulfide bonds that are observed in other immunoglobulin molecules can be explained with the model of Fab’. This observation, together with the similarity of the folding pattern of the homology regions strongly suggest that K and A L chains of different animal species have the same overall threedimensional structure and that the VH and CH regions of different classes of H chain (a,y, p, etc.) also have the same overall pattern of tertiary structure. A large number of the amino acid residues of the V H and VL regions that are invariant or semi-invariant are part of important

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ROBERTO J. POLJAK

structural elements, such as bends in the polypeptide chains, intrachain and interchain contacts, hydrogen bonds, and hydrophobic interactions. Mutations that alter these residues are believed to occur during a process of evolutionary germ-line gene divergence rather than during the course of somatic development. By contrast, the amino acid residues in the hypervariable segments of the L and H chains occur at an exposed end of the molecule, in a region where the coiled polypeptide chains are least subject to structural constraints. The number and nature of the amino acid residues present in this region will determine the antigen binding and the idiotypic specificities of immunoglobulins and antibodies. Hapten binding, which is shown to involve close interactions with side chains and polypeptide chain atoms of this exposed hypervariable region, can be expected to interfere with anti-idiotypic reactions. The arrangement of the Fab’ subunits, linked (V, to CLand V, to CH1)by narrow stretches of polypeptide chain (the switch regions) suggests flexibility and a potential for conformational changes by a relative movement of the homology subunits. Knowledge of the structure of immunoglobulins per se is of great interest to immunologists and immunochemists. It is hoped that such knowledge will provide a frame of reference for detailed correlations between structure and function and for investigations of physiological reactions and mechanisms of the immune response.

ACKNOWLEDGMENTS I am grateful to my colleagues who helped achieve the results summarized above through several years of work in this laboratory. Dr. A. Nisonoff provided the Fab crystals used in the first stages of this work and continued interest and advice. Dr. L. M. Amzel and Mr. F. Saul critically reviewed this manuscript and made corrections and improvements that are incorporated in the final text.

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Buehner, M., Ford, G. C., Moras, D., Olsen, K. W., and Rossmann, M. G. (1973). Proc. Nut. Acad. Sci. U . S. 70, 3052. Capra, J. D., and Kehoe, J. M. (1974). Proc. Nut. Acad. Sci. U . S. 71, 845. Carson, D., and Weigert, M. (1973). Proc. Nut. Acud. Sci. U . S. 70, 235. Cebra, J. J., Ray, A., Benjamin, D., and Birshtein, B. (1971). I n “Progress in Immunology” (B. Amos, ed.), p. 269. Academic Press, New York. Cebra, J. J.. Koo, P. H., and Ray, A. (1974). Science 186, 263. Chen, B. L., and Poljak, R. J. (1974). Biochemistry 13, 1295. Cosenza, H., and Kohler, H. (1972). Science 176, 1027. Crawford, J. L., Lipscomb, W. N., and Schellman, C. G. (1973). Proc. Nut. Acad. Sci. U . S . 70, 538. Dayhoff, M. 0. (1972). “Atlas Protein Sequence and Structure,” Vol. 5. Nat. Biomed. Res. Found., Silver Spring, Maryland. De Preval, C., Pink, J. R. L., and Milstein, C. (1970). Nature (London) 228, 930. Dickerson, R. E. (1964). In “The Proteins” (H. Neurath, ed.), 2nd Ed., Vol. 2, p. 603. Academic Press, New York. Edelman, G. M., and Gall, E. W. (1969). Annu. Reo. Biochem. 38,415. Edelman, G. M., Cunningham, B. A., Gall, E. W., Gottlieb, P. D., Rutishauser, U., and Waxdal, M. J. (1969). Proc. Nut. Acad. Sci. U . S. 63, 78. Eisen, H. N., Little, J. R., Osterlend, C. K., and Simms, E. S. (1967). Cold Spring Harbor Symp. Quant. Biol. 32,75. Eisen, H. N., Simms, E. S., and Potter, M. (1968). Biochemistry 7, 4126. Eisen, H. N., Michaelides, M. C., Underdown, B. J., Schulenburg, E. P., and Simms, E. S. (1970). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 29, 78. Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., and Huber, R. (1974). Eur. J. Biochem. 45,513. Epstein, C. J,, Goldberger, R. F., and Anfinsen, C. B. (1963). Cold Spring Harbor Symp. Quant. Biol. 28, 439. Fleet, G. W. J., Knowles, J. R., and Porter, R. R. (1972). Biochem. J. 128, 499. Francis, S. H., Leslie, R. G. Q., Hood, L., and Eisen, H. N. (1974). Proc. Nut. Acad. Sci. U . S. 71, 1123. Franek, F. (1971). Eur. J. Biochem. 19, 176. Friedenson, B., Appella, E., Takeda, Y., Roholt, 0. A., and Pressman, D. (1973). J . Biol. Chem. 248, 7073. Givol, D. (1973). Contemp. Top. Mol. Zmmunol. 2,27. Givol, D., Strausbauch, P. H., Hunvitz, E., Wilchek, M., Haimovich, J., and Eisen, H. N. (1971). Biochemistry 10, 3461. Goetzl, E. J., and Metzger, H. (1970). Biochemistry 9, 3862. Goldstein, D. J., Humphrey, R. L., and Poljak, R. J. (1968). J. Mol. Biol. 35, 247. Green, N. M. (1969). Aduan. Immunol. 11, 1. Green, N. M., Dourmashkin, R. R., and Parkhouse, R. M. E. (1971). J. M o l . B i d . 56, 203. Grey, H. M., Abel, C. A,, Yount, W. J., and Kunkel, H. G. (1968).J.E x p . Med. 128, 1223. Hadler, N., and Metzger, H. (1971). Proc. Nut. Acad. Sci. U . S . 68, 1421. Haimovich, J . , Eisen, H. N., Hurwitz, E., and Givol, D. (1972). Biochemistry 11, 2389. Hartley, B. S. (1970). Phil. Trans. Roy. Soc. London, Ser. B 257, 77. Hess, M., and Hilschmann, N. (1970). Ho)qie-Seyler’s 2. Physiol. Chem. 351, 67. Hill, R. L., Delaney, R., Fellows, R. E., Jr., and Lebowitz, H. E. (1966). Proc. Nut. A m d . Sci. U . S. 56, 1762.

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Rabbit Immunoglobulin Allotypes: Structure, Immunology, and Genetics THOMAS J. KINDT' T h e Rockefeller University, N e w York, N e w York

I. Introduction . . . . . . . . . . . . . A. Discovery. . . . . . . . . . . . . B. Definitions . . . . . , . . . . . . C. Nomenclature , . . . . . . . . . . . D. Allotypic Groups . . . . . . . . . . . E. Immunizationand Detection Procedures . . . . . . 11. Structural Correlates of Allotypic Determinants. . . . . . A. Structural Features of Rabbit Immunoglobulins . . . . . B. Nature of Allotypic Variation. . . . . . . . . C. Structural Correlates of Heavy-Chain Allotypes . , . . . D. Structural Correlates of Light-Chain Allotypes . . . . . 111. Antigenic Determinants of Rabbit Allotypes . . . . . . A. Allotypic Determinants on Light Chains of Homogeneous Antibodies. B. Subspecificities of Heavy Chain Allotypic Determinants . . . IV. Genetic Relationships among Allotypes , . . , . . . A. LinkageGroups . . . . . . . . . . . B. TheToddPhenomenon . . . . . . . . . . V. Allotypes and the Immune Response . . . . . . . . A. Allelic Exclusion . . . . . . . . . . . B. Allelic Selection . . . . . . . . . . . VI. Allotype Suppression . . . , . . . . . . . VII. Idiotypes and Allotypes . . , . . . . . . . V-Region Allotypes and Idiotypes . , . . . . . . VIII. Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . .

35 35 37 38 40 45 48 48 53 54 60 61 63 64 65 66 67 70 70 72 73 75 76 78 81

I. Introduction

A. DISCOVERY About 20 years ago at the Institut Pasteur in Paris, Oudin (1956) observed that rabbit antibodies were immunogenic when injected into certain other rabbits. Oudin named this phenomen a l l o t y p y , and the intraspecies antigenic determinants on the antibodies he named dotypes. Almost simultaneously in Lund, Grubb (1956) discovered a similar phenomenon for human immunoglobulins. Just prior to these initial observations of allotypy, Slater et aZ. (1955) had reported Established Investigator of the American Heart Association. The author's work was supported by Public Health Service Grants A1 11995 and A1 11439 and by a grant in aid from the American Heart Association.

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THOMAS J. KINDT

the phenomenon of individual antigenic specificity, which was later to be called heterologous idiotypy. Prior to these three key observations, studies on the antigenicity of molecules within a species had been confined almost entirely to the carbohydrate antigens of red blood cells. Graft-versus-host phenomena had not yet been clearly described in immunological terms and histocompatibility antigens were virtually unknown. These discoveries of allotypy and individual antigenic specificity completely laid to rest the notion that serum proteins (or, indeed, any proteins) of individual members of a species were necessarily identical. Equally important, and more relevant to the present discussion, is the fact that these antigenic markers provided invaluable tools for probing the genetic control of antibody synthesis. Easily detected genetic markers were now available for immunoglobulins and studies on their patterns of inheritance could begin in earnest. Within a few years following its discovery, the study of allotypy progressed rapidly. As basic information in immunochemistry became available, studies using allotypes extended this information to the genetic level. Soon after Oudin’s two groups of rabbit allotypes (Oudin, 1960a) were shown to be unlinked (Dubiski et al., 1962), it was shown that the alleles of these two groups represented markers present on the recently described heavy (H) and light (L) chains of IgG molecules (Fleischman et al., 1962; Stemke, 1964). Thus the two chains of the antibody molecules were encoded by genes that assorted independently (Dubiski et al., 1962). Other basic findings in the early studies on allotypy included allelic exclusion in cells producing immunoglobulins (Pernis et d.,1965), symmetry of the multichained immunoglobulin molecules with respect to their allotypes (Dray and Nisonoff, 1963), and the discovery of Todd (1963) that VH allotypes were common to the immunoglobulin classes, which led to the suggestion that a single polypeptide chain may be encoded by more than one gene. From studies such as these it became apparent that allotypy would be a powerful tool in genetic studies of immunoglobulins at the level of the molecule, the cell, and the total organism. Reviews by Kelus and Gel1 (1967) and Oudin (1966) thoroughly cover the early history of allotypy and no attempt will be made to duplicate their efforts. Rather this article represents an attempt to provide the reader with general information on the current status of allotypic studies in the rabbit and to offer more detailed information on several specific topics to which allotypic studies have contributed.

RABBIT IMMUNOGLOBULIN ALLOTYPES

37

B. DEFINITIONS The word allotype is of Greek origin and means “other type” (Oudin, 1960a). Allotypes have been defined as intraspecies antigenic determinants (Oudin, 1966) and were defined by their presence on molecules of the same isotype (Oudin, 1960b) in order not to confuse absence of an allotype with an undetectable level of the particular isotype bearing this marker. Because L chains and their allotypes as well as the V, allotypes are common to all the H-chain isotypes (Oudin, 1966), this restriction must be taken in its current context. Obviously an allotype peculiar to the IgA class could not be observed in a sample that contains no detectable IgA. The antigenic markers of immunoglobulins fall into three categories based on their occurrence in members of a given species (Oudin, 1966). Zsotypes are present in all members of a species and can be detected by heterologous antisera. The determinants of Hchain classes (7, p, a, E) or L-chain types ( K and A) are isotypes and can be detected and differentiated by antisera prepared in other species. Allotypes are present in some, but not all members of a species. Allotypes are detected by antisera prepared by intraspecies immunization. For example, the L-chain allotype b4 can be detected by antisera prepared in rabbits lacking this allotype. Zdiotypes are markers for certain antibodies of an individual or group of individuals. These represent antigenic determinants of the variable region of an antibody of a given specificity and have been shown to include the binding site of the molecule. Idiotypic antisera prepared by injection of specific antibodies into allotypically matched rabbits (Oudin and Michel, 1969a,b) are called homologous idiotypic antisera (Potter and Kunkel, 1971). If a different species is used to prepare the antisera, the term heterologous idiotypy is applied.

1. Allotype or Zdiotype? Although the criteria that distinguish isotype and allotype are for the most part unambiguous, no such sharp distinction can be drawn between allotype and idiotype. These markers are most clearly distinguished on the basis of several experimental tests. Absence of the molecule bearing the questionable determinant in preimmune serum is indicative that it is an idiotype. The presence of antibodies with the nonclassified determinant is not conclusive proof that it is an allotype because it could still be an idiotype present on antibodies raised in response to environmental antigens (sometimes called “natural” antibodies). This criterion of distinction may be further con-

38

THOMAS J. KINDT

fused by the use of heterologous or homologous idiotypic antisera. It was observed that large excesses of certain preimmune IgG’s would inhibit heterologous idiotypic antisera (Eichmann and Kindt, 1971). Absorption of the sample with the antigen against which the immunizing antibody was prepared removes idiotypically reactive molecules. The reaction of antibodies with some idiotypic, but never allotypic, antisera is inhibited by hapten antigens against which the antibodies are directed (Brient and Nisonoff, 1970; Spring et al., 1970; Kindt et al., 1974). All idiotypes are not “ligand modifiable” however (Weigert et al., 1974). Idiotypic determinants are located in the variable region and may require a specific H-L combination for their expression, whereas allotypes are present in either the C or V region on one chain or another (Grey et al., 1965). Idiotypes furthermore may show variable expressivity (Kindt et al., 1973~).This allows a further distinction to be made on the basis of inheritance patterns observed for the markers. Whereas an allotype is inherited as the product of a single autosomal codominant gene, the mode of inheritance for an idiotype may be polygenic. In summary, allotypes and idiotypes can be differentiated by application of the following criteria: (1)presence in preimmune sera; ( 2 ) removal by absorption with antigen; ( 3 ) hapten inhibition; ( 4 ) location of antigenic determinants; and ( 5 ) inheritance patterns.

2 . Nonantigenic Allotypes Although antigenicity results from structural variations, not all structural differences need result in antigenic variation. The question may be raised whether a marker detected by some property other than antigenicity is properly called an allotype. This would include allelic variations in amino acid sequence that fulfill other criteria of allotypy such as an autosomal codominant inheritance. The markers described for L-chain variable regions in the mouse (Edelman and Gottlieb, 1970) and in the rabbit (Thunberg et al., 1973; Thunberg, 1974) as well as the VH marker recently described by Mole (1975) fall into this category.

C. NOMENCLATURE The nomenclature for allotypy agreed upon by the major investigators in the field (Dray et al., 1962) included the symbol A for allotype, a small letter (a, b, c, etc.) to designate the group (or locus), and an Arabic number for the specificity. Genotypes were to be

39

RABBIT IMMUNOGLOBULIN ALLOTYPES

noted as AhlA8 in italics with superscripts, and the specificities in Roman type: AallAb4. As in many fields where there is much active work, this system has not been fully used by all workers. Most commonly the A is omitted and the genotypes written a’a’; b4b4or al,allb4,b4, and the specificity written al,b4 or allb4 with a slash or comma separating the allotypic groups. Newly discovered allotypes are designated as A with a number following. Later these may be assigned to a group and given a small-letter designation. For example, the C Hallotypes 11 and 12 appear in early literature as A l l or A12 and later as d l l and d12. An effort is being made to avoid duplication of numbers so that a given allotype may have a letter indicating group and a number that is not used for allotypes of any other group. Other notations found in early literature on the subject can be translated using tables found in Dray et al. (1962) or Kelus and Gel1 (1967). The most commonly encountered notations are Oudin’s (1956)original designations: b a1 c a2 d a3

a b4 g b5 f b6

The use of the word locus to describe the chromosomal region encoding allotypes is questionable in certain contexts. It is reasonable to state, for example, that the allotypes of two unlinked groups are encoded at distinct loci. This designation becomes unclear, however, when one considers that three different allotypes may exist on a single H chain of the y class (e.g., a single y chain may have allotypes a l , d l l , and e14). Is each then encoded by a separate locus? There is evidence to suggest that V H(group a ) and C H (groups d and e ) regions are encoded by two distinct genes (Mage et al., 1971; Kindt and Mandy, 1972). This has not been proven, but even if it were true, questions may be raised concerning the d and e loci.” Could separate genes encode these C H region polypeptides? Some reports avoid this question and indicate that the a and “de” loci code for the respective V and C regions of the y chain. This is, of course, a further interpretation of the data because it concludes that d and e are present at the same locus. Although this may be true, it is at present unproven. The various different combinations of d and e allotypes that have been observed must then be explained by intracistronic crossovers which occurred at some point or by complex evolutionary pathways. This author prefers to defer such interpretations by ‘I

40

THOMAS J. IUNDT TABLE I ALLOTYPES OF RABBITIMMUNOGLOBULINSN Molecular location

Allotypic group

Heavy chain Variable region Constant region IgC Constant region IgC (a1 only) Constant region IgA, IgA, Constant region IgM

f g

n ms

Light chain K Type h Type Secretory piece

b C

t

Allotypes 1,2,3 32 33 11,12 14,15 A8,AlO 69,70,71,72,73 74,75,76,77 81,82 1,2,3,4,5,6 4,5,6,9 7,21 6 1,62

“ See text for references. The IgA, and IgA, refer, respectively, to papain-resistant and papain-susceptible IgA.

using for the present the more noncommittal designation “groups” for those allotypes that segregate as if encoded by allelic genes.

D. ALLOTYPICGROUPS At the present time allotypes have been reported for the variable region of heavy chains (groups a , x, and y ) , constant regions of y chains (groups d and e , A8 and AlO), a chains (groups f and g ) , P chains (groups n and ms), the secretory component of IgA (group t ) and for L chains of the K (group b ) and h (group c) type. Table I gives a summary of these allotypic specificities.

1. V,Allotypes of Group a The rabbit is unique in having genetic markers present in the variable region of the H chain. Three alleles, a l , a2, and a3, were originally reported for the V Hregion (Oudin, 1956). These group a allotypes were shown to be present in the Fd region of the H chain by their absence on L chains (Stemke, 1964) and by their presence on the Fab or Fd piece (Kelus et al., 1961; Micheli et al., 1968). The direct demonstration of peptides with allotypic activity has further localized these specificities to the V Hregion (Mage et al., 1974; Mole et al., 1975).A more detailed treatment of structural correlations with

RABBIT IMMUNOGLOBULIN ALLOTYPES

41

allotypic markers is presented in the following section for the group a and other allotypic groups.

2 . V HAllotypes of Groups x and y Quantitative allotypic determination on IgG samples from individual rabbits indicated that 10-30% of the molecules did not react with group a antisera (Dray and Nisonoff, 1963; Stemke, 1965). This a-negative population was greatly enriched in serum of rabbits subjected to homozygous allotype suppression in the zygote transfer experiments of David and Todd (1969) and Vice et al. (1970). In addition, a homogeneous antibody to streptococcal carbohydrate lacking all group a markers was produced in large amounts in an immunized rabbit (Kindt et al., 1970b). It was apparent from these observations that V Hregions lacking group a allotypes were being synthesized. Several lines of evidence precluded the possibility that the anegative allotypes were alleles of known group a allotypes. In all breeding studies, group a allotypes have behaved as codominant alleles. Deviations would be expected if alleles existed for which typing reagents were unavailable. For example, the offspring of a cross involving two apparently homozygous animals with allotypes a1 and a3 are always a1a3.If silent group a alleles existed, then one would expect to see offspring rabbits with phenotypes a1 or a3 alone and in some instances, those without a detectable group a allotype. Furthermore, rabbits heterozygous for a allotypes have produced anegative antibodies (Waterfield et d., 1972). The suggestion that the allotypic specificities are masked on such molecules cannot explain the recent findings by Tack et al. (1973a) that a-negative molecules can be distinguished by their amino acid composition and by the sequence of amino terminal peptides. There is in addition serological evidence that a-negative molecules represent two or more allotypic groups separate from the group a. Knight et al. (1971) and Kim and Dray (1972, 1973) have developed antisera against allotypes 32 (group x) and 33 (group y). Immunoglobulin G isolated from the serum of allotypically suppressed rabbits (Section VI) expresses these allotypes on distinct populations of molecules (Kim and Dray, 1973). 3. C , A h t y p e s

In addition to the VH allotypes, markers have been described for the constant regions of H chains of the IgG, IgAI, IgA2, and IgM

42

THOMAS J. KINDT

classes. No CH allotypes have yet been described for antibodies of the IgE class (Lindqvist, 1968) 4 . IgG Allotypes

Mandy and Todd (1968) described an allotype designated A l l ( d l 1)that was detectable by hemagglutination assays. This specificity was shown to be present on the (Fab’)zIgG fragment but not on Fab (Mandy and Todd, 1969), an indication that the determinant involved the hinge region of the molecule. An allele, d12, was discovered soon afterward (Mandy and Todd, 1970). Heavy chains with all combinations of a and d allotypes have been observed, although some are rare, as, for example, the a2-dll association (Mandy and Todd, 1970; Kindt et al., 1970a). A second allotype, e14, for the C , region was reported by Dubiski (1969a) and an allele, e15, has also been described (Dubiski, 196913). Identification of these C , allotypes made possible genetic studies on the inheritance of V, and CHregions of y chains. Two other allotypes have been described for the C , region (Hamers and Hamers-Casterman, 1965). These specificities, designated A8 and A10, were present only on H chains with allotype al. It is not known whether primary structural differences or differences in carbohydrate prosthetic groups are responsible for these antigenic variations.

5. IgA Allotypes There are three allotypic groups peculiar to IgA. They are group f (69,70,71,72,73), group g (74,75,76,77), and group t (61 and 62) (Conway et al., 1969a,b; Knight et al., 1974a). Group fallotypes are present on a chains of the molecules resistant to papain digestion (IgA,); the group g markers on these molecules are sensitive to digestion by this proteolytic enzyme (IgAJ (Hanly et al., 1973). The allotypes of the t group are present on the secretory component of IgA (Knight et al., 1974a). The f a n d g allotypes are closely linked to one another and to allotypes of group a. These linkages give reason to believe that the IgA allotypes are encoded at the gene complex that encodes all immunoglobulin H chains. The t allotypes of secretory component, on the other hand, are not linked to allotypes of the H or the L chain (Knight et al., 1974a). Of the potential combinations of the five group f and the four group g allotypes only the following have been observed:

RABBIT IMMUNOGLOBULIN ALLOTYPES

f

g

71

75

72

74 74 76

73 70 69

43

77

Thus, of twenty possible combinations, only five have so far been observed (Hanly and Knight, personal communication). When papain susceptible IgA was fractionated into Fca and Fab a fragments, it was noted that g74 antisera reacted with a high percentage of the molecules in both fractions. It has since been shown that distinct antigenic specificities are present on these fractions. The g allotypes, therefore, can either be subdivided into two distinct allotypic groups or they may represent more alleles of the same groups (Hanly et al., 1973). This situation may be analogous to the simultaneous presence of both d and e allotypes on the y chain referred to above in the discussion of C, allotypes (see also Section 11,C).

6. IgM Allotypes Allotypes have been described for rabbit IgM (Kelus and Pernis, 1971; Sell, 1966; Kelus and Gell, 1965). Five precipitating allotypes were designated msl through ms5. A sixth (ms6) could be demonstrated only by immunofluorescence techniques. Several of the specificities studied were not detected unless they occurred in combination with specific H- or L-chain allotypes. A second group of IgM allotypes, n81 and n82, has been studied by Gilman-Sachs and Dray (1972). It is not yet known whether these specificities are the same or different from any of the ms allotypes. Genetic studies indicate the presence of at least one more allotype in the n group. These allotypes were detected by precipitin reactions. Linkage to group a allotypes was observed. In their report of IgM allotypes, Gilman-Sachs and Dray (1972) added the precautionary note that closely linked genes may behave as alleles in genetic studies. The validity of their comment is supported by the data obtained on the allotypes of group g (Hanly et al., 1973) as well as those obtained with the c7 and c21 allotypes (Gilman-Sachs et al., 1969).

7 . L-Chain Allotypes a. Kappa Chains. Included in the original six allotypes described by Oudin (1960a) were three allotypes of the b group: 4,5, and 6.

44

THOMAS J. KINDT

These were shown to be on the L chain (Stemke, 1964). An additional allotype, b9, was later added to this group (Dubiski and Muller, 1967; Carbonara and Mancini, 1968). The majority of rabbit K light chains have seven half-cystine residues (KB subtype) (Reisfeld et al., 1968); there is a second smaller population of K chains that has five half-cystine residues (KA subtype). Both of these populations carry the b allotypes (Rejnek et al., 1969a,b; deVries et al., 1969; Zikan et al., 1967). Light chains of the K A subtype as well as A light chains are represented in relatively larger amounts in the IgG from b9 rabbits (Chersi et al., 1970) than from rabbits with other group b allotypes. b. Lambda Chains. A relatively small percentage of molecules with L chains of the A type are present in rabbit serum. These do not carry group b allotypes. Homozygous suppression experiments have allowed an increase in the concentration of A chains to a level that permits comparative allotypic studies (Appella et al., 1968; Rejnek et al., 1969a,b; Chersi and Mage, 1973). Two markers designated c7 and c21 have been described for this L-chain type (Mage et al., 1968). Initially, these were thought to be alleles, but subsequent breeding experiments yielded results inconsistent with this view (Gilman-Sachs et al., 1969). Two animals each with phenotype c7, c21 were mated. Instead of the expected proportions of homozygotes (25% each) and heterozygotes (50%), all animals were c7, c21. This suggests that 7 and 21 are pseudoalleles; that is, they are closely linked but not encoded at homologous DNA regions. The fact that other breeding studies (Mage et al., 1968) yielded expected results suggests that both allotypes c7 and c21 have true alleles that are not being detected by the antisera. 8. Summary From the above discussion and from the information given in Table I, it can be seen that allotypic markers have been described for the V, region and for C Hregions of all rabbit immunoglobulin classes except IgE. There are allotypes for K chains which appear to be common to both K A and KB light-chain subtypes, and there is an unlinked group of allotypes for light chains of the A type. Figure 1 depicts the rabbit immunoglobulin classes and their major allotypes. This representation assumes that VH allotypes of groups x and y will be present on H chains of all classes although this has not been demonstrated. Allotypes A8 and A10 as well as those of the ms and t groups are not represented in Fig. 1.

RABBIT IMMUNOGLOBULIN ALLOTYPES H

45

H f

b orc

b orc

FIG.1. The classes of rabbit immunoglobulins shown with their allotypic groups. All molecules are represented as monomers although IgA may occur as a dimer and IgM as a pentamer. T he light chain allotypes are group b for K chains and group c for A chains; on a single molecule these are mutually exclusive, as are the VH allotypes of groups a , x, and y .

E.

IMMUNIZATION AND

DETECTIONPROCEDURES

1 . Preparation of Allotypic Antisera In general rabbit allotypic antiserum is prepared by immunization of a rabbit with immunoglobulin bearing an allotype that is absent in the injected rabbit. The injected animal must possess all other allotypes present in the injected sample (Oudin, 1960a). The original allotypic antisera were raised (Oudin, 1956) by injection in complete Freund’s adjuvant of specific antibody precipitates formed by reaction of ovalbumin with antiovalbumin. Other injection

46

THOMAS J. KINDT

procedures use antibody agglutinated suspensions of Proteus vulgaris (Dubiski et al., 1959), subcutaneous injection in adjuvant of isolated IgG, or IgG cross-linked with glutaraldehyde to enhance its immunogenicity (Daugharty et ul., 1969). Prior to use, an allotypic antiserum is tested for its reaction with immunoglobulin of the immunizing allotype and for the absence of reaction with all other immunoglobulin allotypes. This may conveniently be done by preparing serum pools that lack only one known allotype to be used as the negative controls. Certain allotypes may show cross-reactions, which can be overcome by preparing the antisera in animals with the potentially cross-reactive allotype. A typical procedure is illustrated, using as an example the preparation of an antiserum to allotype a3. Immunoglobulin G is prepared from the serum of several animals of allotype a3a3/b4b4. The IgG is cross-linked by slow addition of a twenty-fold molar excess of glutaraldehyde to a solution of 10 mg./ml. IgG in 0.1 M phbsphate buffer, pH 7. The clumped protein is dialyzed extensively against saline. One milligram of this material suspended in 1 ml. saline is emulsified with 1 ml. Freund's complete adjuvant. The emulsion is injected beneath the right scapula of a rabbit of allotype u1u2,b4. The injection is repeated monthly, alternating the right and left scapula for injection. Test bleedings are taken prior to each injection. Within 5 or 6 months precipitating antisera to allotype a3 may be produced. If not, the animal is rested for about 4 months and then the injection schedule is resumed. Antisera to allotypes of IgM (Gilman-Sachs and Dray, 1972) have been prepared by induction of IgM cold agglutinins by immunization with Listeria monocytogenes (Costea et al., 1965), and subsequent injection of these into the appropriate rabbits. Immunoglobulin A allotypic antisera are prepared by injection of secretory IgA in Freund's complete adjuvant (Conway et aZ., 1969a,b).

2 . Detection Methods Assays for immunoglobulin allotypes use most standard immunological techniques. Precipitating antisera are produced against most allotypes; notable exceptions being d l l and d12, e l 4 and e15, and ms6. Qualitative tests most commonly used for typing with precipitating antisera are double diffusion in agar or interfacial precipitin (ring) tests. A serum sample can be used for typing allotypes, except for those of IgA because IgA levels in rabbit serum are normally too low for detection of the allotypes, and secretory piece is not present in serum. Saliva, milk, or tears must be collected to detect IgA allotypes (Knight et al., 1973).

RABBIT IMMUNOGLOBULIN ALLOTYPES

47

Tests involving precipitation must be carefully controlled, especially when hyperimmune sera are to be tested for the presence of allotypes. It has been observed that anti-immunoglobulins are present at significant levels in some hyperimmune sera and can possibly interfere with precipitin tests (Bokisch et al., 1972; Kindt et al., 1973~). A hemagglutination method is commonly used for typing d l 1 and d12 allotypes. This involves preparation of rabbit anti-red blood cell group F in a rabbit with the appropriate allotype and the use of these antibodies at subagglutinating titers to actively coat red blood cells. Individual sera are then typed by hemagglutination inhibition (Mandy and Todd, 1968).A hemagglutination test utilizing red blood cells coated with IgG by the chromic chloride method has been described by Steward and Todd (1969). Early studies on the quantitation of allotypes utilized the single diffusion technique of Oudin (1960a,b). Radioprecipitation techniques were soon introduced (Dray and Nisonoff, 1963). A method currently in use involves ethylchloroformate (ECF) solidification of antiallotype sera (Avrameas and Ternynck, 1967) and the use of these ECF antisera in radiobinding or inhibition of radiobinding tests (Tosi and Landucci-Tosi, 1973). Such antisera are especially useful when sequential determinations are carried out on the sample to determine if allotypes are on the same or on different molecules (Landucci-Tosi et d., 1970; Lawton and Mage, 1969). The solidified antisera are also useful for removing molecules with a specific class or allotypic marker from whole antisera (Lindqvist, 1968; Kindt and Todd, 1969). A rapid typing procedure using inhibition of binding of labeled IgG to ECF antisera has been described by Mancini et al. (1970). However, although the ECF antisera are very convenient to use, their preparation requires a fairly large amount of antisera and a long period of time. Recently, a technique has been developed in our laboratory for the preparation of solidified allotypic or idiotypic antisera by a mild and simple procedure. This method involves coupling of whole antisera or specifically isolated antibody fractions to Sepharose beads which have been activated with N-hydroxysuccinimide (Gottlieb et aZ., 1975). The activated Sepharose (HAS) is prepared in large quantities and stored as a lyophilized powder (Cuatrecasas, 1970; Cuatrecasas and Parikh, 1972). This dry material is then reacted with the antisera or specific antibody in aqueous solution at neutral pH. The reaction is stopped by addition of excess glycine and the product is washed, Solid antisera prepared in this manner retain much of their

48

THOMAS J. KINDT

original activity, and no losses of fine specificities have been detected (Gottlieb et al., 1975). Unfortunately the commercially available preparations of activated Sepharose or Agarose have not proven satisfactory in this application. Allotypes d l l and d12 have not been successfully quantitated by radiobinding techniques. However, these allotypes can be measured by quantitation of the 3.5 and 5 S fragments resulting after treatment of IgG with CNBr in dilute acid. Molecules with allotype d l l produce the 3.5 S fragments, those with d12, the 5 S fragments; these can be separated by gel filtration or density gradient centrifugation (Kindt et al., 1970a). A method that utilizes IgG-coated bacteriophages has recently been reported (Maron and Dray, 1973). The authors claim detection of 1 ng. of an immunoglobulin allotype with this method. Luzzati et al. (1973a,b) have determined the allotypes of anti-sheep red blood cell antibodies synthesized in vitro by stimulated lymphoid cell microcultures. A red blood cell overlay technique was used in which allotypic antisera inhibited zones of lysis caused by antibody against which they were directed. Antisera labeled with fluorescent markers have been used in cellular experiments to test for allelic exclusion in single cells (Pernis et al., 1965; Cebra et al., 1966) and, more recently, to search for recombinant cell types (Pernis et al., 1973). II. Structural Correlates of Allotypic Determinants

A. STRUCTURAL FEATURES OF RABBIT IMMUNOGLOBULINS A brief general discussion of the structural characteristics of rabbit immunoglobulins may be useful before data relating to allotypic correlates are presented. There are several structural aspects in which rabbit immunoglobulins differ from the more widely studied human and mouse proteins. One major difference is that, although there is some evidence for the existence of rabbit IgG subclasses (Florent et al., 1973), they have not been described in such detail as in the human (Natvig and Kunkel, 1973). This may be because the sequence information on the constant region of rabbit H chains has been obtained through studies on pooled material (Cebra et aZ., 1968; Hill et al., 1967; Fruchter et al., 1970). Subclasses, present as minor components of the pool, could have been overlooked in these studies. Immunoglobulin G subclasses, if they exist, could be established by utilizing homogeneous rabbit antibodies (Krause, 1970) in the same manner as myeloma proteins were used to establish the human IgG subclasses (Natvig and Kunkel, 1973).

49

RABBIT IMMUNOGLOBULIN ALLOTYPES

Furthermore no VH subgroups (Capra and Kehoe, 1975) have been described for rabbit immunoglobulins, although the H chains with group a allotypes and those with allotypes from the x and y groups may be their counterparts in rabbit immunoglobulins. Prahl et ul. (1973)have demonstrated significant differences in amino acid composition between the V regions of group a and a-negative H chains. 1 . Rabbit H Chain Figure 2 gives a schematic representation of the rabbit IgG heavy chain. Note that there are two intrachain disulfide bridges in the constant Fd region and that the Cys residue involved in the H-L bond is at position 132 or 133 in the rabbit H chain (Fruchter et al., 1970; O'Donnell et al., 1970). Because of their importance in structural studies, the positions of the y-chain methionine residues have been indicated in Fig. 2. Invariant methionine residues are found at positions approximately 249, 350, 367, end 428 residues from the amino terminus. The presence of methionine at 225 has been correlated with allotype d l l (Prahl et al., 1969). In the V Hregion, methionine may be present at positions 34 and 79, either 34 or 79, or the V Hregion may contain no methionine. These V H methionines are present in a significant number of the chains present in an IgG pool. For example, 60-70% of all allotype a1 H chains have methionine at position 34, whereas fewer than 20% have methionine at position 79. On the other hand, 40% of the allotype a3 H chains have methionine at position 34 and 50% at position 79 (Mole et al., 1971). To other H choin

"" "a.

c . . "U

I

1

I

a.r

""

I

""

"a.

I

I

I,

I

I, I

COOH

Reaidue Number

FIG.2. Schematic of rabbit y chain showing the location of Cys and Met residues. The Met residues represented at positions 34 and 79 are not always present; H chains from homogeneous antibodies may lack either or both V-region Met residues. (Adapted from the data of O'Donnell et al., 1970; Prahl et al., 1969.)

ur

0

~

4135 2717 BS-1 BS-5 3315 4 135

~~

~~

0 1 5 10 15 20 A h Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly Gly Thr Val Thr Val Glu-Leu Ser Pro Ala Ala -Val -Val -Gln Ser Ala Ala 25 30 35 40 Ile Lys Cys Gln Thr Ser Gln Ser Ile Asp Asp Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Asx Asx I

2717 BS-1 BS-5

-Ser

33 15

-Asn

4135 2717 BS-1 BS-5 3315

45 50 55 60 Gly Gln Pro Pro Lys Gly Leu Ile Tyr Arg Ala Ser T h r Leu Ala Ser Gly Val Pro Ser Ala Thr Ser Ser Leu LY s Ser Leu Lys A l u Ser -

Ser Thr Lys Ah Ah Ser

Tyr Asx Tyr-Ala Tyr Ser Gly Tyr Asn Gly-Ah Asn Gly Val Tyr Glu Arg

GlxGlx Glx Glx Glx Phe

4135 2717 BS-1 BS-5 3315

65 70 76 80 Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys Thr Glx Leu Val Lys Glu LYs Thr Gln Ser Val Gln-

4135 2717 BS-1 BS-5

85 90 95 100 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Thr Tyr Gly Val Gly Phe Gly Gly ASP Gly Gly Ala Asp Tyr Thr-Tyr Ser Phe-Glx Gly Ser Thr Tyr Gly-Gly Tyr Phe-Glx Gly Ser Asx Tyr Thr-Thr Val Asp Ser Phe Thr

3315

ASP

-

Leu Gly Asn Tyr Asp Cys Ser Ser Gly

FIG.3. Comparison of variable-region sequences of b4 L chain from homogeneous rabbit antibodies. Light 4135 is from an antibody to streptococcal carbohydrate (Chen et al., 1974); L chain 2717 is from an antihapten antibody (Appella et al., 1973); and L chains BS-1 and BS-5 are from antibodies to pneumococcal polysaccharides (Jaton, 1974). L chain 3315 is also from antibody to pneumococcal polysaccharides (Margolies e t a / . , 1974).

52

THOMAS J. KINDT

2. Rabbit L Chain The majority of rabbit IgG molecules have K light chains of the KB subtype (Rejnek et al., 1969a,b). These L chains are different from human and mouse chains in having a third disulfide bridge that links the variable and the constant regions (Poulsen et al., 1972; Strosberg et al., 1972). Sequence data are available for rabbit light chains of the b4 allotype (Appella et al., 1973; Chen et al., 1974; Jaton, 1974; Margolies et al., 1974). A comparison of some of these data is shown in Fig. 3. The sequence comparison of these b4 L chains reveals that no differences are observed after position 98. There are hypervariable positions around residues 30-34 and at 89-97. The second L-chain hypervariable region (Wu and Kabat, 1970) is not evident from these comparisons. Variation also exists in the residues near the amino terminus. This had been earlier observed in amino terminal sequence studies on L chains from homogeneous antibodies (Hood et al., 1970; Braun and Jaton, 1973; Thunberg, 1974; Kindt et al., 1974) and on L chains from IgG pools (Hood et al., 1971; Kindt et al., 1972; Thunberg, 1974). Subgroups of rabbit K chains have been assigned on the basis of differences in chain length at the amino terminus (Hood et al., 1970). The VKIIL Chains have been defined as having the same length as human K chains when aligned for maximum homology. Light chains of the VKI subgroup have an additional amino-terminal residue, light chains lack a residue in the amino terminal posiwhereas VKIII tion (Hood et al., 1970). In addition at least three homogeneous antibodies with blocked amino-terminal residues have been observed (Kindt et al., 1974). Figure 4 gives the sequence from residue 98 through the constant region for b4 L chains from an antibody directed against streptococcal carbohydrate (Chen et al., 1974). An identical C-region sequence has been reported by Margolies et al. (1974) for a b4 L chain from an antibody directed against pneumococcal carbohydrate. Jaton (1974) has reported two b4 L-chain sequences to position 139; these are also identical to the 4135 L chain from position 98-139. Deletions in the sequence depicted in Fig. 4 have been introduced to maximize homology to human K chain. The Cys residue at position 134 forms a disulfide bridge with Cys 194; Cys 171 with Cys 80 in the V region (Fig. 3); and Cys 214 links to the H-chain Cys 132 or 133 (Fig. 2) (Lamm and Frangione, 1972; Strosberg et al., 1972). Different amino acids have been observed for the constant region position 174. Although the 4135 L chain (Fig. 4) has Asn in this posi-

RABBIT IMMUNOGLOBULIN ALLOTYPES

53

Position 98

101

105

110

115

120

Phe-Gly-Gly-Gly-Thr-Glu-Val-Val-Val-Lys-Gly-Asp-Pro-Val-AlaPro-Thr-Val-Leu-Ile-Phe-Pro-Pro121 125 130 135 140 Ala-Ala-Asp-Gln-Val-Ala-Thr-Gly-Thr-Val-Thr-Ile-Val-Cys-Val-Ala-Asn-Lys-Tyr-Phe-

141 145 150 155 160 Pro-Asp-Val-Thr- -Val-Thr-Trp-Glu-Val-Asp-Gly-Thr-Thr-Gln-Thr-Thr-Gly-I~e-G~u161

165

170

175

180

Asn-Ser-Lys-Thr-Pro-Gln-Asp-Ser-Ala-Asp-Cys-Thr-Tyr-Asn-Leu-Ser-Ser-Thr-Leu-Thr181

185

190

20 1 Thr-Thr-Ser-

205 210 Val-Val-Gln-Ser-Phe-Asn-Arg-Gly-Asp-Cys

195

200

Leu-Thr-Ser-Thr-Gln-Tyr-Asn-Ser-His-Lys-Glu-Tyr-Thr-Cys-Lys-Val-ThrGln-Gly-

FIG.4. Amino acid sequence of 4135 b4 L chain from residue 98 to the C terminus. Deletions were introduced to maximize homology to light chains from human myeloma proteins. (From Chen et ul., 1974.)

tion, other workers have found Val or Leu (Appella et al., 1973; Strosberg et al., 1972). The existence of these alternative residues in the constant region of b4 L chains may indicate the existence of polymorphisms within the b4 allele. This may be the case, but differences have not been detected among the allotypic specificities present on b4 L chains from homogeneous antibodies nor has any b4 L chain been shown to be immunologically deficient with respect to pooled b4 chains (Kindt et al., 1972).

B. NATUREOF ALLOTYPICVARIATION Several a priori postulates concerning the nature of substitutions that give rise to allotypic determinants can be made. If allotypes represent primary genetic differences, then there must be differences in primary protein structure associated with allotypic variation. These differences (correlates) must exert positive or neutral effects on the binding and effector functions of the antibodies. The most acceptable explanation for the existence of allotypes is that they represent the accumulation of nondeleterious mutations in replicate copies of an ancestral gene. Whether the mutations occurred and stabilized prior to or after speciation is subject to conjecture (Smith, 1973), as is the exact time sequences in which the different allotypes appeared (Mandy and Rodkey, 1974). Consider an amino acid sequence of an ancestral antibody chain as A A A A as depicted in Fig. 5. If accumulated mutations in the gene encoding this sequence gave rise to three allotypically differ-

54

THOMAS J. KINDT _ _ _ _ _ ~

Sequence of hypothetical precursor Sequences of anticipated allotypic variants

A

A

1

A

B

B 3

C A

A A

A A A E

A

A D A

FIG. 5. Expected variation in amino acid sequence among allotypically distinct peptides evolved from a common precursor. This format assumes that allotypes arose from random mutational events acting on replicates of the gene that encoded the ancestral peptide. In this situation it is not likely that mutations would occur in identical positions in each replicate.

ent genes, the sequences they encode may be as depicted in Fig. 5. Assuming random mutational events, it is more likely that two chains will have the same, and a third, a different amino acid in a given position than that all three will be different. The higher probability is that the two similar chains will display the substitution present in the ancestral sequence. The presence of differences in all three alleles at the same position requires that mutations occurred in the same codon in two copies of the gene. The probability of this being observed may be increased if allowable variations were confined to a relatively few positions. The comparison of amino acid interchanges among subgroups carried out by Novotny (1973) could well be applied to the V-region allotypes. Novotny observed that most differences among subgroups were conservative and that nonconservative changes at one position were compensated by changes at other positions. The problem of dealing with amino acid sequence data to assign allotypic determinants will be complicated by the presence of substitutions which are related to different antibody activities, especially in V-region peptides. It is necessary, therefore, to identify hypervariable positions of the proteins prior to assignment of allotypic correlates (Mole et al., 1971).

HEAVY-CHAIN ALLOTYPES The allotypes for which structural data are available are those of groups a, b, d, and e. Allotypes of groups d and e involve small differences and are structurally well characterized; those of groups a and b appear to involve multiple amino acid interchanges and consequently are not as well defined. c . STRUCTURAL CORRELATES OF

1 . Groups d and e Group d allotypes were shown to correlate with a Met ( d l l ) Thr (d12) interchange at the amino terminal side of the cysteine involved

RABBIT IMMUNOGLOBULIN ALLOTYPES

55

in the inter-H-chain disulfide bond (Fig. 6.) (Prahl et al., 1969). McBurnette and Mandy (1974) have recently demonstrated that a hingeregion peptide that includes the Met substitution has d l l antigenic activity. Group e allotypy was also known to correlate with a single amino acid interchange in the C, region (Appella et al., 1971). These allotypes were correlated with Thr (e14) and Ala (e15)at position N 309. No other differences among tryptic peptides from the Fc fragments were noted. Figure 6 shows the sequence of the peptides carrying the allotypic substitutions for group d and e allotypes. 2. Group a

It was shown that amino acid compositional differences existed among IgG H chains of different allotypes that were matched for antibody specificity (Koshland et al., 1969). It has been further shown that these same differences could be demonstrated among IgM antibody H chains from the same animals. These studies employed highly purified antibodies directed against haptenic antigens. Similar results were obtained in composition studies on Fd fragments from IgG pools (Inman and Reisfeld, 1968). Subsequently Wilkinson (1969a) isolated amino-terminal peptides from H chains of total IgG fractions from rabbits with different group a allotypes and demonstrated differences among these peptides. It was also shown that IgA H-chain peptides were similar to the IgG peptides of the same allotypes (Wilkinson, 1969b). Sequence studies carried out on y chains from pooled IgG of allotype a1 and a3 revealed two major areas of possible allotypic differences (Mole et al., 1971). A partial V Hsequence of an antibody with allotype a2 was available for comparison (Fleischman, 1971).

~

Allotypc

Position in IgC, t 1 chain

dll d12

220 225 230 Thr Cys Ser Lys Pro Met Cys Pro Pro Pro Glu -hT

el4 e 15

305 310 Val Val Ser Thr Leu Pro Ile Thr His Glu Asp Ala ~~

FIG.6. Amino acid sequence variations in the constant region of the rabbit y chain that correlate with allotypes of groups d and e . Group d sequences are from Prahl et ul. (1969); group e sequences are from Appella et ul. (1971). Residues are numbered from the amino terminus of the H chain.

Position

a1 Ab Pool

5 10 15 20 25 30 pClu Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Thr Pro Gly Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Val Val ___ Ala Leu

a2 LysGlu-Gly-Phe Lys ___ Glu-Gly -Phe

Ab Pool (1

Ile Asp-

LysLys-

Asn Thr Asp Thr

Ala

HV HV-

Lys-Gly

Ala Ser

Ah

Asn “YAla

3

Pool

~

Leu

ASP Val

~

FIG.7. Comparison of the sequence at amino-terminal positions 1-30 of rabbit H chains from pooled IgG and homogeneous antibodies (Ah) of different group a allotypes. The a1 Ab sequence is from Jaton and Braun (1972);the a2 Ab sequence from Fleischman (1971); and the pool sequences for al, a2, and a3 are from Mole et al. (1971), Porter (1974),and Mole (personal communication). HV indicates a hypervariable position.

E

% ?

E Z

RABBIT IMMUNOGLOBULIN ALLOTYPES

57

Major differences were observed in the amino-terminal residues and in the area 80-85 residues from the amino terminus. Figure 7 gives amino acid sequence data for the amino-terminal 30 residues of rabbit H chains from IgG pools and from homogeneous antibodies. The pool sequences have been corrected by subtraction of the blank (a-negative) sequence since the peptides isolated from a pool would include those derived from the H chains of groups x and y. The substitutions most likely to be valid correlates of group a allotypy are those at positions 4, 7,9, 11, 12, 15, and 16 from the amino terminus. The differences at position 23 are not related to allotype because the pool and homogeneous antibody sequences are different for both a1 and a2. The differences at 26-28 may arise from the fact that these are hypervariable positions. The original sequences reported for V, positions 79-95 (Mole et al., 1971) have been more recently supplemented by the data of Jaton et al. (1973).These workers found that some of the substitutions thought to be allotypic correlates were unlikely to be so. The a1 sequences reported by Jaton et al. (1973) from position 80 all came from chains with a methionine residue at position 79. As mentioned above, fewer than 20% of a1 H chains have this substitution. In spite of this selection for a minor population, the substitutions at position 84-85 were firmly established as allotypic correlates in this study. Allotype a1 correlates with Thr-Glu; a2 with Ala-Gln and a3 with Ala-Ala. Figure 8 lists sequence variations observed for peptides isolated from IgG and purified antibodies at these positions. In addition to the correlates in the amino-terminal residues and those at position 84 and 85 in the V Hregion, there are also possible

Allotype

H-Chain position 79

ul-pool

80

81 82

83

84 85

86

87

88 89

-Met Leu

u2

a3 FIG.8. Sequence comparison of H-chain positions 79-89 for homogeneous antibodies and pools of allotypes a l , a2, and a3. Lys occurs at position 79 in more than 80% of the a1 H chains from pooled IgG. The residues enclosed in the box are possible correlates of group a allotypy. (Taken from Mole et al., 1971; Jaton et al., 1973; Mole, personal communication.)

58

THOMAS J. KINDT

group a allotypic correlates at position 67-71. The a1 and a3 H chains have the same sequence in this region, whereas both the a2 pool (Mole, unpublished data) and a homogeneous antibody (Fleischman, 1973) have considerably different sequences:

70 al/a3 pool a2 Ab or pool

Phe-Thr-Ile-Ser-Lys Ser-Thr-Ile-Thr-Arg

Some of the well-documented correlates of the group a allotypes are considerable distances apart along the peptide chain, as for example, the amino-terminal substitutions and those at positions 84-85. Examination of a three-dimensional model of an IgG molecule reveals close proximity of positions 15-17 and 84-85 (Poljak et al., 1973). The allotypic determinant may require the presence of both differences for its expression. The fact that H chains with disrupted intrachain disulfide bridges no longer display group a allotypes (Mage et al., 1974) could be taken in support of this. In the same context, Steinberg et al. (1974) have shown that the InV determinants of human K chains involve two correlates, one at position 153 and another at position 191. A three-dimensional model of the L chain shows these positions to be adjacent. In addition to the presence of possible structural correlates, other lines of evidence support the assignment of group a allotypes to the variable region of the H chain. Mole et al. (1975) have prepared Hv fragments by the action of papain on isolated Fd fragments from H chains of allotypes a1 and a3. These fragments had full antigenic activity measured in inhibition of precipitin assays. Mage et al. (1974) have demonstrated activity of tryptic peptides isolated from the Vu region. Comparison of the sequences in the constant portion of the Fd regions of a1 and a3 (Fruchter et al., 1970; Pratt and Mole, 1975) failed to show any structural differences in this region. Assignment of group a allotypic determinants to the V, regions is strongly supported by these direct and indirect data. At this point it is relatively certain that both correlates and determinants of group a allotypes exist in the V,, region. However, it is not yet known which of the correlates are determinative (Todd, 1972) or how much variation can exist within a single allotype (Jaton et al., 1973; Kindt et al., 1973b). An independent confirmation of heritable differences in primary structure of the Vu region has been provided by Mole (1975). Tryptic digests of C1 (an H-chain fragment obtained by CNBr digestion

RABBIT IMMUNOGLOBULIN ALLOTYPES

59

equivalent to the Fd region) from IgG’s and antibodies were carried out on reduced and radioalkylated samples. Radioautography of peptide maps of such digests indicated clear-cut differences between patterns obtained from allotypes a1 and a3. It was shown by breeding studies that the characteristic radiolabeled spots were inherited in a Mendelian manner as autosomal codominant alleles. 3. H Chains Lacking Group a Allotypes

As mentioned above, there are V Hregions lacking the group a allotypic specificities and possessing other allotypic markers (Kim and Dray, 1973). There are no structural data available to identify correlates of group x and y allotypes, but some data are available on general structural aspects of the a-negative H chains. The Fc, region of a-negative chains cannot be distinguished from those of a-positive chains. Peptide maps of the Fc, fragments were shown to be identical for a-positive and a-negative chains (Knight et al., 1971; Prahl and Todd, 1971; Tack et al., 1973b). Further comparisons included examination of CNBr fragments and compositional analysis of a C-terminal octadecapeptide (Tack et aZ., 197313). Again, no differences from a-positive molecules could be established. The a-negative chains were also shown to have the Met-Thr interchanges that properly correlated with their group d allotypes. This evidence speaks strongly against the possibility that a-negative molecules represent subclasses of rabbit IgG. All the structural differences thus far observed between a-positive and a-negative H chains are in the amino-terminal region of the chain. Prahl et al. (1973) demonstrated differences in amino acid composition between the C1 fragment of a-positive and a-negative chains. These differences bear no relationship to those observed by Koshland et aZ. (1969) between H chains differing in group a allotype. One striking characteristic of a-negative H chains is the aminoterminal sequence: whereas a-positive chains begin with pGlu-SerVal or pGlu-Ser-Leu, the a-negative chains begin with pGlu-GluGln. This peptide had been observed in earlier studies on IgG pools (Wilkinson, 1969a,b), and Waterfield et aZ. (1972) found this peptide in the H chain from an antibody lacking the a allotype. Although the total extent of structural difference among V Hregions of H chains of a and x or y allotypes is not yet certain, it is evident from the data on amino acid composition (Prahl et aZ., 1973) that significant differences do exist.

60

THOMAS J. KINDT

D. STRUCTURALCORRELATES OF LIGHT-CHAIN ALLOTYPES Data on the primary structure of rabbit L chains do not allow extensive assignments of group b allotypic correlates. The major reason for this lack of information is that almost all structural studies have been carried out on L chains of the b4 allotype. There are two nearly completed (Appella et al., 1973; Margolies et al., 1974) and one completed sequence (Chen et uLY 1974) of rabbit b4 L chains. In addition, a sequence to position 139 has been assigned for two b4 chains (Jaton, 1974) (see Fig. 3 and 4). Approximately forty sequences for the amino-terminal 23 positions of b4 L chains from homogeneous antibodies have been reported, but only one b5 L chain (Mage et al., 1973b) and five b9 L chains (Thunberg et uLy 1973) have been studied even to this extent. Therefore almost all information on group b allotypic correlates comes from studies on fragments obtained from digests of pooled L chains or from quantitative studies on the amino-terminal sequences of L chains from IgG pools or from hyperimmune animals. Group b allotypes b4, b5, and b6 were correlated with differences at the C terminus of K L chains (Appella et al., 1969; Frangione, 1969). More recently, Goodfleisch (1975) has characterized a similar peptide from b9 and found it to be identical to the b4 peptide. b4 b5 b6 b9

214 Asn-Arg-Gly-Asp-Cys Ser-Lys-AsxSer-Lys-Ser-

Comparison of the sequences of peptides containing other constant region Cys residues shows b4, b5, b6, and b9 to be identical around position 134 (Chen et al., 1974; Goodfleisch, 1975; Lamm and Frangione, 1972). The 194 Cys which, with the 134 Cys, makes up the intra-C-region disulfide bridge shows perhaps one residue difference between b9 and b4 and b5. More extensive variation exists at positions around Cys 171, which participates in the interdomain bridge (Poulsen et al., 1972). 171 b4 Lys-Thr-Pro-Glu-Asn-Ser-Ala-Asp-Cys b5 ASP b9 Thr Ser-Pro-Glu Establishment of the structural correlates of group b allotypes will require further study. It is not certain if the correlates observed

RABBIT IMMUNOGLOBULIN ALLOTYPES

61

in the C region are the only ones existing for the b allotypes. Although there are serological data (Kindt et ul., 1972) (see Section II1,A) to suggest that large sequence variations at the amino terminus do not alter the expression of b4 allotypic specificities on homogeneous antibodies, there may be nondeterminative correlates in the V Lregion or determinative correlates that are in the more constant parts of the V, region.

Allotypes in the V LRegion Although it is not known whether there are amino acid sequence variations in the V, region that correlate with group b allotypy, several studies relevant to this topic have been carried out. Quantitative amino acid sequences of the Kb chains from IgG pools of allotypes b4, b5, b6, and b9 revealed characteristic profiles for yields of selected amino acids at different amino terminal steps (Hood et al., 1971; Waterfield et ul., 1973; Chersi et al., 1971). These variations were sufficiently large to allow the workers to suggest that the group b allotype of a pool or at least the total IgG fraction from an individual could be determined from the amino-terminal sequence data. Some of these differences are most likely due to quantitative differences in the expression of the V, subgroups by L chains of the group b allotypes. Other differences such as the Glu substitution at position 16 for certain b9 L chains from homogeneous antibodies and total IgG fractions are more difficult to explain (Thunberg et al., 1973). An extension of this study on b9 V regions revealed that the Glu substitution at position 16 was related to the b9 allotype of one rabbit family, but not present in another nonrelated b9 line (Thunberg, 1974). It was further observed in this study that there were significant differences between b4 and b9 L chains in the yield of Glu obtained at positions other than 16. Thunberg (1974) showed that it was possible to “type” for b4 or b9 by the presence of Glu at residues 12 and 13 in L chains from total IgG fractions from individual rabbits. More information on the structure and genetics of rabbit L chains will be required to determine the full extent of variation associated with group b allotypy. This information will further resolve questions concerning the relationships between the C and V regions of the rabbit L chains. Ill. Antigenic Determinants of Rabbit Allotypes

It may be inferred from the discussion of allotypic correlates that the determinants of allotypes of groups a and b involve multiple in-

62

THOMAS J. KINDT

terchanges. The question arises whether this complexity may cause variations in the antigenic specificities expressed by the same allotypes on different molecules. For example, is the a1 determinant of one molecule of IgG antigenically identical to that of a second? Early indications of antigenic heterogeneity were given by the presence of double lines in gel diffusion patterns between rabbit sera and allotypic antisera (Oudin, 1960b, 1966). These allotypic specificities were indicated with the prime mark and called subspecificities. The allotypic subspecificities of a1 would be al’, a l ” , etc. Oudin further observed that anti-b5 antibodies occurred in at least two variations: one that would cross-react with b6 light chains, and the other that would not. Anti-b6 had similar populations of antibodies, one showing a cross-reaction with light chains of b5 allotypes, the other not. In certain instances the subspecificities were observed because of the presence of other allotypes on the same chain. For example, it was shown that on the a1 molecules there was a second allotype in the constant region giving rise to the double lines seen in reactions between allotype a1 and anti-a1 (Hamers and Hamers-Casterman, 1965). These are the constant region allotypes, designated A8 and A10. Kakinuma (1971), studying the double specificity of allotype a2 of rabbit immunoglobulin, observed that one of the precipitin lines usually present in the reaction between a2 and anti-a2 was absent when papain-digested IgG was used. On this basis he divided the a2 allotypic specificities into those which are papain-resistant and those which are papain-sensitive. In a more recent report (Kakinuma, 1974), he showed that this phenomenon is not due to an antigenic difference but merely to the complement-binding capacity of the molecules before and after papain treatment. In the latter study, homogeneous antibodies of allotype a2 were used, and these gave double lines, leading Kakinuma to suspect that double lines were not indicative of two allotypically different antibody populations. Because the major rabbit immunoglobulin classes have in common the group a allotypes, the question arises whether allotypic specificities present on the IgG, IgM, and IgA molecules are identical to one another. Todd and Inman (1967) observed in careful quantitative studies that the determinant of IgM was deficient with respect to that of IgG. Seto (1972a,b, 1973) using specifically absorbed allotypic antisera has demonstrated that there are some specificities that are unique to group a allotypes of the two different classes.

63

RABBIT IMMUNOGLOBULIN ALLOTYPES

A. ALLOTYPIC DETERMINANTS ON LIGHTCHAINS OF HOMOGENEOUS ANTIBODIES As pointed out above, rabbit antibody light chains exhibit a great deal of diversity in the sequence of their amino-terminal residues (Hood et al., 1971; Braun and Jaton, 1973; Kindt et ul., 1974; Thunberg, 1974). It was furthermore shown that the group b allotype of IgG from a pool could be ascertained by amino-terminal sequence analysis of this light chain (Hood et al., 1971; Waterfield et al., 1973, Thunberg et al., 1973; Thunberg, 1974). The question then arises whether these substitutions present near the amino terminus of the rabbit light chains have any influence on the antigenic determinant of the group b allotype. A group of homogeneous antibodies with large differences in their amino-terminal amino acid sequences were assembled, and the serological determinants of their group b allotypes compared by quantitative inhibition of radioprecipitation (Kindt et al., 1972). No significant differences were found in the ability of any light chain tested to absorb the antibodies present in the anti-b4 antisera. The reaction between the anti-b4 and the b4 1gc-1251pool was inhibited equally well by each of the homogeneous antibodies even though they differed considerably in their amino-terminal sequences. More recently, Thunberg (1974) has shown that this uniformity of specificity may not be applicable to all group b allotypic determinants. In a study on five homogeneous antibodies of the b9 allotype, at least two different b9 allotypic specificities were observed. The relationship between the specificities seems to be rather simple, where light chains with one specificity are antigenically deficient to those with the other specificity. Table I1 lists inhibition data for b9 TABLE I1 RELATIVE INHIBITION OF BINDINGTO h9 ALLOTYPIC ANTISERA Unlabeled inhibitor" '2"I-Labeledantigen employed in test

b9 Pool

4153-1

4182-1

((k)

(%)

(%)

b9 IgG pool 4153-1 4182-1

100 99

68 100 75

90 100 100

100

All comparisons were carried out using twenty-fold excess of unlabeled inhibitors. Numbers are relative percent inhibition using the inhibition given by the homologous inhibitor as 100%. I'

64

THOMAS J. KINDT

IgG and two homogeneous antibodies of b9 allotype. The reaction between anti-b9 and any of the three lz5I-1abeled preparations is inhibited completely by the b9 pool and by homogeneous antibody 4182-1. That the b9 allotype of 4153-1, on the other hand, is deficient with respect to both 4182-1 and the b9 pool is shown by its inability to absorb completely the anti-b9. These results suggest that b9 light chains of some homogeneous antibodies lack determinants that are present on others and that are represented in the pool. This deficiency has not yet been linked to any structural parameter. Homogeneous antibodies with light chains of allotypes b5 and b6 have not yet been available for such studies. The previous results on the cross-reaction between these allotypes (Oudin, 1966) could be interpreted to indicate the existence of subspecificities on homogeneous antibodies of allotypes b5 and b6.

B.

SUBSPECIFICITIES OF HEAVYCHAIN ALLOTYPICDETERMINANTS

The VH allotypes of group a have also been studied with respect to their diversity of expression. Ten different homogeneous antibodies of allotype a3 were used in a study to determine the extent of the diversity of this allotypic specificity (Kindt et al., 1973b). An inhibition experiment was set up wherein the reactions between 1 2 9 labeled a3 IgG pools and a3 allotypic antisera were inhibited by each of the homogeneous antibodies. The percent inhibitions given by the homogeneous antibodies at thirty-two-fold excess over the a3 pool ranged from 40 to 73%. Under the same conditions, a3 IgG’s and heterogeneous antibodies inhibited this reaction to the extent of 90 or 95%. It is apparent from these results that there is diversity in the a3 allotypic specificities expressed by homogeneous antibodies. Similar diversity has been observed among homogeneous antibodies of allotype a1 and a2, although the variation in inhibition values obtained in these studies was not as marked as in the a3 study (Seide and Kindt, unpublished data). Attempts to enumerate the number of subspecificities present in the a3 allotypic family have not yet been successful. Complementation experiments, carried out by successive addition of different homogeneous antibodies, attempted to reconstruct completely the a3 allotypic spectrum. Successive additions did lead to greater inhibition of the reaction of anti-a3 and a3 IgG-1251,indicatingthat new determinants, that is, those not common to the different homogeneous antibodies, were being added (Kindt et al., 197313).Complete inhibition was never achieved, but this may be because the added anti-

RABBIT IMMUNOGLOBULIN ALLOTYPES

65

bodies represent a limited segment of the entire allotypic repertoire. All antibodies used in this study were directed against microbial carbohydrates. Two explanations for the presence of allotypic subspecificities in the variable region of the rabbit heavy chain have been offered (Kindt et al., 1973b; Thunberg and Kindt, 1974). The first is that the residues involved in the binding site, the hypervariable positions, cause modulation of the antigenic determinant, creating a large spectrum of antigenic specificities. The second possibility is that the allotypic subspecificities result from structural differences present in the constant parts of the variable region of the heavy chain. Such differences could be caused by nondeleterious mutations in replicate copies of the group a allotypic gene. The differences can not be so extensive as to impair recognition of the molecules by the allotypic antisera in the binding reaction. It should be possible by structural and serological experiments to differentiate clearly between these two possibilities. Resolution of the question may come from a structural study of H chains of known serological deficiency. If two V Hregions with different allotypic subspecificities are sequenced, primary structural differences may be observed between the nonhypervariable parts of the variable region (Capra and Kehoe, 1975). On the other hand, if the only V Hdifferences observed are in hypervariable regions, then it could be concluded that modulation of the antigenic determinant is caused by the differences in these regions. In the latter situation a relationship between idiotypes and VH allotypes should exist wherein the subspecificity of the VHallotype should then be the same for idiotypically identical H chains. Another approach may involve production of antisera directed against single subspecificities of the a3 allotypic determinants by adsorption of antiallotypic antisera to homogeneous antibodies. Sequential adsorptions and elutions should result in antibodies that react with an allotypic subspecificity present on one homogeneous antibody of the series. These antisera could be used to “group” antibodies by their allotypic subspecificities. IV. Genetic Relationships among Allotypes

Rabbit allotypes are inherited as if encoded by autosomal codominant genes (Oudin, 1966). Whether the allotypes are encoded by allelic structural genes or are under control of regulator genes has been opened to question by certain experiments (Strosberg et al., 1974; Bell and Dray, 1971). Similar questions have been raised with

66

THOMAS J. KINDT

respect to the allelic nature of the Gm allotypes of man (Rivat et al., 1973) and the C, allotypes of the mouse (Bosma and Bosma, 1974). These data are not as yet sufficient to prove that allotype synthesis is under the control of regulator genes. The genetic relationships discussed here will be relevant even if this is the case. It was shown by Dubiski et al. (1962) that genes coding for Lchain allotypes ( b ) and H-chain allotypes (a) were unlinked. The allotypes of these unlinked groups have been shown to be present in all possible combinations on IgG molecules in the circulation, including combinations not present in parents (Dray et al., 1963). For example, the doubly heterozygous progeny of an u1b4x a2b5mating will have molecules with allotypic combinations alb4, alb5, a2b4, and a2b5, even though the molecules with phenotypic combinations alb5 or a2b4 were not possible in either parent. Individual immunoglobulin molecules will have only one allotype from each group, as shown for the bisymmetrical four-chain structure of IgG (Dray and Nisonoff, 1963) as well as for the oligomeric IgA and IgM molecules (Lawton and Mage, 1969; Schmale et al., 1969). A. LINKAGEGROUPS All of the rabbit allotypic groups thus far reported can be placed at one of four unlinked loci. These include (2) all the H-chain allotypes, (2) the K L-chain allotypes, (3) the A L-chain allotypes, and ( 4 ) the secretory piece allotypes (Mage, 1971; Knight et al., 1974a) (see Table I). The three latter loci are relatively simple because only one or possibly two allotypic groups has been reported for each of these. Contrariwise, the H-chain locus is quite complex with a large number of allotypic groups. Studies on the interaction of the genes in this complex have produced important information on the genetic control of antibody synthesis (Mage, 1971; Todd, 1972). The H-chain linkage group includes allotypes for the V Hregion and the C H region of IgG, IgA, and IgM. No C, allotypes have yet been described for rabbit IgE. Although a large number of combinations of these allotypes are possible, only a few have been observed (Mage et al., 1973a). The combinations of rabbit allotypes are called allogroups (Mage et al., 1973a), and they are analogous to the different haplotypes described for immunoglobulin allotypes of human populations (Natvig and Kunkel, 1973). Figure 9 shows two of the described allogroups. Dubiski and Good (1972, 1974) have studied the combinations of allotypes a l , a2, a3 and e14, el5 in a randomly bred rabbit popula-

67

RABBIT IMMUNOGLOBULIN ALLOTYPES

c,

cf l=k

cr

Ca,

CG2

Ce

.......... CIS

\ \

\

\

TRAVS

9

y l S l x"

I Q '

,

\

\

Choln

Q' d" e"

FIG. 9. Homologous autosomal regions that encode the H chains of a single rabbit. The allotypic combinations shown represent observed H-chain allogroups (Mage et al., 1973a). The dotted and dashed lines represent cis and trans synthesis of two of the IgG molecules possible for a rabbit with this genotype. The arrangement of the genes in the figure is arbitrary.

tion. Of the twenty-one possible genotypic combinations ( ~ ~ e ' ~ / a ~ e ' ~ ; u2e14/a1e15; ~ ~ e ' ~ / u 'etc.) e ' ~ ;only twelve were observed. Six of the

unobserved genotypes were attributed to the complete absence of a single gene pair, u3el4, in the population studied. Absence of the other genotypes was attributed to low gene frequencies of combinations such as a2e14. Similarly, the allotypic combination a2d" is seldom observed (Mandy and Todd, 1970). One example of a rabbit with the genotype has been reported (Kindt et al., 1970a), and it was shown that the rabbit's serum contained approximately equal amounts of a 2 d l l and a2d12 molecules. Reasons for the infrequent occurrence of certain allotypic combinations and the resultant limited number of allogroups are obscure. It is not known whether this phenomenon is related to immunoglobulin synthesis or to the presence of lethal characters in linkage with the allotypes (Dubiski and Good, 1972). B. THETODDPHENOMENON

Allotypes a l , a2, and a3 were among the six originally described (Oudin, 1960a). These are H-chain allotypes and are located in the V Hregion. Detailed evidence for the location of structural correlates of group a allotypes is given in Section II,B. In 1963, Todd observed that group u allotypes were present on molecules of both the IgG and IgM classes (Todd, 1963). This common presence of the same VH marker on H chains of two different classes was also observed for IgA and IgC (Feinstein, 1963).

68

THOMAS J. KINDT

The presence of group a allotypes has been verified for IgM (Todd and Inman, 1967) and for IgA (Lichter, 1967; Pernis et al., 1968; Kindt et al., 1968) by different and more sophisticated techniques than the interfacial precipitin (ring) test used by Todd (1963) in his original observation. It has been further shown that homocytotropic antibodies, which are similar to immunoglobulins of the human IgE class (Lindqvist, 1968), also express the group a allotypes (Kindt and Todd, 1969). Thus, group a markers are present in the variable regions of H chains of each major class. This observation, which has been called the Todd phenomenon, has profound genetic implications (Haber, 1972; Todd, 1972; Cohn, 1967). The Todd phenomenon along with the observation of Hilschmann and Craig (1965) that variability of the L chain was confined to the amino-terminal portion provided strong support for the two gene-one polypeptide chain postulate of Dreyer and Bennett (1965). There are now numerous examples of data that are best explained by the interaction of two genes prior to synthesis of a single polypeptide chain (Haber, 1972). Although no adequate molecular mechanism can be postulated for these gene interactions, the findings of Fleischman (1967) and Knopf et al. (1967) that the rabbit H chain has a single growing point limit the possibilities to those involving events occurring prior to translation. This postulated gene interaction, called translocation, has been discussed in detail by Gally and Edelman (1970). More recently, extensions of this concept to include possible translocations within the V region have been discussed by Capra and Kindt (1975). Recombination between V, and C Hallotypes Further information on antibody biosynthesis has been gained by a study of the allotypes present in the C region in combination with the group a allotypes. The two allotypic groups in the C region of y chains, the d group (Mandy and Todd, 1969, 1970) and the e group (Dubiski, 1969a), are linked to the group a allotypes (Zullo et al., 1968; Dubiski, 196913). Recombinations have been observed between group a and group e allotypes (Mage et al., 1971) and between group a and group d allotypes (Kindt and Mandy, 1972). The frequency of recombinational events between VH and CHallotypes has been estimated at 0.3% (Mage et al., 1973a). It was shown that the immunoglobulins synthesized by the rabbit with u2e14recombinant allotypes (Mage et al., 1971) had H chains with the proper molecular weight (Reynolds et al., 1973) and that the

RABBIT IMMUNOGLOBULIN ALLOTYPES

69

Fd fragments had amino acid compositions that correlated with group a allotypes (Carta-Sorcini et al., 1973). The structural correlate of el4, Thr at position 309, was shown to be present on these molecules (Carta-Sorcini et al., 1973). The molecules synthesized by the rabbit with the a3d12recombinant allotypes (Kindt and Mandy, 1972) lacked Met at position 225 which correlates with d l l . The presence of the hinge-region Thr (the d12 correlate) was not demonstrated for these H chains (Kindt and Mandy, 1972), although there is no reason to doubt its presence. It has been observed that the allotypic combinations present on the majority of H chains reflect the parental linkage groups of VH and CHallotypes. For example, if the a2 and d12 allotypes are inherited from one parent and a3 and d l l from another, the majority of molecules isolated from the serum will have H chains with allotypes a2d12 or a 3 d l l (Kindt et al., 1970~).This was similarly shown for group a and group e using specific immunoabsorbent techniques (Landucci-Toci et al., 1970). There is always a small percentage of molecules that have allotypes of the “recombinant” type, that is, one allotype from each of the two parental linkage groups. Cell staining techniques have given an estimate of 1% for recombination of VH and C, allotypes (Pernis et al., 1973). This closely agrees with the figure obtained by Landucci-Tosi and Tosi (1973). These findings indicate that the majority of y chains are synthesized utilizing information on a chromosome derived from one parent for both V and C regions, that is, in a cis as opposed to a trans fashion. In the example depicted in Fig. 9, cis synthesis would result in H chains with the allotypic combinations a3, d l l , el5 or a2, d12, e15, whereas trans synthesis would yield a3, d12, el5 or a2, d l l , e l 5 H chains. Combinations of group a and group g allotypes present on IgA molecules of heterozygous rabbits of defined genotypes were more recently studied by Knight et al. (197413). The percentage of recombinant molecules found in this study was higher than the 1% observed for VH-C, allotypes, averaging around 2 or 3% for the five IgA samples studied. The existence of so-called recombinant molecules may be taken as evidence that at least two genes are involved in synthesis of a single rabbit H chain. The interaction of the genes on different chromosomes may take place subsequent to somatic recombination events. Alternatively, DNA from the different autosomes may be physically joined prior to transcription, or selective transcription (copy choice) may operate as either an intra- or interchromosomal

70

THOMAS J. KINDT

mechanism. If either of the latter mechanisms is operative, the data given above suggest that the interchromosomal events would be the less frequent. Other recent evidence suggesting that two genes are involved in the synthesis of a single chain comes from studies on the common idiotypes of biclonal myelomas (Natvig and Kunkel, 1973). Structural studies showing the association of the same V Hsubgroups with all Hchain classes (Putnam et al., 1972) as well as V, subgroups with different L-chain subtypes (Hood, 1972) also support the two gene-one polypeptide chain hypothesis. V. Allotypes a n d the immune Response

The immunoglobulins present in the circulation of a normal rabbit presumably represent the animal’s response to the antigens of its environment. The allotypic markers of these immunoglobulins may be related by genetic linkage or by some other association to their antigen-binding specificities, Alternatively, allotypes may have no relationship to the antibody-binding functions and may represent markers on nonfunctional portions of the immunoglobulins. Studies directed toward the determination of relationships between allotype and immune response have revealed that certain antigenic stimuli evoke antibody responses comprised of molecules with a limited number of the possible allotypes. Before discussing this phenomenon, which may be called allotypic or allelic selection, the relevant but more general phenomenon of allelic exclusion will be discussed.

A. ALLELICEXCLUSION As mentioned in Section IV,A, individual immunoglobulin molecules display only one allotype from each group. It follows therefore that any truly homogeneous immunoglobulin or antibody will show allelic exclusion. This selectivity of gene expression has been demonstrated for human myeloma proteins (Harboe et al., 1962) and for certain homogeneous antibodies in humans (Mannik and Kunkel, 1963) and in rabbits (Gel1 and Kelus, 1962; Nisonoff et al., 1967; Rodkey et al., 1970; Kindt et al., 1970b).Allotype exclusion has been given as one of the criteria for homogeneity of rabbit antibodies (Krause, 1970). Allelic Exclusion in Immunoglobulin-Producing Cells The majority of rabbit lymphoid cells that produce immunoglobulins have been shown to display only one allotype from

RABBIT IMMUNOGLOBULIN ALLOTYPES

71

each group (Pernis et al., 1965; Cebra et al., 1966). A detailed study carried out by staining cells with different allotypic antibodies labeled with fluorescent and radioiodine markers revealed that only a small percentage of cells carried two allotypes from the b group (Davie et al., 1971). Although a simiIar conclusion was reached in studies using mixed fluorescent labels (Pernis e t al., 1970), much higher estimates of “double producers” have been made by workers utilizing other techniques. Sell et al. (1970) using blast transformation and thymidine uptake found that a significant fraction of peripheral blood lymphocytes required reaction with two allotypic antisera for stimulation. Wolf et al (1970), using a mixed antiglobulin reaction, estimated that more than 50% of circulating lymphocytes from adult heterozygous rabbits had surface determinants of two group b allotypes, Jones et al. (1973, 1974) have separated peripheral blood lymphocytes from heterozygous (b5b9)rabbits on the fluorescence-activated cell sorter on the basis of their reaction with either anti-b5 or anti-b6 sera. Allelic exclusion of the separated cells and their progeny was observed. Allelic exclusion with respect to allotypes of antibodies produced by single cells has been observed for both the mouse (Weiler, 1965) and the rabbit (Ingraham et al., 1967). Ferrarini et al. (1973), studying rosette-forming cells, also concluded that they could detect only one allelic allotype per cell. Although it appears that the majority of lymphocytes produce immunoglobulins of a single allelic type, the failure to detect the alternative marker in these studies may be related to the sensitivity of the methods or to the type of cell studied. Allelic exclusion has been demonstrated for genes located on the X chromosome (Lyon, 1961). The immunoglobulins represent the only known example of allelic exclusion of autosomal gene products. If allelic exclusion is the rule for all immunoglobulin allotypes, then genes or gene complexes in three different linkage groups must be inactivated in an antibody-producing cell. With our present knowledge of this subject, it is difficult to propose a mechanism for allelic exclusion in immunoglobulin-producing cells. Observations of allotype exclusion pose interesting genetic questions, but they do not actually give information on the relationship between allotypes and antibody specificities unless exclusion on the same allele frequently occurs among antibodies of a given specificity. A related phenomenon that is concerned with this relationship is allelic or allotypic selection.

72

THOMAS J. KINDT

B. ALLELICSELECTION Early studies (Gel1 and Kelus, 1962; Rieder and Oudin, 1963; Lark et al., 1965) involved examination of allotypic markers on specific antibodies. No clear tendency for antibodies of certain allotypes to be synthesized in response to the antigens was noted. Catty et al. (1969) immunized twenty heterozygous ( b4b5)rabbits with pneumococcal polysaccharide and measured the ratios of these allotypes in the antisera. There was a general preference for antibodies with allotype b4 although in some rabbits a reversal was noted. More dramatic examples of allotype selection have been noted in recent years. Zimmerman and Haurowitz (1974) have shown that certain u1a3 rabbits synthesize antibodies against p-azophenylarsonate (Ars) that almost completely lack the a3 allotype. Antibodies to other haptens prepared in these same rabbits showed no similar allotypic preference. It was also shown (Zimmerman and Haurowitz, 1974) that this tendency to selection against allotype a3 is inherited in a simple Mendelian fashion. Antistreptococcal antibodies raised in heterozygous (b4bY)rabbits were observed to have preference for L chains with allotype b4 (Kindt, 1974). The rabbits responded to injection with Group C streptococci by formation of an average of 21 mg./ml. of antibody. Quantitative studies on these antibodies revealed that less than 0.3 mg./ml. carried the b9 allotype. Forty-three offspring from brothersister crosses of these heterozygotes were immunized in a similar fashion and the allotypes of their antibodies were studied. In b4bY heterozygous offspring, the preference for b4 was again observed. There was, however, no difference in the amount of antibody produced by the offspring rabbits regardless of their genotype, Rabbits with b9b9genotype responded equally as well as those with b4b4or b4b9genotype. It was concluded that this allelic preference in heterozygotes does not indicate linkage of structural genes to immune responsiveness. The relationship of allotypes to immune responsiveness remains obscure. In the study reported above (Kindt, 1974). the heterozygous rabbits were derived from the cross of an u2b4rabbit that was a high responder to streptococcal antigens (Eichmann et al., 1971) to an a3b9 rabbit that was a low responder (Todd and Kindt, unpublished data). Although the antibodies showed a marked preference for allotype b4, in the F1 generation, the group a allotypes of the antibodies showed, on the average, normal distribution. There was no segregation of high response to streptococcal immunization with the a or b allotypes of the high responder parent.

RABBIT IMMUNOGLOBULIN ALLOTYPES

73

Unequal Expression of Allelic Allotypes Dubiski (1972) has called attention to the fact that heterozygous rabbits synthesize unequal proportions of allelic allotypes. This inequality usually occurs in a predictable direction, which Dubiski calls “pecking order.” For the group b allotypes the order is b4 > b6 > b5 > b9 Thus in heterozygotes, IgG with allotype b4 will be present in a larger proportion that IgG with any other b allele. This hierarchy of expression holds true not only for group b allotypes, but also for Lchain types, K > A (Appella et al., 1968), and for group a and a-negative H chains (Tack e t al., 1973a). In the author’s experience relative ratios of group a allotypes are not as predictable as those of the group b allotypes, although the ratios are relatively constant within a given rabbit family. In general one finds that a1 > a3 > a2 The inequality of expression of allelic allotypes on serum immunoglobulins is reflected in the cells that synthesize them (Chou et al., 1972, 1974). The ratio of cells expressing allelic allotypic markers was measured using several different techniques and was found to agree closely with the ratio of immunoglobulins in the circulation. Thus defective synthesis of allotypes that occur low in the pecking order cannot be the reason for unequal allelic expression. A possible explanation for unequal expression of allelic allotypes as well as for allotype selection by antigens could involve linkage of sets of antibody-binding site genes to the genes encoding the allotypes. Each allotype may have in close linkage a set or “library” of antibody-binding sites. The set associated with a given allotype may include binding sites that better fit the antigens prevalent in a particular environment than does the set associated with a nonselected allotype. Group b allotypes seem to be associated with V regions that are different hom one another at least in quantitative expression of V, subgroups (Hood et d., 1971; Waterfield et al., 1973). There may also be some qualitative differences, that is, certain residues are present at V-region positions of b4 L chains that have not been observed at the same positions of L chains of the b9 allotype (Thunberg et al., 1973; Thunberg, 1974). VI. Allotype Suppression

Allotype suppression involves the in vivo administration of antibodies against a certain allotype to lower or stop the production of

74

THOMAS J. KINDT

that allotype. The immunoglobulin produced by the suppressed rabbit will then express the allele of the suppressed allotype. If there is no alternative allele (as in a homozygote), a different L-chain type or an H chain of another allotypic group will be synthesized. The terms heterozygous and homozygous suppression are used for the different types of allotype suppression. Heterozygous suppression, first reported by Dray (1962), involved neonatal administration of antibodies directed against paternal allotypes, These suppressed allotypes were not expressed until late in the life of the animal, Obviously, the immunoglobulin with the allotype to be suppressed cannot be present in the circulation at the time of injection with allotypic antisera, and it is this complexity that experimentally differentiates homozygous from heterozygous suppression (Mage, 1971). David and Todd (1969) and Vice et al. (1970) have used the technique of zygote transfer to accomplish homozygous suppression. In these experiments, fertilized ova from matings of allotypically matched homozygotes are transferred at about the eight-cell stage to pseudopregnant females of a different allotype. The offspring rabbits can be suppressed for the allctype of their true parents at birth because no immunoglobulin of this type will be present in their circulation. The immunoglobulins present in these animals will be derived solely from the surrogate mother. For more efficient suppression the surrogate mother may also be immunized to produce antibodies against the allotype to be suppressed. Homozygous suppression has also been accomplished by mating a heterozygously suppressed doe to a buck homozygous for the suppressed allotype. The homozygotes among the offspring will be homozygously suppressed; the heterozygotes will be suppressed for the same allotype as their mother (Mage, 1971; Dubiski, 1967). Suppression experiments have provided material for structural and serological studies on immunoglobulin allotypes that are present in low concentrations in normal rabbits. The work on rabbit A chains (Sections I,D and I1,C) and on group a-negative H chains (Sections I,D and I1,B) was made feasible by the use of these techniques. In addition to the immunochemical aspects of suppression, there are more far-reaching implications in the areas of cellular immunology and ontogeny of the immune response, Investigations into the cellular aspects of allotype suppression have revealed that in rabbits suppressed for allotype b5, lymphocytes with this allotype were absent (Harrison et al., 1973a). Further investigations (Harrison et al., 1973b) on rabbits recovering from suppression revealed that

RABBIT IMMUNOGLOBULIN ALLOTYPES

75

lymphocytes with b5 allotype on their membranes appear in considerable numbers prior to the appearance of detectable levels of b5 IgG in the serum. From these findings it was postulated that the cell with detectable membrane IgG may be a precursor of the antibodysynthesizing cell. It has recently been shown by in vitro experiments that allotype suppression can be accomplished with Fab fragments of the allotypic antibodies (Schuffler and Dray, 1974a,b,c). It has been further shown that, whereas antibodies to VH or C, allotypes are effective in suppression, antibodies to C , allotypes are not effective (Mage, 1975). Furthermore, it has been shown that class-specific antisera directed against IgM caused suppression, but anti-IgG had no effect (Schuffler and Dray, 1974a,b,c). Such results may parallel those obtained in suppression experiments on chickens (Kincade et al., 1970) that suggested IgM as the ontological precursor of IgG. The use of suppression techniques has recently been extended to mouse idiotypes. Such experiments open the possibility that specific responses can be inhibited with no general immunosuppressive effect, Hart et al. (1972, 1973) found that mice suppressed for the idiotypes of antihapten antibodies produced antibodies of the same specificity but with different idiotypes. No significant differences in the magnitude of the response was noted between the suppressed and control animals. Cosenza and Kiihler (1972) used a mouse myeloma protein with activity phosphorylcholine to prepare idiotypic antisera and found that this would inhibit plaque formation by spleen cells from animals immunized with phosphorylcholine polysaccharides. McKearn (1974) used idiotypic antisera to suppress the formation of antibodies that react with alloantigens in the rat. VII. ldiotypes and Allotypes

Because the general subjects of idiotypy and antibody V regions have been extensively reviewed in the preceding volume of this series (Capra and Kehoe, 1975), this section will be concerned only with the relationship of allotypes and idiotypes in the rabbit. Idiotypic antisera may be prepared in different ways, and the method of preparation may influence the specificity of the antisera (Potter and Kunkel, 1971). The two major variables are the species in which the antiserum is prepared and the heterogeneity of the antibody injected to raise the idiotypic response. When a rabbit is injected with rabbit antibody the term homologous idiotypy is used. When a different species, generally a guinea pig or goat, is injected, this is called heterologous idiotypy (Potter and Kunkel, 1971). The

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THOMAS J. KINDT

preparation injected may be homogeneous antibody (Eichmann and Kindt, 1971; Kindt et al., 1974), or the total antibody directed against a given specificity from an individual (Daugharty et al., 1969; Oudin and Michel, 1969a,b), or even the total IgG fraction from an individual (Schade and Nisonoff, 1972). Heterologous idiotypic antisera must be absorbed with pooled IgG to remove antibodies directed against common antigenic determinants, Reactions of guinea pig antisera prepared against homogeneous rabbit antibodies were inhibitable by IgG from various rabbits (Eichmann and Kindt, 1971). It was shown that large differences existed in the ability of different IgG samples to remove the antibodies in the guinea pig antisera. Preimmune IgG samples from the donor rabbit and its siblings were most efficient, whereas absorption with certain homogeneous antibody preparations from other rabbits would not remove this activity to any degree. In the preparation of homologous idiotypic antisera, allotypes are matched to insure against the possibility of allotypic responses in the injected rabbits. This does not, of course, rule out the possibility that unknown allotypes will be present on the antibodies in the injected sample. This difficulty can be minimized by the use of homogeneous antibodies as immunogens and by properly controlled quantitative measurements of idiotypic antibody (Kindt et al., 1973~).As mentioned previously (Section I,C), IgG from preimmune bleedings of the donor rabbit will most likely contain some immunoglobulin of any allotypic specificity that the rabbit can produce. A more imaginative way to eliminate the possibility of unknown allotypic differences has been described by Rodkey (1974). Antibodies isolated from rabbits were injected into these same rabbits after a rest period of more than 1 year. Idiotypic antisera raised by those autoimmunizations were shown by antigen inhibition experiments to be directed against binding site determinants (Rodkey, 1974). Antisera prepared in this fashion may be said to recognize isologous or autologous idiotypes.

V-REGIONALLOTYPESAND

IDIOTYPES

Studies that utilize V-region allotypes and idiotypes have the potential to answer basic immunogenetic questions concerning the number of genes involved in the synthesis of a single antibody V region. The allotypes are markers for what may be considered framework residues, whereas the idiotypes are markers for the binding site-associated hypervariable positions (Capra and Kehoe, 1974). The schematic drawing of the V, region in Fig. 10 shows the loca-

77

RABBIT IMMUNOGLOBULIN ALLOTYPES

I 0

I

I

10

20

I

30

I

40

1

I

I

I

1

SO

60

70

80

90

Residue Number

Group

[I

Allotypic Correlates

Hypervariable Repions

I

100

Ilb

L.:.'.::::,..', '',':I

FIG.10. Diagram of rabbit V Hregion indicating positions of group u allotypic correlates (Section II,C) and hypervariable positions. (Adapted from data of Mole et al., 1971; Mole, personal communication.)

tions of correlates for group a allotypes (Section I1,C) and the hypervariable positions which may be regarded as idiotypic correlates (Capra and Kehoe, 1974). If V regions are encoded by single germline genes, then allotypes and idiotypes should be in stable association; both the framework residues and the binding site of any single V Hregion would be encoded by the same gene. A study on the inheritance of the idiotype of an a2/b4 homogeneous antibody directed against streptococcal Group C carbohydrate (Kindt et aZ., 1973c; Kindt and Krause, 1974) in a rabbit family gave evidence of linkage of this idiotype to allotype a2. At least one exception to the association was observed in that an a3a3 rabbit produced antibodies with the proband idiotype. In another study, an identical idiotype was observed on two different antibodies that differed in V, allotype; one was allotype a3, the other was negative for group a allotype (Waterfield et aZ., 1972; Kindt et al., 1973a). These observations as well as the results of other recent studies keep open the possibility that the information for each V, region is encoded by two or more distinct genes (Capra and Kindt, 1975). Although the rabbit L chain has no known V-region allotype, the V,, subgroups, or more precisely, the amino-terminal sequences of homogeneous L chains can tentatively be used as markers for the framework residues of the L chain. Even among antibodies directed against streptococci, sufficient variety of different amino-terminal sequences has been observed to make this a useful marker (Kindt et d., 1974; Thunberg, 1974). There are, however, not yet sufficient data on VL-region sequences to allow identification of subgroup-

78

THOMAS J. KINDT

specific residues at positions other than at the amino-terminal23 residues. Light-chain amino-terminal sequences have been determined for antibodies to Group C streptococcal carbohydrate with idiotypic cross-reactivity (Klapper and Kindt, 1974). All the antibodies tested had the same amino-terminal sequence as the proband antibody. This VKIIl sequence Ile-Val-Met was represented at less than 5% in the L chains from preimmmune IgG isolated from these rabbits. It was shown, however, that other antibodies with the same L-chain sequence did not carry this common idiotypic determinant (Kindt e t al., 1973~).The same amino-terminal sequence was also observed among L chains from antibodies to streptococci of another group, namely Group A variant (Braun and Jaton, 1973). Two antibodies isolated from the same rabbit with L-chain sequences that differed by one residue among the amino-terminal 23 residues (Ser/Lys at position 12) were idiotypically cross-reactive (Thunberg and Kindt, 1974). By the use of H-L recombinants prepared from these two molecules, it was shown that, whereas the H chains were idiotypically cross-reactive, the L chains were idiotypically indistinguishable. These and other data on idiotypic crossreaction obtained in the author’s laboratory (Kindt et al., 1974) and from the studies of Braun and Kelus (1973) suggest that, although idiotypes may show linkage to V-region markers, these do not appear to be relationships of such stability that would permit one to conclude that they are encoded by the same gene. VIII. Conclusion

Almost 20 years have passed since the appearance of the first reports describing intraspecies antigenic differences among immunoglobulins of the rabbit (Oudin, 1956) and of man (Grubb, 1956; Slater et al., 1955). In the time following these key observations, much of our knowledge of immunogenetics has been gained through studies utilizing these genetic markers. Although this review has placed special emphasis on the rabbit allotypes, the developments with human genetic markers have also been especially informative (Natvig and Kunkel, 1973). Before concluding, it may be helpful to highlight the major advances in immunogenetics that have been achieved through the use of allotypes and to indicate areas where fruitful investigations with allotypes and idiotypes are currently in progress. Although there is a paucity of structural information on rabbit immunoglobulins, an optimal amount of genetic information has been obtained in studies on this species by the use of the immunoglobulin

RABBIT IMMUNOGLOBULIN ALLOTYPES

79

antigenic markers. Despite the fine discriminations that can be obtained by the use of radioimmunoassays in allotypic studies, serological markers remain an indirect way to examine determinants that are ultimately directed by the primary structure of proteins. Homogeneous rabbit antibodies (Krause, 1971; Greenblatt et al., 1973) directed against microbial carbohydrates can now be obtained in sufficient quantities for detailed structural analysis. It should be possible to obtain structural information on antibodies preselected for genetic interest on the basis of their allotypic and idiotypic specificities. Many studies concerning the nature and inheritance of antibody-binding sites that were previously difficult or impossible to undertake because of antibody heterogeneity can now be accomplished. One of the exciting initial discoveries in the area of genetic control of antibody synthesis was the occurrence of rabbit V-region allotypes on H chains of different classes (Todd, 1963; Feinstein, 1963). The one gene-one polypeptide chain hypothesis was to be challenged by this observation. More recently, studies describing recombinations of C, and V, allotypes suggest that different regions of the same polypeptide chain are encoded by closely linked genes (Mage et al., 1971; Kindt and Mandy, 1972). More detailed analyses have placed constraints on the mechanisms by which these genes might interact prior to the synthesis of an antibody H chain (Pernis e t al., 1973; Landucci-Tosi and Tosi, 1973; Knight et al., 197413). The possibility has been raised that rabbit immunoglobulin allotypes are not products of allelic structural genes but rather are products of regulator genes (Bell and Dray, 1971; Strosberg et al., 1974). If this were the case, each individual would possess an identical complement of immunoglobulin structural genes and expression of the genetically determined allotypes would depend on inheritance of regulator genes. To explain allotypic phenomena adequately, it would be necessary for these regulator genes to be involved in the linkage, somatic recombination, and selective gene expression that have been observed in genetic studies employing allotypes. It is difficult to visualize these complex genetic relationships in terms of the repressors and corepressors that have been described for nonmammalian systems involving regulation (Jacob and Monod, 1961). Such speculation best awaits further evidence rigorously demonstrating the existence of regulator genes for immunoglobulins. Allelic exclusion in antibody-producing cells provided the first observation of selective expression of autosomal genes (Pernis et al., 1965; Weiler, 1965). It is not certain that all cells that produce antibodies always show selective expression of allelic genes (Davie et

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al., 1971; Wolf et al., 1971). Allelic exclusion may be limited to certain cell types or, alternatively , to certain developmental stages for all antibody-producing cells. Certainly a description of allelic exclusion in biochemical terms would enrich our knowledge of mechanisms in mammalian genetics. Recently described cell separation techniques may expedite a more complete description of this phenomenon (Jones et al., 1973, 1974). Allotypic and idiotypic suppression have been employed in studies in cellular immunology and ontogeny of the immune response (Dray, 1962; Mage, 1971; Harrison et al., 1973b). Recent studies on the suppression of specific immune responses which may lead to allograft rejection (McKearn, 1974) suggest new and practical roles for this experimental tool. Clearly the ability to suppress specifically deleterious immune responses has obvious and important medical application. The use of idiotypic markers in conjunction with V, allotypic markers should help define the relationships between these V-region markers and should give new information concerning the generation of antibody diversity. Idiotypes may be considered markers for hypervariable regions of H chains (Capra and Kehoe, 1974, 1975), while VHallotypes have their origins in the more constant portions of the V region. Although there are indications that idiotypes and V-region markers are linked (Kindt et al., 1973c; Klapper and Kindt, 1974), other studies have shown that the association between these two Vregion determinants is not sufficiently stable to conclude that they are encoded by the same genes (Kindt et al., 1973a, 1974). It has been suggested that separate genes encode binding sites (idiotypes) and framework residues (allotypes or V, subgroups) of the V-region (Kindt et al., 1974; Capra and Kehoe, 1975; Capra and Kindt, 1974). It may be concluded that studies on allotypes and idiotypes have made significant contributions to our knowledge of genetic control of immunoglobulin synthesis, knowledge that is, in many instances, relevant to more general biological topics. It should, furthermore, be evident from the above survey that these genetic markers will be of continuing value in basic and possibly applied immunology. Not only will new questions arise from these studies but the genetic markers of immunoglobulins will continue to provide the basis for formulating experimental approaches to the answers.

ACKNOWLEDGMENTS The author thanks Drs. J. D. Capra, J. M . Kehoe, D. G . Klapper, R . M . Krause, L. E. Mole, A. D. Strosberg, A. L. Thunberg, and C. W. Todd for their valuable suggestions and criticisms. I am grateful to Drs. R. Goodfleisch, W. C. Hanly, K. L. Knight, M. Margolies, and L. E. Mole for allowing me to quote from their unpublished results

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and to Dr. M. Mudgett for imaginative help with descriptive material. The expert editorial and secretarial assistance of Ms. Marie Kindt is gratehlly acknowledged.

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Stemke, G. W. (1965). lmmunochemisty 25,359. Steward, M. W., and Todd, C. W. (1969). Proc. Soc. E x p . Biol. Med. 131, 1393. Strosberg, A. D., Fraser, K. J., Margolies, M. N., and Haber, E. (1972). Biochemistry 11, 4978. Strosberg, A. D., Hamers-Casterman, C., Van der Loo, W., and Hamers, R. (1974). J. Immunol. 113, 1313. Tack, B. F., Feintuch, K., Todd, C. W., and Prahl, J. W. (1973a). Biochemistry 12,5172. Tack, B. F., Prahl, J. W., and Todd, C. W. (197313).Biochemistry 12,5178. Thunberg, A. L. (1974). Ph.D. Thesis, Rockefeller Univ., New York. Thunberg, A. L., and Kindt, T. J. (1974). Eur. J. lmmunol. 4,478. Thunberg, A. L., Lackland, H., and Kindt, T. J. (1973).J . lmmunol. 111, 1755. Todd, C. W. (1963). Biochem. Biophys. Res. Commun. 11, 170. Todd, C. W. (1972). Fed. Proc., Fed. Amer. Soc. E x p . Biol. 31, 188.. Todd, C. W., and Inman, F. P. (1967). lmmunochemisty 4,407. Tosi, R., and Landucci-Tossi, S. (1973). In “Contemporary Topics in Molecular Immunology” (R. A. Reisfeld and W. J. Mandy, eds), pp. 79-93. Plenum, New York. Vice, J. L., Gilman-Sachs, A., Hunt, W. L., and Dray, S. (1970).J . Zmmunol. 104, 550. Waterfield, M. D., Prahl, J. W., Hood, L. E., Kindt, T. J., and Krause, R. M. (1972). Nature (London), New Biol. 240, 215. Waterfield, M. D., Morris, J. E., Hood, L. E., and Todd, C. W. (1973). J. Immunol. 110, 227. Weigert, M., Raschke, W. C., Carson, D., and Cohn, M. (1974).J. E x p . Med. 139, 137. Weiler, E., (1965). Proc. Nut. Acad. Ssi. U . S . 54, 1765. Wilkinson, J. M. (1969a). Biochem. J. 112, 173. Wilkinson, J. M. (1969b). Nature (London)223, 616. Wolf, B., Janeway, C. A., Jr., Coombs, R. R. A,, Carry, D., Cell, P. G. H., and Kelus, A. S. (1971). Immunology 20, 931. Wu, T. T., and Kabat, E. A. (1970).J . E x p . Med. 132, 211. Zikan, J., Skarova, B., and Rejnek, J. (1967). Folia Microbiol. (Prague) 12, 162. Zimmerman, S., and Haurowitz, F. (1974).lmmunochemisty 11, 403. Zullo, D. M., Todd, C. W., and Mandy, W. J. (1968). Proc. Can. Fed. Biol. SOC. 11, 111.

Cyclical Production of Antibody as a Regulatory Mechanism in the Immune Response' WILLIAM 0. WEIGLE Department o f Immunopathology, Scripps Clinic a n d Research Foundation, l a Jolla, California

I. Introduction

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11. Cycling in the Immune Response . . . . . 111. Synchrony of Appearance of Antibody-Producing Cells

IV. Conclusions References

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I. Introduction

As important as the mechanisms that initiate an immune response are the mechanisms responsible for the regulation of immunity once it has been initiated. The cessation of antibody production induced by antigens that are rapidly catabolized may result from failure of a persistent stimulation of antigen-reactive cells. However, it is more difficult to explain regulation of an immune response to antigens that persist in lymphoid tissue for even relatively short periods of time. It is accepted that such control lies within the immune system itself and that both antibody and suppressor cells can be responsible for regulation. Since the initial observations of Uhr and Baumann (l),it has become well documented that the primary immune response can be readily inhibited by injection of specific antibody (reviewed in ref. 2) and that the temporal relationship between the injection of antigen and antibody is critical, Of further importance is that the secondary response is relatively resistant to inhibition by antibody. These experimental results suggest a role for circulating antibody in the regulation of the immune response, but the mechanism by which this phenomenon occurs is not clear. However, it was demonstrated by several workers (3-6) that the inhibition probably resulted from covering of specific antigen determinants thus inhibiting their stimulation of specific lymphocytes. It appears that the B lymphocytes may I This is Publication No. 879 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation. The author's experimental work presented here was supported by the U. S. Public Health Service Grant AI-07007, American Cancer Grant IM-42D, Atomic Energy Contract (04-3)-410, and U. S. Public Health Service Research Career Award 5-K6-GM-6936.

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be the cells for which stimulation is inhibited, since passive antibody does not inhibit priming of T cells for a secondary response (7). Although there is little question that passive antibody can interfere with the expression of an immune response, only sparse information has been obtained demonstrating that the antibody produced during an antibody response has any role in controlling that same response, The best evidence supporting this latter point is the enhancement of the immune response after removal of circulating antibody. In these experiments antibody was removed specifically in two ways: first, by successive exposure of aliquots of plasma taken from immunized animals to solid-phase immunoadsorbents followed by return of the adsorbed plasma (8); second, by exchange transfusion of animals immunized to two different antigens with blood containing antibody specific to only one of the antigens, thereby specifically deleting the other antibody (9). After the removal, there was a marked rise in serum levels of specific antibody, which frequently reached peak titers exceeding those prior to removal. Such observations strongly implicate circulating antibody in the regulation of the immune response. These studies led to the proposal by Bystryn et uZ. (10) that the regulation of antibody production to both persisting and readily catabolized antigens is controlled by antibody via a dynamic equilibrium among circulating antibody, antigen, and antigen-antibody complexes throughout the extracellular compartment. Thus, alterations in circulating antibody levels may shift the equilibrium toward free antigen or complex formation, with a consequent increase or decrease in stimulation of antibody formation. With readily catabolized antigens the need for regulation may end with the elimination of the antigen, whereas continuing control may be necessary with persisting antigens. As is discussed below, the mechanism presented above is not compatible with data related to an antigen that has been shown to persist in the spleen. Suppression of the immune response may occur after specific interaction of anti-idiotypic antibody with specific idiotypes, where the idiotype is a specific determinant in the variable region of the antibody molecule. This model represents another mechanism for specifically inhibiting the formation of a selected antibody population without generalized suppression of the immune response. The possibility of such a mechanism was demonstrated by Hart et aZ. (1l),who observed that injection into A/ J mice of rabbit anti-idiotype antibody directed to A/J antiphenylarsonate antibody suppressed almost completely the subsequent production of antibody of the corresponding idiotype. Recently, it has been shown that such anti-idiotype an-

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tibody reacts with idiotypic determinants on Ig receptors of normal B lymphocytes (12) and that these idiotype-carrying cells are sensitive to lysis by anti-idiotypic serum. Anti-idiotype antibody prepared in F, hybrids to a parental alloantibody directed to the other parent inhibits the graft-versus-host (GVH) reaction (13) and may well be antibody to the idiotype of T-cell receptors. Thus, it seems reasonable that an individual could possibly produce antibody to idiotypes produced by specific clones of cells. Jerne (14) has recently postulated a mechanism of specific anti-idiotype suppression of the immune response. He suggested that the control of the immune response is brought about by balance of a network of regulatory mechanisms in the immune system involving stimulation and suppression by antiidiotype antibody produced in situ. Thus, there would be a stimulatory effect when idiotypes are recognized by antibody as receptors on the cell surface and a suppressive effect when idiotypes of the Ig receptors on the surface of cells are recognized by free antibody. Over the past few years a large volume of data has been collected which strongly suggests that part of the regulation of the immune response occurs at the cell level (reviewed in refs. 15-17) by a subpopulation of T cells. It appears that one population of T cells acts as helper cells and enhances the immune response, whereas the other population suppresses the response. Such suppressor T-cell activity has been observed with many antigens (16), even with those antigens that are thymus-independent (18, 19). In specific regulation of the immune response, specific suppressor activity is apparently a direct result of antigen stimulation (16). It is of interest that helper and suppressor cell activities appear to play interacting roles resulting in an actual regulation. An example is the GVH reaction in mice (in which parental spleen cells are injected into F, hybrids). When the responding host cell is highly active, the net interaction effect is suppression, whereas when the responding host cell is relatively inactive, the net interaction effect is enhancement (20). Although it appears that the suppressive effect lies in a subpopulation of T cells (T,) with specific properties (21-23) and the helper cell lies in another subpopulation (T,) with different properties, the possibility that suppressor and helper activities are present in the same cell has not been ruled out. Many of the studies that have shown suppressor cell activities involve depletion of T cells by injection of antilymphocyte serum or after adult thymectomy (18, 19, 21, 23), whereas other studies have shown that T cells from immunized animals suppress the responses of normal animals (15, 20, 24-26). Taniguchi and Tada (27), using

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procedures that resulted in depletion of T cells in the rabbit, implicated the thymus in the maturation of both helper and suppressor T cells. Not only has production of IgG and IgM been shown to be affected by suppressor T cells, but IgE production has also been shown to be regulated by T cells. The homocytotropic (IgE) antidinitrophenyl (anti-DNP) response of rats immunized with DNP-Ascaris could be increased by a number of maneuvers that deleted T cells (25, 28, 29). This enhanced response could be abolished by injection of thymus cells from syngeneic rats hyperimmunized to Ascaris or DNP-Ascaris, but not by cells from rats hyperimmunized to DNP conjugated to an unrelated carrier (29), suggesting that it was the T cells and not the B cells that caused suppression. Droege (24) showed that there are three T-cell types in the chicken’s thymus. One T-cell type present in 16-week-old bursectomized chickens immunized with Brucella abortis had specific suppressor activity and was not one of the cell types involved in the GVH reaction. The suppressor T cell was the main T-cell type present in young chickens (0-2 weeks), whereas old chickens (8 months) had no suppressor T-cell activity. Thymus cells from normal (unimmunized) chickens possessed helper activity. Suppressor T cells have been implicated in certain types of immunological tolerance (30-34), but not when a complete unresponsive state is present in both B and T cells (35,36). It is not certain whether both the T and B cells are affected by suppressor T cells or how the suppressor cells function; however, supernatants from T cells stimulated in vitro are capable of either enhancing (37-45) or suppressing (44-49) the immune response. Thus, it appears that the immune response is capable of being regulated through at least two active processes: one involves a B-cell product and the other a T-cell product. It may be that during any immune response both mechanisms are at play, although one may be more pronounced than the other. The present review concerns the regulation of the immune response following a single injection of antigen. II. Cycling in the Immune Response

It has been known for a number of years that fluctuation in the levels of antibody can occur after a single injection of antigen. Moller (50) observed two peaks of antibody-producing cells in spleens of mice receiving a single injection of lipopolysaccharide (LPS) from Escherichia coli. These two peaks were separated by a period of time when fewer antibody-producing cells were present. The antibody-

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producing cells were enumerated by a modification of the Jerne and Nordin (51)hemolytic plaque assay in which the number of plaqueforming cells (PFC) to LPS was determined. Britton and Miiller (52) observed cyclical appearances of direct PFC to LPS in CBA mice given a single injection of E . coli bacteria. Four individual peaks of PFC were apparent during a period of 46 days after the injection. These findings were interpreted as evidence that IgM antibody produced endogenously at various phases of the immune response blocked the antigen from stimulating competent cells. After antibody coating the antigen was catabolized, the free antigen could again stimulate cells, thus leading to a cyclical production of antibody. A less dramatic fluctuation was also observed in the production of hemolytic antibody to LPS. In the above experiments, only the kinetics of IgM antibody production was monitored. A similar fluctuation in the IgM response to the 0 antigen of Salmonella adelaide was observed in chickens (53).At least five peaks were observed in a 65-day period after a single injection of S. adelaide. No IgG antibody could be detected during this period of time. A biphasic IgM antibody response was recently noted with another thymus-independent antigen. Schlegel (54) studied the kinetics of the response to the hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) coupled to polymerized flagellin (POL) in CBA mice after a single intraperitoneal injection. The IgM PFC response to NIP peaked on the third day after injection, and then decreased markedly on day 4. The lowest number of PFC was on the fifth day. The second peak of IgM PFC appeared between days 6 and 8. No cyclical phenomenon was observed during the secondary response. A number of workers have observed cycling during the immune response to a single injection of sheep red blood cells (SRBC). Wortis et al. (55) noticed a biphasic appearance of both direct (IgM) and indirect (IgG) PFC in spleens of CBA mice each given a single injection of SRBC. These peaks occurred on days 4-6 and days 9-11 with a smaller number of PFC being present at times between the peaks, Dual peaks occurred over a six log,, range in the number of SRBC injected. The appearance of the second peak was more obvious after intravenous injections than after intraperitoneal injections. It was suggested that the biphasic appearance of PFC might result from antibodies of different molecular Ig subclasses or with different avidities. The importance of antibody avidity for suppression of the immune response has been well documented (56). Sell et a2. (57) also noticed biphasic appearances of PFC in the spleens of Balblc mice after each received a single injection of SRBC. By using spe-

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cific amplifying sera, they were able to study the kinetics of the appearance of PFC of IgM, IgA, and IgG classes and IgG subclasses. Only a single peak was observed with IgM PFC. However, two peaks each of IgA, IgGl, IgGea, and IgG2b were observed. The first peak appeared 5-6 days after injection, and by day 7 the PFC had disappeared from the spleens. Plaque-forming cells reappeared with a peak response on either day 8 or 9. These results differ from those of Wortis et al. (55) in that no cycling was observed with IgM, the intervals between the peaks were slightly shorter, and there was a complete disappearance of PFC between the two peaks. Since the antigen dose and route of injection were the same, the differences might be attributed to differences in the CBA and Balb/c strains of mice. Sell et aZ. (57) offered several possible explanations for the biphasic response, but favored control by a homeostatic mechanism involving inhibition of the antigen by endogenous antibody. Stimpfling and Richardson (58) reported a fluctuation in the production of hemagglutinating antibody to SRBC in B10.D2 mice. Four peaks appeared over a period of 30 days. However some of these peaks were subtle increases in antibody, and no attempt was made to determine the classes of antibody involved. The failure to observe cyclical appearances of PFC of IgM classes cannot be readily explained. The importance of the route of injection is evidenced by the observation of cycling in a noninbred strain of Swiss white mice after a single injection of SRBC (59). Intravenous injection resulted in a fluctuation in the PFC response in the spleen, but only a single peak appears in the renal nodes. However, intradermal injection into the footpad resulted in a biphasic IgG response (peaked at days 5 and 10) in the draining popliteal node, but only a single peak of PFC appeared in distal lymph nodes or the spleen. Compatible with the observations of Sell et aZ. (57) a biphasic IgM response was not observed after injection by either route. In addition to LPS and SRBC, a number of other antigens have been reported to cause fluctuations in immune responses. A cyclic production of immunity has been shown to occur after immunization with alloantigens. Congenic strains of mice differing in an H-2 antigen were injected intraperitoneally, and hemagglutinating antibody to the H-2 antigens was determined (60). In most instances 20-30 days separated the peaks of hemagglutinating antibody. A fluctuation in the immune response to H-2 antigens has also been observed in which four peaks occurred within 35 days (58). The period of time separating these peaks was only 8-10 days. In the latter experiments, A/WySn thymus cells were injected into the peritoneal cavities of C57BL/10 ScSn mice. A cyclic resistance to transplantable allogeneic

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tumors was shown to occur after a single subcutaneous injection of nontumor cells of the same type as the tumor (61,62). In uiuo assays for tumor resistance in one study (61) demonstrated a biphasic resistance separated by 18 or 19 days, whereas in another study (62) similar assays showed four peaks of resistance over a period of approximately 45 days. The latter two studies are difficult to interpret, since one does not know the mechanism of in uiuo destruction of transplantable allogeneic tumor cells. Although cell-mediated immunity appears to be responsible for destruction of autochthonous tumors, the destruction of transplantable allogeneic tumor cells may involve other mechanisms that require the participation of antibody. Denham e t al. (63) observed two peaks in the development of cell-mediated immunity to tumor allografts injected intraperitoneally into C57BL mice. The first peak appeared between days 6 and 8, and the second peak of activity was present between days 14 and 18. A similar cycling of cell-mediated immunity to xenografts was demonstrated in the spleen cells of mice injected with hamster tumor cells (64). Cell-mediated immunity to the tumor peaked on day 10 in both the spleens and lymph nodes. Additional peaks of cytotoxic activity were seen at days 18 and 42 in spIeens, but not in lymph nodes. It may be that two functionally different cell types were involved in the first two peaks, since they appeared to differ in their resistance to irradiation and mitomycin. Recently, Britton (65) has reported a biphasic response for GVH reactions, F, hybrid mice (A/sn x C57BL) were injected with A/sn parental cells. The mice developed cytotoxic cells that killed i n uitro both the host cells and YAC lymphoma cells carrying the same histocompatibility antigens as the host cells. The cytotoxic cells appeared on day 2 after injection, and peak activity reappeared on day 14. No cytotoxic activity was present on day 5 and only a trace amount was detectable on day 9. However, the cytotoxicity was resistant to anti-8 and complement. It was also resistant to rabbit anti-B lymphocyte antibody, and removal of adherent cells had no effect. Thus, this response might be a particular manifestation of the GVH reaction and could possibly require the participation of antibody. Gillespe and Barth (66) observed a cyclical pattern in the cytotoxicity response of spleen cells of C57BL/6 mice rejecting A/J skin grafts. By using a microcytotoxicity assay, they observed three peaks showing cytotoxicity of C57BL/6 lymphocytes to A/J cells. The first peak appeared 9-10 days after grafting and was followed by two additional peaks of activity on days 14 and 17. The cytotoxicity was not the result of antibody, and suppressor cell activity could not be demonstrated.

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WILLIAM 0. WEIGLE

----

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6 10 14 18 Day After Injection

lnllrtcl PFC O i r ~ e PI I C

22

FIG. 1. Kinetics of the appearance of plaque-forming cells (PFC) in spleens of rabbits after a single intravenous injection of 2 mg. aggregated human IgG. [Reprinted from Romball, C. G., and Weigle, W. 0. (1973).J . E r p . Med. 138, 1426.1

The cyclical appearance of antibody has recently been reported after intravenous injections of heat-aggregated heterologous yglobulins. A single intravenous injection of 2 mg. aggregated human IgG (AHuIgG) into rabbits resulted in a cyclical appearance of PFC to HuIgG in the spleen (67).After an initial peak of PFC on day 5, two subsequent peaks of PFC were observed at 8-day intervals, with a marked decrease in the number of PFC detected during these intervals (Fig. 1). The peaks were sharp, showing dramatic increases and decreases in the number of PFC. A similar response was observed in the spleens of rabbits injected intravenously with 2 mg. of aggregated turkey IgG (Fig. 2). Indirect PFC reached a peak 43 days

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FIG.2. Kinetics of the appearance of plaque-forming cells (PFC) in spleens of rabbits after a single injection of 2 mg. of aggregated turkey IgG. [Reprinted from Romball, C. G., and Weigle, W. 0. (1973).J . E x p . Med. 138, 1426.1

CYCLICAL ANTIBODY PRODUCTION AND IMMUNE RESPONSE

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after injection. Only a few PFC were present on day 8, and by day 12, a second peak of PFC was observed. As with AHuIgG, the interval between peaks was approximately 8 days. Direct PFC reached a peak on day 5, but no second peak was observed. The failure to observe a second peak of direct PFC might have been due to the insensitivity of the assay system. By reduction with dithiothreitol and alkylation with iodoacetamide within the assay system before developing the plaques with complement, it was shown that direct PFC and indirect PFC were the result of IgM and IgG antibody, respectively. The cyclical appearance of PFC was reflected in the level of circulating antibody. Peak antibody was present in the serum 3 days after the first peak of PFC, and then the level decreased steadily until after the appearance of the second peak PFC when the level of antibody again began to increase (Fig. 3). The relationship of antibody to the cycling of PFC is more dramatically observed in individual animals. An occasional rabbit will not give a significant second peak of PFC; in these cases, there is a rapid loss of circulating antibody (Fig. 4).By contrast, in the rabbits showing a second peak of PFC, antibody levels remain high. There was a markedly enhanced PFC response (on day 3 ) after a second injection of AHuIgG; however, no subsequent peak occurred. Biphasic responses to heat-aggregated bovine y-globulin (ABGG) have been seen in mice. CBA mice injected with 1 mg. of ABGG showed a peak of PFC in their spleens on days 5 and 10 after injection (68). As previously observed by Sell et al. (57) with SRBC, the biphasic peak was seen with IgG but not IgM antibody. However, mice make only meager IgM PFC responses to aggregated y-globulins (69). The cyclical appearance of antibody after a single injection of an-

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FIG.3. Relationship between plaque-forming cells (PFC) in spleens of rabbits and precipitating antibody (AhN) in peripheral blood after a single injection of 2 mg. aggregated human IgC. [Reprinted from Romball, C. G., and Weigle, W. 0. (1973). J . E x p . Med. 138, 1426.1

WILLIAM 0.WEIGLE

96 "o]

Rabbit # 1

i?\

180

t14

Day After Injection

FIG.4. Relationship between plaque-forming cells (PFC)and precipitating antibody (AbN) in peripheral blood after a single injection of 2 mg. aggregated human IgG. The data are from representative rabbits that were bled 25 ml. of blood (cardiac) on the respective days. [Reprinted from Romball, C. G., and Weigle, W. 0. (1973). J . E x p . Med. 138, 1426.1

tigen probably depends on persistence of antigen in the organ producing the antibody. The persistence of antigen in the spleens of rabbits injected with AHuIgG was shown both functionally and directly (67). Functional persistence of antigen was demonstrated by the stimulation of sensitized cells in irradiated recipients injected with AHuIgG before transfer of the cells. Using similar cell transfer procedures, Britton et al. (70) have demonstrated the persistence of SRBC for at least 14 days and LPS for at least 45 days. Persistence of antigen was shown directly by determining organ distribution of AHu1gG-l2T. Injection of AHuIgG-12sI resulted in localization of some trichloroacetic acid-precipitable '9 activity in most of the tissues tested; however, the major localization was in the lung (as would be expected after an intravenous injection) and spleen (Table I). The labeled AHuIgG was rapidly eliminated from the lungs and other tissues, but persisted in the spleen with a half-life of 13 days. Localization of antigen after various routes of injection has been studied and reviewed by Nossal and co-workers (71, 72). In studies with AHuIgG, radioautography demonstrated dense localization of AHuIgG-lZ5Iin the germinal centers of the spleen 10 days after injec-

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TABLE I ORCAN DISTRIBUTION OF ACCHECATED HUMANICG-'"1(2 MC.) I N RABBITS AFTER INTRAVENOUSINJECTION ON DAYO"?" Fg. AHuIgG-lLsII/gm.tissue" Organ

Day 2

Day 10

TI,, (days)d

Lung Kidney Liver Spleen Mesenteric lymph nodes Thymus

9.80 0.26 0.15 1.14 0.09

0.86 0.03 0.02 0.74 0.01 0.01

2.3 2.5 2.7 12.8 2.5 -

-

Reprinted from Romball, C. G., and Weigle, W. 0. (1973).J . Exp. Med. 138, 1426. represent averages of 3 animals for each day. ' Trichloroacetic acid (TCA) precipitable. " T h e TI,, value was calculated between D2 and D10 and was based on TCAprecipitable activity.

'' Values

tion (Fig. 5), but no such localization was observed at this time in the mesenteric lymph nodes. Failure to observe such localization corresponds with the failure to observe PFC in the nodes on day 13, which further indicates the need of persistent antigen for cyclical production of antibody. The route of injection may determine organ localization of antigen and, thus, determine whether cyclical production of antibody occurs. Intraperitoneal injections of tumor xenografts also resulted in cyclical production of cell-mediated immunity in the spleen, but not in the lymph nodes (67). On the other hand, an intradermal injection of SRBC resulted in a biphasic immune response in the draining lymph node, but not the spleen, whereas intravenous injections showed a biphasic response in the spleen, but not in the lymph nodes (59). It is not clear whether localization of AHuIgG in the germinal centers was induced by antibody, as has been demonstrated with soluble antigens, or whether the AHuIgG bound to cells of the follicles directly through the Fc fragment and complement. Direct binding is supported by the observation of Brown et ul. (73) who demonstrated that aggregated human y-globulin, but not the monomeric form, rapidly localized in the germinal centers of the lymph nodes after intradermal injection into guinea pigs. It may be that localization of aggregated y-globulins in lymphoid tissue is due to their affinity for lymphocytes (74, 75). Failure of cycling in the secondary response to

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WILLIAM 0. WEIGLE

FIG.5 . Radioautographic evidence of follicular localization of aggregated human IgG-lzsI in the spleens of rabbits. Stained with hematoxylin. Magnification: (a) x50; (b) x320.

AHuIgG (67)and possibly NIP-POL (54) may result from the lack of persisting antigen, since it has been shown that there is a more rapid disappearance of antigen injected into previously stimulated animals. Although persistence of antigen appears to be correlated with cyclical antibody production, insufficient data about either persisting or nonpersisting antigens are available to warrant such a generalization. It would be of interest to know the long-term kinetics of the response to antigens that persist for long periods of time, such as pneumococcal polysaccharides. Because of the apparent lack of

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appropriate enzymes, this antigen is not catabolized (76). Chen et al. (77) did observe cyclical variations in the immune response of rabbits given periodic injections of Type I11 or Type VIII pneumococcal bacteria throughout their study. Rabbits received three weekly injections for the first month and then weekly injections were given indefinitely. The time of cycling varied from one rabbit to another and differed from the cycling phenomenon previously discussed in that long intervals (up to 12 weeks) of depressed responsiveness occurred between the peaks. As suggested by the authors, this type of cyclical variation must be complex and is the result of an interplay of several competing and cooperating mechanisms. It seems most likely that antibody plays a major role in the cyclical production of antibody. Certainly it is well documented that passive antibody can inhibit the induction of a primary response (2), and after the antibody is catabolized, the exposed antigen can stimulate the animal to antibody production. However, other mechanisms have been suggested. The secondary peaks could be from a release of stored antigen by macrophages (52); even though this seems unlikely in light of the available data. It was also suggested that cycling might result from the presence of different subclasses of Ig-forming cells, each having its own response time (55). This possibility is not in conflict with the current concept of antibody-mediated regulation, since appearance of such antibody could be the consequence of suppression. It has been suggested that the various peaks might be the result of responses to different determinates at different times (55). This seems unlikely, since the second peak could be suppressed by passive antibody produced during the first peak (52). Although the evidence that antibody is directly responsible for the cyclical production of immunity is impressive, it is possible that regulatory T cells play a role at least in some of the cases. It has been adequately demonstrated that T cells can both help and suppress the immune response, and in all likelihood they play a major role in regulation of immunity. Once the immune response is stimulated and reaches a critical level, suppressor T cells may be stimulated and competent B and/or T cells would be suppressed (16). With a decrease in the activity of antibody-producing cells, helper cell activity might be activated, and another immune phase would be initiated resulting in a second peak of immunity. This cycling may then continue as long as antigen persists. Regulatory T cells may be involved in the sporadic variations in immune responses to pneumococcal polysaccharide, in which mul-

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tiple injections of the bacteria are given to rabbits over a long period of time, and long intervals of unresponsiveness are observed between peaks of responsiveness (77).T-cell regulation may also be involved in regulation of cell-mediated immunity to allogeneic (63) and xenogeneic tumors (64); however, the recipients do contain circulating antibody. In the case of responses to xenogeneic tumors in which peaks were observed at days 10, 18, and 42, antibody may have been responsible for suppressing the first peak which reappeared 8 days later, whereas suppressor T cells may have caused the suppression of the second peak that remained suppressed for 24 days. T cells may be more important for regulation of secondary or hyperimmune responses, since suppressor T cells are found in abundance in such stimulated animals (16), and it is difficult to suppress the secondary response with passive antibody. The T regulatory cells may be involved indirectly in cyclical production of immunity. It was suggested that antigen-antibody complexes in antigen excess might cause regulatory T cells to enhance the B-cell response, yet during antibody excess suppression would occur (14). In the case of cyclical production of antibody to AHuIgG, it appears that the second and third peaks of PFC are the result of stimulation of memory cells (67). It is unlikely that the precise timing of the peaks could be the result of synchronized recruitment of virgin precursor cells. In agreement with the suggestion that memory cells primed during the first peak are responsible for the second peak is the fact that the dose of antigen affects the ratio of PFC in the first and second peaks (Fig. 6). This result suggests that with higher doses of AHuIgG more cells had sufficient contact with the antigen to cause exhaustive differentiation to PFC, thus leaving fewer memory cells. By contrast, with lower doses of antigen, cells were stimulated to differentiate, yet with most cells not enough hits per cell occurred to complete the pathway from precursor to PFC. This would explain why, after injection of extremely small amounts of AHuIgG (0.02 mg.), the ratio of the first to second peaks was small. If the above postulate is valid, then the affinity of the antibody produced during the subsequent peaks should be greater than that produced during the second peak. Ill. Synchrony of Appearance of Antibody-Producing Cells

The cyclical appearance of antibody-producing cells after a single injection of AHuIgG into rabbits seems to be synchronized to 8-day cycles (67). Similar synchrony with multiple peaks was observed with the antibody response to H-2 antigens in mice and to a lesser

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201 * 0

9 0.2-

0

-

I

I

-

I

I I I

0.02-

i

extent to SRBC (58). In the latter studies, 8-10 day intervals separated the peaks. A cyclical production of PFC to LPS after a single intraperitoneal injection of certain doses of Escherichia coli appeared to be synchronized to some degree, in that four peaks, also with 8-10 day intervals between the peaks, were observed. Furthermore, a cyclical production of IgM antibody in chickens appeared to be synchronized in that each of the five peaks observed after a single injection of LPS was separated by approximately 5 days of a repression in the response (53).Other studies in which assays for antibody or antibody-producing cells were not carried out beyond a second peak yielded no information on synchrony of the cyclical production of antibody. However, it is likely that synchronous production of antibody is more common than realized in the past. It is much easier to understand cyclical production of antibody after a single injection of persisting antigen than it is to understand the regulatory mechanisms resulting in synchrony. Although circulating antibody of relatively high affinity is probably responsible for the inhibition of the immune response after it has been initiated, it appears unlikely to be involved in the precise synchrony observed in the cyclical production of antibody after a single injection of AHuIgG. If circulating antibody is responsible for the cyclical synthesis of antibody, the time of appearance of the subsequent peaks would be expected to vary with the level of antibody present in the

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circulation either after the first peak or just before the subsequent peak. However, when a ten-fold or more difference was observed in levels of circulating antibody, there were no differences in the appearance of subsequent peaks (67). The synchronous cycling of antibody-producing cells thus appears to be independent of the levels of circulating antibody. Several other possible mechanisms, which involve suppression by antibody, may be at play. First, the amount of antibody required may be extremely small, and any detectable level is capable of shutting off antibody synthesis. However, some event other than antibody inhibition would have to be postulated in order to account for the synchronous appearance of the subsequent peaks. Second, a class or subclass of immunoglobulin, other than IgG or IgM, which is not detected by conventional assays, may be responsible. Third, the precise cyclical appearance of PFC after a single injection of AHuIgG could be explained if the antibody was effective locally at the site of production (67). Antibody regulation of the immune response at the site where antigen stimulates the cells involved in antibody production would explain why the level of circulating antibody does not determine the intervals between the appearance of peaks of PFC after injection of AHuIgG. The local concentration of antibody in the sites of antibody formation would be the same in the spleens of all animals, and would be independent of the total amount in either the intra- or extravascular compartments. Although local control would explain the rapid decrease in the number of PFC that occurs regardless of the concentration of antibody in the serum, it cannot entirely explain synchrony. The precise interval between the appearance of peaks of PFC could be explained by control mechanisms that have been described in other systems in which regulation of cell renewal can be achieved by extending the length of the GI period of the cell cycle (78,79).In addition, cells from a variety of sources can be stimulated to begin dividing again, after a period in which DNA synthesis has been arrested (80,81). From incorporation studies of cells blocked at different phases of the mitotic cycle, it has been determined that, upon appropriate stimulus, cells can reenter the cycle from GI or, although less likely, from G, (80). In spleens of rabbits producing PFC to AHuIgG, inactivation of antigen by antibody produced locally could result in cells being arrested in some phase of the cell cycle, Whether a cell was arrested or continued to develop into a PFC might depend on the number of contacts it made with antigen. Cells that developed beyond a certain point would continue to differentiate to PFC, whereas cells that had developed to a lesser

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degree would be blocked temporarily from further division and differentiation (Fig. 7). In order to explain the synchronous production of antibody, one could predict that a precise latent period would be required before the cells could again be stimulated. Upon termination of this latent period, cells would again be stimulated in the spleen by persisting antigen. Thus, as a result of a transient curtailment of stimulation by antigen, cells that had not completed enough differentiation to become PFC without further stimulation might be arrested temporarily. When the cells are again capable of responding, antigen would be available in the germinal centers in the spleen to restimulate them, and synchronization of PFC could occur. In any event, it may be that control of the latent periods and the synchrony of cyclical production of antibody are exercised through some event in the G , phase of the cell cycle, The effect on GI may be the result of an indirect effect of antibody present at or near the site of its production. The mechanism suggested in the preceding may not be applicable to all systems in which multiple peaks are observed. In the case of the response to AHuIgG in rabbits, preliminary studies demonstrated that antibody resulting from the first peak of PFC was incapable of suppressing antibody production when passively transferred to other rabbits (82). On the other hand, Britton and Miiller (52) were able to inhibit the anti-LPS response in mice injected with

Arrested

Stage 1

Arrested

Stage 2

Stages 01 Cell Differentiation (During Division]

FIG.7. Possible events resulting in arrest or extension of the G , phase of the life cycle of stimulated B cell which may account for the latent period and synchrony in the cyclical production of antibody. (a, b, c) Number of contacts of the cell with antigen.

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E . coli with passive antibody produced by the first peak of PFC. This difference may be owing to differences in affinity of the antibody. In any event, in the former situation circulating antibody appears not to

be involved in either cyclical production of antibody or the observed synchrony, whereas in the latter experiments it may indeed be the controlling factor. The basic mechanism in the synchronous peaking of PFC is further complicated by both T and B cells being required for the response to AHuIgG. Although it has been assumed that the B cell is regulated indirectly by antibody (2),there are no available data that implicate the T cell in synchrony. In fact, when passive antibody is used to inhibit the response to SRBC, T cells become primed (83). This does not rule out a role for T cells in the synchronous appearance of PFC to AHuIgG, since T cells may play an active role in several ways. As already mentioned, T cells do play an important role in the regulation of the immune response by acting as both helper and suppressor cells in the production of antibody by B cells. The synchronous appearance of PFC could be explained if helper activity was responsible for the appearance of PFC and suppressor activity was responsible for the intervals in between the peaks. If suppressor T cells are involved, their effectiveness would have to be relatively short-lived, since in the rabbit the peaks seem to reappear every 8 days. This would be compatible with suppressor T cells being the short-lived TI cells as suggested by their elimination by adult thymectomy (21, 22). The C3 component of complement has been implicated in both antibody production in general (84,85) and in the cyclical production of antibody (53).Injections of LPS not only result in the cyclical production of IgM antibody in both mice (50)and chickens (53),but they also activate the alternate pathway of complement through the C3 component (88).Furthermore, a cyclical production of IgM antibody to SRBC in chickens does not occur unless cobra venom factor (CVF) is also injected simultaneously. It has been postulated by Nielsen and White (53)that activation of C3 by CVF converts thymus-dependent responses to thymus-independent responses. Thus, the switchover from an IgM to an IgG response may be a complementdependent process selectively inactivated by CVF or LPS. In the absence of IgG synthesis, it was hypothesized that normal negative feedback does not occur and, as a result, a single injection of antigen causes a series of cyclical fluctuations in IgM antibody synthesis. This reasoning certainly cannot explain the simultaneous cyclical production of both IgM and IgG to a thymus-dependent antigen

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(AHGG) (67). Although the C3 component of complement may not be involved in the cyclical production of antibody as has been postulated, its implication in antibody biosynthesis has to be seriously considered. Pepys (86) has shown that the depletion of C3 by prior injections of CVF inhibits the immune response to thymus-dependent but not to thymus-independent antigens. In addition, Dukor and coworkers (87, 89) have implicated C3 as a second signal required for B-cell activation. However, what role, if any, complement plays in cyclical production of antibody is speculative at the present time. The kinetic pattern of the appearance of PFC in lymphocytes of the blood after an intravenous injection of AHuIgG is identical to that observed in the spleen (Fig. 8) (67).Because of the identical patterns in the spleen and blood, it is most likely that the PFC in the blood originated from the spleen. The lymph nodes apparently do not contribute a significant number of PFC to the blood, since the ratio of spleen to blood PFC is approximately the same during the first and second peaks, and a second peak of PFC does not occur in lymph nodes. Although it has been demonstrated that the blood does contain antigen-binding lymphocytes (86, 87), there is no detectable antigen in the circulation at the time of the second peak. Thus, it is unlikely that the PFC in the blood are stimulated in the blood. That stimulated B cells can leave the spleen and enter the circulation is supported by the observation of Strober (go), who demonstrated a qualitative change in B cells after stimulation that permits them to migrate. He observed that thoracic duct cells from primed donors were capable of responding to the same antigen when transferred to irradiated recipients after passage through an intermediate host. On

-

Indirect PiC/106 Spleen Cells

---. Direct pFc/lO6 Spleen

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CIHS

Indirect P i C / 1 0 6 Blond Cells

&---

Oirsct P i C / 1 0 6 Blond Cells

Day A f t e r Injection FIG. 8. Kinetics of the appearance of plaque-forming cells (PFC) in spleens and peripheral blood of rabbits after a single intravenous injection of 0.2 mg. aggregated human IgG. [Reprinted from Romball, C. G., and Weigle, W. 0. (1973)./. Ex?,. Med. 138, 1426.1

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the other hand, normal (nonprimed) thoracic duct cells, when transferred to irradiated recipients, were not able to respond to the antigen after passage through an intermediate host. Nonprimed cells were capable of responding in irradiated recipients if not first passed through an intermediate host. The differences in the ability of normal and primed passed cells to respond were attributed to a change in the B cells, in which the primed B cells (B,) acquired the ability to recirculate, but the virgin B cells (B,) could not. In the studies with AHuIgG, the ability of B2cells to enter the circulation may have depended on the loss of effective receptor sites during differentiation, thus freeing them from further antigen stimulation. Depending on the degree of stimulation, B, cells, while in the circulation, may either differentiate into PFC or, if not sufficiently stimulated, they may return to the antigen depots in the spleen (Fig. 9) where, after a precise latent period, they can be stimulated further. Retention of specifically sensitized cells from the circulation by lymphoid tissue containing the antigen was demonstrated by Ford (91) and Durkin and Thorbecke (92). Furthermore, it has been demonstrated that trapping of specific lymphocytes by the spleen occurs after intravenous injection of antigen at the expense of depletion of such lymphocytes from the peripheral blood, lymph nodes (93), and thoracic duct (94). It is likely that such trapping by the spleen is the result of the persistence of antigen in this organ after intravenous injection. Thus, although specific lymphocytes to AHuIgG can leave the spleen upon antigenic stimulation, they do have an affinity for antigen localized in the spleen, The above postulation is supported further by the recent demonCirculation

Yimari

I

Spleen

I

FIG. 9. A schematic representation of a possible migration pattern of B lymphocytes after stimulation with antigen localized in the follicles of the spleen.

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stration by Strober and Dilley (95)that hapten-primed cells (B, cells) do migrate from the spleen to the circulation. They also showed that memory cells for the hapten could be eliminated from the spleen by prolonged thoracic duct drainage. Migration of stimulated cells was also observed by Cannon and Wissler (96), who demonstrated 3Hthymidine-labeled cells in the blood of spleen-shielded, irradiated recipients after stimulation with antigen. It is obvious that not all cells stimulated by antigen leave the spleen, since a relatively large number of PFC are found in the spleens of rabbits injected intravenously with AHuIgG (67). Possibly after these cells lose receptor sites, they differentiate into PFC before they can migrate from the spleen. Other factors may play a role in whether a B, cell leaves the spleen and enters the circulation. Rabin and Rose (97) suggested that there may be two populations of B lymphocytes in the rat- one that leaves the spleen and circulates after stimulation with antigen and another population that remains in the spleen after antigenic stimulation. IV. Conclusions

It appears that cyclical production of antibody after a single injection of antigen is more common than has been realized in the past. Cyclical production of antibody has become obvious with the use of the hemolytic plaque assays to determine PFC, since the appearance and disappearance of PFC occur much more rapidly than those of antibody in the serum. The reason for such cell cycling is not clear; however, it is associated with antigen that persists in the host and apparently plays a role in the body’s defense mechanisms. In most cases, it is likely that antibody regulates the cyclical production of antibody, but it is not clear whether circulating antibody or whether antibody produced locally at the site of interaction between antigen and competent cells is more important. It has also been suggested that antibody produced in situ to the idiotype of the specific antibody may be responsible for specific regulation of the immune response. Furthermore, a role for suppressor T cells in certain cases of cycling cannot be eliminated. Little is known about cyclical production of antibody in human diseases or what influence such a mechanism might have on the disease. It is not unlikely that such cycling occurs in infections with persisting agents such as mycobacteria. Cycling may be necessary in order to prevent exhaustive differentiation of competent lymphocytes as a result of constant stimulation by antigen. There is little available evidence that cyclical production of antibody occurs in autoimmune

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diseases. Even if such fluctuations do occur, they would be difficult to interpret, since such fluctuation may be the result of either change in available antigen or removal of antibody by autoantigen. There is evidence of regulation of autoimmunity in the lupuslike disease in New Zealand mice. However, there is no evidence that the autoimmune response is cyclical, and it appears that the regulation is dependent on the presence or absence of suppressor T cells rather than on antibody (98, 99). The antibody response to homologous thyroglobulin, in rabbits developing thyroiditis after a series of injections of aqueous preparations of heterologous (cross-reacting) thyroglobulin (loo), has a cyclical component that is dependent on the autologous thyroglobulin present in the thyroid gland (101).After the last of a series of injections of rabbits with aqueous preparations of bovine thyroglobulin, peaks of PFC to both bovine and rabbit thyroglobulin appeared in the spleen. Without additional injections of bovine thyroglobulin, a peak of PFC to rabbit thyroglobulin appeared 7 days later in the thyroid gland. Because of free rabbit thyroglobulin present in the gland, B, cells stimulated by the response in peripheral lymphoid tissue are probably absorbed by the gland. The 7-day latent period before these absorbed lymphocytes respond to produce PFC may involve the same mechanism as that responsible for the latent periods between the peaks of PFC appearing after the injection of rabbits with AHuIgG. However, there are no available data that permit any conclusion as to the cellular events involved in thyroiditis in the human. There is some evidence that a cyclical cell-mediated response can occur with transplantable allogeneic tumors in experimental animals (61,62), but at the present time one cannot justify the extrapolation of these observations to spontaneously arising tumors in humans. Thus, although the cyclical nature of the immune response to certain antigens is a real biological phenomenon repeatedly observed in experimental animals, its implication in human diseases has not been established. Although it is difficult to envision the advantage of cyclical production of antibody, the existence of regularly timed cycles is even more difficult to understand. Cycling in the immune response may be a requisite for conservation of competent memory cells and yet assure that circulating antibody is continuously present in order to keep persisting infections in check. If a cell is stimulated by antigen, but because of the presence of newly synthesized antibody the stimulation is insufficient for the cell to differentiate into an antibodyproducing cell, it may be necessary for that cell to become arrested in some phase of the cell cycle in order to survive. The precise latent

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period required before the cell can respond may be the time necessary for reorganization of the intracellular apparatus in order that the cell may successfully respond after free antigen again becomes available. It is obvious that there is very little, if anything, known about either the cellular or the subcellular mechanisms involved in the cyclical production of antibody and any further insight into this phenomenon will require more sophisticated approaches. ACKNOWLEDGMENTS The author would like to acknowledge the excellent assistance in preparation of the manuscript by Miss Carole Romball, Mrs. Phyllis Minick, and Mrs. Linda Norwood.

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66. Gillespe, G. Y., and Barth, R. F. (1974). Cell. Zmmunol. 13,472. 67. Romball, C. G., and Weigle, W. 0. (1973).J. Ex;). Med. 138, 1426. 68. Dresser, D. W. (1974). In “Immunological Tolerance: Mechanism and Potential Therapeutic Application” (D. H. Katz and B. Benacerraf, eds.). Academic Press, New York. 69. Hahicht, G. S., and Chiller, J. M. (1970). Unpublished observations. 70. Britton, S., Wepsic, T., and Miiller, G. (1968). Zmmunology 14, 491. 71. Nossal, G. J. V., Austin, C. M., Pye, J., and Mitchell, J. (1966). Znt. Arch. Allergy App1. Immunol. 29, 368. 72. Nossal, G. J. V., and Ada, G. L. (1971). “Antigens, Lymphoid Cells, and the Immune Response.” Academic Press, New York. 73. Brown, J . C., Schwab, J. H., and Holborow, E. J. (1970). Immunology 19, 401. 74. Basten, A., Miller, J. F. A. P., Sprent, J., and Pye, J. (1972).J. E x p . Med. 135, 610. 75. Dickler, H. B., and Kunkel, H. G. (1972).J . Ex),. Med. 136, 191. 76. Stark, 0. K. (1955)./. Immunol. 74, 130. 77. Chen, F. W., Strosberg, A. D., and Haber, E. (1973).J . Zmmunol. 110, 98. 78. Prescott, D. M. (1968). Cancer Res. 28, 1815. 79. Cameron, I. L., and Greulich, R. C. (1963).J. Cell B i d . 18, 31. 80. Gelfant, S. (1966). Methods Cell Physiol. 2, 359. 81. Gelfant, S., and Smith, J. G., Jr, (1972). Science 178, 357. 82. Romball, C. G., and Weigle, W. 0. (1972). Unpublished observations. 83. Miiller, E., and Greaves, M. F. (1971). In “Cell Interactions and Receptor Antibodies in Immune Responses” (0.Miikelii, A. Cross, and T. U . Kosunen, eds.), p. 101. Academic Press, New York. 84. Pepys, M. B. (1974).J. E x ) ) . Med. 140, 126. 85. Dukor, P., and Hartmann, K. U. (1973). Cell. Zmmunol. 7,319. 86. Dwyer, J. M., and Mackay, I. R. (1970). Lancet 1, 164. 87. Bankhurst, A. D., Torrigiani, G., and Allison, A. C. (1973). Lancet 1, 226. 88. Lachmann, P. J., and Nichol, P. (1973). Lancet 1, 465. 89. Dukor, P., Schumann, G., Gisler, R. H., Dierich, M., Konig, W., Hadding, U., and Bitter-Suermann, D. (1974).J . EX?,. Med. 139, 337. 90. Strober, S. (1972).J . E x p . Med. 136, 851. 91. Ford, W. L. (1972). Clin. E x p . Zmmunol. 12, 243. 92. Durkin, H. G., and Thorbecke, G. J. (1973). In “Microenvironmental Aspects of Immunity” (B. Jankovic and K. Isakovic, eds.), p. 63. Plenum, New York. 93. Emeson, E. E., andThursh, D. R. (1974).J. lmmunol. 113, 1575. 94. Sprent, J., and Miller, J. F. A. P. (1974).J . Exp. Med. 139, 1. 95. Strober, S., and Dilley, J. (1973).J . Ex),. Med. 137, 1275. 96. Cannon, D. C., and Wissler, R. W. (1965). Nature (London)207,654. 97. Rabin, B. S., and Rose, N. R. (1973).Cell. Zmmunol. 7,389. 98. Barthold, D. R., Kysela, S., and Steinberg, A. D. (1974). J . Zmmunol. 112, 9. 99. Steinberg, A. D., Law, L. W., and Talal, N. (1970). Arthritis Rheum. 13, 369. 100. Weigle, W. O., and Nakamura, R. M. (1967).J. Irnmicnol. 99, 223. 101. Clinton, B. A,, and Weigle, W. 0. (1972).J. E x ) ) . Med. 136, 1605.

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Thymus-Independent B-Cell Induction and Paralysis1 ANTONIO COUTINHO AND GORANMOLLER Division o f Immunobiology. W o l l e n b e r g Laboratory. Karolinska lnrfitute. Stockholm. S w e d e n

I . Introduction . . . . . . . . . . . . . A . BCells . . . . . . . . . . . . . B. Polyclonal B-Cell Activators . . . . . . . . . I1 . Hypotheses for Immune B-Cell Activation . . . . . . . A . The One Specific Signal Theory . . . . . . . . B. Antigen Presentation by T Cells and Pattern of Antigen Presentation . . . . . . . . . C Two-Signal Hypotheses . D . The Quantitative Concept . . . . . . . . . E . The Cross-Linking Concept . . . . . . . . . F. One Nonspecific Signal Hypothesis . . . . . . . I11 . Some Important Technical Considerations . . . . . . . A . Detection of B-Cell Activation by Means of a Sensitive Hemolytic Plaque Assay . . . . . . . . . . . . . . . . B. Misinterpretation of Antihapten Plaque Assays . C . Serum-Free Cultures . . . . . . . . . . D . Requirement for Polyclonal B-Cell Activators in the Mishell-Dutton . . . . . . . . . . . . System . E . A Quantitative Concept for B-Cell Activation . . . . . IV . Critical Evidence Supporting the One Nonspecific Signal Hypothesis . A . Activation of B Cells Directly and Nonspecifically by Thymus. . . . . . . . . Independent Antigens . B. Specific Thymus-Independent Immune Responses Induced by . . . . Polyclonal Activators-The Paradoxical Reality . C Synergism of Thymus-Independent Antigens and Polyclonal Acti. . . vators in Induction of Specific Immune Responses . D . Need for Functional Mitogenicity of the Antigen for Induction of Thymus-Independent Specific Responses . . . . . . E . No Detectable Signal from Interaction of Antigen with Ig Receptor . . . . . . . . . Site on Resting B Cells . V . Basis of Thymus Independence (Direct, Specific B-Cell Activation): . . . . . . . . . . Competing Concepts . A . The Associative Recognition Model . . . . . . . B. Other Two-Signal Hypotheses . . . . . . . . C . The Pattern of Antigen Presentation . . . . . . . D . Positive and Negative Views on the Catabolic Behavior of Antigen . VI . Molecular Basis of B-Cell Activation . . . . . . . . A . Involvement of the Specific Receptor-Combining Site: The Antigen . . . . . . . . . Presentation Concept .

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114 115 116 119 120 121 122 124 125 125 126 127 130 132 135 140 145 145 152 157 158 164 167 168 170 181 186 191 191

The work reported in this review was supported by grants from the Swedish Cancer Society .

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. . B. Second Messengers and Two-Signal Hypotheses. C. Involvement of the Immunoglobulin Surface Receptor Independently . . . . of Its Specific Site: The Cross-Linking Concept . D. Lack of Requirement for Polymeric Molecules in B-Cell Activation: Reinterpretation of the Cross-Linking Concept in the Light of Multipoint Binding . . . . . . . . . . . . VII, B-Cell Paralysis in Thymus-Independent Responses . . . . A. One Nonspecific Signal Concept and Thymus-Independent Paralysis . . . . B. Nonimmunogenic but Paralytogenic Molecules . C. Nonimmunogenic but Paralytogenic Thymus-Independent Antigen Concentrations . . . . . . . . . . . D. A “Two-Signal” Comment on Thymus-Independent Paralysis . . E. Increased Numbers of Antigen-Binding Cells in Thymus-Independent Paralysis . . . . . . . . . . . VIII. B-Cell Induction and Paralysis by Nonpolyclonal Activator Molecules (Thymus-Dependent Antigens) . . . . . . . . . IX. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .

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I. Introduction

The specificity of the immune response has been recognized since the days of Metchnikoff, much before antibody activity was ascribed to serum globulins. At that time, the basic principle, which still governs modern immunology, was layed down, namely the selective theory of antibody diversity (Ehrlich, 1900; Jerne, 1955; Burnet, 1957; Talmage, 1959).Antibody-forming cells are distributed in a range of different clones, each of them displaying a distinct immunological specificity (the combining site of the cell-associated or secreted antibody molecule). Regardless of the extent of this diversity, preformed clonal distribution ensures the specificity of responsiveness characteristic of the system, The antigen-specific binding receptor exposed by the competent cell on the surface membrane is identical to the antibody molecules that the cell is capable of secreting after activation. The antigen selects the clone(s) of cells competent to recognize and bind the antigenic determinants (only a small fraction of all immunocompetent cells). After relevant binding of immunogenic molecules, the cell becomes activated to expand clonally and to differentiate into a state in which it can secrete large amounts of specific antibody. In recent years, evidence has been produced for the separation of lymphocytes, previously recognized as the immunocompetent cells (Gowans and McGregor, 1965), into two classes with sharply different functional capacities. Thus, it is well established that only one

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class of lymphocytes (B cells) is competent to synthesize and secrete antibodies (Moller, 1969; Roitt et al., 1969).2

A. B CELLS We define B cells as lymphocytes capable of synthesizing Ig molecules (Vitetta et d., 1972, 1973). The B cells are derived from lymphoid-hematopoietic precursors in the adult marrow and may or may not require a peripheral lymphoid organ for full differentiation in mammals. This lymphocyte population is quite heterogeneous, ranging from primitive precursors to fully differentiated plasma cells, with concomitant alterations in the surface markers expressed (Bianco et al., 1970; Takahashi et al., 1970; Eden et al., 1971; Raff et al., 1971; Unanue et al., 1971; Basten et al., 1972; Snell et al., 1973; Sachs and Cone, 1973) in patterns of recirculation (Sprent et al., 1971; J. C. Howard, 1972; Strober, 1972) and homing (LindahlKiessling et aZ., 1971) into the secondary lymphoid organs, in lifespan (Miller and Sprent, 1971a), in class of Ig produced (Weissman et aZ., 1973), and, most important, in ability to respond by proliferation and/or antibody secretion after interaction with appropriate ligands (Gronowicz and Coutinho, 1974). Due to lack of functional characterization of distinct B-cell subpopulations, we will distinguish the following types of B cells. 1. Precursor B cells: Ig-negative cells of lymphoid lineage, that will differentiate into mature B cells (Gronowicz et al., 1974a). 2. Resting B cells (Go or “antigen-sensitive” in the commonly used nomenclature): small, Ig-positive lymphocytes that are competent to synthesize Ig molecules and to deposit them on the plasma membrane to serve as antigen-binding receptors (Wigzell and Andersson, 1969; Miikelii et al., 1969; Moller, 1970), which are turnedover slowly (20-80 hours half-life) (Melchers et al., 1974; Melchers and Andersson, 1973). 3. Activated B cells: large lymphoblasts or plasma cells that develop after activation of resting B cells and are able to synthesize antibody molecules at a high rate; the latter are exported from the The T Cells do not secrete antibodies, even though phenomenological evidence has been interpreted to support such a conclusion. Whether T cells possess Ig receptors on the surface is not settled as yet, and different findings have been reported (for a review, see Mdler, 1973). In this context it is sufficient to state that T cells can specifically bind antigens and haptens (for a review, see Bullock and Miiller, 1974), the nature of the binding receptor being controversial.

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cells in large amounts (Melchers et al., 1974; Melchers and Andersson, 1973). Most of the discussion centers on the small, resting B cells since these are the lymphocytes that can be activated into specific product formation. The B-cell response to immunization is characterized by the appearance of an increased number of cells, morphologically defined as plasma cells (Gudart et al., 1971a,b), in the secondary lymphoid organs, as well as by a sharp increase in the numbers of cells functionally defined by high-rate synthesis and secretion of antibodies specific for the antigenic determinants present on the immunogenic molecules (Ellis et al., 1967; Jerne et al., 1974). This results in an analogous increase in serum antibody titers (Eisen, 1966). Therefore, the response is characterized by division and clonal expansion of the original set of antigen-reactive B cells, as well as by a qualitative change in the phenotypic expression of these cells. Independently from other alterations in the surface mosaic (Takahashi et al., 1970; G . Moller, 1974), the activated B cell changes from a resting small lymphocyte into a typical secretory cell (Melchers et al., 1974; Moller, 1973). B. POLYCLONALB-CELLACTIVATORS The mechanisms by which the immunocompetent B cell becomes activated upon binding an immunogenic molecule have been the subject of a large number of experimental studies and theoretical speculations. It is well established that antigen recognition by B cells is not sufficient for activation to occur, because induction does not always follow the binding of immunogenic molecules and in some cases requires the help of other cell types, mainly T cells (Moller, 1969; Claman et al., 1966; Miller and Mitchell, 1968; Taylor and Wortis, 1968) and macrophages (Mosier, 1967). On the other hand, some ligands always activate suitable target lymphocytes upon binding to the surface membrane. These are commonly termed lymphocyte mitogens or polyclonal B-cell activators (PBA), and have been extensively used, over the past few years, in studies of the mechanism of lymphocyte activation (Moller, 1972). The monotony of phenotypic expression in immunocompetent cells has been used to support the studies using mitogens as a model for immune triggering. In other words, mitogen-activated immunocompetent cells perform equally well all the functions that antigen-activated cells can perform: B cells divide and differentiate into high-rate antibody-secreting cells (Anderson et al., 1972; Melchers and An-

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dersson, 1973; Greaves and Janossy, 1972a,b; Janossy et al., 1 9 7 3 ~ ) ~ and T cells divide, kill any target they get in close contact with, and function as helper cells for the induction of antibody production in B cells (Smith, 1972; Perlmann and Holm, 1969; Mdler et aZ., 197213; Asherson et al., 1973; Sjiiberg et al., 1973). This is to be expected from our present knowledge of cell physiology, since information theories of immune responsiveness are now excluded. Once activated, the lymphocyte expresses the functions f o r which it is genetically programmed and of which it is capable according to its stage of differentiation. 1 , Direct Activation of B Cells b y B-Cell Mitogens Znteracting with the Cell Surface Membrane It has been shown in several systems and species that B-cell mitogens activate suitable target B cells directly in the absence of any accessory cells, such as T cells (Greaves and Janossy, 1972a; Andersson et al., 1972c,d) or macrophages (Coutinho, 1974; Lemke et al., 1975; Melchers, Sprent, and Andersson, personal communication; Yoshinaga et al., 1972). This statement of principle does not exclude the possibility that these ligands may have activating properties for other cell types, in particular macrophages, as has been demonstrated (Blythman and Waksman, 1973; Nelson, 1973), and even for nonimmunological cells, such as fibroblasts (Vaheric et al., 1973; Persson and Miiller, unpublished). The demonstration that each PBA seems to activate a distinct subpopulation of B cells (Gronowicz and Coutinho, 1974, 197%; Gronowicz et al., 1974a) makes it essential to interpret carefully findings on the failure to activate a certain subset of B cells by a certain B-cell mitogen. So far only one experimental report (Kagnoff et al., 1974) has appeared claiming that B cells require accessory cells for activation by lipopolysaccharide (LPS). However, the weakness of the results makes us doubt that, even for this particular subset of B cells (Peyer’s patches), any conclusions can be drawn with regard to their responsiveness to LPS. It appears likely that the study reflects conditions for in vitro survival of Peyer’s patches B cells rather than for activation of B cells. In ours and in other hands, Peyer’s patches B cells indeed respond to LPS (Gronowicz and Continho, 1975c; Janossy, personal communication). Direct activation of macrophages by LPS is well documented, but the effect of LPS on T cells is more controversial. There are reports that general immunological effects of LPS in uivo and in vitro require the presence of T cells for optimal expression (Allison and Davies, 1971; Hamaoka and Katz, 1973; Armerding and Katz, 1974a;

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Kreisler and Moller, 1973). However, all attempts to demonstrate direct activation of T cells at different stages of differentiation by LPS have been completely unsuccessful so far. The problem of LPS and T cells is discussed below (Section V,B,4,a), since it involves synergy of antigens and mitogens in B-cell activation. It should be pointed out here that all the present evidence is compatible with the possibility that T cells can be activated by products released from macrophages after activation by LPS, and that the presence of T cells is, indeed, required for optimal expression of adjuvant effects, simply because the antigen by itself is completely incapable of B-cell activation, but may induce cooperating T cells to act synergistically with LPS (see Section V,B,4,a). Only two claims have been made for the internalization of the mitogen as a necessary step in B-cell triggering (Diamantenstein et al., 1973; Vogt et al., 1973; Adler et al., 1972), of which only one is supported by indirect experimental evidence (Adler et al., 1972). Because insolubilized ligands, e.g., lectins (Greaves and Bauminger, 1972; Andersson et al., 1972a; Andersson and Melchers, 1973) and LPS (Moller et al., 1975), can activate B cells as well, internalization as a necessary or even optimizing event in B-cell triggering is excluded. Therefore, the internalization of the ligand must reflect some general phenomenon common to all cell surface events after binding of multivalent ligands. 2. Independence of B-Cell Activation by Mitogens from I g Receptor Specificity of Responding Cells The various antibody activities that have been studied after activation of a B-cell population by a B-cell mitogen have all been found to be increased. Furthermore, the number of B cells activated by a particular mitogen is several orders of magnitude higher than the number of precursor cells reported for each particular clone. The variety of ligands capable of activation, as well as the multiplicity of the antibody specificities found upon stimulation, definitely establish that B-cell activation by PBA develops independently of the specific Ig receptor site. This is in marked contrast to the postulates in the “specific” hypotheses (Section II,A,B,C) because they are all based on the concept that only an interaction with the specific receptor site can activate resting B cells. Therefore, as a rescue operation, another postulate was made, namely that the responsive B cells are, indeed, memory cells (Schrader, 1974c) or else had received a specific signal by interacting with self-components (Watson et al., 1973b), but this did not result in induction of tolerance as would be

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expected from the hypothesis (Bretscher and Cohn, 1970). However, the same polyclonal responses can be obtained in newborn and germfree animals in serum-free culture systems (Gronowicz and Coutinho, unpublished; Moller, unpublished). It is difficult, therefore, to accept such postulates, mainly because they represent an obvious conceptual contradiction in a hypothesis known for its elegant explanation of self-nonself discrimination. B-Cell activation by PBA results in cell division and “reprogramming” (Tsanev, 1973) into preferential synthesis and secretion of immunoglobulin. An activated B cell is qualitatively different, in morphology and function, from a resting B cell (Melchers et al., 1974; Melchers and Andersson, 1973; Greaves and Janossy, 1972b). Since B-cell activation may result in division, immunoglobulin synthesis, or both (Gronowicz and Coutinho, 1974, 1975a), depending on the degree of differentiation of the responsive cell (Gronowicz et al., 1974a), the commonly used term mitogen, should be replaced by activator. Actually, in many instances, activation results in increased antibody synthesis in the absence of cell division or DNA synthesis. Some ligands only marginally activate mitosis in target B lymphocytes but cause a pronounced increase of antibody synthesis (Coutinho and Moller, 1973~). The reverse situation occurs with other ligands (Coutinho et al., 1974c; Gronowicz et al., 1974a). Therefore, the only common denominator of these ligands is the fact that they directly activate B cells having a large variety of Ig specificities and we, therefore, propose that “polyclonal B-cell activators” replace the commonly used term mitogen.

3. B-Cell Activation by PBA As a Concentration-Dependent Phenomenon For a particular B-cell population responding to a certain PBA,

the dose-response curve is always bell-shaped (Andersson et al., 1972c,e; Coutinho and Moller, 1973d; Coutinho et al., 1973a). Below triggering concentrations, the B cell maintains the resting state; above a certain threshold, the cell is paralyzed rather than induced. The end results are dependent only on the quantity of PBA reacting with the corresponding B cells. II. Hypotheses for Immune B-Cell Activation

Since it is impossible to review all the relevant work in this field during the past 10 or 20 years, the present discussion inevitably treats our own findings to an excessive extent. We use these findings to support our recently proposed hypothesis for B-cell activation

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which is the leitmotiv of the paper. However, relevant work produced in other laboratories, as well as some competing concepts, are analyzed, both at the experimental and conceptual level. To a large extent the discussion is restricted to the induction of primary IgM responses by thymus-independent (TI) antigens. However, a uniting conceptual framework of B-cell induction and paralysis is kept in mind throughout the discussion, and a detailed model will be presented later. The reader should keep in mind that the great variety of experimental systems employed in producing the evidence discussed may create illusionary discrepancies among findings. In several cases, the experiments have been carried out in highly artificial in vitro systems, and it will certainly be dangerous to extrapolate results from a particular experimental system to a general theoretical concept. As stated above, many reported facts concerning B-cell activation will not be included in this discussion, but an interested reader will easily find them elsewhere. We first present an uncritical outline of several different hypotheses proposed to explain the mechanisms by which cell surface ligands generate signals capable of turning B cells on and off. These hypotheses have been selected according to our subjective view of their importance, fashion, and completeness. It is taken for granted that selective theories of antibody responses are correct (Ehrlich, 1900; Jerne, 1955; Burnet, 1957; Talmage, 1959).

A. THE ONE SPECIFICSIGNALTHEORY The “one specific signal” hypothesis (Mitchell et al., 1972c; Mitchell, 1975) recognizes the ability of B cells to be directly activated by any type of antigen that can be recognized and bound to the specific Ig receptors. The T-cell helper effects reflect the release of B cells from “blockade,” which was induced by excess antigen-binding and may operate either directly or via T-cell-mediated activation of phagocytic or other ancillary cells. Triggering signals are generated by dissociation of antigen-receptor aggregates and redistribution of receptors, which is induced by T-cell-mediated digestion or removal of antigen. High-affinity cells are usually paralyzed after confrontation with antigen, in particular when this is a polymeric, multivalent ligand. In addition, these TI antigens do not activate T cells and, therefore, high-affinity cells to TI antigens cannot be released from paralysis, and only low-affinity IgM antibody appears in the response. All IgM antibody, as well as all primary responses are accepted to be TI in general, whereas IgG antibody and secondary responses are highly thymus-dependent (TD). T-cell activity can

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only result in a positive effect on antibody responses (in particular for high-affinity antibodies), and, therefore, suppressor T cells cannot exist. Paralysis is evidently postulated to be reversible.

B. ANTIGENPRESENTATION BY T CELLS AND PATTERN OF ANTIGENPRESENTATION Basically, theories of this type also accept that B cells can be triggered only by the interaction between the antigen and the specific Ig receptors on the cell surface (Mitchison, 1971; Mijller, 1970c; Taylor and Iverson, 1971; Mitchison et al., 1970; Feldman, 1972a,b; Feldman and Nossal, 1972; Greaves et al., 1974). Since the affinity of the interaction between antigenic determinants and the Ig-combining sites is generally low, it is postulated that the cell will only be triggered if the avidity of the reaction is increased by means of a multipoint interaction. This is achieved in the case of large polymeric immunogens by the antigenic molecules themselves (TI) but requires several helper mechanisms, all based on a passive presentation or concentration mechanism in case of nonpolymeric molecules (TD). These mechanisms may vary depending on the approaches of different authors, but quite often involve T cells, either directly -via specific surface receptors for antigenic determinants on the same molecule that react with B cells-or indirectly -via the release of receptors that complex the antigen and bind to the Fc receptors on the macrophage surface. Other postulated mechanisms of achieving a multipoint presentation include the concentration of antigen directly on macrophage surfaces, the natural or artificial concentration of antigens on cells or inert surfaces, and lattice formation by multivalent antibodies directed against multivalent antigens. These hypotheses explain paralysis in terms of excessive levels of interaction, mediated by signals generated only at the Igcombining site. The concept of “pattern of antigen presentation” seems to involve something else than these straightforward thermodynamic rules, because it makes use of the additional concept of cross-linkage. As noted below, this implies that signals are (also) generated at other levels of the membrane, which are the direct result of the crosslinkage of membrane components, and these are not necessarily Igspecific receptors. Here resides the subtle difference that we can perceive between hypotheses of antigen concentration and those of pattern of antigen presentation, because the latter accepts that triggering is determined not only by specific signals generated at the combining site but also by signals resulting directly from the cross-

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linkage of membrane components, However, since it postulates that Ig receptors are necessarily involved, it appears either that only these structures are competent to generate “cross-linking signals,” or else that they are not effective when generated by the cross-linking of other structures. In the latter case, pattern of antigen presentation is actually a two-signal hypothesis. C. TWO-SIGNAL HYPOTHESES Various two-signal hypotheses (Bretscher and Cohn, 1970; Cohn, 1972a,b; Bretscher, 1972; Watson et al., 197313; Dutton et al., 1971; Britton, 1972; Dukor and Hartmann, 1973; Schrader, 1973a,b) share two basic postulates: (1) B cells cannot be directly activated by a single type of signal generated at the membrane and (2) one signal (insufficient for triggering to occur) is delivered to the cell via specific Ig receptors after combination with the antigen. The nature of the postulated second signal varies for each model, from a specific signal mediated by a T-cell antibody, to a nonspecific T-cell product, or a B-cell mitogen. A detailed model for the operational mechanism of this second signal has been proposed (activated complement component C3 interacting with the C3 receptor on immunocompetent B cells) (Dukor and Hartmann, 1973). Paralysis is seriously considered only in the “two specific signal” hypothesis by postulating that the first signal, generated by the combination of antigen with the specific receptor, results in tolerance rather than induction, except if the cell is simultaneously exposed to the second specific signal, generated by the recognition of the antigen by associative antibody. This model of associative recognition is designed in order to explain self-nonself discrimination and denies the existence of truly TI antigens (Bretscher and Cohn, 1970; Cohn, 1972a,b; Bretscher, 1972), as well as the possibility of triggering B cells by any mechanism not involving the first specific signal. It also postulates that the second signal has to be delivered to the antigensensitive cell undergoing associative recognition via the first signal. The two-signal hypothesis based on a nonspecific thymus-derived mediator (Dutton et al., 1971; Schimpl and Wecker, 1972) differs from the original Bretscher and Cohn model mostly in the nonspecificity of the postulated second signal. Furthermore, a mechanism for paralysis induction has not been proposed, and the time sequence of the two signals required for activation was not specified. Recently, however, two of the groups who have presented much of the support for nonspecific T-cell factors in vitro, presented experimental evidence indicating that antigen might be competent to in-

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duce division in B cells, whereas the thymus-derived mediators would function by differentiating the expanded clone of antibodyforming precursors into mature plasma (antibody-producing) cells (Hunter and Kettman, 1974; Dutton, 1974; Hunig et al.,1974). This idea of the importance of T-cell factors for the differentiation of plaque-forming precursors has been proposed before (Britton, 1972a). Another second-signal source, which has received some attention, is activated macrophages. Evidence has been presented for the exclusive role of adherent cells in the secretion of the relevant, cooperative nonspecific factors (Britton, 1974; Schrader, 1973a,b) as well as denying such an idea (Wecker, personal communication; Sjoberg, personal communication). Moreover, some other experiments seem to indicate a great heterogeneity of active soluble mediators, with sharply different functional properties (Watson, 1973). However, here again, the agreement is not general and definite conclusions must wait for further experimentation. Nonspecificity of the second signal also characterizes hypotheses ascribing this role to B-cell mitogens or other activators (Schrader, 1973a,b; Watson et al., 197313; Dukor and Hartmann, 1973). Only the more elaborate models of this type, derived from Bretscher and Cohn’s original hypothesis (Watson et al., 1973b), try to explain induction of B cells by the second signal alone, in the absence of antigen. This is actually the main difficulty with all two-signal theories, as can be concluded from various attempts to explain the “abnormal induction” caused by mitogens. With time, the positions have changed from complete rejection of the available evidence, supported by reports on the failure of achieving mitogen-induced antibody production in B cells (Watson et al., 1973a), to a much more negotiable position, where abnormal induction is accepted as a fact (Watson et al., 197313). In the latter case, mitogen-activated cells are believed to have received the first (specific) signal by interaction with cross-reactive self-antigens. The mechanism by which a mitogenic signal can be effective was first ascribed to associative antibody which could function by recognizing determinants on the antigen or on the antigen-sensitive cell itself (as a surface-bound ligand would be) (Cohn, 1972a,b). Now, it seems that the mitogen signal is considered to act directly on the membrane of the responding B cell (Watson et al., 1973b). Some concepts that a priori include a basic statement of two signals, also emphasize the importance of multipoint binding (Schrader, 1973a,b). However, it is not clearly indicated which of the

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mechanisms are fundamental and which are accessory in the triggering process, Sometimes, a number of possible different ways of achieving B-cell activation are proposed in the same model, and it is implied that they are all operating at the same time (Schrader, 1973b; Gisler et al., 1973). Apart from the lack of a theoretically consistent framework and a clearly defined hypothesis, such models imply great difficulty in the achievement of B-cell triggering, even though they rely on experimental claims of activation which are “too easy” to be acceptable as operative on physiological grounds (Schrader, 1973b).

D. THE QUANTITATIVE CONCEPT With the quantitative concept (MBller, 1970a; Andersson et al., 1972d; Sjoberg et al., 1972b) an important feature is introduced, namely the suggestion that B-cell activation can be due to signals generated at membrane sites other than the Ig receptors. This hypothesis accepts that Ig receptors are competent structures for the generation of triggering signals by combination with the antigen but does not postulate that specific signals are strictly required for activation to take place. B Cells do not distinguish the quality of the stimuli, but are only capable of counting the number of interacting surface sites. Activation results when a minimal threshold of triggering signals is reached, and paralysis occurs because of an excessive number of signals. This hypothesis, primarily derived from experiments with nonspecific ligands, or with specific and nonspecific ligands operating synergistically in the induction of specific responses, accepts, therefore, basic postulates from antigen concentration as well as from two-signal hypotheses. Thymus-independence is still explained in terms of multipoint binding to Ig receptors, but it is accepted that induction by TD antigens can be achieved if further nonspecific stimuli are provided to the specific B cells, such as nonspecific thymus-derived mediators or B-cell mitogens, which will add to the insufficient stimuli generated at the specific receptor sites, to reach threshold levels of activation. Furthermore, activation thresholds can be reached in the complete absence of specific interaction with the antigen, by stimulating “virgin” B cells with competent ligands - B-cell mitogens. It does not specify which structures on the cell membrane may be responsible for delivering triggering signals, not does it postulate any requirements for efficient interaction, namely cross-linkage of surface components.

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E. THE CROSS-LINKING CONCEPT In a list of decreasing “specificity” requirements of the postulated triggering mechanisms, the cross-linking concept (see Greaves and Janossy, 1972a) could be placed either at the top or at the bottom. It has already been noted that this concept is used in specific theories that explain activation via specific Ig receptors. However, the strongest evidence for a necessary cross-linkage of B-cell surface components as the mechanism of triggering is not derived from experiments with TI polymeric antigens, but from experiments involving nonspecific mitogens. In this case, activation is achieved in the complete absence of specific signals. This concept does not actually constitute a hypothesis of B-cell activation but, rather, a general biological idea which may include other concepts, such as the importance of multipoint interaction for stabilization of binding, redistribution of membrane structures, restriction of the bilayer fluidity, and shedding of receptors. Therefore, it seems convenient to search for a general definition of the basic assumption contained in the concept when applied to B cells. It seems that the characteristic feature of the concept is that relevant triggering signals are generated when surface structures are cross-linked by multivalent ligands and that this step must occur at some time during the activation process (see Greaves and Janossy, 1972a; Greaves et d.,1974). Based on this postulate, there may be divergent viewpoints when considering the relevant surface structures that trigger the cells after cross-linking. Thus, the concept could be divided into subgroups of decreasing specificity requirements, postulating activation as resulting from cross-linkage of (I) only specific Ig receptors, (2) some surface structures including Ig receptors, ( 3 )some surface structures other than Ig receptors, ( 4 ) any surface structure, (5) any surface structure other than Ig receptors.

F. ONE NONSPECIFIC SIGNALHYPOTHESIS No signal is generated by the interaction between antigenic determinants and the combining site of the specific Ig receptors on the B-cell surface (Coutinho and MGller, 1973a,c,d, 1974; Coutinho et d.,1974a,b). Triggering signals are generated at other surface structures (mitogenic receptors) that are not clonally distributed and, therefore, will be called nonspecific, and only at these sites. The result of the confrontation of B cells with surface ligands competent to interact with mitogenic receptors is controlled by

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purely quantitative rules. Two thresholds of interaction are decisive. The cell is activated only above the first threshold of efficient interaction, the resting state being maintained below this threshold. If the level of interaction rises above a second threshold, the cell is paralyzed; induction of paralysis requires nonspecific overstimulation. It is not clear whether the same B cell expresses only one type of mitogen receptor or more than one structurally different receptor. However, it is the quantitative level of interaction with these receptors, and not the quality of the molecules competent to interact that determines activation and paralysis. Nothing is known about either the physicochemical structure of the mitogen receptor or the biochemical events underlying activation and paralysis. Different subsets of B cells differ in the availability or in the number and/or types of mitogen receptors. The activated B cell performs all the functions for which it is genetically programmed and according to its stage of differentiation at the time it is activated. The level of induced mitosis, as well as the ability of the cell to reach a state of high antibody production upon activation, depend, therefore, on the differentiation level of each activated cell and are not determined by the quality of the nonspecific stimuli. Little is known about the stages along the differentiative pathway that determine the competence of the B lymphocyte to be triggered to each different function, but different ligands show distinct capacities to interact efficiently with different B-cell subpopulations. The function of Ig-specific receptors, expressed on the surface of immunocompetent resting B cells, is to focus on the membrane of the cells (which specifically recognize antigenic determinants on immunogenic molecules) the mitogenic potential carried on the same molecules and/or generated by them in the microenvironment. The activation proceeds via nonspecific mitogen receptors. However, since binding is selective, it results in antibodies identical to the specific receptors that were competent to focus efficiently the triggering signals, and only to these. Several other mechanisms, both humoral and tissular, play an important role in optimizing the monoclonality of the response. Ill. Some Important Technical Considerations

As pointed out above, most of the discussion is centered around our own results. Therefore it is important to analyze our methods. This also gives us a chance to discuss interpretations of experimental results in well-known systems, such as the hemolytic plaque assay,

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as well as to introduce some fundamental concepts of B-cell activation in connection with methods of i n vitro induction of specific and polyclonal antibody responses.

A. DETECTION OF B-CELLACTIVATION BY MEANS OF A SENSITIVE HEMOLYTIC PLAQUEASSAY B-Cell mitogens such as LPS are well characterized by their ability to activate B lymphocytes to divide and to differentiate morphologically (Greaves and Janossy, 1972b; Janossy et al., 1973c) and functionally (Melchers and Andersson, 1973; Andersson et al., 1972e; Parkhouse et al., 1972) into high-rate antibody-secreting cells. Mitogenic activation involves a large fraction of all immunocompetent B cells (Janossy et al., 1973a,c; Shands et al., 1973; Parkhouse et al., 1972) and is independent of the clonal specificities of the acExpertivated cells (Andersson et al., 1972e; Coutinho et al., 1974~). imentally, activation can be easily detected in tissue culture systems by assaying division or antibody production. Normal B-cell populations, exposed in vitro to suitable concentrations of a mitogen, exhibit increased incorporation of radioactive thymidine into acidprecipitable material (Andersson et al., 1972c; Peavy et al., 1970; Janossy and Greaves, 1971; Sultzer and Nilsson, 1972), as well as increased numbers of cells detected as plaque-forming cells (PFC; high-rate antibody producers) in the hemolytic plaque assay (Andersson et al., 1972e; Nilsson et al., 1973). In the latter case, since the activation is polyclonal, any antigen used in the plaque assay system can presumably detect increased numbers of PFC. The need for great sensitivity in the detection methods, as well as for maximum efficiency and reproducibility, led us to use a standard plaque assay, originally developed by Bullock (Bullock and Miiller, acetyl 1972), which employed highly (4-hydroxy-3,5-dinitrophenyl) (NNP)-substituted sheep red cells (SRC) as targets. Several practical and theoretical considerations justify this choice. The polyclonality of activation induced by B-cell mitogens had already been demonstrated for two different non-cross-reacting red cells (Anderson et al., 1972e), as well as for the hapten trinitrophenyl (TNP) (J. Anderson, 1972) bound to erythrocytes. It had also been shown that the PFC produced against a certain antigen after mitogenic activation were mainly of low affinity for the antigen, whereas an antigenic challenge resulted in a preferential increase in high-affinity PFC (Nilsson et al., 1973; J. Andersson, 1972). Furthermore, it was found that normal (unprimed) spleen cells, as well as mitogenically activated cells, exhibited a much higher number of PFC against

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hapten-coated red cells than against uncoated erythrocytes. This fact, together with the simplicity and high reproducibility of the coupling of the hapten NNP to red cells, would make it convenient to use highly hapten-substituted SRC as standard targets for detecting mitogen-induced activation of normal spleen cells in vitro. Extensive studies by Pasanen demonstrated that when red cells coated with NNP were used as targets in the plaque assay, increasing epitope densities on the indicator cells allowed the detection of antihapten antibodies of decreasingly lower affinity (Pasanen and Makela, 1969; Pasanen, 1971a,b). It was demonstrated in a number of different ways that this was not due to increased fragility of the target red cells. It follows that red cells coated with a high hapten density would be suitable targets for the detection of polyclonal activation in the hemolytic plaque assay. Most of the antibodies detected would be of very low affinity for the hapten, too low to be considered specific and to be produced by cells that could possibly respond to an immunogenic challenge with that hapten. Most likely, PFC detected under these conditions would represent a range of different specificities and, therefore, can be regarded as an indication of polyclonal activation. These considerations are supported by direct experiments. By using this standard plaque assay, the number of background PFC detected in spleens of normal (unprimed) mice of various strains was about 50/106 cells. If it is assumed that only half of the cells are B cells (Raff et al., 1971) and, of these, only 1-2% are high-rate antibody producers in normal mice (Melchers et al., 1974), the conclusion will be that this assay detects 0.5-1% of all PFC present in these cell populations. This value is far too high to be accepted as the frequency of specific hapten-sensitive B cells (Quintins and Lefkovits, 1973; Pasanen, 1971b),even if all of them are high-rate antibody producers. When the majority of the spleen PFC precursors are activated by a suitable mitogen to develop into PFC in vitro, the numbers of PFC detected in this assay is around 3000-5000/106 cells (Nilsson et al., 1973; Gronowicz and Coutinho, 1974). This also gives a value of around 1% of the total number of activated B cells. This subject has been developed further in connection with the affinity distribution of B cells detected as antihapten PFC (using different epitope densities on the target red cells) after activation by different concentrations of the same ligand. It was shown that a high epitope density allows the detection of PFC activated by concentrations of the mitogen that result in polyclonal activation, whereas a low epitope density exclusively detects PFC of high affinity, developing after antigen activation (Coutinho et al., 1974a).

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Thus, a plaque assay employing highly hapten-substituted target cells is suitable as a measure of polyclonal activation for various reasons: (1) since activation is achieved in the absence of antigen, one hapten is as suitable as any other antigen for the detection of polyclonal activation; (2) a high epitope density allows detection of PFC of very low affinity, presumably belonging to several different clones; and ( 3 )the substantially higher numbers of PFC detected, as compared to those when nonhapten-coated red cells are used, greatly increases the sensitivity of the method. Only direct PFC can be observed under these conditions, both in normal and in mitogen-activated spleen cell populations. This is in agreement with several reported observations and with the fact that only IgM is detected after mitogen-induced activation of normal spleen cells by pokeweed mitogen (PWM) (Parkhouse et al., 1972), LPS (Melchers and Andersson, 1973), or insoluble concanavalin A (Con A) (Anderson and Melchers, 1973). We can find no explanation for the observation reported by Armerding and Katz (1974a) that LPS could induce the appearance of IgG PFC among normal spleen cells, except for the possibility that the developing serum did not suppress the IgM PFC efficiently. Indeed, the number of IgG PFC was always lower than the number of IgM PFC (Armerding and Katz, 1974a). However, the question of Ig class produced upon PBA activation is far from solved. Increased levels of serum IgG were found after LPS challenge of newborn mice (Kolb et al., 1974). Moreover, the failure to induce IgG PFC by PBA in normal spleen cell populations in vitro is not due to an intrinsic inability of these ligands to activate 7-PFC precursors, since this is readily achieved in primed spleen cell populations (Kreisler and Moller, 1973). Therefore, it is possible that differences in the peripheral distribution of precursor cells of each class of immunoglobulin are responsible for these findings. This is discussed below in connection with B-cell subpopulations, but it is noteworthy that, with the exception of one group of workers (Pierce et al., 1971), IgG PFC have not been obtained by primary antigenic challenge of spleen cells in vitro. Very recently, increased synthesis of IgG and IgA has been reported in the mouse after in vitro activation by LPS and PWM (Janossy, personal communication). Although no increase in secretion could be demonstrated, the observation appears extremely important because the activation was polyclonal and TI. For every PBA so far studied, fetal calf serum (FCS), Con A-activated T cells, LPS, purified protein derivative (PPD) (Coutinho et al., 1973a), pneumococcal polysaccharide (Coutinho and Moller, 1973c,d), polymerized flagellin (POL), levan, polyvinylpyrrolidone

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(PVP), and dextran (Coutinho and Miiller, 1973), other antigens were used as indicators to ensure that the results were not restricted to the particular hapten used in the standard plaque assay. The variety of mitogens, as well as the variety of antigens or haptens used as targets [SRC, horse red cells (HRC), NNP, TNP, 4-hydraxy-3-iodo-5nitrophenylacetic acid (NIP) (Coutinho and Moller, 1973c,d; Coutinho et al., 1973a), fluorescein isothiocyanate (FITC), pencillin ) ~ an extensive list of red cells (PEN) (Coutinho et al., 1 9 7 4 ~ plus reported to cross-react with paralytogenic doses of LPS in vivo (Rank et al., 1972)], together with several other reasons discussed below, make it highly unlikely that cross-reactions between a particular mitogen and any of the antigens used in the plaque assay were responsible for the findings, The use of plaque assay to detect mitogen-induced B-cell activation offers several other advantages. When compared to other methods of detecting nonspecific activation of immunoglobulin synthesis, such as radioactive biosynthetic labeling and specific precipitation, it was found to be more sensitive (J. Anderson, personal communication), although it correlated well with total IgM secretion (Melchers and Andersson, 1973; Andersson et aZ., 1972e). It provides a quantitative estimation of antibody secretion at the cellular level and allows the enumeration of activated cells. This may be of value in other situations, namely kinetic studies of activation, since it is accepted that B cells are activated to produce antibody in an all-ornothing mode (Melchers et aZ., 1974). Due to the heterogeneity of spleen cell populations, even in T-cell-deprived animals (Raff, 1973), any assay system that detects a B-cell function exclusively is advantageous. The plaque assay is further suitable since it only detects high-rate antibody-secreting B cells and not resting B cells that shed 7 S subunits from the membrane (Jerne et aZ., 1974; Jerne and Nordin, 1963; Jerne, 1967). It is clear that a PFC is a B cell activated to high-rate antibody production and could never be just a resting lymphocyte living better (Anderson et aZ., 1974b; Melchers and Andersson, 1974). This conclusion is important in negating arguments that PBA may merely provide the cultured cells with a type of metabolic help or feeder effects. B. MISINTERPRETATION OF ANTIHAPTENPLAQUE ASSAYS

The plaque-forming cell assays with hapten-coated red cells have been widely (and wildly) used during the last years for the detection of claimed “specific” antihapten antibody responses. The standard procedure consists of determining the numbers of PFC detected

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against hapten-coated or uncoated target red cells. By subtracting these two values, the numbers of specific antihapten PFC are easily obtained. However, this immunomathematical operation is quite often incorrect, as can be demonstrated theoretically as well as experimentally. As we have just seen, highly substituted red cells are suitable targets for PFC assays detecting polyclonal activation. Among the many reports on this subject, the degree of hapten substitution is well established only in few instances (Strausbauch et al., 1970), and, since only one epitope density is used in each investigation, it is difficult or impossible to evaluate the specificity of the PFC detected. Even with low levels of hapten substitution, there is a similar problem. It is well known that many specificities available in the B-cell repertoire are able to bind nitrophenyl groups (Schuberg et al., 1968; Jormalainen and Makela, 1971; Varga et al., 1973). Therefore, even at low degrees of hapten substitution there are more haptenic determinants than natural red cell determinants on the target and, consequently, a large excess of PFC can always be detected in normal, unstimulated B-cell populations against hapten-coated red cells, as compared to uncoated targets (Bullock and Miiller, 1972; Coutinho et al., 1973a). Thus, when normal spleen cells are polyclonally activated by a mitogen, the fraction of cells that can be detected with that specificity is also much larger. It follows that after polyclonal activation the increase in hapten-specific PFC is much greater than the increase detected with uncoated red cells. It is now easy to see that by simply following the widely used method of subtracting the background PFC from the number of hapten PFC, a hapten-specific response would appear to have been induced by a polyclonal mitogen (see Coutinho et al., 1973a, where this result is obtained after 24 hours in culture with several mitogens). This type of experiment requires special care in interpretation when the “specific” responses are obtained with the help of polyclonal stimulants (such as mixtures of antigen-mitogen or antigen-adjuvant) in in &To cultures supplemented with “good” batches of FCS (Coutinho et al., 1973a; Watson et al., 1973b; Miiller et al., 1975). Moreover, the fact that TI antigens display polyclonal activating properties necessitates careful specificity controls, These can be achieved either by using a similar degree of target cell substitution with a non-cross-reacting hapten (which is difficult because most haptens used are cross-reacting nitrophenyl compounds) or else by determining in every experiment the avidity distribution of the activated cell populations (see, e.g., Coutinho et al., 1974a). Unfortunately this precaution is very often forgotten, frequently leading to misinterpretations of the results.

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C. SERUM-FREE CULTURES Experimental systems dealing with activation of mouse lymphocytes in vitro face the paradox of the impressive suppressor effect of even low amounts of homologous serum (Bullock and Moller, 1972). This led to the introduction of heterologous serum supplementation, the most commonly used serum being FCS. However, heterologous serum supplementation may have several disadvantages. Thus, different batches of serum vary in their suppressive or stimulatory properties, and the use of serum involves the exposure of cells to a number of different antigens and other serum factors with unknown properties. When in uitro systems aim at clarifying triggering mechanisms of lymphocytes, short-term cultures are suitable instruments, and this suggested the possibility of avoiding these disadvantages by simply deleting serum supplementation. Some reports on lymphocyte responses in serum-free media were already available (Kirchner and Oppenheim, 1972; Mackler et al., 1972; Levy and Rosenberg, 1972; Visher, 1972a,b,c) but all were fragmentary and exclusively concerned with DNA synthetic responses. Our main purpose was to study the role of B-cell mitogens in the induction of specific antibody responses, and, therefore, we employed the Mishell-Dutton system. This system had been extensively used in many laboratories and provided the experimental basis for a number of hypotheses concerning the development of a normal immune response. However, all conclusions reached with this system depend on selected “good” batches of FCS used to supplement the cultures (Mishell and Dutton, 1967; Watson and Thoman, 1972; Watson and Epstein, 1973). Therefore, it was important to elucidate the role of FCS. In addition, studying lymphocyte activation required better in vitro conditions than cultures supplemented with FCS in which lymphocytes exhibit “spontaneous” activation. Thus, a ten- to twenty-fold polyclonal increase in the numbers of background PFC was found when normal mouse spleen cells were cultured for 3 days in the Mishell-Dutton system (Bullock and Miiller, 1972; Mishell and Dutton, 1967; Pasanen and Virolainen, 1971; Jennings and Rittenberg, 1971). Similar increases were also detected when DNA synthesis was measured (Gazit and Harris, 1972; Zimmerman and Kern, 1973). This activation in the absence of added antigen or any apparent stimulus was observed after the original description of the induction of primary anti-SRC responses in this system (Mishell and Dutton, 1967), but it did not attract much attention, since the background activation was low compared to the specific response. This

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was due to the low sensitivity of the assay for anti-SRC PFC and to the different kinetics of background and antigen-induced responses, respectively. The “spontaneous” increase was maximal on day 3 of culture, whereas antigen-specific responses peaked later, at a time when the background activation had disappeared to a large extent (Bullock and Mijller, 1972). For our particular study of B-cell activation by mitogens, this background activation was particularly disturbing, because control (“unstimulated”) cultures were actually stimulated to a polyclonal response with kinetics similar to mitogeninduced responses. Taken together, all these considerations led us to undertake a comprehensive study of the serum requirements for mouse spleen lymphocytes cultured in uitro. The initial studies on T- and B-cell activation by mitogen (Coutinho et ul., 1973a) showed that good cell survival and excellent induction could be achieved in serum-free cultures, thus providing a well-defined, simple, and reproducible culture system. The most important conclusion from this investigation was the finding that the spontaneous appearance of background PFC observed in cultures of normal mouse lymphocytes was not a necessary feature of the in uitro methods, but was due to the presence of B-cell mitogens in FCS. Normal mouse spleen cell cultures in serum-free media were not spontaneously activated but maintained the functional state they had at the start of the culture [99% of B cells being resting cells (Melchers et ul., 1974)l. A recent observation (Schrader, 1974c) is in disagreement with this fact, and it was claimed that background PFC arise in culture even in the absence of serum supplementation. However, this statement was not supported by the results, because the numbers of PFC were not determined at the start of the culture. Therefore, the correct conclusion from that observation is that PFC survive when cultured for 3 or 4 days in serum-free media, as we reported before (Coutinho et al., 1973a). Indeed, when PFC of any specificity are determined at the start of the serum-free culture and regularly thereafter, there is no increase in numbers with time of culture, simply because no activation is provided for the cultured cells (Coutinho et al., 1973a). By completely avoiding activation in nonstimulated cultures, it became possible to use a proper control for mitogen-induced stimulation in uitro. Furthermore, the mitogen-induced responses developed to the same extent in serum-free as in serum-supplemented cultures and, therefore, the absence of background stimulation increased the sensitivity of the system. In this way, it could be shown that B-cell mitogens induced polyclonal antibody synthesis in

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normal B cells in the absence of serum antigens or other factors in the culture media. This response, as measured by DNA or antibody synthesis peaked on day 2 of culture. An important initial finding in this system was that the addition of the T-cell mitogen, Con A, to normal spleen cells induced a polyclonal, T-cell-mediated antibody response, whereas previous studies on this problem in serumsupplemented cultures had been negative or produced very low levels of stimulation (Greaves and Janossy, 1972a,b; Janossy et al., 1973a,c; Vischer, 197213). Actually, these discrepancies in results are at least partially caused by the presence of serum (Coutinho, unpublished). Optimal concentrations of Con A, a mixed-leukocyte culture (MLC) reaction, or nonspecific T-cell factors obtained from MLC supernatants added directly to cultures, all can be shown to increase the number of PFC of various specificities in the absence of antigen (G. Moller and Coutinho, unpublished), as well as total immunoglobulin production (Coutinho et a1., 197313; Sjoberg, 1975), but only in serum-free media (Fig. 1).When parallel experiments are carried out in the presence of FCS, suppression or no effect usually occurs. The interpretation of these findings is discussed below, but it

DAYS IN CULTURE

FIG. 1. Normal ( A X B10.5M)FI spleen cells were cultured in serum-free medium (10 x los cells/ml./culture dish), in the presence of T-cell-replacing factors (TRF) (0.5 dilution) ( 0 ) control ; supernatants (0.5 dilution) (0);or fresh medium (A). Polyclonal activation was assayed by measuring anti-(4-hydroxy-3,5-dinitrophenyl) acetyl plaque-forming cells (PFC) after various days in culture. The TRF and control supernatants were 24-hour supernatants from spleen cell cultures of A and B10.5M, mixed or unmixed, respectively.

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is unlikely that the effect of FCS is simply to reduce the sensitivity of detecting activation. It is more likely that the simultaneous stimulation by FCS mitogens and T-cell factors actually suppresses B cells by an overdose effect or that B-cell repressors are present in FCS (Bullock and Andersson, personal communication). D. REQUIREMENTFOR POLYCLONAL B-CELLACTIVATORSI N THE MISHELL-DUTTON SYSTEM The finding of PBA in FCS is important for an understanding of immunocyte activation in vitro. It is well known, that primary immunization of dissociated spleen cell cultures by SRBC can be achieved only when the culture medium is supplemented with a “good” batch of FCS (Mishell and Dutton, 1967). The good batches are currently selected on the basis of their capacity to “support” on anti-SRBC PFC response which peaks on days 4-5 and is dependent on the presence of T cells. Indeed, some particularly good batches of FCS can actually substitute for T cells (Byrd, 1971). In other words by selecting batches of FCS according to different criteria, one can have the commonly accepted situation of primary, anti-SRBC TD responses, which increase to days 4-5, or else a TI response with earlier or later peaks. The similarity with the property described for LPS as being capable of substituting for T cells in the system (Sjoberg et al., 1972b) also suggested that serum PBA could be involved in the induction of the in vitro primary anti-SRBC responses. For these reasons, the requirement for FCS in the induction of primary and secondary responses to TD and TI antigens was investigated. A striking conclusion from these studies was an absolute requirement for B-cell mitogens in all the specific B-cell responses. In summary, specific B-cell responses only occurred in the presence of nonspecific PBA, and these could be provided in a number of different ways: by adding to the cultures a “good” batch of FCS, LPS, or a large number of specifically (in primed cell populations) or nonspecifically (by Con A or by a MLC reaction) activated T cells. Thus, the serum dependence of primary responses to SRC was due to the presence of B-cell mitogens in FCS, since no response could be induced in the absence of serum or when the mitogens in a good batch of serum had been inactivated. However, a specific anti-SRC response could develop in serum-free cultures if some type of nonspecific PBA were provided, and a specific anti-LPS response could always be induced, the B-cell mitogen being, in this case, the antigen itself (Coutinho and Moller, 197313). Since the Mishell-Dutton system has been used for the demon-

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stration of specific B-cell responses and is generally considered to reproduce the normal physiological process of B-cell induction operating in vivo, it is important to discuss the absolute requirement for nonspecific mitogens in the system, and to evaluate the significance of experimental results derived from what appears to be an artificial in vitro situation. In optimal conditions, the system allows primary immunization of dissociated spleen cells by antigens present on red cells (Mishell and Dutton, 1967), or else, by soluble antigens if they are highly TI (Diener and Feldman, 1970; Sjoberg and Britton, 1972). In the case of primary responses to red cell antigens, three types of cells are needed for induction, namely macrophages (Mosier, 1967), T cells (Chan et al., 1970; Sjoberg et al., 1972a), and B cells, but only IgM antibodies are regularly induced (Mishell and Dutton, 1967). Furthermore, induction of responses necessarily requires the supplementation of the cultures with 5 to 10% of selected batches of FCS (Mishell and Dutton, 1967; Watson and Thoman, 1972; Watson and Epstein, 1973). The fact that red cell antigens were the only antigens effective in inducing primary antibody responses in this system has been the subject of several speculations. One common concept is that primary in vitro responses are, in fact, secondary, due to the presence of memory cells in unprimed mice which have been immunized by cross-reacting antigens. However, this interpretation does not fit with the exclusively IgM response obtained in normal spleen cells, nor with the responsiveness observed in germ-free animals. On the other hand, with the increasing list of antigens tested in the system, it is evident that primary responses to other antigens are also possible and, in fact, are always obtained if the antigen exhibits some degree of thymus independence. This fact, taken together with the complete failure to induce primary responses to highly TD serum proteins (Feldman, 1972a), raise the question whether primary anti-SRC responses occur under the Mishell-Dutton conditions, because of the relative degree of thymus independence of this antigen. It is well known that reasonable IgM responses can be induced by SRC in thymus-deprived mice in vivo (Pantalouris and Flisch, 1972), or in cultured spleen cells (Feldman et al., 1972). Furthermore, the macrophage (Feldman and Palmer, 1971) and T-cell independence (Byrd et al., 1974) of the in vitro responses to lysates of SRC reinforces the idea that the immune response to this antigen is at least partially TI. Other characteristics of red cells may be of importance, namely their particulate nature and the fact that cooperative nonspecific factors bind readily to erythrocytes ( H o h a n n and Dutton, 1971; Anderson

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et al., unpublished). The latter point will be discussed below and might actually be a clue to understanding these observations. However, if relative T independence is the reason for the effectiveness of SRC in the Mishell-Dutton system, it is reasonable to ask why anti-SRC responses are TD. Furthermore, since it is known that LPS can substitute for T cells in the anti-SRC responses in v i t m (Sjoberg et al., 1972b), it may appear strange that antibody responses to TD antigens in the Mishell-Dutton system are still TD, in spite of the absolute requirement for the presence of PBA. The answer appears to rely on a quantitative concept of B-cell activation. If the available evidence concerning B-cell activation in the Mishell-Dutton system is put together in a conceptual scheme, the following picture emerges, which may help in the interpretation of the above findings. Cooperation occurs rather poorly when spleen cells are dissociated and cultured in vitro. This may be due to, among other reasons, destruction of the normal splenic architecture, preferential loss of certain cell types, or deprivation of normal, regulative serum factors. Therefore, B-cell induction via the physiological and antigen-induced cooperating cell system is impaired and insufficient to result in activation of specific B cells. No experimental methods performed in well-defined, serum-free culture conditions, are available to assess primary T-cell immunization in v i t m . However, from the findings obtained in secondary responses (Coutinho and Moller, 1973b), we assume that antigen-induced activation of antigen-sensitive T cells occurs. Therefore the failure of physiological cooperation results either from any of the above reasons, or from the small numbers of T cells present in culture. Since T cells are known to recirculate (Miller and Sprent, 1971a), the proportion of antigen-sensitive T to B cells is likely to be greatly altered in cultures deprived of the circulating cell pool. It follows that the difficulty in inducing an antibody response in this system increases with the requirements for cell cooperation in B-cell activation by each particular antigen. Highly TI antigens, which do not require cooperation because they can immunize B cells directly, are capable of inducing optimal responses in vitro in the absence of any further helper activity, either physiological [T cells (Feldman and Basten, 1971) and macrophages (Diener et al., 1970)], or artificial [(FCS supplementation (Coutinho and Moller, 197313; Coutinho et al., 1974a))l. This is the case with LPS, for instance, which is capable of inducing primary antibody responses in “nude” spleen cells cultured in serum-free media (Coutinho et al., 1974a). This finding strengthens the conclusions that B-cell performance is unimpaired in serum-free media (Coutinho et

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d.,1973a) and that the failure in inducing TD responses depends on deficient conditions for cooperation, independent of serum supplementation. Antigens (such as SRC) that display a certain degree of thymus independence can induce a primary antibody response only if some further nonspecific stimuli are provided to the B cells, because of the defect in physiological cooperation. Under normal Mishell-Dutton conditions, B-cell immunization by SRC is achieved by the addition of several factors: the direct (TI) stimulus provided by the antigen itself, mitogenic factors present in suitable batches of FCS, and mitogenic factors released by antigen-activated T cells and macrophages. All these mitogenic factors are required for quantitative reasons. Depletion of T cells or macrophages results in reduced or abolished responses (Chan et al., 1970), and absence of supplementation by a suitable batch of FCS abolishes the response (Coutinho and Moller, 1973b). Therefore, the response to SRC appears to be TD to a large extent and requires further addition of mitogens in the absence of serum. However, due to the nonspecificity and the quantitative basis of B-cell activation, different mitogenic factors can substitute for each other. Thus, large numbers of nonspecifically activated T cells substitute for FCS (Coutinho and Moller, 197313; Moller and Coutinho, 1973), and selected batches of particularly active FCS substitute for T cells (Byrd, 1971). Moreover, each of these sources of mitogenic factors can be replaced by another B-cell mitogen (LPS) (Sjoberg et d.,1972b; Coutinho and Moller, 197313). However, if the mechanisms responsible for B-cell activation are nonspecific and activate B cells polyclonally, it is reasonable to ask why the responses still appear as specific. This phenomenon, commonly termed synergy of antigen-mitogen, has been reported for several different combinations of antigen-mitogen (Coutinho and Moller, 1973b; Moller and Coutinho, 1973; Kreisler and Moller, 1974; Dennert and Lennox, 1973; Schrader, 1973a; Watson et al., 1973b) and appears to be of central importance for an understanding of B-cell activation by TD antigens. Synergy will be extensively discussed below in the light of further evidence for the nonspecificity of B-cell triggering. It is enough to stress here that several reasons other than the bridging of T to B cells by antigen are likely to be involved and that synergy seems to operate in serum-free cultures only with antigens that express a certain degree of thymus independence (SRBC in particular). Moreover, it should be pointed out that there is a strict correlation between the level of nonspecific activation induced in cultures as measured on day 2 or 3 (Coutinho

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and Moller, 1973b) and the level of antigen-specific responses induced in the same cultures, even if the response appears to be specific at later periods of culture, after the polyclonal activation has declined. Highly TD antigens cannot induce primary immune responses in normal Mishell-Dutton cultures, even with good batches of FCS (Feldman, 1972a). Since these TD antigens completely lack the capacity to immunize B cells directly, the main mechanism that is presumably responsible for the synergy of SRBC-PBA (namely relative TI of the antigen) cannot function. However, other mechanisms of synergy can be optimized in extreme situations and result in an antibody response to a TD soluble protein. Invariably, these seem to involve activation of macrophages by the presence of a PBA in addition to FCS [LPS or POL (Schrader, 1973a,b, 1974a,b; Watson et al., 1973b)l or products released by activated macrophages (Schrader, 1973b; Munder et al., 1973). In secondary responses, a much larger number [a ten-fold increase as compared to primary responses (Miller and Sprent, 1971b; Mitchell et al., 1972a)l of antigen-sensitive T cells is present in the culture. Therefore, nonspecific factors released after antigen activation of T cells are competent to provide sufficient B-cell activation in the absence of serum in responses to SRC (Coutinho and Moller, 197313). In the presence of serum mitogens, even responses to TD proteins are possible in such situations (Feldman, 1972c; Bullock and Rittenberg, 1970a,b; Feldman and Nossal, 1972). It is plausible that another factor is responsible for the “easier” induction of secondary in vitro responses, namely the higher sensitivity to triggering displayed by the B-cell population responding to a secondary antigen challenge. Thus, activation of IgGsecreting “memory” cells by LPS in the absence of antigen is achieved by mitogen concentrations 10-100 times lower than the concentrations required for activation of IgM in primed or unprimed cells. Also the synergy of antigen-mitogen was shown to require lower concentrations of mitogen to be optimally expressed in primed cells (Kreisler and Moller, 1974). Another possible mechanism by which PBA, LPS in particular, might be argued to act in the induction of antibody responses to a TD antigen by T-cell-deprived cultures invokes the claimed capacity of LPS to induce the appearance of T-cell surface markers in these cultures (Scheid et al., 1973). The functional properties of such cells, however, are far from firmly established (Scheid et al., 1973). Clearly this mechanism cannot be operative for the simple reason that spleen cell cultures with a normal content of T cells cannot mount a primary

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response in the absence of nonspecific PBA (Coutinho and Moller, 1973b). Nevertheless, differentiation of T-cell precursors should be kept in mind as a possible explanation of the excellent synergy obtained when heterologous or homologous sera are used in systems where LPS is completely negative (Bullock, unpublished).

E. A QUANTITATIVE CONCEPT

B-CELLACTIVATION It can be concluded from these in vitro studies that there is an absolute requirement for the presence of B-cell mitogens for the induction of specific antibody responses. When a B-cell population is stimulated to respond, the limiting variable appears to be the quantity of stimuli available. The nature of the triggering stimuli seems to be entirely nonspecific (this will be discussed in detail, but a number of reasons have already been outlined). By increasing the quantity of stimuli (but independently of their quality), increasing responses occur, until the total number of B cells of that specificity are activated. Above this level of stimulation, suppression occurs. The dose-response curve of any B-cell population to any competent stimulus is a bell-shaped curve, therefore, and the ultimate limiting variable in the system is always the total number of B cells present in the population having the particular specificity detected in the assay system. This theoretical extrapolation of the experimental data may be of some value for understanding B-cell induction and paralysis in specific responses. Let us stress before going further that factors produced by T cells or macrophages upon specific or nonspecific T cell activation are polyclonal mitogens for B cells and do not require antigen to express mitogenicity. This has been demonstrated in serumfree cultures (Coutinho et al., 1973a,b; Vischer, 1972a,b; Sjoberg, 1975), but several positive reports in other culture systems have also been published (Geha et al., 1973; Rich and Pierce, 1973a; Armerding and Katz, 197413; Katz and Benacerraf, 1974). Furthermore, it is well known that the “allogeneic effect” increases background PFC, in the absence of antigenic challenge in primed animals (Katz and Benacerraf, 1972) and recent findings demonstrate that induction of a graft-versus-host (GVH) reaction in a normal, unprimed animal also results in polyclonal increase in the number of splenic PFC (G. Moller, unpublished; Orvellas and Scott, 1974), indicating that these factors are also active in uiuo. The importance of macrophages for immune induction in the Mishell-Dutton system is clear but the mechanism not fully understood. Macrophage dependence of antigen responses is strictly correlated with thymus dependence, suggesting that the importance of these cells may be related to T-cell activation FOR

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by antigen (Rosenthal and Shevach, 1973; Shevach and Rosenthal, 1973). However, it seems possible that (activated) macrophages also release some type of activating substances for B cells, which would function not only by providing a feeder effect but may actually be B-cell mitogens (Schrader, 197313; Munder et al., 1973). This activity may not be restricted to macrophages, since fibroblasts, which can be substituted for adherent cells in anti-SRC responses, also release mitogenic factor(s) allowing the primary immunization of nonadherent spleen cell populations by SRC (G. Moller, unpublished; Moller and Coutinho, 1975). Since adherent cells are strictly required for the expression of synergy of SRC-PBA (Kreisler and Moller, 1974) and the soluble products that they release upon activation and since they are fully active in binding to the SRC (HofFmann and Dutton, 1971), it seems plausible to ascribe tentatively to macrophages the roles of antigen presentation to T cells and secretion of B-cell-activating substances. With these facts in mind, let us now analyze the interaction among different nonspecific stimuli in the Mishell-Dutton system, in the light of a quantitative concept. It is evident that the response induced by SRC in normal (T, B, and adherent cells) spleen cultures supplemented with a good batch of FCS is not optimal. Indeed, the responses can be increased further by addition of other nonspecific stimuli, such as LPS (Sjoberg et al., 1972b), PPD (Kreisler and Moller, 1974), Con A (Sjoberg et al., 1973; Dutton, 1972; Rich and Pierce, 1973a), small numbers of H-2 incompatible cells (Britton, 1972a; Mdler and Coutinho, 1973), or large numbers of non-H-2 incompatible cells (Britton, 1972a). Therefore, it is clear that under “normal” Mishell-Dutton conditions, the system is not saturated and the response can be maximized by several manipulations, all of which result in increased levels of nonspecific B-cell-activating stimuli in culture. This maximum corresponds, of course, to the activation of all the PFC precursors with detectable anti-SRC specificity, as well as to maximal clonal expansion. If the levels of B-cell stimulation are further increased, above the saturation limit of the system, the responses are suppressed, by turning off B cells submitted to overactivation. This is readily achieved by the same manipulations used to enhance the responses, simply by increasing their quantity-LPS (Sjoberg et al., 1972b), PPD (Kreisler and Moller, 1974), Con A (Sjoberg et al., 1973; Dutton, 1972; Rich and Pierce, 1973a), phytohemagglutinin (PHA) (Britton, 1972a), H-2 incompatible cells (Britton, 1972a), antigen-activated T cells (Feldman and Basten, 197213). The quantitative rules that govern the system are clearly demon-

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strated by experiments performed under obviously subnormal conditions. Namely, if most of the T cells or the FCS mitogens are omitted from the cultures, virtually no response is obtained. In such cases, the B-cell-responsive capacity is far from saturated, and a great deal of further nonspecific stimulation should be required to bring the response to an optimum. Indeed, high concentrations of LPS (Coutinho and Moller, 1973b; Sjoberg et al., 1972b), PPD (Kreisler and Moller, 1974), Con A (Sjoberg et al., 1973; Dutton, 1972; Rich and Pierce, 1973a), PHA (Britton, 1972b), H-2 incompatible cells (Rich and Pierce, 1973a; Moller and Coutinho, 1973), which are highly suppressive for serum-supplemented normal spleen cell cultures, provide good stimulation in T-cell- or serum-deprived cultures. These findings constitute a strong argument against claims for the existence of a separate subpopulation of T cells exclusively involved in suppression of B-cell activity (Dutton, 1972, 1973; Rich and Pierce, 1973b; Baker et al., 1973). This concept was derived from experiments in which overstimulation of B cells with consequent suppression was always present. The claimed direct evidence for a suppressive subpopulation of T cells in this particular system of antiSRC responses is very weak (Dutton, 1972; Rich and Pierce, 197313). In &TO experiments with Con A provide good evidence against “suppressor” T cells, even though this has been used to support the opposite conclusion (Dutton, 1972, 1973). As discussed above, nonspecific stimulation of SRC-specific B cells in primary responses is nearly optimal in the presence of the antigen itself, FCS mitogens, and a very small number of antigen-responsive T cells (50-500 cells, to l W antigen-sensitive T cells). The accepting a frequency of response can be further increased to optimal levels when all SRCspecific B cells are optimally activated by the addition of small numbers of antigen-activated T cells (lo5) (Feldman and Basten, 1972b), small numbers of allogeneic cells (resulting in around lo4activated T cells, if 1-2% are considered to react in an allogeneic interaction) (Moller and Coutinho, 1973; Britton, 1972a), small numbers of Con A-activated spleen cells (105-10s)(Rich and Pierce, 1973a), or low concentrations of Con A (Sjoberg et al., 1973; Rich and Pierce, 1973a). If larger amounts of activated T cells are present, or if higher concentrations of Con A are used, the response is suppressed (Sjoberg et al., 1973; Rich and Pierce, 1973a,b) because the optimal level of stimuli for saturating the system is overcome. However, this could never be due to suppressor cells, because if the system is deprived of a large fraction of the initial stimulation, by omitting FCS, the postulated suppressor T cells even in very large amounts

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(Coutinho and Miiller, 197313; Moller and Coutinho, 1973), as well as optimal doses of Con A (Coutinho and Moller, 1973b), which presumably activated most of the T cells present, are no longer suppressive. Another argument is the fact that nonspecific T-cell factors obtained from MLC supernatants are often more active when diluted than when added undiluted to T-cell-deprived cultures (Watson, 1973; A. S. Rubin et al., 1973). Both observations suggest that the distinction between activation and suppression is due to purely quantitative factors and not to the existence of a separate subpopulation of suppressor T cells. The responses to immunogenic concentrations of other antigens, competent to induce directly nearly optimal responses in B cells (TI), will be much more easily suppressed by further “help.” This is clearly the case for anti-LPS in vitro secondary responses. We have shown before (Coutinho and G. Miiller, unpublished) that the characteristics of the in vivo responses to LPS depend on the immunogenic form which is used for priming. Mildly inactivated Escherichia coli bacteria induce an anti-LPS response, consisting of IgM and IgG antibodies, the latter being TD. Soluble LPS or a standard E . coli vaccine induces a TI response that is primarily restricted to IgM antibodies. Because the IgM response to the mildly inactivated bacteria in thymus-deprived mice is comparable to the IgM response to soluble LPS in normal or “B” mice, it appears that a Tcell-dependent IgG response is, in that case, superimposed onto a basic TI response, induced directly in B cells by the LPS molecule in both situations. The helper T-cell activity is likely to be induced by bacterial proteins present in the mildly inactivated preparation of E . coli. The in vitro secondary responses follow exactly the same pattern observed in vivo. This is an example of a clearly TI immune response that can be augmented by the addition of T cells. However, in this case, due to the higher degree of thymus independence of LPS (the system is nearly optimally saturated by nonspecific stimuli provided to specific B cells by the antigen itself), addition of FCS to cultures where a good response occurred is highly suppressive. Moreover, the addition of primed T cells to the cultures greatly enhances the suppression observed with FCS supplementation, as compared to homologous serum [Table I (A)]. On the other hand, removal of T cells from secondary cultures results in enhancement of the responses observed in the FCS-supplemented cultures, indicating again the suppression induced by overactivation [Table I (B)]. A similar explanation may be used in situations where suppressor T cells are readily demonstrated, such as in the in vivo responses to

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TABLE I EFFECTS OF FETAL CALF SERUM (FCS) IN THE in Vitro SECONDARY ANTI-Escherichia coli LIPOPOLYSACCHARIDE RESPONSES

SUPPRESSIVE

A. Day 4 plaque-forming cell (PFC) responses by spleen cells from T X B or T X B mice cultured in normal mouse serum (NMS) or FCS-supplemented cultures:

Ratio

of PFC responses

direct PFC Experiment

TXB

+T

indirect PFC

TXB+T

TXB

TxB+T

104.2 59.5 38.1

2.1 2.7 1.1

11.3 24.0 14.7

~~

1 2 3

9.4 2.8 1.6

B. Effect of a n t i 4 serum treatment in the day 4 PFC responses by normal spleen cells cultured in NMS or FCS supplemented cultures: Percent of control responses NMS

FCS

Experiment

d.PFC

i.PFC

d.PFC

i.PFC

1 2

87.5 107.4

42.5 106.1

98.8 1331.0

124.0 2060.1

~~

other highly TI antigens, e.g., SIII (Baker et al., 1970, 1973) and PVP (Kerbel and Eidinger, 1972; B. Andersson, 1972). The prediction would be that B-cell mitogens, other than antigen-activated T cells, would give the same results in thymus-deprived mice. B-Cell inductionlparalysis is further complicated by the fact that different B-cell subpopulations are involved which respond differently to the same stimulus and might respond selectively to a certain available stimulus. Some of these aspects will be discussed below. However, it should be stressed here that the bell-shaped dose-response profile is also obtained when polyclonal responses of the whole B-cell population are studied (Coutinho et al., 1973a; Coutinho and Moller, 1973d; Andersson et al., 1972c,e), similar to the curve discussed for a particular clone responding to selective stimulation. Actually, the bell-shaped curve is characteristic of the responses to all B-cell mitogens by any B-cell subpopulation, in which each point represents the sum of the individual cell responses to each particular concentration of mitogen. Therefore, the curve represents the Gaussian distribution of B cells in a population, ac-

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cording to the degree of sensitivity to triggering and paralysis, or else, a Poisson distribution, provided that all cells are equally sensitive to activation and that triggering is a one-hit phenomenon. The unknown properties of FCS that make it highly effective in this system may very well be due also to factors other than its B-cell mitogenicity, as suggested by the different levels of responsiveness obtained in FCS-supplemented cultures and in cultures supplemented with well-defined B-cell mitogens. This could be due to the drop in cell survival, observed in later periods of serum-free cultures when antigen responses are measured. However, it seems more likely that other regulatory factors present in serum are responsible for the modulation of the responses and for an optimizing effect in the mechanisms responsible for the synergy of mitogen-antigen, and, therefore, for the amplification of specific responses induced by nonspecific stimulation. One should keep in mind that the selection of a certain batch of FCS for culture supplementation is a selection a priori of a certain type of results (before doing the experiments). Experimental support for these assumptions is now available, and all the evidence suggests that the immune system is prepared to respond specifically, even if the actual mechanism of triggering is nonspecific. The artificiality of the in vitro systems does not obscure a comparison between the models discussed above and the in v i m situation, in which terms such as “good” and “bad” immunogens are currently employed, and where adjuvants are extensively used for immunization. IV. Critical Evidence Supporting the One Nonspecific Signal Hypothesis

In this section we shall review in detail the main experiments which constituted the basis for the suggestions of the one nonspecific signal hypothesis, as well as some more recent evidence which directly support it.

A. ACTIVATION OF B CELLS DIRECTLYAND NONSPECIFICALLY BY THYMUS-INDEPENDENT ANTIGENS In order to study direct activation of a certain cell by any ligand it is necessary to exclude the participation of other cell types in activation. In the case of spleen B-cell responses to B-cell mitogens, the influence of T cells had already been excluded in several systems (Greaves and Janossy, 1972a; Andersson et d,1972c,d). However, the participation of other cell types, in particular macrophages, had not been explicitly excluded, although some reports were compatible

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with no requirement for adherent cells in LPS activation (Coutinho, 1974; Yoshinaga et al., 1972). In view of recent claims for the importance of macrophages in any type of B-cell induction, suggesting that B-cell mitogens may actually induce B cells via macrophages (Watson et al., 1973b; Schrader, 1973b; Dukor et al., 1974; Schumann et al., 1974) it seemed pertinent to test whether B cells could be induced to proliferate and differentiate into high-rate antibodyproducing cells in the absence of functionally active macrophages. Depletion of adherent cells from normal spleen cell populations, at all the mitogen concentrations tested, did not interfere with the ability of nonadherent cells to be activated by LPS to DNA and polyclonal antibody synthesis (Lemke et al., 1975). These results do not exclude the possibility that B-cell mitogens, LPS in particular, may also activate macrophages (Blythman and Waksman, 1973; Nelson, 1973), nor that the results of such activation (e.g., the release of soluble factors) may have some importance in B-cell induction. What was shown was that activation of spleen B cells by LPS proceeds at normal levels after depletion of adherent cells. It is well known that there are no reliable methods available to deplete a cell population completely of macrophage or macrophage-like cells (Greenberg et al., 1973). The degree of residual contamination may vary, but some of these cells are always left. The functional test for depletion (abolishment of primary SRC responses) is commonly used (Mosier, 1967), but it implies that the same number of macrophages are required to support primary anti-SRC responses as are needed to help B-cell activation by LPS. This is not necessarily the case, and it could be argued that a few remaining macrophages could efficiently participate in LPS-induced responses but still be incapable of supporting a primary anti-SRC response. To exclude this argument we serially diluted twice-purified, nonadherent, spleen cell populations in a microculture system, in order to increase the possibility of achieving completely macrophage-free cultures. Nevertheless, LPS induced B-cell activation in these cultures that contained as few as 2.5 X lo4cells developed at normal levels. Moreover, the lowest cell density necessary for mounting a response was always recorded in the macrophage-depleted cell suspensions. Other experiments that support consistently negative effects of macrophages in B-cell activation by LPS were performed by reconstituting responding nonadherent populations with normal or LPS-activated macrophages or else with supernatants from adherent cell cultures. In all instances, only suppressive effects could be .demonstrated at any concentration of the adherent cells or of the supernatants. These conclusions apply

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to normal or nude spleen cells, to serum-free or serum-supplemented cultures (Lemke et al., 1975), and also to activation of spleen B cells by other B-cell mitogens, such as dextran, dextran sulfate of high and ) ~ PPD (Gronowicz and Coulow MW (Coutinho et al., 1 9 7 4 ~and tinho, unpublished). The slightly decreased responses observed in some experiments, in particular with dextran sulfate, may be explained by the removal of target B cells displaying increased adherence at some stage of differentiation (Greaves and Hogg, 1971; Julius et al., 1973). Even if it is likely that some B cells are removed by the carbonyl-iron or other adherence techniques, it seems established that LPS-sensitive B cells are not removed. This piece of information (although not unexpected from previous reports) strengthens the point about direct B cell activation by these ligands and makes it highly unlikely that activation proceeds via macrophages or macrophage products. Since the experiments were carried out in serum-free media, B-cell activation via C3 receptors becomes much less likely (Dukor and Hartmann, 1973). Probably the clearest evidence for direct B-cell activation by LPS comes from experiments with adherent cell-depleted, thoracic duct lymphocytes of nude mice. These cell populations respond perfectly to LPS and contain practically 100% of Ig-positive small lymphocytes (Melchers et al., personal communication). Extensive experimental evidence shows that TI antigens are also competent to induce B cells directly in vivo or in vitro (Moller and Michael, 1971; Feldman and Basten, 1971; Wrede et al., 1972; Sela et al,, 1972; Anderson and Blomgren, 1971; Howard et al., 1971b; Miranda, 1972; Diener et al., 1970), whereas other immunogenic molecules (TD antigens) and haptens are not. However, all these different types of ligands are equally recognized by B cells and bind to surface Ig-specific receptors with identical affinity, strongly suggesting that molecular properties other than the antigenic determinants and the capacity to interact with Ig receptors on immunocompetent B cells are responsible for the outcome of that interaction. This was already known to be the case, since haptens or even large proteins became TI when coupled to a TI carrier (Feldman, 1972a; Guercio and Leuchars, 1972; Wrede et al., 1972; Mitchell et al., 1972c; Feldman and Nossal, 1972). Findings of this type had been interpreted on the basis of the polymeric structure of these antigens, which should make it possible for the antigen to induce a suitable pattern of cross-linkage of the Ig receptors on the specific B cells. Nevertheless, it is very difficult to idealize a similar epitope repetition when a small hapten (Feldman, 1972a) or a large

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protein molecule (Feldman and Nossal, 1972) is coupled to the same carrier. However, if the triggering mechanism was completely nonspecific, as suggested by the experiments reported above, and independent of Ig receptors, the activating properties of these molecules should also be expressed on B cells possessing Ig receptors that could never be cross-linked by the antigen, because they were not specific for any determinants present on the antigen molecules. Six different TI antigens (LPS, POL, SIII, levan, dextran, and PVP) were tested for their capacities to activate B cells directly and nonspecifically. All were shown to be competent in inducing T-celldepleted spleen cells to increase division and Ig secretion. It is most important that this activation was shown to be polyclonal, because antibodies of many different specificities were produced after stimulation (Coutinho and Moller, 1973c,d). The critical direct test for this hypothesis was performed. The conclusion is clear: TI antigens can activate B cells directly, not only independently of their antigenic determinants, but also independently of their pattern of presentation to the surface Ig-specific receptors, simply because no Ig specificities are involved. Recently, it has been argued that high levels of mitogenicity, as measured by DNA synthesis, can only be obtained with the particular culture conditions we employed, the activation levels being very low otherwise (Greaves et al., 1974). For a number of reasons, this criticism is very weak. First, our experiments have shown PBA activity of TI antigens mostly on the basis of their capacity to induce polyclonal antibody production, since it is now well established that mitosis (measured by DNA synthesis) and antibody secretion are not necessarily linked (Nilsson et al., 1973; Coutinho et al., 1973a,b; Melchers et al., 1974; Anderson and Melchers, 1974) and ligands are known that selectively induce only one of these patterns of response (Gronowicz and Coutinho, 1974). Therefore, such criticism is pertinent when considering the capacity of some TI antigens to induce cell division, but there is no question about their capacity to activate B cells directly into polyclonal antibody production. Second, even when only mitogenesis is considered, these critics fail to present a serious experimental alternative to our findings. When using our culture conditions, these authors have actually repeated our findings “at the count.” However, by changing two parameters in the culture system the mitogenicity of two TI antigens (SIII and levan) is not comparable to that of LPS. These modifications involve the supplementation of the cultures with FCS, and the use of a tritiated thymidine of lower specific activity (Greaves et al., 1974). The

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claim is that by using a high specific activity thymidine as we did, the responses lose quantitative value (Janossy e t al., 1973a) and " weak" mitogens appear as potent as LPS. However, the real proof for this claim has not been presented, namely the use of a low specific activity radioactive precursor in a serum-free medium! Indeed, as we have repeatedly pointed out, the use of FCS in culture supplementation might obscure completely the outcome of an experiment (Coutinho e t al., 1973a), either by inducing high levels of "background" activation with serum mitogens, which may be actually activating the same cell population as the mitogen to be tested, or else by introducing factors suppressive of cell activation or response. In the experiments we are discussing, this latter point is made very evident by supplementing the cultures with a special batch of FCS that, indeed, suppresses SIII or levan-induced responses but is also highly suppressive for the background DNA synthesis as well as for the LPS-induced responses (Greaves et al., 1974). The argument about the specific activity of the thymidine is actually contradicted by the results presented by these authors. The increased responses to SIII and levan obtained under our culture conditions parallel exactly the increases in the responses to LPS and lipid A, showing that the radioactive precursor is not limiting but simply that our culture systems are actually the .best suited to assay B-cell mitogenicity in vitro. We do not want to argue about a well-demonstrated point (the advantageous use of serum-free cultures) but we just want to add that several of these TI antigens, SIII in particular, have been tested for PBA activity in v i v o with clear-cut positive results (Coutinho and Moller, 1973d). This is probably the basis for its in v i v o adjuvant activity in antibody production (Persson and E. Moller, personal communication; Fellows and Taussig, personal communication).

1 . Cross-Reactivity Two points deserve further discussion. The first concerns the possibility that Ig-specific receptors were actually involved in cell activation, and the results obtained were due to cross-reactivity between all the TI antigens and all the antigens tested in the plaque assay. Some further information concerning this point will be added here. The list of determinants that have been tested with similar results is long (NNP, DNP, NIP, FITC, penicillin, TNP, SRC, HRC, FyG), some of them being completely non-cross-reacting (Coutinho et al., 1974~). Moreover, a very complete list of different red cell antigens has been reported as cross-reacting with highly tolerogenic doses of LPS in v i v o (Rank e t al., 1972). Cross-reactivity can be fur-

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ther excluded on the basis of the following considerations (see also the discussion about the hemolytic plaque assay in Section 111,A). 1. Antibody production induced by B-cell mitogens has been detected against every antigen so far tested in the plaque assay and correlates with total secretion of IgM in activated cultures (Melchers and Andersson, 1973; Andersson et al., 1972e). 2. A large number of cells is activated in culture in order to account for the increased DNA synthesis detected. In our systems, a minimum of 2% to the total number of cells must divide for significant activation to be detected, and large numbers of cells have been shown to divide after mitogenic activation (Janossy et al., 1973c; Shands et al., 1973; Parkhouse et al., 1972). 3. Activation is achieved by all TI antigens so far tested, but only by these (not by serum proteins or by red cell stroma, for instance). 4. Antibody production against these unrelated test antigens is induced by concentrations of the mitogen that are highly tolerogenic for the responses directed against determinants present on its own molecule (Coutinho and Miiller, 1973d; Coutinho et al., 1974a; Andersson et al., 1972e). No specific antibodies are available for crossreaction in the detection assay, 5. High-affinity antibodies directed towards determinants present on the mitogen molecule do not cross-react at all with antigens commonly used to detect polyclonal activation (Coutinho et al., 1974a) as would be expected (Eisen, 1969) if cross-reactivity was involved in polyclonal responses. 6. When the responsive cell population is depleted of cells bearing a certain specificity, no antibodies of this specificity can be detected after stimulation (J. Andersson, 1972). The first three points argue against cross-reactivity at the level of cell induction, and the last three at the level of antibody detection. 2. Are Thymus-Independent Antigens Contaminated by LPS?

The second point to discuss concerns the possible contamination of all these different antigen preparations with bacterial endotoxins. This arguement was discussed at the time when PPD tuberculin was described as a B-cell mitogen (Sultzer, and Nilsson, 1972). The argument, in extreme form, assumes that there is only one possible, “universal” B-cell mitogen (LPS from gram-negative bacteria) that can contaminate all TI but no TD antigens. It is then stressed that bacterial products are likely to be contaminants. However, one of these antigens is not a bacterial product, PVP, and three others are not derived from gram-negative bacteria (SIII, levan, and dextran). A frequent claim concerns the high concentrations of PBA (TI antigen)

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required to induce polyclonal activation. However, as discussed elsewhere (Coutinho et al., 1974a), this should actually be expected and is observed with LPS as well. It does not seem very profitable to discuss LPS contamination of POL or SIII, because they are as effective as, or even more potent than LPS at the same concentration range. Carbohydrate contamination in three different batches of POL used in this study is lower than 0.2% (Ada et al., 1964), which could not explain the effectiveness of POL concentrations ranging from less than 1-500 pg. even if all the carbohydrate were LPS. Furthermore, it has been shown that trypsinization destroys the mitogenicity of POL, but leaves LPS activity unchanged (Schrader, 1974b). The same reasoning about dose-response profile applies to SIII. In addition, this mitogen is derived from gram-positive bacteria and reported to be free of LPS (Howard et al., 1971). Furthermore, the adjuvant activity of SIII is sharply distinct from that of LPS (Persson and E. Moller, personal communication; Fellows and Taussig, personal communication), also indicating that its biological properties are not due to endotoxin. The argument for Contamination apparently becomes somewhat stronger when the other antigens are considered (levan, PVP, and dextran) which are clearly less effective than LPS, POL, or SIII as nonspecific PBA. However, if contaminating endotoxin was the mitogen in these preparations, it should be expected that by increasing the concentrations of the contaminated antigens, the observed responses would also rise to reach levels comparable to those of LPS-induced responses. This is definitely not the case. A clear o p timal concentration is reached above which suppression is observed (Continho, unpublished). The only exception to this rule is dextran (Coutinho et al., 1974c), but a plausible explanation is the fact that, for solubility the maximal concentrations that can be used are only 100-fold higher than minimal activating concentrations (suppression is observed with LPS at concentrations 5000 fold higher than minimal effective concentrations). In the case of dextran, which was extensively studied, several other findings argue strongly against LPS contamination (Coutinho et al., 1974~).First, the patterns of genetically controlled “high” and “low” responsiveness are different for dextran and for LPS in several strains of mice. Second, there is a semilinear relationship between molecular weight and mitogenicity, which shows that this property is a characteristic of the dextran molecule, Finally, by introducing different chemical groups into the dextran molecule, mitogenicity is enhanced to such an extent that LPS contamination can no longer be considered. A final argument against endotoxin contamination applies to all

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mitogens so far studied in detail. As pointed out (Coutinho and Moller, 1973c) and extensively demonstrated recently (Gronowicz and Coutinho, 1974), the relative ability of each mitogen to activate DNA synthesis in spleen B cells does not parallel its relative ability to activate antibody synthesis. This phenomenon is not dose-dependent, and each mitogen can be shown to induce a typical pattern of responses, It seems likely that this is owing to the fact that different mitogens activate different subpopulations of B cells in the spleen (Gronowicz and Coutinho, 1974) and in other lymphoid organs as well (Gronowicz and Coutinho, 1975c), which rules out LPS as responsible for mitogenic activity of these antigen preparations. B. SPECIFICTHYMUS-~NDEPENDENT IMMUNE RESPONSES INDUCED BY POLYCLONAL ACTIVATORSTHE PARADOXICAL REALITY

The discovery of polyclonal B-cell mitogenicity in TI antigens appeared to contrast with the well-known capacity of these ligands to induce specific immune responses. To reconcile all the available evidence concerning specific and polyclonal B-cell induction in the same conceptual scheme, the following reasoning evolved (Fig. 2) (Coutinho and Moller, 1973a,c, 1974; Coutinho et al., 1974b). The basic mechanisms of B-cell triggering are essentially nonspecific. The binding of antigenic determinants to the Ig receptors is irrelevant for the triggering. Direct B-cell induction by TI antigens is due to nonspecific activating properties of these molecules, which are lacking in TD antigens. The function of the specific Ig receptors is passive and consists of concentrating PBA molecules on the membrane of the specific B cells. By means of this focusing function of the Ig surface receptors, B cells that specifically recognize antigenic determinants on the PBA molecules always bind more PBA, compared to nonspecific cells, which are only capable of nonspecific binding. The nonspecific binding of PBA depends exclusively on the PBA concentration in the medium, and all cells have an equal chance to bind. Therefore, the preferential binding of PBA by specific cells occurs at any concentration of the PBA molecules. It follows that, at concentrations of the PBA in the medium too low to reach triggering concentrations on nonspecific cells, the specific B cells are confronted with effective, high “membrane concentrations” of the PBA and are, consequently, triggered by the activating properties of the surface-bound molecules. For the same reason, when PBA concentrations are increased and reach triggering levels on the membrane of every cell, specific B cells are confronted with an excess of

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PBA molecules, and specific paralysis follows. If PBA concentrations are even further increased, nonspecific cells bind too many molecules, and the characteristic polyclonal suppression of the responses by high concentrations of any B-cell mitogen is observed. The specificity of TI immune responses could be explained on the basis of a selective, specific binding, rather than on a specific mechanism of triggering. Since any hypothesis requires that binding and recognition must necessarily precede triggering, the present concept accounts for specificity as adequately as any other. Actually, this concept has had direct experimental support. Thus, a well-known TI antigen and B-cell mitogen (LPS) was found, both in vivo (Rank et al., 1972) and in vitro (Anderson et al., 1972e), to induce specific antibody responses at low concentrations and polyclonal antibody production at high concentrations which were supANTIGEN-SPECIFIC B CELLS

NON-ANTIGEN-SPECIFIC B CELLS

SPECIFIC

ANTIGENIC

ACTIVATION

CONCENTRATIONS

MITOGENIC CONCENTRATIONS

FIG.2. Schematic outline of the proposed hypothesis showing the focusing function of specific Ig receptors. At low (antigenic) concentrations of thymus-independent antigens (+ V +) that display both antigenic determinants (V)and mitogenic properties (arrows), the specific Ig receptors on antigen-sensitive cells preferentially concentrate the mitogenic molecules on their surface. Therefore, the threshold level of mitogen molecules required for triggering is reached only on the membrane of these specific cells. Consequently the response is detected as antigen-specific and thymusindependent. High (mitogenic) concentrations of the same molecules bind nonspecifically to all B cells and induce polyclonal responses, but a t the same time they reach the paralytic threshold on antigen-specific cells, which always bind an additional amount of molecules by the presence of specific Ig receptors.

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FIG. 3. Results of one experiment testing the hypothesis outlined in Fig. 2. Normal mouse spleen cells were cultured in the presence of different concentrations of (4-hydroxy-3,5-dinitrophenyl)acetyl-lipopolysaccharide(NNP-LPS) in serum-free medium. After 2 days the responses induced in these cultures were measured. Activation of DNA synthesis as well as induction of antibody production against an irrelevant antigen [sheep red cells (SRC)] was determined. In addition, high- and low-affinity antibody synthesis against the specific hapten NNP was measured. Background values in unstimulated cultures were subtracted from each experimental group, and the net responses were plotted in a Hewlett-Packard 9820A calculator, using a program for least squares fit. (A)= cpmlculture (Y :O to 84,000); ( 0 )= anti-SRC plaqueforming cells (PFC)/culture (Y :O to 300) (*) = high-avidity anti-NNP PFC/culture (Y: O to 1500);and (0)= low-avidity anti-NNP PFC/culture (Y :O to 6000).

pressive for the specific response. We extended these findings to another TI antigen (SIII) which behaved in vivo in exactly the same way (Coutinho and Moller, 1973d). In order to demonstrate further the relationship between the affinity of binding and antibody produced after nonspecific induction, we used the well-characterized PBA (LPS), and coupled an antigenic determinant to it (the hapten NNP), which we could deal with in determinations of antibody avidity at the single-cell level. We could demonstrate that the same molecule (NNP-LPS) was competent to induce either specific high avidity anti-NNP antibody responses or polyclonal antibody responses in the same cell population, depending on the concentrations that reacted with the responsive cells (Coutinho et al., 1974a)

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(Fig. 3). By determining the affinity distribution of the responsive cell populations for each concentration of the mitogen, it was possible to demonstrate that a gradual change in the dose-response curve took place for cell populations having decreasing affinity for the hapten, until complete nonspecificity was reached. Also, in this way it was possible to show that suitable concentrations of the conjugate induced high-affinity cells exclusively, as would be expected, and as had been found for other TI antigens (J. Anderson, 1972). Therefore, the difficulty in detecting high-affinity antibodies in the in vivo response to these antigens, seems to be due to reasons other than the inability of high-affinity cells to be induced in the absence of T-cell activity. These other reasons may include the following: paralysis of high-affinity cells, readily achieved by a strong mitogen ; of the antigen in vivo (Sela et al., (Mitchell et al., 1 9 7 2 ~ )persistence 1972), causing a persistent paralysis mechanism; the characteristic IgM response induced by these antigens (Britton and Miiller, 1968; Howard et al., 1971a; Miranda, 1972), the lack of high-affinity cells merely reflecting the absence of IgG-secreting cells; difficulty in determining affinity for these antigens. Whatever the reason, the main conclusion is that a PBA is competent to induce directly a specific, high-affinity TI response in B cells. Finally, we could show that the preferential activation of the hapten-specific cells at low conjugate concentrations, due in the light of this concept, to preferential binding of PBA molecules, could be inhibited by inhibiting that preferential binding with free hapten in the cultures. Another interesting finding was that high-affinity NNP-specific cells could be induced by NNP-LPS, even in the presence of high concentrations of free hapten. It is likely that the polymeric conjugate would compete very efficiently with the free hapten for the specific Ig sites on the cell receptors (Hammarstrom, 1973). However, it should be noted that free hapten did have an effect when added to the cultures, because the preferential activation and paralysis of the specific cells reverted to polyclonal levels of activation. It seems logical to conclude, therefore, that the excess of free hapten, compared to mitogen-bound hapten, sufficed to compete for the Ig sites successfully and that these were occupied by free hapten at the time when the B cells were activated by high concentrations of NNP-LPS (Pasanen and Virolainen, 1971). The addition of free hapten did result in the abolishment of the function of the specific Ig receptors, since the specific cells now behaved nonspecifically. However, the absence of function of the specific receptors did not prevent the cell from being activated to the same extent as when

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they possessed functional (nonblocked) receptors. It seems established that free hapten only abolished the focusing function of these receptors and therefore, the specific cells were unable to concentrate the mitogen, but not unable to be activated. One other important conclusion can be drawn from these experiments, concerning the kinetics of mitogen-induced activation. It has been postulated (namely in different two-signal hypotheses) that the second signal is only competent to differentiate B cells to antibody production, but is restricted in its capacity to induce B-cell division and clonal expansion (Britton, 1972a; Hunter and Kettman, 1974; Dutton, 1974; Hunig et al., 1974). The latter ability should be provided by the first, specific signal. Since B-cell mitogens are often considered good second-signal generators (Watson et al., 1973b), the scheme seems attractive in view of the sharp peak of mitogen-induced responses in early periods of culture (Coutinho et al., 1973a; Coutinho and Moller, 1973d) compared to the maximal antigen responses occurring later (Coutinho and Moller, 197313). However, it is clear that an earlier peak should actually be expected to occur in polyclonal responses, as compared to monoclonal responses, and that the differences seem to depend on the number of activated cells (30-50,000 times higher numbers of activated cells by a mitogen), rather than on the quality of the activating stimuli [compare 30-50% of all spleen cells (Shands et al., 1973) with 0.001% of all B cells (Quinths and Lefkovits, 1973)]. When only low numbers of cells (anti-NNP-specific) were activated by the mitogen (NNP-LPS), the peak responses did, indeed, occur at later periods of culture. The limitation to continuous expansion of responses that involve a large fraction of the cell population need not necessarily be due to deficient culture conditions, since the same phenomenon is observed in vivo (Gronowicz and Coutinho, 1974) and may depend on other regulatory factors present in the organism or generated by the extensive cell activation. Once more, the evidence suggests that the immune system is selectively adapted to respond in a clonal mode to nonspecific stimuli and that factors other than the focusing function of the Ig receptors may be responsible for this regulation. These findings were important in order to establish the one nonspecific signal hypothesis for activation of B cells in specific TI responses by a nonspecific mechanism. We cannot think of any good reason why LPS would lose its nonspecific PBA properties when focused on specific antihapten cells. Since we know that mitogen-induced B cells act exactly like B cells involved in specific immune responses (Melchers and Andersson, 1973), it seems plausible that

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the mitogenicity of the molecule is the only factor responsible for the triggering process. All the parallel situations involving molecules of known PBA properties, e.g., hapten-substituted TI antigens and the natural antigenic determinants of these ligands, can now be explained in the same way. Namely, the mechanism operating in the induction of specific anti-DNP responses by DNP-POL (Feldman, 1972a), DNPlevan (Guercio and Leuchars, 1972), DNP-dextran (Wrede et al., 1972), and DNP-SIII (Mitchell et al., 1972c) is the activation of hapten-specific cells by the mitogenic properties of the conjugate, the Ig receptors being passive focusing devices. C. SYNERGISM OF THYMUS-INDEPENDENT ANTIGENS AND POLYCLONAL ACTIVATORS IN INDUCTION OF SPECIFIC RESPONSES IMMUNE Our hypothesis is a minimal model (one signal) based on what we surely know is sufficient stimulation for B-cell induction to occur, namely the interaction with a PBA. We shall now discuss whether it is operative and necessary or whether specific B-cell activation may proceed or always proceeds via other mechanisms. Because TI antigens were capable of direct B-cell activation in specific responses, as were PBA in polyclonal responses, it was important to establish whether the ability of direct B-cell activation displayed by TI antigens was due to an intrinsic property which was independent of antigenic determinants and therefore, of the Ig receptor specificity of the activated cells. If this was so, then these molecules should also be competent to activate nonspecific B cells. However, our finding that TI antigens were, indeed, PBA simply expanded the list of these substances. It excluded specific B-cell triggering involving lg receptor specificities in polyclonal activation, but it did not demonstrate that specific (monoclonal) B-cell activation could be achieved by the same nonspecific mechanism as is operative in polyclonal activation. Thus, it could not be excluded that B-cell mitogenicity and T independence of specific immune responses might be coincidental. Therefore, the next question to be asked should be the reverse of the first one: Can a nonspecific mitogen induce a monoclonal, specific high-affinity response in B cells against antigenic determinants present on the mitogen molecule? The answer to this question would also provide the explanation for the apparent paradox that polyclonal mitogens are capable of inducing specific immune responses. The demonstration that the same molecule (NNP-LPS) could induce polyclonal activation at high con-

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centrations and high-affinity specific responses at low concentrations further strengthens the concept of nonspecific triggering and constitutes experimental support for the “passive focusing function” of specific Ig receptors in the process of B-cell activation (Coutinho et al., 1974a). These experiments, although suggestive, are not conclusive of an entirely nonspecific mechanism of immunocyte triggering for induction of specific responses. We had demonstrated previously that the same cell population can be induced either by the specific or by the nonspecific ligand (Coutinho et al., 1973a, 1974a), showing that the nonspecific signal might be suflicient. We should now demonstrate that it was operative. If this was the case, the specific ligand and the nonspecific PBA would synergize in the process of generating nonspecific signals at surface sites other than Ig. Normal spleen cells were cultured with various concentrations of NNP-LPS, which had been demonstrated to be either optimal or suboptimal for the induction of specific anti-NNP immune responses. Parallel cultures were also given LPS in concentrations suboptimal for polyclonal induction. If the triggering of NNP-specific cells by NNP-LPS was due to the mitogenic properties of the carrier (LPS) and not to the antigenic determinants present on the molecule, then addition of free LPS to the cultures would cause the induction of hapten-specific cells at lower concentrations of the conjugate than the concentrations required for induction in the absence of LPS. This would occur only if the mitogenicity of the free carrier (LPS) added to the mitogenicity of the conjugated carrier on the membrane of specific cells, operating by the same mechanism in the process of triggering. Results shown in Fig. 4 clearly demonstrate that this was the case (Coutinho et al., 1975a). The same type of additive effects would be expected for paralysis induction in specific cells and was also found to be the case (Coutinho et al., 1975a). The complete nonspecificity of the system was further demonstrated by replacing LPS with another PBA, pentosan sulfate, and showing exactly the same additive effects with NNP-LPS both in induction and in paralysis of NNP-specific cells (Coutinho et al., 1975a).

D. NEED FOR FUNCTIONAL MITOGENICITYOF THE ANTIGEN FOR INDUCTION OF THYMUSINDEPENDENT SPECIFIC RESPONSES The foregoing experiments demonstrate that nonspecific signals are operative in specific immune responses. To decide whether nonspecific signals are strictly necessary for induction to occur, experi-

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FIG. 4. Dilutions of (4-hydroxy-3,5-dinitphenyl)acetyl-lipopolysaccharide (NNP-LPS) were incubated with normal spleen cells (B10.5M)alone (x) or in the presence of 0.1 pg./ml. of LPS (A)or of 10 pg. of LPS (0). Two days later, the numbers of high-avidity anti-NNP plaque-forming cells (PFC) were assayed. Values observed in cultures given 0.1 or 10 pg. of LPS alone are also indicated.

ments should be designed in which the mitogenicity of the ligand could be destroyed or prevented from being expressed. Three distinct sets of this type of experiment are available at present. 1 . lnhibition of Specific Responses by Hapten-PBA Substances Previous studies on the molecular basis of B-cell activation suggested that some compounds were capable of interacting with the mitogen receptor but with such a low efficiency that they actually inhibited cell triggering by more efficient ligands (Coutinho, unpublished). Unsubstituted dextrans display PBA properties only above a minimal molecular weight (70,000) (Coutinho et al., 1974~). Low molecular weight fractions can be shown to act as haptens to the mitogen receptor and to inhibit the polyclonal activation induced by high molecular weight dextrans as well as by other PBA, LPS being included to a variable extent. Other examples of these PBA inhibitor substances are polyacrylic acid (Coutinho et al., 197413) and hydrolyzed LPS (Coutinho, unpublished). If the specific antihapten

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TABLE I1 INHIBITIONOF HIGH-AVIDITY HAPTEN-SPECIFICB-CELL RESPONSES BY INHIBITINGTHE “MITOGEN RECEPTOR”” Mitogen

NNP-LPS PHA

-

NNP-LPS PHA

-

NNP-LPS PHA

NNP-LPS NNP-LPS NNP-LPS NNP-LPS NNP-LPS

Inhibitor (mg./ml.)

PFC/culture Experiment 1

23 + 4 1.250-C 133 N.D.

10+7 305 f28 N.D.

10 -t2 368 f50 N.D. Experiment 2 68 +- 17

2.550f215 90 + 15 1.490f59 148 + 28 1.135& 172 60 + O 1.238+ 101 68 -t 20 1.570-C 144

CPM/culture

1.058+ 29 N.D.

+

23.795 4.587 1.800+ 30 N.D.

70.585f7.080 2.688+ 889 N.D.

75.081+ 2.421 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

% Control

100 100 -

24.0 237 -

29.4 249

100 -

56 -

40

-

47 -

61

Normal B10.5M spleen cells were stimulated for 3 days with optimal (Exp. 2)or suboptimal (Exp. 1) concentrations of (4-hydroxy-3,5-dinitrophenyl)acetyl(NNP)Iipopolysaccharide(LPS). To some cultures, two different concentrations of polyacrylic acid(PAAC) and dextran(D) (mol. wt. 5 x lo5 or 7 X lo4)were added at the start of the cultures. Toxic effects of such high concentrations of PAAC were monitored by measuring phytohemagglutinin(PHA) responses in parallel cultures. N.D., not determined; PFC, plaque-forming cells.

responses induced by NNP-LPS were dependent on a nonspecific signal delivered by the LPS molecule, it should be possible to inhibit them with these PBA inhibitor molecules. Results of two experiments in which low molecular weight dextran and polyacrylic acid were used as inhibitors of NNP-LPS-induced monoclonal responses are shown in Table 11. Although antigen presentation to hapten-specific cells was presumably not affected by these substances, NNP-LPS-induced specific responses were inhibited to a large extent. This was not due to deleterious effects of dextran or polyacrylic acid on the cultures since PHA

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responses induced in parallel cultures were normal or even enhanced. Since these same substances could also be shown to inhibit LPS-induced polyclonal responses, the inhibition observed in the specific responses was nonspecific. Furthermore, no cross-reactivity could be demonstrated between NNP and antigenic determinants present on the inhibitory molecules. It is noteworthy that these inhibitors were also competent to abolish TD antibody responses, clearly suggesting that cooperative signals were also operating on B cells by similar nonspecific mechanisms (Coutinho et al., 1974b).

2 . Incompetence of Nonmitogenic Hapten - LPS Conjugates in Induction of Antihapten Specific Responses in Vitro Very elegant evidence demonstrating the nonspecificity of B-cell activation was recently presented by Jacobs and Morrison (1975). By using a TNP-LPS conjugate, they first demonstrated the mitogenicity of this ligand and its ability to induce antihaptenspecific responses at low concentrations thus confirming our previous observations (Coutinho et al., 1974 a). Moreover, these authors have shown that a TNP conjugate of nonmitogenic LPS [treated to remove the lipid A moiety (Anderson et al., 1973)], substituted at the same hapten ratio as the active conjugate, was no longer immunogenic in vitro. Regardless of whether the nonmitogenic conjugate is immunogenic at all in vivo, its lack of in vitro immunogenicity clearly excludes thymus independence and strongly suggests that the mitogenic properties of LPS in the hapten conjugate are responsible and strictly necessary for the triggering of hapten-specific cells. These experiments appear to us as a clear-cut inescapable demonstration of the one nonspecific signal hypothesis.

3. lnability of Geneticully Nonresponder Mice to Mitogen LPS to Mount TI Responses to LPS or to Haptens Coupled to LPS Finally, direct evidence for the strictly necessary role of p l y clonal B-cell activator properties for the induction of TI immune responses comes from studies with genetically nonresponder mice. As Sultzer first pointed out (Sultzer and Nilsson, 1972), C3H/HeJ mice do not respond to LPS as a B-cell mitogen. This unresponsiveness can be shown (Coutinho et al., 197513; Coutinho and Gronowicz, 1975b; Chiller, personal communication; Sultzer, personal communication) to be a defect of B cells in their capacity to recognize LPS as a PBA, and it does not depend on in vivo or in vitro elimination or inactivation of the mitogen by other cell types, nor

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does it depend on the activity of any suppressor cells. Whether these mice lack the subpopulation of B cells that selectively responds to this PBA or whether the LPS-sensitive B cells are present and the defect depends on their capacity to recognize the mitogenic determinants of the ligand are questions requiring further investigation. These mice, however, have normal numbers of Ig-positive cells, as well as of cells bearing surface receptors for complement or IgG Fc. Moreover, in antigen-binding studies, there is no indication of any defect involving the V-gene repertoire or its expression on B cells (Coutinho and Gronowicz, 1975a,b). This appears to be a perfect experimental situation in which to perform a direct test of the one nonspecific signal hypothesis that PBA properties are strictly required for the induction of TI immune responses. The prediction should be that LPS or hapten-LPS conjugates should behave like TD antigens in C3H/HeJ mice, since the mitogenicity of these molecules cannot be expressed, due to the animals’ genetic B-cell defect. The experiments show very clearly (Coutinho et al., 197413; Coutinho and Gronowicz, 1975a) that NNP-LPS conjugates -TI antigens in high-responder mice in vitro - are not immunogenic in vitro for low responders and that concentrations of LPS that are optimally immunogenic in vivo for the induction of TI anti-LPSspecific responses in high-responder mice, are not immunogenic in the nonresponders. However, these mice have no defect in the expression of the antibody receptor repertoire but only in the LPS-induced, direct B-cell triggering, because responses are obtained to the same or related haptens coupled to other carriers, as well as to the LPS determinants themselves, when LPS is conjugated to proteins. In fact, a very good TD antibody response is induced in vivo (Coutinho and Gronowicz, 1975a). These experiments extend earlier observations by Chiller et al. (1974) who have shown that the ability of LPS to interfere with the B-cell-tolerant state to human y-globulin (HGG) in vivo is not observed in these mice and also requires intact mitogenicity. Moreover, these authors have recently shown (Chiller, personal communication) that hydrolyzed LPS (nonmitogenic) is a poor immunogen in vivo and that there is no difference in the responsiveness of high- and low-responder mice, whereas a clear-cut high and low response is obtained when the specific antibody responses are developed to the intact (mitogenic) LPS molecule. Since there is no defect in Ig receptor expression of these mice, antigen presentation hypotheses are clearly excluded in TI responses. On the other hand, since the genetic defect is a well-

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defined defect of B cells and no accessory cells are involved either in the high responder’s induction or in the low responder’s lack of induction, two-signal hypotheses are also excluded. 4. Impossibility of Inducing Tl-Specific Responses with PVP in Very Young Mice Different PBA appear to activate distinct subpopulations of B cells in the peripheral lymphoid organs of mice (Gronowicz and Coutinho, 1974, 1975c; Greaves et al., 1974). The different outcome of activation by each ligand with regard to division, antibody synthesis, or both (Gronowicz and Coutinho, 1974, 1975c; Janossy, personal communication; Lamelin, personal communication) suggested that these subpopulations would include B cells at different stages of differentiation, which was already shown to be the case (Gronowicz et al., 1974a). Precursor B cells (13day-old fetal liver cells) respond readily to dextran sulfate but require 6 days of further differentiation in irradiated hosts to respond to LPS and about 11days to respond to PPD (Gronowicz et al., 1974a). In this type of experiment (Gronowicz and Coutinho, unpublished), it was found that PVP was a very “late” PBA that was only effective 3 weeks after transfer. This observation was confirmed in intact young mice and was in agreement with the finding that PVP was much more effective in inducing polyclonal antibody synthesis than cell division (Coutinho and Moller, 1973~). In light of the one nonspecific signal model, it would be expected that the cell population responding specifically to PVP as a TI antigen should also belong to a rather mature subset of B cells, since these are the only cells that PVP can activate directly (and nonspecifically). Several facts were already available pointing in this direction, namely the overlap of a PVP-sensitive B-cell population and B cells responding to cooperative nonspecific factors, which we consider to be mostly highly differentiated B cells (Gronowicz, Coutinho, and Moller, unpublished). This theory was strengthened by the ease of demonstrating suppressor T-cell activity in anti-PVP responses (Kerbel and Eidinger, 1972; B. Andersson, 1972; Rotter and Trainin, 1974) and the well-defined IgG TI response to this antigen (B. Andersson, 1972). Direct evidence, however, was recently obtained by B. Andersson (personal communication) who showed that in young mice the specific anti-PVP antibody response is TD. These findings cannot be interpreted in terms of the maturation of accessory cell activity. The experiments show that PVP is not competent to direct B-cell activation in young mice, neither with respect to

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specific nor to polyclonal responses, strongly suggesting the requirement for functional PBA properties in TI responses.

E.

NO DETECTABLE SIGNAL FROM INTERACTION OF IG RECEPTORSITE ON RESTINGB CELLS

ANTIGEN

WITH

In our attempts to document the one nonspecific signal hypothesis, we proceeded as follows: (1) it was first demonstrated that an entirely nonspecific signal was sufficient for polyclonal activation of B cells (Coutinho et al., 1973a); ( 2 ) it was shown that all TI antigens display PBA properties and are capable of directly activating B cells by a nonspecific mechanism (Coutinho and Moller, 1973c,d), and therefore, one nonspecific signal might be sufficient for the induction of specific TI responses as it would also be expressed at the level of the specific antigen-binding cells; (3) the direct test of this assumption was the demonstration that the same molecule could induce either polyclonal or specific direct B-cell activation (Coutinho et al., 1974a); ( 4 ) next experimental e'vidence was presented demonstrating that, in TI-specific responses, the nonspecific signal is operative (Coutinho et al., 1975a) and (5) actually strictly necessary for B-cell induction to take place (Coutinho et al., 197413; Coutinho and Gronowicz, 1975a; Jacobs and Morrison, 1974; Chiller, personal communication). It should now be shown that one nonspecific signal is functionally sufficient, i.e., no other (specific) signal is required, for optimal induction of B cells of a certain specificity. It could, indeed, be argued by two-signal model believers that, in TI-specific responses, antigen binding cells do receive signal 1via Ig receptors, and the PBA properties of the antigen simply provide signal 2 which, although operative and required would not be as efficient as in the presence of signal 1. [This argument has not actually been applied to TI responses, since all the phenomenological evidence (synergy of antigen-mitogen) for this type of proposal is bound to be obtained with T D ligands.] Moreover, if PBA-induced PFC that arise in culture were, indeed, the result of signal 2 delivery to cells that had been previously exposed to some level of signal 1by cross-reacting self-determinants, it would be expected that signal 1 was limiting. Therefore, if this hypothesis was correct, higher levels of responsiveness should be obtained by providing the responding cells with optimal levels of signal 1. We designed experiments to test these assumptions directly (Moller et al., 1975; Coutinho et al., 1974b). Since TD proteins or haptens are not competent to induce primary in vitro responses, we have used normal spleen cells throughout these experiments. These

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were very simple. The cells were given the postulated signal 1 by addition of a specific ligand to the cultures. Simultaneously or a few hours later, signal 2 was provided, by giving the cultures LPS. Responses were measured after various times in culture by assaying PFC specific for the signal 1 antigen and for any other unrelated specificity. Two different haptens were used as signal 1 generators-NNP and FITC, either soluble or coupled to HSA or HGG. The conjugates were, in turn, either soluble or precipitated or were cross-linked on the surface of Sepharose beads. The responses were measured after 2 to 5 days in culture with serum-free or in serumsupplemented media. Positive controls were always included, namely soluble or Sepharose-bound hapten-LPS conjugates at antigenic concentrations. The results were invariably the same in all these experiments: (1) none of the haptens or hapten-protein conjugates, in any form of presentation to the cells, were capable of inducing an antibody response, whereas the hapten-PBA conjugates could readily achieve this; and (2)the addition of the specific ligand (signal 1) was not detected to interfere with the PBA-induced levels of PFC of that specificity. Results of one experiment are shown in Table 111. Normal spleen cells were given the postulated signal 1 -the hapten-NNP in concentrations known to bind efficiently to specific Ig receptors (Pasanen and Virolainen, 1971; E. Miiller et al., 1973). Effec-

SIGNALS

TABLE I11 ARE NOT GENERATEDBY REACTION BETWEEN ANTIGEN AND

COMBINING

SITE OF IMhfUNOGLOBULIN RECEPTORS ON SPECIFIC CELLS"

PFC/106 cells PFC specificity

Concentration of NNP-cap in culture: 3 x 10-4 3 x 10-5 3 x 10-8

LPS

-

Anti-SRC

-

High-avidity anti-NNP Low-avidity anti-NNP

-

25 ?2 93 15 27 a 5 175 a29 393 a41 1.613+- 156

+

+

+ -

+

~~

33 f 5 113 40 10 f 10 140 f25 545 f64 1.908f 206

+

33 f 3 110 f 9 20 f 3 175 k21 483 f54 1.823f82

33 +-6 80 -C 14 30 & 4 188 k 15 540 & 46 1.392f 143

~

Normal B10.5M spleen cells were cultured in serum-free medium (lo' cells/ml./ culture) with or without 100 pg. lipopolysaccharide(LPS) after the addition of the indicated concentrations of (4-hydroxy-3,5-dinitrophenyl)acetyl(NNP)-cap.Numbers of plaque-forming cells(PFC) were determined after 2 days against sheep red cells (SRC) and NNP(high and low avidity). "

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tive concentrations of LPS were added to the cultures, thus providing the postulated second signal, and the responses of haptenspecific (high- and low-avidity) and nonspecific cells were measured after 2 days. It is evident that only LPS achieved cell activation. More important, the postulated signal 1 did not interfere in any detactable way with cell activation by the competent ligand. Specific cells became neither activated nor paralyzed after reacting with the hapten, and they responded to the mitogen exactly as did cells that had not bound and recognized the specific ligand. These experiments demonstrate that no detectable signal is generated by the specific recognition of the antigen by the Ig receptor on B cells. Specific interaction does not result in activation nor in paralysis. Moreover, they show that B cells are activated by PBA via surface sites other than Ig-combining sites, since PBA were fully active even when the Ig receptors were occupied by the specific ligand. Since specific, high-affinity TD immune responses can be induced by a PBA molecule that activates both the specific and the nonspecific cells by surface structures other than Ig receptors, and no detectable effects result from the specific recognition of antigenic determinants, we conclude that all the available evidence strongly suggests one nonspecific signal as the mechanism responsible for B-cell induction in TI responses. The biological significance of the proposed mechanism for direct B-cell activation is evident. It is worthwhile to recall the following: (1)the danger to life in any infection is primarily due to the systemic spread of the pathogen; (2) IgM is the first class of antibody to appear in evolution (Clem and Small, 1967) and to be produced upon infection (Svehag and Mandel, 1964); (3)IgM is the only class of antibody that circulates in peripheral blood at higher concentrations than in the tissues (Kaartinen et al., 1973; Vaerman and Heremans, 1970); (4) the clearance of microorganisms from the circulation is to the largest extent due to specific antibody, with or without enhanced phagocytosis, due to antigen-antibody complexes; (5) the spleen is the only secondary lymphoid organ inserted in the bloodstream, and it is the main site for IgM production; and (6) IgM is the prevalent class of antibody secreted by B cells activated by a TI process (Britton and Moller, 1968; Howard et al., 1971a; Miranda, 1972). It would seem that the immune system is especially prepared to react promptly to generalized infections by IgM production induced directly in spleen B cells, whereas local infections would probably be more efficiently controlled by IgG (or IgA) antibody, which displays clear advantages for tissue diffusion and may be produced locally in peripheral lymph

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nodes, mostly by TD B-cell activation. The reasons for the differences in “strength” among the different B-cell mitogens may also be explained in terms of the frequency or importance of aggressions by each bacterial species, if the case for biological relevance of B-cell mitogens were to be stressed. Regardless of the preceding speculations, these differences probably rely on the variable affinity of mitogenic moieties of each substance for the mitogen receptor and on the avidity of the interaction. The latter presumably depends on other properties too, such as multivalency (Hammarstrom, 1973), nonspecific capacity of surface binding to any cell membrane (Anderson et al., 1973; Chiller et al., 1973; Peavy et al., 1973), and net charge (Diamantenstein et al., 1973). Negative or toxic effects that these substances may have on lymphocytes may also play a role. It is worth pointing out that the simple existence of “good” and “bad” immunogens would be in itself another argument to support the concept that B-cell activation is not carried out via immunoglobulin receptors, because it cannot be explained in terms of antibody diversity, in situations where no evidence is available to suggest differences in clone sizes or in the persistence of antigen or macrophage involvement. V. Basis of Thymus Independence (Direct, Specific B-Cell Activation): Competing Concepts

Accepting that TI antigens do induce specific B cells directly, one cannot exclude that at least some of them also activate other cell types, including T cells (Baker et al., 1973; Kerbel and Eidinger, 1972), nor that some or possibly all of them interact with macrophages (Nelson, 1973).We suggest that the effects on other cell types represent trivial or optimizing events on top of the basic, fundamentally effective mechanism of direct B-cell activation. All these non-Bcell effects, as well as the particular molecular (polymeric structure) and biological (undegradability and complement-activation) characteristics of TI antigens have been used as support for different hypotheses explaining the particular immunological behavior of these antigens. We want to stress that all of these properties might be phenomenological side effects of a certain molecular conformation, and that the chemical structure must be delineated in support of a welldefined mechanism of action. Considering these problems, Mitchell (1975) asks the question whether C3 activation, B-cell mitogenicity, and T independence are simply assays for polymeric structure. It is suggestive, indeed, that these three properties may be found

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together in a number of polymeric molecules, suggesting the possible advantage for direct mechanisms of response to dangerous (bacterial) aggressions. Mitchell’s question simply states the facts and does not ascribe any causal relationship to any of the “assays” (nor does it try to segregate fundamental from accessory properties). We are convinced that T independence is, indeed, an assay for PBA activity and that this property is not necessarily dependent on a polymeric structure. Moreover, the PBA properties of TI antigens, which we use as the primary evidence for our model of immune B-cell activation, cannot be considered in these terms and appear to us as the functionally relevant characteristic of these antigens, simply because TI and PBA properties are both expressions of direct B-cell activation, and both phenomena represent the same biological property. THE ASSOCIATIVE RECOGNITIONMODEL As a first step in discussing concepts of thymus independence, we shall analyze that concept which simply refuses to accept the existence of TI antigens (Bretscher and Cohn, 1970; Bretscher, 1972; Cohn, 1972a,b). The extensive experimental evidence for TI behavior of certain antigens (Moller and Michael, 1971; Feldman and Basten, 1971; Wrede et al., 1972; Sela et al., 1972; Andersson and Blomgren, 1971; Howard et al., 1971b; Miranda, 1972; Diener et al., 1970) does not fit this model. However, the argument will always be intellectually legitimate that, for reasons related to the “foreignness” of these antigens, very low levels of associative antibody (produced by residual T cells in every animal) are highly efficient, and it is impossible to detect them by our present methods. Therefore, other arguments may be worth bringing up. It is well known that TI and TD immune responses differ by characteristics other than the moreor-less marked requirement for T cells. Differences include the prevalent class of antibody produced in vivo after immunization (Britton and Moller, 1968; Howard et al., 1971a; Miranda, 1972; Taylor and Wortis, 1968), the establishment of a memory (Miller and Sprent, 1971b); Mitchell et al., 1972a; J. G . Howard, 1972), and the maturation (increase in affinity) of antibody with time after immunization (Britton, 1969a; Gronowicz and Moller, 1972). If some of these differences can be ascribed to special properties of the antigen, such as persistence, the existence of fundamentally different patterns of response cannot be ignored. On the other hand, some TI antigens seem to be completely “ignored” by T cells as if the latter lacked receptors capable of recognition (McDevitt and Landy, 1972; McA.

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Devitt and Benacerraf, 1969). However, when T-cell activity is provided in an artificial way, the response to these very same antigens changes radically, and assumes characteristics of TD immune responses (IgG antibody, memory, etc.) (Ordal and Grumet, 1972). This demonstrates that, at least in this case, the TI characteristics of the response are not due to any other properties of the antigen, but only to the lack of T-cell involvement in the responses (Mitchell et al., 1972b). If the very same mechanism of B-cell induction (associative recognition) was actually operating with different levels of efficiency for the induction of both types of responses, it would be expected that the characteristics of the response are similar. On the other hand, if associative antibody was a necessary requirement for B-cell activation, and it was present at very low levels for TI antigens, it is difficult to understand how this group of antigens can be capable of polyclonal activation (Coutinho and Moller, 1973~).Since it is postulated that associative antibody against PBA also participates in polyclonal activation of a large fraction of all B cells, much higher levels should be expected for TI antigens than for TD antigens, but this is in contrast both to the primary postulate of this hypothesis and to all the experimental evidence concerning true TI responses. Moreover, if associative antibody was operative and produced or released in culture, it would be expected that prolonged culture periods increase the effectiveness of a PBA added later, However, the opposite conclusion has been reached experimentally in different situations (Schrader, 1974a; Persson, Coutinho, and G. Miiller, unpublished). As discussed above, it is actually the PBA side of the TI phenomenon that constitutes the main difficulty with the associative recognition model. Indeed, induction of antibody production by PBA in pure populations of B cells involving no specific signal contradicts the hypothesis that requires two specific signaZs. Claims were first made - again refusing to accept the reality of these findings -that LPS was not competent to activate polyclonal antibody synthesis (Watson et al., 1973a). Shortly afterwards, however, the statement was withdrawn (Watson et al., 197313).Lipopolysaccharide was also reported to be incompetent in activating DNA synthesis in a certain subset of B cells (Peyer’s patches) (Kagnoff et al., 1974), but again others find this observation incorrect (Janossy, personal communication; Gronowicz and Coutinho, 1975~). “Abnormal induction” is now accepted as a fact (Watson et al., 1973b), and it is explained as the delivery of signal 2 to a cell that previously received signal 1 by cross-reactive self-antigens or culture

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GORAN MOLLER

medium antigens, As seen above, newborn and germ-free mice also develop polyclonal antibody responses in serum-free medium (Gronowicz and Coutinho, unpublished; G. Moller, unpublished). Therefore the postulate for a previous signal 1 received by PBA-activated cells must be restricted to self-antigens. In this case, signal 1, because of low affinity, does not result in paralysis but rather in an induced state of responsiveness to a signal 2 delivered later. However, the fundamental point in the explanation of self-tolerance by the associative recognition model was that paralysis was thought to be easier to achieve than induction and to be the only possible outcome of antigen recognition in the absence of associative antibody. The explanation of self-tolerance actually constituted the reason to postulate two signals for B-cell induction. When this fundamental point is now denied, the model loses much of its attractiveness and elegance. Moreover, it was originally postulated that the second signal had to be delivered “to the antigen-sensitive cell undergoing associative recognition via the first signal.” This is in obvious contradiction with the last concept of abnormal induction (Watson et al., 1973b).

B. OTHERTWO-SIGNAL HYPOTHESES All other two-signal hypotheses relate activation to a nonspecific second signal competent to induce B cells that had received the specific signal 1 (Schrader, 1973a,b; Watson et al., 1973a,b; Dukor and Hartmann, 1973; Dukor et al., 1974; Dutton et al., 1971; Schimpl and Wecker, 1972; Britton, 1972a). It is obvious, therefore, that all these hypotheses have the same problems as the associative recognition model in explaining B-cell activation in the absence of the specific signal 1. All the arguments raised above are also valid here. Models of this type accept that TI antigens have an intrinsic capacity to generate signal 2 in the surroundings of the specific B cells which have already received signal 1by antigen recognition. In the presence of these two signals, B-cell activation takes place without the requirement for T cells, which are the second-signal generators in TD responses. Let us consider these proposals in detail only for the sake of phenomenological classification and not for a conceptual argument, since we consider them excluded on the basis of the reasons already stated. 1 . Necessary Involvement of Activated Complement Components in B-Cell Activation This concept of B-cell activation (Dukor and Hartmann, 1973; Dukor et al., 1974) is based on the capacity of most TI antigens to ac-

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tivate complement, and it is postulated that this is the reason for their immunological behavior. The interaction of activated complement with the surface receptor for these products is considered to be a necessary step in B-cell activation. This model has the merit of putting together a number of facts into a working hypothesis (Dukor and Hartmann, 1973), such as the presence on the B-cell surface of a receptor for C3, the capacity of most TI antigens and PBA to activate complement and the fact that nonspecific T-cell factors display the same property. At the time the model was built, many facts that disprove it were still unknown. However, even then some points cast doubt on this model. First, not all TI antigens described are capable of complement activation (Schumann et al., 1974; Dukoret al., 1974). Second, it is, indeed, questionable whether the B-cell complement receptor can ever be considered as a functional receptor in vivo except for its existence as a defined structure on the B-cell surface, involved in decisions that depend on the surface mosaic of the cells (Parrott et al., 1966). Thus, it has been demonstrated (Eden et al., 1973) that low concentrations of fresh mouse serum in vitro completely inhibit the binding of immune complexes to this receptor. It is plausible, therefore, to question whether any interaction can actually occur in the presence of 100% fresh mouse serum, as in the in vivo situation. The critical experiments, however, came later. It was first demonstrated that induction of polyclonal antibody synthesis could take place in serum-free medium (Coutinho et al., 1973a) giving the same or even better responses than in serum (complement)-supplemented medium, The same was found to be true for specific TI antibody responses (Coutinho et al., 1974a)-findings now confirmed in other laboratories (Schrader, 1974c; Feldman and Pepys, 1974). The requirement for serum supplementation considered to be necessary by this model was excluded in all cases involving direct B-cell activation and was well understood in primary, anti-SRC, in vitro responses (Coutinho and Miiller, 1973b) (see Section 111,D). Furthermore, the claim that these responses were due to macrophage synthesis of complement in cultures (Schumann et al., 1974; Dukor et al., 1974) was also excluded for polyclonal and specific responses (Lemke et al., 1975).The presumed failure of LPS to activate certain B-cell subsets (lymph node B cells ) in serum-free medium (Dukor et al., 1974) was also found to be incorrect (Lemke et al., 1975; Gronowicz and Coutinho, 1975~).On the other hand, polyclonal B-cell activation developed at normal levels in the presence of anticomplement antibodies (Janossy et al., 1973b), and the same was

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found for specific TI responses (Feldman and Pepys, 1974). The lack of complement involvement in TI responses was recently extended to in vivo situations (Pepys, 1974). Also Dukor has reported analogous findings (Dukor et al., 1974) and accepts C3 involvement as an auxiliary mechanism which seems to be operating in TD antibody responses (Feldman and Pepys, 1974; Pepys, 1974). The reason for the complement dependence of TD responses might be explained in terms of important binding functions among the cells that cooperate (macrophages, T cells, and B cells), keeping them in close contact and thereby allowing maximal effectiveness of the cooperative factors. Our reasons for questioning a direct triggering role of C3 on B cells involved in TD responses, rested on experiments in which we failed to activate B cells by immune complexes of TD antigenantibody-complement. Moreover, the accessory binding function is logical, since all cooperative cells have surface complement receptors (Bianco et al., 1970; Nussenzweig, 1974; Roitt, personal communication) and it might be of importance to explain the synergism of TD antigen and PBA. 2. T-cell or Macrophage Products As Second-Signal Substances In this section we discuss a rather heterogeneous group of experiments and models that have in common a two-signal background and are not concerned with TI responses. Actually, it is because only non-PBA molecules (TD antigens) are studied in these experiments that the role of accessory cells is given so much importance. Even when PBA are used as second-signal generators, they are claimed to act via macrophage activation for the secretion of the relevant second-signal substances (Schrader, 1973a,b; Watson et al., 197313). All these proposals also have a common experimental basis, namely the phenomenon of synergy of antigen and PBA. We shall discuss this phenomenon in some detail since it has been frequently interpreted a vol d’oiseau (Dennert and Lennox, 1973; Schrader, 1973a,b; Watson et al., 1973b; Coutinho and Moller, 1973b). Evidence from experiments in which soluble nonspecific cooperative factors have been shown to “help” the induction of a primary antibody response to red blood cell antigens will be considered separately. a. Synergy of Antigen and PBA. Two very important points should be stressed. (1) So far, it has not been directly demonstrated that there is one specific signal generated by the combination of the antigen with the specific receptor (see above), and all the evidence put forward is phenomenological and circumstantial. (2) All second-

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signal substances have been shown without exception to be nonspecific PBA which can induce B-cell activation in the absence of antigen, demonstrating that the postulated first signal is not required for induction to occur. The phenomenological evidence simply shows that in particular experimental conditions the presence of the specific ligand “amplifies” the nonspecific PBA-induced responses. Basically, the experiments demonstrate that the simultaneous addition of antigen and PBA to a spleen cell population results in an increased response, as compared to the responses induced by either alone. Since the specific ligand is a TD protein devoid of PBA activity (Schrader, 1973a,b) or else a nonimmunogenic hapten (Watson et al., 1973b), the findings appear to contrast with the postulate that B cells can be activated only via surface sites other than Ig receptors (Coutinho et al., 1974b). However, these findings argue against twosignal theories, as well as against one-signal hypotheses, whether this signal is specific or not. Thus, the two-signal concept implies that each signal is qualitatively different from the other and that each signal alone can neuer result in cell actiuation. However, the findings are that signal 2 is always effective, even in the complete absence of signal 1. This cannot be explained by two-signal theories. Moreover, most of these observations are derived from experiments in which a certain level of signal 2 was always present, provided by the FCS mitogens. Therefore, no conclusions concerning signal 1 can be obtained, However, since these observations do not take this fact into consideration, they cannot either explain the existence of “background” responses that appear in cultures only given signal 1 and FCS. This further demonstrates the intrinsic contradiction between these experimental findings and the two-signal theories. Only one experimental situation was claimed to be an exception to this rule, namely that no antigen-specific PFC could be found when either signal was given alone (Schrader, 1973a,b). However, it is now recognized to be identical to all the others, probably because of an increase in sensitivity of detection methods (Schrader, 1974~). Since none of the present models can actually explain the phenomenon, it appears to us that it is caused by trivial events, none of which involve the generation of any signal at the specific receptor site, Let us first say that this synergy is not a reproducible finding. It has been reported to occur in vitro between fowl y globulin (FyG) and POL (Schrader, 1973a,b) and between soluble haptens or hapten-protein conjugates and LPS (Watson et al., 1973b). However, a specific suppression of the LPS-induced responses, rather than synergy, had been reported to be induced by hapten-HGG con-

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jugates under more controlled culture conditions (Bullock and Andersson, 1973). As discussed in Section IV,E, we have not been able to find any significant interference of specific TD ligands (haptens, hapten-protein conjugates, soluble or cross-linked) with the induced responses in serum-free or serum-supplemented cultures of normal spleen cells (Moller et al., 1975), and a similar lack of synergy was obtained by Waldman, and Munro (personal communication) in FCSsupplemented cultures, using DNP-keyhole limpet hemocyanin (KLH) or DNP-FyG as antigens and LPS or POL as PBA. The only antigen consistently showing synergy in combination with several different PBA is SRC both in serum-free (Coutinho and Moller, 1973b) or in serum-supplemented cultures (Watson et al., 1973b; Kreisler and Moller, 1974; Armerding and Katz, 1974a). However, this phenomenon has been conclusively shown to be strictly dependent on the presence of macrophages [or adherent cells (Kreisler and Moller, 1974)], strongly suggesting that the mechanisms operating do not concern B cells directly. Actually, it is very important to point out that no other experiments have been performed so far in purified populations of B cells. Frequently the responsive cell populations contain T cells and always macrophages. There are, indeed, claims that macrophages are absolutely necessary for the expression of the phenomenon (Schrader, 1973a,b). It appears to us that a number of trivial mechanisms are involved in this type of experiment, the relative importance of each varying from one system to the other. There are several possibilities. 1. Synergy occurs via accessory cells. It has been shown that T cells are required for optimal expression of synergy (Kreisler and Moller, 1974; Armerding and Katz, 1974a) and that adherent cells are necessary for the phenomenon to occur (Kreisler and Moller, 1974), whereas PBA are fully active in the absence of any helper cells (see Section IV,A). Therefore synergy appears to be due to effects of antigen or PBA on helper cells and not on B cells. First, antigen and PBA may be concentrated on macrophage surfaces. If so, specific antigen-sensitive cells, binding the macrophage-bound antigen are exposed at the same time to higher mitogen concentrations. Second, PBA activate macrophages to secrete soluble factors that, in turn, activate B cells nonspecifically, as was demonstrated for POL (Schrader, 1973b). Macrophage-secreted factors were shown to substitute for the PBA itself (Schrader, 197313) as well as for adherent cells ( H o h a n n and Dutton, 1971; Moller and Coutinho, 1975). Again, these factors may be concentrated on antigen-binding B cells

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preferentially, because antigen may bridge macrophages (having cytophilic antibody) and specific B cells. A very similar situation, which actually supports this argument, involves antigens that behave as TI and macrophage-dependent, namely rabbit Fab fragments (Hunter and Munro, 1972) and DNP-D-GL (Mosier, personal communication). These antigens do not activate B cells directly but do induce a TI response, presumably because they are competent to activate macrophages and to bridge them to the specific B cells. Third, when present, T cells may be activated by the antigen, resulting in a similar situation, namely a T-cell secreted PBA to which specific B cells are preferentially exposed. This will be particularly evident when secondary TD responses are studied (Armerding and Katz, 1974a). 2. The antigen binds to the PBA. If so, the conjugate preferentially binds to the specific cells, resulting in preferential activation, even though the Ig receptors do not generate any specific signal. The situation is comparable to that of the hapten-PBA conjugates that induce specific antihapten responses as discussed in Section IV,B. An example is found in a commonly used synergistic system-LPS and SRC. It is well known that LPS binds readily to SRC and this actually constitutes the method for detecting anti-LPS PFC (Moller, 1965). Moreover, it has been shown that LPS coating of SRC makes this antigen TI (Moller et al., 1972a), most likely because SRC-sensitive B cells binding the complex via Ig receptors are exposed to LPS and triggered mitogenically; this is exactly the mechanism we are proposing. The same situation has been described for POL-FyG conjugates (Feldman and Nossal, 1972). In the experiments reported for FyG and POL (Schrader, 1973a,b), as well as for free haptens and LPS (Watson et al., 1973b), this possibility has not been critically excluded and it is actually known to occur to some extent (Watson, personal communication). It is obvious that the same mechanism may be operating when the second signal (PBA) is a cooperative soluble factor. It has been demonstrated that macrophage-produced factors do, indeed, bind to SRC ( H o b a n n and Dutton, 1971) and that these “coated” SRC can induce an antibody response independently of adherent cells. With PBA present in culture, activating macrophages for the secretion of such factors (Schrader, 1973b), it is certain that this mechanism will be highly efficient. 3. The antigen contains a PBA. As discussed in Section III,D, antibody responses to SRC are partially TI, both in vivo (Pantalouris and Flisch, 1972) and in vitro (Feldman e t al., 1972). Moreover, SRC

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lysates are macrophage- (Feldman and Palmer, 1971) and T-cell independent (Byrd et al., 1974) in vitro, suggesting that they have the capacity to activate B cells directly. It is, indeed, possible to demonstrate that concentrated soluble extracts of SRC are strong PBA (Coutinho and Gronowicz, unpublished; Cohn, personal communication) capable of inducing, polyclonal antibody and DNA synthesis. Therefore, the synergy of SRC and PBA may be another example of additive effects between two nonspecific PBA at suboptimal concentrations (see Section IV,C). These effects would obviously be preferentially expressed on SRC-binding B cells. In support of this assumption is the fact that synergy is much more evident at suboptimal concentrations of PBA (Coutinho, unpublished; Watson, personal communication). Evidently the same explanation applies to all experiments in which hapten-coated red cells were used as antigens (Watson et al., 1973b). 4. Antigen bound to specific B cells may amplify mitogenic effects on these cells by several mechanisms. Antigen may redistribute cell-surface components on the antigen-sensitive cells and cause an exposition of mitogen receptors or modify the turnover of these structures. In essence, such a cell would bind more PBA and, therefore, be selectively triggered. Antigen may stabilize the binding of the PBA to the cells. Since most PBA appear to bind weakly to the B-cell surface, it seems possible that antigen bound to a specific B cell may cause a more efficient binding of the mitogen by sterically preventing it from dissociating from the cell. Antigen may displace normal serum suppressors from the cell surface. It has been adequately demonstrated that normal sera of different origins contain serum suppressors that markedly inhibit B-cell activation. These suppressors bind to B cells and suppress their ability to become triggered (Bullock and Miiller, 1972; Bullock and Andersson, 1973). Antigen bound to Ig receptors may possibly displace these serum suppressors from the B-cell surface. Such cells would then be much more readily accessible to activation by B-cell mitogens. This hypothesis is confirmed by experimental findings showing that the synergism between SRC and LPS is increased ten-fold when very low concentrations of mouse serum are added to the cultures (Bullock, unpublished). As noted above, this may constitute one of the mechanisms by which the immune system optimizes specific responses induced by nonspecific triggering signals, in particular via repressors of B-cell activation. Moreover, since these repressors are also present in FCS (Bullock and Andersson, personal communication), it is likely that

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this mechanism constitutes one of the reasons for the findings reported on synergism obtained in FCS-supplemented cultures that cannot be repeated in serum-free systems (cf. Watson et al., 1973b; Bullock and Anderson, 1973; Miiller et al., 1975). The particular circumstances of synergism with haptens must be interpreted with further care, because of the sensitivity of the methods used for detection of hapten-specific PFC (see Section 111,B). Because of the high degree of hapten substitution or because of the well-known fact that many of the specificities available in the B-cell repertoire are able to bind nitrophenyl groups (Schuberg et al., 1968; Jormalainen and Makelii, 1971; Varga et al., 1973), a large excess of PFC can be detected against hapten-coated red cells, as compared to uncoated targets. Therefore, when one stimulates normal spleen cells with a polyclonal mitogen, the detected increase in hapten-"specific" plaques is much greater than the increase detected with uncoated red ceIIs (Coutinho and Mdler, 1973c,d; COUtinho et al., 1973a; Coutinho et al., 1974~). It follows that by simply subtracting numbers of PFC detected with uncoated targets, a hapten-specific response would appear to have been induced. Since no experiments on synergy so far reported have determined the affinity distribution of the PFC detected, it is impossible to ascertain the pertinence of this comment. However, its rationality has been experimentally confirmed. When studying in vitro synergy between LPS and hapten-protein conjugates (Miiller et al., 1975), it was found that, in the complete absence of hapten-specific synergism, increased numbers of PFC could be detected if the hapten coating of the target cells in the assay was considerably increased. These PFC, however, could not be triggered by a suitable immunogen, demonstrating their low affinity and/or nonspecificity. Many of the possibilities listed probably do occur, and a few have been directly demonstrated in experiments. Most of the possibilities are individually adequate to explain synergy, but it is not unlikely that several mechanisms operate at the same time. Therefore, until these trivial explanations are ruled out, it does not appear fruitful to base concepts of immunocyte activation on synergy between mitogen and antigen, Experiments have been reported on synergy in vivo (Chiller and Weigle, 1973; Louis et al., 1973; Schmidtke and Dixon, 1972). In many instances, the use of adjuvants in antibody production merely reflects a pragmatic precursor of these two-signal experiments. It is obvious that the far more complicated in vivo situation makes it difficult to dissect the evidence at the cellular level. However, some of these experiments have been of great value for the un-

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derstanding of some general aspects of synergy, such as the strict requirement for functional B-cell mitogenicity of the second signal (Chiller et al., 1974) and the requirement for non-B cells in the expression of synergy (Allison and Davies, 1971; Dresser and Phillips, 1973; Hamaoka and Katz, 1973). b. T-cell Replacing Factors, Macrophage and Fibroblast Factors. Soluble, nonspecific factors secreted by accessory cells have been found to activate B cells specifically in the presence of the antigen (Dutton et al., 1971; Schimpl and Wecker, 1972; Britton, 1972a; Gorczynski et al., 1973; Watson, 1973). The synthesis of these factors has been currently attributed to T cells specifically or nonspecifically activated (Katz and Benacerraf, 1972; Ordal and Grumet, 1972; Hartmann, 1970; Schimpl and Wecker, 1972; Gorczynski et al., 1973; Britton, 1972a; Dutton et al., 1971; Dennert and Lennox, 1973; Sjoberg et al., 1973; Watson, 1973). However, recently it was claimed that macrophages synthesized the T-cell replacing factors (TRF) (Britton, 1974). In other systems, however, similarly produced TRF has been found to have properties quite different from a macrophagesynthesized soluble factor (Schrader, 1973b). The question does not appear to be solved since it is possible that macrophages are required only for T-cell activation. There are no published experiments that directly demonstrate the capacity of macrophage-depleted T cells to synthesize such factors. The first point to stress here has already been discussed, namely the PBA properties of TRF expressed in the absence of the antigen, As pointed out (Section II1,C) the presence of FCS mitogens in the assay cultures obscures the experimental results. It is easy to demonstrate a polyclonal increase in numbers of PFC (Coutinho et al., 1973a,b) (see Fig. 1) or total Ig production in serum-free cultures (Sjoberg, 1975), whereas this is consistently negative in FCSsupplemented cultures. Also a sensitive PFC assay (with highly hapten-coated target red cells) is frequently required for detecting these low levels of activation. Mitogenicity, as measured by increased DNA synthesis, cannot be detected, even under serum-free conditions, by using crude MLC supernatants. However, after purification of these supernatants, consistent increases of DNA synthesis can also be shown in various subpopulations of B cells, the stimulation indexes often being ten- or twenty-fold greater than background (Coutinho and Gronowicz, unpublished). This indicates that the failure to detect antigen-independent B-cell activation by TRF should be interpreted with care (Askonas et al., 1974). However, even under similar suboptimal conditions, others have found strong antigen-in-

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dependent activation of background PFC (see, e.g., Armerding and Katz, 1974b) which appeared to be related to the degree of purification of the TRF used. It might also be of importance to mention that TRF substitutes for FCS mitogens in the induction of primary antiSRC responses in &To (Moller and Coutinho, 1973) as do other B-cell mitogens (Coutinho and Mijller, 1973b; Kreisler and Moller, 1974). Soluble nonspecific factors secreted by purified, activated macrophages, which are also active in similar antigenic systems (Schrader, 197313; Britton, 1974), have many of the properties attributed to TRF. We have recently analyzed them (Moller and Coutinho, 1975).They include (1)capacity to substitute for adherent cells in primary antiSRC responses, (2) PBA activity as measured by stimulation of DNA or polyclonal antibody synthesis in unprimed spleen cells, and ( 3 ) substitute for FCS mitogens in primary anti-SRC responses. It is important for the establishment of the nonspecific nature of such soluble factors to realize that precisely the same effects can be obtained with supernatants from cultures of growing fibroblasts (Moller and Coutinho, 1975). The nature of the cooperative capacity of TRF has a striking characteristic, namely that in unprimed cells it has only been demonstrated in responses to red cell antigens. [There is actually a report on xenogeneic cooperative TRF in the response to a nonimmunogenic hapten (Dennert and Lennox, 1973). Unfortunately, specificity controls were not included and since the “cooperative responses” were not greater than those that can be obtained with TRF alone, we need not further consider these findings in this discussion,] It has actually been demonstrated that TRF does not cooperate with soluble proteins or conjugates (Feldman and Basten, 1972a; Schrader, 1973b). As we have already seen, red cell antigens display other unique properties that are important for the interpretation of TRF mechanism of action: ( 1 ) red cells are the only antigen that consistently shows synergy with several PBA in serum-free cultures (Coutinho and Moller, 1973b; Kreisler and Moller, 1974); (2) red cells are the “unique” TD antigen in the original MishellDutton system (Mishell and Dutton, 1967) [(the most widespread example of synergy, FCS being the mitogen (Coutinho and Moller, 1973b)l; (3)red cells are partially TI and contain a PBA (see above); ( 4 ) red cells are particulate and, therefore, are likely to activate macrophages; (5) red cells readily bind cooperative factors, both macrophage (Hoffmann and Dutton, 1971) and T-cell produced (Andersson et al., unpublished).

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It is important to analyze the possible reasons for the unique properties of the red cell antigens. For reasons discussed above, we cannot accept the idea that primary anti-SRC responses are actually secondary and that this is the reason for the TRF “amplification” of the response (Schrader, 1973b). T-Cell-replacing factor-coated red cells appear to be the most likely explanation for the operational mechanisms, similarly to LPScoated red cells that also behave as a TI antigen (Moller et al., 1972a). The system might actually involve several steps. (a) Interaction of SRC with macrophages in culture initially activates these cells to secrete nonspecific factors (PBA) that easily adsorb to the red cells and are optimally concentrated on the surface of SRC-binding B cells. This step can be directly by-passed by preincubating SRC with macrophage supernatants. (b) Specific B cells are, therefore, simultaneously exposed to PBA and to the intrinsic activating properties of the red cells (partially TI) and the response is initiated. [It is likely that the intrinsic PBA properties of SRC are not strictly required, but only accessory, since the same results can be obtained with a completely TD molecule- FyG (Schrader, 1973a). The important functional property, therefore, appears to be the capacity to bind macrophage-secreted factors and in this way focus them onto the specific B cells.] (c) T-Cell-replacing factor can now influence a B-cell population that has previously been triggered and is supporting clonal expansion as well as driving the responsive population to a further stage of “reprogramming” for IgM synthesis. The quality of the PBA properties of TRF suggests that it is competent to activate very differentiated B cells into antibody secretion, but little DNA synthesis (see above) (Gronowicz and Coutinho, 1974, 197%). In studying activation of B cells at different stages of differentiation, we showed that B cells exposed to an “early” B-cell mitogen that induces extensive cell division with little antibody synthesis greatly enhances the Ig production by these already activated cells in response to a “late” B-cell mitogen (Gronowicz and Coutinho, 1974, 1975~).It is likely that such sequential activation of SRC-binding B cells by nonspecific PBA takes place in this system, because it has been shown that TRF is most effective when added to the cells 48 hours after initiation of the cultures (Hunig et al., 1974; Askonas et al., 1974). There are no experiments available at present to support critically or exclude the foregoing scheme. However, we have observed a counterpart of this sequentially additive PBA-induced activation-differentiation of B cells when studying polyclonal B-cell activation (Gronowicz and Coutinho, 1974, 1975a). Furthermore, the “coating” of red cells with the PBA cooperative factors makes it easy to understand selective optimal expression at the level of the specific antigen-binding clone,

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without generation of specific signals via the Ig receptors. Moreover, experiments that provide direct support for this concept have been reported. Thus, the major part of clonal expansion in macrophage B-cell mixtures, given red cell antigens, occurs before the addition of TRF, which is delayed for 48 hours (Hunter and Kettman, 1974; Dutton, 1974; Hunig et al., 1974). In addition, if TRF is not provided at all, very few of these dividing cells synthesize antibody. Although suggestive, the evidence provided by these experiments is not conclusive. In one set of experiments with radioactive killing of dividing cells (Dutton, 1974), one of the positive controls necessary to exclude nonspecific effects of DNA precursors did not function. Earlier experiments with radioactive labeling and radioautography of PFC, actually had the opposite result (Sulitzeano et al., 1973). It remains unknown whether the experimental conditions in the original observation selected for unlabeled PFC (Sulitzeano et al., 1973) or whether the positive results were obtained under conditions that selected for labeled PFC (Hunig et al., 1974). The inhibitory effect of the radioactive precursor in the PFC responses further complicates the interpretation of these results. As previously stated, we do not find it fruitful to base various hypotheses of B-cell triggering on experiments of this type. We have tried to indicate that many possible interpretations can be based on the available evidence. It is certainly too much to ask for the repetition of these experiments with true TD proteins, due to the limitations of the experimental systems. However, it is certainly not too much to wish to see the results of similar experiments performed in purified populations of B cells in well-defined culture conditions (serum-free media). We base our belief concerning B-cell triggering on the latter type of experiments.

c. THE PATTERN OF ANTIGEN PRESENTATION The original evidence for the one nonspecific signal hypothesis (Coutinho et al., 1974a), although suggestive, does not conclusively establish an entirely nonspecific mechanism of immunocyte triggering. Let us consider competing arguments raised by theories of antigen presentation. Hypotheses of this type seem to ignore the fact that B cells can be nonspecifically triggered by mitogens. At least, no attempt has been made to explain these facts in terms of the hypotheses. It is clear that mitogen-induced B-cell activation is incompatible with any scheme of antigen presentation, simply because no antigen is involved. Even so, PBA-induced activation of B cells was known at the time some of these models were proposed. The concept of antigen concentration was built to a large extent

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on experiments with TI antigens and on their characteristic polymeric structure. However, their mitogenic properties had been ignored, with one exception-LPS. Polymerized flagellin is an example of this neglect: only very recently was a statement made about its mitogenicity (Schrader, 1973a, 1974c; Nossal et al., 1973). Therefore, it is reasonable to expect a reinterpretation of the results or at least a restatement of the theory. For a believer in antigen presentation, the obvious claim would be that the mechanism actually operating for the induction of hapten-specific cells by low concentrations of hapten-PBA conjugates was the presentation of hapten epitopes in a suitable pattern to the Ig receptors of these cells. The polyclonal activation at higher conjugate concentrations or by TI antigens would probably be labelled as mitogenic and left aside. The fact that specific cells can be activated by the hapten-PBA conjugate even in the presence of high concentrations of free hapten (Coutinho et al., 1974a) would not necessarily disturb the argument, since multipoint presentation of the antigen could still be explained. Thus, it could be argued that a multivalent conjugate would compete efficiently with free monovalent hapten for the occupation of specific Ig receptor sites on the cell surface (Hammarstrom, 1973). This reasoning, however, does not consider the fact that, in the experiments we are discussing, free hapten did have an effect, namely to revert the paralyzed state of specific cells to a normal level of PBAinduced polyclonal response, comparable to that of nonspecific cells. As pointed out above, this could only happen if the free hapten was, indeed, occupying the specific receptor sites at the time the cell was activated by other surface interactions. Polymeric structure is the basis for the concept of B-cell activation induced by a suitable “pattern” of epitope presentation (Feldman, 1972a; Feldman and Nossal, 1972). As pointed out above, this concept seems to involve something other than the requirement for a polymeric structure, since it is stated that some patterns of polymeric structures only tolerize and never induce, whereas others always induce and never tolerize. The concept implies that specific Ig receptors on the B-cell surface generate the triggering signal when they have interacted with the antigenic determinants in a certain polymeric pattern. The available evidence, however, does not support the concept that repeated presentation of epitopes is the basis for TI B-cell activation. Thus, a high density of repeated epitopes on a TD carrier does not lead to TI responses (Aird, 1971; Feldman, 1972a), whereas a low density of epitopes on a TI carrier does (Feldman, 1972a; Guercio and Leuchars, 1972; Wrede et al.,

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1972; Mitchell et al., 1972~).When comparing the abilities of two different hapten-carrier immunogens to induce TI anti-DNP responses, it was found by Feldman (1972a) that DNP,,oo-KLH was entirely TD, where DNP,.,-POL was TI. The molecular weights of KLH and POL were about 1 X lo6 and 4 x lo4 x n ( n being variable but certainly above loo), respectively. The ratios of hapten substitution in these two conjugates, independently of the molecular weight of the molecules, were 1 haptenic group per 555-mol. wt. fraction of the carrier for the TD conjugate, and 1 haptenic group per 57,000-mol. wt. fraction of the carrier for the TI conjugate. In another set of experiments (Guercio and Leuchars, 1972), in which bovine y-globulin (BGG) and levan were compared, both carriers had similar molecular weights (around 150,000and 100,000, respectively). The conjugation ratios were, in this case, 1 DNP group per 3000-mol. wt. fraction of the carrier for the TD conjugate, and 1 DNP group per 20,000-mol wt. fraction of the carrier for the TI conjugate. Although possible differences in the availability of the haptenic groups in the conjugate, as well as different spatial conformations of the carrier molecules, should be kept in mind, these results argue strongly against the concept of repeated epitope presentation as the mechanism for B-cell activation if the fluidity of the cell membrane is considered (Singer and Nicolson, 1972). Rather, they suggest that thymus independence is due to properties inherent in the carrier molecule, independently of the epitope density. Moreover, when large protein molecules are bound to a TI carrier, the response to the protein also becomes TI (FyG-POL conjugates) (Feldman and Nossal, 1972). It is difficult to understand how FyG molecules could be repeated on a POL backbone in a “pattern” similar to small haptenic molecules (DNP-POL). The difficulty with the concept of a pattern of repeated determinants becomes even more evident when corpusculate antigens (SRC or HRC) are coated with a TI antigen (LPS). Also in this case the immune response to the red cell antigens becomes TI (Moller et aZ., 1972a). Now it is definitely impossible to argue for the repetition of SRC epitopes in an inducive pattern on a backbone of LPS. These findings very strongly suggest that TI molecules have the intrinsic ability of directly activating B cells, independently both of epitope Presentation and of the specificity of the activated B cell. In all these examples the presence of a TI molecule bound to the specific cells causes TI activation of these cells. However, since the activating carrier has no specificity for the activated cells, the mechanism of activation has to be nonspecific. We also know (Coutinho and Moller, 1973c) that all TI antigens do express

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these PBA properties in B cells of all specificities and that nonspecifically PBA-activated B cells express the specific phenotype exactly as if they had been specifically activated. Thus, evidence can be found that argues against a pattern of antigenic presentation, but no findings are available so far to disprove the one nonspecific signal concept, Moreover, the latter is simpler (it actually constitutes a minimal model) and is based on well-defined functional properties of TI antigens. These conclusions, in our opinion, rule out all concepts of antigen presentation as the basis for TI B-cell activation. Another basic finding used to establish the pattern of antigen presentation hypothesis has also been recently disproved. Thus, Langman et al. (1974) have shown that the TI behavior of POL and MON does not depend on the pattern of epitopes on the molecule as accepted before (Feldman and Nossal, 1972). Antigenic preparations from some bacterial strains are always TD, regardless of whether they are monomeric (MON) or polymeric (POL). The opposite can be found with other preparations, MON as well as POL behaving as T I antigens. These findings directly exclude repetition of epitopes as the basis for TI. Other experiments that argue against this concept were presented in Section IV,C and D. Thus, it was shown that a nonspecific signal was operating in TI-specific responses and that this signal was strictly necessary. Trinitrophenyl conjugates of hydrolyzed (nonmitogenic) LPS are not TI antigens, whereas TNP-mitogenic LPS conjugates are, although the degree of hapten conjugation and pattern of epitopes is similar in both cases (Jacobs and Morrison, 1975). Hapten-LPS conjugates also do not induce TI-specific responses in mice that do not respond to LPS as a mitogen. However, the same conjugates (the same pattern of determinants) are TI antigens in mice that respond to LPS as a mitogen (Coutinho et al., 1974b; Coutinho and Gronowicz, 1975a). The same LPS nonresponder mice respond normally to conjugates of the same haptens on other carriers. In spite of all the available evidence that excludes this concept, some recent experiments have still been interpreted along the line of pattern of antigen presentation (Greaves et al., 1974). Hapten-KLH conjugates are not competent to induce a primary antihapten in vitro response. However, they are immunogenic and TI when bound (cross-linked) to the surface of Sepharose beads. As suggested by the authors themselves, this set of experiments provides little evidence for the pattern of presentation concept. Thus, KLH has been described as an antigen that behaves as TI at sufficiently high concentrations and, therefore, can immunize B cells directly (Torrigiani, 1972). At low concentrations, T-cell activity provides further help,

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presumably because the clones of spedific B cells are not optimally stimulated by the intrinsic PBA properties of KLH alone. The direct capacity to activate B cells is further suggested by the ease of demonstrating suppressor T-cell activity in these responses (Kerbel and Eidinger, 1971) in analogy with other TI antigens (Baker et al., 1970; Kerbel and Eidinger, 1972). Moreover, as Osborne and Katz (1973) recently demonstrated, KLH is mitogenic for unprimed spleen cells. High concentrations of KLH were shown to induce DNA synthesis in unprimed cells. The increments above background values were as high as when primed cells were used and secondary responses studied. We have actually repeated these experiments (Gronowicz and Coutinho, unpublished) and confirmed the PBA properties of KLH, which were also expressed by a marked induction of polyclonal antibody synthesis. This PBA activity could be shown to be at least partially independent of endotoxin contamination, which was already suggested in the Osborne and Katz (1973) experiments by the lack of activity displayed by low molecular weight fractions of KLH. It is clear that the cross-linkage of KLH on Sepharose beads only provided the specific cells with a very high local concentration of the antigen. Under these conditions, it would be expected to be mitogenic and therefore TI, as in Torrigiani’s experiments (1972). These observations appear to argue against a presentation mechanism rather than to support it. Thus, since KLH is a very large molecule (more than lo6 mol. wt.), it is difficult to see how the crosslinkage of a hapten conjugate on very big particles could affect the pattern of hapten presentation to the surface of cells that are orders of magnitude smaller than the particle. Similar experiments were performed with a hapten directly coupled to polyacrylamide beads (Feldman, personal communication). In this case the critical point to exclude seems to be the properties of the unconjugated “carrier” beads which appear to be competent to activate complement and to induce polyclonal antibody secretion nonspecifically in unprimed B cells, although to a limited extent (Greaves, personal communication). However, these experiments are preliminary. Experiments of this type raise a general question, namely how “strong” the activating properties of a ligand expressed at the polyclonal level on non-antigen-specific binding cells should be in order to consider it important in the physiological induction of TI responses (Greaves et al., 1974). As discussed below the immune system appears to be prepared in a number of ways to respond specifically to nonspecific triggering mechanisms. W e actually consider thymus-independence to be the best test for nonspecific €3-cell

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activating properties of a ligand, since it is the physiological expression of direct B-cell activation. Although we believe that with sensitive enough techniques it is always possible to demonstrate PBA properties in any TI molecule expressed at the nonspecific cell level, it is possible theoretically that some ligands are such weak PBA that their intrinsic mitogenicity can only be expressed when they are very firmly bound to the cell surface. This situation is physiologically achieved via specific, Ig-mediated binding: high affinity of interaction and high density of receptor sites on the specific cells. Therefore, it is conceivable that TI antigens that do not express PBA properties on nonspecific cells could exist. For all these reasons it does not appear profitable to strengthen arguments about how "weak" a PBA is, since these nonspecific properties are certainly preferentially expressed on specific binding cells. The experiments with unsolubilized antigens are also defective in the sense that little or no information is available concerning the pattern of presentation of the particle-bound materials. A better approach appears to be the use of antigens with very well-defined structures (such as synthetic polypeptides). In this case, the available evidence strongly argues against an epitope presentation concept. Thus, it was found by Sela et al. (Mozes and Shearer, 1971; Shearer et al., 1972; Sela et al., 1972) that several different polypeptides, all of them composed of well-known repeating antigenic determinants behaved quite differently in the induction of specific immune responses. Some of these compounds were TI, but others with similar characteristics in that respect were TD, as opposed to what would be expected if B cells were activated by the pattern of antigen presentation. These experiments elegantly confirm the available evidence and show that high epitope densities on TD (non-PBA) carriers does not confer to the conjugate the capacity to activate B cells directly (Aird, 1971; Feldman, 1972a; Guercio and Leuchars, 1972; B. Rubin et at., 1973; Klauss and Humphrey, 1974). As discussed in Section IV,E, we have failed to repeat experiments of this type with unsolubilized antigens, using true TD proteins or haptens bound to Sepharose beads (Moller et al., 1975).

D. POSITIVEAND NEGATIVE VIEWS ON THE CATABOLIC BEHAVIOROF ANTIGEN 1 . Slow Catabolism and TI Responses A concept introduced by Sela suggests that some immunogens are TI because they cannot be metabolized and, therefore, are persistent

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and stable in the organism (Sela et al., 1972). Like Cohn (1972b), we classify this suggestion as a statement of “fact” in the absence of a conceptual framework, because it does not advance any mechanism of immune induction. However, unlike Cohn, we do not label it “misleading” because it may actually provide the basis for some further insight into the problem. The idea is derived from a series of experiments in which many different synthetic polypeptides of welldefined structure were used as antigens (Mozes and Shearer, 1971; Shearer et al., 1972; Sela et al., 1972). A clear-cut correlation was found between TI and the D-amino acid composition of the polypeptides. Since these compounds are known to be slowly and incompletely metabolized, this was taken as evidence for a causal relationship (Sela et al., 1972). This reasoning is not justified, since we might be dealing with coincidental properties of the same molecules, and there is (1) no evidence to support the assumption directly nor (2) an outline of the possible mechanism of B-cell activation into which these findings would fit. However, since some of the observations seem to argue against the mechanism of B-cell activation that we are proposing, it is worth discussing them. Thus, it was reported (Sela et al., 1972) that the same antigen (Phe-G)-Pro-L induced antibody responses that were TI to the Pro-L part of the molecules but T D to the (Phe-G) moiety. This contrasts with the postulate that B cells participating in TI responses are activated by nonspecific properties of the ligand that is bound to the cell surface via Ig receptors. Indeed, the (Phe-G)binding B cells would presumably also be exposed to the PBA properties of the Pro-L moiety. However, it is not known whether B cells binding the TD moiety are confronted with the TI (PBA) part of the molecules. Thus, the conjugate could be rapidly split upon injection and the (Phe-G)-binding B cells would never have the chance to bind enough PBA for activation to occur. Actually the same experiments provide evidence for this assumption, When the whole macromolecule is not significantly digested, the presence of a TI moiety does induce a TI response to another region of the molecule known to be T D antigen otherwise (Sela et al., 1972). Furthermore, another mechanism of responsiveness might be involved, namely the capacity of the TD moiety to be recognized (and activate) T cells. The antibody responses in these experiments were very low (one to three log 2 dilutions above control in hemagglutination assays), and only total antibody titers were measured without indication of classes. Thymus dependence or independence was ascertained by the percent of irradiated mice responding in a bone

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marrow (with and without thymocytes) transfer. Therefore, it is possible that the higher percent of responders in the presence of thymocytes was due to a TD response (presumably of IgG class) superimposed on a basic TI IgM response. A very similar situation was described for (T-G) A-L in responder mice, -I which thymectomy abolished the IgG antibody response, leavii the ZgM at control levels. The antibody titers, therefore, were harply reduced after thymectomy (Mitchell et al., 1972b). Sela’s concept is incompatible with the T independence and T dependence of different antigens when they irre compared in vitro. Furthermore, it cannot explain why rapidly catabolized antigens cannot immunize B cells directly, but are still persistent enough to ( 1 ) immunize T cells and (2) assist in T-B ce!l. cooperation. Actually for any model of TD B-cell activation, antigsn has to be present during the entire process (either to produce specific signal 1 or else to bridge the cooperating cells). It must be further postulated that TI B-cell activation takes a longer time than the TD counterpart, but this is known to be incorrect. An explanation should also be given for the fact that during the long period of persistence the molecules never activate T cells. The concept, therefore, appears inconsistcnt, and the slow metabolism of TI antigens, D-aminO acid po1ypepti:le.s in particular, might simply reflect another side of a characteristic common to these substances-all are very “foreign” (different from self) to the animal. The slow catabolism simply indicates the lack of enzymatic specificities to recognize and split these molecules and does not appear to be relevant to the mechanisms operating in B-cell activation. However, it should be kept in mind that the persistence of antigen may be the basis for some characteristics of TI responses, such as antibody class (Howard et al., 1971a; Miranda, 1972), absence of memory (J. G. Howard, 1972), fluctuation in numbers of splenic PFC (Britton and Miiller, 1968), and lack of maturation (Britton, 1969a).

2. Mitchell’s Hypothesis Sela’s concept (Sela et al., 1972) implies that the function of helper cells in the response to TD antigens is somehow to stabilize the antigen or to prevent it from being degraded rapidly. This is exactly opposite to Mitchell’s proposal for the function of helper cells and the mechanism of cell cooperation (Mitchell et al., 1972c; Mitchell, 1975). This hypothesis is derived from results primarily (exclusively?) concerned with IgG TD responses. Therefore, it is to some extent outside the scope of this review. Although IgG antibody

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responses that appear to be TI have been described (B. Andersson, 1972; Sela et al., 1972; Mosier et al., 1974), the prevalent finding of exclusively IgM responses in both specific and polyclonal direct (non-T-cell-mediated) activation of B cells cannot be ignored. We agree that all IgG sponses are basically TD, whereas all IgM production is basic; ty TI (Mitchell, 1975). Furthermore, we are prepared to accept ti. it induction of IgG precursors is controlled by signals that might be, if not radically different, at least obeying distinct rules as compared to the signals that operate in IgM precursors. Apart from the strict ‘thymus dependence, there is very little if any direct experimental evidence that can be used in the delineation of any detailed model fdr IgG induction. This is presumably the result of the failure to obtaiih consistently i n vitro, primary, IgG antibody responses, as well as’ of the fact that B-cell activation by PBA does not result in IgG secretion. It might, of course, be argued that the failure to obtain IgG secretion by mitogen activation of B cells simply results from the fact that all the PBA so far studied in this respect [PWM, LPS, and insolubilized Con A (Parkhouse et al., 1972; Andersson et al., 1972; Andersson and Melchers, 1973)] activate a subpopulation of B cells that contain the IgM but no? the IgG precursors. We have recently developed this argument coHcerning B-cell subpopulations (Gronowicz et al., 1974a). The possibility was advanced that some TI antigens, such as PVP and Ficoll, which appear to induce specific TI IgG responses and certainly activate a subpopulation of B cells different from the LPS-sensitive population, would be likely candidates for polyclonal inducers of IgG secretion. It is attractive to apply all the evidence concerning IgM induction to IgG (namely a one nonspecific signal concept) by simply postulating that the IgG precursor subpopulation of B cells is selectively activated by cooperative T-cell nonspecific factors that are far more effective in this respect than any other PBA. However, we have found that primed spleen cell populations are readily activated to IgG secretion by LPS (Kreisler and Mdler, 1974), an argument against that assumption. These findings could lead to the idea that PBA do turn on IgG precursors directly, but they are not present on unprinied spleen cell populations. Polyclonal IgG induction i n viuo by lipid A (Kolb et al., 1974), recirculation of memory B cells (Strober, 1972), and the appearance in the spleen of IgG-producing cells that migrated from the lymph nodes upon immunization (Weissman et al., 1973), all support this suggestion. In this case, the reason why TI responses are exclusively IgM in nature would have

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to be interpreted in terms of paralysis of the specific IgG precursors induced by the TI ligands. This is exactly Mitchell’s proposal, and we did find that IgGproducing cells in primed spleens were, indeed, ten- to a hundredfold more sensitive to LPS than IgM-secreting cells, both with regard to induction and to paralysis (Kreisler and Moller, 1974). If the PBA is firmly bound to the specific cell surface via Ig receptors, that operational difference in sensitivity is certainly greatly enhanced, and even further so if the avidity of interaction with IgG receptors is much higher than with IgM receptors (Mitchell et al., 1972~).It should be kept in mind that all the evidence produced in these experiments (Mitchell et al., 1972c) can be interpreted in terms of the phenomenon recently described as “effector cell blockade” (Schrader and Nossal, 1974). On the other hand, experiments in which T-cell activity is artificially provided in a TI response, resulting in IgG production (Ordal and Grumet, 1972; Klauss and McMichael, 1974; Coutinho and Moller, unpublished), are more easily interpreted by attributing the absence of IgG in TI responses to the lack of the T-cell derived PBA that selectively activate those precursors. However, it is possible to accept Mitchell’s idea that T-cell activity releases IgG precursors from paralysis and that this mechanism is normally absent in TI responses because these antigens lack the ability to be recognized and to activate T cells. This postulate is more difficult to accept on basis of ( I ) the T-cell activity demonstrated (as suppressor effects on IgM production) in anti4111 responses without resulting in IgG synthesis (Baker et al., 1970,1973); (2)the fact that most TI antigens appear to activate macrophages that are postulated to be the effector cells in the release from paralysis, upon activation by antigen stimulated T cells (Mitchell et al., 1972~); ( 3 )the strict time requirements for providing extraneous T-cell activity, in order to get high effectivity (Ordal and Grumet, 1972; Katz and Benacerraf, 1972). Release from paralysis induced by T I ligand by excess T-cell factors is not in agreement with a quantitative concept of B-cell activation. If both TI antigen and cooperative factors are considered to be PBA and the B cells are, indeed paralyzed, no release should be expected to occur in the light of a one nonspecific signal hypothesis. This appears to be the case of IgM producers and emphasizes that it is not safe to extrapolate results or concepts from IgM to IgG induction and paralysis. According to recent observations (Janossy, personal communication), LPS does activate intracellular synthesis of IgG2 in resting splenic and lymph node B cells. Around 20% of all LPS-ac-

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tivated cells appear to be IgG2 synthesizing cells. However, this Ig is not exported from the cells and it is impossible to detect it in the supernatant fluids, in contrast to IgM. Although this observation demonstrates that IgG precursors can be triggered nonspecifically, it suggests that either this mechanism is not physiological and the LPS activation is abortive, or else more than one signal is involved in inducing IgG precursors to the complete expression of the activated phenotype. Mitchell’s hypothesis derived from experiments involving IgG production was also applied to IgM synthesis. We certainly cannot agree with these postulates. All the evidence presented throughout this review against the involvement of Ig surface receptors in B-cell activation of IgM responses applies to the one specific signal theory. Mitchell’s concept actually contains an internal conceptual contradiction. Thus, IgM precursors interacting with multivalent TI antigens are postulated to be induced rather than paralyzed because, for reasons of receptor affinity, receptor redistribution by dissociation of the ligand-critical for triggering to occur-takes place in the absence of T cell-macrophage activity. It should be expected that TD nonmultivalent ligands would more easily dissociate permitting receptor redistribution and, therefore, should require less T-cell help than TI ligands. If the mechanism of T cell-macrophage cooperation for IgM induction were to clear the antigen-binding B-cell surface, this requirement should be much greater for large multivalent TI molecules. VI. Molecular Basis of B-Cell Activation

We believe that lymphocyte activation is a cell-surface phenomenonal though the opposite has been claimed by several authors (Diamantenstein et al., 1973; Vogt et al., 1973; Adler et al., 1972). Our assumption is based primarily on experiments with insolubilized ligands and also on current knowledge of cell activation and membrane function (Greaves and Janossy, 1972a; Greaves and Bauminger, 1972; Andersson et al., 1972a; Andersson and Melchers, 1973; Levine et al., 1973; Oka and Topper, 1971; Moller et al., 1975).

A. INVOLVEMENT OF THE SPECIFICRECEPTOR-COMBINING SITE: THE ANTIGEN PRESENTATION CONCEPT Since the immune response is specific, it follows that the combining site of the surface Ig receptor of immunocompetent B cells must be involved in triggering. However, involvement does not nec-

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essarily mean direct participation in the triggering even, although this has been taken for granted in all “specific” hypotheses of B-cell activation (see Section 11). The generation of a triggering signal b y the combination of the specific receptor site with the antigen has been accepted without discussion, except in very critical reviews (Singer, 1974). However, the direct evidence for the involvement of Ig receptors in the process of triggering is scarce or nonexistent. Binding of haptens or TD antigens to Ig receptors neither seems to induce any alteration in the resting state of the specific B cell (Watson et al., 197313; Schrader, 1973a; Moller et al., 1975) nor to interfere in any detectable way with the triggering by a competent ligand that acts via other surface structures. Phenomenological evidence reported in support of one-signal generation by the recognition of an antigenic determinant (Watson et al., 197313) has not been confirmed in a number of other experiments and laboratories (Bullock and Andersson, 1973; Waldman and Munro, personal communication; Dutton, personal communication; Moller et al., 1975) (see Section V,B for discussion of synergy of antigen-mitogen). On the other hand, studies of antigen-antibody interactions in solution, showing an alteration in the conformation of the Ig molecule in regions other than the combining site, are few and contradictory (Metzger, 1970; Callahan et al., 1973). Even if such a change occurred, it could be irrelevant to the generation of any triggering signal. The current view is actually strongly suggestive that no antigen-induced alterations occur in the Fc region of the antibody molecules (Metzger, 1974a,b) and that the dynamic equilibrium between hapten and antibody involves a single step (Pecht, 1974). In terms of membrane physiology, it is not definitely established whether Ig receptors are peripheral or integral proteins of the lymphocyte plasma membrane (Wernet et al., 1973; Singer, 1974). This question is important, since it is generally accepted that the properties and interactions of peripheral proteins do not seem to be relevant to membrane structure and function (Singer and Nicolson, 1972) and that only integral proteins seem to participate in thermodynamically relevant lipid-protein interactions in the membrane (Singer and Nicolson, 1972). However, this classification is based on operational definitions, and the question concerning Ig receptors is far from settled. Even i f l g receptors were integral B-cell membrane proteins, all the available evidence is against the involvement of the speci$c receptor site in the generation of a triggering signal, thus denying all two-signal hypotheses. On a highly speculative basis it is

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possible to argue that if Ig receptor-combining sites were involved in triggering, this should be mediated by modifications induced in the Fc piece of the molecule by antigen binding; but this is not found experimentally (Metzger, 1974a,b; Pecht, 1974). On the other hand, it is accepted that the evolution of immunglobulins proceeded mainly by selective pressures for different effector mechanisms mediated by the Fc fragment (Marchalonis, 1972). It would seem unlikely that one of the effector mechanisms would be cell triggering. Furthermore, different Fc pieces would possibly imply distinct membrane mechanisms of signal generation for different B cells or for successive stages in the life history of the same cells. Experiments involving stimulation of cells by anti-Ig sera are not relevant in this connection, because most likely these sera do not contain antibodies direct against the combining site of the Ig receptor but rather against other parts of the molecules. They will be discussed below in the general context of cross-linkage. All “specific” hypotheses also accept that the postulated first signal is not enough to trigger the cell. This necessarily complicates an understanding of the activation events. However, the original antigen presentation concept postulates that the specific signal is enough if generated in a sufficient number of receptors at the same time (Moller, 1970~).Therefore, multipoint binding may have an accessory stabilizing function in the triggering process, exclusively due to signals generated at the specific sites. Multipoint presentation, however, is always necessary, because the affinity of the reaction event at repeated single receptor sites is too low for induction (Moller, 1970~).Other concepts derived from antigen presentation theories, such as the pattern of determinant presentation, commonly make use of the cross-linking concept which is considered in Section V1,C. It is not clear in these models, whether the authors postulate generation of “microsignals” at the receptor-combining site or “macrosignals” that are also generated at the level of the membrane by multiple Ig receptors interacting with the antigenic structure, or else, whether both mechanisms are supposed to operate (Bell, 1975). It is clear, however, that only the cross-linkage of Ig receptors in a suitable pattern is postulated to be competent to trigger the cell.

B. SECONDMESSENGERSAND TWO-SIGNAL HYPOTHESES In terms of two-signal hypotheses, new difficulties continually arise in defining a mechanism for the generation of the second signal and for the interaction and cooperation of both signals. The problem

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becomes more complicated when distinct end results are ascribed to each signal, such as paralysis for signal 1, no effect for signal 2, and induction for signals 1 plus 2. Recently an attempt was made to identify the intracellular pathways of paralysis and activation by using two most fashionable ) ~ this deserves some “second messengers” (Watson et al., 1 9 7 3 ~and comments. The idea is not new and has been proposed before to explain triggering of lymphocytes by mitogens or polyvalent ligands (Greaves and Janossy, 1972a; Braun, 1972; Hadden et al., 1972). In the present interpretation, signal 1, generated by the combination of the antigen with the specific cell receptors is postulated to result in increased activity of the adenyl cyclase system. We assume, as implicit postulates, that antigen binding induces some modification in the membrane-bound piece of the Ig receptor molecule and that, in addition, the adenyl cyclase system is closely related to the Ig receptors in the membrane and is sensitive to such postulated changes in conformation. The first assumption has been shown to be wrong. The second one has not been studied so far. Two important points deserve further comments. First, the mechanism proposed to operate in lymphocytes, namely the opposite effects of cyclic adenosine 5‘-monophosphate (CAMP)and cyclic guanosine 5’-monophosphate (cGMP), where cAMP displays negative effects in induction, is not at all unique for antigen-sensitive cells, as stated, and, therefore, cannot reveal a differentiative pathway unique to these cells, as claimed (Watson et al., 1973c; Estensen et al., 1973; Editorial, 1973). Second, it is not at all evident how increased cAMP levels can trigger the intracellular paralytic pathway. Does it result in an active synthesis of repressors or in an increased efficiency of the existing repressors that must be inactivated for the cell to be activated to the expression of the activated phenotype [the number of messenger RNA’s (mRNA’s) in the activated B cell is several orders of magnitude higher than in the resting B cell (Melchers et al., 1974; Jerne, 1967)]? Are these postulated CAMP-induced negative effects the new molecules characteristic of the paralyzed state of the cell or is the phenomenon explained on a purely quantitative basis? These alternatives appear difficult to correlate with recent results (Melchers et al., 1974), suggesting that the synthesis of surface Ig receptors by resting (Go)B cells is actinomycin D-sensitive and that the cell may not need more than one RNA actively engaged in H p-chain synthesis. Is it the balance between the intracellular concentrations of cAMP/cGMP that regulates activation versus paralysis? If so, cGMP generated by signal 2 could alone be expected to

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be effective in suitable concentrations in the complete absence of cAMP generated by signal 1. This possibility has not been excluded, and results obtained from mitogen activation of lymphocytes (Hadden et al., 1972) suggest that this is actually the case. Even in the case of new molecular species induced by the generation of increased concentrations of CAMP, or of a mechanism operating in the “paralytic pathway” induced by increased concentrations of cAMP (different from the inductive pathway), which could provide support for an active state of tolerance, it was accepted that an increase of cGMP can reverse this state on a simple quantitative basis (Watson et al., 1973~). Therefore, the only possible conclusion is that signal 2 alone is the only mechanism capable of induction. In conclusion, this interpretation fails to support a two-signal concept, and the experimental findings provide good evidence and a chemical intracellular mediator for hypothesized lymphocyte activation resulting from one nonspecific signal. Furthermore, no evidence is provided to show that the intracellular levels of CAMP are increased after antigen binding and, therefore, the experiments fail to support the concept that antigen recognition has a negative effect in cell activation. Thus, the postulate that no signal is generated by the combination of the antigen with the specific sites on the Ig surface receptors can be maintained, since there is no critical evidence from any experiments to support the idea that a signal is generated because of antigen recognition by the specific combining site of the Ig surface receptor on B cells. However, there is much support for the contrary view. More recently, experimental evidence was presented against cyclic nucleotide involvement in the B-cell triggering processes (Greaves et al., 1974). Namely, cAMP was found to be suppressive of both T- and B-cell proliferative responses to mitogens, but CAMP-induced suppression could not be reversed by cGMP in contrast to the above observations. All procedures that raised intracellular concentrations of cGMP were incompetent to initiate or to amplify a B-cell response, thus denying the above observations (Watson et al., 1973~). These contradictory findings cannot be explained at the moment. However, a last point of caution should be raised, namely that even if the demonstration of cyclic nucleotide activity in B-cell triggering turns out to be correct, there is no evidence of a physiological role for these messengers in this particular respect. B-Cell activation with the necessary involvement of C3 receptors on the cell surface (Dukor and Hartmann, 1973) is another model that proposes a detailed mechanism. The above discussion on this hy-

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pothesis and the direct experimental evidence which disproves it need not be further developed. C. INVOLVEMENT OF THE IMMUNOGLOBULIN SURFACE RECEPTOR INDEPENDENTLY OF ITS SPECIFIC SITE: THE CROSS-LINKING CONCEPT From all the evidence of B-cell activation by nonspecific ligands, it is safe to conclude that triggering is entirely independent of the specificities of the Ig receptor-combining sites (see Section IV,A above), Let us now discuss the possibility that the surface Ig receptors are actually involved in the generation of triggering signals although not at the specific combining site. Indeed, the possibility exists that triggering signals do not originate at the single receptor level but rather at the bilayer level by interacting Ig receptors with multivalent ligands thus disturbing their free mobility and the fluidity of the membrane (Singer, 1974). This brings the discussion to the cross-linking concept. The polymeric structure of antigens known to be capable of direct B-cell activation (TI) and the idea of antigen presentation in general, as well as the fact that the first B-cell mitogens described were also polymeric molecules, probably constitute the origin of the widespread concept of cross-linking (see Greaves and Janossy, 1972a). Experiments in which insolubilized selective T-cell mitogens were shown to activate B cells (Greaves and Bauminger, 1972; Andersson et al., 1972a; Andersson and Melchers, 1973) further supported the idea and brought to light the basically different requirements for activation in T and B cells. In its simplest (pure?) form this concept assumes that B-cell activation is the result of a suitable degree of crosslinking of any (or some) surface structures. Probably because of its highly dynamic and, therefore, attractive character, the “fluid mosaic’’ composition of the cell membrane (Singer and Nicolson, 1972)as applied to the lymphocyte (Moller, 1961; Taylor et al., 1971; Unanue et al., 1972; Loor et al., 1972), is frequently used in support of the cross-linkage concept. [It is worth noting that this type of description currently ignores the basic distinction between integral and peripheral membrane proteins, each of which can move equally well (Singer and Nicolson, 1972; Karnovsky et al., 1972).] The mechanism by which cross-linking of the relevant structures results in cell activation has been explained in various terms. The simplest approach would be that restriction in mobility or “freezing” of a critical area of the cell surface, disturbing the physiological fluidity, at any site on the membrane would trigger membrane-bound mechanisms to initi-

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ate the process of activation. This idea, however, is not acceptable because of the failure to activate B cells by cross-linking of several different surface structures. Therefore, cross-linkage of some but not of every surface structure may lead to activation. Furthermore, aggregation of intramembranous particles (IMP) has been shown to parallel inhibition of cell proliferation, rather than cell triggering, and no redistribution of IMP was observed in dividing cells (Scott et al., 1973; Marchesi et al., 1972). Several other possible alternatives have been discussed (see Greaves and Janossy, 1972a; Singer, 1974), such as modification of receptor turnover and conformational changes induced in crosslinked receptors (Wilson and Feldman, 1972) with or without redistribution, alteration of the mobility of the bilayer (Berlin et al., 1974), and interference with critical transport mechanisms across the membrane (Singer 1974). However, it should be kept in mind that changes in the cell membrane mosaic, even if they reflect a functional reactive modification, may occur without triggering cell division and “reprograming” (Tsanev, 1973). The fact that “capping,” which is an induced active energy-dependent phenomenon and, therefore, constitutes a cellular response, has been shown in a number of situations not to be correlated with cell activation supports this reservation. Furthermore, it was recently found that redistribution of surface proteins in lymphocytes is not parallelled by redistribution of IMP (Wofsy, 1974), which makes the capping phenomenon lose much of its attraction. This constitutes one of the reasons to reject a recent model for lymphocyte triggering in which results of T-cell activation were interpreted in terms of alterations of this type induced in B cells (Edelman et al., 1973). The basic differences in the process of activation between T and B cells (Greaves and Janossy, 1972a; Andersson et al., 1972a) and the lack of reproducibility in the results used in support of the model (Wofsy, 1974; Raff and DePetris, 1974) are other reasons for that critical attitude. In cases of T-cell activation by Con A, cross-linkage seems to be responsible for paralysis rather than for triggering. Thus, divalent Con A is as active as the tetramer for activation but is much less competent to turn off the responses at high concentrations (Edelman et al., 1973; Trowbridge, 1973). The next question at this point concerns the plausibility of the concept as applied to lymphocytes, namely; Is there any correlation between redistribution and capping of surface components and cell activation? The answer is clearly negative. The following are some examples concerning different types of membrane structures:

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1. Surface structures, such as Con A receptors, known to be involved in triggering, Thus, Con A caps surface receptors on both T and B cells but only T cells are activated, and optimal and supraoptimal Con A concentrations induce capping to a comparable extent (Anderson et al., 1972a,c; G. Moller et al., 1973). Furthermore, only a fraction of Con A-responsive T cells show receptor redistribution in the presence of optimal concentrations of the mitogen. Other lectins are known (Hammarstrom et al., 1973) which bind and cap on the lymphocyte membrane but never activate the cell. 2. Integral membrane proteins, such as histocompatibility antigens (Singer and Nicolson, 1972; Wernet et al., 1973). Redistribution and “capping” of these structures by antisera does not result in cell triggering (Ceppellini et al., 1971; Thorsby et al., 1973). 3. Immunoglobulin surface receptors. Redistribution of Ig receptors on mouse B cells by antisera does not lead to activation (Katz and Unanue, 1972; Greaves and Janossy, 1972a; Elson et al., 1973; Greaves et al., 1974). In other species the reported results are somewhat more variable. Clear stimulation of lymphoid cells by anti-Ig sera has been shown essentially in rabbits (Sell and Asofsky, 1968; Sell, 1970; Fanger et al., 1970) and chickens (Skamene and Ivanyi, 1969; Alm and Peterson, 1969; Weber, 1973a,b).Very low levels of stimulation have also been reported in some experiments with human and guinea pig lymphocytes (Greaves et al., 1969; Greaves, 1970; Oppenheim et al., 1969; Adinolfi et al., 1967; Frgjland and Natvig, 1971, 1973; Daguillard et al., 1969). In none of these situations were the responsive cells shown to be B cells. It is an established fact that in species where B cells are well characterized, no such response has ever been detected. Actually, the available evidence shows that, at least in the well-known stimulation of rabbit peripheral blood lymphocytes by antiallotypic sera (Sell, 1970), the responsive cell is a T lymphocyte (Sell and Sheppard, 1973). Rabbit peripheral blood lymphocytes contain more than 80% of Ig-positive cells by direct fluorescence methods (Pernis et al., 1970) or electron microscopy (Linthicum and Sell, 1974). However, only 20% of the cells in this population are complement-receptor lymphocytes (Elfenbein et al., 1973), markers for B cells (Nussenzweig, 1974). Moreover, the surface Ig is distributed in patches and does not appear to be more than 3000-12,000 molecules per cell, in contrast to the characteristics of well-identified mouse B cells (Raff, 1970). These cells are clearly distinct in ultrastructural studies from the rabbit spleen Ig-positive cells (Sell and Sheppard, 1973), the latter presumably being B cells. However, the most important findings suggesting that

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these lymphocytes are actually T cells are the fact that more than 80% of the cells in these populations show blast transformation after 18 hours in culture with soluble Con A-a selective T-cell mitogen (Sell and Sheppard, 1973; Greaves and Janossy, 1972a; Anderson et al., 1972~).These Con A-induced blasts are Ig-positive. Moreover, these cells do not show specific product formation upon activation, a necessary criterion to classify them as B cells (Sell, personal communication). The assumption that cells responding to anti-Ig sera are indeed T cells is supported by the fact that these cells respond readily to antibodies directed to cell-surface components, such as antilymphocyte sera (Greaves and Janossy, 1972a; Janossy and Greaves, 1971). The particular behavior of anti-Ig in the rabbit and chicken might be explained in these terms. If the T-cell surface Ig is cytophilic, bursectomy in the chicken should be expected to abolish it as if the responsive cells were B cells (Skamene and Ivanyi, 1969; Weber, 1973a). Therefore, there is no direct evidence to support the assumption that B cells are activated b y redistribution of surface I g receptors. The low levels of stimulation reported in other species are presumably due to contamination of the anti-Ig sera by antilymphocyte antibodies that activate T cells as a rule. This is certainly known to occur in the mouse with impure anti-Ig sera (Greaves, personal communication). It is important to stress that anti-Ig sera do not activate B cells even after they have been cross-linked and presented on the surface of Sepharose beads (Greaves, personal communication). Some conclusions concerning hypotheses of B-cell activation can be made from these observations. Sometimes the concept of crosslinking is used as a synonym of multipoint binding. This may be misleading, and we prefer to separate the two concepts. When triggering signals are considered to be independently generated at individual sites on the membrane, multipoint binding, though essential, only increases the efficiency (avidity) of the interaction as postulated by antigen concentration theories (Miiller, 1970~). The concept of crosslinking would imply (Greaves and Janossy, 1972a; Bell, 1975) that signals are also (or exclusively) generated at the membrane level, because several receptors are simultaneously interacting. The difference between these concepts becomes more evident when each type of postulated signal is placed in different categories, such as microsignals and macrosignals (Bell, 1975). The hypothesis of pattern of determinant presentation (Feldman, 1972a; Feldman and Nossal, 1972) appears to require both types of signals or else it

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ascribes unique properties to Ig receptors as membrane proteins. Thus, it postulates that the cross-linkage of I g receptors (and only of these structures) in a suitable pattern leads to cell actiuation. We have already presented evidence against the participation of the specific binding site in the Ig receptor in the triggering process, thus excluding antigen presentation and two-signal hypotheses. The failure to activate B cells by redistribution and cross-linkage of surface Ig receptors certainly argues strongly against hypotheses of determinant presentation that cannot explain B-cell activation via other surface sites (PBA induced), New cross-linking concepts in which activation of B cells can take place both by determinant presentation to Ig receptors (in TI responses) and by cross-linkage of nonspecific sites (in PBA activation) (Greaves et al., 1974) also find that failure to be a strong argument against the involvement of Ig surface receptors in triggering, even when considered to be independent of the specific binding site. Recent experiments (Andersson et al., 1974a) cast some doubt on the above assumption by introducing evidence for the nonspecific involvement of surface Ig receptors in LPS-induced B-cell activation. These authors had previously found that as soon as 15 minutes after adding LPS to resting B-cell cultures, the surface Ig appeared to be clustered in large aggregates (Melchers and Andersson, 1973). These experiments, however, did not distinguish between a general disturbance of the B-cell surface after interaction with the mitogen, common to all membrane proteins, and a more specific phenomenon restricted to the Ig receptors. Moreover, the results were obtained by calculating the size of aggregates obtained after precipitation with specific antisera, but direct fluorescence (Greaves et al., 1974) did not confirm them. In a new type of experiments the influence of antiIg pretreatment of B cells on LPS activation was assessed. Elson et al. (1973) had already worked out a similar system and showed that anti-Ig sera-induced redistribution of surface Ig on B cells did not interfere with LPS activation, as measured by increased DNA synthesis. Andersson et al. (1974a) confirmed these observations but found that activation of antibody production, as measured by numbers of polyclonal PFC detected in these cultures, was consistently depressed as compared to untreated cultures. Antisera with specificities for other surface components did not exhibit this effect. How ever, it is difficult if not impossible, to achieve quantitatively similar amounts of bound antibody when other specificities are used, due to the number of Ig molecules present on the B-cell surface. These experiments are open to two interpretations, that is, interference

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20 1

with the process of activation or with the expression of Ig secretion. Although the antiserum was present only before activation, it was certainly internalized, and this process has recently been shown to result in a similar “blockade” for secretion of the specific Ig after incubation with antigen (Klaus and Humphrey, 1974; Schrader and Nossal, 1974). If the inhibition operates at the induction level, the question is why cell division is not inhibited. Two alternatives are possible, namely that two different subpopulations of B cells are activated by LPS, one selectively responding by division and the other exclusively by antibody production. This explanation finds some support in recent studies on B-cell subpopulations responsive to PBA (Gronowicz and Coutinho, 1974, 197%; Gronowicz et al., 1974a). If this is the case, and since all the LPS-activated B cells are Ig-positive at the surface (G. Moller, 1974; Gronowicz et al., 1974a) in the dividing subpopulation, activation occurs completely independently of Ig receptors but not in the Ig-secreting population. This would support the assumption that the inhibition operates by blockade of the expression of Ig secretion. The alternative explanation would be that the same B cell can be induced to divide or to secrete IgM independently and via distinct surface phenomena and/or sites. Although the first statement is known to be correct (Nilsson et al., 1973; Coutinho et al., 1973a; Melchers et al., 1974, Andersson and Melchers, 1974), there is no evidence for the second one, and for obvious reasons discussed previously (Gronowicz and Coutinho, 1974) it does not appear likely. After the extensive list of negative findings presented above relating activation with cross-linking, it seems reasonable to ask whether there is any direct evidence for the cross-linking concept, namely whether the cross-linkage of any surface structure might lead to cell activation. An entirely different picture emerges from the work with B-cell activation by mitogens, strongly supporting the cross-linking concept, but firmly excluding Ig surface receptors from the list of possible candidates responsible for the generation of triggering signals after cross-linkage, because of its polyclonal characteristic. Although the molecular weight of PBA is usually relatively low, most of them are polymers made up of repeated identical units, more or less complicated in structure (LPS, POL, SIII, levan, PVP, dextran) (Coutinho and Miiller, 1973~).Furthermore, the requirement for a certain molecular weight has been demonstrated both in TI-specific responses (Andersson, 1969; Miranda, 1972) and in polyclonal activation (Coutinho et al., 1974~).Although the repeated units may be very different from one mitogen to another (compare,

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e.g., POL with levan or dextran) both in structure and in size, such findings support the idea that several identical moieties in the mitogen molecule must simultaneously react with the relevant surface receptor (and cross-link them) for activation to occur. B-Cell activation by locally concentrated lectins (Greaves and Bauminger, 1972; Andersson et al., 1972a; Andersson and Melchers, 1973) further supports the concept.

D. LACKOF

REQUIREMENT FOR POLYMERIC

MOLECULESIN

B-CELLACTIVATION:REINTERPRETATIONOF THE

CROSS-LINKING CONCEPT MULTIPOINT BINDING

IN THE

LIGHTOF

From the evidence presented so far it is reasonable to conclude that cross-linking of some B-cell surface structures (e.g., PBA receptors) but not of all structures (e.g., Con A receptors, H-2 antigens, Ig receptors) results in cell activation. However, it appears to us that the cross-linking concept may be reinterpreted in terms of multipoint binding. Available evidence strengthens this interpretation. The lipid A molecule of LPS is a glucolipidic nonpolymeric structure that is very efficient in B-cell activation (Andersson et al., 1973; Chiller et ul,, 1973; Peavy et ul., 1973). Although this example is not entirely satisfactory, because the molecule is highly hydrophobic and presumably always occurs in aggregates, this finding led to the assumption that B-cell activation could proceed independently of any receptors (Andersson et al., 1973). The insertion of the fatty acid side chains in the bilayer would disturb its mobility enough for triggering to occur. Our own findings (Coutinho et al. 1974c) as well as those of others (Diamantstein et al., 1973; Vogt et al., 1973) with sulfate-substituted polysaccharides demonstrate that cross-linkage of any surface receptor does not seem to be an essential, initial requirement for B-cell activation. Sulfate derivatives of pentose or glucose with less than a dozen of units proved to be efficient mitogens (Coutinho et al., 1974c; Diamantstein et al., 1973). Furthermore, these compounds are strongly hydrophilic and activation is observed in macrophagedepleted B-cell populations as well (Coutinho et al., 1974~).It may, of course, be argued that these molecules are still competent to cross-link some (at least two) surface structures, which would initiate activation. However, it seems more likely that these ligands interact with high affinity with individual receptor sites. These findings exclude that an extensive cross-linkage is initially required for triggering, regardless of the fact that changes may be expected to spread eventually to larger areas on the membrane at later phases of the process.

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The requirement for polymeric structures in B-cell activation may, therefore, be interpreted in terms of accessory “helper” effects of multipoint binding leading to increased avidity of the very low-affinity interaction between the competent receptor on the B-cell surface and the complementary groups on the PBA molecules. Ligands capable of high-affinity interaction with these “PBA receptors, would not require multipoint interaction and could therefore be nonpolymeric molecules [pentosan or dextran sulfates (Coutinho et al., 1974c; Diamantstein et al., 1973)l. It may be advantageous that the reaction between triggering receptors on the B-cell membrane and competent ligands is of low affinity as a rule, in order to prevent sterile nonsense activation of B-cell clones. Therefore, efficient interaction and triggering can only be achieved in t w o major ways: either by high concentrations of polymeric molecules (usually demonstrated in vitro by polyclonal activation) or else (at physiologic antigen concentrations in vivo) by stabilizing the interaction by an accessory high-affinity binding. The latter is the situation observed during the induction of specific immune responses by TI antigens, where the specific recognition of antigenic determinants by Ig receptors with a comparatively much higher affinity provides a strong stable binding of the mitogen to the surface of specific B cells. If this hypothesis is correct and multipoint binding is an accessory mechanism for causing an increase in the avidity of the reaction rather than a strict requirement for cross-linkage, it can be predicted that when a strong stable interaction results from the binding of PBA molecules by the Ig receptor, the requirements for a specific molecular weight would be less strict. It has been confirmed experimentally that the expression of PBA activity as measured by polyclonal activation requires a higher molecular weight than the expression of PBA activity as measured by specific activation [W. Richter, personal communication; Coutinho et al., 1974c (for dextran); Andersson, 1969; Coutinho, unpublished (for PVP)]. In other words, the same substance can be shown to activate cells that are capable of additional (Ig receptor-mediated) binding, although the substance is still incapable of activating nonspecific cells because of low molecular weight. The activation of the latter cells can be obtained by the same substance but requires not only higher concentration but also higher molecular weight. Due to the variety of different molecules that are known to activate B cells (different PBA) the postulate of a PBA receptor might appear irrelevant without further definition of how many different PBA receptors there are (if more than one) and what they have in common, The finding that each PBA seems to activate a distinct

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subset of B cells (Gronowicz and Coutinho, 1974, 1975c) further complicates the problem. A striking characteristic common to most PBA is their polysaccharide structure, which would indicate a receptor for saccharide molecules as the surface site relevant for activation of B cells, Thus, with few exceptions (e.g., POL, PWM, and PVP), all PBA are sugar polymers, and, since very low-affinity interactions are involved, it seems possible that groups with similar stereochemical configurations may be found among these exceptions. In the dextran system that we have used (Coutinho et al., 1 9 7 4 ~ ) ~ well-defined substitutions of this very simple molecule were studied with respect to their capacity to enhance or confer PBA activity to the poorly mitogenic unsubstituted dextran. It was found that molecular weight was only important when a very low affinity of the interaction with the PBA receptor was to be expressed, as in the unsubstituted dextran fractions. Furthermore, lipid substitution did not increase mitogenicity, whereas sulfate or phosphate (Coutinho, unpublished) substitution transformed dextran into a very potent mitogen. This was not due to anionic properties of the molecules, because other negatively charged groups (carboxymethyl) did not have this capacity. Therefore, it appears that the PBA receptor interaction with dextran derivatives shows a very high affinity for sulfated or phosphated glucosyl residues. As we have pointed out (Cou) ~ is a striking structural similarity between tinho et al., 1 9 7 4 ~there lipid A-the mitogenic moiety in LPS-and these compounds [lipid A contains phosphated glucosamine residues and fatty acids (Andersson et al., 1973)l. Since these dextran derivatives contain no lipid at all, it appears that the active group in the lipid A moiety is the phosphated glucoseamine rather than the fatty acids. They could display accessory functions of binding to the hydrophobic bilayer. Other molecules of similar structure - hyaloronic acid and chondroitin sulfate -have recently been tested with positive results (Coutinho and Gronowicz, unpublished). The question remains open whether only one or several structures on the B-cell surface are competent to deliver triggering signals. It is easy to see from the foregoing assumptions that, although the operating mechanism of triggering is nonspecific, the immune system seems to be prepared to respond specifically. It is worth recalling the well-known fact, discussed below, that TI B-cell tolerance can never be induced by subtriggering concentrations of the antigen. In this context it is easy to understand why B-cell induction always precedes tolerance, because TI B-cell responses are elicited by potentially dangerous products (bacterial antigens) the defense

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against which depends mostly on an efficient antibody response. The mechanism of direct B-cell activation ensures a prompt and efficient response, but the low affinity of interaction between PBA receptors and PBA sites prevents sterile activation of nonspecific cells. The specific recognition (Ig-mediated) enables the relevant clones to respond efficiently at low concentrations of the antigens. When the characteristics of the pathogen (eg., virus) make antibody responses ineffective or self-aggressive (e.g., lymphocytic choriomengitis) no such direct mechanism of direct B-cell activation should be expected. It is well known that B-cell-deficient patients die from bacterial infections, whereas T-cell deficiency leads to frequent viral infections (Rosen, 1971), and genetic control of antiviral reactivity seems to be expressed at the T-cell level (McDevitt and Landy, 1972). It is possible that other naturally occurring devices, characteristic of TI antigens, are also operating to facilitate a direct efficient response in the B-cell compartment. An example of this may be the lipid moiety in LPS and the etherification of the sugar residues in other bacterial polysaccharides. A more efficient binding to the cell surface seems to occur in both cases, one involving gram-negative and the other grampositive bacterial products. The last question concerns the possible intracellular mediators or signal molecules that must carry the triggering message from the cell membrane to the nucleus. As accepted above, it seems likely that one single pathway is involved and that “second messengers” are likely to operate, possibly in the way proposed in the Yin-Yang model (Editorial, 1973). A monodirectional system could be effective as well, since it is known that this is the case for certain hormones that do not induce changes in the cAMP content of the cell, but stimulate exclusively cGMP levels (Illiano et al., 1973). It seems difficult for any of these alternatives to account for the suppressive effects of overstimulation induced by the same ligand that promotes activation. Possibly this phenomenon is dependent on the exhaustion of receptors for the competent ligand with consequent failure to generate the intracellular messenger, as described in other situations (Franklin and Foster, 1973). However, very little evidence actually supports the concept that CAMP,cGMP, or both are second messengers in lymphocytes, and there is no evidence to indicate that they are generated by any ligand competent to induce B-cell activation. Furthermore, other systems involving clonal proliferation and differentiation by altered transcription, as well as B-cell activation, are known to be mediated by ligands that do not operate via a cAMP message.

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We would like to finish this part of the discussion by describing a phenomenon that does not fit the above assumption, namely B-cell activation by locally concentrated lectins (Greaves and Bauminger, 1972; Andersson et al., 1972a; Andersson and Melchers, 1973). These findings are clearly in support of a cross-linking concept of B-cell activation, because the soluble lectin binds very firmly to the B-cell surface (a more stable binding than any PBA) (G. Moller et al., 1973) but is incapable of triggering the cell. Several other interpretations are still possible, none of which supports a cross-linking concept, but they are not confirmed by experimental evidence and are highly speculative, It is difficult to evaluate the possible role of soluble mitogen “leaking” from the insoluble matrix. In one set of experiments with Con A (Andersson et at., 1972a), it was claimed that T cells were completely unresponsive to the cross-linked lectin and this was used as an argument for the absence of soluble Con A in the system. Actually, soluble Con A was shown to inhibit B-cell activation by the insolubilized form (Andersson et al., 1972a). However, it was later found that insolubilized Con A actually leaked at a rate amounting sometimes to 10% of the insolubilized material per day (G. Moller et al., 1973). In the other system (PHA) (Greaves and Bauminger, 1972), reasonable T-cell stimulation was consistently obtained by the cross-linked lectin. On the other hand, it has been reported that B cells may be triggered by soluble Con A in the presence of humoral factors released by activated T cells (Andersson et al., 1972b). Such mechanisms could operate under optimal conditions when both cells are firmly bound to a large surface (the bead or the bottom of the petri dish). It should also be shown that the “inert” surfaces, used for supporting the matrix of lectin, are not responsible for activation, the lectin providing nothing more than stable binding to the surface. A similar effect of the lectin could be due to the “polymerization” of polysaccharides leaking from the beads by the soluble or by the insolubilized lectin, activation resulting from a “two-layer” matrix. As shown in Table 11, addition of a ligand with very low affinity for the B-cell triggering receptor to spleen cell cultures stimulated by PHA, greatly increases the level of response. One last possibility could result from the immobilization of the lectin molecules on the matrix, exposing a suitable group for B-cell activation, which cannot be expressed in soluble form because binding to the cell membrane by higher affinity sites prevents this possibility. The latter would imply that the site on the lectin molecule responsible for T-cell activation is not the same as for B-cell activation. This should be considered, because the PBA receptor postulated above seems to be a

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receptor for saccharide structures. This would account for a welldefined symmetry in triggering receptors and ligands, between T and B cells, because all T-cell mitogenic lectins are sugar-binding proteins. Possible implications for the mechanism of T and B cell cooperation are evident in this assumption: one cell (B) is activated via a receptor for sugar, the other (T) via a sugar-containing receptor. More explicitly, the same molecule that functions as a triggering receptor on T cells would be capable of interacting with the relevant B-cell receptor and could function as the T-cell-derived PBA in cell cooperation. Actually there are molecules that appear ( 1 ) to be glycoproteins and (2) to display both of these functions (McDevitt et al., 1974; Katz, personal communication), namely, the Ia antigens. VII. B-Cell Paralysis in Thymus-Independent Responses

Interaction between antigen and antigen-sensitive cells may or may not result in changes in the functional state of the cell. If a response is induced, it may be activation, resulting in division and/or reprogramming for a different pattern of phenotypic expression, or paralysis, resulting in a state of unresponsiveness to a competent stimulus, Although activation may also result in the establishment of cellular unresponsiveness to further stimulation (Gronowicz and Coutinho, 1974), the activated cell is actively engaged in division and/or Ig secretion, whereas the paralyzed cell apparently maintains the resting unproductive state. Paralysis to TI antigens has certain distinctive features, as compared to paralysis to TD antigens, and is better understood. The main reason for this is that paralysis can be induced to TI antigens in vitro (Coutinho et al., 1974a; Diener and Feldman, 1972; Kotlarski et al., 1973; Britton, 1969b), whereas this is so far not possible with TD antigens (Dresser and Mitchison, 1968; Weigle et al., 1972; Weigle, 1973). Therefore, all experimental systems used to study induction of tolerance to TD antigens at the immunocompetent B-cell level are inadequate. Reasons for care in interpreting results concerning in vivo systems at the cellular level will be discussed below. It is enough to keep in mind that the failure to induce tolerance to TD antigens in vitro parallels the failure to activate in vitro with the same antigens (Coutinho and Miiller, 1973~). The situation concerning TI antigens is different both for paralysis and activation. A. ONE NONSPECIFIC SIGNAL CONCEPT AND THYMUSINDEPENDENT PARALYSIS The main general conclusion concerning induction of paralysis by TI antigens is that specific B cells are turned o f f a t higher concentra-

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tions of the TI antigen than those causing activation. The other important point to bring up at this time is the parallel turning-off of the polyclonal response by nonspecific mitogens. Again, this only occurs at superoptimal concentrations of the mitogens (Coutinho et al., 1973a, 1974a; Coutinho and Moller, 1973d). Below effective concentrations the resting state is maintained, and there is no evidence that B cells exposed to subtriggering concentrations of B-cell mitogens are in any way defective in their capacity to respond fully to a competent stimulus by optimal concentrations of the mitogen. Since we know that all TI antigens are competent to activate B cells polyclonally and that this nonspecific property seems to be responsible for induction of specific cells, it is likely that the inactivation of competent specific cells is also a result of the interaction between nonspecific surface receptors and the competent ligand, similar to polyclonal activation. As shown (Coutinho et al., 1974a), the same molecule that is able to turn off the response in nonspecific cells is also competent to inactivate the specific cell population. In both cases, inactivation occurs at ligand concentrations higher than the concentrations required for activation. Paralysis of specific cells, however, is observed at much lower concentrations than polyclonal paralysis, as would be expected from the focusing function of the Ig receptors on specific cells. This was also demonstrated when paralysis induction was prevented by inhibiting the preferential binding of mitogen to the specific cells. Therefore, we assume that B cells are paralyzed by a mechanism that does not involve antigenic specificities and is caused by mitogenic overstimulation when the interaction between the nonspecific receptor sites and the competent (TI) ligands markedly exceeds the threshold levels. Below the paralytic threshold, only activation can occur. The hypothesis of antigen concentration also explains paralysis in terms of excessive levels of interaction, but between Ig receptors and antigen (see Section 11,B). This postulate is difficult to accept because of the failures to induce tolerance in vitro with highly TD proteins, irrespective of the concentrations used (Diener and Feldman, 1972; Dresser and Mitchison, 1968; Weigle et al., 1972; Weigle, 1973). However, when haptenic determinants are coupled to TI antigens (which have the capacity to paralyze B cells directly and nonspecifically), tolerance to these determinants is easily induced (Feldman, 1972a; Feldman and Nossal, 1972). Again the capacity of an antigen to paralyze (or activate) B cells seems to depend on molecular characteristics and sites other than the antigenic determinants as is even more apparent for large protein molecules (FyG) (Feldman and Nossal, 1972). The mechanism of paralysis induction,

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therefore, must involve surface receptors other than Ig receptor-combining sites, since no specificity is involved. The pattern of antigen presentation hypothesis postulates more than simple thermodynamic rules for induction and tolerance. Some patterns are considered to be only immunogenic, independent of the actual concentration of determinants presented to the B cells, whereas other patterns are always tolerogenic (Feldman, 1972a). It is clear (Coutinho et al., 1974a) that the same molecule is either activating or paralyzing, independent of the pattern of presentation, the only variable that accounts for the amount of mitogen present on the cell membrane. Furthermore, as discussed above, addition of a PBA to cultures that have been optimally activated by antigenic concentrations of the hapten-PBA conjugate, results in suppression of the response by paralyzing the specific cells (Coutinho et al., 1975a). However, nonspecific cells are activated because the concentration of mitogen is not high enough to reach paralytic levels by itself, whereas this could be achieved in specific cells already exposed to optimal concentrations of PBA on the surface. This finding definitely demonstrates that the PBA properties of the antigenic molecule are operative in paralysis induction and must be responsible for turning off the specific cells. According to this view, it follows that induction must necessarily precede paralysis. Implications of this assumption must be considered when discussing the self-nonself discrimination. B. NONIMMUNOGENIC BUT PARALYTOGENIC MOLECULES The discussion first centers on situations in which the reverse of the foregoing prediction has been obtained experimentally, namely induction of paralysis by antigenic molecules or concentrations that are incompetent to induce immunity.

1 . A Low Molecular Weight Levan Fraction The first observation concerns a low molecular weight preparation of levan (mol. wt. less than lo4), which is tolerogenic but very poorly immunogenic (Miranda et al., 1972; Kotlarski et al., 1973). A parallel decrease in immunogenicity and tolerogenicity with decreasing molecular weight was previously found for SIII in a similar system (Howard et al., 1971c), and this is in agreement with the above assumptions and with the molecular weight requirements in B-cell induction (Coutinho et al., 1974~).In the levan system, a decrease in molecular weight of the antigen is followed by a decrease in immunogenicity, although this is much less apparent when high doses are used for immunization (Miranda et al,, 1972). Doses

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of levan, from 100 to 1000 times higher than optimally immunizing doses, were shown to be tolerogenic in a transfer system, with several striking characteristics, such as a very rapid induction of tolerance and a very long persistence (up to 150 days, even after transfer). However, the low molecular weight fraction, which was a very poor immunogen, was capable of tolerance induction although at even higher concentrations. Tolerance induction in vitro required only a short incubation time and occurred at 4°C. (Kotlarski et al., 1973). These findings suggest that this particular antigen displays a very high degree of cell-surface binding and is possibly transferred on the cell surface and, therefore, maintains the state of unresponsiveness. This conclusion had actually been reached by comparing the behavior of levan with SIII in vivo and in vitro, but it was suggested that levan displayed a “more potent avidity for immunoglobulin receptor sites” (Kotlarski et al., 1973). However, this inference is likely to be wrong, because it only involves differential binding capacities of levan and SIII to the Ig receptor sites, excluding the possibility that the nonspecific stickiness of levan to cell surfaces in general may be entirely responsible for the findings. It is difficult to understand how two different polysaccharide molecules can be distinguished by their capacities to be recognized and bound by specific receptor sites. It is logical to assume, therefore, that the differences are due to nonspecific properties of levan. The inhibition of tolerance induction by anti-Ig sera (Kotlarski et al., 1973) does not at all exclude this assumption, since all of us believe that Ig receptors focus antigenic molecules on specific cells. It should be expected that, with sufficiently high concentrations, tolerance could be achieved even in the presence of anti-Ig sera, but it would be nonspecific and polyclonal, in this case. No experiments are available to support or exclude this possibility. Therefore, the question is, Why is the low molecular weight preparation of a sticky substance (levan) capable of inducing tolerance? From the above considerations it seems likely that the low molecular weight preparation simply blocks the interaction between the competent, high molecular weight ligands and the receptors on the cell surface, since it is by itself incapable of activation and remains firmly bound to the cell. This could actually be expected from the preceding discussion about molecular weight requirements for the expression of mitogenicity. Thus, it is very likely that such coating of the cell surface does not provide an efficient interaction with the mitogen receptors, or any interaction at all, because the binding may be established via irrelevant receptors, most likely the Ig receptors. This set of observations argues against

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the concept of excessive cross-linking as the mechanism of paralysis induction. Thus, it is difficult to explain how a low molecular weight molecule can induce more extensive cross-linking than a molecule of higher molecular weight (immunogenic levan molecules used for challenge), when the patterns of antigen presentation are obviously the same and independent of their sizes. The fact that “tolerance” can be induced at 4°C. strongly argues against an active phenomenon in induction of tolerance (Kotlarski et al., 1973) and supports the assumption that these cells are simply blocked.

2. D-GL A similar situation, with the nonimmunogenic molecule DNP-DGL (GL = copolymer of glutamic acid and lysine), has been described in guinea pigs (Katz et al., 1971) and mice (Katz et al., 1972) as an “intracellular mechanism of inactivation of hapten-specific precursors of antibody forming cells.” Although the “tolerant” state was not broken by in vivo transfer and resisted trypsin treatment, some evidence for a blocking mechanism was inferred from the fact that hapten-specific cells were shown to be responsive when T-cell activity was provided under suitable conditions (Katz et al., 1971). Furthermore, the conjugate appeared to have some nonspecific (toxic?) effects, because it greatly affected, at least in some experiments, irrelevant control responses (Katz et al., 1971). This was impossible to ascertain in the last observations reported, because no specificity controls were included (Katz et al., 1972; Nossal et al., 1973). Blocking has been excluded on the basis of repeated transfers and trypsinization experiments of tolerized cells (Katz et al., 1972). However, no attempt was made to show whether or not D-GL molecules were left on the surface of the paralyzed cells after these procedures. Trypsinization experiments are not convincing because this enzyme does not seem to strip efficiently the Ig receptors from the B-cell surface (Julius and Herzenberg, personal communication; Moller and Gronowicz, unpublished). More than 50% of the original number of cells that stain for surface Ig was still found after treatment with trypsin concentrations ten-fold higher than those used here. Thus, trypsin treatment removes blocking only when a trypsinsensitive antigen is used [the positive control is therefore irrelevant (Katz et al., 1972)l. Strong support for a blocking mechanism is the finding that the “tolerant” state can be induced by in vivo incubation with low concentrations of the conjugate (1 pg./ml.) for very short periods (30 minutes) at 4°C. (Nossal et al., 1973). Also in this case, the unre-

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sponsiveness induced by some concentrations of the conjugate could be reversed to normal control levels by the B-cell mitogen POL, demonstrating that the specific B cells were functional (Nossal et al., 1973). The failure of specific cells to respond to POL after incubation with higher concentrations of the “tolerogen” presumably reflects complete coating [to use the author’s expression (Nossal et al., 1973)] of the B-cell surface, preventing any binding of the nonspecific competent ligand. On the other hand, it is not strange that specific cells fail to respond to an immunogenic low concentration of a TI antigen in this case, because the focusing function of the specific Ig receptors has been inhibited by the binding of nonimmunogenic hapten. Exactly the same situation was shown (Coutinho et al., 1974a) when the preferential activation of hapten-specific cells was inhibited by free hapten. As pointed out, specific cells are activated at higher concentrations of the mitogen, because they are not paralyzed at all but only their capacity to concentrate preferentially the mitogen on the surface is blocked. If the hapten is very firmly bound to the cell surface, as seems to be the case of DNP-D-GL, the Ig receptors are certainly blocked to a much greater extent and for much longer periods of time. If the D-GL molecule is, indeed, nonimmunogenic, the reasons must be that it is not capable of direct B-cell activation (is not a PBA) and that it is not recognized by T cells, as postulated above. This situation may actually be found with many molecules, more or less sticking or blocking. The D-GL system might be more complicated than suggested here. It has recently been found that if mouse spleen cell cultures are immunized with macrophage-bound antigen, a good antihapten PFC response is obtained which appears to be TI (Mosier, personal communication). Since soluble antigen inhibits this response, these experiments further support the blocking concept. Thus, the immunogenicity of the macrophage-bound antigen shows that there are no intrinsic paralytogenic properties as such. Its thymus independence would probably rely on a capacity to activate macrophages. There is actually another antigen that has been described as TI and macrophage-dependent (Hunter and Munro, 1972). 3. Free Reactive Forms of Haptens The previous examples were included in this part of the discussion because the molecules involved have great similarities to TI antigens. Therefore, induction of unresponsiveness by a free reactive form of a hapten (Fidler and Golub, 1973; Gronowicz et aZ., 1974b; Gronowicz and Coutinho, 1975c) or a hapten-autologous protein

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conjugate (Aldo-Benson and Bore], 1974) would seem more appropriately discussed together with TD unresponsiveness. However, we have shown that specific TI antibody responses are also abolished in this system (Gronowicz and Coutinho, 1975c), and because the mechanism of unresponsiveness is now clear, the example appears very instructive. Mice injected with a reactive form of the hapten NNP (NNPazide) show specific unresponsiveness in vivo and in vitro to both TD (NNP-HRC) and TI (NNP-LPS) conjugates of the hapten (Gronowicz et al., 1974b). Since the role of suppressor cells in this system cannot explain the total unresponsiveness achieved (Gronowicz and Coutinho, 1 9 7 5 ~ it ) ~would appear, therefore, that we were dealing with a clear-cut B-cell effect. In addition, we had found earlier that hapten-specific cells could persist at normal levels in tolerized mice, thus excluding toxicity and killing of specific cells by the tolerogen. Moreover, spleen cells from paralyzed mice could be activated to antihapten antibody production by LPS in vitro to a roughly normal extent, in apparent contradiction with the one nonspecific signal hypothesis. Indeed, this hypothesis states that functional expression of the PBA properties of the antigen on specific cells is all that is required for induction of TI responses. In this situation, the findings indicated that the specific cells were there and they were not prevented from participating in the polyclonal response to LPS. Therefore, the only possibility left to explain TI unresponsiveness was that NNP-LPS could not be selectively concentrated on the specific cell surface via Ig receptors, due to blocking of the specific focusing function. After the failure to remove Ig receptors by trypsin treatment, other methods were tried, and an effective way of breaking blocking was found to be the culture of tolerized cells for 24 hours with extensive washings. Presumably, in this situation the Ig receptors that had been turned over were regenerated in vitro in the absence of tolerogen (blocker). It was then possible to demonstrate that these “deblocked” cells from paralyzed mice could mount perfectly normal responses to the TI antigen NNP-LPS, in parallel with the polyclonal responses to LPS (Gronowicz and Coutinho, 1975c) (see also Aldo-Benson and Bore], 1974, for a similar mechanism of unresponsiveness in TD responses).

4 . Highly Substituted Hapten-PBA Conjugates If blocking of the mitogen sites on B cells by ligands incapable of interacting with these receptors (Sections VII,B,l and 2) or block-

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ing of the focusing function of Ig receptors (Section VII,B,3) explains a large part of the reported data on B-cell “tolerance,” another set of experimental observations must be concerned with true B-cell paralysis, We think that in the situations discussed next, specific B cells are, indeed, paralyzed by overstimulation by competent TI ligands. The experiments to test this assumption should be the demonstration in every case that it is impossible to turn on these cells by a competent B-cell mitogen. The hypothesis of one nonspecific signal for induction and tolerance of B cells apparently is contradicted by the claimed evidence for certain haptenmitogen conjugates (DNP-POL) that they can be made only immunogenic and never tolerogenic, or in the reverse situation, only tolerogenic and never immunogenic by varying the degree of hapten substitution (Feldman, 1972a). Thus, low degrees of hapten substitution yield conjugates capable of inducing immunity, but never capable of inducing specific paralysis. The first point to stress is the fact that even nonsubstituted POL can be easily shown to induce either immunity or paralysis to DNP, simply because it behaves exactly as expected for a PBA and induces polyclonal activation and paralysis, the anti-DNP-specific cells obviously being included. Feldman (1972a) reported that 100 Fg. of unconjugated POL are incapable of activating an anti-DNP PFC response. However, following our original description (Coutinho and Moller, 1973c) it is now generally accepted that POL gives rise to a good anti-DNP response even at lower concentrations, because it increases nonspecific “background” PFC in culture (Schrader, 1973a, 1974c; Nossal et al., 1973). Furthermore, it can be shown that concentrations higher than those required for optimal activation readily suppress the polyclonal response including the anti-DNP response. According to our outline of the focusing function of the Ig receptors, it seems evident how a low degree of hapten substitution shifts the dose-response curve observed for specific cells [as shown for NNP-LPS (Coutinho et a,?., 1974a)l to a variable extent, depending on the degree of substitution. The higher the degree of substitution, the larger the preferential concentration of mitogen on the membrane of specific cells which can bind the mitogen-coupled haptenic determinants. Therefore, with conjugates of very low degree of substitution, the suppression of the responses observed at high concentrations with higher hapten substitutions should be expected to be found at much higher concentrations, similar to the concentrations required to suppress polyclonal responses. It is not surprising that this fact is not reported (Feldman, 1972a) since polyclonal activation was not mentioned either.

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The reverse situation, in which high degrees of hapten substitution lead to conjugates capable only of inducing tolerance but never immunity, is also understandable on a similar basis. Since there is no experimental indication of the minimal number of POL molecules required to activate or to paralyze a B ceI1, the discussion at this point must be based on assumptions. It was not shown (Feldman, 1972a) that the response to the highest hapten-substituted conjugates could not occur at concentrations even lower than the concentrations used, which were the same as the minimal concentrations required for inducing a response with less substituted conjugates. The prediction would actually be that the response occurs at much lower concentrations because this should be expected from the focusing function of the Ig receptors. However, accepting the reported facts, it can be argued that two or more DNP groups per monomeric unit of POL would keep a number of POL molecules on the surface of DNPspecific B cells, with a very high affinity and efficient interaction sufficient to paralyze the cell. This is not difficult to accept, since the repeating unit of POL (MON) is supposed to reappear many times in the molecule and since the affinity of interaction between specific, focusing Ig receptors and antigenic determinants must be several orders of magnitude higher than the affinity of the reaction between the mitogenic sites and receptors on the cell surface. For every new bond in the interaction, the avidity of the reaction is squared (see above). The explanation proposed for these findings is a simple restatement of the facts and the postulate of “immunogenic patterns” as opposed to “tolerogenic patterns.” However, it is recognized that “the degree of DNP conjugation was important, but operated independently of DNP concentration” (Feldman, 1972a). It is evident that the variable was the concentration of the carrier (mitogen) at the surface of the specific cells rather than the concentration of the epitope. Accepting the fluid mosaic model of cell membranes (Singer and Nicolson, 1972), it is difficult to understand why the surface receptors would not move on the plane of the membrane to fit any possible antigen presentation and therefore, equalize all different patterns of determinant presentation. Furthermore, experiments with mixed conjugates of two different haptens that have the same site of conjugation on the POL molecule (Feldman, 1972a) suggest that no pattern of determinants is involved, since the conjugation of the second hapten could be expected to interfere with the sites on the POL molecule by which DNP commonly binds to POL and, consequently, would alter the pattern of DNP determinants in the con-

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jugate. However, no change was observed in the response, presumably because no change in the amount of mitogen bound to the specific cells occurred as expected if the number of DNP determinants per mitogen molecule was the same (Feldman, 1972a). The conclusion from these observations, in the light of our present knowledge of the strong B-cell mitogenicity of POL, is that there is a critical amount of surface-bound mitogen that controls induction of immunity or tolerance in B cells, The induction of tolerance to FyG by FyG-POL conjugates (Feldman and Nossal, 1972) is a confirmation of the assumption, since repetition of epitopes can definitely not be accepted in this case. Moreover, the “pattern of presentation” model does not explain the turning-off of responses by superoptimal concentrations of an immunogenic conjugate. It is well known and generally accepted that there is an optimal concentration of every immunogenic molecule above which the response is suppressed (see, e.g., Coutinho et al., 1974a). It is evident that the pattern of determinant presentation is exactly the same for optimal or supraoptimal concentrations, and it has to be accepted in this situation that “the degree of DNP conjugation is not important and operates dependently on DNP concentration,” the latter apparently being the relevant variable. Again the functionally important variable seems to be the amount of surface-bound PBA, which operates either by increasing the avidity of the interaction by multipoint binding (by increasing epitope density) or else by increasing concentrations in the medium of conjugates with the same degree of substitution. As noted above, recent experiments showing that thymus independence of MON and POL preparations does not depend on the pattern of determinants cast further doubts on the whole concept (Langman et al., 1974).

5. Antibody-Induced Concentration of Sur$ace-Bound Antigens Another experimental system used in support of the cross-linking concept as the mechanism of tolerance induction employs antigenantibody complexes for induction of paralysis (Diener and Feldman, 1970,1972; Feldman and Nossal, 1972). In this case, all the evidence is for a concentration mechanism mediated by a bivalent antibody polymerization of the antigen, at the surface of the specific cells. Therefore, as is expected, monomers of TI antigens or TI antigens are shown to be capable of tolerance induction (Diener and Feldman, 1972) at “membrane concentrations” higher than the concentrations required for immunity. The differences in interpreting these results, according to the antigen presentation hypothesis or to

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the hypothesis defended in this discussion have been already delineated. The slower receptor turnover after binding of antigenantibody complexes (Wilson and Feldman, 1972a),as well as the uncertain efficiency of trypsinization techniques, cast some doubt on the true state of tolerance in the specific cells, leaving the possibility of a simple blocking of the surface-binding receptors. Because of the TD characteristics of the antigen, this appears to be the most likely explanation for the tolerance induced by anti-FyG, FyG. Here again, mitogenic activation of the “tolerant” cells should be critical for an understanding of the phenomenon. The concept of antibody-induced “central” suppression of the B-cell responses has been used in another context which does not seem to involve excessive cross-linking. It has been postulated that in TD responses, the mechanism of B-cell immunization is mediated by antigen-T-cell receptor complexes bound to the macrophage surface (Feldman, 1972b). However, activation may fail when the complexes interact directly with the B cells. In this case, B cells are postulated to be paralyzed (Feldman and Nossal, 1972), but the mechanism of tolerance induction cannot be the same as above, since a greater extent of cross-linking must be expected when the complexes are presented in a matrix on the macrophage membrane. No mechanism has been proposed for this antibody-induced tolerance. No explanation is required by us, since this is merely a postulate, although blocking of the Ig receptor function that bridges the B cell to PBA-secreting cells [macrophages or T cells (Schrader, 1973b; Vischer, 1972a; Coutinho et al., 1973b)l could possibly be responsible for these observations.

c. NONIMMUNOGENICBUT PARALYTOGENIC THYMUSANTIGEN CONCENTRATIONS INDEPENDENT The one nonspecific signal hypothesis predicts that in a TI system, immunity always precedes paralysis, because this is necessarily the result of overactivation. Therefore, reports on induction of paralysis by subimmunogenic concentrations of TI antigens directly contradict the hypothesis. The only clear-cut situation of this type that has been described concerning TI antigen is with SIII (Baker et al., 1974). Priming with nonimmunogenic doses of the antigen leads to unresponsiveness to a challenge with optimally immunogenic doses. However, this phenomenon has been conclusively demonstrated to be the result of suppressor T-cell activity and, therefore, it does not involve B cells

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directly, Thus, this “low zone unresponsiveness” is not observed in “nude” mice and is sensitive to antilymphocyte serum treatment in normal mice. It simply provides one new piece of information concerning T-cell activity in the anti4111 antibody responses. T Cells are obviously “primed” by antigen concentrations that are lower than those required for direct induction of B cells. As discussed (quantitative concept of B-cell induction and paralysis), when the specific B-cell clone is maximally stimulated by an optimally immunogenic dose of antigen, further T-cell help results in suppression. When PBA synergizes with a TI antigen for induction of paralysis in specific B cells (Coutinho et al., 1975a), the phenomenon is entirely nonspecific and is governed by purely quantitative rules. A similar effect of “priming suppressor T cells” can also be obtained in anti-PVP responses (B. Andersson, personal communication). The partial unresponsiveness to DNP-levan conjugates obtained by priming with an immunogenic dose of the antigen (Klaus and Humphrey, 1974) may be related to the long persistence of this antigen in the organism (Miranda et d.,1972) and to the fact that, in the continuous presence of levan, antigen-sensitive B cells are activated and very efficiently driven to an end stage of PFC. The second antigenic challenge finds a much smaller pool of indubicle (resting) B cells than a primary antigenic challenge, Somewhat different results with identical antigenic conjugates have also been reported (Guercio et al., 1974). However, in these experiments, the safest conclusions concern only the long persistence of levan in vivo, and the stickiness of this antigen to spleen cells, both of which had already been demonstrated (Miranda, 1972; Kotlarski et aZ., 1973). We have reported a parallel situation at the polyclonal level in the responses to high concentrations of LPS in vivo and in vitro (Gronowicz and Coutinho, 1974). Therefore, the nonspecificity of the mechanisms involved in this process seems firmly established. Experiments of this type might provide further insight to the problem of immunological memory. It appears that a large fraction of the B-cell population responding to PBA, either specifically or polyclonally, is driven to become end cells secreting antibody at a high rate. Here again the B-cell subpopulation responding to cooperative factors appears to have markedly different properties. However, we have found that in vivo or in vitro exposure of bone marrow or spleen cells to the very “early” PBA dextran-sulfate (which activates precursors and very immature B cells) greatly enhances the responsiveness of these cell populations to challenge by LPS. Therefore, the phenomenon may not be restricted to B cells sensitive to cooperative factors, or else these cells are actually at a rather primitive stage

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of differentiation. From these experiments the main conclusion we obtained was that there is a critical level of differentiation for cells involved in IgM synthesis that the cell must reach before it acquires the capacity to be “reprogrammed” for high-rate antibody synthesis. This stage reached, the result of activation appears to be necessary reprogramming for Ig synthesis with very restricted division. Before that critical stage, activation appears to always result in extensive mitosis without Ig secretion. Since different PBA interact with distinct B-cell subpopulations at various stages of differentiation, the behavior of TI antigens in this respect may be expected to be rather heterogeneous (Gronowicz et d.,1974a). D. A “TWO-SIGNAL’’ COMMENT ON THYMUS-INDEPENDENT PARALYSIS It was originally claimed by Guercio and Leuchars (1972) that induction of specific tolerance to levan would abolish the TI antibody responses to a DNP-levan conjugate. This observation was taken up by two-signal believers as a strong argument against direct activation of B cells by TI ligands and for the involvement of accessory signal (cells?, antibody?) in these responses. However, from a careful look at the results obtained in these experiments, it was obvious that the concIusions were wrong (Guercio and Leuchars, 1972). Thus, the anti-DNP-levan responses in levan-paralyzed mice were, indeed, reduced, but the control responses to DNP-BGG were also depressed to a comparable extent in those mice. Since the responses to DNP-levan were lower than the responses to DNP-BGG in normal mice, the same degree of nonspecific suppression in levan-tolerized mice appeared to be complete and specific for the DNP-levan conjugate. Klauss and Humphrey (1974) recently repeated these experiments and could very elegantly demonstrate that in the TI responses to DNP-levan or DNP-SIII conjugates, paralysis to the carrier did not affect antihapten responses, nor did the paralysis to the hapten affect the anticarrier responses. The original observation, therefore, was shown to be incorrect. However, those experiments do demonstrate a nonspecific polyclonal effect of high doses of levan (depression of the anti-DNP-BGG responses).

E. INCREASED NUMBERSOF ANTIGEN-BINDING CELLS IN THYMUS-INDEPENDENT PARALYSIS One last point concerning tolerance to TI antigens deserves discussion, namely the reported evidence for increased numbers of antigen-binding cells in tolerant animals, as compared to normal con-

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trols (Howard et al., 1969; J. G. Howard, 1972; Sjoberg and Moller, 1970; Sjoberg, 1971; Moller and Sjoberg, 1972). Tolerized animals, which show no increase in PFC after an immunogenic challenge, exhibit higher numbers of rosette-forming cells (RFC) than untreated controls, but lower than normal animals challenged with immunogenic doses. Apart from trivial explanations, such as macrophage RFC, which will not be discussed, two possibilities should be considered. One explanation concerns the levels of sensitivity of the tests used to assay immunity and tolerance, namely PFC and RFC assays. It is well known that antigen-binding cells, can be demonstrated by the rosette technique which are specific but of such a low avidity for the antigen that they are never triggered upon antigenic challenge, (E. Moller et al., 1973; E. Moller, 1974). Numbers of RFC found in normal unimmunized animals can be over 10 times the number of PFC detected with the same epitope density in the target red cells for both tests. Also, the increase after immunogenic challenge is much greater for PFC than for RFC, supporting the above assumption (E. Moller et al., 1973; E. MGller, 1974). When tolerance is induced by a TD (non-PBA) substance, no increase should be expected for either PFC or RFC, because the mechanism of tolerance induction presumably is deletion of normal cell cooperation by blocking or by tolerizing T cells. In any case, B-cell activation does not take place, regardless of specificity or avidity of different cell populations. However, when tolerance to TI antigens is induced, according to the present hypothesis of one nonspecific signal, the same dose of antigen that tolerizes high-avidity cells (PFC) always triggers other cells of lower avidity because of the PBA properties of the antigen, which can be expressed in every B cell. This situation is observed (Coutinho et al., 1974a), when successive paralysis of progressively lower-avidity cells takes place and at the same time cells of even lower avidity (and different specificity) are activated. These lowavidity antigen-specific cells are not detected as PFC because of the avidity requirements of the assay, but RFC are included in the group of low-avidity cells that accounts for the ten-fold difference in background numbers detected by each method. Therefore, an increase in RFC without a change in PFC should be expected to occur every time a large dose of a PBA is injected, showing that TI antigens are indeed PBA capable of triggering nonspecific cells at appropriate concentrations. Moreover, as we have previously reported (Coutinho and Moller, 1973d) for SIII, such paralyzed animals exhibit a clear increase in numbers of PFC against other specificities, because con-

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centrations required for polyclonal activation are reached. It should be expected that by further increasing the antigen doses, the next cells to be paralyzed after high-avidity specific cells (PFC) should be the low-avidity specific cells (RFC), and no increase over normal controls should be found in this situation. Under special conditions, where the levels of sensitivity of detection are very high (red cells coated with a high hapten density), a similar, although less pronounced situation, can also be found for TD antigens (Moller et d., 1971), as is discussed briefly below. The other explanation is more speculative. It concerns various subpopulations of B cells that are competent to respond to an antigenic challenge. Little evidence is available regarding the degree of differentiation B cells must reach in order to be sensitive to a triggering signal (Lafleur et al., 1972, 1973) or the pattern of responsiveness that B cells can mount in response to competent stimuli, at successive stages of differentiation. It is likely that B cells at a rather primitive stage of differentiation are competent to divide extensively after activation, but they are not mature enough to differentiate into high-rate antibody-forming cells, whereas more differentiated cells, which are always “reprogrammed” upon activation, are restricted in their mitotic capacity. If this is the case, and some evidence is now available in support of this assumption (Gronowicz and Coutinho, 1974, 1975c Gronowicz et al., 1974a), it would be expected that after receiving tolerogenic concentrations of the TI antigen, the less differentiated cells, being less sensitive to triggering (and paralysis), divide, accounting for the increase in RFC, but they are incapable of reprogramming to PFC. VIII. B-Cell Induction a n d Paralysis b y Nonpolyclonal Activator Molecules (Thymus-Dependent Antigens)

It is not the purpose of the present review to consider TD B-cell activation. Therefore, this part of the discussion is limited to some general implications of the one nonspecific signal hypothesis, as applied to TD immune responses, and to some considerations on tolerance to TD antigens. For reasons already discussed we want to make it clear that the extension of these concepts to TD B-cell triggering is restricted to IgM induction. Thymus dependence or thymus independence of an immune response is most often an operational classification, and therefore it might be misleading if it is not defined according to well-characterized properties of the antigens involved. Thus, as shown above, some antigens considered to be TI benefit by T-cell helper activity

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and cause an increased response in the presence of activated T cells (see Table I). In this particular case, LPS could receive help from determinants other than those for the LPS itself present on the bacteria. However, the phenomenon is not unique to LPS and has been described for anti-POL (Feldman and Basten, 1972a; Schrader, 1973b) and KLH (Kerbel and Eidinger, 1971) responses too. In some other cases, further T-cell activation results in suppression, as in the case of SIII (Baker et al., 1970, 1973) and of PVP (Kerbel and Eidinger, 1972; B. Anderson, 1972), in which T cells may recognize and be activated by determinants present on the same molecule that activates B cells directly. Differences in behavior may also be dosedependent phenomena, as demonstrated for KLH (Torrigiani, 1972), which is completely TI at high concentrations (when direct activation of B cells saturates their responsive capacity) but TD at lower concentrations. Confusing questions can be raised based on these phenomenological considerations. Is a TI antigen a substance the response against which can be greatly enhanced or suppressed by the presence of T cells (LPS, KLH, PVP, POL)? Is a TD antigen a substance the response against which is greatly impaired in the absence of T cells in some strains, but is still as good as the TI response observed in other strains [(T,G)-A--L] (Mitchell et al., 1972b)T The basic mechanisms responsible for thymus dependence and thymus independence of a certain molecule should be considered in order to define the characteristics of its immunogenicity. Because of the characteristics of the Ig repertoire, the B-cell system has the capacity to recognize any antigenic determinant. Therefore, thymus independence cannot be dependent on antigenicity for B cells but rather on the ability to activate B cells directly. On the other hand, it seems possible that molecules that are recognized by T cells can always activate them (McDevitt and Landy, 1972). Therefore, the outcome of the immune response to a certain immunogen fundamentally depends on its capacity to activate B cells directly and to be recognized by T cells. It is evident that in the light of a quantitative concept of B-cell activation (overactivation resulting in suppression), the response to an immunogen which displays both properties depends on the quantity of these. What predictions can be made about T-cell recognition or lack of recognition of antigens in relation to its thymus independence? Since the poor degradability of some TI antigens simply reflects the lack of enzymatic specificities capable of recognizing and splitting the molecule, complete thymus independence indicates (1)the lack of T-cell specificities for the recognition

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and/or responsiveness to this molecule and (2) the capacity to activate B cells directly. T Cells are largely responsible for the selfnonself discrimination (immune responsiveness controlled by genes linked to the genes governing histocompatibility antigens) primarily at the level most similar to self (a large number of T cells react against histocompatibility antigens) and in particular at the level of fine specificities (tumor-specific and virus-determined antigens). It would appear, therefore, that the “foreignness” of TI antigens is so evident that T-cell-dependent recognition of nonself is not required. Since foreignness can be expected to parallel dangerous aggressions, there is a selective advantage for a mechanism of direct B-cell activation. A completely TD antigen is a molecule that displays the reverse properties, namely total lack of PBA activity and the capacity to be recognized by T cells (more similar to self). If the one nonspecific signal hypothesis is applied to B-cell induction by non-PBA molecules, two basic assumptions must be made. 1. Since TD antigens completely lack the capacity to activate B cells directly, and B-cell activation must always proceed via nonspecific triggering signals, the basis for helper cell activity is the secretion of nonspecific triggering substances (PBA) by the COoperating cells. 2. Since TI and TD immune responses are qualitatively different, B cells responding to the cooperative mitogens must belong to another subset of antibody-forming cells that differs partially from the cells participating in TI responses. This latter point may actually constitute an argument for the nonspecificity of triggering and a challenge to all “specific” theories of B-cell activation. These hypotheses postulate that B cells are activated via the combining sites of Ig surface receptors, by one common mechanism for TI and TD responses (e.g., pattern of antigen presentation or associative recognition). Since the Ig-combining sites are clonally distributed and identical for all B cells of the same clone at different stages of maturation, it should be expected that no differences could be found in the pattern of responses to TD or TI antigens (the same cell population would be activated via the same surface receptor and by the same mechanism). On the other hand, by the present hypothesis, triggering can only be achieved by nonspecific ligands that interact with surface structures other than Ig receptors, These PBA receptors are not clonally distributed but, most likely, change during the life cycle of every B cell (Gronowicz et al., 1974a), in analogy with other changes on the surface mosaic (Takahashi et al., 1970; G. Moller,

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1974; Forman and Moller, 1974). Therefore, it should be expected that a distinct pattern of response can be found for each particular ligand. Moreover, if the cooperating system provides one single type of PBA, it follows that, whereas TI responses could be expected to be rather different from each other, the responses to highly TD antigens should be radically different from TI responses, but very similar for every TD antigen. The available evidence appears to support this assumption, in particular the selective activation of various B-cell subpopulations by different B-cell mitogens (Gronowicz and Coutinho, 1975c; Coutinho et al., 1974b; Gronowicz et al., 1974a). Let us see how TD immune responses are interpreted in other models before analyzing these postulates. As seen above, the theories of antigen presentation explain B-cell induction by the interaction of antigenic determinants with specific Ig receptors. When nonpolymeric molecules are involved (TD antigens), multipoint binding or the correct immunogenic pattern is achieved via binding of the antigen by T cells (Mitchison, 1971; Moller, 1970c; Taylor and Iverson, 1971) or via binding of antigen-T-cell receptor complexes to the macrophage surface (Feldman, 1972b; Feldman and Nossal, 1972). The basic postulate, therefore, is that a TD antigen, when bound to a T cell or to a macrophage, becomes TI and capable of direct B-cell activation. The mechanism of cell cooperation is completely passive, and T cells or macrophages do not contribute in an active way to the ultimate triggering of B cells, The basic mistake in this type of theory is the assumption that T-cell binding of antigen is more stable than the binding of the antigen by B cells or macrophages. Independent of the recent claim that T cells are only capable of binding antigen via cytophilic antibody (Simonsen, 1972; Webb and Cooper, 1973; Crone et al., 1972), there is a large body of evidence demonstrating that antigen binding by T cells is clearly “weaker” and less stable than B-cell binding (Roelants and Rydkn, 1974; Hammerling et al., 1973; Lamelin et al., 1972). T Cells themselves seem to be capable of being activated only when the antigen is presented on macrophages (Rosenthal and Shevach, 1973; Shevach and Rosenthal, 1973). At least for in vitro experiments, there seems to be no reason why B-B cell cooperation or macrophage-B cell cooperation should not be more efficient than T (macrophage)-B cell cooperation if a simple passive mechanism of antigen presentation is involved. Thus, the much greater affinity and/or stability of antigen binding to B cells would make it the perfect cooperative situation for antigen presentation. Furthermore, a direct test of these hypotheses was performed in uivo, by injecting T cells capable of passive an-

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tigen presentation but incapable of any active role (Miller et al., 1971). It was demonstrated that these antigen-presenting T cells were completely devoid of cooperative activity. If cooperation is assumed to be mediated via macrophage-bound complexes (Feldman, 1972b; Feldman and Nossal, 1972), one further factor of instability is added to the reaction, namely the dissociation of the complexes from the Fc receptor on the accessory cell’s surface. Another finding against a passive role of antigen presentation is the fact that antibody responses to TD antigens in thymus-deprived animals are depressed both in quantity and in quality (Taylor and Wortis, 1968; Gershon and Paul, 1971). If a passive mechanism was involved the reverse should be expected, namely that low-avidity B cells would be triggered by T-cell help. However, this argument does not take into account the difference in antibody class that constitutes the prevalent response in either case. As pointed out, antigen presentation hypotheses do not account for the markedly different patterns of responses observed for TD or TI antigens. In view of the available evidence, we think that a passive function of antigen presentation by accessory cells can be excluded as a major mechanism of cell cooperation for B-cell induction. The T cell-macrophage system appears to function by an active mechanism, providing the B cells with some fundamental signal for activation. Two questions are pertinent, namely, How specific is the signal? and Which cell type is responsible for delivering it? These questions are considered in detail by two-signal hypotheses. When the second signal is postulated to be specific (associative antibody), it is also postulated that its main source is T cells (Bretscher and Cohn, 1970; Cohn, 1972a,b), although it has been recently accepted that other cells (such as macrophages) may provide the associative signal (Watson et aZ., 1973b). In this case, signal two is not at all specific and may even result from mitogen-induced activation of adherent cells (Schrader, 1973b). Therefore, even the most “specific” hypotheses now accept that signal two may be completely nonspecific. It is obvious that the accumulated evidence for the nonspecificity of cell cooperation is enough to support these assumptions. Thus, it has been shown in a number of different systems that helper cell activity can be bypassed by providing the specific B cells with nonspecific triggering signals, such as B-cell mitogens (Byrd, 1917; Sjoberg et aZ., 197213; KreisIer and Moller, 1974; Schrader, 1973a; Watson et al., 1973b; Moller et al., 1972a; Chiller and Weigle, 1973) or products of nonspecific T-cell activation (Katz and Benacerraf, 1972; Ordal and Grumet, 1972; Hartmann, 1970; Schimpl and Wecker, 1972; Britton,

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1972a; Dutton et al., 1971; Dennert and Lennox, 1973; Gorczynski et al., 1973; Sjoberg et al., 1973; Watson, 1973). It is evident that the focusing function of the Ig receptors on specific B cells operates by concentrating the cooperative PBA via antigen bridging to T cells or macrophages. It should be stressed that all the available facts in TD responses can be interpreted in the light of this minimal concept without postulating any other triggering signal. The implications of a one nonspecific signal hypothesis for tolerance induction by TD antigens will be outlined simply. From the foregoing postulates, it follows that TD antigens are also incompetent to paralyze B cells, since they lack the ability to activate them directly, and threshold levels of paralysis are higher than activation levels. Therefore, a true state of B-cell paralysis for TD antigens can occur only by overstimulation mediated by cooperative PBA. This is probably a rare event in responses to highly TD antigens, because it would imply excessive levels of T-cell activity, which are not likely to be reached under physiologic conditions, due to the low thresholds of tolerance induction in T cells (Weigle, 1973). However, it may be the mechanism responsible for the effect of “suppressor” T cells. The well-known phenomenon of suppression of specific responses by a graft-versus-host reaction may also be interpreted in these terms. Most of the situations reported from in vivo experiments, which demonstrate a lack of antibody response to a TD antigen, have been interpreted as states of B-cell tolerance, but other mechanisms that do not reflect a true state of tolerance among the specific B cells are likely to be involved. Thus, T-cell tolerance, of course, results in the lack of antibody responses. Furthermore, blocking of cell cooperation by excess antigen is likely to be more frequent than is commonly accepted. The critical test of these assumptions is always the failure or success of activating the “paralyzed” B cells with a competent ligand (PBA). The solution of problems concerning self-nonself discrimination, which required the original postulate of two signals in B-cell induction, appears to have no room in the one nonspecific signal model, since it postulates that induction precedes tolerance. However, only one postulate is necessary to solve the problems, namely that all selfdeterminants are TD antigens, as they should be expected to be. IX. Concluding Remarks

As pointed out at the beginning of this review, this is not an exhaustive review of all the reported facts concerning B-cell activation. The phenomenology and the theories were, indeed, selected

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from the fantastic volume of work that has been produced in this field during the last few years. It is also true that Cyrus, king of the Persians, who could call every soldier in his armies by name, was not a very capable thinker, because to think is to overlook differences and seek general conclusions. We might have selected from the available evidence, those facts that appeared particularly suitable to developing arguments for the present theoretical framework. We might have left aside arguments or facts that others would consider of primary importance. However, as Me1 Cohn often says, it is more important to be clear than to be right. We definitely pushed some arguments too far and spent too much time interpreting the data of others. Like all the heretics in history, we could not avoid proselytism. Finally, should we become convinced that some other model explains more facts concerning B-cell activation and paralysis, we shall defend it with equal enthusiasm.

ACKNOWLEDGMENTS The technical assistance of Miss Yrsa Avellan, Miss Lena Lundin, and Miss Susanne Bergstedt is gratefully acknowledged. We are deeply grateful to Dr. Eva Gronowicz for her participation in experiments and in the elaboration of many of the ideas expressed in this review.

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SUBJECT INDEX A Allotypy, structural correlates, heavy chain, 54-59 light chain, 60-61 nature of variation, 53-54 rabbit immunoglobulin, 48-53 Amino acid(s), sequence of L and H chains from IgG New, 9-11 Antibodies, polypeptide chain structure, 2 Antibody-producing cells, synchrony in appearance, 100-107

B %cells, activators, polyclonal, 116-119 basis of thymus independence, 167168 associative recognition models, 168-170 other two-signal hypotheses, 170181 pattern of antigen presentation, 181-186 positive and negative views on catabolic behavior of antigen, 186-191 evidence for one nonspecific signal hypothesis, activation by thymus-independent antigens, 145-152 need for mitogenicity of antigen, 158-164 no detectable signal from interaction of antigen, 164-167 specific thymus-independent immune responses, 152-157 synergism of thymus-independent antigens and polyclonal activators, 157-158 technical considerations in activation, 126-127 detection by hemolytic plaque assay, 127-130 misinterpretation of antihapten plaque assay, 130-131

quantitative concept, 140-145 requirement for polyclonal activators, 135-140 serum free cultures, 132-135 hypotheses for activation, 119-120 antigen presentation by T cells, 121- 122 cross-linking concept, 125 one nonspecific signal, 125-126 one specific signal, 120-121 quantitative concept, 124 two-signal hypotheses, 122-124 induction and paralysis by nonpolyclonal activator molecules, 221226 molecular basis of activation, antigen presentation concept, 191193 cross-linking concept, 196-202 lack of requirement for polymeric molecules, 202-207 second messengers and two-signal hypotheses, 193-196 paralysis in th ymus-independent responses, comment on two-signal paralysis, 219 increased numbers of antigenbinding cells, 219-221 nonimmunogenic molecules, 209217 nonimmunogenic thymus-independent antigen concentrations, 217219 one nonspecific signal concept, 207209 types of, 115-116 C Crystallographic analysis, techniques, 2-4 D Disulfide bonds, interchain and intrachain, 15-17

I Immune response, cycling in, 90-100 Immunoglobulins, 237

238

SUBJECT INDEX

high resolution X-ray diffraction studies, amino acid sequence of L and H chains from IgG New, 9-11 changes in conformation, 27-29 homology subunit structures, 11-15 hypervariable regions and active site structure, 18-23 interchain and intrachain disulfide bonds, 15-17 isotype and allotype location markers, 17-18 ligand-Fab' complex marker, 23-26 patterns of change, 26-27 shape, dimensions and symmetry of Fab', 6-9 low resolution X-ray diffraction studies, 4-7 polypeptide chain structure, 2 Immunoglobulin fold, X-ray diffraction studies, 11-15 R Rabbit immunoglobulin allotypes, allotypic groups, 40-45 antigenic determinants, 61-62 heavy chain subspecificities, 64-65 light chains of homogeneous antibodies, 63-64 definitions, 37-38 discovery, 35-36

genetic relationships, 65-66 linkage groups, 66-67 Todd phenomenon, 67-70 idiotypes and, 75-78 immune response, allelic exclusion, 70-71 allelic selection, 72-73 immunization and detection techniques, 45-48 nomenclature, 38-40 suppression, 73-75

X X-ray diffraction studies, high resolution, amino acid sequence of the L and H chains from IgG New, 9-11 changes in conformation, 27-29 homology subunit structures, 11-15 hypervariable regions and active site structure, 18-23 interchain and intrachain disulfide bonds, 15-17 isotype and allotype location markers, 17-18 ligand-Fab' complex marker, 23-26 patterns of change, 26-27 shape, dimensions and symmetry of Fab', 6-9 low resolution, results, 4-7

Contents of Previous Volumes Volume 1 Transplantation immunity a n d Tolerance

M. HASEK, A. LENGEROV~, AND T. HRABA immunological Tolerance of Nonliving Antigens

RICHARD T. SMITH Functions of the Complement System

ABRAHAMG. OSLER I n Vitro Studies of the Antibody Response

ABRAM B. STAVITSKY Duration of Immunity in Virus Diseases

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

WILLIAM 0. WEICLE Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. GELL

AND

B. BENACERRAF

The Antigenic Structure of Tumors

P. A. GORER AUTHOR INDEX-SUBJECT INDEX Volume 2 immunologic Specificity a n d Molecular Structure

FREDKARUSH Heterogeneity of y-Globulins

JOHN L. FAHEY The immunological Significance of 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

CHARLESG. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICKROWLEY 239

240

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOULSBY Embryological Development of Antigens

REED A. FLICKINGER AUTHOR INDEX-SUBJECT INDEX Volume 3 I n Vitro Studies of the Mechanism of Anaphylaxis K. FRANKAUSTEN AND JOHN H. HUMPHREY The Role of Humoral Antibody i n the Homograft Reaction

CHANDLER A. STETSON Immune Adherence

D. S. NELSON Reag ini c Anti bodies

D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms DAN H. CAMPBELLAND JUSTINE S. GARVEY Blood Groups in Animals Other Than Man W. H. STONE AND M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R. JENKIN

AUTHOR INDEX-SUBJECT INDEX Volume 4 Ontogeny and Phylogeny of Adaptive Immunity ROBERT A. GOOD AND BEN PAPERMASTER

w.

Cellular Reactions in Infection EMANUELSUTER AND

HANSRUEDY bMSEIER

Ultrastructure of Immunologic Processes

JOSEPH D. FELDMAN Cell W a l l Antigens of Gram-Positive Bacteria MACLYN MCCARTYAND STEPHEN

I. MORSE

Structure and Biological Activity of Immunoglobulins SYDNEYCOHENAND RODNEY R. PORTER

CONTENTS OF PREVIOUS VOLUMES Autoantibodies and Disease

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

J. MUNOZ AUTHOR INDEX-SUBJECT INDEX Volume 5 Natural Antibodies and the Immune Response

STEPHEN V. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAEL SELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIP Y. PATERSON The Immunology of Insulin

c. G.

POPE

Tissue-Specific Antigens

D. C. DUMONDE AUTHOR INDEX-SUBJECT INDEX Volume 6 Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMIL R.

UNANUE AND

FRANKJ. DIXON

Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSONAND ROBERT A. GOOD Nucleic Acids as Antigens

OTTOJ . PLESCIAAND WERNER BRAUN In Vitro Studies of Immunological Responses of Lymphoid Cells

RICHARD W. DUTTON Developmenta I Aspects of Immunity

JAROSLAVSTERZL

AND

ARTHUR M. SILVERSTEIN

Anti -antibodies

PHILIP G. H. GELL

AND

ANDREW s. KELUS

Conglutinin a n d lmmunoconglutinins

P. J. LACHMANN AUTHOR INDEX-SUBJECT INDEX

24 1

242

CONTENTS O F PREVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins SYDNEYCOHENAND CESARMILSTEIN Genetics of Immunoglobulins in the Mouse MICHAEL POTTERAND ROSE LIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues

JOHN B.

ZABRISUE

Lymphocytes and Transplantation Immunity DARCYB. WILSON AND R. E. BILLINGHAM Human Tissue Transplantation

JOHN P. MERRILL AUTHOR INDEX-SUBJECT INDEX Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. MULLER-EBERHARD Regulatory Effect of Antibody on the Immune Response JONATHANW. UHR AND GORAN MOLLER The Mechanism of Immunological Paralysis D. W. DRESSERAND N. A. MITCHISON I n Vitro Studies of Human Reaginic Allergy

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

THOMAS B. TOMASI,JR.,

AND

JOHN BIENENSTOCK

Immunologic Tissue Injury Mediated by Neutrophilic leukocytes

CHARLESG. COCHRANE The Structure and Function of Monocytes and Macrophages

ZANVIL A. COHN The Immunology a n d Pathology of NZB Mice J. B. HOWIE AND B. J. HELYER

AUTHOR INDEX-SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 10 Cell Selection by Antigen i n the Immune Response

GREGORYW. SISKINDAND BARUJBENACERRAF Phylogeny of Immunoglobulins

HOWARDM. GREY Slow Reacting Substance of Anaphylaxis ROBERT P. ORANGE AND K. FRANK AUSTEN Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response

OSCARD. UTNOFF Antigens of Virus-Induced Tumors

KARL HABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS AUTHOR INDEX-SUBJECTINDEX Volume 1 1 Electron Microscopy of the Immunoglobulins

N. MICHAEL GREEN Genetic Control of Specific Immune Responses HUGH 0. MCDEVITTAND BARUJBENACERRAF The Lesions i n Cell Membranes Caused by Complement JOHN H. HUMPHREYAND ROBERTR. DOURMASHKIN Cytotoxic Effects of Lymphoid Cells I n Vifro PETER PERLMANNAND G ~ R A HOLM N Transfer Factor

H. S. LAWRENCE Immunological Aspects of Malaria Infection

IVOR N. BROWN AUTHOR INDEX-SUBJECTINDEX Volume 12 The Search for Antibodies with Molecular Uniformity RICHARDM. m U S E Structure and Function of yM Macroglobulins

HENRYMETZGER

243

244

CONTENTS OF PREVIOUS VOLUMES

Transplantation Antigens R. A. REISFELD AND

B. D.

UHAN

The Role of Bone Marrow in the Immune Response NABIH I. ABDOU AND MAXWELL RICHTER Cell Interaction in Antibody Synthesis

D. W. TALMAGE, J. RADOVICH, AND H, HEMMINGSEN The Role of Lysosomes in Immune Responses GERALDWEISSMANNAND PETER DUKOR Molecular Size and Conformation of Immunoglobulins

KEITH

J. DORRINGTON AND

CHARLES

TANFORD

AUTHOR INDEX-SUBJECT INDEX

Volume 13 Structure and Function of Human Immunoglobulin E HANS BENNICH AND GUNNARJOHANSSON

s.

Individual Antigenic Specificity of Immunoglobulins

JOHN E. HOPPERAND ALFRED NISONOFF I n Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions

BARRY R. BLOOM Immunological Phenomena in Leprosy and Related Diseases J. L. TURKAND A. D. M. BRYCESON Nature and Classification of Immediate-Type Allergic Reactions

ELMERL. BECKER AUTHOR INDEX-SUBJECT INDEX

Volume 14 lmmunobiology of Mammalian Reproduction

ALAN E. BEER

AND

R. E. BILLINGHAM

Thyroid Antigens and Autoimmunity

SIDNEY SHULMAN Immunological Aspects of Burkitt's Lymphoma

GEORGEKLEIN Genetic Aspects of the Complement System CHESTER A. ALPER AND FRED

s. ROSEN

CONTENTS OF PREVIOUS VOLUMES

245

The Immune System: A M o d e l f o r Differentiation i n H i g h e r Organisms L. HOODAND J. PRAHL

AUTHOR INDEX-SUBJECT INDEX Volume 15 The Regulatory Influence of Activated T Cells on B Cell Responses t o Antigen

DAVIDH. KATZ

AND

BARUJ BENACERRAF

The Regulatory Role o f Macrophages i n Antigenic Stimulation

E. R. UNANUE Immunological Enhancement: A Study o f Blocking Antibodies

JOSEPH

D. FELDMAN

Genetics a n d Immunology o f Sex-Linked Antigens

DAVIDL. GASSERAND WILLYSK. SILVERS Current Concepts o f Amyloid

EDWARDc. FRANKLINAND DOROTHEAZUCKER-FRANKLIN

AUTHOR INDEX-SUBJECT INDEX Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, a n d ldiotypes

J. B. NATVIG AND H. G. KUNKEL Immunological Unresponsiveness

WILLIAM0. WEIGLE Participation o f Lymphocytes i n V i r a l Infections

E. FREDERICKWHEELOCKAND STEPHEN T. TOY Immune Complex Diseases i n Experimental Animals a n d M a n C. G. COCHRANE AND D. KOFFLER The lmmunopathology of Joint Inflammation i n Rheumatoid Arthritis

NATHAN J. ZVAIFLER AUTHOR INDEX-SUBJECT INDEX Volume 17 Antilymphocyte Serum

EUGENEM. LANCE,P. B. MEDAWAR,AND ROBERT N. TAUB

246

CONTENTS OF PREVIOUS VOLUMES

In Vitro Studies of Immunologically Induced Secretion of Mediators from Cells and Related Phenomena

ELMERL. BECKER AND PETER M. HENSON Antibody Response to Viral Antigens

KEITH M. COWAN Antibodies to Small Molecules: Biological and Clinical Applications VINCENT P. BUTLER, JR., AND SAM M. BEISER

AUTHOR

INDEX-SUBJECT

INDEX

Volume 18 Genetic Determinants of Immunological Responsiveness DAVIDL. GASSERAND WILLYS K. SILVERS Cell-Mediated Cytotoxicity, Allograft Rejection, and Tumor Immunity JEAN-CHARLES CEROTTINI AND K. THEODORE BRUNNER Antigenic Competition: A Review of Nonspecific Antigen-Induced Suppression HUGH F. P ~ o s sAND DAVIDEIDINCER Effect of Antigen Binding on the Properties of Antibody

HENRY METZGER lymphocyte-Mediated Cytotoxicity and Blocking Serum Activity to Tumor Antigens KARL ERIK HELLSTROM AND INGEGERD HELLSTROM

AUTHOR INDEX-SUBJECTINDEX Volume 19 Molecular Biology of Cellular Membranes with Applications to Immunology

S. J. SINGER Membrane Immunoglobulins a n d Antigen Receptors on B and T lymphocytes

NOEL L. WARNER Receptors for Immune Complexes on Lymphocytes

VICTOR NUSSENZWEIG Biological Activities of Immunoglobulins of Different Classes a n d Subclasses

HANSL. SPIEGELBERG SUBJECTINDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 20 Hypervariable Regions, idiotypy, and Anti body-Combi ni ng Site

J. DONALDCAPRA AND J. MICHAEL KEHOE Structure and Function of the J Chain

MARIAN ELLIOTTKOSHLAND Amino Acid Substitution and the Antigenicity

of Globular Proteins

MORRIS REICHLIN The H-2 Major Histocompatibility Complex and the I Immune Response Region: Genetic Variation, Function, and Organization DONALDC. SHREFFLERAND CHELLAS. DAVID Delayed Hypersensitivity in the Mouse

ALFRED J. CROWLE SUBJECT

A 5 8 6

c 7 D 8

E 9 F C H 1 1

O 1 2 3 4

INDEX

247

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