Advances in Immunology Volume 27

ADVANCES IN

Immunology

V O 1 U M E 27

CONTRIBUTORS TO THIS VOLUME

PETERC. DOHERTY EDWARDJ. GOETZL JON LINDSTROhf

IAN F. C. MCKENZIE TERRYPOTTER PETERF. WELLER ROLF M. ZINKERNAGEL

ADVANCES IN

Immunology EDITED B Y

HENRY G. KUNKEL

FRANK J. DIXON

The Rockefeller University New Yo&, New Yo&

Scripps Clinic and Research Foundation 10 Jolla, California

V O L U M E 27

1979

ACADEMIC PRESS

New York

Sun Francisco

A Subsidiary of Harcourf Brace lovanovich, Publishers

London

COPYRIGHT @ 1979, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 6 1- 17057 ISBN 0-12-022427-5 PRINTED IN THE UNITED STATES OF AMERICA 79 80 81 82

9 8 7 6 5 4 3 2 1

CONTENTS LIST OF CONTRIBUTORS PREFACE . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii ix

Autoimmune Response to Acetylcholine Receptors in Myasthenia Gravis and Its Animal Model JON LINDSTROM

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . I1. Neuromuscular Transmission . . . . . . . . . . . . . . . . 111. Molecular Properties of the Acetylcholine Receptor (AChR) . .

. . . IV. Clinical Features of Myasthenia Gravis . . . . . . . . . . . . . V. Experimental Autoimmune Myasthenia Gravis (EAMG) in Rats .

VI . VII . VIII . IX .

. . . . .

. . . . .

Experimental Autoimmune Myasthenia Gravis in Other Species Autoimmune Response to AChR in Human MG . . . . . . . . . . Other Autoimmune Anti-Receptor Diseases . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 5 11 14 27 33 42 43 44

MHC-Restricted Cytotoxic T Cellr: Studies on the Biological Role of Polymorphic Major Transplantation Antigens Determining T-cell Restriction.Specificity. Function. and Responsiveness

ROLF M. ZINKERNACEL AND PETERC . DOHERTY

I . Introduction . . . . . . . . . . . . I1. Virus-Specific Cytotoxic T Cells . . 111. Definition of Target Antigens . . .

............... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. Ontogeny of Effector Cells: The Role of the Major Histocompatibility Gene Complex in Defining T-cell Specificity during Ontogeny . . . V. Role of the Major Histocompatibility Gene Complex in Determining T-cell Responsiveness . . . . . . . . . . . . . . . . . . . . . . VI . Interpreting MHC Restriction and Ir Regulation of T Cells . . . . . VII . I n Vivo Relevance of MHC-Restricted Cytotoxic T Cells . . . . . . VIII . Finale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations Used in the Text . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52 59 74 96 109 118 128 141 141 142

Murine lymphocyte Surface Antigens IAN F. MCKENZIEAND TERRYPOTTER

c.

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Classification of Alloantigenic Determinants . . . . . . . . . . .

111. Production and Testing of Antisera

. . . . . . . . . . . . . . . . . .

V

181 182 187

vi IV. V. VI . VII . VIII . IX . X. XI * XI1 . XI11. XIV.

xv.

CONTENTS Characterization of Antisera . . . . . . . . . . . . . . . . . . . . Histocompatibility ( H ) Loci-CMAD of General Distribution . . . . Lymphocyte Alloantigens . . . . . . . . . . . . . . . . . . . . . Erythrocyte Alloantigenic (En) Loci . . . . . . . . . . . . . . . . Miscellaneous Antigens . . . . . . . . . . . . . . . . . . . . . . Xenoantisera Recognizing Lymphocyte Cell-Membrane Determinants Relationship of Murine Leukemia Virus (MuLV) and CMAD . . . . Functional Studies with Serological Markers . . . . . . . . : . . . CMAD in Studies of T-cell Ontogeny and Differentiation . . . . . . CMAD in B-Cell Differentiation and Ontogeny . . . . . . . . . . . Expression of CMAD on Mouse Leukemias and Lymphomas . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations Used in the Text . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . .

195 201 205 254 258 266 272 283 303 309 313 320 322 322 338

The Regulatory and Effector Roles of Eorinophilr

PETER F. WELLERAND EDWARDJ . GOETZL

I. I1. I11. IV. V. VI . VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Eosinophil Production and Distribution . . . . . . . . . . . . . . Cellular Properties of Eosinophils . . . . . . . . . . . . . . . . . General Functions of the Eosinophil . . . . . . . . . . . . . . . . Involvement of Eosinophils in Immunological Responses . . . . . . The Role of the Eosinophil in the Host Response to Helminthic Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . ... References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . CONTENTSOF PREVIOUSVOLUMES. . . . . . . . . . .

......... .........

339 340 347 349 354 360 364 365 373 377

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

PETERC . DOHERTY, The Wistar Institute, Philadelphia, Pennsylvania (51) EDWARDJ . GOETZL,The Howard Hughes Medical Znstitute Laboratory at Harvard Medical School, and Departments of Medicine, Harvard Medical School and the Robert B. Brigham Hospital Division of the Afiliated Hospitals Center, Inc., Boston, Massachusetts (339) JON

LINDSTROM,The Salk Institute, Sun Diego, California 921 12 ( 1 )

IAN F. C . MCKENZIE,Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria 3084, Australia ( 179)

TERRYPOTTER,Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria 3084, Australia ( 179) PETER F. WELLER,The Howard Hughes Medical Znstitute Laboratory at Harvard Medical School, and Departments of Medicine, Harvard Medical School and the Robert B. Brigham Hospital Division of the Afiliated Hospitals Center, Znc., Boston, Massachusetts (339) ROLF M. ZINKERNAGEL, Department of Zmmunopathology, Scripps Clinic and Research Foundation, La Jolla, California (51)

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PRE FAC E

It is never possible to predict explicitly the directions that science will take in its probing of the unknown. This is certainly the case with inimunology and the last few years, particularly, have seen some surprising developments. Who could have predicted that hybridomas would spell the doom of the rabbit as the primary antibody producer? The utility of this system is already apparent in several of the reviews in this volume. In the area of human disease, etiological mechanisms of immune dysfunction are uniquely unpredictable and even more difficult to document. However, the recent exciting developments regarding myasthenia gravis discussed in this volunie probably represent a major exception; a prediction almost 20 years ago that it might be caused by antibodies to acetylcholine receptors appears to have been fulfilled. The first article, by Lindstrom, covers these recent developments regarding myasthenia gravis. He and his associates made the surprising finding that acetylcholine receptors isolated from the electric eel produced myasthenia symptoms when injected into rabbits. Similar observations were later made in the rat and in other species, and the disease appears to result from the production of antibodies to the electric eel receptors that cross react with the animal’s own receptors. Patients with myasthenia gravis have similar antibodies that can be quantitated by radioimmune assays using labeled receptors obtained from human muscle. Some gaps still remain concerning this disease, such as the stimulus giving rise to the antibodies, but the extraordinary similarity between the natural disease and the experimental model indicates a common mechanism. Zinkernagel and Doherty have written the second article on the subject of what is now generally described as the ZinkernagelDoherty-Shearer phenomenon. They made the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the MHC. The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems such as T N P coupled to cells, minor histocompatibility antigens, and the H-Y model. The significance of this work can scarcely be overestimated, since it goes far in explaining disease associations with histocompatibility antigens, the extreme polymorphism of the MHC system, and the strong linkage disequilibrium among the MHC genes. ix

X

PREFACE

The third article, by McKenzie and Potter, is an in-depth review of the multiple lymphocyte surface antigens of the mouse. A wide range of different antigens is described in detail, ranging from those that have a broad tissue distribution to those highly restricted to specific lymphocytes and including such others as viral-associated antigens. Special emphasis is placed on the Ly markers, in view of their extreme utility in defining functional subsets of lymphocytes. However, the new Qa antigens and the Za system are also discussed in detail. This article should prove to be a very valuable compendium of this highly specialized field that has assumed such broad significance. The final article, by Weller and Goetzl, covers the regulatory and effector role of eosinophils. Much has been learned recently about this long mysterious cell associated with certain immunological reactions. Surprising findings such as the passive transfer of eosinophilia by T cells and the special role of the stimulated mast cell in local eosinophilia have come to the fore. Factors also have been obtained that are preferentially chemotactic for eosinophils as compared to other types of leukocytes. Most importantly, the long-noted association of eosinophilia with infection by helminths has been given special significance by increasing evidence for the concept of a protective effector function of these cells in parasitic infections. The Editors wish to express their gratitude to the publishers for their constant cooperation in producing this volume. H. G. KUNKEL F. J. DIXON

.

ADVANCES IN IMMUNOLOGY. VOL 27

Autoimmune Response to Acetylcholine Receptors in Myasthenia Gravis and Its Animal Model JON LINDSTROM The Salk Institute. San Diego. California

I . Introduction .......................................................... I1. Neuromuscular Transmission .......................................... 111. Molecular Properties of the Acetylcholine Receptor (AChR) ............. A . Electrophysiological Properties ..................................... B. Biochemical Properties ............................................ C . Synthesis and Destruction .......................................... IV. Clinical Features of Myasthenia Gravis ................................. V. Experimental Autoimmune Myasthenia Gravis (EAMG) in Rats .......... A . Immunization with AChR .......................................... B . Acute and Passive EAMG .......................................... C . Chronic EAMG ................................................... D . Effects of Bound Antibody on AChR Function ....................... E . Effect of Complement on Amount of AChR .......................... F. Effect of Antigenic Modulation on Amount of AChR ................. VI . Experimental Autoimmune Myasthenia Gravis in Other Species ......... A . Rabbits ........................................................... B. Mice, Guinea Pigs, Goats, Monkeys, and Frogs ...................... VII . Autoimmune Response to AChR in Human MG ........................ A . Antibodies to AChR ............................................... B. Pathological Mechanisms Impairing Transmission .................... C . Cause of the Autoimmune Response to AChR ....................... VIII . Other Autoimmune Anti-Receptor Diseases ............................. IX. Concluding Remarks .................................................. References ...........................................................

1 3

5 5 6 9 11 14 14 17 22 24 25 26 27 27 31 33 33 36 39 42 43 44

.

I Introduction

Myasthenia gravis (MG) is a disease characterized by weakness and fatigability of voluntary muscles . It was long suspected that the weakness resulted from impaired transmission of signals from nerve to muscle. but until recently it was not clear whether transmission was impaired by a defect in the nerve ending or in the muscle The similarity of the features of myasthenic weakness to curare poisoning. thymic abnormalities. the occasional presence in serum of antibodies reacting with muscle striations. and the occasional occurrence of a transient form of myasthenia in newborn babies of myasthenic mothers suggested to some workers years ago (Simpson. 1960) that MG was an autoimmune disease mediated by antibodies to acetylcholine recep-

.

1 Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved . ISBN 0-12-022427-5

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JON LINDSTROM

tors that competed for binding with acetylcholine, but methods were not available to test this hypothesis and it was later rejected (Simpson, 1971). It is now known that MG is an autoimmune disease in which transmission is impaired by an autoimmune response to acetylcholine receptors in the postsynaptic membrane of the muscle. The antibodies to receptor do not act as competitive antagonists of receptor, but impair transmission primarily by causing reduction in the amount of receptor. Biochemical study of pathological mechanisms in MG has been dependent on development of methods for quantitating and purifying acetylcholine receptors (AChRs). Studies of AChR biochemistry have grown to some extent synergistically with studies of MG, since antibodies to AChR are proving to be useful reagents for studies of AChR. The seminal experiment that directly implicated a postsynaptic defect in MG was the observation that in these patients the ultrastructure of the postsynaptic membrane was altered (Engel and Santa, 1971). The seminal experiments that directly implicated an autoimmune response to AChR as a cause of MG and initiated the necessary methodology for subsequent tests of this implication were done in rabbits (Patrick and Lindstrom, 1973; Patrick et al., 1973). A protein capable of specifically binding cholinergic ligands was purified from the electric organs of electric eels (Lindstrom and Patrick, 1974). Rabbits immunized with this protein produced antibodies to it that inhibited AChR activity in electric organ cells. This showed that the protein comprised at least part of the physiologically significant AChR. Surprisingly, the immunized rabbits also weakened and died. The rabbits had a flaccid form of paralysis and decrementing electromyograms, and both the paralysis and decrement were relieved by inhibitors of acetylcholinesterase. These are diagnostic features of MG and showed that neuromuscular transmission was impaired. An autoimmune response to muscle AChR seemed an obvious mechanism. Subsequently, the model of MG produced by immunizing animals with purified AChR was termed experimental autoimmune myasthenia gravis (EAMG).The seminal experiment suggesting the importance of AChR loss to the pathology of MG was the observation that fewer AChR binding sites could be detected in muscle from MG patients (Fambrough et al., 1973). The pathological mechanisms impairing neuromuscular transmission in MG and EAMG have turned out to be quite similar. In both cases the primary effect of the immune response is not to produce antibodies to AChR that block AChR activity, but rather to produce

AUTOIMMUNE RESPONSE TO ACETYLCHOLINE RECEPTORS

3

antibodies to AChR that cause a decrease in the amount of AChR. In addition, postsynaptic membrane structure is altered and receptor function somewhat impaired. The net effect of all these assaults to the muscle is to reduce its sensitivity to acetylcholine released from the nerve endings and thereby to inhibit effective transmission of signals fi-om nerve to muscle. In order to understand the pathological mechanisms involved in MG and EAMG it will be first necessary to briefly review the process of neurotransmission as well as AChR structure and function. Then studies of the autoimmune response to AChR in MG and its animal model will be reviewed. II. Nauromuscular Transmission

Transmission of signals from motor neurons to striated muscle fibers is an archetypic example of chemically mediated neurotransmission (reviewed in Katz, 1966).A motor neuron cell body in the spinal cord extends a myelinated axon to its ending adjacent to a specialized area of membrane at a single point on a muscle fiber, usually near the middle of the fiber. A synapse between a motor neuron and a striated muscle fiber is called an endplate. Impulses are conducted from the neuron cell body along the axon by means of action potentials, which involve the propagation of signals by successive waves of opening and closing ion channels. The opening and closing of these channels is regulated by the electrical potential across the membrane. A depolarization of the normally negative membrane potential is produced when an action potential invades the nerve ending. In the ending, acetylcholine is stored in 500-A vesicles containing around lo4acetylcholine molecules (Kuffler and Yoshikami, 1975). Depolarization results in exocytosis of these acetylcholine “quanta” from specialized areas of the presynaptic membrane adjacent to areas in the postsynaptic membrane where receptors are most concentrated (Heuser et al., 1975).At a nerve ending on a human intercostal muscle fiber, for example, about 60 quanta are released per impulse (Lambert and Elmquist, 1971). Acetylcholine released from the presynaptic membrane of the nerve diffuses across a 600-A gap to the postsynaptic membrane where acetylcholine receptors are located (Fertuck and Salpeter, 1974). The postsynaptic membrane is organized in a regular array of folds typically 0.5-1 pm deep (Fertuck and Salpeter, 1976). AChR are concentrated in the tips of these folds, where they are the principal membrane protein (30,000 sites/p2) (Fertuck and Salpeter, 1976). Binding of acetylcholine to an AChR triggers the transient opening of a cation-

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JON LINDSTROM

specific channel through which sodium and potassium ions passively flow according to their concentration gradients across the cell membrane (generation of endplate potentials is reviewed in Gage, 1976). The resulting decrease in potential across the postsynaptic membrane is conducted electrotonically along the membrane to areas outside the endplate that are electrically excitable. If enough AChRs are activated to reduce the membrane potential in the electrically excitable regions below threshold, an action potential is triggered that is propagated along the muscle and activates the contractile machinery. If too few AChRs are active, transmission fails. Normally, the amount of acetylcholine released and the number of AChRs activated is much larger than the minimum necessary. This provides a large safety factor ensuring effective neuromuscular transmission. Transmission is terminated by removal of acetylcholine from the cleft by diffusion and destruction by acetylcholinesterase. Acetylcholinesterase is associated with basement membrane and is localized over the whole surface of the postsynaptic membrane (McMahan et al., 1978). Endplate potentials are produced in muscle fibers by the cascade of quanta released by nerve stimulation after a delay of about 0.7 msec required for release, diffusion, and binding of acetylcholine. The endplate potential develops rapidly, and within 0.5 msec the membrane potential changes from something like -70 mV to -50 mV or less, which is sufficient to trigger an action potential (reviewed in Katz, 1966). The endplate potential decays over a few milliseconds. After inhibition of acetylcholinesterase, local depolarization may be prolonged to 100 msec (Katz and Miledi, 1973a). Miniature endplate potentials (of about 1 mV) have a similar time course to endplate potentials. Miniature endplate potentials are produced by the spontaneous random release of single quanta of acetylcholine at the rate of about 1 per second. When it was initially observed that miniature endplate potentials in muscle from MG patients were abnormally small (Elmquist et al., 1964), it was erroneously concluded that this was due to a defect in the nerve ending, resulting in packaging of insufficient acetylcholine into quanta. This misled many into thinking that the lesion in MG was presynaptic. However, later the observation that the postsynaptic membrane of MG patients contained fewer and smaller postsynaptic folds (Engel and Santa, 1971) showed that there was a postsynaptic lesion. We now know that the decreased amplitude of miniature endplate potentials found in MG and EAMG (Lambert et al., 1976) results primarily from decreased acetylcholine sensitivity due to reduced numbers of AChR (Fambrough et al., 1973; Engel et al., 197713; Lindstrom and Lambert, 1978).

AUTOIMMUNE RESPONSE TO ACETYLCHOLINE RECEPTORS

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Electromyography uses extracellular needle electrodes to measure action potentials in muscle fibers near the electrode. It is a much cruder technique than the intracellular microelectrodes used in the studies previously discussed, but it can be used clinically. At low rates of nerve stimulation (for example 5 per second) the amplitude of an electroymyogram response is normally constant, because at each nerve stimulation an action potential is triggered in every fiber. In MG patients or animals with EAMG, the electromyogram response decreases on successive stimuli (Seybold et al., 1976)because with succeeding stimuli an increasing proportion of fibers do not have action potentials. This is thought to be the electrophysiological analog of the fatigability that is characteristic of myasthenic weakness. The cause of the decrement is not known with certainty, but such a decrement can be produced simply by reducing the number of active AChRs by use of a specific AChR antagonist (Satyamurti et al., 1975). Nerve endings are known to release decreasing amounts of acetylcholine on successive stimuli (Otsuka et al., 1962). In normals, the amount of AChR may be sufficient to efficiently respond to this acetylcholine and produce an endplate potential that in every fiber is sufficient to trigger an action potential. In MG and EAMG the amount of AChR may be reduced to the point where a slight reduction in the amount of acetylcholine released produces endplate potentials in many fibers that are too small to trigger action potentials. Also, desensitized AChR may accumulate on successive stimuli, reducing the amplitude of the endplate potential. Use of antiesterase drugs eliminates the decrementing electromyogram response in MG and EAMG (Seybold et al., 1976) by prolonging the action of acetylcholine. 111. Molecular Properties of the Acetylcholine Receptor (AChR)

A. ELECTROPHYSIOLOGICAL PROPERTIES Binding of acetylcholine to AChR causes an increase in cation permeability of the membrane via mechanisms that have been much studied but are little understood. However, as a result of many elegant studies, the AChR is by far the best characterized neurotransmitter receptor both electrophysiologically and biochemically. In the resting state, AChR molecules apparently have a relatively low affinity for acetylcholine (K, low4to 10-5M) (Grunhagen et al., 1977; Dionneet al., 1978)which is, however, quite sufficient to ensure binding of acetylcholine at the high local concentrations present in the synaptic cleft to M ) (Kuffler and Yoshikami, 1975). Because of the high

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JON LINDSTROM

local concentration of AChRs, the amount of acetylcholine in a single quantum does not saturate all the AChRs in the 1 p2 area it affects (Kuffler and Yoshikami, 1975). Binding of one or more acetylcholine molecules per AChR rapidly alters it to an active conformation with higher binding affiity (KD 10+ M ) and opens a cation-specific ionophore through which sodium, potassium, and larger ions can passively flow according to their concentration gradients across the membrane (Stevens, 1976; Dionne et al., 1978). The activated conformation is transient, normally lasting about 1 msec, but channel opening time is affected by temperature (Katz and Miledi, 1972), local anesthetics (Ruff, 1977), use of agonists differing in structure from acetylcholine (Colquhaun et al., 1975), or the binding of antibodies (Heinemann et al., 1977). Normally, the current that flows through an open channel corresponds to about 50,000 monovalent ions (Katz and Miledi, 1972). Thus, it is evident that the net effect of the chemical step in transmission between nerve and muscle is to greatly amplify the currents involved in the action potential propagated along the small nerve axon so that the currents are sufficient to trigger an action potential in the much larger muscle fiber. An activated AChR, especially after prolonged exposure to high concentrations of acetylcholine, may relax into a “desensitized” state characterized by a much M ) (Grunhagen et al., 1977; higher affinity for agonists (KD Dionne et al., 1978). The desensitized state is thought to relax over seconds through an unliganded conformation and back to the resting state. Competitive antagonists of AChR, like curare and cobra venom toxin, are classically thought to act by binding to the acetylcholine binding site of AChR and stabilizing the bound AChR in the resting conformation. In fact, it has recently become apparent that local anesthetics, agonists, and antagonists, which are all cations, may also to various extents enter the open ion channel of the activated AChR and transiently block it (Neher and Steinbach, 1978). In fact, quite large organic cations can pass through activated AChR channels (Huang et al., 1978). Local anesthetics may also allosterically affect AChR, stabilizing the desensitized conformation (Sugiyama and Changeux, 1975).

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8. BIOCHEMICAL PROPERTIES Biochemical studies of AChR have depended greatly on two zoological curiosities. One of the types of small protein toxins present in the venom of cobras and kraits binds with great affinity and specificity to the acetylcholine binding site of AChR (Lee, 1972). Thus, toxin labeled with lZ5I(Fertuck and Salpeter, 1976) or peroxidase (Engel et

AUTOIMMUNE RESPONSE TO ACETYLCHOLINE RECEPTORS

7

al., 1977b) can be used to localize and quantitate AChR in tissue sections. Radioactive toxin is routinely used to identify and quantitate solubilized AChR, and toxin conjugated to agarose can be used as an affinity adsorbent for purifying AChR (Lindstrom and Patrick, 1974; Eldefrawi and Eldefrawi, 1973; Biesecker, 1973). AChR can also be purified using synthetic acetylcholine analogs (Changeux et al., 1976; Raftery et al., 1976; Chang, 1974; Karlin et al., 1976). Amounts of AChR are usually expressed as moles of toxin binding sites. The use of toxin solved the problem of readily identifying AChR in membrane fragments or solution, where its electrophysiological function was not easily monitored. Another problem is the small amount of AChR in muscle. Although AChR are packed at a density of 2 to 4 x 104 sites/ pm2 (Fertuck and Salpeter, 1976) at the tips of junctional folds, an endplate on a muscle fiber contains only 4 to 6 x lo7 molecules of AChR (Fambrough et al., 1973), making a total of only about 1 x lo-'' mol per gram of muscle (Lindstrom, 1977a) or about 6 x lo-" mol per rat (Lindstrom et al., 1976a). Fish electric organs use AChR directly in generating the current for their electrical discharges (Bennett, 1970), so electric organs contain much higher concentrations of AChR than mol of AChR muscle. Torpedo electric organ contains about 1 x per gram (Changeux et al., 1976). Electric organ is such a rich source of AChR that membrane fragments can be prepared that contain 50% or more of their protein as AChR (Sobel et al., 1977). These fragments form closed vesicles that retain 22Na+,and AChR activity can be measured in witro as agonist-sensitive zzNa+efflux from these membrane vesicles (Kasai and Changeux, 1971; Popot et al., 1976; Hess and Andrews, 1977). The AChR is an integral membrane protein that spans the postsynaptic membrane, extending out around 15 A on the intracellular surface of the membrane, nearly 55 A on the extracellular surface (Ross et al., 1977),with an overall length of 110 A. AChRs are approximately 85 A in diameter viewed from the extracellular surface (Cartaud et aZ., 1978). From this view, an AChR appears as a rosette of several subunits with a hydrophilic negatively staining center. It is not known whether this center staining corresponds to the ionophore. AChR solubilized in detergent looks like the AChR seen in membrane fragments. AChR purified from electric organs is a glycoprotein composed of several dissimilar polypeptide chains (Karlin et al., 1976; Raftery et al., 1976; Lindstrom et al., 1979b). Its isoelectric point is -5.0, and its amino acid composition is unremarkable. AChR purified from Torpedo californica contains four kinds of polypeptide chains of apparent molecular weights variously reported to be about 38,000, 50,000, 57,000

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JON LINDSTROM

and 64,000 (Raftery et al., 1976; Karlin et al., 1976). These chains are referred to as a,p, y, and 6, respectively. The mole ratio of a :/3 :y : 6 is probably 2 : 1: 1: 1 (Reynolds and Karlin, 1978; Lindstrom et al., 1979a). The a chain is known to contribute to the acetylcholine binding site because it is &nity labeled with a specific antagonist (Karlin et al., 1976). Functions of the other chains are not known, though it is thought that some might be components of the ionophore. Detergent solubilized AChR exists as 9.5 S monomers of -250,000 MW (Reynolds and Karlin, 1978) and dimers formed by disulfide bridges between the 6 chains (Chang and Bock, 1977; Hamilton et al., 1979). AChR monomers have 2 acetylcholine binding sites (Damle and Karlin, 1978). There is, in fact less general agreement about AChR structure than the preceding paragraph suggests. It has been reported that membrane fragments can be prepared from Torpedo marnorata electric organ that contains only two sizes of polypeptide chain of molecular weights 41,000 and 43,000 (Sobel et al., 1977). The 41,000 MW chain is reported to be the receptor per se, and the 43,000 MW chain is thought to be the ionophore it regulates (Sobel et al., 1978). These components are separated when the membranes are solubilized in Triton X-100 (Sobel et al., 1978; Eldefrawi et al., 1977). Both the membrane fragments and soluble receptor purified from Torpedo marnorata appear by electron microscopy to be identical to those from Torpedo californica (Cartaud et al., 1978; Ross et al., 1977), and the monomer size on sucrose gradients is the same. Although the receptor from T. marmorata is reported to be composed solely of identical 41,000 MW chains, its specific activity for toxin binding is less than or equal to that from T. californica, which is composed of not only the corresponding a chain, but also p, y, and 6 chains (Sobel et al., 1977; Karlin et al., 1976; Raftery et al., 1976). Another similar conflict in the literature is that AChR purified from cultured muscle cells (Merlie et al., 1977) or from denervated rat muscle (Dolly and Barnard, 1977) has been reported to consist only of 41,000 MW chains, whereas other groups have purified AChR from cultured cells (Boulter and Patrick, 1977) or from denervated rat muscle (Forehner et al., 1977) at similar specific activity and found it to be composed of several polypeptide chains, rather like Torpedo californica AChR. It seems unlikely that everyone is right. One possibility is that, on solubilization from the membrane, AChR is irreversibly associated in mixed detergent micelles with contaminating proteins. However, highly purified membrane fragments from Torpedo californica, which appear by electron microscopy to be composed of nearly side-by-side AChR, are composed prominently of all 4

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chains (Hucho et al., 1978; Hamilton et al., 1979). Also, the same 4 chains are observed in purified AChR when detergents as different as Triton X-100 and sodium cholate are used for solubilization (Lindstrom et al., 1979a). Another possibility is that the preparations that appear to be composed of predominantly 41,000 MW chains are the result of proteolytic (and perhaps other lytic) damage during purification. It is known that proteolysis of AChR from electrophorus can reduce it to a single component on acrylamide gels (Lindstrom et al., 1976~). Antisera specific for all 4 polypeptide chains in Torpedo califormica have been prepared. It was observed that rats immunized with any of the polypeptide chains developed EAMG (Lindstrom et al., 1978b). Antisera to each torpedo chain showed specific cross-reaction with AChR from human muscle. Further, each subunit of AChR purified from electric eels cross-reacts with antibodies to a different subunit of AChR purified from torpedo (Lindstrom et al., 197913). Together these results suggest that AChR from electric organs and muscles have a similar subunit composition including some functionally important determinants conserved over wide evolutionary distances. Until recently (Epstein and Racker, 1978; Schiebler and Hucho, 1978), it has not been possible to reproducibly reconstitute AChR into an artificial membrane in such a way that addition of agonists causes an increase in cation conductance, and it has not yet been possible to reproducibly reconstitute purified AChR (Briley and Changeux, 1977). Thus it is not known with certainty whether purified AChRs contain ionophores, but evidence suggests that they do (Anholt et al., 1979). Histrionicotoxin behaves as a local anesthetic (Kato and Changeux, 1976) and is reported to affect ionophore function (Eldefrawi et aZ., 1977) by stabilizing the desensitized conformation of the AChR (Burgermeister et al., 1977). Components that bind histrionicotoxin and are distinct from purified AChR have been reported (Eldefrawi et aZ., 1977; Sobel et al., 1978).Whether these components are ionophores remains to be proved. Monospecific antibodies to components of purified polypeptides from electric organ membranes might prove to be valuable reagents for identifying the ionophore and characterizing its function. Figure 1 depicts the possible structure of the AChR.

c. SYNTHESIS AND DESTRUCTION Synthesis of AChRs has been studied using muscle cells in tissue culture (Devreotes and Fambrough, 1975; Merlie et aZ., 1975; reviewed in Fambrough et al., 1978).These cells resemble muscle cells

T

FIG.1. Structure ofthe acetylcholine receptor (AChR). This is a fanciful depiction of some features of AChR structure. Many of these features are generally agreed on, but others, the subunit structure in particular, are not. Some of the features are supported by facts, others only by reasonable speculation. The AChR molecule is represented as an integral membrane protein spanning the membrane. It is composed of several distinct polypeptide chains. Each polypeptide chain is at least partially exposed on the extracellular surface, and at this surface each has some carbohydrate bound. Each AChR has two acetylcholine binding sites, which regulate a single ionophore by way of a small conformational change.

in fetal animals before innervation (Burden, 1977) or muscle fibers in mature animals after the nerve has been cut (Chang and Huang, 1975) in that in all these cases AChRs are relatively abundant and found scattered over the surface membrane, and these AChRs turn over with the relatively short half-life of about 22 hours. This contrasts with mature innervated muscle fibers, which contain less than one-tenth the amount of AChR, and this is concentrated only at the endplate in patches of specially architectured postsynaptic membrane. Junctional AChR at endplates turns over much more slowly than extrajunctional receptor, having a half-life in excess of 5 days (Berg and Hall, 1974; Hogan et al., 1976). There is evidence for a small, as yet undefined, chemical difference between junctional and extrajunctional AChR (Brockes and Hall, 1975). Synthesis of membrane proteins is only beginning to be understood

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(Blobel and Doberstein, 1975; Rothman and Leonard, 1977; reviewed in Lodish and Rothman, 1979). It seems likely that this may be accomplished by membrane-bound ribosomes that extrude the newly synthesized polypeptide chain through the membrane N-terminal first. In the case of a large, multisubunit glycoprotein like AChR, substantial postsynthetic modifications are probably involved, in addition to whatever cleavage of “pre” and “pro” sequences may be involved. In muscle cells in tissue culture approximately 20-30% of AChR are not exposed on the surface membrane (Devreotes et al., 1977; Patrick et al., 1977). Newly synthesized AChR are localized in the Golgi apparatus, and about 3 hours are required before they appear in the plasma membrane (Devreotes et al., 1977; Fambrough and Devretoes, 1978). In culture, synthesis of AChR occurs at a constant rate, which somewhat exceeds a constant rate of destruction. Synthesis and destruction are not closely coupled (Devreotes and Fambrough, 1975). AChR appear to be selected randomly for destruction, which occurs by a process involving internalization, proteolysis, and release of degraded amino acid residues into the medium (Devreotes and Fambrough, 1975; Merlie et al., 1975). The processing time between removal from the surface membrane and release of degraded residues is of the order of 40-50 minutes. The AChR content of muscle cells in tissue culture appears to be normally regulated through control of the rate of AChR synthesis rather than AChR degradation (Merlie et ul., 1975; Devreotes and Fambrough, 1975). As will be discussed later, this contrasts with what happens after the AChR have reacted with antibodies. Then the rate of AChR destruction is increased, resulting in a net decrease in AChR content (Heinemann et al., 1977; Kao and Drachman, 1977; Appel et al., 1977; Heinemann et al., 1978). It is not known whether normal turnover of AChR and antigenic modulation occur by similar mechanisms, although similar processing times are involved, the same inhibitors affect both, and the release of degraded amino acid residues results in both cases. IV. Clinical Features of Myasthenia Gravis

The most characteristic clinical feature of MG is muscle weakness and fatigue. [Clinical features and treatment have recently been well and concisely reviewed in Drachman (1978).] A decrementing electromyogram response to repetitive nerve stimulation, which is relieved by administering a short-acting inhibitor of acetylcholinesterase like tensilon, is a frequently used objective diagnostic criterion. As will be discussed later, radioimmune assay of anti-AChR antibodies in

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serum can provide an equally sensitive objective diagnostic technique (Lindstrom, 1977a; Lindstrom et al., 1976d; Appel et al., 1975; Mittag et al., 1976; Aharonov, 1975a; Monnier and Fulpius, 1977), which, if the assay were routinely available to clinicians, might provide for more convenient diagnosis. The clinical picture in MG is varied, often complicating diagnosis. In the mildest forms extraocular muscles are most affected, resulting in drooping eyelids (ptosis) and double vision (diplopia). In more generalized cases, other muscle groups may be affected in the trunk or extremities. Which muscles are most severely affected varies among patients. And, as in some other autoimmune diseases, the severity in an individual may vary with time, resulting in spontaneous remissions and exacerbations. The most common form of therapy for MG patients is treatment with inhibitors of acetylcholinesterase. These are quite effective in many patients. Treatment with esterase inhibitors is thought to increase the effective concentration and duration of acetylcholine in the synaptic cleft, and may permit it to diffuse over a wider area. At endplates in an MG patient, where the amount of AChR is reduced (Fambrough and Drachman, 1973; Engel et al., 1977, Lindstrom and Lambert, 1978) and where many of the AChR that remain have antibodies bound (Engel et al., 1977a; Lindstrom and Lambert, 1978) and may not be advantageously arrayed owing to alteration of postsynaptic membrane structure (Engel et al., 1977b),the effect of antiesterase treatment is to produce greater numbers of activated AChRs, thus increasing the probability that enough will be activated to trigger an action potential in the muscle. Control of dosage is critical because underdosage results in weakness and overdosage may also result in weakness and even death. This is because prolonged exposure to high concentrations of acetylcholine results in the accumulation of desensitized AChRs that are inactive. In laboratory animals, prolonged treatment with high doses of antiesterase agents also results in simplification of postsynaptic membrane structure and loss of AChR (Wecker et al., 1978; Fambrough and Drachman, 1973; Chang et al., 1973). Given variation in weakness due to activity during the day as well as spontaneous remissions and exacerbations, determination of dosage is highly empirical. Thymectomy is frequently used to treat patients who are not well controlled on antiesterase drugs alone. About 10-15% of MG patients have a thymoma, and thymic hyperplasia or germinal centers are observed in 7 5 4 5 % (Castleman, 1966).Thymectomy is reported to have some beneficial effects, especially in younger patients, though these benefits are thought to accrue over several years (Simpson, 1958; Buckingham et al., 1976). The mechanism by which thymectomy may

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produce any beneficial effects is not known. Although it was reported that thymectomy caused a decrease in serum anti-AChR titer (Scadding et al., 1977), we have found no correlation between thymectomy and anti-AChR titer (Seybold et al., 1978). Steroids are also used to treat MG patients (Mann et al., 1976; Kjair, 1971; Seybold and Drachman, 1974; Howard et al., 1976). These are effective in many cases. Dosage is varied during the course of treatment and is often given on alternate days to minimize adverse side effects of steroid therapy. Steroids may have some direct effects on neuromuscular transmission (Wilson et al., 1974). Some patients undergoing altemate-day prednisone therapy show decreased serum anti-AChR antibody concentration (Seybold and Lindstrom, 1979), and a cell-mediated response to AChR decreases after steroid therapy (Abramsky et al., 1975b),but the effects of steroids on the pathology of MG are not yet well characterized. Azathioprine and 6mercaptopurine are immunosuppressive drugs that have been effective also in the treatment of MG (Mattell et al., 1976). Clinical improvement concomitant with prolonged azathioprine treatment is associated with decreased concentrations of anti-AChR antibody in serum (Hertel, 1979). Plasmapheresis (plasma exchange) coupled with immunosuppressive drug therapy (Pinching et al., 1976; Dau et al., 1977, 1979; Newsome-Davis et al., 1978)and thoracic duct drainage (Mattell et al., 1976; Bergstrom et al., 1975) have been used to treat severely affected patients resistant to other forms of therapy. Thus far only small numbers of patients have been investigated, but plasmapheresis appears to be quite effective in many cases (Pinching et al., 1976; Dau et al., 1977, 1979). Titer of serum anti-AChR decreased during treatment preceding clinical improvement, and increases in titer were associated with clinical exacerbations (Dau et al., 1977; Newsome-Davis et al., 1978). Electromyographic studies (Denys et al., 1979) showed that improved neuromuscular transmission occurred concomitant with decreased antibody concentration. A minimum delay of 2 days was observed between decrease in anti-AChR titer and subsequent clinical improvement (Newsome-Davis et al., 1978), suggesting that clinical improvement resulted from the accumulation of newly synthesized AChR after a decrease in the rate of antibody-mediated AChR loss. Following thoracic duct drainage, reinfusion of plasma was reported to produce clinical deterioration (Bergstrom et al., 1975).Together, these results correlate well with additional evidence, to be discussed later, suggesting that the immune attack on AChR in MG is usually mediated primarily by antibodies rather than cells.

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JON LINDSTROM V. Experimental Autoimmune Myasthenia Gravis (EAMG) in Rats

A. IMMUNIZATION WITH AChR

EAMG has been produced in Lewis rats by immunization with AChR purified from the electric organs of electric eels and torpedos (Lennon et al., 1975; Lindstrom et al., 1976c) and with AChR purified from normal Lewis rat muscle (Lindstrom et al., 1976a) and fetal calf muscle (Merlie and Lindstrom, unpublished). Denaturation of AChR by sodium dodecyl sulfate (SDS) (Lindstrom et al., 1976c), heat, or urea (Lindstrom, unpublished) greatly reduces its immunogenicity. Thus, in rats, conformationally dependent determinants on AChR are the most immunogenic. Each of the subunits composing AChR is also myasthenogenic. Each of the polypeptide chains composing AChR from Torpedo californica has been purified in denatured form by preparative electrophoresis in SDS (Lindstrom et al., 1978b, 1979b,c). Although they are much less immunogenic than native AChR, immunization with any of these four denatured polypeptide chains produces EAMG. The a and 6 chains were most effective. Antibodies raised to the polypeptide chains are directed at determinants that normally account for little of the immunogenicity of native AChR, since antibodies to native AChR show little reaction with the polypeptide chains, whereas antibodies to the chains react quite well with native AChR. The polypeptide chains are immunologically distinct from one another. Antisera to the chains show specific cross reaction with native AChR from other sources. These experiments have important implications for AChR structure: (1) AChR from both electric organ and muscle show antigenic similarities at several sites corresponding to each of the polypeptide chains in AChR purified from Torpedo californica, and (2 )at least part of each of the 4 polypeptide chains is exposed on the extracellular surface of the AChR molecule in order to permit cross reaction in vivo. These experiments also have important implications for EAMG and MG: (1)there is no single determinant responsible for immunological cross-reaction of AChRs; (2) there is no single myasthenogenic determinant responsible for induction of EAMG. This result is consistent with others to be discussed subsequently, showing that the mechanisms by which transmission is impaired in EAMG and MG need not depend strongly on where antibodies bind to the AChR molecule. EAMG is produced in rats by immunization at multiple intradermal sites with AChR emulsified in complete Freund’s adjuvant. Single injections of 1-100 pg of purified electric organ AChR will produce

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chronic EAMG (Lennon et al., 1975; Lindstrom and Einarson, 1979), and even lower doses of muscle receptor are effective (Lindstrom et aE., 1976a). Maximum titer is usually achieved 3 0 4 0 days after a single injection (Lindstrom et al., 1976~). Multiple doses of 15-30 p g at 2- to 3-week intervals are used to obtain very high titer sera. Torpedo AChR is the easiest to purify in quantity and produces the highest titers of anti-electric organ AChR antibodies, though these antibodies in general are more species specific than those produced by immunization with AChR from electric eels (Lindstrom et al., 1978a). Antibodies to AChR are usually quantitated by radioimmune assay (Patrick et al., 1973; Lindstrom, 1977a). Detergent-solubilized AChR are labeled by incubation with excess 1251-labeleda-bungarotoxin. Specificity of labeling is established by controls incubated with acetylcholine and esterase inhibitor, or an antagonist like benzoquinonium, to inhibit specific toxin binding. Because of the specific [1251]toxin labeling, the AChR used as antigen need not be pure, and crude detergent extracts of muscle can be used (Lindstrom et al., 1976a). Note also that antibodies directed at the acetylcholine binding site cannot be detected by this method, since the site is occupied by toxin. As will be discussed later, this is not a problem, since most anti-AChR antibodies are directed at other determinants. Fixed amounts of [1251]toxin-labeledAChR are incubated with increasing amounts of serum. After overnight incubation, anti-Ig is added to precipitate Ig along with any bound [1251]toxin-AChR. After centrifugation, lZ5Iin the washed pellets is measured. The result is a curve that increases linearly while antigen is in large excess. Titer is the slope of this line. The curve plateaus in antibody excess, giving a measure of AChR concentration in the extract. Antibody-AChR complexes extracted from the muscles of an immunized rat can be detected by labeling the extract with [1251]toxinand then precipitating with anti-Ig (Lindstrom et al., 1976a). As a quantitative example, consider a group of 3 rats immunized on day 0 with 60 p g of Torpedo californica AChR (-5 x mol of toxin-binding sites) in complete Freund's adjuvant and given pertussis at other sites (Lindstrom and Einarson, previously unpublished results). At sacrifice on day 36 the titer of their serum averaged 3.1 ? 0.5 x lop6mol of torpedo AChR [1251]toxin binding sites of AChR precipitated per liter of serum. Titer against AChR from eel electric organ was only 6.3 ? 2 x lO-'M. Titer against AChR from rat muscle was 1.6 2 0.5 x lO-'M, giving a total of about 8 x mol of anti-rat AChR per rat. AChR content of the muscles of these rats was reduced 64% from normal. Whereas normals contained 3.9 2 0.05 x lo-" mol per rat, the

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rats with EAMG contained only 1.4 2 0.02 x lo-" mol per rat. None of the AChR from the normal rats could be precipitated by anti-Ig, but 81% of that from the rats with EAMG had antibodies bound. Thus the rats with EAMG retained only 7% of their normal content of unaffected AChR. As will be described later, serial measures of antibody titer and content of AChR and antibody-bound AChR have been made throughout the course of EAMG (Lindstrom et aZ., 1976a,b), and similar techniques have been applied to sera (Lindstrom, 1977a) and muscle biopsies from MG patients (Lindstrom and Lambert, 1978). Because of the high dose of AChR and extra adjuvant, these rats showed relatively high cross reaction with rat AChR, yet this was only 5% of the titer against torpedo AChR. However, because of the small amount of AChR present, antibody to rat AChR was present in 57,000-fold excess over the amount of AChR remaining. Given this excess, it is surprising that even 20% of the AChR remained unlabeled by antibody. This suggests that these AChR may be newly synthesized and still on their way to the surface membrane or newly arrived. Antibodies to AChR are quite species specific. Antibodies to AChR from eel or torpedo electric organ cross react better with AChR from the other electric organ (1-29%) than with AChR from several species of muscle (1-19%) (Lindstrom et al., 1978a). Rat antisera to syngeneic AChR show extensive (5-80%) cross reaction with AChR from both muscle and electric organs (Lindstrom et aZ., 1978a). In a rat immunized with eel AChR, the serum antibodies that cross-react with rat muscle AChR also react with eel AChR and are adsorbed by eel electric organ membrane fragments at the same rate (Lindstrom et al., 1976~). Thus, as will be discussed later, the muscle AChRs that are objects of the autoimmune response to EAMG do not appear to contribute significantly to autoimmunization. Most of the antibodies to AChR are directed at determinants on the molecule other than the toxin binding site, and this applies to crossreacting antibodies. This is shown by the observation that, depending on the species immunized, it makes little (Patrick et al., 1973) or no (Lindstrom, 1976)difference whether [1251]toxin-labeledAChR is used as antigen or whether [3H]acetylAChR having a free toxin binding site is used as antigen. Even in uiuo, where AChR is exposed to large excesses of antibodies, small fractions of antitoxin binding-site antibodies are not selected out, since antibody-AChR complexes extracted from muscle retain the ability to bind toxin (Lindstrom et al., 1976b). The observation that more peroxidase-labeled toxin is bound and more antibody is seen bound at endplates by peroxidase-protein A staining in mild MG and less toxin and antibody is seen in severe MG

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(Engel et al., 1977a) shows that AChR loss is real rather than apparent, owing to blockage of toxin binding sites by antibodies. Most of the anti-AChR antibodies observed in rats with EAMG (Lindstrom et al., 1976c), as in humans with MG (Toyka et al., 1975, 1977), are 7 S IgG. Even by 10 days after immunization two-thirds of the serum anti-AChR is 7 S and only one-third is 19 S (Lindstrom et al., 1976~). Production of anti-AChR is a T cell-dependent response not observed in rats depleted of T cells by neonatal thymectomy and X-irradiation (Lennon et al., 1976). Response to AChR is reconstituted by B plus T cells, but not B alone. Adult thymectomy of immunized animals does not prevent induction of EAMG. B. ACUTE AND PASSIVEEAMG Between 8 and 11 days after immunization with AChR in complete Freund’s adjuvant, an “acute” phase of muscular weakness is observed if pertussis is included as an additional adjuvant (Lennon et al., 1975), but not in its absence (Lindstrom, unpublished). Thus it is uncertain whether this stage is altogether an artifact of the use of pertussis, or whether pertussis simply makes processes that would occur in any case more concerted. The characteristic feature of the acute phase is a massive phagocytic invasion (Engel et al., 1976a,b). Whether or not an acute phase is observed, a chronic phase of EAMG begins some 30 days after immunization. The chronic phase is characterized by high serum anti-AChR concentrations (Lindstrom et al., 1976c) and postsynaptic membrane simplification in the absence of phagocytic cells (Engel et al., 1976a,b). In both acute and chronic EAMG, weakness is associated with loss of AChR (Lindstrom et al., 1976a,b; Engel et al., 197713). There is no obvious parallel to acute EAMG in human MG, which closely resembles chronic EAMG. The features of acute and chronic EAMG are summarized in Fig. 2. Electrophysiological findings during acute EAMG differ somewhat from those in chronic EAMG. Both compound action potentials and muscle twitch are reduced in forelimb muscles, but the muscles respond to direct electrical stimulation and nerve conduction is normal (Lambert et al., 1976).The electromyogram response decrements, and the decrement is prevented by esterase inhibitors (Seybold et al., 1976). Hindlimb muscles and diaphragm are less severely affected than forelimb muscles, and ptosis is not observed clinically (Lambert et al., 1976). Thus, as in human MG, different muscle groups are differentially affected, although the pattern of muscles affected in rats does not exactly mimic that in humans, where ptosis is a frequent

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FIG.2. The course of experimental autoimmune myasthenia gravis (EAMG) in rats. (A) A normal neuromuscular junction. 1, Acetylcholine vesicles in the nerve ending; 2, the presynaptic membrane of the nerve ending; 3, the synaptic cleft; 4, the tip of a fold in the postsynaptic membrane which is packed with acetylcholine receptor (AChR). (B) The earliest phases of EAMG 6 or 7 days after immunization. 1, Antibodies bound to a small fraction of the total AChR; 2a, the fixation of complement, which results in the focal lysis shown at the next junctional fold; 2b, the release of complement fragments that promote migration of phagocytes. (C) The phagocytic invasion characteristic of the acute phase of EAMG, which occurs between 8 and 11 days after immunization. The phagocytes interact with the postsynaptic membrane through Fc and C 3 receptors and destroy large areas of it. Many more AChRs are destroyed than are labeled with antibodies. The phagocytes may also interrupt transmission by interposing between nerve and muscle. The result in many fibers is transient denervation. (D) The simplified postsynaptic membrane structure characteristic of chronic EAMG some 30 days after immunization. No phagocytes are seen. 1,The simplified postsynaptic membrane; 2, the few remaining AChR, mostly labeled with antibodies; 3, complement-mediated focal lysis resulting in the shedding, into the synaptic cleft, of membrane fragments containing AChR, antibody, and C3; 4, loss of AChR, which occurs also through antigenic modulation involving antibody cross-linking of AChR, internalization, and proteolysis; and 5, synthesis of new AChR, which may partially compensate for the ongoing loss of AChR through complement-mediated lysis and antigenic modulation.

feature. Direct measurements with microelectrodes show that up to

90% of forelimb muscle fibers are functionally denervated during the acute phase (Lambert et al., 1976). The phagocytic invasion of forelimb and diaphragm muscles is so intense that a yellowish colora-

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tion of the endplate zone is visible to the naked eye. At fibers where innervation remains intact, miniature endplate potentials are reduced both in number and amplitude. Acetylcholine sensitivity is so low even in these fibers that in many cases nerve stimulation does not produce endplate potentials large enough to evoke an action potential. Despite the ravaging of the postsynaptic membrane by phagocytes (Engel et al., 1976a), the resting potential of the muscle fibers is only slightly reduced (Lambert et al., 1976). Thus the effects of the phagocytic attack are quite localized, perhaps owing to the complex internal membrane system of the muscle fiber, which may somehow rapidly seal off the phagocytic lesion preventing generalized ionic leakage from the fiber or generalized lysis. After the phagocytic invasion and associated denervation return to normal, the decrease in miniature endplate potential amplitude persists during the return to apparent clinical normality about day 11 and on through the chronic phase of weakness starting about day 30. Microscopic studies show that the acute phase is associated with a massive phagocytic invasion of the endplate region (Engel et al., 1976a,b). From animals sacrificed on the first day of weakness, electron microscopy reveals some endplates where phagocytic invasion has not yet occurred, but focal lysis of the tips of postjunctional membrane folds is seen, evidently owing to the local effects of bound antibodies and complement (Sahashi et al., 1978). Membrane fragments are shed into the synaptic cleft. Mononuclear cells invade the endplate region in large numbers and engulf the postsynaptic membrane. Segmental necrosis of some fibers by macrophages is centered on the endplate region. Large segments of material that stains for acetylcholinesterase are separated from nerve and muscle by the invading cells. These may correspond to the structured areas of basement membrane containing acetylcholinesterase that persist after loss of postsynaptic membrane following chronic denervation (McMahon et al., 1978). The nerve terminals show little effect. The nerve ending membrane is not attacked, but the area of the ending decreases, and the concentration of cholinergic vesicles increases slightly (Engel et al., 197613). After the phagocytic invasion the nerve endings become reapposed to the simplified postsynaptic membrane. Nerve sprouts and immature endings are occasionally observed, suggesting that some new endings are formed in response to the functional denervation accompanying destruction of the old endings. Serum concentration of anti-AChR antibodies is low during the acute phase, and the degree of cross-reaction with muscle AChR is lower than at later stages (Lindstrom et al., 1976b,c). This may in part be due to depletion of the small amounts of cross-reacting anti-AChR

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present by adsorption onto AChR in muscle. Cross-reacting anti-AChR has been first detected 3 days after immunization, and antibodylabeled AChR in extracts of muscle has been detected first on day 6. By day 10 approximately equal amounts of cross-reacting anti-AChR are found in serum and bound to AChR in muscles. At the height of the acute response on day 8, there is a decrease in the total amount of AChR by 50% (Lindstrom et al., 1976a).Of AChRs that remain, only 1 or 2% have antibodies bound. This decrease in total AChR content is quickly followed by, and probably to some extent obscured by, an increase in AChR content to nearly double the normal amount by about day 12. This no doubt results not only from repair synthesis of junctional AChR, but also from synthesis of extrajunctional AChR in response to denervation. Extrajunctional AChR have been detected after acute EAMG (Engel et al., 1978). Termination of denervation contributes to the sudden decline in AChR content that follows, but the content of AChR continues to decrease below normal levels on succeeding days as the animals progress toward the chronic phase (Lindstrom et al., 1976a). The amount of antimuscle AChR antibody increases with time after immunization, exceeds the amount of AChR in muscle after about day 15, and increases rapidly thereafter. The fraction of AChR complexed with antibodies gradually increases from the time of immunization through the chronic phase, when nearly all of the AChR remaining may have antibodies bound. EAMG can be passively transferred from a rat with chronic EAMG to a normal rat with serum anti-AChR antibodies (Lindstrom et al., 1976b; Lennon et al., 1978, Engel et al., 1978). Passive transfer is very efficient and signs of mild muscular weakness can be produced after 1 day in a rat containing 5 x lo-" mol of AChR by as little as 1 x lo-" mol of antirat AChR (Lindstrom et al., 1976b). The efficiency of transfer results from a massive phagocytic invasion of the endplates that accompanies the transfer (Lindstrom et al., 197613). This invasion closely resembles that seen in acute EAMG (Engel et al., 1978) and indicates that the cellular invasion in acute EAMG is not produced by anti-AChR cells, but rather by nonspecific cells responding to bound antibody and complement. By 6 hours after injection, antibodies are found on the tips of postjunctional folds (Engel et al., 1978). By 24 hours membrane fragments containing AChR, antibody, and C3 are shed into the synaptic space (Engel et al., 1978),resulting in net AChR loss (Lindstrom et al., 1976b). The postsynaptic membranes at many endplates are destroyed by phagocytes on day 2, but effective contacts are restored by day 5 (Engel et al., 1978). Total AChR content is greater than twice normal by day 5 (Lindstrom et al., 1976a),yet AChR

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content at endplates is still slightly reduced (Engel et al., 1978), showing that most of the AChRs are extrajunctional. Extrajunctional AChRs were also directly observed (Engel et al., 1978). Effectiveness of transmission parallels the changes in AChR content (Lindstrom et al., 1976a; Engel et al., 1978). Increased AChR, antibody-bound AChR, and structural abnormalities persist after 10 days, although the rats are clinically normal. If rats are depleted of C3 by cobra venom factor treatment, even large doses of anti-AChR which bind 67% of their AChR do not produce phagocytic invasion, AChR loss, muscle weakness, or electromyogram decrement (Lennon et al., 1978). This is an important experiment. It shows that the phagocytic invasion depends on bound C3. It shows that AChR loss, not direct inhibition of AChR function by antibodies, is responsible for impaired transmission. Depletion of C3 also inhibits the acute phase of EAMG produced by immunization with AChR. These results suggest that C3-mediated lysis and/or phagocytic attack are important in the immune assault on the endplate prior to the chronic phase. What terminates the phagocytic invasion of acute or passive EAMG? In chronic EAMG there are more antibody-bound AChRs than during acute EAMG (Lindstrom et al., 1976a), and large amounts of C3 are bound as well (Sahashi et al., 1978). During the acute phase, but not thereafter, positive skin tests are obtained by intradermal injection of AChR (Lennon et al., 1976). If the skin test response in some way parallels the cellular invasion of the endplates, termination must be a systemic problem. On the other hand, if this delayed-type hypersensitivity response is irrelevant, as is likely, a more specific solution may be sought. Because of the small amount of AChR present, it seems unlikely that sufficient antigen-antibody complexes could be shed from endplates to saturate all potentially sensitive cells. If a more local explanation is sought, the decrease in both amount and concentration of AChR is a possibility. Perhaps after the initial phagocytic response neither the amount nor organization of AChR permit effective cellular interactions (Lennon et al., 1978). However, the phagocytic invasion may be self-limiting for other unknown reasons. Both acute and passive EAMG are associated with rapid changes in antibody concentration and initial trace antibody labeling of AChR, so dynamic considerations might also be important. The phagocytic invasion in acute and passive EAMG depends on the C3 component of complement as well as bound antibody (Lennonet al., 1978). Although bound C3 is present at the endplate in the acute phase, (Sahashi et al., 1978), much of this may be inactivated. Perhaps the rate of complement activation sig-

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nificantly exceeds the rate of inactivation only when antibody deposition at the endplate is first begun, but not in the chronic state. To what extent are the morphological alterations observed in chronic EAMG the persistent artifacts of the phagocytic destruction that occurs during the acute phase? Since AChR content of muscle can change rapidly following denervation or reinvervation (Lindstrom et a1., 1976a; Berg and Hall, 1975; Burden, 1977) and since AChR turnover in cell or organ culture (Devreotes and Fambrough, 1975) is rapid, it seems likely that AChR content if not detailed postsynaptic architecture should be in a vital dynamic equilibrium between destruction and synthesis. The ongoing focal lysis observed at endplates in chronic EAMG (Engel et al., 197613) certainly suggests a dynamic system, and suggests a mechanism for sustaining simplified membrane architecture in the absence of continued phagocytic attack. Endplates of rats examined 54 days after passive transfer (Engel et al., 1978) had regained junctional folds but still showed some alteration in postsynaptic morphology and some decrease in AChR content. So the outside possibility must be admitted that phagocytic invasions that were very infrequent, and hence not observed, could contribute to the simplified morphology in chronic EAMG or human MG. But it seems more likely that focal lysis and, perhaps, antigenic modulation could independently sustain the altered morphology and perhaps create it independently of phagocytes. Rats were tested long after passive transfer of EAMG to determine whether the muscle AChR lost during the acute response were immunogenic, leading to a chronic, self-sustaining autoimmune response (Lindstrom et al., 1976b). No clinical signs of chronic EAMG were observed, and serum anti-AChR was not detected. More than 4 x lo-" mol of AChR were lost from muscle to phagocytic destruction and only 2.6 x lo-" mol of purified rat AChR suffice to induce chronic EAMG (Lindstrom et al., 1976a). Since chronic EAMG was not observed, this suggests that normal AChR in postsynaptic membrane fragments is not immunogenic, even when fed directly into phagocytes in rats given pertussis as additional adjuvant. C. CHRONICEAMG Beginning approximately 30 days after immunization with purified AChR, a chronic phase of muscular weakness occurs (Lennon et al., 1975). This weakness persists until the animal dies, or, if it survives, until more than 80 days (Lindstrom, 197713) after immunization, when the immune response has diminished substantially. The features of chronic EAMG in rats closely resemble those of MG in humans.

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In chronic EAMG, acetylcholine sensitivity is decreased (Bevan et

a1., 1976) and miniature endplate potential amplitude is decreased

(Lambert et al., 1976). Decrementing electromyograms are observed (Seybold et al., 1976). There is no sign of the denervation frequently observed during the acute phase. In rat diaphragm or forelimb nearly 200 quanta are released per impulse (Lambert et al., 1976) as compared with 60 in human intercostal muscle (Lambert and Elmquist, 1971).The large amount of acetylcholine released may help to account for the observation that obvious weakness is not observed until nearly 90% of the AChRs are either lost or coupled with antibodies (Lindstrom and Lambert, 1978). As in human MG (Lindstrom et al., 1976d; Lindstrom and Lambert, 1978), concentration of anti-AChR in rats with chronic EAMG does not correlate well with miniature endplate potential amplitude or severity of weakness (Lambert et al., 1976). It should be pointed out that in all these rats the amount of anti-AChR was well in excess of the amount of AChR. Anti-AChR is probably the most important factor causing AChR loss. Yet it is not known whether the limiting factor determining the relationship between antibody concentration and AChR loss is antibody specificity, access of antibodies to AChR, complement, rate of AChR synthesis, a combination of these factors, or something else altogether. Simplified postsynaptic membrane architecture is the characteristic morphological feature of chronic EAMG (Engel et d., 1976b). Phagocytic cells are not observed. Focal lysis of the membrane is observed. AChR can be localized by staining with peroxidase-conjugated toxin (Engel et al., 1977b), and antibody and the C3 component of complement can be localized by rabbit anti-rat Ig followed by staining with peroxidase-conjugated protein A and peroxidase-conjugated anti-C3, respectively (Sahashi et al., 1978). Membrane fragments are observed in the synaptic cleft, which can be stained for AChR, antibody, and C3. Relative quantitation of AChR is achieved by measuring the length of postsynaptic membrane stained with peroxidase-toxin relative to the length of presynaptic membrane. There is a direct correlation between the amount of AChR estimated in this way and acetylcholine sensitivity measured b y miniature endplate potential amplitude. As in human MG (Engel et al., 1977a), the amount of AChR, bound antibody, and bound C3 was greatest in the least severely affected rats (Sahashi et al., 1978).This is consistent with the idea that loss of AChR is the most important factor impairing transmission. Curiously, although the presynaptic membrane appears to be unaltered, cholinergic vesicles are significantly more concentrated and abundant than normal (Engel et al., 1976b). It has been observed that muscle

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biopsies from MG patients contain even more acetylcholine than normal (It0 et al., 1976), and suggested that this might be somehow compensatory for the decreased postsynaptic sensitivity (Cull-Candy et al., 1978). This might be the morphological expression of a similar phenomenon, although neither quantum content of the endplate potential nor quantum store in these rats was increased significantly over normal (Lambert et al., 1976), and resting and stimulated acetylcholine release was also normal (Kelly et al., 1978). High concentration of anti-AChR in serum, low amount of AChR, and antibodies bound to a large fraction of the remaining AChR are characteristic features of chronic EAMG (Lindstrom et al., 1976a). AChR content decreases to a minimum value about one-third normal. Then increasing severity of weakness is associated with the accumulation of an increasing fraction of the remaining AChR labeled with anti-AChR (Lindstrom and Lambert, 1978).Nearly all AChR extracted from muscle may be labeled with antibodies. Whereas AChR extracted from normal rat muscle sediments at 9.5 S on a sucrose gradient, AChR extracted from the muscle of rats with chronic EAMG sediments in aggregates of 18 S or larger (Lindstrom et al., 1976a), indicating that several antibodies and AChRs are aggregated together. Cross-linking of AChR on muscle cell membranes by antibodies from rats with EAMG triggers antigenic modulation of AChR that causes a decrease in total AChR content (Lindstrom and Einarson, 1979). The factors proposed to contribute to impaired neuromuscular transmission in chronic EAMG, in approximate order of importance, are (1) loss of AChR, (2) inhibition of AChR activity by bound antibodies, and (3)alteration of postsynaptic membrane structure so that sites of acetylcholine release and AChR concentration are not optimally juxtaposed. The following sections review evidence for impairment of acetylcholine sensitivity by (1)inhibition of AChR function by bound antibodies, (2) loss of AChR due to complement-mediated destruction, and (3)loss of AChR due to antigenic modulation.

D. EFFECTSOF BOUNDANTIBODYON AChR FUNCTION Antisera to electric organ AChR raised in rats or other animals directly affect AChR function in electric organ cells or muscle to which they are added, independent of complement or antigenic modulation (Patrick et al., 1973; Lindstrom et al., 1976c, 1977; Karlin et aZ., 1978; Bevan et al., 1977). Greater than 80% blockage of the depolarizing response of eel electric organ cells to applied carbamylcholine is blocked by preincubation with anti-AChR (Patrick et al., 1973; Lindstrom et al., 1976c, 1977). Under these conditions, a large fraction

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of the AChR in the electric organ cells are bound with antibodies and linked into aggregates, but there is little or no impairment of ‘“I-toxin binding and no decrease in the amount of AChR (Lindstrom et al., 1977). Antibodies bound to electric organ tissue can be visualized directly and by using peroxidase or ferritin as labels (Karlin et al., 1978; Tarrab-Hazdai et al., 1978). Application of anti-AChR to muscle decreases its acetylcholine sensitivity (Bevan et al., 1976), but part of this loss can be prevented at low temperature or by inhibitors of energy metabolism (Bevan et al., 1977, Heinemann et al., 1977), indicating that this results from antigenic modulation of AChR, as will be described later. Studies of acetylcholine noise in rat muscle cells in culture exposed to anti-AChR show a decrease in the mean conductance of the activated AChR by 15% and a decrease in the mean open time of 23% (Heinemann et al., 1977), and similar results have been observed with human muscle cells in culture (Bevan et al., 1978). No detectable changes in these parameters were observed in endplates from MG patients (Cull-Candy et al., 1978). Unfortunately, the number of patients, their severity, or the fraction of their AChR labeled with antibodies in this study is unknown. Because of the large safety factor for neuromuscular transmission in the rat (Lambert et al., 1976) and the net 38% inhibition of AChR function by bound antibodies (Heinemann et al., 1977), 67% of muscle AChR can be labeled with anti-AChR without producing detectable weakness or electromyogram decrement (Lennon et al., 1978). In rats the acetylcholine binding site is not directly obscured by bound antibody (Lindstrom et al., 1976a). In summary, then, these results are consistent with the idea that anti-AChR antibodies bind to sites on the AChR molecule other than that for acetylcholine binding, and that antibodies bound to at least some of these sites have an allosteric effect on AChR function. The impairment of function by anti-electric organ AChR antibodies on electric organ AChR may be greater than the impairment of muscle cell AChRs by cross-reacting antibodies. The impairment might be produced by some combination of effects on ligand binding, ionophore regulation, and ionophore function, but this remains unknown. E. EFFECTOF COMPLEMENT ON AMOUNTOF AChR As previously described, acute and passive EAMG depend critically not only on binding of anti-AChR, but also on binding of the C3 component of complement (Lennon et al., 1978). Depletion of C3 in mice by cobra venom factor also impairs passive transfer of MG to mice with anti-AChR from MG patients, whereas C5 deficient mouse stains are as

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susceptible as normals (Toyka et al., 1977). In both chronic EAMG (Sahashi et al., 1978) and MG (Engel et al., 1977a) C3 is bound to the postsynaptic membrane and to fragments shed from it. Thus some, but not necessarily all, of the proteins potentially involved in the complement cascade are implicated in both acute and chronic EAMG. In the acute phase, bound C3 may be important for binding of phagocytes (Lennon et al., 1978), whereas in chronic EAMG and MG it may be involved in focal lysis (Engel et al., 1976b; Sahashi et al., 1978). Because development of an immune response to cobra venom factor limits depletion of C3 to a few days, it has not been possible to deplete C3 long enough to observe an increase in AChR content in rats with chronic EAMG (Lindstrom, Einarson, and Lennon, unpublished). This could be either because the change in AChR content per day is too small to accumulate a significant change in 3 or 4 days or because C3 is not especially important in chronic EAMG.

F. EFFECTOF ANTIGENIC MODULATIONON AMOUNT OF AChR Addition of anti-AChR antibodies to rat or human muscle cells in tissue culture causes the rate of AChR destruction to increase by 2- to 6-fold (Heinemannet al., 1977; Kao and Drachman, 1977; Appelet al., 1977). This has been termed antigenic modulation of AChR. AChR turnover is measured by loss of [1251]toxinbinding sites (Kao and Drachman, 1977; Appel et al., 1977)or by release of [12SI]tyrosinefrom cells whose AChR were initially specifically labeled with [1251]toxin (Heinemann et al., 1977). Antigenic modulation of AChR is independent of complement but inhibited by low temperature and inhibitors of energy metabolism like DNP and NaF (Heinemann et aZ., 1977; Kao and Drachman, 1977) and by colchicine and cytochalasin B, which affect the cytoskeleton (Appel et al., 1977). Both the AChR degradation that occurs normally in the course of AChR turnover and the accelerated degradation that occurs after binding of antibodies are blocked by inhibitors of lysozomal proteases (Merlie et al., 1979b). Cross-linking of AChR is a necessary requirement, since the F(Ab)l fragment of anti-AChR binds, but does not induce modulation unless it is crosslinked by anti-Ig (Lindstrom and Einarson, 1979; Drachman et al., 1978a). AChRs newly incorporated after modulation of AChR are degraded at the normal rate (Drachman et al., 1978a). This has been interpreted as showing that only cross-linked AChRs are degraded at the accelerated rate. But the data do not exclude that adjacent unlinked AChRs and other membrane components are simultaneously accelerated in their degradation by endocytosing with cross-linked AChRs, while newly synthesized AChRs incorporated subsequently are not. After addition of antibody there is a delay of about 2 hours

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before [12511tyrosinefrom degraded toxin is released (Heinemann et al., 1977; Bevan et al., 1977; Lindstrom and Einarson, 1979); during this time antibody presumably binds to AChRs, aggregates them, and causes them to be internalized and degraded in lysozomes. In tissue culture there is no compensatory increase in AChR synthesis (Drachman et al., 1978b). Muscle cells in culture turn over their AChR very quickly ( t l R= 20 hours: Fambroughet al., 1978),whereas normal muscle in organ culture does not (tlA= 158 hours: Merlie et al., 1979b). Thus AChR synthesis in cell culture may already be proceeding maximally, and it is not known whether in vivo the increased rate of AChR degradation caused by antibody cross-linking is accompanied by an increase in AChR synthesis. Antigenic modulation is an appealing mechanism for helping to explain the loss of AChR observed in chronic EAMG and MG. It is not a pecularity of AChR on muscle cells in culture. Antigenic modulation can be demonstrated using rat and mouse diaphragms in organ culture (Heinemann et al., 1978; Reiness et al., 1978; Stanley and Drachman, 1978). Addition of anti-AChR antibody to rat diaphragm muscle in organ culture increases the rate of destruction of extrajunctional AChR in denervated muscle by 2-fold (from tl12= 15 hours to t l R= 8 hours) and also increases the rate of destruction ofjunctional AChR in muscle that had been normally innervated by 2-fold (from tl12= 190 hours to t l a = 88 hours) (Heinemann et al., 1978).More important, it has been found that both junctional and extrajunctional AChR in diaphragms removed from rats with EAMG are being destroyed at approximately twice the normal rates (Merlie et al., 1979a). In muscle from rats with EAMG, degradation of AChR occurs at approximately the rates observed when diaphragms are exposed to anti-AChR antibodies in vitro (tllPjunctional AChR = 72 hours; t I l 2extrajunctional AChR = 7-13 hours). These rates of destruction are not further increased by addition on anti-AChR antibodies in vitro. This AChR destruction, like antigenic modulation in vitro, and unlike complement-mediated destruction, is blocked by inhibitors of metabolism and lysosomal proteases. These results suggest that antigenic modulation occurs in vivo in EAMG. If there is no compensating increase in AChR synthesis, the observed rates of antigenic modulation can account for most of the AChR loss observed in EAMG (Lindstrom and Einarson, 1979). VI. Experimental Autoimmune Myasthenia Gravis in Other Species

A. RABBITS EAMG was first described in rabbits, and was subsequently studied in rabbits by several groups (Patrick and Lindstrom, 1973; Patrick et

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al., 1973; Sugiyama et al., 1973; Heilbron and Mattson, 1974; Aharonov et al., 1975b; Sanders et al., 1976; Penn et al., 1976; Berti et al., 1976; Green et al., 1975). EAMG in rabbits differs from that in rats in that no similar distinction between an early acute phase and a later chronic phase has been made, and that EAMG in rabbits often occurs suddenly 24-26 days after the first immunization and is often rapidly fatal rather than progressively chronic, as in rats at about this period. However, careful comparisons between the species have not been made, pertussis has not been used as an additional adjuvant, and rabbits have often been immunized with large and repeated doses of AChR, rather than the small single doses used in most of the studies with rats. So some of the differences between the course of EAMG in rats and rabbits may be more apparent than real. Decrementing electromyograms and small miniature endplate potentials are observed in rabbits as in rats. It has been reported that traces of anti-AChR can be found in the cerebrospinal fluid of immunized rabbits and that this is associated with electroencephalogram abnormalities (Fulpius et al., 1977); however, chronic intracranial electrodes were used, and it seems likely that serum anti-AChR could have entered around these wounds along with other factors that could account for these abnormalities. AntiAChR is not significantly elevated in the cerebrospinal fluid of MG patients in our studies (Keesey et al., 1978), despite another report (Lefvert and Pirskanen, 1977), and there is no evidence of central dysfunction in MG. Head droop and shaking are observed in rats with EAMG (Lennon et al., 1975), but this is probably due to weakened neck muscles. Electron microscopic observations of immunized rabbits have also been performed (Thomell et al., 1976) although the serial studies, histiometric measurements, and localization of AChR, antibodies, or C3 that have been done on rats (Engel et al., 1976a,b, 197%; Sahashi et aZ., 1978) have not been done on rabbits. Postsynaptic membrane structure is altered in rabbits with EAMG, but massive phagocytic invasions have not been observed (Penn et al., 1976; Heilbron et al., 1976; Green et al., 1975). Studies of serum anti-AChR in rabbits are similar to those in rats, as already discussed, although an inverse proportion between anti-AChR titer and number of toxin binding sites/ endplate in the immunized rabbits was reported (Green et al., 1975). Rabbit anti-AChR serum inhibits AChR activity in eel electric organ cells (Patrick et al., 1973; Karlin et al., 1978) rat, and frog muscle cells (Green et al., 1975; Bertie et al., 1976). Serum from rabbits with EAMG contains small amounts of antitoxin binding site antibody, but

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most of the antibodies are directed at other determinants on the molecule (Patrick et al., 1973; Karlin et al., 1978). EAMG has been induced in rabbits by immunization with native AChR from eel and torpedo electric organs (Patrick and Lindstrom, 1973; Sugiyama et al., 1973; Heilbron et al., 1976; Aharonov et al., 1975b; Sanders et al., 1976; Penn et al., 1976; Berti et al., 1976; Green et al., 1975), with AChR purified from fetal calf muscle (Merlie and Lindstrom unpublished), or from a tissue mouse muscle cell line (Boulter and Patrick, 1977). Antibodies to the immunogen, but no evidence of EAMG, have been reported after immunization of rabbits with SDS-denatured polypeptide chains from eel or torpedo AChR (Lindstrom et al., 1976c; Valderama et al., 1976; Claudio et al., 1977; Lindstrom et al., 1978b) or with whole torpedo AChR denatured by reduction and carboxymethylation (Bartfield and Fuchs, 1977). Antigenic specificity of reduced and carboxymethylated AChR differs from that of native AChR in that some antigenic determinants are lost, but no new sites are generated. Antiserum to native AChR shows traces of antibodies that inhibit the binding of toxin to AChR, but antiserum to reduced and carboxymethylated AChR shows far less (Bartfield and Fuchs, 1977). This was taken to imply that reduction and carboxymethylation destroys antigenic determinants on AChR, including those at the acetylcholine binding site [where a disulfide bond is known to be located (Karlin et al., 1976)] and that this site is critical for the development of EAMG in rabbits. Unfortunately, cross-reaction of these sera with rabbit AChR was not measured, so it is not known whether cross-reacting antibodies were formed that could bind but were not effective in inducing EAMG. Reduction and carboxymethylation may have destroyed all antigenic determinants on the extracellular surface of the torpedo AChR capable of cross-reacting with rabbit muscle AChR, in which case no antibodies could be bound to muscle AChR of the immunized rabbits. In these experiments, neither muscle AChR content nor antibody-AChR content was measured, so it is not known whether subclinical EAMG was provoked. The interpretation of these experiments, that there is a single myasthenogenic antigen, conflicts with what is known to be the case in rats. In rats, denaturation of AChR in SDS destroys all the normally immunogenic determinants, but other determinants shared by native and denatured AChR account for the low residual immunogenicity of denatured AChR (Lindstrom et al., 1978b). This denatured AChR induces EAMG in rats (but not rabbits). Each of the polypeptide chains of AChR is immunologically and biochemically distinct (Lindstrom et id., 1978b, Lindstrom et al., 1979a,b), yet each can induce EAMG.

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Thus in rats, if not perhaps in rabbits, several different determinants can induce EAMG. This is consistent with the evidence that in rats (and humans) the pathology in MG results primarily from AChR loss due to complement-mediated destruction and antigenic modulation, processes that do not depend strongly on where antibodies bind to the AChR molecule, and is not due to direct inhibition of AChRs by antibodies, a process that depends strongly on where antibodies bind to AChR. It has been reported that in 50% of rabbits preimmunized with reduced and carboxymethylated AChR and then immunized with native AChR, onset on EAMG was delayed or prevented, and that in some cases EAMG could be cured by injecting reduced and carboxymethylated AChR (Bartfield and Fuchs, 1977; Fuchs et al., 1978). The mechanism of these effects was presumed to be alteration of antigenic specificity of the antibodies produced. The idea that only certain anti-AChR specificities can produce disease is appealing, since this could account for the observation that in MG severity does not correlate closely with antibody titer (Lindstrom et al., 1976d). The idea that one might treat EAMG or MG by administering a modified antigen is appealing, since this might allow specific immunosuppression of only the pathological immune response without the generalized immunosuppression produced by drugs and without the side effects of the drugs. However, in rats, at least, no single myasthenogenic determinant is involved, and the pathological mechanisms apparently acting in human MG patients do not seem to require a single determinant. An interesting experiment would be to see if EAMG induced in rabbits by immunization with AChR from electrophorus could be suppressed by the modified torpedo AChR antigen. This might be the case if in rabbits only one antigenic determinant were shared by AChR from rabbit muscle and AChRs from electrophorus and torpedo electric organs. Evidence suggests that cross-reaction between AChRs occurs at several determinants (Lindstrom et al., 1978a,b), so a likely result would be that EAMG induced by electrophorus AChR could not be suppressed by modified torpedo AChR. Another potentially interesting experiment, but one best left to speculation, would be an attempt to suppress MG in a human with modified torpedo AChR. This would be unlikely to be effective, first because there is very little cross-reaction between the anti-human AChR in MG patients and AChR from torpedos (Lindstrom et al., 1978a). Second, it seems likely that immunization of an MG patient with AChR from torpedo would probably induce EAMG rather than cure the MG. It is known that AChRs from various species share antigenic determinants that are not

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normally immunogenic. For example, anti-AChR antibodies from MG patients show little cross-reaction with AChR from eel electric organ, yet animals immunized with AChR from eel electric organ show significant cross-reaction with AChR from human muscle (Lindstrom et al., 1978a). Also, MG patient sera show limited cross-reaction with AChR from normal rat muscle, but rats immunized with syngeneic AChR show substantial cross-reaction with human AChR (Lindstrom et al., 1978a). Macrophage cytophilic anti-AChR antibodies are found in rabbits with EAMG (Martinezet al., 1977).These are measured by binding of [12SI]AChRand have been detected in the alveoli of sick rabbits. Their role, if any, in the impairment of neuromuscular transmission in these rabbits is not known. Similar experiments have not been done in rats, but in rats it is clear that during the acute phase the pathologically important sequence of events is binding of antibodies to AChR on the postsynaptic membrane, then fixation of complement, and then invasion of phagocytes capable of recognizing bound complement and antibodies (Engel et al., 1976b; Lindstrom et al., 1976b; Lennon et al., 1978, Engel et al., 1978). Development of EAMG is suppressed in rabbits treated from the time of immunization with hydrocortisone and azathioprine (Abramski et al., 1976). This is not surprising, since these are well known immunosuppressive drugs. These drugs have been used successfully in treating patients with MG (Mattell et al., 1976; Hertel, 1979). One difficulty in using animals with EAMG as models for studying the effect of immunosuppressive drugs is that the autoimmune response in these animals is a matter of cross reaction with a foreign immunogen, and the autoimmune response declines as the response to the foreign immunogen diminishes, whereas humans with MG have an autoimmune response stimulated through unknown mechanisms, presumably by an endogenous immunogen, and their disease usually persists over prolonged periods, sometimes with repeated spontaneous remissions and exacerbations.

B. MICE, GUINEAPIGS, GOATS,MONKEYS, AND FROGS EAMG has been induced in mice by immunization with AChR purified from torpedo electric organ (Fuchs et al., 1976) and denervated rat muscle (Granato et al., 1976; Fulpius et al., 1976). EAMG in mice shows no acute phase, though pertussis as additional adjuvant was not tested. The disease in mice in most strains develops slowly and is often relatively mild, though it may be lethal. As in rats (Lennon et al., 1976), this is a T cell-dependent response (Fuchs et al., 1976).

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Fourteen strains of mice were immunized twice with 10 pg of torpedo AChR at 9-week intervals. Although all developed anti-torpedo AChR antibodies, DBA/l, SWR, SJL/J, and ASW failed to show clinical signs of EAMG (Fuchs et al., 1976). We have immunized mice with larger doses of the same antigen and succeeded in inducing clinical signs of EAMG in Balb/c, DBA/15, C57BL/6J, and B6D2F1 (Lindstrom and Einarson, previously unpublished). By day 100, after 5 doses of 15 pg, all 4 strains showed decreases in AChR content by 30-63%, and 1642% of this AChR had antibodies bound (Lindstrom and Einarson, previously unpublished). Concentration of serum antibodies to torpedo AChR ranged from 2.9 to 6.3 x M, while concentration of antibodies cross-reacting with AChR from mouse muscle was only 4.0 to 9.9 x M, and concentration of antibodies cross-reacting with AChR from rat muscle ranged from 2.2 to 12 x lovsM. Thus, we found no genetic nonresponders among those tested, and found that in general it was much more difficult to induce severe EAMG in these mice than in Lewis rats. Balb/c mice immunized with of the order of 5 doses of 10 pg of AChR purified from denervated rat muscle developed clinical EAMG by 40 days (Granato et al., 1976). Concentration of antibodies to rat AChR in serum was 3 x M, and concentration of antibodies cross-reacting with mouse AChR was 2 x M. This high degree of cross-reaction resembles that seen with rats immunized with syngeneic AChR (Lindstrom et al., 1976a, 1978a) or fetal calf muscle AChR (Merlie and Lindstrom, unpublished). It may be a fairly general phenomenon that the immunogenic determinants in mammalian AChR are very conservative ones common to AChR from many sources. Anti-idiotype antibodies to anti-AChR antibodies have been produced in mice (Schwartz et al., 1978).Thus far, these have been of low concentration and ineffective in suppressing EAMG. EAMG has been induced in guinea pigs by immunization with AChR from eel and torpedo (Lennon et al., 1975; Tarrab-Hazdai et al., 1975). An acute phase was not a characteristic feature. EAMG can be transferred to normal animals with low efficiency and long delay by lymph node cells (Tarrab-Hazdai, 1975a). As in the passive transfer of EAMG with lymph node cells in rats (Lennon et al., 1976),this leaves the question of whether the transferred cells participated in a cellmediated response or simply produced antibodies to AChR that directly affected the response. Since EAMG can be passively transferred by antibodies very efficiently and without the delay characteristic of the cell-transfer experiments (Lindstrom et al., 1976b), it seems likely that transfer of lymph node cells probably results in transfer of cells producing antibodies to AChR.

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EAMG can be induced in goats (Lindstrom, 1976; Lindstrom et al., 1978a). Immunization with AChR from eel electric organ is more potent at inducing EAMG, even though immunization with AChR from torpedo electric organ results in the rapid development of very high titers of antibodies to torpedo AChR (Lindstrom et al., 1978a). Goat antibodies to torpedo AChR are very highly species specific. EAMG can be induced in rhesus monkeys by immunization with AChR from torpedo electric organ (Tarrab-Hazdai et al., 1975b). Four doses of 100 p g over 12 weeks produced clinical signs of EAMG. This is of interest primarily because the structural similarities between primates makes the clinical similarities between EAMG and MG strikingly evident. In the monkeys signs of ptosis, facial diplegia, jaw paralysis, and severe dysphagia are evident. A few lymphocytes and a lymphorrhage were observed in a leg muscle. Squirrel monkey AChR shows a high degree of cross-reaction with human AChR (Lindstrom et al., 1978a). It seems likely that a human similarly immunized would be indistinguishable from a severely affected MG patient, except in having higher concentrations of antibodies to torpedo AChR than human AChR and, probably, in recovering completely if he survived. EAMG has been induced in frogs by immunization with torpedo AChR (Nastuk et al., 1979). Four to six doses of 100 pg over 4-6 months were required. The slow development of EAMG was paralleled by at least an &month persistence. The primary interest in frogs is that the electrophysiology and morphology of neuromuscular transmission has been very well studied in this animal. No phagocytic invasion was noted. As in rats, postsynaptic membrane structure is simplified (Rutherford et al., 1978) and miniature endplate potential amplitude is decreased.

VII. Autoimmune Response to AChR in Human MG

A. ANTIBODIES TO AChR Antibodies to AChR have been detected in the sera of MG patients by radioimmune assay using as antigen [1251]toxin-labeledAChR from human muscle (Lindstromet al., 1976d; Lindstrom, 1977a; Monnierand Fulpius, 1977) denervated rat muscle (Appel et al., 1975) and other sources (Lindstrom et al., 1978a). Antibodies to AChR in MG patient sera have also been detected by complement fixation using AChR purified from torpedo (Aharonov et al., 1975a) and by inhibition of ['251]toxin-labeledrat AChR to concanavalin A columns (Mittag et al.,

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1976). Because these antibodies, like antibodies to electric organ AChR, are highly species specific (Lindstrom et al., 1978a), the most sensitive detection is achieved using human AChR. Radioimmune assay is sensitive and quantitative and measures in the same units of moles specific toxin binding sites also used in EAMG to measure antibody and AChR. Antibodies to human AChR can be detected in 87% of patients diagnosed as having MG (Lindstrom et al., 1976d). The remaining 13% may be composed of patients with low titers, patients with muscular weakness arising from other causes, and neurasthenics. Antibodies to AChR are not found in diseases involving muscular atrophy and breakdown or as an epiphenomenon of other autoimmune diseases (Lindstrom et al., 1976d). In all sera tested, anti-AChR were 7 S IgG (Lindstrom, unpublished). Antibodies to AChR in the sera of MG patients have also been detected by their ability to impair binding of [1251]toxinto solubilized AChR (Almon et al., 1974) or to sections of tissue (Bender et al., 1975, 1976).These methods are neither so sensitive, quantitative, nor easy as radioimmune assay using [1251]toxin-labeledAChR as antigen. On the one hand, they might give information about a particularly pathologically significant subfraction of anti-AChR antibodies; but on the other hand, these methods might give misleading information. Most antibodies to AChR do not directly impair toxin binding (Lindstrom et al., 1976d). Demonstrating impairment of [1251]toxinbinding to solubilized AChR (Almon et al., 1974) requires preincubation of the AChR with huge excesses of anti-AChR, so any inhibition of binding that is observed could as likely result from impaired access of [1251] toxin to aggregated AChR as from specific anti-site antibodies (Lindstrom et al., 1976d).Demonstration of impairment of toxin binding to tissue sections was done by incubation first with MG serum, then with toxin, then antitoxin, and finally peroxidase anti-antibody (Bender et al., 1976). Given the dense packing of AChR at the tips of the postsynaptic folds (Fertuck and Salpeter, 1976) and the conglomeration of basement membrane and acetylcholinesterase in the synaptic cleft (McMahon et al., 1978), it would be surprising if the inhibition observed by such an indirect method were a good predictor of the accessibility of the acetylcholine binding site of the AChR to the 182dalton molecule of acetylcholine. In a more direct assay, only 2 of 28 sera from MG patients caused any detectable inhibition of [1251]toxin binding to rat muscle membrane fragments (Mittag et al., 1976). The amount of antibody bound to the postsynaptic membrane in MG patients is less in the more severely affected patients (Engel et al., 1977a). This is the opposite of what would be expected if acetyl-

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choline sensitivity were impaired by the accumulation of anti-site antibodies, and this is precisely the observation which would be expected if acetylcholine sensitivity were impaired primarily by the loss of AChR. Evidence of cells sensitive to AChR has also been obtained in MG patients (Abramskyet al., 1975b; Richman et al., 1976; Conti-Tronconi et al., 1977). These cells are assayed by increased [3H]thymidine incorporation in the presence of purified electric organ AChR. The effects are relatively small and scattered, and electric organ AChR must be used because purified human AChR is not available. It is not evident whether the cells detected are involved in the endplate pathology or whether they are involved in antibody production. The cellular response diminishes in patients who improve clinically after prednisone treatment (Abramsky et al., 1975a). The concentration of anti-AChR in MG patients varies widely from a minimum of 0.6 x lo+' M to a maximum in excess of 1000 X M with an average around 50 x M (Lindstrom et al., 1976d). Although patients with only ocular signs show statistically significant lower titers, among patients with generalized MG, antibody titer does not correlate closely with severity of disease. The observations that MG can be passively transferred to mice with anti-AChR antibodies (Toyka et al., 1975, 1977) and that decrease in antibody concentration by plasmapheresis is associated with clinical improvement (Dau et al., 1977; Newsome-Davis et al., 1978) argue for the importance of antiAChR in the pathology of MG. Also, anti-AChR antibodies are found at endplates in MG patients (Engel et al., 1977a), but lymphocytes or phagocytes are not (Engel and Santa, 1971; Engel et aZ., 1977b). Thus, if antibodies to AChR are important in causing the observed loss of AChR, as seems likely, then either patients must differ one from the other in the specificities of anti-AChR that they produce, and these different anti-AChR must differ in their ability to cause loss of AChR, and/or patients must differ substantially one from the other in immunological or muscle parameters that govern the effect of bound antibodies. From studies of cross reaction with AChR from various sources (Lindstrom et al., 1978a) and other studies (Mittag et al., 1976), it is evident that patients produce anti-AChR in differing arrays of specificity. It has not yet been possible to correlate any particular specificity with a functionally specific effect. The simplest form of antigenic specificity in the anti-AChR antibodies in MG patients has not yet been assayed for. It is not yet known what fraction of the antibodies are directed at determinants expressed on the external surface of the AChR molecule. Clearly, even an awesome concentration of

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antibodies directed at intracellular determinants would be without effect in vivo , Sera from most MG patients have higher titers by up to 2-fold when assayed using extrajunctional AChR from denervated rat muscle than when assayed using junctional AChR from normally innervated rat muscle (Weinberg and Hall, 1979). This is because extrajunctional AChR has unique determinants recognized by sera from MG patients in addition to determinants shared with junctional AChR. Antisera to native AChR from eel, torpedo, or denervated rat muscle could not distinguish between junctional and extrajunctional AChR. These very interesting experiments suggest that the immunogen in MG might resemble extrajunctional AChR (Weinberg and Hall, 1979).This might give some clue as to the mechanism by which the immune response to AChR in MG is triggered. Extrajunctional AChR might be expected to be present on myoid cells in the thymus or as a fetal antigen. Unfortunately, there is only a small amount of cross-reaction between antibodies to human AChR from MG patients and rat AChR (Lindstrom et al., 1978a), thus these experiments examined only a minor fraction of the anti-AChR antibodies present. Experiments using junctional AChR from normal human muscle and extrajunctional AChR from the denervated muscle of amyotrophic lateral sclerosis patients might be especially interesting. We have observed that antisera to the denatured a chains of torpedo AChR, like sera from some but not all MG patients, and unlike antisera to native torpedo AChR, has a higher titer against AChR from denervated muscle (Lindstrom et al., 197913).This suggests that the antigenic difference between junctional and extrajunctional AChR may be located at least in part on their a chains, and that the determinants unique to extrajunctional AChR may more closely resemble denatured than native AChR.

B. PATHOLOGICAL MECHANISMS IMPAIRINGTRANSMISSION In biopsies of intercostal muscle from MG patients, acetylcholine sensitivity is reduced (Rash et al., 1976; Albuquerque et al., 1976), as reflected in decreased amplitude of miniature endplate potentials (Elmquist et al., 1964; Lambert and Elmquist, 1971), and acetylcholine content is equal to or greater than normal (It0 et al., 1976). Postsynaptic membrane structure is simplified (Engel and Santa, 1971),and antibodies and the C3 and C9 components of complement are bound to the postsynaptic membrane (Engel et al., 1977a; 1979). Structure of the nerve ending is fundamentally normal, and vesicle size and number are normal, although area is slightly decreased (Engel and Santa, 1971). Presynaptic membrane structure is unaltered.

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Together, these results indicate that the defect in transmission in MG results not from a lesion in the nerve ending, but instead from a defect in the postsynaptic membrane of the muscle, which reduces its sensitivity to acetylcholine. Muscle from MG patients contains reduced amounts of AChR (Fambrough and Drachman, 1973; Engel et al., 197%; Lindstrom and Lambert, 1978). In four patients the average number of toxin binding sites per endplate was reduced by an average of 82% to 0.7 x lo7 (Fambrough and Drachman, 1973). In 11 MG patients, the amount of AChR extracted from intercostal muscle was reduced by an average of 64% from a control value of 28.6 x mol per gram of muscle to 10.3 x moYgm, and an average of 51% of the remaining AChR had antibodies bound (Lindstrom and Lambert, 1978).AChR remaining free of antibody (and total AChR) increased in direct proportion to acetylcholine sensitivity, as measured by miniature endplate potential amplitude. AChR identified electron microscopically by staining with peroxidase toxin is decreased in amount and altered in distribution (Engel et al., 1977b). The amount of AChR measured in this way also increases in direct proportion to miniature endplate potential amplitude. The most severely affected patients have the least residual AChR (Engel et al., 1977a,b; Lindstrom and Lambert, 1978). Thus, loss of AChR appears to be the primary lesion impairing neuromuscular transmission in MG. To what extent is AChR activity in MG patients directly impaired by the binding of antibodies? No change in channel opening time or conductance was observed in biopsies from MG patients (Cull-Candy et al., 1978). Unfortunately, the number of patients, disease severity, or fraction of AChR bound by antibodies was not reported, so it is difficult to make much of this findlig. Using human muscle cells in culture, antibody was observed to slightly decrease both channel opening time and conductance (Bevan et al., 1978). In rats with EAMG there are small but measurable decreases in both channel opening time and conductance (Heinemann et al., 1977). AChR whose function was completely or nearly completely blocked by antibody would not be detected by the methods used in these studies. One of the patients biopsied had antibodies bound to all the AChR extractable from the biopsy (Lindstrom and Lambert, 1978), and in this respect it resembled some of the most severely affected rats with chronic EAMG (Lindstrom et al., 1976a). Since some AChR activity was detectable, bound antibody clearly did not completely inhibit the function of the remaining AChR. Anti-AChR specificity as well as concentration is known to vary widely among patients (Lindstrom et al., 1976d, 1978a;

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Mittag et aZ., 1976).The extent to which AChR function is impaired by bound antibodies probably varies somewhat among patients, depending on the specificities of antibodies produced, from perhaps 0 to 30%. As indicated by experiments with rats (Lennon et al., 1978), the safety factor for transmission is normally sufficiently large that binding of antibodies alone would not impair transmission; however, after reduction of the safety factor by extensive AChR loss, a further decrement in acetylcholine sensitivity due to the direct effects of bound antibody might be critical in determining whether or not transmission would succeed. Antibody-dependent, complement-mediated focal lysis of the postsynaptic membrane probably contributes to the observed loss of AChR in MG. C3 and C9 is observed bound to the postsynaptic membrane and bound to AChR containing fragments shed from it (Engel et al., 1977a; 1979). Studies in rats show the functional importance of C3 (Lennon et al., 1978). Increased turnover of AChR induced by antibody binding probably also contributes to the observed loss of AChR in MG. Anti-AChR antibodies from MG patients increase the rate of AChR turnover in human (Bevan et al., 1977)and rat (Kao and Drachman, 1977; Appel et al., 1977) muscle cells in culture. As previously discussed in connection with chronic EAMG, the relative contribution of antigenic modulation and complement-mediated destruction to the observed loss of AChR is not certain, but antigenic modulation appears to be very important. Loss of AChR due to phagocytosis is probably not important in most MG patients. Phagocytic invasion of endplates has not been reported (Engel and Santa, 1971), and there is reason to believe that postsynaptic membrane is sufficiently dynamic that persistent alteration of its structure is unlikely to be due to very infrequent phagocytic invasions. The transient form of myasthenia gravis that is sometimes observed in the newborn of mothers with MG is probably caused by transplacental transfer of anti-AChR antibodies. Anti-AChR antibodies have been detected in the serum of newborn babies with neonatal MG (Lindstrom et al., 1976d), and anti-AChR concentration in such babies has returned to normal over 4-6 weeks as the babies have returned to normal (Keesey et al., 1977; Masters et al., 1977; Nakao et al., 1977). MG can be passively transferred to mice with anti-AChR antibodies (Toyka et al., 1975, 1977), and EAMG can be similarly transferred to normal rats (Lindstrom et al., 1976b). In rats with EAMG, antibodies can also be transferred to newborn via milk (Sanders et al., 1977). We have not detected anti-AChR in the breast’milk of several myas-

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thenic mothers with detectable serum concentrations, but our antihuman IgG sera probably would not have precipitated IgA anti-AChR if it had been present (Lindstrom, unpublished). We have also detected anti-AChR in babies without obvious weakness born to mothers with MG, but have not yet been able to set an absolute cutoff value for anti-AChR titer that will predict whether neonatal MG will occur (Keesey et al., 1977). Thus, neonatal MG, like the adult form, does not show a close correlation with absolute antibody titer, probably explaining why only 1 in 8 babies of myasthenic mothers show weakness. C. CAUSEOF THE AUTOIMMUNE RESPONSETO AChR What triggers the autoimmune response to AChR in MG is not known. The immunogen is probably modified human AChR. Syngeneic AChR solubilized in detergent from normal rat muscle and administered in complete Freund’s adjuvant causes EAMG in rats (Lindstrom et aE., 1976a), but there is some evidence in rats that normal AChR in phagocytized membrane is not immunogenic (Lindstrom et al., 1976b). Anti-AChR antibodies in humans do not occur as an epiphenomenon of degenerative neuromuscular disease in humans (Lindstrom et al., 1976d). These results suggest that to become autoimmunogenic AChR must be modified somewhat in structure and/or shed from the membrane. Some clues about the origin of the immune response in MG may be found by looking upon the anti-AChR antibodies in these patients as fossil templates of the triggering (or sustaining) immunogen. The antibodies to AChR in MG patients react best with human AChR, nearly as well with primate AChR, less well with other mammalian AChR, and least well with AChR from fish electric organs (Lindstrom et al., 1978a). These results suggest that the immunogen in MG closely resembles human skeletal muscle AChR. Sera from many MG patients react better with extrajunctional than with junctional AChR from rat muscle (Weinberg and Hall, 1979). This might indicate that the immunogen in MG is often extrajunctional AChR. Antisera to denatured a subunits of torpedo AChR react better with extrajunctional AChR, whereas antisera to native torpedo AChR do not distinguish between these AChR (Lindstrom et al., 1979b). This might indicate that the determinants unique to extrajunctional AChR resemble denatured structures and are, at least in part, located on the a chains. AChR from human muscle and eel electric organs share antigenic determinants that are not immunogenic in MG patients (Lindstrom et al., 1978a). This finding suggests that, if human AChR is the immunogen in MG,

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either some determinants are not immunogenic in humans, or the immunogen in MG has (or retains) only some of the antigens of native AChR. It is not known whether anti-AChR antibodies in M G patients are directed only at determinants on the extracellular surface of the AChR molecule, or whether intracellular determinants are also involved. If intracellular determinants were involved, it would suggest that the immunogen in MG were shed from cells. What little is known is consistent with the idea that the immunogen in M G is human AChR, perhaps slightly modified in conformation or structure. Another possibility, which seems somewhat less likely, is that the immunogen in MG is a bacterial or viral antigen that crossreacts with AChR. Still another possibility is that the autoimmune response to AChR in M G arises not from a stimulatory event, but from a failure of normal inhibition of cells responding to AChR. There is as yet no information about whether any population of AChR in MG patients is biochemically distinguishable from normal. Such an observation would allow discrimination between these possible mechanisms. If human AChR were the immunogen, where is the immunogen located and what makes it immunogenic? The high incidence of thymoma, thymic hyperplasia, and germinal centers has drawn attention to the role of the thymus in M G patients. Thymectomy may be beneficial for M G patients (Buckingham et al., 1976). Approximately 10-15% of MG patients have thymomas (Castleman, 1966). These patients have serum antibodies detectable by immunofluorescence, which cross-react with striations in muscle cells and with certain cells in thymus termed myoid cells (Strauss and Kemp, 1967). Such antibodies are also found in some thymomatous patients who do not have MG. Traces of AChR can be extracted from the thymuses of calves (Aharonov et ul., 1975c) and rats (Lindstrom et al., 1976~). Muscle cells bearing AChR have been cultured from dissociated rat thymus tissue and thymus tissue from an M G patient (Kao and Drachman, 1976). It has been suggested that muscle cells in thymus may arise from pluripotent stem cells capable of differentiation into many specialized cell types (Wekerle et al., 1975).This intriguing possibility suggests that many autoantigens may be available in thymus. That the primary immunogen in MG might be AChR on thymic myoid cells seems a reasonable hypothesis. Since thymectomy does not produce dramatic cures in all M G patients, the thymus clearly is not the sole repository of immunogen. What would make AChR on myoid cells immunogenic? A viral infection is one reasonable mechanism by which surface proteins, including AChRs, might be modified. This is attractive because, if in 10%of infections the virus transformed

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the infected myoid cells or cells nearby, it could account for the high incidence of thymoma in MG patients. There is no evidence that thymomatous cells exhibit any differentiated features of musclelike striations. The increased frequency of other autoimmune diseases noted in MG patients (Simpson, 1960) could result either from modification of other antigens by such a virus or from a genetic predisposition to altered immune response. Thymic hyperplasia occurs in about 75% of MG patients (Castleman, 1966), and some anti-AChR antibody can be detected in 70% of thymus extracts (Mittag et al., 1976). Synthesis of anti-AChR antibody has been detected in one of four thymic tissues and three of five thymic lymphocyte populations removed from MG patients (Vincent et al., 1978a). Thymectomy may be beneficial for some MG patients early in the disease (Buckingham, 1976), but it is not accompanied by a consistent decrease in anti-AChR titers (Seybold et al., 1978; Mittag et al., 1976). Anti-AChR production in rats is dependent on T cell-B cell cooperation (Lennon et al., 1976). Together these results suggest that although it is possible that the thymus or cells derived from it might have roles in the induction or regulation of the autoimmune response to AChR, in the ongoing disease the thymus is not directly a critical source of immunogen or antibody. There is an abnormally high frequency of HLA-B8 and DRW3 in MG patients, and HLA-B8 frequency is also elevated in autoimmune diseases involving other antigens (Feltkamp et al., 1974; Fritz et al., 1973). Patients with either of these histocompatibility antigens tend to have significantly higher titers of anti-AChR antibodies than MG patients lacking both (Naeim et d., 1978). Although this by no means indicates an absolute requirement for a particular genetic background in MG patients, it does suggest that their response to a triggering environmental influence could be influenced by a genetic predisposition. Frequency of HLA-B8 and DW3 is also elevated in Graves’ disease andjuvenile onset diabetes in whites (Thomsonet al., 1975; Grumet et al., 1974). In Japanese, other HLA determinants are associated with these diseases (Yoshida et al., 1977). If histocompatibility genes are associated with immune response genes, then this might indicate that altered immune response is associated with increased susceptibility to autoimmune disease, but not necessarily with triggering an immune response to AChR in particular. This seems a reasonable hypothesis in the case of an autoimmune disease like MG. Clinical signs of MG have been reported in patients with rheumatoid arthritis after treatment with prolonged high doses of penicillamine (Bucknall et al., 1975). Signs of muscular weakness re-

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mitted after penicillamine therapy was discontinued. In similar patients antibodies to AChR were detected in serum (Masters et d., 1977; Russell and Lindstrom, 1978; Vincent et al., 1978b). In these patients anti-AChR titer decreased after discontinuance of penicillamine. This is strong evidence that penicillamine occasionally induces an autoimmune response to AChR in reheumatoid arthritis patients. Antistriational antibodies have been noted in l l of 56 patients treated with penicillamine (Masters et al., 1977).Chronic treatment of normal rats with high doses of penicillamine resulted in no detectable induction of anti-AChR antibodies (Lindstrom, Lennon and Seybold, unpublished). By what mechanisms penicillamine might act to induce autoimmune responses to muscle antigens in some rheumatoid arthritis patients remains an intriguing question. An answer might shed light on the mechanisms by which MG is induced in other patients. Since penicillamine has been associated with autoimmune responses to other antigens (listed in Russell and Lindstrom, 1978), it might be that penicillamine affects an immunoregulatory process rather than a particular immunogen. Preliminary experiments suggest that MG patients are deficient in mitogen-induced suppressor cell activity (Mischack et aZ., 1979). Whether this is indicative of a generalized defect in suppressor cell activity responsible for susceptibility to autoimmune responses remains to be determined. VIII. Other Autoimmune Anti-Receptor Diseases

Other autoimmune diseases involving hormone receptors have been identified that show interesting parallels and contrasts with the autoimmune response to AChR in MG. In Graves’ disease serum antibodies to the receptor for thyroidstimulating hormone (TSH) are found (Rees Smith, 1976; Adams and Kennedy, 1971; Peterson et al., 1977; McLachlan et al., 1977). By contrast with anti-AChR, some of these antibodies competitively inhibit TSH binding and activate the TSH receptor, while other antibodies bind but do not activate. As with anti-AChR, cross-reaction with receptors from other species is seen, but the antibodies are relatively species specific, especially those that are not agonists. As in MG, a transient form of neonatal Graves’ disease occurs as a result of transplacental transfer of anti-receptor antibodies (Dirmikis and Munro, 1975).In malignant exophthalmos, antibodies afFect the TSH receptor in retroorbital tissue, but not the TSH receptor in thyroid tissue (Winand and Kohn, 1975). This is similar to the case in MG in that

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antibodies to the nicotinic AChR of skeletal muscle do not affect the muscarinic AChR in smooth muscle and cardiac muscle. An interesting similarity to the high incidence of thymoma in MG is the high frequency of antibodies to TSH receptors in patients with thyroid carcinoma (Rees Smith, 1976). Certain patients with insulin-resistant diabetes have antibodies to insulin receptors (Flier et al., 1976; Jarrett et al., 1976; Flier et al., 1979).By contrast with MG, antibodies in most patients bind at or near the site on the receptor for insulin binding and inhibit insulin binding while in some cases acting as partial agonists. Peptide hormones like TSH and insulin may have vastly larger binding sites on their receptors than does a small molecule like acetylcholine, which may help to explain the apparent lack of antigenicity of the acetylcholine binding site. Antibodies to insulin receptors show fairly extensive interspecies cross-reaction. The effect of antibody on amount of TSH or insulin receptor is not yet known. Since insulin is among the many hormone receptors whose amount is down-regulated by the presence of hormone, it seems quite possible that receptor amount in this case might potentially be influenced by anti-site antibodies through this mechanism as well as by complement-mediated destruction and antigenic modulation. IX. Concluding Remarks

Evidence suggests that the muscular weakness and fatigability characteristic of myasthenia gravis results from impaired neuromuscular transmission due to an autoimmune response to AChR in the postsynaptic membrane of muscle fibers. Antibodies rather than cells are the primary effector of the immune assault. The primary lesion impairing transmission is loss of AChR due to complement-mediated focal lysis and antigenic modulation, Direct inhibition of AChR function by bound antibodies and alteration of postsynaptic membrane architecture also contribute to impaired transmission. What triggers the autoimmune response to AChR initially, or how to specifically inhibit it, are as yet unknown. However, recent studies have gone far toward explaining the mechanisms of the pathology at the endplate. A new diagnostic method has been developed. These studies have provided improved rationale for the use of therapeutic methods long in use, like antiesterase drugs, steroids, and thymectomy, and have provided the rationale for the application of new methods like plasmapheresis. EAMG is increasingly well understood both qualitatively and quantitatively and provides a model for further study of MG that may be

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relevant in some respects also to other autoimmune diseases involving receptors. As it becomes possible to purify the receptors for other neurotransmitters and hormones, immunization with these proteins may provide models for diseases as yet unrecognized. Antibodies to AChR are proving to be valuable probes for the study of AChR structure, function, and metabolism, and these studies may provide models for the study of other receptors.

ACKNOWLEDGMENTS Research in the author’s laboratory is supported by grants from the National Institutes of Health (NS11323),the Muscular Dystrophy Association, Sloan Foundation, and the Myasthenia Gravis Foundation of Los Angeles.

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ADVANCES IN IMMUNOLOGY, VOL. 27

MHC-Restricted Cytotoxic T Cells: Studies on the Biologica I Role of Po Iymorp h ic Ma ior Transpla ntat ion Antigens Determining T-Ce I I Restriction-Specificity, Function, and Responsiveness ROLF M. ZINKERNAGEL A N D PETER C. DOHERTY Department of Immunopdhology, Scripps Clinic and Research Foundation, La Jolla, California, and The Wistar Institute, Philadelphia, Pennsylvania

I. Introduction . . . . . . . . . . . . . . . . . . . . . . 11. Virus-Specific Cytotoxic T Cells . . . . . . . . . . . . . . . . . .. . . . . . . . . 59 A. Generation of Effector Cells and Assay . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 1. Induction of Primary and Secondary Effector Lymphocytes in Vivo ......................................................... 59 2 . Stimulation of Primary or Secondary Virus-Specific Cytotoxic T Cells i n Vitro 61 3. Release Cyt the Assay Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 4 . I n Vivo Correlates ..................... 67 B. Characterization of E ................................... 67 1. Surface Markers of Effector Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 2. Specificity and Clonality of Effector Cells . . . . . . . . . . . . . ...... 68 C. Evidence for MHC Restriction in Other Species and Outbre Populations . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 D. Models for Recognition by Effector T Cells . . . . . . . . . . . . . . . . . . . . . . . . . 72 111. Definition of Target Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 A. Nahire of the Restricting Self-MHC Determinants . . . . . . . . . . . . . . . . . . . 74 1. Genetic Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 . . . . . . . . . . . . . . . . . . . 75 2. Antibody Blocking.. . . . . . . . . . . . . . . . . . . . . . . 3. Restriction Specifici ..................................... 76 B. Nature of Virally Indu e n s . . . . . . . . . . . . .. . .. .... .. . . . . .. . ... 80 1. Minimal Requirements for Target Cell Induction . . . . . . . . . . . . . . . . . 81 2. Virus Mutants . . . . . . . . . . . . . ............................... 83 3. Comparison of Serological and Cytotoxic T-cell Specificity . . . . . . . . 84 4. Antibody Blocking.. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 88 . . . . . . . . . . . . . . . . . 88 5. Tumor-Associated Viruses . . . . C. Evidence for Interaction of Self-H ens . .. . . . . .. . . . . 91 D. The Special Case of Alloantigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 . . . . . . . . . . 95 E. Conclusion . . . ... .. . . . .......... IV. Ontogeny of Effector Cells e Major Histocompatibility Gene Complex in Defining T-cell Specificity during Ontogeny . . . . . . . . . . 96 A. Differentiation of T-cell Restriction-Speci . .. . ...... . . . . . . . . . .. . 97 1. Early Studies of Chimeras ...................... 97 2. Neonatally Tolerant Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 3. Irradiation Chimeras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 4. Thymus-Craft Chimeras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

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ROLF M. ZINKERNAGEL AND PETER C. DOHERTY

B. Role of Lymphohemopoietic Cells in T-cell Maturation and Antigen Presentation ............................................... 1. Postthymic T-cell Maturation .................................... 2. Antigen Presentation ............................................ C. MHC Incompatible Chimeras ...................................... D. Negative Selection Experiments .................................... 1. The Transferred T Cells and the Irradiated Recipients Need Not Share I-Region Determinants .................................... 2. Negatively Selected T Cells Interact with Vaccinia Virus in the Context of Some H-2K Determinants Not Encountered in Thymus ........................................................ E. Concliision ........................................................ Role of the Major Histocompatibility Gene Complex in Determining T-cell Responsiveness ................................................ A. Evidence for MHC-Coded Ir Genes Regulating the Expression of Cytotoxic T Cells ............................................... B. Influence of Thymic Selection of T-cell Restriction Specificities on Responsiveness during T-cell Ontogeny ......................... C. Conclusion.. ...................................................... Interpreting MHC Restriction and Ir Regulation of T Cells .............. I n Viuo Relevance of MHC-Restricted Cytotoxic T Cells ................ A. Immune Protection ................................................ B. T Cell-Mediated Immunopathology ................................. C. MHC Polymorphism., ............................................. D. MHC-Associated Diseases . . . . . . . . . . . . . ......................... E. Conclusion .................................................. Finale ................................................................ Abbreviations Used in the Text . . . . . . . . . . . . .................... References ...........................................................

104 104 104 105 106 107

108 109 109 110 115 118 118 128 129 132 135 139 140 141 141 142

I. Introduction

The study of infectious disease has been central to the development of immunology, starting with the use of poxvirus for vaccination by Jenner (1798; see Jenner, 1975) and becoming a scientific discipline with the work of Pasteur, Koch, and Ehrlich. The ravages of plague (Defoe, 1722; see Defoe, 1908), smallpox, or even measles (Bumet, 1962) in a previously unexposed population are such that infectious diseases must be considered a major evolutionary force that has probably been instrumental in the development of immune mechanisms characteristic of higher vertebrates. This may explain the vertebrates’ extraordinary immunological reactivity against extracellular and intracellular parasites. Specific immunity is by no means the only solution for overcoming infectious disease; as an example, crayfish (Tyson and Jenkin, 1974) are infected by viruses and are apparently able to control infection without a conventional immune system. Still, as a means of coping with these problems in animals that are large and long-lived, the vertebrate immune system is a fascinating evolutionary adaption that is generally very efficient.

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Some aspects of immune response, in particular antibody, have now been analyzed at the molecular and genetic levels (for references, see Weigert et al., 1975; Hood et al., 1978; Tonegawa et al., 1977). Immunity to extracellular bacteria is mediated at least in part by antibodies and complement and is reasonably well understood. However, cellmediated immunity, which is a major factor in the elimination of intracellular parasites, such as viruses or intracellular bacteria, offers many enigmas. We know that the thymus-derived lymphocytes (T cells) orchestrate the cell-mediated immune response, but we do not yet understand either the nature of the T-cell receptor or the configuration of the antigenic determinants recognized. Furthermore, it is now apparent that major histocompatibility’ antigens are involved in all T-cell interactions with other cells, which complicates these issues, but offers the most fascinating possibilities for the eventual comprehension of both the evolution and nature of cell-interaction mechanisms. The biological function of major transplantation antigens has been a puzzle since the discovery of alloreactivity. Much work has been done, both because the problem is experimentally accessible and because of the clinicians’ hope that organ transplantation would prove feasible. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics (Gorer, 1936; reviewed in Klein, 1975; Shreffler and David, 1975; Snell et al., 1976).The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. The murine major histocompatibility gene complex (MHC) is designated H - 2 and is located on chromosome 17, which is subdivided into several subregions, K , I , S , G, and D in order of increasing distance from the centromere (reviewed in Shreffler and David, 1975; Klein, 1975) (Fig. 1). The gene region spans about 0.5 recombination units (centimorgans),approximately the length of the genome ofEscherichia coli (Cohn and Epstein, 1978). The human MHC ( H L A ) contains regions of comparable character, but in slightly different order. The I-region equivalent, D, is closest to the centromere, followed by the B

* Major histocompatibility or transplantation antigens are polymorphic cell-surface antigens that are encoded by genes mapping to the major histocompatibility gene complex. Transplanted cells or tissues that differ from the graft recipient with respect to major histocompatibility antigens induce a strong immune reaction (antibodies and cytotoxic T cells) and are rejected rapidly within 7 to 12 days. They are therefore called major (or strong) transplantation antigens, in contrast to a variety of minor (or weak) transplantation antigens that are encoded by genes distributed through the entire genome. Transplanted tissues expressing only minor transplantation antigen differences are generally rejected more slowly and do not readily induce primary immune responses that are detectable by in uitro assay procedures.

54

ROLF M. ZINKERNAGEL AND PETER C. DOHERTY MAJOR HISTOCOMPATIBILITY COMPLEX

u

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.

17TH CHROMOSOME

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6TH CHROMOSOME

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NONLYTIC T

FIG.1. The major histocompatibility gene complex (MHC) maps to chromosome 17 in mice and to chromosome 6 in humans. The small portion of the chromosome is magnified to show the known regions within the MHC: K,I (I-A, I-B, I-], I-E, I-C ), S, D (including L ) in H-2,D, DR,A, C, B in HLA.The functions of some of the gene products of these regions are summarized in the brackets underneath. TA stands for target antigen, CTL for cytotoxic T lymphocytes, "Ir" for immune-response gene regulating expression of CTLs; MLR stands for mixed lymphocyte reaction, Ir for immune-response genes (or restricting target antigen) regulating responsiveness of nonlytic T cells; C stands for complement factors coded within the MHC.

and A regions; the C region located between A and B has no equivalent in H-2 as yet ( reviewed in McDevitt and Bodmer, 1974; Snell et al., 1976) (Fig. 1).These subregions code for different classes of cellsurface antigens. The K, D (A, €? ) loci code for the classical, serologically defined major transplantation antigens and for the target antigens for cytotoxic T cells. The D region codes for the D structure and the L structure (D6mant et al., 1978, 1979). The Z ( D )region contains the immune-response (Zr) genes (McDevitt et al., 1972; Benacerraf and Germain, 1978) and encodes the serologically defined Ia antigens (Shreffler and David, 1975; Klein, 1975), which may also serve as targets for alloreactive cytotoxic T cells in some situations (Wagner et al., 1975, 1977; Nabholz et al., 197513; Klein et al., 1976). Much of the I-region function has been analyzed by T-cell proliferation in alloreactions (reviewed in Bach et al., 1973, 1976, 1977), in MHC-restricted interactions between T cells and bone marrow-derived lymphocytes (B cells) (Katz and Benacerraf, 1975)and interactions between T cells

MHC-RESTRICTED CYTOTOXIC T CELLS

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and antigen-presenting stimulator cells (Shevach and Rosenthal, 1973; Rosenthal and Shevach, 1973). The present knowledge of the MHC has been reviewed comprehensively b y Shreffler and David (1975), b y J. Klein (1975, 1976), and by Snell et al. (1976). Much had been done to define the function of the I region before any clear evidence emerged to link the otherH-2 genes with cell-mediated immunity in the context of “Self.” Levine et al. (1963)and McDevitt and Sela (1965) described antibody responses that were regulated by Ir genes, which later were shown to map to the MHC (McDevitt and Chinitz, 1969). Shortly thereafter, McCullagh (1972) found that a graft-versus-host reaction could reverse tolerance to sheep red blood cells in rats, and Katz et al. (1971) discovered that the requirement for T-cell help for the induction of antibodies against T-dependent antigens could be replaced by unprimed allogeneic and alloreactive T cells. This “allogeneic effect,” or abnormal induction (Katz, 1972; Bretscher, 1972; Cohn, 1972) of B cells, became better understood when Kindred (1971), Kindred and Shreffler (1972), and subsequently Katz, Hamaoka, and Benacerraf and co-workers (Hamaoka et al., 1973; Katz et al., 1973a; Katz and Benacerraf, 1975) showed that T help could be delivered only to H-2-compatible B cells. Independently, Shevach and Rosenthal found that antigen-specific T-cell proliferation was dependent on T cells and macrophages sharing the same I-region genes (Shevach and Rosenthal, 1973; Rosenthal and Shevach, 1973). The idea that Self major histocompatibility antigens (Self-H) are involved in normal cell-mediated immunity has historically been the subject of some speculation. For example, Mitchison speculated in his classic paper on adoptive transfer of the rejection reaction by lymphocytes that cellular immune recognition o f skin-sensitizing antigens, such as tuberculin or chemical allergens, occurred only when these antigens appeared on cell membranes, thus resembling foreign transplantation antigens (Mitchison, 1954). Some years later, Lawrence, expanding on Thomas’ surveillance hypothesis (Thomas, 1959),intuitively proposed that immune lymphocytes evolved to combat intracellular parasites and recognized the parasite antigen (called X) expressed on macrophages only in association with a Self antigen (Lawrence, 1959, 1973). This Self plus X hypothesis preempted many of the principles later defined experimentally for interactions between T cell-mediated immunity, intracellular parasites, and MHC. The discovery that susceptibility to tumor induction was ljnked to the MHC (Sjogren and Ringertz, 1962; Lilly et al., 1964; Lilly and Pincus, 1973) was not interpreted in this way. More direct indications were provided by the experiments of Bryere and Williams (1964), SvetMoldavsky and co-workers (Svet-Moldavsky et al., 1964, 1967), and later Holterman and Majde (1969, 1971), who discovered that the re-

56

ROLF M. ZINKEFWAGEL AND PETER C. DOHERTY

jection of syngeneic virus-infected cells, or tissue grafts, resembled allograft rejection. The latest addition to this sequential uncovering of the MHC’s role in T cell-mediated immunity was the discovery that the cytotoxic activity of virus-specific T cells was H-2-restricted in mice injected with lymphocytic choriomeningitis virus (LCMV) (Zinkernagel and Doherty, 1974a,b, 1975a; Doherty and Zinkemagel, 1974, 1975a,b). Independently, Shearer and co-workers found that trinitrophenyl (TNP)specific cytotoxic T cells were similarly restricted (Shearer, 1974; Shearer et al., 1975). The finding that virus-specific cytotoxic T cells are dually specific for virus and for a Self cell-surface antigen encoded by the MHC was essentially the result of serendipity. Oldstone et al. (1973) had found that susceptibility to LCMV-induced disease varied slightly with H-2 type, a finding that has not been satisfactorily confirmed (Oldstone, 1976). We thus set out to test the hypothesis that highly susceptible mice generate greater LCMV-specific cytotoxic T-cell activity than the less susceptible mice, because much experimental evidence from Traub (1936, 1939), Haas (1941), Lillie and Armstrong (1945), Rowe (1954), Hotchin (1963, 1971), Rowe et al. (1963), Hotchin and Sikora (1964), Gledhill(1967), Hirsch et al. (1967), Johnson and Mims (1968), Cole and co-workers (Cole et al., 1971, 1972; Gilden et al., 1972a,b; Johnson and Cole, 1975), and ourselves (Zinkernagel and Doherty, 1973; Doherty and Zinkemagel, 1974) suggested that fatal lymphocyticchoriomeningitis (LCM)was causedbyvirus-immuneTcells. Mice of various H-2 types were injected intracerebrally (i.c.) with LCMV, and all showed symptoms of severe neurological disease 7 days later. Some were killed, and the cytotoxic activity of their spleen cells was tested on virus-infected L cells, a fibroblast cell line of C3H ( H - 2 k ) origin. Surprisingly, only spleen cells from mice possessing the H-2 haplotypes lysed these targets specifically (Zinkernagel and Doherty, 1974a,b; Doherty and Zinkernagel, 1974,1975a). This result was compatible with experiments published by Oldstone and Dixon (1970) and Marker and Volkert (1973a,b),who always used syngeneic lymphocytetarget cell combinations for assaying virus-specific cytotoxicity, and explained why lysis obtained in H-2-incompatible combinations was marginal (Cole et al., 1973a,b; Gardner et al., 1974a,b). The observation made with LCMV also seemed to explain earlier findings of de Landazuri and Herberman (1972) that when murine sarcoma virus (MSV)-immune cytotoxic T cells were tested, H-2incompatible or xenogeneic tumor target cells expressing the Friend-Maloney-Rauscher virus antigens were inefficient in competing with H-2-compatible targets, and allogeneic target cells were lysed

MHC-RESTRICTED CYTOTOXIC T CELLS

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poorly by MSV-immune cytotoxic T cells (Herberman et al., 1973; Leclerc et al., 1973; Lavrin et al., 1973; Senik et al., 1975).The failure to recognize the H-2 restriction phenomenon may reflect the lesser sensitivity of the MSV system and the restricted target cell repertoire available. The H-2 restriction finding was soon confirmed for ectromelia (mouse pox) virus (Gardner et al., 1975; Blanden et al., 1975a,b), vaccinia virus (Koszinowski and Thomssen, 1975; Koszinowski and Ertl, 1975a,b), Sendai virus (Doherty and Zinkernagel, 1976; Koszinowski et al., 1977; Ertl and Koszinowski, 1976a; Starzinski-Powitz et al., 1976a; Schrader et al., 1976, Schrader and Edelman, 1977; Sugamura et al., 1977), LCMV (Pfizenmaier et al., 1975, 197613; Marker and Andersen, 1976), influenza virus (Yap and Ada, 1977, 1978a-d; Effros et al., 1977; Doherty et al., 1977c, 1978; Zweerink et al., 1977a; Ennis et al., 1977a,b,c; Biddison et al., 1977a; Ada and Yap, 1977; Braciale, 1977a,b), herpes virus (Pfizenmaieret al., 1977a,b; Rollinghoff et al., 1977a), Simian virus 40 (SV40) (Trinchieri et al., 1976; Pfizenmaier et al., 1978),rabies virus (Wiktoret al., 1977), vesicular stomatitis virus (VSV), (Zinkernagel et al., 1977d, 1978f; Hale et al., 1978), Coxsackie virus (Wong et al., 1977a,b,c), cytomegalovirus (Quinnan et nl., 1978), and alpha viruses (Miillbacher and Blanden, 1979a,b). The phenomenon was also shown for tumorassociated viruses, such as Rauscher leukemia virus (Schrader et al., 1975), Friend virus (Blank et al., 1976; Blank and Lilly, 1977), MSV (Gomard et al., 1976, 1977a; Plata et nl., 1976; Holden and Herberman, 1977), and, at least in part, for the response against murine mammary tumor virus (Stutman et al., 1977; Stutman and Shen, 1978) and adenovirus (Inada and Uetake, 1978a,b). Shearer’s results with TNP-specific cytotoxic T cells were confirmed b y Forman (1975a, 1976) Dennert (1976), and Dennert and Hatlen (1975). Other hapten-specific cytotoxic T cells were detected for various nitrophenyl compounds (Rehn et al., 1976a,b), for N-(S-nitro-4hydroxy-5-iodophenyla~etyl)-~-alanylglycerylglycyl-modified cells (Koren et al., 1975; Rehn et al., 1976a) and related compounds (Rehn et al., 1976b), fluorescein-isothiocyanate-conjugated cells (StarzinskiPowitz et al., 1976b), and dinitrophenyl (DNP) (Forman, 197%; Schmitt-Verhulst and Shearer, unpublished; Dennert, unpublished). Subsequently, the same restriction was found for cytotoxic T cells directed against minor transplantation antigens (Bevan, 1975a,b) and against the male H-Y antigen by Simpson and co-workers (Gordon et al., 1975, 1976). The MHC restriction of cytotoxic T cells directed against weak transplantation antigens has been extended to Lyt antigen (Rollinghoffet al., 197%) and possibly to Thy-1 (Zatz, 1978). In addition, H-2-restricted cytotoxic T cells have been detected for anti-

58

ROLF M. ZINKERNAGEL AND PETER C. DOHERTY

gens in fetal calf serum that may become inserted into the cell membrane during in uitro culture (Peck et al., 1977b) or during autosensitization in uitro (Ilfeld et al., 1975; Fomi and Green, 1976; Goldstein et al., 1978; Levy and Shearer, unpublished). MHC restriction of effector lymphocytes in uitro has also been documented for the following species: rats (Marshak et al., 1977; Zinkemagel et al., 1977c; Jungi and McGregor, 1978a,b); humans, for the male H-Y antigen (Goulmy et al., 1977), for virus (McMichael et al., 1977; McMichael, 1978; Tursz et al., 1977; Biddison and Shaw, 1979) and DNP or TNP (Dickmeiss et al., 1977; Shaw and Shearer, 1978, Shaw et al., 1978);chickens (Wainberg et al., 1974; P. Toivanen et al., 1974a,b; A. Toivanen et al., 1977a,b). Also, MHC restriction of T cells was soon shown for a variety of in viuo situations. Early experiments with the virus models demonstrated that operation of H-2-restricted T cells was central to both the induction of fatal neurologic disease and the severity of disease in LCM (Doherty and Zinkemagel, 1975a; Doherty et al., 1976a) and to clearance of virus from liver or spleen (Blanden et al., 1975b; Kees and Blanden, 1976; Zinkernagel and Welsh, 1976). The same was true for delayed-type hypersensitivity to soluble proteins, chemically modified cells (Miller et al., 1975, 1976; Vadas et al., 1977), and virus (Zinkemagel and Doherty, 197%). Findings were similar for T cells involved in activating macrophages to increased bactericidal activity during an immune response against Listeria monocytogenes, a facultative intracellular bacterium (Zinkemagel, 1974; Zinkemagel et al., 1977a), or for T cells mediating various forms of suppression (Claman et al., 1977; Cohn and Epstein, 1978).Thus, MHC restriction of T cells seems to apply generally to effector functions tested so far in mice, and probably universally in higher vertebrates. The experimental evidence for MHC-restricted cytotoxic T cells has been reviewed extensively during the past 3 years (e.g., Doherty et al., 1976c; Shearer et al., 1976,1977; Forman, 1976; Blanden et al., 1976b; Munro and Bright, 1976; Schrader et al., 1976; Koszinowski et al., 1976; von Boehmer et al., 1976; Paul et al., 1976; Paul and Benacerraf, 1977; Zinkemagel and Doherty, 1976a,b, 1977a; Shearer and SchmittVerhulst, 1977; von Boehmer, 1977; Simpson and Gordon, 1977; Katz, 1977; Miller and Vadas, 1977; McKenzie et al., 1977; Zinkemagel, 1978a,b; Miller, 1978; Sprent, 1978c; Bevan and Fink, 1978; Langman, 1978; Cohn and Epstein, 1978; Thorsby, 1978). The present paper concentrates mainly on the virus model to avoid duplication of a recent review in this series by Shearer and Schmitt-Verhulst (1977).

MHC-RESTRICTED CYTOTOXIC T CELLS

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II. Virus-Specific Cytotoxic T Cells

A. GENERATION OF EFFECTORCELLS AND ASSAY

1 . Induction of Primary and Secondary Efector Lymphocytes in Vivo The kinetics of viral growth and induced immune response are interrelated and generally follow a uniform sequence [summarized by Fenner (1949), Mims (1964), and Blanden (1974)l: When injected into the footpad of a mouse, poxvirus localizes and replicates in regional lymph nodes. The virus then passes via the lymph to the blood, with resultant seeding into target organs, such as liver, spleen, or skin. If unchecked, further virus growth ultimately results in death. The developing immune response, as measured by delayed-type hypersensitivity, cellular cytotoxicity, or transferable cellular immunity, usually becomes detectable at about the time that virus titers start to decrease and reaches highest levels soon after the virus is cleared from the organism. All these manifestations of cell-mediated immunity fade rather rapidly, whereas, in a typical acute virus infection, antibody responses or antibody-dependent cell-mediated cytotoxicity become measurable only when the virus is no longer detectable and remains at substantial levels long after infection is apparently terminated. Virus is usually injected intravenously ( i.v.), intraperitoneally (i.p.), or intracerebrally (is.) in laboratory experiments. Peak cytotoxic T-cell activities are reached by about 5-9 days after infection for viruses which cause acute infections, depending on the route of injection, the dose and virus used, and the strain of mice (Marker and Volkert, 1973b; Gardner et al., 1974a,b; Koszinowski and Thomssen, 1975; Doherty and Zinkemagel, 1976; Starzinski-Powitz et at., 1976a; Pfizenmaier, 1977a; Wong et al., 1977a,b,c). Induction of cytotoxic T cells by tumor-associated viruses such as MSV is slower and peaks between 10 and 14 days after infection (Leclerc et al., 1973; Lamon et al., 1973; Herberman et al., 1973).Induction of secondary responses in vivo using MSV is difficult, whereas challenge with great numbers of tumor cells usually yields high cytotoxic T-cell activities in the local lymph nodes. In contrast to MSV, neither Friend, Rauscher, or Maloney virus induce appreciable primary cytotoxic T-cell responses, whereas injection of virus-expressing tumor cells does. These viruses do, however, prime mice in vivo so that their lymphocytes are susceptible to secondary sensitization in vitro with stimulating tumor cells (Levy and Leclerc, 1977). Some non-tumor-associated viruses do not cause generation of readily measurable cytotoxic T cells in uivo, for example, herpes virus. However, these lymphocytes can often be

60

ROLF M. ZINKERNAGEL AND PETER C. DOHERTY

boosted to relatively high activity by culturing local lymph node cells soon after in uiuo infection (Starzinski-Powitz et al., 1976b; Rollinghoff et al., 1977a) or restimulation in vitro in secondary mixed lymphocyte cultures (Senik et al., 1975a,b; Plata et al., 1975, 1976; Gardner and Blanden, 1976; Dunlop and Blanden, 1976). In some cases a suppressor mechanism may be responsible for the lack of generation of high cytotoxic T-cell activity. Rollinghoff et al. (1977a) showed that low doses of cyclophosphamide, which usually eliminate suppressor cells, may increase relative cytotoxic T-cell activities generated during local herpes virus infection in mice. It is not yet clear whether this is a general explanation for absence of a detectable primary response in some in uivo models. Whether or not inactivated virus can induce virus-specific cytotoxic T cells in viuo in the same manner as live virus, apparently depends on the characteristics of the particular virus. In general, a noninfectious virus that can fuse with cell membranes is able to trigger an immune response; examples are Sendai virus inactivated by ultraviolet light (UV) (Schrader et al., 1976; Schrader and Edelman, 1977; Ertl and Koszinowski, 1976a; Palmer et al., 1977; Koszinowski et al., 1977; Gething et al., 1978) or vaccinia virus (Hapel et al., 1978).A primary response may also be generated with P-propiolactone-inactivated rabies virus (Wiktoret al., 1977). However, with other viruses, such as VSV, influenza, or LCMV, in vivo induction of measurable primary cytotoxic T-cell responses has not been possible with UV- or formalininactivated virus (Zinkemagel et al., 1978f; Reiss and Schulmann, 1979). However, formalin-inactivated influenza does, but disrupted virus or isolated, aggregated influenza glycoprotein did not induce secondary cytotoxic T-cell responses in uiuo, if the primary infection was with live virus (Reiss and Schulman, 1979). We do not know if these differences are only quantitative, or qualitative as well. Secondary responses have been induced in viuo in two ways. (1) Challenge with the same virus used for the original primary infection usually results in early generation of cytotoxic T cells of lower relative activity than those seen during a primary response (Gardner and Blanden, 1976; Dunlop and Blanden, 1976; Dunlop et al., 1976, 1977; Schrader and Edelman, 1977).Whether this reflects more rapid antigen elimination by T cells or antibody (Dunlop and Blanden, 1977a) or incorrect antigen presentation due to concurrent presence of neutralizing antibody (Effros et al., 1978)is unclear. However, for some viruses the latter possibility seems more likely, since massive secondary cytotoxic T-cell responses that are totally cross-reactive for the viruses being used are found within 3 days after challenging influenza-primed mice with heterologous influenza A viruses. These do not share serologically defined hemagglutinin specificities with the original

MHC-RESTRICTED CYTOTOXIC T CELLS

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immunogen ( D o h e m et al., 1977c; Effros et al., 1978). ( 2 ) Memory spleen and lymph node cells can also be injected into lethally irradiated, virus-infected recipient mice (Zinkernagel and Dohem, 1974c, 1975a; Dunlop et al., 1976, 1977). The relative cytotoxic activities at 4-6 days after this challenge are comparable to those obtained for in vitro secondary mixed lymphocyte cultures, and possible problems due to presence of fetal calf serum are avoided (Peck et al., 1977b).

2 . Stimulation of Primary or Secondary Virms-Speci$c Cytotoxic T Cells in Vitro This technique has been refined and analyzed extensively (Alter et al., 1973; Widmer et al., 1973; Bach et al., 1976, 1977; Peck et al., 1977a; Cerottini and Brunner, 1974; MacDonald et al., 1974; Miller and Dunkley, 1974; Henney, 1977) since the observation of Hayry and Defendi (1970) that memory lymphocytes primed against alloantigens could be restimulated in vitro with the same alloantigens or stimulator cells. Similar procedures have also been applied for the restimulation of cytotoxic T cells specific for minor alloantigens (Bevan, 1975a,b; Gordon et al., 1975, 1976), since, with very few exceptions (e.g., Goldberg et al., 1972; Botzenhardt et al., 1978), it has been difficult to induce measurable cytotoxic T-cell activities during in vivo primary responses against weak transplantation antigens (Matzinger and Bevan, 197%). Virus-specific memory cytotoxic T cells have been similarly restimulated in vitro against MSV (Senik et al., 1975a,b; Plata et al., 1975); Rauscher leukemia virus determinants (Schrader et al., 1975); live or UV-inactivated poxvirus (Gardner and Blanden, 1976, Pang and Blanden, 1976a; Pang et al., 1977; Hapel et al., 1978); LCMV (Dunlop and Blanden, 1976); herpes virus (Pfizenmaier et al., 1977a,b; Rollinghoff et al., 1977a); live, UV- or P-propiolactoneinactivated Sendai virus (Schrader and Edelman, 1977; Schrader et al., 1976; Koszinowski et al., 1977; Sugamura et al., 1977; Pfizenmaier et al., 1 9 7 7 ~ )and ; hemagglutinin plus fusion protein subunits of Sendai virus (Gething et al., 1978; Sugamura et al., 1978; Finberg et al., 1978a) and influenza virus (Yap and Ada, 1977; Ennis et al., 1977a,b,c; Doherty et al., 197713,~).Both UV-inactivated influenza virus and the hemagglutinin subunits of influenza virus have also been used to stimulate secondary cytotoxic T cells in vitro (Zweerink et al., 1977b; Braciale and Yap, 1978; Reiss and Schulman, 1979). Secondary restimulation has the advantage of generating extremely potent T-cell populations that may then be used to analyze the role of various weak transplantation antigens and viral antigens and to investigate minimal antigenic requirements and cross-reactivities. The methods have been studied and described extensively by Simpson et

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ROLF M. ZINKERNAGEL AND PETER C. DOHERTY

al. (Gordon et al., 1975, 1976; Simpson and Gordon, 1977), by Bevan (1975a,b, 1976a,b,c) and von Boehmer (von Boehmer and Haas, 1976, von Boehmer, 1977, von Boehmer et al., 1977),who used weak transplantation antigens, by Senik et al. (1973, 1975a,b) and Plata et al. (1975, 1976), and by Blanden and co-workers (Gardner and Blanden, 1976; Pang and Blanden, 1976a,b; Dunlop and Blanden, 1976, Dunlop et al., 1976,1977; Blanden et al., 1977a; McKenzie et al., 1977; Panget al., 1977), who worked with restimulation in the virus models. The disadvantages obviously are the increased experimental effort required and the potential hazard that new artifacts may be introduced. For example, serum components may induce some antigenic changes or be incorporated into cell membranes, and immunogenic determinants may stimulate serum-specific cytotoxicT cells in cultures and also serve as target antigens (Ilfeld et al., 1975; Forni and Green, 1976; Peck et al., 1977b; Wagner et al., 1978; Goldstein et al., 1978). Experimentation with in uitro primary or secondary induction must therefore include proper specificity controls and should always be approached somewhat circumspectly. The protocols and requirements for in uiuo induction of cytotoxic T-cell responses directed at weak transplantation antigens (reviewed in Simpson and Gordon, 1977; von Boehmer, 1977; Bevan, 1976c) usually consist of injecting mice with lymphocytes carrying the appropriate antigens or applying a skin graft some 2-50 weeks before the spleen cells of these animals are restimulated in uitro. The possible influence of allogeneic effects (Katz, 1972) occurring when parental lymphocytes are used to sensitize F, recipients, or in completely allogeneic sensitization procedures, does not seem to have been adequately analyzed. In contrast, the potential role macrophages may play by reexpressing phagocytized minor histocompatibility antigens, called “antigen processing,” has been studied by Bevan and coworkers. Bevan (1976a,b) reported that F1 mice sensitized in uiuo against cells bearing one parental H-2 type, plus minor histocompatibility antigens could be restimulated in uitro by minor transplantation antigens presented in the context of both parental H-2 haplotypes. Since in uiuo induction of F, cytotoxic T cells against parental cells results in generation of two subpopulations of F, cytotoxic T cells that lyse one or the other parental targets expressing relevant minor antigens, Matzinger and Bevan (197713) favored the interpretation that the recipient F, macrophages reprocessed minor transplantation antigens of parental cells to express them in the context of both H - 2 haplotypes in the F1. This interpretation has lately been supported by elegant studies with irradiated bone marrow chimeras (Fink and Bevan, 1979). Such cross-priming was not generally observed for H-Y (Gordon et al., 1976; Simpson and Gordon, 1977). In the virus model cross-priming

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has been described for Sendai virus (Palmer et al., 1977) and for MSV (J. P. Levy, personal communication), but not for vaccinia virus (Bennink and Doherty, 1978a). Methods and requirements for the secondary in vitro induction of virus-specific cytotoxic T cells and evidence that these cells are active when transferred back to mice (Kees and Blanden, 1977; Dunlop, 1978) have been elaborated very carefully and have been summarized for poxvirus by McKenzie et al. (1977). Macrophages seem to be a primary requirement. They operate partly as antigen presenting cells (a function that can also be fulfilled by other cells derived from lymphohemopoietic stem cells) and partly, in some obscure way, as conditioners for the culture (Davidson, 1977; Pang et al., 1976b; Pettinelli et al., 1978).Infected peritoneal exudate cells are notably good stimulators in the poxvirus and LCMV models, particularly if used at a ratio of about 10 (range 3-100) responder lymphocytes to 1 macrophage (Gardner and Blanden, 1976; Pang and Blanden, 1976; Dunlop and Blanden, 1976b), whereas high macrophage concentrations tend to be “suppressive,” i.e., result in low yields and activities (Davidson, 1977; Pang and Blanden, 197613). However, stimulator macrophages are not required for secondary Sendai virus-specific cytotoxic T-cell induction (Koszinowski and Simon, 1979), although, when heat-inactivated stimulator cells or cells fragmented by freeze thawing are used, they are mandatory (Koszinowski et al., 1978). The antigenic requirements for secondary in vitro responses have been studied extensively with respect to the foreign antigenic determinant, as will be discussed later. However, it is still unclear whether, in addition to H-2K7D structures, other MHC products are necessarily involved. Some experiments concerned with the stimulatory capacity of virus-infected fibroblasts revealed only a poor response of questionable significance (Dunlop and Blanden, 1976). This contrasts with a report by Hapel et al. (1978), who indicated that UV-inactivated vaccinia virus presented on H-2-compatible7but not H-2-different7 fibroblast cell lines were able to restimulate virus-specific cytotoxic T cells. The latter experimental protocol used controls convincingly to allay the possibility that it was not virus transferred from the fibroblast onto lymphoid cells but virus on the fibroblast that was stimulatory. Also, glutaraldehyde- or formaldehyde-fixed, infected macrophages induced good secondary antiviral responses (Dunlop and Blanden, 1976; Koszinowski and Simon, 1979). This is compatible with Forman’s (1977a) finding that similarly fixed TNP-modified lymphocytes were still capable of inducing anti-TNP-cytotoxic T-cell responses. To observe these effects, fixation is usually minimal and therefore the relevance of these experiments is unclear. Primary virus-specific or minor transplantation antigen-specific

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cytotoxic T cells are not readily inducible in vitro (Botzenhardt et al., 1978)-a situation that is quite distinct from the relative ease of inducing specific responses to different major transplantation antigens. Reports on induction of primary virus-specific cytotoxic T cells (against poxvirus or LCMV) first came from Blanden's laboratory (Blanden et al., 1977a; Dunlop and Blanden, 1977b; Dunlop et al., 1977). However, as stressed by these authors, the relatively weak primary responses obtained under optimal conditions are highly variable and subject to influences not yet fully understood. Primary in vitro responses to Friend-Moloney-Rauscher virus-expressing lymphomas are regularly obtained under well-controlled culture conditions (J. P. Levy, personal communication). Similar reports on generation of primary cytotoxic T cells against Sendai virus (Schrader and Edelman, 1977; Jung et al., 1978) did not include adequate controls to exclude the strong probability that the mice from which the lymphocytes were obtained had prior contact with Sendai, a very difficult problem indeed, except in germ-free mice. In fact, results obtained by Finberg et al. (1978a) suggest that environmental exposure to Sendai virus may prime mice and thus explain why some laboratories have observed primary in vitro responses to Sendai virus. They have found that spleen cells from mice just arriving from commercial breeding colonies generated minimal cytotoxic responses after in vitro stimulation with Sendai virus-coated syngeneic cells. However, if these mice remained in the laboratory's animal colony for 2 weeks or more, spleen cells from these mice gave strong cytotoxic responses after stimulation with Sendai virus-coated cells. Nevertheless, in a recent paper, Koszinowski and Simon (1979)presented some evidence that the primary response to Sendai virus is different from a secondary response.

3. 51CrRelease Cytotoxicity Test in Vitro and Limitations of the Assay Met hods Experimental results are no better than the method used to obtain them. The more experimental manipulation, the greater the likelihood of introducing artifacts that blur the relevant issues. In this respect, the "Cr release assay is reasonably satisfactory since, as stated by Cerottini and Brunner (1974), this assay is simple, sensitive, precise, quantitative, reproducible, and independent of target-cell multiplication. However, care must be taken in the interpretation of results. Qualitative and quantitative comparisons of various T killer and target cell combinations may offer very reliable information, but only when one compares the linear portion of the dose-response curve of logarithmic number of cytotoxic T cells versus the percentage of 51Cr released (reviewed in Cerottini and Brunner, 1974; Henney, 1971;

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Miller and Dunkley, 1974; Bevan et al., 1976; Zeijlemaker et al., 1977). In such plots, both the slope and the point of interception with an arbitrarily chosen line of 33% lysis are analytically useful. Parallel dose-response lines reveal identical cytotoxic mechanisms with variations in lymphocyte frequency, whereas different slopes infer that the specificity of the effector mechanism is not identical. The latter possibility must be considered, particularly in long-term assays when antibody production may enhance antibody-dependent cell-mediated cytotoxicity or when release of interferon may induce natural killer cells (Takasugi et al., 1973; Rosenberg et al., 1974; Koszinowski and Volkman, 1974; Herberman et al., 1975; Kiessling et al., 1975, 1976; Wolfe et al., 1976; Levy and Leclerc, 1977; Welsh and Zinkernagel, 1977; Welsh, 1978a,b; Trinchieri et al., 1978a,b; Santoli et al., 1978; Gidlund et al., 1978; Henin et al., 1978). Specificity controls should, for the latter reason, include both uninfected and MHC-incompatibleinfected target cells. The reader is referred to the excellent review of Cerottini and Brunner (1974) for questions of choice of radioactive label, test duration, target-cell sensitivity and relative quantitation of cytotoxic activity (see also Henney, 1971, 1977; Bevan et al., 1976; Miller and Dunkley, 1974; Zeijlemaker et al., 1977). Here only some limitations particular to the virus-specific cytotoxicity assays will be discussed. A more complete account of methods is given by Doherty et al. (1977d). Speel et al. (1968) and Lundstedt (1969) were the first to describe that virus-immune lymphocytes destroyed target-cell monolayers of virus-infected cells in uitro, as assessed in a microcytotoxicity assay. This observation was soon extended by Oldstone and Dixon (Oldstone et al., 1969; Oldstone and Dixon, 1970) to show that cytotoxic activity can be monitored by 51Crrelease in vitro, They used techniques originally introduced by Rosenau and Moon (1961) and Brunner and coworkers (Brunner et al., 1967, 1968, 1970) to measure lymphocyte activity directed against foreign major transplantation antigens. The, assay method was further developed to detect tumor virus-specific cytotoxic T cells (de Landazuri and Herberman, 1972; Lavrin et al., 1973; Herberman et al., 1973; Wright and Herberman, 1973; Leclerc et al., 1973; Lamon et al., 1973) or T cells raised during conventional, acute virus infections (Marker and Volkert, 1973a,b; Cole et al., 1973a,b; Gardner et al., 1974a,b; Zinkernagel and Doherty, 1973, 1974a, 1975a; Doherty et al., 1974; Koszinowski and Thomssen, 1975). There is no doubt that the in vitro 51Crrelease assays, characterized by their reliability and simplicity, are the prime reason for the rapid development ofthe field reviewed here. We do not consider in detail other means of measuring cytolytic activity, such as microcytotoxicity assays (Takasugi and Klein, 1970), since these methods are cumbersome,

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much less reliable, and only rarely used (e.g., Lundstedt, 1969; Lamon et al., 1973; Wright and Herberman, 1973; Plata et al., 1974; Levy and Leclerc, 1977). Similarly, we do not discuss antibody-dependent cell-mediated cytotoxicity (for reviews see Perlmann et al., 1972; Cerottini and Brunner, 1974) or “natural killer” cell function [reviewed in Welsh, 1978b and Zrnrnunol. Rev. 44(1979)] that have no known bearing on the H-2 restriction phenomenon. a. Target Cells. Not all established cell lines are susceptible to infection by viruses. Primary or secondary fibroblast cultures prepared by trypsinization of tissues from embryos or young adults have been used extensively (Oldstone et al., 1969; Oldstone and Dixon, 1970; Cole et al., 1973a,b; Gardner et al., 1974a,b; Koszinowski and Ertl, 1975a,b; Wonget al., 1977a,b,c). Such fibroblasts seem, for reasons that are poorly understood, to be insensitive target cells; the level of specific lysis is low, while spontaneous release is high. Macrophages obtained by peritoneal washing of normal (Drizlikh et al., 1975; Zinkernagel and Doherty, 1974b, 1975b) or thioglycolate-stimulated (Argyris, 1967) mice have been particularly helpful for mapping H-2 restriction when no established cell line was available. Macrophages are susceptible to infection by many viruses, but have the disadvantage that they are very fragile and tend to show high levels (2-4% per hour) of spontaneous 51Crrelease. Lectin or lipopolysaccharide-activated lymphocytes are the preferred target cells to test TNP (Shearer, 1974; Forman, 1975; Dennert and Hatlen, 1975) or minor-transplantation-antigen-specific(Bevan, 1975a,b; Gordon et al., 1975) or Friend-Moloney-Rauscher-virusspecific (J. P. Levy, personal communication) activity. The advantage of using established tissue culture cell lines to test virus-specific activity is, in most cases, the relatively low spontaneous release, availability, and reproducibility; for most tumor-associated virus models these are the only target cells readily available. Considerable success has been achieved recently with SV40-transformed (e.g., Pfizenmaier et al., 1978; Doherty et al., 1978; Zinkernagel et al., 1978d) or methylcholanthrene-inducedcell lines (P. A. Klein, 1975). b. Viral Cytopathogenicity and Test Duration. This is obviously no great problem when one uses viruses that are not cytopathogenic or only poorly so (e.g., LCMV, MSV, Friend virus). However, with cytopathic viruses the timing of infection and the test duration become critical. Another consideration that is important when interpreting cytotoxicity tests is the rate of spontaneous release of the radioactive label. The introduction of radioactive labels that have a slow spontaneous release may well satisfy the need for assays of long duration. However, without careful analysis of related events, much confusion may result (e.g., Stutman et al., 1977; Holden et al., 1977; Levy and Leclerc, 1977; Henin et al., 1979).

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4 . In Vivo Correlates The strength of experiments on cell-mediated immunity to intracellular parasites summarized here results in great part from the fact that, first, these responses can be monitored so easily in uitro, second, and most important, results obtained in uitro can be tested relatively directly for relevance in uiuo. The adoptive transfer of an immune function to naive recipients by injecting them with immune lymphocytes is much more complex than measuring in uitro cytotoxic activity. Nevertheless, the work of Mackaness (1964, 1969), Blanden (1970, 1971a,b, 1974), Lane and Unanue (1972), North (1973), and others has established simple experimental rules for measuring protective activity of T cells in uiuo. Optimally, both the amount of virus used and the numbers of lymphocytes injected should be titrated to establish conditions in which a clear doseresponse relationship can be determined in a short-term (24 hours) adoptive transfer assay (Reviewed in Blanden, 1974). For logistic reasons, this is usually done only for one dose of virus. The involved effectors have been defined as T cells, and the possibility that secreted antibodies may be responsible for a substantial part of the observed protection has been ruled out in at least two murine model systems, ectromelia virus and LCMV (Section VII,A). The adoptive transfer of T-cell-mediated immunopathology is limited to noncytopathic viruses, since the cytopathic effect of the virus itself and cell destruction induced by T cells may not otherwise be distinguished. The model of adoptive induction of acute LCM disease by immune lymphocytes was introduced by Cole and co-workers (Cole et al., 1971, 1972; Gilden et al., 1972a,b; Johnson and Cole, 1975). Their protocol was modified subsequently to demonstrate that H-2-compatible immune T cells could induce LCM disease in an accelerated fashion (Doherty and Zinkemagel, 1975c,d). The interval between i.c. injection of virus and systemic injection of immune lymphocytes is also crucial, although no detailed analysis has been made. Under optimal conditions, immune T cells can induce acute disease and/or death within 24-36 hours after transfer (Doherty and Zinkernagel, 1975d; Doherty et al., 1976~).

B. CHARACTERIZATION OF EFFECTORCELLS

1 . Surface Markers of Efector Cells Virus-specific cytotoxic lymphocytes induced in viuo are nonadherent, sensitive to anti-8 plus complement (Marker and Volkert, 1973b; Leclerc et al., 1973; Lamon et al., 1973; Herberman et al., 1973; Doherty et at., 1974; Gardner et al., 1974a; Koszinowski and Thomssen, 1975; Pfizenmaier et al., 1976b; Doherty and Zinkernagel, 1976), insensitive to anti-mouse immunoglobulin (Ig) plus complement, and

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absent from the lymphocyte population that can form rosettes with anti-mouse Ig-coated sheep red blood cells (Blanden et al., 1975b; Doherty et al., 1976a). Furthermore, direct contact is required for cytotoxicity (Zinkemagel and Doherty, 1974a; Gardner et al., 1974a,b), which excludes a mandatory role for lymphokines (Gardner et al., 1974a,b; Zinkemagel and Doherty, 1974a). Mice that are deficient in T cells such as adult-thymectomized, lethally irradiated and bone marrow-reconstituted (ATXBM) or nude mice (Doherty and Zinkemagel, 1974; Cole and Nathanson, 1975) are unable to generate virus-specific cytotoxic T cells. However, B cell-deficient (Welsh, 1978a) or anti-p-treated mice are unimpaired in this respect. The Lyt phenotype of cytotoxic precursor T cells is probably Lyt 1+(2,3)+;the Ly type of effector in the virus system has been described as Lyt 1+ (2, 3)+but mainly Lyt (2,3)+for in vivo primary cytotoxic T cells and as Lyt (2, 3)+ for secondary in vitro restimulated cytotoxic T cells (Pang et al., 1976; Koszinowski and Simon, 1979; Cantor and Boyse, 1977; Leclerc and Cantor, 1979).All these properties are characteristic of classical T cells. (For reviews see Blanden, 1974; Doherty et al., 1976c; Blanden et al., 197610; Zinkernagel and Doherty, 197713.) 2. Specijicity and Clonality of Efector Cells Virus-specific syngeneically restricted cytotoxic T cells are monospecific for both viral antigens and the restricting K or D structure, i.e., one particular LCMV-specific T cell from an H-2k mouse lyses LCMVinfected, but not poxvirus-infected, targets and in addition operates only in the context of either the Kkor the D k structure, but not both. The clonal character of these monospecificities has been demonstrated formally by von Boehmer et al. (1979) and implied both from experimental results enumerated below and from models used to study other T-cell specificities (Dennert and De Rose, 1976; Fathman and Hengartner, 1978; DiPauli and Langman, 1979; Nabholz et al., 1978). The antigen-specificity of virus-specific cytotoxic T cells has been tested in various ways. LCMV-immune T cells do not lyse poxvirus-infected target cells or vice versa (Doherty et al., 1974). Under optimal test conditions, cross-reactivity is below the 1% level. Similarly, the specificity of paramyxovirus-immune T cells is comparable to that of poxvirus or LCMV (Doherty and Zinkemagel, 1976; Starzinski-Powitz et al., 1976a). More revealing studies of the antiviral specificity of cytotoxic T cells as compared with serological specificities will be discussed in Section II1,B. The monospecificity (and implied clonal character) of K- versus D-restricted cytotoxic T cells has been deduced from several pieces of

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evidence. First, LCMV-specific 51Crrelease from infected targets expressing H-2K” but not H-2D” cannot be blocked by excess unlabeled infected D ktargets, but is blocked very efficiently by “cold” infected K ktargets (Zinkernagel and Doherty, 1975a; Plataet al., 1976; Gomard et al., 1976,1977). Similar results have been found for the TNP model (Sheareret al., 1975; Forman, 1975) and for minor transplantation antigens (Bevan, 1976a; Gordon et al., 1976). Second, anti-K antisera block only K-restricted virus-specific lysis but not D-restricted T cell-target cell interactions (Koszinowski and Ertl, 1975a; Lindahl, 1975; Germain et al., 1975; Schmitt-Verhulst and Shearer, 1976; Gomard et al., 1976, 1977a,b). Third, in homozygous mice, the two K- and D-restricted specificities can be boosted independently by secondary restimulation in vivo or in vitro (Zinkernagel and Doherty, 1974c, 1975a; Blanden and Gardner, 1976; Gardner and Blanden, 1976; Dunlop et al., 1977; Bevan, 1976a,b). Fourth, using a “suicide” technique both Schmitt-Verhulst and Shearer (1977) and Janeway et al. (1978) demonstrated that in F1heterozygote mice at least two subpopulations of TNP-reactive T cells could be induced independently to commit suicide with 5-bromodeoxyuridine and light. Fifth, virus-specific cytotoxic F, T cells specific for one parental H-2 haplotype can be absorbed on infected macrophages of the same, but not on those expressing the second parental H-2 type (Kees et al., 1978). Sixth, and most crucial, cloning experiments with anti-H-Y specific cytotoxic T cells formally demonstrated clonality of restricted T cells (von Boehmer et al., 1979). C. EVIDENCEFOR MHC RESTRICTION IN OTHERSPECIESAND OUTBREDPOPULATIONS MHC restriction of cytotoxic effector T cells has been found in several species other than mice. It is not surprising that T cells from other rodents, such as rats, are MHC (Ag-B)-restricted, as shown in two independent studies. Marshak et al. (1977) defined effector thoracic duct lymphocytes as T cells and documented their Ag-B-restricted activity for both minor histocompatibility antigens and influenza virus. Ag-B restriction of effector T cells was also found by injecting vaccinia virus and LCMV into the footpads of rats and harvesting the local popliteal lymph nodes (Zinkernagel et al., 1977~). A note of caution must be sounded concerning these experiments with rats. MHC restriction is most clearly shown when either thoracic duct populations or lymphocytes that have been restimulated in vitro or otherwise enriched in T cells are used as effectors. Moreover, other cell-mediated cytotoxicity mechanisms also operate simultaneously in rat spleens and lymph nodes.

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Experiments with hamsters have so far failed to show any evidence for MHC restriction, even between different species of hamsters. There is only limited cross-reactivity between hamsters and mice (Zinkernagel et al., 1978~). Also, using vaccinia virus in rabbits, Woan et al. (1978) found that immune lymphocytes did not lyse infected xenogeneic target cells but lysed autochthonous and allogeneic infected rabbit target cells to comparable extents. Studies in cattle (Rouse and Babiuk, 1977) or dogs (Ho et al., 1978) have not given any clear evidence that virus infection induces MHC-restricted T cells. Obviously, part of the reason is that in none of these species are reliable T-cell markers available. Analysis is therefore complicated both by the lack of defined effector cell populations and by the fact that serological typing of major transplantation antigens is not well developed. Wainberg et al. (1974) investigated in microcytotoxicity and absorption assays the cytotoxic, or cytostatic, effect of lymphocytes obtained from Rous sarcoma virus-infected outbred chickens and found a small but significant preference for autochthonous versus nonautochthonous lymphocyte-target interactions. These earlier studies with Rous sarcoma virus were confirmed and extended more recently by using inbred lines of chickens (McBride, personal communication). This fits well with the observation of Toivanen et al. (1974a,b, 1977a,b) that chicken T cells cooperate preferentially with B cells of the same MHC (B locus) type. The first example of HLA-restricted cytotoxic T cells was found in humans for anti-H-Y responses when Goulmy et al. (1977) restimulated lymphocytes from a woman who had rejected bone marrow from her brother. This observation was then confirmed for virus-specific cytotoxicity by in vitro stimulation of peripheral blood lymphocytes with influenza virus (McMichael et al., 1977; McMichael, 1978; McMichael and Askonas, 1978; Biddison and Shaw, 1979; Shaw and Biddison, 1979). Similar results were recorded for human T cell response to measles virus (Kreth et al., 1979) or infectious mononucleosis (Tursz and Fridman, 1978). The HLA-A2 antigen first appeared to be outstanding, since for H-Y and DNP the most clear-cut restriction had been observed with this antigen. However, Biddison et al. (1979) and McMichael and Askonas (1978)clearly documented the restriction for many HLA-A and B products in the influenza system. These are undoubtedly secondary responses, because everyone has been exposed to influenza and measles, or to measles vaccine. However, in humans no evidence has been obtained so far that MHC-restricted cytotoxic T cells are generated during the process of infection. The only possible exception is some published evidence by Ter Meulen

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and co-workers using peripheral blood leukocytes from children undergoing acute measle infections (Kreth et al., 2979).All other attempts to demonstrate cytotoxic T cells directly in the peripheral blood have failed so far. This is true for rubella (Steele et al., 1973), for poxviruseven after secondary infection by revaccination (Perrin et al., 1977b)or for measles virus infection (Perrin et al., 1977a). In all these instances, the only antibody-dependent cell-mediated lysis found was not HLA-restricted in uitro. Several reports on generation of TNP-specific cytotoxic T cells with human peripheral blood leukocytes have been published (Newman et al., 1977; Shaw et al., 1978; Shaw and Shearer, 1978). The very extensive and detailed studies by Shaw and Shearer suggest that human T cells recognize TNP in association with at least three classes of cellsurface determinants: (a) determinants that are common among humans, (b)HLA-A and B-linked determinants that are polymorphic, and (c) determinants that are HLA-linked, but do not seem to correlate with the serologically defined A and B products (corresponding possibly to unrestricted TNP-I region killing in mice). Perhaps these results from the TNP system indicate that the repertoires of anti-Self-H specificities overlap much more in the outbred mouse as compared to the inbred mouse or rat models. However, Biddison and Shaw (1979) used the same families tested in the experiment with TNP to assess MHC restriction in the influenza model and found marked specificity for HLA-A and B antigens. The previously cited data are still fragmentary, but raise many questions. A very obvious one is whether the rather clear-cut results obtained in inbred mice can be readily translated to outbred individuals? For example, is the serological definition of major transplantation antigens sufficient to predict cross-reactivities or restrictions? This is somewhat doubtful, since, even in the murine model, exceptions exist. Thus, the original type H - X b and the mutant H-2Kbm1(or H z l or Kba) can be distinguished serologically only with difficulty (Klein et al., 1974; J. Klein, 1978a). Nevertheless, at least for poxvirus and LCMVspecific cytotoxic T-cell activity, there is no substantial cross-reactivity (Blanden et al., 1976c; Zinkemagel, 197613).There is also the possibility that in an unexplained manner the process of inbreeding has narrowed the recognition repertoire with respect to the MHC. Furthermore, it is difficult to overestimate the technical problems in studying MHC restriction in man, although now it is clearly established that HLA-restricted T-cell killing can be shown with the influenza model. Studies in random-bred strains of mice, such as Swiss mice, WEHI or Quaggenbush mice, revealed that all members of small groups of one strain cross-reacted mutually in terms of restriction specificity,

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whereas the different random-bred strains showed reciprocal specificity (Zinkemagel et al., 1975). However, this indicates only that closed mouse populations maintained other than by sibling mating are substantially inbred. D. MODELSFOR RECOGNITION BY EFFEC~OR T CELLS Virus-specific cytotoxic T cells express specificity for Self-H and for virally induced antigens. The dual specificity of effector T cells can be discussed from the point of view of a T cell and its receptor(s) or, alternatively, of a target cell. Many models have been proposed to explain the dual specificity. Considering nonlytic proliferative or helper T cell function, Katz, Hamaoka, and Benacerraf (Katz et al., 1973a), and Rosenthal and Shevach (1973) postulated that T cells interact with other cells via a “physiological” interaction of Self-H with Self-H. Although these authors favored a self-self-like interaction, they did include the possibility that T cell interactions may occur via complementing interaction of receptor for Self-H. The findings with restricted cytotoxic T cells were interpreted to reflect either some form of mutual interaction of Self-H as had been proposed for nonlytic T cells to allow intimate contact between killer T cell and target cell, or, alternatively, that T cells had receptors for “altered self.” The latter model stated originally that T cells recognize either modified Self-H or a complex of viral and H-2 antigens (Zinkernagel and Doherty, 1974a,b; Shearer, 1974). These possibilities of the altered-self idea were defined more stringently as follows. Modified self was redefined as neoantigenic determinant resulting from the interaction of Self-H and X (Bevan, 1975a,b). The alternative possibility that T cells recognize a bit of Self-H and a bit of viral antigen essentially states that Self-H and viral antigen are recognized in association or coupled. Since this latter statement implies that the specific receptor part for Self-H is allelically excluded, it is essentially a two-receptor model (Zinkernagel and Doherty, 1975a; Doherty and Zinkernagel, 197513; Shearer et al., 1975). From these various speculations the two alternative models for T cell recognition evolved: 1. The two recognition sites model states that T cells possess two separable recognition sites that are specific for two separate antigens on the target cells; one receptor site binds to the restricting Self-H, the other receptor binds to the foreign (viral) cell surface antigen X. 2. The single recognition site model states that T cells express a single receptor site that is specific for a single neoantigenic determinant formed by the complexing of Self-H with the foreign antigen X on

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the target cell. As pointed out by Langman (1978a; and in discussions) and by Cohn and Epstein (1978), the term neoantigenic determinant as used here must obey strict rules that guarantee restricted recognition. The neoantigenic determinant XIH a formed between Self-Ha plus foreign antigenic determinant X1 must not equal either any of the possible foreign antigens X,,X,, or X,,or any of the neoantigenic determinants formed between any possible X, and allogeneic H". Neoantigenic determinants obeying these rules can well be constructed when one considers a very limited number of possible X or H antigens; as soon as great numbers of possible X and H antigens are considered, this proposal becomes, according to Langman, logically untenable. However, all immunological specificity is relative and therefore any such rule will be only relatively strict. Furthermore, all specificity is measured at the level of effector function and, therefore, evidence for, or rules about, specificity cannot readily be expected to apply equally to binding of Self-H, of X, or of neoantigenic determinants. Obviously, analysis of the T-cell receptor(s) in molecular terms may well provide the major source of information for understanding these problems. Functional analyses are also necessary and have provided most of the stimulus to date, but structural definition may not result from such an approach. No distinction can be made between a single T cell receptor for a neoantigenic determinant formed between Self-H and X antigens, on one hand, and two T-cell receptor sites that are on one molecule, or on two molecules but linked in some way, on the other hand, since they are functionally identical, particularly with respect to clonality or specificities. This point did not become clear until it was realized that clonally restricted specificity of cytotoxic T cells could reflect allelic exclusion of all but one of the possible anti-Self-H receptors which might be expected in a particular T-cell population (Zinkemagel and Doherty, 1974b,c, 1975a; Shearer et al., 1975; Langman, 1978). This idea took some time to emerge because the K and D products are expressed codominantly on T cells, as detected serologically (Cullen et al., 1972; Shreffler and David, 1975; Klein, 1975) or by alloreactive cytotoxic T cells directed against both primary or secondary syngeneically restricted virus-specific cytotoxic T-cell populations (Davidson et al., 1976). At least, these results excluded the possibility that K and D products of T cells (as detected by these methods) are interacting with K, D products of the target cells in a like-like interaction and indicated that the recognition is unidirectional, as in an antibody-antigen interaction. However, since these studies of clonality did not provide

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more insight into the nature of T-cell receptors, efforts were made to define Self-H antigen and the virally induced antigens expressed on target cells or alternatively to determine whether a complex antigen formed between X plus Self-H antigens could be demonstrated. 111. Definition of Target Antigens

A. NATURE OF THE RESTRICTJNG SELF-MHC DETERMINANTS

1. Genetic Mapping Use of H-2 congenic mouse strains quickly revealed that the specificity for Self-H antigen was coded by the MHC and that, for lysis of uninfected cells to occur, virus-specific T cells and infected target cells must be identical at either K or D,but not necessarily at Z (Doherty and Zinkernagel, 1975a; Blanden et al., 1975a; Zinkernagel and Doherty, 1975a). Therefore, the restricting cell surface structure is either identical with the serologically defined major transplantation antigens or encoded by closely linked genes. The same is true for cytotoxic T cells directed against TNP-modified target cells or against minor histocompatibility antigens (Shearer et al., 1975; Bevan 1975a,b, 1976a; Gordon et aZ., 1975, 1976). This quality separates syngeneically restricted cytotoxic T cells from noncytotoxic T cells, since the latter were found to be restricted to H-21 (Shevach and Rosenthal, 1973; Rosenthal and Shevach, 1973; Katz et d., 1975; Miller et al., 1975; Schwartz and Paul, 1976; Thomas et al., 1977; Zinkernagel et al., 1977a). This point fits well with the fact that alloreactive cytotoxic T cells are generally specific for the gene products of the K or D regions (Brondz and Snegirova, 1971; Alter et al., 1973; Widmer et al., 1973; Nabholz et al., 1974). Even so, Z region-specific alloreactive cytotoxic T cells have also been found by some investigators (Wagner et al., 1975; Nabholz et al., 1975b; Klein et al., 1976). More recently, J. Kleinet aZ. (1977)and Billingset al. (1977)described that alloreactive, as well as TNP-specific cytotoxic T cells (Wagner et aZ., 1977), may be specific for Z region and/or determinants coded by the Qa or TL, T, or other regions closely linked to H-2D (Forman and Flaherty, 1978; Lindahl, 1978; Klein and Chiang, 1978) without being restricted to either K or D. Experiments with influenza virus indicated that the same may be true for the L molecule, which is closely linked to H-2D (Biddison et al., 1978).These findings suggest that the generally true and simple notion that only K or D products serve as receptors for lytic signals may

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be incorrect. These mapping studies also established that identity of multiple shared or public K, D-antigenic specificities is not sufficient and that lysis depends upon virus-specific cytotoxic T cells and targets expressing K or D antigens bearing the same private specificities (Gardner et al., 1975; Zinkemagel and Doherty, 1977b). Again, some doubt is thrown on the generality of this phenomenon by the finding that the L molecule may possibly act as a restricting antigen in the influenza model (Biddison et al., 1978). Expression of the K and D molecules is essential as shown by using stable cell lines lacking detectable major transplantation antigens, such as the teratoma cells F9 isolated by Jacob (Artzt and Bennet, 1975; Jacob, 1977) or the T lymphoma RTL described by Hyman (Hyman and Stallings, 1977). TNP-modified F9 cells were not susceptible to TNP-specific cytotoxic T cells (Forman and Vitetta, 1975), nor were vaccinia- or LCMV-infected F9 cells lysed significantly by virusspecific cytotoxic T cells (Zinkemagel and Oldstone, 1976; Doherty et al., 1977a). The RTL variant cell line lacking K and D was similarly resistant to lysis by cytotoxic T cells specific for minor histocompatibility antigens (Bevan and Hyman, 1976) or TNP (Dennert and Hyman, 1977). Interestingly, these targets were susceptible to other forms of immune cytolysis, such as antibody plus complement or antibodydependent cell-mediated cytotoxicity (Zinkemagel and Oldstone, 1976; Dennert and Hyman, 1977).This may indicate the operation of a variety of lytic mechanisms or, alternatively, that the cell lines studied lacked only the restricting Self-H, but not the lytic pathway, which may be common to all situations. 2. Antibody Blocking The involvement of K or D products encoded by identical or closely linked genes to those encoding the serologically defined major transplantation antigens was also supported by blocking studies with antibodies directed against private specificities of K or D or other products of subregions ofH-2 haplotypes. Early experiments of Shevach et al. (1972) and Shevach and Rosenthal (1973) demonstrated that antiMHC sera blocked macrophage-mediated T-cell stimulation in guinea pigs and mice. Similarly, Gardner et aZ. (1974b) and Koszinowski and Ertl (1975a) found blocking of killing by antisera to H-2 for conventional lytic viruses and Germain et aZ. (1975), Schrader and Edelman (1976), Gomard et al. (1976, 1977a), and Blank and Lilly (1977) documented such effects for tumor-associated viruses. These studies indicated that blocking was probably associated with H-2 antigens expressed on the target cell but not on the T cell. For example, the lysis

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of H-2*-cytotoxicT cells directed against TNP-H-2b but cross-reactive against TNP-H-2k was blocked by the anti-H-2k antibodies but not by anti-H-2b (Burakoff et al., 1976a; Germain et al., 1975; Schrader et al., 1975). Blocking with anti-H-2 antisera also revealed differences in responsiveness of K versus D restricted T cells, e.g., anti-Kk blocked H-2 restricted killing of vaccinia virus-infected target cells completely, implying that no D k-restricted killing occurred (Koszinowski and Ertl, 1975b; Lindahl, 1975), or anti-Db but not anti-Kb blocked anti-MSV responses (Gomard et al., 1977a,b; Bubbers et al., 1977). 3. Restriction Specijicity Specificity for Self-H-2K or D was shown by titration to be exquisite for virus-specific cytotoxic T cells (Doherty and Zinkemagel, 1975a; Bevan, 1975a,b, 1976a; Zinkernagel et al., 1977e). In contrast, TNPreactive cytotoxic T cells were much less specifically restricted (Shearer et al., 1975, 1976; Burakoff et al., 1976b; Lemonnier et al,, 1977; Billings et al., 1978a,b), as discussed in the review by Shearer and Schmitt-Verhulst (1977). Even so, the seemingly precise discriminatory capacity of virus-specific cytotoxic T cells does not imply that there is absolutely no overlap of the anti-Self-H repertoire of, for instance, H-2k and H-24 particularly if, under conditions of alloantigen tolerance, minor clones can be boosted to detectable levels. This may, perhaps, explain results obtained in negative selection experiments (Wilson et al., 1977; Bennink and Doherty, 1978b; Doherty and Bennink, 1979) or with certain irradiation bone marrow chimeras (Matzinger and Mirkwood, 1978), as will be discussed in Section IV,D. However, whether the generally less absolute specificity of restriction, and the strong cross-reactivity of TNP-specific cytotoxic T cells with alloantigens, necessarily indicate that cytotoxic T cells against TNPmodified Self-H react via a single receptor for altered Self-H that cross-reacts with alloantigen, is still not clear. a. H-2 Mutants. Many H-2 mutant mice (Bailey and Kohn, 1965; Egorov, 1967; Blandova et al., 1975; Melvold and Kohn, 1975, 1976) differ from the original strain of mice with respect only to a single or a few amino acid changes (Brown and Nathenson, 1977; Brown et al., 1978) of the serologically defined K or D products. Additional mutations in or deletions of other H-2 (e.g., L) products have been detected more recently in 2 of some 25 mutant strains (Demant et al., 1975; Hansen et al., 1977; Blanden et al., 1977b; Biddison et al., 1978); for extensive review, see McKenzie et al., 1977; J. Klein, 1978a). Mhual testing of virus-specific cytotoxic T cells from mutant and original-type mice on infected original-type or mutant targets reveals a gradient of

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cross-reactivity among the various strains. For example, poxvirus immune T cells from H-2bm1 (formerly H z l or H-2ba) lyse infected wild-type K b targets only marginally (Blanden et al., 1976c; Zinkernagel, 1976b), whereas H-2 bh cross-reacts substantially with K b , and H-2bs1 lymphocytes lyse infected K b targets as well as do H - 2 b T cells. Other mutant mice have virus-specific cytotoxic T cell activity that cross-reacts to a variable extent with wild-type targets and vice versa. In general, the tendency of mutant T cells to cross-react in the virus system is inversely related to the rapidity with which skin grafts are mutually rejected (reviewed in McKenzie et al., 1977; and Klein, 1978a). The situation is a little less clear with LCMV or Sendai virus, but the general picture of virus-specific cross-reactivities between mutant and H-2 b-type cytotoxic T cell-target cell combinations seems to be similar to that observed with poxviruses. However, since proper titrations have not been performed with either of the viruses when using established cell lines rather than macrophage targets, the issue is still open until these experiments are completed. The results obtained in experiments with viruses differ from those with TNP, since complete cross-reactivity has been observed between mutant and wild-type TNP-specific cytotoxic T cells (Forman and Klein, 1977). However, the cross-reactivity patterns for minor alloantigens resemble those found in the virus models (Klein and Chiang, 1978; Simpson et al., 1978a). If the cross-reactivity patterns were to vary with the examined virus, this finding might suggest that either the particular virus defines the restriction specificity, or, alternatively, the complexing of viral antigen with the K or D product involves different parts of the MHC antigen causing varying degrees of cross-reactivity of altered self. But, if the cross-reactivity patterns between mutant and original mice were to be really constant, irrespective of the examined virus, this finding would tend to substantiate the proposition that T cells recognize an invariant part of a K or D product independent of the particular viral antigen that is recognized (Zinkernagel and Doherty, 1976a,b; Zinkemagel and Klein, 1977). More recently, Blanden and co-workers (Blanden et al., 1977b; Blanden and Kees, 1978) reported that the H-2db mutant reflects a deletion in the H-2D region and codes for an H-2D-like product close to D , is highly cross-reactive with targets bearing the original H - 2 type, and vice versa, in the poxvirus or minor alloantigen model. They therefore concluded that no L-restricted virus or minor alloantigen specific cytotoxicity was in force or at least detectable (Blanden and Kees, 1978). However, when tested for their stimulatory capacity in

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secondary antiviral responses in uitro, stimulator cells bearing this mutation were much less efficient than those from wild-type mice (Blanden et al., 197%). However, Biddison et al. (1978) found evidence in the influenza model that L may also code for a restricting element. The implications of these contradictory findings are not understood. The experiments with mutant mice reveal two relevant points. First, alloreactive T cells raised in mixed lymphocyte reactions between various mutants and original mouse strains recognize some 10-20 distinct allospecificities (Melief et al., 1977; Forman and Klein, 1975), even in strain combinations for which restriction specificities of T cells are indistinguishable. One may therefore conclude that only one, or maybe a few, of H-2K- or D-coded antigenic determinants serve as restricting Self-H antigens. Second, since some mutant H-2K products cannot be distinguished readily from original H-2K products by serological means, serologically defined private major transplantation antigen specificities may not always be identical with the restriction specificity (Blanden and Kees, 1978; Simpson et al., 1978a) or the discriminatory capacity of the effector T cell may simply be greater than that of a polyclonal antibody response. b. Modulation, Repression, or Derepression of MHC Antigens. That virus infection may influence the concentration of major transplantation antigens on cell surface has been documented for VSV (Hecht and Summers, 1972, 1976) and vaccinia virus (Koszinowski and Ertl, 197513; Ertl and Koszinowski, 1976b). Although this decrease in H2-coded antigens might be thought to reflect the effects of VSV replication on host-cell protein synthesis, this possibility was not considered to be a valid explanation by Hecht and Summers (1972). However, Ertl and Koszinowski (1976b) demonstrated that this mechanism could explain the decreased expression of MHC products on vaccinia virusinfected cells. They eliminated H-2 products by enzymic treatment, infected the cells with vaccinia virus, and showed that H-2 was detectable on uninfected, but not infected targets within a few hours. Even so, it is by no means clear whether this metabolic effect is the only mechanism influencing H-2 antigen expression after virus infection. For example, H-2 antigen may also be lost because it is incorporated into the viral envelope of budding VSV (Hecht and Summers, 1976). Also, Friend leukemia virus circulating in serum may contain some but not all of the available H-2 molecules, and incorporation may be correlated with whether or not a T-cell response is seen in the context of a particular H-2 allele (Bubbers and Lilly, 1977; Bubbers et al., 1978). Alternatively, direct binding of virus (or mycoplasma) to

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MHC products may influence their detectability by specific antisera or their turnover rate (Helenius et al., 1978). Bodmer (1973) proposed that the multiple allelic forms of the MHC are not really alleles, but products of a multigenic complex, and the MHC phenotype is determined by regulatory mechanisms (See also Martin, 1975; Hood et al., 1977). Results in support of such a concept have been found by several groups looking for unexpected serological specificities on transformed or tumor cell lines grown in uitro (Invernizzi and Parmiani, 1975; Invernizzi et al., 1977; Pellegrino et al., 1976; Rajan, 1977; Carbone et al., 1978) or in uiuo (Chang et al., 1972; Bowen and Baldwin, 1975; Martin et al., 1976,1977; Wrathmell et al., 1976) or on cells superinfected with lytic viruses in uivo (Garrido et ul., 1976a,b, 1977; Matossian-Rogers et al., 1977). The concept of repression and derepression of MHC products proposes that the process of virus infection disturbs normal regulation of MHC antigen expression and causes repression, or derepression, of similar (but antigenically different) gene products. For example, methylcholanthrene-induced ( H - 2 d )tumor cells infected in uiuo with vaccinia virus react with anti-D.32 (a private H-2Db specificity), which is not normally expressed in this cell line (Garridoet al., 1976a,b, 1977; Matossian-Rogers et al., 1977). In contrast, similar infection of a chemically induced lymphosarcoma (Gardner,H - 2 ’) apparently suppressed expression of the H-2Kk molecule. More recently, these results were confirmed with different antisera by using differential absorption or alloreactive T cells as methods of detection { Matossian-Rogers et al., 1977). However, we (Zinkemagel et aZ., 1977c) could not detect changes in the MHC phenotype of virus-infected target cells by cellmediated cytotoxicity. Alloreactive T cells specific for H-2 ’, H - 2 b, H - 2 d target cells infected with poxvirus, LCMV, VSV, or Sendai virus were screened for either loss or gain of alloantigenic determinants, but no significant variation from the normal was demonstrated. Furthermore, Flaherty and Rinchik (1978) could not confirm some of the serological findings. Even so, the phenomenon should not be disregarded, because there may be subtle, but presently unidentifiable, variations in the methods used. If true, the concept is an important one. c. Possible Relationships between K , D (or I ? ) and the Receptor for the Lytic Signal. Mapping of the restriction specificity of syngeneic or of alloreactive cytotoxic T cells predominantly to K and/or D leaves open the question of whether these gene products or genetically and/ or physically linked structures mediate the lytic message. These possibilities were clearly stated in the early papers (e.g., Doherty and Zinkernagel, 1975a; Blanden et aZ., 1975a), although the point has not

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been belabored, The issue is unresolved, despite its conceptual importance for certain theories of MHC restriction (Langman, 1978; Cohn and Epstein, 1978). Actually, little experimental evidence addresses this question. Some rare tumor cell lines express serologically defined K or D products, but cannot be lysed readily by alloreactive cytotoxic T cells (e.g., methylcholanthrene-induced (Garrido et al., 1976a,b; Matossian-Rogers et al., 1977; Koszinowski and Ertl, 1977). Other well-defined established cell lines lack serologically defined K and/or D products, but are reportedly lysed by cytotoxic T cells in an antigen-specific fashion with or without phytohemagglutinin (Wagner et al., 1977; Goldstein et al., 1976; Goldstein et al., 1978). These examples may indicate that the K and D alloantigens are only linked to a synapse-like lytic “channel.” This model, proposed by Langman (1978),may be used to explain the finding that I-, L-, Qa-, or TL-specific cytotoxic T cells are not K or D restricted. The postulate is then that these molecules are sufficiently close to the lytic channel so as not to require the hypothetical synapse-adjusting structures K or D. ANTIGENS B. NATURE OF VIRALLY INDUCED So far it has been difficult to define the antigenic moiety recognized by syngeneically restricted cytotoxic T cells. Despite the fact that TNP is a simple haptenic group, this antigen has proved to be as difficult to assess as complex antigens like virus or minor alloantigens. The experiments attempting to analyze whether TNP is seen in isolation (Dennert and Hatlen, 1975) in association with its proteinic carrier (Rehn et al., 1976a,b; Henkart et al., 1977; Schmitt-Verhulst et al., 1978) or only indirectly via altered Self-K or D (Burakoff et al., 1976a,b; Lemonnier et al., 1977; Forman et al., 1977a,b) have been summarized recently (Forman, 1976; Dennert, 1976; Shearer et al., 1975, 1976, 1977), particularly by Shearer and Schmitt-Verhulst (1977) in this series. At present it seems that, depending on the induction procedure used, anti-TNP-specific cytotoxic T cells reflect a mixture of the enumerated possible specificities. The probability that TNP may, at least under some conditions, be recognized as such is favored by the finding that anti-TNP antibodies block cytolysis (Schmitt-Verhulst et al., 1976; Burakoff et al., 1976a). Also, Forman (1977b) and others (Dennert, unpublished, Shearer, unpublished) found that cytotoxic T cells activated against TNP- or DNP-modified cells do not cross-react. One of the problems of such analyses is that the procedures used do not allow one to distinguish between hapten specificity and responses that are specific for changes induced by the modification procedure. The more recently developed methods with which TNP or otherwise

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modified soluble proteins can be adsorbed b y cells to render them immunogenic or susceptible to lysis may open the way for a more rigorous analysis of cytotoxic T-cell specificity (Schmitt-Verhulst et al., 1978; Janeway et al., 1978). The situation is even less clear for minor alloantigens. Except for H-Y, and a few experiments with H-3, H-4, and H-7 minor alloantigen-congenic strains of mice, little is known about the target antigens recognized (Bevan, 1976d; Wettstein and Frelinger, 1977; Wettstein et al., 1977; Hauptfeld and Klein, 1977). The fact that the minor transplantation antigens are not readily defined serologically does not facilitate the search. Antibodies to H-Y exist (Gasser and Silvers, 1972), but recent evidence indicates that the serologically defined antigen and the target antigen recognized by T cells may be different (Melvold et al., 1977). Virus-induced cell-surface antigens have been studied for many years, and for certain viruses (see Kilboume, 1975; and Wagner, 1975) detailed analyses on the antigens expressed on both the inner and outer aspects of the cell membrane exist. The application of this knowledge to solving some of the questions concerning the specificity of virus-specific cytotoxic T cells is a slow process. Efforts have concentrated on the analysis of minimal temporal and synthetic requirements for viral antigen induction (Adaet al., 1976; Jackson et al., 1976; Koszinowski et al., 1977; Sugamura et al., 1977, 1978; Hapel et al., 1978). Other studies involve mutant viruses that have defined defects in the expression of certain viral antigens (Koszinowski and Ertl, 1977; Hale et al., 1978; Zinkemagel et al., 19780. Attempts have also been made to block killing with specific antisera and to assess crossreactivities between unrelated, or closely related but serologically distinct, viruses by direct testing of cytotoxic cross-reactivity (Effroset al., 1977; Bennink et al., 1978; Ennis et al., 1977a,b,c; Braciale, 1977a,b; Gomard et al., 1977; Zinkemagel et al., 1977d). Additional analysis has involved selective restimulation of T-cell subspecificities with serologically or genetically defined recombinant viruses or antigens derived from these (Ennis et al., 1977a,b,c; Benninket al., 1978; Effros et al., 1978; Zweerink et al., 1977a,b). 1 . Minimal Requirements for Target-Cell Znduction How much of the virus replication cycle is necessary before virally induced antigens recognized by cytotoxic T cells appear on target cells? Providing that there is sufficient infecting virus, no replication at all may be necessary in some systems. This is certainly the case for Sendai virus, a parainfluenza virus with a great capacity to fuse with

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cell membranes (Schrader et al., 1976; Koszinowski et al., 1977; Sugamura et al., 1977).UV-inactivated Sendai virus does not generate infectious progeny, but can still render target cells susceptible to virus-specific lysis (Schrader and Edelman, 1976; Koszinowski et al., 1977; Pfizenmaier et al., 1977~). Mere absorption of Sendai virus is not sufficient; i.e., fusion is mandatory for inactivated Sendai virus glycoprotein to render targets susceptible to lysis (Gething et al., 1978; Sugamura et al., 1978). Thus, ptoper insertion of the hemagglutinin into the cell membrane by fusion is the crucial factor in this model; whether, in addition to the hemagglutinin, the fusion protein itself also represents a major antigenic determinant for cytotoxic T cells is not clear (Finberg et al., 1978a). These results establish that, for certain viruses at least, the relevant antigenic determinant is present in the virus particle and can be inserted externally; actual infection to induce expression of viral antigen from within the cell is not'necessary. This conclusion put one of many nails into the coffin of the idea that MHCrestriction reflects the activity of glycosyltransferases or other cell enzyme systems that modify viral antigens during synthesis (Blanden et al., 1976a; Rothenberg, 1976, 1978). The alternative concept that viral infection alters these mechanisms specifically so as to change the glycosylation pattern of the Self-H-2 antigens is also effectively buried (Blanden et al., 1976a; Rothenberg, 1976, 1978). It is, however, unclear what the fusion process itself entails and whether cell-membrane components or their relative association patterns are modified in a specific way recognizable as different by cytotoxic T cells. This general possibility has been discussed by Cohen and Eisen (1977),Tauber (1977), and Hood et al. (1978). Evidence compatible with that available for the Sendai virus system has also been obtained by several groups working with poxviruses (Ada et al., 1976; Jackson et al., 1976; Koszinowski and Ertl, 1976; Hapel et al., 1978). Ada and co-workers used inhibitors of protein synthesis to demonstrate that target antigen induction occurred during the initial 30 minutes or so after virus infection and did not need active DNA synthesis (Ada et al., 1976; Jackson et al., 1976). Koszinowski and Ertl (1977) used specific antisera against early or late vaccinia virus-induced cell-surface antigens or vaccinia proteins induced early or late to show that only antisera specific for early viral antigens could block cytotoxic T-cell activity. This evidence conflicts somewhat with data from Hapel et al. (1978), who extended the observation of Ada et al. by showing that mere fusion of sufficient viral envelope glycoprotein rendered cells susceptible to lysis. Apparently, if the viral input is sufficient, even protein synthesis of the early type is not necessary.

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With LCMV or VSV, target cells could not be rendered susceptible to T-cell lysis by inactivated virus even when very high multiplicities of infection were used (Zinkernagel et al., 19780. The simplest explanation for this failure is that neither of these viruses fuses substantially with the cell membrane, and insertion of viral antigen into the cell surface must, therefore, occur from within. This notion is supported by the fact that, with temperature-sensitive (ts) mutants, raising the temperature interferes with viral replication and inhibits the appearance of appropriate target antigens, as in the case of VSV (Hale et al., 1978; Zinkernagel et al., 19780. Thus, the infecting virus may either leave direct “antigenic footprints” on the cell surface or induce such changes rapidly during infection and consequently render the host cells susceptible to attack by T cells before viral progeny assemble (Ada et al., 1976) during the so-called eclipse phase of viral infection. Obviously, considering the multiplicities of virus available, the second mode would be the normal sequence in uiuo. The capacity of T cells to recognize these early cell-surface changes has been formally shown in uitro with vaccinia virus. Virus progeny appear about 3-4 hours after infection of L-cell fibroblasts. When these targets are exposed to cytotoxic T cells during the eclipse phase, i.e., the initial 4 hours, most of the incorporated 51Cr is released and few viral progeny are produced. In contrast, if the targets are not exposed to cytotoxic T cells until more than 4 hours after infection begins, cytolysis is still extensive, but there is no demonstrable effect on release of viral progeny (Zinkernagel and Althage, 1977).This sequence of events may be of importance in uiuo once the inflammatory process is established, and suggests that at least some of the antiviral activity of cytotoxic T cells is mediated via target cell killing during the eclipse phase of the virus (see also Section VI1,A).

2 . Virus Mutants A different way of analyzing the relative significance of various virally introduced (or induced) target antigens is to use ts mutant viruses that either fail to express certain viral antigens, express a mutated variant of a normal antigen, or have other defects defined at the molecular level. Koszinowski and co-workers used this tool to analyze target antigens induced by vaccinia virus (Koszinowski and Ertl, 1976; Koszinowski and Ertl, 1977). More recently, Hale and co-workers (1978) and Zinkernagel et al. (19780 used VSV-Indiana ts mutants to study the nature of viral target antigens. VSV virus codes for 3 major viral proteins, the nucleocapsid, the matrix (M) protein, and the glycoprotein; the latter is the only one expressed on the cell surface (Wagner,

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1975). Two groups of ts mutants were of particular interest: ts M 301 (Knipe et al., 1977a,b; Hale et al., 1978)and ts G31 (Pringle, 1970) fail to express the matrix protein at the nonpermissive temperature of 3133"C, but do express both nucleocapsid antigen and the glycoprotein. Cells infected with these mutants at permissive or nonpermissive temperature were lysed by VSV-Indiana immune T cells, although to a slightly lesser extent when infected at the nonpermissive temperature. Whether this difference, which was obvious at lower multiplicities of infection with t s G31, is due to a less efficientadsorption and infection at the lower temperature or reflects the possibility that faulty array (or absence) of the matrix protein underneath the cell membrane prevents optimal distribution of the glycoprotein is unresolved. The second group of mutants, t s M501 (Knipe et al.,1977a,b; Hale et al., 1978) and ts 045 (Lafay, 1974), failed to bring the glycoprotein to the cell membrane at the nonpermissive temperature, while expressing matrix and nucleoprotein antigens. Target cells infected with these mutants at moderately low multiplicities of infections (20-3 : 1) and at the nonpermissive temperature resisted virus-specific T-cell lysis. Under these conditions such target cells were also quite resistant to lysis by anti-VSV antibodies plus complement. Analysis of an additional mutant tl 17 (Zavada, 1972) with a mutant glycoprotein was unrevealing, since VSV-Indiana wild-type immune T cells lysed the targets infected with tl 17 as well as wild-type VSV infected targets (Zinkemagel et al., 19780. These results taken together support, but do not prove unequivocally, the idea that the glycoprotein is a major target antigen for VSV-specific cytotoxic T cells. 3. Comparison of Serological and Cytotoxic T-cell Specijicity The nature of the viral antigen recognized by virus-specific cytotoxic T cells is poorly understood. In many ways, cytotoxic T-cell specificity seems to be comparable to serological specificity. Since quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results should be interpreted with great reservation. Several methods have been used. It has been known for some time that ectromelia virus-immune T cells are not protective in LCMV infection (Mims and Blanden, 1972). The same lack of cross-reactivity has also been shown for ectromelia virus and LCMV-immune cytotoxic T cells (Doherty et al., 1974). This study was later extended to include Sendai virus (Doherty and Zinkemagel, 1976). However, Gardner et al. (1974b) found that ectromelia and vaccinia virus, which are serologically closely related, cross-react greatly at the target cell level.

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Influenza viruses are excellent tools for more detailed analysis of antigen specificity. The reason for using the influenza viruses is that they are extremely well defined in both molecular and. serological terms (for review see Kilbourne, 1975). The influenza A viruses all share internal matrix and ribonucleoprotein antigens that are serologically similar and until recently were thought to be expressed only within the infected cell and the virus particle. Both matrix antigens and ribonucleoprotein antigens differ for influenza A and B viruses. The surface of each strain of the influenza virus is dominated by the glycoprotein hemagglutinin spikes, which are the antigens traditionally associated with protection in the serological sense. Also exposed is the neuraminidase antigen, which may function by facilitating elution of the budding virus particle from the cell membrane and may also be involved, in a relatively minor way, in protection. The influenza A viruses are fascinating in the present context in that they apparently offer a system for assessing fine specificity in the cytotoxic T-cell response (Cambridge et al., 1976). This analysis is still proceeding and involves some controversy. Influenza A viruses are subject to two forms of variation, antigenic drift and antigenic shift (Reviewed by Laver, 1973). Antigenic drift is probably mutational and reflects progressive change within a particular subtype. Antigenic shift is thought to occur as a result of recombination or repackaging between human and animal viruses and is readily induced in the laboratory because of the segmented nature of the influenza genome (e.g., concurrent infection with wild-type viruses HONl and H3N2 may also give rise to HON2 and H3N1 (H = hemagglutinin antigen, N = neuraminidase antigen). The three research groups working with cytotoxic T cells and acutely infected tumor-cell lines or lymphoblasts as target cells found that there is complete mutual specificity between the influenza A and B viruses (Yap and Ada, 1977; Effroset al., 1977; Dohertyet al., 1977a; Zweerink et al., 1977a; Braciale, 1977a). However, a major component of the cytotoxic response to any one influenza A virus is totally cross-reactive for targets infected with any other influenza A virus. This may be directed against the shared matrix protein, which is now known to be present on cell surfaces (Biddison et al., 1977b; Braciale, 197%; Ada and Yap, 1977, 1979). A second population of influenza-immune cytotoxic T cells is apparently hemagglutinin specific. This may be inferred from “cold-target’’ competitive inhibition experiments and is established with greater precision by the fact that secondary stimulation in vitro with isolated hemagglutinin or inactivated virus leads only to the emergence of the virus-specific T-cell subset (Zweerink et al., 1977b; Braciale and Yap,

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1978). Secondary stimulation with a different influenza A virus leads, both in uitro and in d u o , to a massive secondary cytotoxic T-cell response that is totally cross-reactive and of rapid onset (Doherty et al., 1977a). The use of closely related influenza A viruses has failed to reveal anything equivalent to the “original antigenic sin” phenomenon that has been studied extensively for the antibody response (Fazekas de St. Groth and Webster, 1966; Effros et al., 1978). This phenomenon is the only known instance when viruses that do not cross-neutralize at all [e.g., NPuerto Rico/8/31 (HON1) and NNorthern Territory/60/68 (H2N2)] do cross-prime for a secondary cytotoxic T-cell response. A fourth group of investigators failed to see the cross-reactive cytotoxic T-cell response consistently and considered it to be of no biological significance (Ennis et al., 1977a,b,c). There is a major technical difference in that all of the other groups studying this problem use cell lines or lymphoblasts that have been infected with influenza virus for a maximum of 12-15 hours at the assay’s completion, whereas the work of Ennis et al. (1977a) is done with mouse kidney cells that are first infected with influenza virus for 18 hours, then labeled with Na 51Cr, and then incubated with lymphocytes for an additional 18 hours (Ennis et al., 1977a). Braciale (1979) has attempted to repeat these observations by using primary mouse kidney cells acutely infected with influenza A viruses. He found that these targets, like many other primary cells (Section 11,A73),were not particularly susceptible to lysis in an 8-hour assay, but were killed in a highly crossreactive way by influenza-immune lymphocyte populations that were stimulated secondarily in uitro. The viral matrix protein has also been precipitated from the surface of these productively infected mouse cells (Braciale and Higgins, 1979) and is serologically detectable on the surface of productively infected chick embryo fibroblasts (Biddison, Doherty and Webster, in preparation). Even so, the experiments of Ennis et al. (1977a,b,c) and Zweerink et al. (197%) indicate that the virus-specific component of the response may recognize a very limited site on the hemagglutinin molecule. Cytotoxic T cells are apparently able to distinguish between hemagglutinin antigens that show extensive serological crossreactivity. Our failure to find anything equivalent to the original antigenic sin in the influenza-immune T-cell response (Effros et al., 1978) also supports this idea. These latter experiments raise the question, however, of whether the hemagglutinin specific T-cell subset is ever detectable following infection with two heterologous influenza A viruses. Restimulation of human lymphocytes in uitro with influenza

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A-infected stimulator cells has, to date, generated potent HLArestricted cytotoxic T-cell populations (McMichael et al., 1977; McMichael and Askonas, 1978) that are totally cross-reactive when assayed on lymphoblast targets infected with any influenza A virus (Biddison and Shaw, 1979). More recently, experiments with the two serologically different wild-type VSV strains, Indiana and New Jersey, revealed that virusspecific cytotoxic T cells generated during these infections have specificities that parallel the serological classification (Zinkernagel et al., 1978f; Rosenthal et al., unpublished). Observed, however, was an unexplained asymmetry of specificity, which seemed to depend on the H-2 type of the mice studied. For example, H - 2 b VSV-Indiana immune T cells lysed not only Indiana strain-infected targets, but also substantially lysed VSV-New Jersey strain-infected targets. This crossreactivity was less in an H - 2 d system. T cells from VSV-New Jersey immune H - 2 * or H - 2 d mice lysed VSV-New Jersey-infected targets much better than VSV-Indiana-infected targets. The reasons for these asymmetries are not understood and contrast with the fact that antisera to these two strains neither cross-neutralize (Cartwright and Brown, 1972; Wagner, 1975) nor promote complement-mediated lysis of infected target cells (Zinkernagel et al., 19780. In contrast, Buchmaier et al. (see Oldstone, 1979) found no crossreactivity between cytotoxic T cells generated after infection with LCMV or Pichinde viruses; the latter do not cross-react serologically with LCMV, as measured by neutralization or complement fixation (Reviewed in Lehmann-Grube, 1971). Gomard et al. (1978) studied the cross-reactive cytotoxic reactivity by T cells from mice with murine sarcoma virus-induced tumors. Since they found no apparent specificity when T cell specificities were compared, the main antigen appears to be a determinant common to the Friend-Moloney-Rauscher virus complex; this notion is supported by the finding that occasionally anti-gp70 antibody blocked all cytotoxic activities. In summary, the evidence so far suggests that virus-specific cytotoxic T cells generally express a specificity spectrum that, in part, is not too different from, but also not identical with, the familiar serological one. The exception may be the influenza A viruses, although cross-reactivity could reflect recognition of the shared viral matrix protein that is detectable serologically on cell surfaces (Biddison et al., 1977b; Braciale, 1977b; Ada and Yap, 1977; Braciale et d., 1978). We may conclude that general patterns of specificity and crossreactivity for T cells are difficult to assess until more-sensitive tech-

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niques become available. A promising technique, the clonal analysis of influenza-immune T-cell populations, is currently under investigation by Marbrook and colleagues. 4 . Antibody Blocking Blocking of virus-specific cytotoxic T-cell activity with antiviral antibodies presents some difficulties and is not readily achieved in the ectromelia virus (Gardner et al., 1974a,b; Blanden et al., 1976b), influenza virus (Braciale, 1977a), or LCMV models (Doherty et al., 1976~).Nevertheless, some evidence of blocking has been found in several experiments with antisera of variable quality and specificity for vaccinia virus (Koszinowski and Ertl, 1976) and for VSV (Zinkemagel et al., 1977e; Hale et al., 1978). Blocking of VSV-specific cytotoxicity was mediated strongly by an antiglycoprotein serum (Hale et al., 1978).These data are compatible with evidence from the mutant virus studies (Section III,A,3,a) that the glycoprotein is crucially involved in defining the antigenic entity recognized by T cells, although steric hindrance may also be involved. These findings, or the fact that TNPspecific cytotoxic T-cell interactions are blocked rather efficiently b y anti-TNP antibodies (Burakoff et al., 1976a; Schmitt-Verhulst and Shearer, 1976; Shearer et al., 1976) may also be explained by steric hindrance. It is intriguing that antibodies to TNP are not efficient in blocking T-cell proliferation induced by TNP-pulsed macrophages (Thomas et al., 1978). Very recent experiments with monoclonal hybridoma antibodies that bind to the influenza virus hemagglutinin antigen have shown excellent blocking of the hemagglutinin-specific component in the influenza-immune T-cell response (Effros et al., 1979). This procedure, together with use of mutant viruses immunoselected in the presence of hybridoma antibody in vitro, will allow a very clear analysis of the significance of antibody blocking phenomena, especially with respect to whether steric inhibition is an important factor.

5. Tumor-Associated Viruses These tumor models are considered separately because in many interesting ways the MHC restriction phenomena pose somewhat distinctive insights. MHC restriction of tumor virus-specific cytotoxic T cells has been described for MSV (Landazuri and Herbeman, 1972; Herberman et al., 1973; Leclerc et al., 1973; Gomard et al., 1976, 1977a,b; Plata et al., 1975, 1976), Rauscher sarcoma virus antigenpositive T-cell lymphoma EL 4 (Schrader et al., 1975), Friend leukemia virus-induced target cells (Blank et al., 1976; Bubbers and

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Lilly, 1977; Bubbers et al., 1977, 1978), SV40-transformed tumors (Trinchieri et al., 1976; Pfizenmaier et al., 1978), and mammary tumor virus (Stutman et al., 1977; Stutman and Shen, 1978). The role of the MHC in T cell responses to tumor-associated viruses has been studied most extensively for the exogenous oncornaviruses such as MSV, Friend, or Moloney. It is important to point out that the various models do differ, mainly with respect to the induced pathology, but that the antigens involved in cellular immune responses may be the same. They all have in common that they infect and involve lymphohemopoietic cells; these infected lymphohemopoietic cells or tumor cells are usually the best target cells for cytotoxic T cells in these models. The exogenous viruses induce rather sizeable T cell responses, a fact that contrasts with endogenous C-type viruses, that in general only give very weak or no measurable T cell responses (Duprez et al., 1978). But, in general, there are no apparent fundamental differences between the cytotoxic T cell responses again& tumorassociated exogenous C-type viruses and nontumorigenic viruses (Levy and Leclerc, 1977). Tumor virus-specific cytotoxic T cells are induced in vivo either by injecting mice with virus (Leclerc et al., 1973; Herberman et al., 1973) or with tumor cells expressing virus (Schrader et al., 1975; Blanket al., 1976; Bubbers et al., 1977, 1978). The kinetics of such cytotoxic T-cell generation in mice are protracted and are usually substantially weaker when compared with models of acute virus infection. Peak activities are reached by 10-14 days after immunization (compared with 5-8 days in acute infection) (Leclerc et al., 1973; Lavrin et al., 1973). Because of the relatively low activity, secondary in vitro mixedlymphocyte stimulation methods were developed with resultant generation of highly active cytotoxic T cells (Senik et al., 1975a,b; Plata et al., 1975, 1976). Qualitatively, the cytotoxic T-cell activity observed in these tumorvirus models is comparable to that found for acute viruses. However, issues that need to be discussed in some detail are the nature of effector cells, their degree of H-2 restriction, and the evidence for particular relationships between viral or tumor-associated antigen and MHC products. Different lymphocyte subclasses may be involved in tumor elimination in vivo. Some of these may be identical to those operating in vitro to cause 51Crrelease or microcytotoxicity (reduction in cell numbers). In short term W r release assays (Herberman et al., 1973; Leclerc et al., 1973; Plata et al., 1974), the effector cell is a T cell, as shown by sensitivity to anti-T cell serum plus complement but insensitivity to comparable treatment with anti-Ig. This is established for MSV (Her-

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berman et al., 1973; Leclerc et al., 1973), Friend leukemia virus (Blank et al., 1976), SV40 virus (Trinchieri et al., 1976),and mammary tumor virus (Stutman and Shen, 1978). The effector cells are virusspecific, since they do not lyse unrelated tumor cells, and are usually MHC-restricted to D or K for MSV (Plataet al., 1975,1976; Gomard et al., 1976, 1977a,b, 1978), Friend leukemia virus (Blank et al., 1976; Bubbers et al., 1978; Plata et al., 1978), SV40 virus (Trinchieri et al., 1976; Pfizenmaier et al., 1978) and mammary tumor virus (Stutman et al., 1977). Because the cytotoxic activity of immune spleen cells generated in vivo against MSV or Friend virus-transformed cells tends to be low, the in vitro 51Crrelease assays may be prolonged for 16-24 hours. Furthermore, in long-term microcytotoxicity assays, both T cells and non-T cells seem to be involved (Plata et al., 1974). Therefore, the possibility that cytotoxicity mechanisms other than effector T cells may be involved must be considered when discussing the validity of MHC restriction in these tumor systems (Holden and Herberman, 1977; Gomard et al., 1977; Stutman et al., 1977; Stutman and Shen, 1978; Burton et al., 1977; Henin et al., 1979). This has been a subject of some controversy, particularly for MSV (Holden and Herberman, 1977; Ting and Law, 1977). However, the studies of Plata et al. (1976) and Gomard et al. (1977, 1978), who used highly enriched MSV-specific cytotoxic T cells in a 6-18-hour assay, clearly showed that MHC restriction operates at levels comparable to those found for the acute virus-models (Section 11,A). The lesson to be learned is that no general conclusions concerning the specificity of cytotoxic T-cell activity should be drawn in the absence of either careful lymphocyte titration or time-course experiments in a reasonably short-term assay (Henney, 1971; Cerottini and Brunner, 1974; Miller and Dunkley, 1975; Bevan et al., 1976). An interesting observation has been reported by Stutman et al. (1977; Stutman and Shen, 1978). By using murine mammary tumorimmune lymphocytes, these authors demonstrated that within the first 6 hours of testing, H-2 restriction governs effector-target cell interaction, but during the next 10-20 hours an unrestricted mechanism is also responsible for target-cell lysis (see also Holden and Herberman, 1977; Burton et al., 1977; Henin et al., 1979). Treatment with anti-T cell serum plus complement before the test eliminates both cytotoxic activities. However, it is unclear whether the second phase is directly caused by T cells or is only T cell-dependent but mediated by antibodies or other possibly T-dependent mechanisms. Even so, it is possible that MHC restriction may be less rigorous for cytotoxic T cells specific for tumor-associated compared to acute vi-

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ruses. Perhaps if careful comparative titrations were made for tumor virus-specific cytotoxic T-cell activity versus the activity for, say, vaccinia virus used to infect the same target cells, differences would emerge that might be meaningful.

c.

EVIDENCEFOR INTERACTION OF SELF-H AND FOREIGN ANTIGENS

Ever since the discovery of MHC restriction of cytotoxic T cells specific for virus or minor transplantation antigens, evidence has been sought for some structural interaction between foreign antigen and Self-H. None of the evidence for or against this idea has been convincing. The present discussion will concentrate mainly on minor transplantation and viral antigens, since the situation for TNP has recently been reviewed (Shearer and Schmitt-Verhulst, 1977). Suffice it to state that there is some evidence that TNP must be coupled to K or D products to serve as targets (Forman et al., 1977a,b),but TNP can also be introduced on proteinic carriers that absorb onto the cell membrane (Schmitt-Verhulst et al., 1978). The association of viral antigen with K or D products has been investigated in several ways. Schrader et al. (1975) and Zarling et al. (1978) reported that capping of H-2 antigens on Rauscher virus-infected E L 4 lymphoma cells entailed cocapping of viral antigen gp70. Antisera to both antigens, particularly that directed against H-2 antigen, had been characterized (Henning et ul., 1976) to exclude the possibility that contaminating antibodies against endogenous virus (Klein, 1975; Nowinski and Klein, 1975)might be responsible for the phenomenon. However, this is a very difficult task and some possibility for criticism remains. In more recent studies with vaccinia virus, Senik et al. (1979) found that either anti-H-2Kd or anti-H-2Dd antibodies alone caused complete redistribution of viral antigens; however, not all H-2K or D molecules were complexed with viral antigens, since free K or D molecules were found to move independently on the cell surface and capping of vaccinia virus-induced antigens failed to induce redistribution of all K or D molecules. Another asymmetry of redistribution has been found for VSV viral antigens and K or D (Geiger et al., 1979). Here, capping with anti-H-2Kb, but much less or not at all with antiDb,induced co-capping of VSV antigens. Capping with anti-H-2Kb or anti-H-2Db failed to cause co-redistribution of VSV-induced cell surface antigens. These results must now be considered in the light of recent elegant studies by Singer and co-workers (Bourguignon et al., 1978). They demonstrated that cell-surface antigens on independent structures may cocap merely because these antigens are anchored to

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the actin-myosin cell skeleton and may thus move passively with the actively capping structures, This adds another dimension to the interpretation of cocapping data. Absence of cocapping has been observed by Geib et al. (1977) for the Y antigen and H-2D b. This negative result certainly suggests that the major portion of H-Y is not complexed with Db. However, the sensitivity of the method leaves the remote possibility that a very minor, but crucial, portion is complexed and constitutes the relevant target determinant of cytotoxic T cells. A more serious challenge to any speculation is the recent finding that the serologically defined H-Y antigen and the cytotoxic T-cell target antigen for graft rejection may not be identical (Melvold et al., 1977). Similarly, no obvious cocapping has been observed for measles antigen and HLA (Oldstone et al., 1976; Haspel et al., 1977), for LCMV and H-2 (Oldstone, 1979) nor for influenza antigens and H-2 (Biddison et aZ., 1977; Doherty, unpublished observation) or for Friend-Moloney-Rauscher gp70 antigen and H-2 (Gomard et al., 1978). Several attempts at demonstrating coprecipitation of viral antigens with MHC determinants seem to have produced some evidence that compleaes between X and Self-H antigens, which are probably noncovalent, may form in cell membranes and withstand the usual solubilization procedures. Callahan and Allison (1978)have documented coprecipitation of tumor-associated antigens and H-2K or D products, and Zarling et al. (1978) have similar results with an MSV-tumor cell line. However, Fox and Weissman (1979), using similar techniques, failed to find any association between Moloney virus-induced cell surface antigens and H-2 molecules. At present, no comparable evidence has been published for conventional infectious viruses. An intriguing example of interactions of tumor-associated viral antigens with MHC products has been studied by Gomard and Levy (Gomard et al., 1977a,b; Duprez et al., 1978) and by Lilly, Bubbers, and Blank using the T cell-immune response to Friend leukemia virus-induced leukemias as a model. They made the fascinating observation that cytotoxic activity generated in d u o was associated preferentially with particular H-2K or H - 2 D alleles. For example, H-2* mice generated only D b-restricted Friend virus-specific cytotoxic T cells as shown by mapping (Gomard et al., 1977a,b; Blank and Lilly, 1977) and by the effect of anti-D versus anti-K antisera blocking of cytotoxicity (Gomard et aZ., 1977a,b; Bubbers et al., 1977). When Friend leukemia virus isolated from sera of the infected mice was purified, disrupted, and tested for content of MHC products, Bubbers and Lilly (1977) and Bubbers et al. (1978) found that the preparation’s antigenic material could absorb anti-K or anti-D antisera of the host

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H-2 type. The MHC products incorporated in the virion were not accessible to antibodies in a preparation of intact virions (Bubbers and Lilly, 1977; Bubbers et al., 1978).Interestingly, this absorptive capacity for virus grown in H - 2 H - 2 ’, and H - 2 d mice was specific for D and for Kk, but not for Kb, Dk, Kd or Dd. These findings strikingly parallel both the specificity expressed by cytotoxic T cells generated in vivo for D b and the absence of H-2d restricted cytotoxic T cells in H-2d mice (Gomard et al., 1977a,b; Duprez et al., 1978; Bubbers et al., 1978). The authors interpreted these results, and the fact that D b and Kk, but none of the other four D or K markers studied, cocapped when combined with anti-Friend leukemia virus antibodies (Bubbers et al., 1978) to indicate selective complexing between Self-K or D and viral antigens. The fact that these associations are so selective seems to rule out the possibility that contaminating antibodies may explain the results unless unorthodox possibilities, such as MHC restriction of antibody specificity, are invoked. The significance of these findings is not yet clear. Together with the cocapping (Schrader et al., 1975) and coprecipitation studies mentioned (Callahan and Allison, 1978; Zarling et al., 1978), this is the strongest evidence produced so far suggesting that some viral antigens may complex with MHC products. D. THE SPECIAL CASE OF ALLOANTIGENS Alloreactive cytotoxic T cells are obviously not restricted to SelfMHC. However, their specificity is for foreign H-2 antigen, and they are therefore restricted to MHC determinants. The difference from the Self situation is that the alloantigen represents both the restricting and foreign antigenic determinant X combined. The question then is whether the Self plus foreign antigen X concept is no more than a complex way of stating a relatively simple reality. Are alloreactive cytotoxic T cells a special case, or do they just reflect a functional requirement for focusing T cells on MHC antigen? The functional demarcations between MHC subregions tend to be less clear-cut for alloreactivity than for syngeneic interactions, a fact stressed mainly by Klein (1975,1976,197813).For example, cytotoxic T cells have been raised against Z-region antigens (Wagner et al., 1975; Nabholz et ul., 1975b; Klein et al., 1976). These alloresponses to I are not restricted to K or D (Klein et al., 1977; Billings et al., 1977); the target antigen maps to Z-A or to Z-C (Klein et al., 1976; Klein, 1978b). Part of this response has more recently been attributed to cytotoxic T-cell activity directed not at I but at Qa, Tla, or T (Klein and Chiang, 1978). Similarly, cytotoxic T cells specific for the new L determinant (reviewed in Demant et al., 1978, 1979) are not restricted to K or D

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(Blanden et al., 1978; Hansen and Levy, 1978). Thus, in alloreactions Z-region determinants or other MHC molecules encoded near D may function in the same way as K or D. However, in syngeneic reactions these determinants generally do not seem to serve as receptors for lytic signals. One exception is TNP-specific cytotoxic T cells and H-21 (Billings et al., 1977), possibly because at least a portion of the TNPinduced cytotoxic T cells are in fact alloreactive cytotoxic T cells specific for altered I determinants. The difference between K, D, and I structures in the virus models is emphasized by the fact that antiviral protective activity in v i m of Z-region-compatible immune spleen cells is at least lo4 times lower than for K - or D-region-compatible effector cells (Kees and Blanden, 1976; Zinkernagel and Welsh, 1976). This conclusion is valid since these latter studies involved the same Zk-region haplotype used to assess I-specific alloreactivity or TNP-Z region-restricted lysis. These data so far suggest that there seems to be a definite difference in how Z-region determinants are handled in strictly syngeneically restricted versus allogenetically restricted models. Although Z-region differences are responsible for most of the proliferative responses measured in mixed lymphocyte reactions, K - or D-region differences alone may substantially stimulate proliferative responses (reviewed in Klein, 1978b). The same applies to antigenspecific T cells; e.g., TNP-derivatized cells best stimulate proliferation if the Z region is compatible with the responder cells, but D compatibility also induces some proliferation (Schmitt-Verhulst and Shearer, 1977). The main question then is how alloreactivity is mediated. Is alloreactivity equivalent to recognition of altered Self-H via a single receptor site (Zinkernagel and Doherty, 1974b,c; Shearer, 1974; Doherty and Zinkernagel, 19751b; Schrader et al., 1975; Bevan, 1975a,b; Burakoff et al., 1976a,b; Matzinger and Bevan, 1977a; Lemonnier et al., 1977; Doherty and Bennink, 1979)? Alternatively, in a tworeceptor model, is one of the receptors silent; i.e., is alloreactivity mediated via the anti-X receptor site (Langman, 1978; Cohn and Epstein, 1978) or via anti-allogeneic Self-like receptor sites that have emerged from a germline gene repertoire (Janeway et al., 1976; Doherty et al., 1976b; Blanden and Ada, 1978)? Yet another possibility is that alloreactive T cells use both receptors in a reverse mode so that the anti-Self-H receptor now recognizes foreign minor transplantation antigens on targets, whereas the anti-X (when operating in the context of Self) receptor is now specific for alloantigen (a modification of the Matzinger and Bevan proposal, 1977a). These questions are not

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settled experimentally, but are of major conceptual interest and are dealt with in Section VI. In light of these difficulties, it is impossible to explain satisfactorily the cross-reactivity between various specificities of syngeneically restricted and/or alloreactive cytotoxic T cells. The most prominent example of such a cross-reaction is that of alloreactive T cells lysing TNP-modified targets syngeneic with the effector T cells (Schmitt-Verhulst and Shearer, 1975; Lemonnier et al., 1977); the reverse is not found. Similarly, virus-immune cytotoxic T cells can sometimes (Starzinski-Powitzet al., 1976a),but not generally (Biddison et al., 1977), lyse TNP-modified syngeneic target cells. Again, the converse has not been found. Thus, LCMV or vaccinia virus, but not Sendai virus-immune cytotoxic T cells, lyse TNP-modified syngeneic target cells, but not vice versa. More recently, Bevan (197%) demonstrated that cytotoxic T cells specific for minor transplantation antigens could also lyse target cells bearing foreign alloantigens. Also, anti-Sendai virus-specific cytotoxic T cells have been shown to lyse allogeneic uninfected target cells (Finberget al., 1978b; Burakoff et al., 1978). An interesting point is the finding that elimination of TNP-reactive cytotoxic T cells by a suicide technique did not eliminate the alloreactive cytotoxic T cells that lysed the syngeneic TNP-modified targets (Shearer and Schmitt-Verhulst, 1978). This finding is not readily compatible with cold target competition experiments in which allogeneic cells blocked TNP-specific killing as well as did TNP-modified syngeneic cells (Lemonnier et al., 1977).

E. CONCLUSION The analysis of target antigens for virus-specific MHC-restricted cytotoxic T cells has not led to the unequivocal definition of either the Self-H and the foreign antigen X, on the one side, or of a neoantigenic determinant resulting from the complexing of Self-H antigens with foreign antigens. The majority of the results, including the most recent ones for monoclonal antibodies, suggest but do not prove that T-cell recognition and discrimination potential is generally comparable to that of B cells. The finding that inactivated fusing virus can induce target antigens, even when inserted into appropriate lysosomes, puts some restraints on any model. Therefore, the postulate that direct chemical modification of Self-H by viral influence, or of viral antigens by host-cell influence, is a very unlikely explanation for the immunological phenomenology. This leaves the possibility that viral antigens complex in a noncovalent fashion to create neoantigenic determinants made up by parts of both Self-H and viral antigen or via allosteric changes in Self-H or in the viral antigen. Interpretations of

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antibody blocking experiments, particularly with monoclonal antibody, suffer from the fact that the importance of steric hindrance cannot be assessed properly. The discovery of complexes in tumor models may indicate two possible explanations: either these models exemplify what happens in the acute virus models, or, alternatively, these models reflect that the viruses are so much more integrated into the vertebrate organism that they behave somehow as Self-H in an ecological niche; this would be an easy way to beat immunological surveillance. Thus far, the available data do not explain alloreactivity in a satisfactory way, and the examples of cross-reactivity cannot be taken as sufficient evidence for a single-receptor model. IV. Ontogeny of Effector Cells: The Role of the Major Histocompatibility Gene Complex in Defining T-cell Specificity during Ontogeny

The MHC exerts at least two major influences on T cells: coding for T-cell restriction specificity and coding for Zr genes. As summarized in Section 111, the nature of the target antigens recognized by virusspecific cytotoxic T cells is not yet definite. In addition, the molecular nature of the T cells’ receptors is still unclear. However, some information on these subjects has emerged from studies on the ontogeny of virus-specific cytotoxic T cells designed to analyze how restriction specificities differentiate or are acquired by T cells during ontogeny and to determine whether restriction is imposed at the level ofantigen presentation or is independent of antigens. Analysis focused on whether T cells acquire and express restriction specificities before they encounter foreign antigen X; i.e., are T cells biased a priori to recognize Self-H antigen, or is development and triggering of restriction specificities induced only at the level of antigen presentation when antigen is complexed (or presented) in the context of a certain MHC determinant? If restriction is antigendependent, the prediction is that, under conditions of tolerance to allogeneic MHC determinants, T cells of H-2k haplotype tolerant to H-2b should be receptive to sensitization against, and respond specifically, to virus-infected H-2 * target cells. However, if restriction is antigen independent, this phenomenon is not expected and H - 2 k T cells tolerant to H-2b should react only in an H-2k, but not H-2b, restricted fashion. Starting from these considerations, several models of alloantigen tolerance were studied. The systems used were based on the classic experiments on transplantation tolerance as follows: 1. Chronic unresponsiveness was induced by (a) establishing lymphohemopoietic chimeras before immunocompetence developed by

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transfusing immunoincompetent (Owen, 1945) or competent but not alloreactive (F,) lymphohemopoietic cells (Billingham et al., 1953; Streilein and Klein, 1977) (these chimeras are referred to henceforth as neonatally tolerant mice), (b) constructing zygote fusion chimeras (Mintz, 1967); or, alternatively, ( c ) making adult bone marrow chimeras by using lethal irradiation to eradicate immuno.competent cells and subsequently reconstituting the host’s lymphohemopoietic compartment with defined mixtures of presumed immunoincompetent lymphohemopoietic stem cells (Gengozian et al., 1965; Loughman and Nordin, 1973; Loughman et al., 1973; Urso and Gengozian, 1973, 1974; von Boehmer and Sprent, 1976); 2. Acute unresponsiveness to MHC antigens was achieved by (a) filtering out alloreactive cells in appropriate irradiated recipients (Ford and Atkins, 1971; Sprent and von Boehmer, 1976; Wilson et al., 1977) or (b)suiciding alloreactive T cells with radioactive nucleic acid analogs (Schmitt-Verhulst and Shearer, 1977; Thomas and Shevach, 1977; Janeway et al., 1978). A different protocol was used in studies of the role of the thymus in T-cell differentiation. In the early 1960s it had become clear that the thymus played a decisive role in the differentiation of T-cell immunocompetence. Therefore, thymus and T cell-deficient hosts were reconstituted with thymus grafts of various origins, and reconstitution of immunocompetence (reviewed by Dalmasso et al., 1963; Miller and Osoba, 1967; Davies, 1969) was studied extensively to learn more about T-cell maturation and its relationship to the T-cell restriction specificity (for recent reviews of these subjects see Howard, 1978; Bevan and Fink, 1978; Zinkernagel, 197813; Sprent, 1978~).

A. DIFFERENTIATION OF T-CELL RESTRICTION-SPECIFICITY 1 . Early Studies of Chimeras The previously described models have been used either unknowingly or deliberately in the past to study differentiation of MHC restriction of T cells. Gengozian and co-workers developed chimeras reconstituted with H-2-incompatible bone marrow and found that immunocompetence was fragmentary, but that foreign skin grafts could be rejected and that antibodies to sheep red blood cells were generated. These data were compatible with experiments of Miller and co-workers (reviewed in Miller and Osoba, 1967), who demonstrated that a mouse without a thymus and deficient in T cells regained full immunocompetence only after reconstitution with an H-2-

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compatible thymus graft, These recipients of allogeneic thymus grafts eventually rejected skin grafts, but the interval until rejection was often prolonged. These mice were also deficient in that their antibody responses were definitely weaker than in recipients of H-2compatible thymus grafts. Similar results have been obtained more recently by Kindred and co-workers using nude mice lacking both thymuses and functional, mature T cells (Kindred and Loor, 1974; Kindred, 1975). After the discovery of Zr genes and their mapping to the MHC, McDevitt and co-workers used irradiation bone marrow chimeras to analyze whether the MHC of lymphohemopoietic stem cells or that of the irradiated host determined the responder phenotype (Tyan et al., 1969; Tyan and McDevitt, 1970). Results from these studies were not conclusive, but strongly suggested that the responder type was determined by the genome of the donor lymphohemopoietic stem cells. By using zygote fusion chimeras or tetraparental mice that combined responder and nonresponder mice, the same group of researchers found that the antibody response was composed of great amounts of antibodies of the responder allotype, but that significant levels of nonresponder allotype antibodies were also generated and were specific for the antigen under Zr control (Bechtol et al., 1974a,b; Bechtol and McDevitt, 1976).This result suggested that responder T cells cooperated with nonresponder B cells to produce antibodies of the nonrespander allotype. The result was apparently in contrast to the subsequently emerging rule that T and B cells must be MHC-compatible for cooperation to occur (Kindred, 1971; Kindred and Shreffler, 1972; Katz et al., 1973a). Although later experiments and ongoing studies have failed to substantiate the original results, this work was very important in the evolution of our understanding of MHC restriction. To analyze restriction of T-B collaboration, Sprent and von Boehmer used double bone marrow chimeras that were irradiated (H-2k x H-2b)F1mice reconstituted with anti-&treated bone marrow stem cells from both parents. In cultures, lymphocytes of one parental haplotype were then eliminated by antibodies to H-2 plus complement after sensitization with sheep red blood cells (von Boehmer and Sprent, 1976; von Boehmer et al., 1975a,b). The surviving parental H-2b T cells could then cooperate in vitro with B cells of both parental types. This result was compatible with experiments of Bechtol et al. (1974a,b) and was interpreted by the authors as suggesting that allotolerance allowed T cells to recognize antigen-modified all0 MHC products, or B cells, or macrophages, thus showing cooperation across the MHC barrier. Both sets of data seemed to add a new dimension to the then emerging rule

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that T cells and the cells with which they interact must be MHCcompatible. This discrepancy was interpreted by Katz and Benacerraf (1976) to mean that in these composite mice, lymphocytes learned to interact with each other under the chimeric conditions encountered during either physiological differentiation or more acute experimental manipulation (Skidmore and Katz, 1977), so that the foreign MHC determinants thus functioned as Self-H. This postulate was called “adaptive differentiation” of lymphocytes and was considered to apply to both B and T cells. Since it is difficult to explain how allogeneic chimeric lymphocytes can interact, particularly in a model of physiological interaction that favors like-like interaction of MHC products, these results are more readily incorporated into either a single receptor model for T cells or a dual receptor model in which the anti-Self receptor is expressed clonally. These latter two explanations were favored to explain data obtained in irradiation chimeras formed between parental H-2k anti-$treated bone marrow cells and irradiated (H2 k x H-2*)F1recipients. When stimulated with TNP-modified F1cells (Pfizenmaier et aZ., 1976a; von Boehmer and Haas, 1976), or when injected with virus (Pfizenmaier et aZ., 1976a; Zinkemagel, 1976b,c), these chimeras generated measurable cytotoxic activity against infected H-2b targets mediated predominantly by T cells of the H-2’ type, as shown by anti-H-2 plus complement treatment of effector cytotoxic T cells. Cold-target cell-blocking experiments were used to demonstrate that these chimeric T cells’ restriction specificities for Self H-2k and for tolerated H-2b were expressed by two distinct subpopulations of T cells of donor parental type (Zinkernagel, 1976b). Therefore, cells from such Parent + F1 chimeras either operated as separate sets of T cells with single receptor specificities for either altered H-2k or for altered H-2b, or as two clones of T cells expressing two receptors, one for virus and.one specific for Self-H-2kand the other for Self-H-2b. This implied a unidirectional recognition of Self in an antibody/antigen-like interaction and effectively excluded early ideas of Self-self-like interaction (Katz and Benacerraf, 1975). Such lymphohemopoietic chimeras offered a means of analyzing the following questions: (1)Do tolerance and the degree of chimerism play roles in dictating the MHC restriction specificity of T cells? Neonatally tolerant mice have a low degree of chimerism, whereas irradiation chimeras have a high degree of chimerism. (2) At what level of differentiation of T cells can the restriction specificity be influenced experimentally? (3)What is the role of the thymus in determining the restriction specificity?

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2. Neonatally Tolerant Mice That tolerance to alloantigen was a necessary condition, but not a sufficient condition by itself to allow expression of restriction specificity across the MHC barrier, was first observed when using neonatally tolerant mice. An elegant study by Kindred (1975) showed that T cells from C57BL/6 (H-2b)mice that were made tolerant t 0 H - 2 ~ by neonatal injection of (H-2bx H-2d)F1spleen cells and subsequently failed to reject H-2d skin were not able to restore T help for antibody responses when transferred to nude BALB/c (H-2d)mice. Similarly, A.AL mice (KkZkDd)that were neonatally tolerant to Ks, or H-2a mice neonatally tolerant to H-2b, generated virus-specific cytotoxic T cells operating only in the context of Self-H-2, not of the tolerated H-2 type (Zinkernagel et al., 197713, 1978a,b,c). This lack of responsiveness to the tolerated target did not reflect insufficient stimulation by the few chimeric antigen-presenting cells of the tolerated H-2 type, since appropriate sensitization in an acutely irradiated and infected stimulator mouse expressing both H-2 types did not reveal significant activity to the target of tolerated H-2 type. In a similar study of mice from the same origin, T cells from K k mice neonatally tolerant to Ks were found to react against the TNP-modified Ks tolerated target (Forman et al., 1977c), whereas in other haplotype combinations, little or no TNPspecific activity for the tolerated H-2 type target was found (Forman et al., 1979). 3. Irradiation Chimeras Irradiation chimeras differ from neonatal mice in that the extent of lymphohemopoietic chimerism is usually between 90% and 100%. Such chimeras have been constructed according to two different protocols. After lethal irradiation, recipient mice have been reconstituted with untreated bone marrow cells which, as predicted from the genetic combination used, usually results in graft-versus-host disease. Some animals survive and become reconstituted by donor-type lymphohemopoietic stem cells (Gengozian et al., 1965; Urso and Gengozian, 1973, 1974; Gengozian and Urso, 1976; Slavin et al., 1978a,b). Alternatively, irradiated recipients are reconstituted with liver cells from 15-day-old fetuses or with anti-T cell serum plus complementtreated bone marrow (Loughman et al., 1973; Loughman and Nordin, 1973; Dauphinee and Nordin, 1974; Nordin and Farrar, 1974; von Boehmer et al., 1975a,b; von Boehmer and Sprent, 1976; Waldmann, 1977; Zinkernagel et al., 1978a,b,c). The latter mice do not usually undergo overt or recognizable graft-versus-host disease. Survival is

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generally high for Parent + F1 or F, + Parent chimeras, but low for allogeneic combinations, although this is again influenced by the H-2 combinations employed. Loss of allogeneic chimeras apparently results from intercurrent infection during the period immediately after irradiation and reconstitution; in no case has evidence for ongoing graft-versus-host disease been observed. So far, at least two factors have been found to be crucial in influencing the results obtained with irradiation chimeras: (1)the dose of irradiation determines how many contaminating host lymphohemopoietic stem cells survive to later provide mature T cells that may even differentiate to effector cytotoxic T cells; and (2) contamination of donor bone marrow stem cells with even a few immunocompetent T cells either induces a graft-versushost disease, or in F, + Parent chimeras, eventually provides sufficient lymphocytes to express the T restriction specificity of the second parent. For example, and as an extreme case, irradiation chimeras made b y reconstituting irradiated parental mice with adult spleen cells from F, donors always express both parental restriction specificities, even after chimeric existence is prolonged for up to one year. F1+ Parent bone marrow irradiation chimeras were formed and some 6-24 weeks later were tested for the restriction specificity expressed by the F1chimeric T cells in the minor transplantation antigen system (Bevan, 1977; reviewed in Bevan and Fink, 1978) and in the virus and TNP models (Zinkemagel et al., 1978a,b; reviewed in Zinkemagel, 1978~). Immunologically competent T-cell populations were detected within 6 weeks of reconstitution. The surprising finding was that the restriction specificity was preferential for the H-2 type of the parental recipient. Both for the virus model and for minor transplantation antigens, the cross-reactivity of primary cytotoxic T cells from F, + Parent chimeras does not to date seem to differ markedly from that of unmanipulated parental mice (Zinkemagel et al., 1978b,c; Fink and Bevan, 1978). The degree of restrictiveness expressed by chimeric T cells has been discussed repeatedly and will be considered in more detail in Section VI. Subsequently, comparable results have also been found for cross-reactive TNP-specific cytotoxic T cells (Billings et al., 1978a,b), for T helper cells (Sprent, 1978a,c; Waldmann et al., 1978a; Kappler and Marrack, 1978; Katz et al., 1978),and for cytotoxic T cells specific for the male H-Y antigen (von Boehmer et al., 1978b). The high degree of restriction specificity in the virus model was further apparent from F, + Parent chimera experiments using the mutant H-26m1( H x l or H-2”) and the original H - 2 b mouse. The H-2K molecules of these mice differ by one or two amino acids, but so far as

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is known share the rest of the H-2 gene complex. As described in Section III,A,3,a, T cells from H-26m1mutant and H - 2 b do not crossreact at the target cell level when vaccinia virus-specific cytotoxic T cells are assayed. Lymphocytes from F,+H-2b chimeras, but not F, + H-2bm1or H - 2 b + H-2bm1(Zinkernagel et al., 1978h) lyse infected K b targets. The H-2Kbm'and H-2Kb molecules share most target antigens, as defined serologically and by alloreactive T cells (Nabholz et al., 1975; Forman and Klein, 1975; Klein, 1978a; Melief et al., 1977, 1978). These results suggest, therefore, as did others reviewed in S e c tion III,A,3,a, that the restriction specificity of T cells may be only one of many possible antigenic determinants on K- or D-region products. The necessary interpretation within a single-receptor model would be that the determinants involved in virus-H-2 complex formation have mutated, and that from this point of view Kbml may in fact be as different from K b as Kd or Kk. The alternative idea is that T cells have two receptors, anti-Self-H being specific for a particular antigenic determinant defined by the point of mutation in the H-2bm1mouse. The experiments with F, + Parent chimeras discussed so far demonstrate that (1)the chimeric host MHC, not the reconstituting stemcell MHC, selects the restriction specificity expressed by the stem cells; (2) overlap of restriction specificities is probably small; (3)once the restriction specificity of T cells is determined, it does not change when such T cells are transfused into irradiated recipients. Is it possible that this host-dependent selection of the restriction specificity reflects positive suppression that is in some way specific for H-2 alleles expressed other than on radiation-resistant thymus cells? Attempts at demonstrating such suppression have failed so far (Fink and Bevan, 1978; Bevan and Fink, 1978; Zinkernagel and Althage, 1979a). Acute sensitization of normal F, spleen cells mixed with F1+ Parent chimeric spleen cells at various ratios by acute adoptive transfer into irradiated and infected F, recipients revealed no suppression of the cytotoxic activity restricted to the other parental H-2 type. Also, irradiated (500 rad or 950 rad) normal F, mice were reconstituted with chimeric spleen cells or bone marrow cells and tested for generation of virus-specific cytotoxic T cells at various times after reconstitution. The mice that had received the lower dose of irradiation did not show suppression. The lethally irradiated mice that received chimeric spleen cells, or bone marrow cells, first showed only the restriction specificity expected for the chimera, but later also developed the second alternative restriction specificity. Thus, such suppression of a restriction specificity either is absent, short-lived, perhaps occurring

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only in thymus, or it cannot be demonstrated by the methods used (Zinkemagel and Althage, 1979a,b). 4 . Thymus-Graft Chimeras The experiments with chimeras indicated that the MHC antigens expressed on radioresistant host cells in some way determined the T-cell restriction specificity. Since the host exerted its influence only on immature stem cells, not on mature T cells, the possibility that the thymus was involved was investigated. Initially, irradiated (875-900 rad) parental thymus grafts were used to reconstitute adult thymectomized, lethally irradiated and bone marrow-reconstituted (ATXBM) mice. The restriction specificity of lymphocytes measured 2-5 months after thymic reconstitution was for the MHC of the reconstituting thymus graft (Zinkemagel et al., 1978a,b). However, when adult thymectomized parental mice were reconstituted with F1 bone marrow and F1 thymus tissue, the restriction specificity was for both parental H-2 types (Zinkernagel et al., 1978a). Similar results have been obtained since for the minor transplantation model (Fink and Bevan, 1978), for T helper cells (Waldmann et al., 1979) and for T cells involved in delayed-type hypersensitivity (Miller et al., 1979). More recently, neonatal thymuses yielded comparable results (Fink and Bevan, 1978) as did similar experiments in nude mice (Zinkemagel et al., 1979; Zinkernagel and Althage, 1979b). The radioresistant portion of the thymus seems to dictate the restriction specificity of maturing T cells. The role of thymic hormones versus that of the epithelial cells is unclear, but should be amenable to experimental analysis by classical histology or transplantation of thymic epithelial cell cultures (Rouse et al., 1978a,b). Also unclear is the role that macrophages or antigen-presenting cells may play in the process of selection of the restriction specificity. So far there is only some circumstantial evidence that they are probably not involved. In F1+ Parent chimeras, or thymic chimeras of F1 type reconstituted with parental irradiated thymus grafts, only the puental restriction specificity appears. This indicates either that F1 antigenpresenting cells that may repopulate the thymus are not the selecting population, or that the thymic macrophages are different from the rest of the antigen-presenting cells in that they are radioresistant in the long term and are not replaced by donor type antigen-presenting cells. Several experiments indicate, however, that lymphohemopoietic cells, including antigen-presenting cells, may well be involved in the full maturation of T cells and, more important, in selecting the restric-

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tion specificity of effector T cells from the available restrictionspecificity repertoire selected in the thymus, as will be explained in the following section. B. ROLE OF LYMPHOHEMOPOIETIC CELLSIN T-CELLMATURATION AND ANTIGEN PRESENTATION

1. Postthymic T-cell Maturation

The results from irradiation bone marrow chimeras and the thymus chimeras suggested that the thymic MHC was necessary for selection of the restriction specificity of maturing T cells. Was this process also sufficient for T-cell maturation to occur? The work of Stutman, Good, and co-workers (reviewed in Stutman, 1977) has suggested for some time that thymic maturation must be followed by a postthymic phase before T cells are fully functional. In an attempt to probe this question, nude (ATXBM) mice of one parental haplotype were reconstituted with thymus transplants from neonatal F1 mice. After 2-3 months these mice were infected with virus, and the restriction specificity of the cytotoxic T cells generated was then tested. These chimeric T cells responded only against infected targets of the recipient parental H-2 types, even after adoptive sensitization in infected, irradiated F, stimulator mice (Zinkernagel, 197813).If suppression cannot be considered responsible for this phenomenon, as experiments performed so far would indicate, this result suggests that the thymus environment is essential, but not sufficient, for selection of the restriction specificity. Lymphohemopoietic cells of the same H-2 type as the thymus seem necessary for full T-cell maturation, and it is assumed but not yet formally demonstrated that this requirement is valid for K-, D-, and I-restricted T cells. It is unclear where the block in the maturation of these T cells lies. Is it at the level of the generation of diversity of the anti-X receptor or is the increase in numbers of available precursor cells dependent on some I-dependent T help (Zinkernagel et al., 1979)? 2. Antigen Presentation Parent + F, chimeras made with a higher irradiation dose (950 rad) than in the initial studies (900 rad) (Pfizenmaier et al., 1976a; Zinkernagel, 1976a,b) failed to express significant levels of cytotoxic T-cell activity specific for targets of the second parental H-2 type. If, however, such chimeric lymphocytes were sensitized in irradiated and infected F1 stimulator mice, both restriction specificities were apparent (Zinkernagel et al., 1978~). Similar observations were made subsequently

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for Parent += Fl chimeras in terms of responsiveness of T helper cells (Katz et al., 1978; Erb et al., 1978, 1979; Sprent, 1978a,c). In view of the established role of the thymus in selecting the restriction specificity, this result was unexpected, since more than 95% of the chimeric host’s cells were of both H-2 types, and many of these were infected with virus. These results were interpreted to mean that chimeric cells (other than of donor lymphohemopoietic origin) were not efficient in presenting antigen in an immunogenic way, and that in the acutely irradiated F1-sensitizing host, adequate numbers of stimulating cells of the relevant H-2 types were present. This interpretation still left the discrepancy that Parent + F1 chimeras could generate reactive T cells restricted to the second nondonor H-2 type, although always somewhat less markedly than for the donor parent type, whereas nude or ATXBM mice reconstituted with F, thymus tissues could not. Since suppression does not seem to be responsible for the phenomenon, the most likely explanation appears to be that irradiation Parent +. Fl chimeras are not “clean” with the methods used; a small number of lymphohemopoietic stem cells seem to survive irradiation at least long enough to allow some postthymic maturation to occur (Zinkernagel, 1978b; Zinkernagel et al., 1979b). C. MHC INCOMPATIBLE CHIMERAS Irradiation bone marrow chimeras formed between anti4 plus complement-treated bone marrow of H - 2 k type and irradiated recipients of H-2* type (or vice versa) are for all practical purposes immunoincompetent. They fail to (a) generate significant levels of virusspecific cytotoxic T cells; (b) respond to virus infections with a T cell-dependent inflammatory response; (c) rapidly eliminate poorly cytopathogenic LCMV; and (d) develop high rates of complement-fixing antibodies during infection (Zinkernagel, unpublished). Attempts to map the minimum MHC compatibility requirements between stem cells and recipient host that still result in an immunocompetent chimera have indicated that sharing of Z-A plus either K or D is mandatory for chimeras to generate functional, virus-specific cytotoxic T cells. Chimeras formed with stem cells compatible only at D have so far been incapable of generating H-2D-restricted virusspecific cytotoxic T cells, even after adoptive sensitization in appropriate F, hosts (Zinkernagel et al., 1978~). These results contrast with more recent findings for allogeneic bone marrow chimeras, mostly between H - 2 d and H-2*, that were tested for their reactivity against minor histocompatibility antigens (Matzinger and Mirkwood, 1978). In these experiments, the effector cytotoxic T cells were shown to be

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of donor H-2 type, and reactivity was preferential for the recipient H-2 type target, but significant also for the donor H-2 type. The reason for this difference is not yet clear. Possible explanations are as follows: (1) The different H - 2 haplotypes used may be more or less overlapping in their anti-Self-H restriction specificities so that sharing H - 2 d and H-2b may develop some immunocompetence, whereas H - 2 k and H - 2 b do so much less readily. Cold target competition experiments performed to analyze such potential cross-reactivities of anti-Self-H-2d and anti-Self H-2b do not, however, support this interpretation. (2) the in uiuo primary, in uitro secondary, and sometimes tertiary stimulation may reveal very minor immune reactivities that may not be detectable in a primary or secondary in uiuo situation used for viral responses. To explain the marked lack of immunocompetence in allogeneic chimeras, the following explanations have been formulated: (1)Lack offunctional help. This is based on the idea that T help is necessary to generate an appreciable cytotoxic T cell response and would, if the situation is analagous to that for B cells, require that appropriate I-region restriction specificities be recognized (von Boehmer et al., 1978b). In the latter case, I-restricted T help may not be deliverable in allogeneic chimeras because the restriction specificity selected in the host thymus does not fit the genetically determined I structures expressed on the donor cells derived from lymphohemopoietic stem cells. (2) Block in T-cell maturation. As is suggested by the experiments in which nude or ATXBM parental mice were reconstituted with F, thymus and failed to generate cytotoxic T cells restricted to the other parental H-2 type, lymphohemopoietic stem cells and thymic H-2 must be compatible for T cells to mature fully (Zinkernagel et al., 1979b). Since this condition is not fulfilled in allogeneic chimeras, T-cell maturation may be blocked, as discussed in the previous section.

D. NEGATIVESELECTIONEXPERIMENTS The question of whether T cells’ restriction specificity for Self-H differentiates independently of antigen was also investigated by using a protocol in which tolerance to alloantigens is produced more acutely than in chimeras. The negative selection procedure (Ford and Atkins, 1971; Sprent, 1978a,b,c) offers the possibility of examining T-cell populations that are acutely depleted of alloreactive potential. The basic protocol is that F1 mice are irradiated (950 rad) and injected i.v. with large numbers of T cells from one parent on the following day. Thoracic duct lymphocytes are then drained from these mice for 24-42 hours after cell transfer via cannulas inserted into the cistema chylae.

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Sprent (1978a,b) has shown that such thoracic duct lymphocytes are devoid of both blast cells and alloreactivity for the other parent. These negatively selected T cells are then injected i.v. into other groups of irradiated recipients (950 rad, -24 hours) that are inoculated with virus 3 hours later. Spleen cell populations are then tested for cytotoxic Tcell activity on day 5 (influenza virus) or day 6 (vaccinia virus). These experiments are technically difficult. However, some points of interest have emerged so far and will be summarized in the next section.

1 . The Transferred T Cells and the Irradiated Recipients Need Not Share I-Region Determinants Appropriately filtered T cells from B10.D2 ( K d I-Ad D d ) or (C3H X BALB/c)F, ( K kI-AhD kX K d I-Ad D d )mice generate a strong vaccinia virus-specific cytotoxic response at H-2Ddwhen stimulated in recipients (Bennink and Doherty, BlO.A(SR)or BlO.A(5R) (KbZ-AbDd) 197813). In these strain combinations, there is a lack of identity from H-2Kb to I-J or I-E. The need for homology at I-C or S is ruled out by the finding that negatively selected BlO.A(SR) T cells respond to vaccinia virus in the context of H-2Db when primed in C57BLJ6 recipients, the only H-2 compatibility in this system being at H-2D9 Therefore, these results oppose the idea that I region-restricted T-cell help stimulated by the recipient is necessary for the generation of a strong virus-immune cytotoxic T-cell response at H-2D. In other words, if T-cell help operates in this system, it apparently obeys different rules from those governing T cell-B cell collaboration (Sprent, 1978a,b; Bennink and Doherty, 1978a,b). One objection that may be raised to this interpretation is that there is some form of allogeneic effect mediated via radiation-resistant T cells in the stimulator environment (Katz, 1972). Although we have not been able to generate experimental support for this idea (Bennink and Doherty, unpublished data), we would expect that such a phenomenon should also operate in the chimera experiments in which similar strain combinations are used and no cytotoxic T-cell response to vaccinia virus is detected at H-2D (Section IV,A). The idea of T-cell help may still be retained if the helper T cells are considered to interact directly with the cytotoxic T cells, perhaps via idiotype recognition without involvement of an intermediary stimulator cell in the recipient. Alternatively, it may be possible that H-21 region-related T help is cross-reactive for these acutely selected thoracic duct lymphocyte populations (see following section), whereas this is not the case for the persistently tolerized chimeric lymphocytes (Section IV,A).

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2 . Negatively Selected T Cells Znteract with Vaccinia Virus in the Context of Some H-2K Determinants Not Encountered in Thymus When negatively selected BALB/c ( K d Z-Ad D d ) T cells are stimulated in BIO.A (Kk Z-Ak D d ) vaccinia virus-infected recipients, a strong vaccinia-specific cytotoxic T-cell response is generated to both H-2Kk vaccinia virus and H-2Dd vaccinia virus (Doherty and Bennink, 1979). Similarly, filtered C57BL16 ( K bZ-AbDb)thoracic duct lymphocytes can be sensitized to vaccinia virus presented in the context of both H-2Kk and H-2Dd when sensitized in a BlO.A(4R) ( K k Z-Ak D b ) environment (Bennink and Doherty, 1979).Also, BALB/c lymphocytes can be induced to recognize H - 2 K 8 vaccinia virus following priming in (A.TL x DBA/2)F1 ( K8 Z-Ak Dd x Kd Z-Ad D d ) irradiated mice. However, the converse does not necessarily apply: BlO.A(BR)( K k Z-AkD b ) T cells apparently do not interact with H-2Kb vaccinia virus when stimulated in irradiated C57BL16 ( K b Z-AbD b )recipients, and we have not yet been able to induce H - 2 k T cells to lyse vaccinia-infected H - 2 0 r H - 2 ~target cells. In all cases, there is no concurrent alloreactivity for uninfected targets. This “aberrant recognition” of H-2Kk vaccinia virus by H - 2 b or H - 2 d T cells is restricted to the stimulating H-2Kk determinant and is not obviously cross-reactive with the response at H-2Dd, H-2Db, H-2Kd, or H-2Kb.The favored interpretation (Doherty and Bennink, 1978)is that the aberrant recognition of H-2Kk vaccinia virus by H - 2 d thoracic duct lymphocytes is mediated via a single T-cell receptor that would normally interact with H-2Kd virus X or H-2Dd minor H antigen, the specificity of physiological H-2 restriction being determined in the thymus. Any potential effector population of this type would need to be either suppressed or deleted in the thymus of an H-2k”d F, +H - 2 d chimera to account for the difference in findings for the negativeselection and chimera models (Section IV,A). The irradiation chimeras have been immensely valuable as tools for the analysis of H-2 restriction. However, the limitation they suffer is that responsiveness in the absence of alloreactivity can be assessed only in the context of non-Self encoded H-2 antigens that are encountered throughout the process of physiological T-cell development in the thymus. This may explain why findings generated in irradiation chimeras, neonatal chimeras, and negatively selected mature T cells sometimes differ (Sections IV,A,l-3). The experiments with lymphocytes from inbred mice that are tolerant toward alloantigens suggest that in general tolerance alone is necessary, but not sufficient, for T cells to react in a restricted way with the tolerated H-2 types and with a comparably broad spectrum of activity. There may, however, be some overlap of restriction specificities be-

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tween various K or D alleles, and the experimental conditions that are discussed here have uncovered some of these overlaps for a few H - 2 haplotype combinations. Since for many other H-2 combinations, restriction specificity assayed after negative selection is exquisite, the importance of “aberrant recognition” is not clear as yet. Whether the shared restriction specificities can also be demonstrated directly after in uivo immunization of unmanipulated mice remains to be seen. We do not yet know whether the aberrant recognition phenomenon associated with some H-2K alleles may also occur for H-2D. Also, it is not clear if the capacity of H-2d and H-2* T cells to respond to H-2K vaccinia virus is recognized if the thoracic duct lymphocytes are stimulated in a completely allogeneic environment; all experiments to date use either F, mice or strains which share at least one H-2D allele in common with the T cells. Is aberrant recognition at H-2K restricted to H-2D-compatible interactions? There is a need to determine whether aberrant recognition occurs for other viruses. The only information available concerning specificity is that H-2k T cells may be induced to recognize H-2b TNP but not H-2b vaccinia or influenza virus (Wilson et al., 1977; Bennink and Doherty, 1978a,b). Negative selection of antigen-specific T-cell subsets by filtration through irradiated virus-infected recipients has not yet proved to be technically feasible, probably because of the combined deleterious effects of the virus and the i.v. inoculation of large numbers of cells. Even so, it is already obvious that the negative selection experiments provide a valuable counterbalance to the work with chimeras and may help us to arrive at a valid general conclusion.

E. CONCLUSION Thus far, experiments with chimeras and lymphocytes that are acutely depleted of alloreactive cells have provided the following evidence: (1) Selection of T cells’ restriction specificity is for Self-H as expressed in the radioresistant portion of the thymus and is independent of antigen. ( 2 ) Full maturation of virus-specific T cells depends on some lymphohemopoietic cells and the thymus being MHCcompatible both for Z and K or D.(3) Restriction specificities seem to overlap between certain H - 2 haplotypes. (4)There is some evidence emerging for T helper cells being involved in the generation of cytotoxic effector cells. V. Role of the Major Histocompatibility Gene Complex in Determining T-cell Responsiveness

The MHC exerts two major influences on T cells. First, it determines the restriction specificity of T cells, and second, it influences the

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T-cell responder phenotype. Since the selection of the restriction specificity expressed by T cells is crucially determined by the thymus, it was of interest to learn whether the capacity of T cells to respond was also dictated by the MHC of the thymic environment (von Boehmer et al., 1978b; Billings et al., 1978b; Zinkernagel et al., 1979). This section reviews the experimental evidence for Zr genes regulating generation of syngeneically restricted cytotoxic T cells either via T helper cells or suppressive T cells, or, more importantly, by direct influence on expression of cytotoxic T cells. The main finding is that Zr genes generally seem to map to the same type ofH-2 region as do the genes coding for the restricting Self H. Recent experiments support this notion and indicate that the thymic MHC environment dictates both restriction specificity and Zr phenotype. We define Zr genes as genes that map to the MHC and code for regulatory influences that determine the capacity of T cells to respond to a particular antigen. Although most classical Zr genes regulate antibody responses and map to the Z region ofH-2, other Zr-gene-like influences regulating proliferation of T cells or delayed-type hypersensitivity have been recognized and mapped toH-21 (reviewed in Thomas et al., 1977, 1978; Miller and Vadas, 1977). It is debatable whether one should name genes that regulate responsiveness of cytotoxic T cells but map outside of the Z region (e.g., K or D genes regulating responsiveness of virus-specific cytotoxic T cells) Zr genes. There .is, however, no doubt about the fact that at least some of these K, D regulatory influences on expression of cytotoxic T cells resemble those of classical Zr genes that regulate the interaction of T cells with macrophages and/or B cells, and in this sense an extension of the meaning of Zr gene seems warranted.

A. EVIDENCEFOR MHC-CODEDzr GENESREGULATING THE EXPRESSION OF CYTOTOXICT CELLS Phenomena concerning Zr genes that regulate immune responsiveness as measured by antibody responses have been reviewed extensively (Benacerraf and McDevitt, 1972; McDevitt and Bodmer, 1974; Benacerraf and Katz, 1975; Benacerraf and Germain, 1978). These classical Zr genes have the following characteristics: (1)they regulate antibody production (Levine et al., 1963; McDevitt and Sela, 1965); (2) their effect is antigen-dose dependent; (3)they map to the MHC (McDevitt and Chinitz, 1969), mainly to H-2 Z-A (McDevitt et al., 1972), although complementary genes may map to Z-E or Z-C (Dorf and Benacerraf, 1975; Munro and Taussig, 1975); (d) high response, measured as the capacity of a mouse to produce antibodies to the antigen

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under Zr gene control, is dominant. Both Katz et al. (1973b) and Shevach and Rosenthal(l973) showed that F1 heterozygotes between high and low responders generated T cells that could cooperate only with B cells or macrophages of the responder, but not of the nonresponder, parental H-2 type. These experiments showed, therefore, that Zr genes act at the level of the B cells or macrophages. However, these results may also be interpreted as meaning that Zr genes act on T cells. Apparent Zr gene regulation of the generation and/or expression of syngeneically restricted cytotoxic T-cell activity was first described for TNP-specific cytotoxic T cells (Shearer et al., 1975; Schmitt-Verhulst and Shearer, 1975, 1976). Since these studies have been reviewed recently in this series (Shearer and Schmitt-Verhulst, 1977), their evidence will be summarized only briefly. The following characteristics for Zr gene regulation of responsiveness of Dd TNP were noted: (1) high responsiveness is dominant (Schmitt-Verhulst and Shearer, 1975); (2) two genes may be involved, one mapping to the Z region between I-A and Z-J, and the other to the left of I-A, possibly K (Schmitt-Verhulst and Shearer, 1976). The most extensive study of Zr genes regulating syngeneically restricted cytotoxic T cells has been performed on responses to the male H-Y antigen. Based on earlier in uiuo studies of the capacity of female mice to reject male skin grafts, it was concluded from in uitro cytotoxicity tests that H - 2 b mice were high responders and all other H-2 haplotypes tested ( H - 2 d , H - z k , H-2a, H-29) were low responders (Gordon et al., 1975; Simpson and Gordon, 1977; von Boehmer, 1977; von Boehmer et al., 1977; Hurme et al., 1977, 1978a,b). F1offspring of H - 2 b responder and H-2k nonresponder parental mice generated different clones of H-Y-specific cytotoxic T cells restricted to H-2k and to H-2b (von Boehmer, 1977; Simpson and Gordon, 1977; Gordon et aZ., 1977; Hurme et al., 1978a). Further analysis of the restriction specificities expressed in H - 2 b mice and various F1mice revealed that cytotoxic T-cell responses were generated for H-Y plus Db,Kk,and Dk but not for Kb, Kd, or Dd (Hurme et al;, 1977, 1978a,b; Gordon and Simpson, 1977; Simpson and Gordon, 1977; Gordon et al., 1977; von Boehmer, 1977; von Boehmer et al., 197813). Haplotypes other than H-2Zb, e.g., I d (in CSH.OH, K d ZdDk),have not generally been found to generate anti-H-Y cytotoxic T cells (restricted to Dk in C3H.OH) (Gordon et al., 1977; Gordon and Simpson, 1977; Matsunaga and Simpson, 1978). As previously mentioned, gene complementation that allowed nonresponder H-2d, H-2k, H-28 or other strains to express responsiveness could be mediated by the H-2 haplotype by Simpson and Gordon (1977)and von Boehmer et al. (197813).Other examples of complemen-

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tation in the F, hybridbetween twolow responders (e.g.,H-2” x H-28F,) have been found only by Simpson and Gordon (1977). Whether this discrepancy reflects the differing schedules (time, numbers, use of irradiation, and F1 or parental origin of male cells) used by the two groups to immunize F1 mice is currently unresolved (see Langman, 1978b). The fact that complementing F, combinations that do not include H - 2 b fail to reject male skin grafts has been interpreted as indicating that Zr genes regulating expression of cytotoxic T cells and those involved in skin graft rejection may not be identical. The Ir gene regulating cytotoxic T-cell activity against H-Y has been mapped to Z-A, whereas the Zr gene controlling male skin graft rejection maps to I-B (Hurme et al., 1978a,b). However, the last word on the in v i m model may not be available in view of the experimental evidence that splenectomized H-2b female mice reject male skin much faster than those with spleens and that splenectomized H - 2 k female mice do reject male skin whereas females with spleens do not (Coons and Goldberg, 1978).Whether the particular transplant technique used may explain some of the differences remains to be seen (Johnson, 1978). The various examples of complementations found so far in the TNP and the H-Y models may reflect several different possible mechanisms. One is that Zr genes are expressed in different T-cell subsets (Cantor and Boyse, 1977) that must interact, cooperate, or suppress for the generation of cytotoxic T effector cells. This would imply that a nonresponder status may result from various combinational possibilities: (a) Zr defect at the level of T help alone such as Zk; (b)Zr defect at the level of cytotoxic T-cell induction or effector cells as with Kb, Kd, D d ) ; (c) Zr defects in both T-cell populations; (d) high or low responder status at the level of suppressor T cells that may act alone or be superimposed on the previous mechanisms. An unexplained finding in the H-Y model, sometimes paralleled in the regulation of cytotoxic T-cell responsiveness against viruses, is that F, (CBA x C57BL110) (H-2” x H-2b) females immunized against F, male cells generate anti-H-Y cytotoxic T cells restricted toH-2” but not H-2 (Gordon et al., 1977).Whether this “immunodominance” reflects Zr gene regulation or not is unclear. Zr phenomena have also been described that regulate expression of cytotoxic T cells specific for tumor-associated viruses. Some aspects have already been discussed briefly in Section 111,B). With Friend virus, responsiveness for the H-2 haplotype is restricted to Db but not to Kb (Gomard et al., 1977a,b; Duprez et aZ., 1978). In contrast, H-2” mice are low responders whereas (H-2” x H-2b) mice immunized with either H - 2 k or H-2” x H-2b tumor cells respond very well to H-2k Friend virus-infected tumor cells (Gomard et al., 1977a,b; Blanket aZ., 1976). Lack of the necessary H-2 recombinant BALB mice and/or ap-

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propriate tumor target cells has rendered further analysis of the genes involved difficult. In particular, the responder status 0 f H - 2 ~mice has not been investigated to date (Bubbers et al., 1978). Studies on the selective blocking of Friend virus-specific cytotoxic T-cell activity by anti-H-2Db antisera, as well as cocapping induced by anti-Friend virus selectively for Db, all suggest that there is a special relationship between Db and virally induced antigens (Gomard et d . , 1977a,b; Bubbers et al., 1977; Duprezet al., 1978).This indication and the evidence that budding Friend virus seems selectively to incorporate Dbor Kkbut not Kb, Dk,Kd or Dd, (Bubbers and Lilly, 1977; Bubbers et aZ., 1977; Freedman et al., 1978) correlate well with the expressed responder restriction specificities and indicate that the interaction of virus and K or D products may influence responsiveness. A more intricate type of Zr-regulated cytotoxic T-cell response has been described recently by Billings et al. (1978).Lymphocytes other than those of the H-2k haplotype that have been stimulated against TNP-modified syngeneic stimulator cells express a high degree of cross-reactivity for all TNP-modified target cells, irrespective of their H-2 type. However, anti-TNP-H-ekresponses are highly restricted to H-2k. The tendency toward cross-reactivity is dominant in the F1 and seems to be controlled by a gene mapping to K and/or Z-A. An example of Zr regulation by suppression, influenced b y the Z-J region, or expression of cytotoxic T-cell activity is exemplified by a model in which the cytotoxic T-cell responses against an AKR tumor cell were investigated in F, hybrid mice (AKR x various C3H or C57BW10-derived H-2 recombinant mouse strains) (Meruelo et al., 1977). The authors attributed lack of responsiveness measured by cytotoxic activity of T cells to either dominance of suppression, recessive responsiveness or an unknown mechanism mapping to I-]. Evidence that cytotoxic T cells generated during acute virus infection are also under MHC-coded Zr gene control has been first demonstrated for vaccinia, influenza Sendai, and LCM virus (Doherty et al., 1978a; Zinkernagel et al., 1978d) and subsequently for Sendai (Kurrle et al., 1978) and alpha viruses (Miillbacher and Blanden, 1979a). Some variability in T cell responses to poxviruses had been observed earlier but was overlooked (Kaszinowski and Ertl, 1975b; Lindahl, 1975) or thought to reflect classical Zr genes, mapping to H-21 (Blanden et al., 1975a); however, a search for H-21 influence on generation of virus-specific cytotoxic T cells failed to reveal supporting evidence (Zinkernagel et al., 1976). Despite the fact that all of the mouse strains examined respond to LCMV, vaccinia, Sendai or influenza viruses tested so far, as measured by capacity to express cytotoxic T-cell activity, there are great differences in the levels of responsiveness when K- and D-restricted activities are tested separately

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on appropriate H-2 recombinant targets. This has now been shown for LCMV, vaccinia, Sendai, and influenza viruses. Since all strains of mice respond to either K plus virus or D plus virus, it seemed possible that these Zr defects probably operated not at the T-help level, but at the cytotoxic T-cell level. This argument assumes conventional I-restricted T help specific for antigens. However, it does not exclude the possibility that K or D plus virus-specific T help could function to regulate responsiveness in a K, D, and virus-specific manner. This could occur if T cells are specific for an altered Self antigen or if they are recognizing T-cell receptor idiotypes. The fact that T help restricted to the Z region has not yet been shown to be responsible for any of the known Zr effects on expression of virus-specific cytotoxic T cells is very interesting and may suggest that (1)T help is not necessary to induce virus-specific cytotoxic T cells; or (2) regulation of T help for B cells is different from that for K, D restricted cytotoxic T cells, which is also indicated by the fact that K is duplicated in D, whereas such duplication is apparently lacking for I-A. Recently, Mullbacher and Blanden (1979a,b) have studied the cytotoxic T cell response against alpha viruses:A measurable response was found for H-2Dkplus alpha virus; mouse strains not expressing Dk failed to respond. No complementing H-2 haplotype combination has been found so far. It is unknown whether these examples resemble the findings for H-Y (Simpson and Gordon, 1977; von Boehmer, 1977), where very few responder alleles of D or K and very few responder I alleles for T help exist (von Boehmer et al., 1978b; Matsunaga and Simpson, 1978). Two types of MHC-coded Zr effects regulating responsiveness of cytotoxic T cells have been observed: (1)Stimulation with virus in the context of a particular K or D allele does not allow generation of cytotoxicity in T cells restricted to the same K or D allele. For example, there is no response to Kb plus influenza or Dk plus vaccinia or Sendai virus or Kb, Kd, and Dd plus H-Y; however, both K b and D k are high-responder alleles for LCMV (Zinkernagel et al., 1978d; Doherty et al., 1978a; Kurrle et al., 1978). ( 2 ) A K allele determines whether a particular D-restricted virus-specific response can be expressed; e.g., Kk allows low response, whereas Kq, K b and K S allow high response to Db plus vaccinia virus; in contrast, Kk allows high response to Dbplus LCMV. These Zr genes have the folIowing characteristics: (a) They are virus-specific, at least to a certain degree. (b) They map to H-2K or D. (3) The low response linked to a particular K orD allele has a dominant character. (4) The low-responder status is not absolute, particularly in the case of K k regulated D-restricted nonresponsiveness to Db vaccinia virus (Zinkemagel et al., 1978d;

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Doherty et aZ., 1978a). This low response can be converted to a high response if such lymphocytes are selectively restimulated with K-incompatible plus Db-vaccinia infected stimulator cells (Zinkernagel et al., 1978d), or if negatively selected T cells are stimulated in an environment expressing H-2D4 but not H-2Kk (Bennink and Doherty, 1979). In these restimulation experiments possible allogeneic effects have not yet been formally excluded. However, despite active search, no positive evidence for allogeneic effects in these models has been found so far. An Zr-like gene has been described by Egorov et al. (1977) that influences the capacity of parental lymphocytes to induce graftversus-host reactions when injected into F1 recipients, and has been mapped to K. It is unclear how this phenomenon relates to the examples of Zr genes that map to K or D and influence responsiveness of restricted cytotoxic T cells. There is no obvious unifying concept that could readily explain all the Zr phenomena. In fact, particular examples may reflect combinations of several possible mechanisms. The various hypotheses, such as an MHC-dependent tolerance (Snell, 1968), MHC as virus receptors (Snell, 1968; Ohno, 1977; Helenius et al., 1978), MHC products may or may not complex immunogenically with virally induced antigens (Doherty and Zinkernagel, 1975b), the Langman preclusion rule (Langman, 1978a),the modified Jerne hypothesis (von Boehmer et al., 1978b),and other ad hoc rules that certain anti-Self-H cannot combine with certain anti-X specificities or, alternatively, that Zr phenomena may reflect that various K or D products are expressed differently quantitatively (O’Neill and Blanden, 1979) (reviewed in Zinkemagel, 1978b), will be discussed in more detail in the last part of this review together with models of T-cell recognition.

B. INFLUENCE OF THYMIC SELECTIONOF T-CELL RESTRICTION SPECIFICITIES ON RESPONSIVENESSDURING T-CELL ONTOCENY Once the crucial role of the radioresistant host and its thymus were realized, it was immediately clear that older data on chimeras made with mice expressing differing Zr genes for a particular antigen could be reinterpreted or tested more directly (Tyan et al., 1969; Tyan and McDevitt, 1970; Bechtol et al., 1974a,b). Does the MHC type of the irradiated host or of the thymus dictate the Zr phenotype, or is it determined by the MHC genotype of the lymphohemopoietic stem cells? Independent evidence from at least seven different laboratories indicates that the thymic MHC environment defines the Zr phenotype when it selects the restriction specificity of T cells whose responsive-

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ness is regulated (Press and McDevitt, 1977; Warner et al., 1978; von Boehmer et al., 1978b; Billings et al., 1978b; Zinkernagel et al., 1978g; Kappler and Marrack, 1978; Miller et al., 1979). In all protocols, irradiation bone marrow chimeras or zygote fusion chimeras were made from responder and nonresponder mice. Warner et al. (1978) demonstrated that these chimeras generated antibodies of the responder allotype alone. Press and McDevitt (1977) used tetraparental bone marrow chimeras and had results that supported those of the study of Warner et al., but differed from previous work by Bechtol et al. (1974a,b). Such chimeras formed by reconstituting irradiated F, (responder x nonresponder) with responder bone marrow plus nonresponder bone marrow cells only generated Ig of the responder allotype for the antigen under Ir control. Five years earlier, these data would have been taken as evidence that only histocompatible T and B cells could cooperate. Although not formally shown in these studies, one would expect that both responder and nonresponder T cells can cooperate only with B cells of the responder H-2 haplotype to induce production of responder allotype-positive antibodies. Recent experiments on cytotoxic T cells support such an interpretation. Von Boehmer et al. (1978b) showed that stem cells of either I-dependent or K, D -dependent nonresponder helper or cytotoxic T cells that differentiated in an irradiated recipient of responder H-2 type could respond to H-Y in the context of the responder-type restriction marker. Lymphohemopoietic stem cells of responder MHC genotype expressed the nonresponder phenotype when they matured in irradiated recipients of nonresponder MHC type. Similarly, when matured in an H-2 K d Id Dd mouse ( K d and Dd are responder alleles for vaccinia virus), stem cells from Kd Id Dk nonresponders to Dk plus vaccinia virus could express responsiveness to Dd plus vaccinia virus and vice versa (Zinkernagel et al., 1978g). Identical results have been found for alpha viruses (Mullbacher and Blanden, 1979b). Billings et al. (1978b) found the same evidence for the cross-reactive tendency of TNPimmune T cells. H-2k (non-TNP cross-reactive) bone marrow stem cells maturing in (H-2kx H-2b) F1 TNP-cross-reactive irradiated recipients generated H - 2 k T cells that expressed the Ir phenotype of TNP cross-reactivity. Similarly, the thymus influences the Zr phenotypes of T cells involved in help (Kappler and Marrack, 1978) or delayed-type hypersensitivity (Miller et al., 1979). The H-Y example studied by von Boehmer et al. (197813) is of particular interest because it combines an Ir defect at the T-helper level (as formally demonstrated in mixing experiments by von Boehmer and Haas, 1979) with an Zr defect at the level of cytotoxic T-cell expression. BlO.A(5R) female mice are low responders to H-Y

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because they lack expression of cytotoxic T cells that can react against either Kb plus H-Y or Ddplus H-Y. CBA/JH-2kmice are low responders, perhaps because they cannot generate I-region-restricted T helper cells specific for I k plus H-Y. BlO.A(5R) stem cells maturing in an [CBNJ x C57BL/6 ( H - 2 k x H - 2 * ) ] F1 host generate H-Y-specific cytotoxic T cells restricted to H-2Db or H-2kbecause both responder Ib-restricted T helpers and H-2k or Db-restricted cytotoxic T effector cells have differentiated in this chimera. In contrast, CBNJ stem cells differentiating in the same type recipient F1 mice do not generate H-Yspecific cytotoxic T cells possibly, as discussed in a previous section, because of lack of appropriate Ik-restricted help. This prevents a response despite the presence of potentially triggerable Kk or Dkrestricted H-Y-specific cytotoxic T cell precursors. The Zk allele is a nonresponder for anti-Y responses, and Ib-restricted T help acquired in this chimera cannot be delivered in an appropriate way to lymphocytes bearing H-2k. Similar experiments in the H-Y model were reported later by Matsunaga and Simpson (1978), who, in contrast to von Boehmer et al. (1977, 1978b), found that H-21d could also induce appropriate T help sufficient to generate H-Y-specific, restricted activity, for example to Dk in C3H.OH ( K dI d Dk)mice. Zygote fusion chimeras (H-2d H - 2 k ) and irradiation bone marrow chimeras of (H-2d + H-2k) + (H-2d x H-2k)F1generated both H-2k and H - 2 d presumed helper cells restricted to the responder allele I d , but no T cells restricted to Zk nonresponder alleles. In addition, from H - 2 k and H - 2 d stem cells, Kk and Dk as well as Kd- or Dd-restricted precursor cytotoxic T cells should have been generated in these chimeras. After challenge, anti-H-Yspecific cytotoxic T cells developed that were restricted only to responder H-2k-Self. These H-2k-restricted cytotoxic T cells carried the H - 2 d haplotype. This finding supports the proposal that only I d (responder allele)-restricted T help is operative; therefore, help is delivered only to cytotoxic T cells of the H - 2 d type. Negative selection experiments have provided the further insight (Bennink and Doherty, 1979) that filtered C57BU6 T cells generate a strong vaccinia virus-specific T-cell response at H-2Db when stimulated in a BlO.A(4R) environment, and BlO.A(SR) lymphocytes can also develop potent virus-immune cytotoxic T-cell populations at H-2Db when primed in C57BL16 mice, but this is not true for BlO.A(4R) recipients. The capacity to respond to vaccinia virus presented in the context of H-2Db is, thus, ultimately independent of the context in which the precursor cell encounters H-2Db in the thymus. Low responsiveness at H-2Db depends on: (a) lymphocytes recognizing H-2Kk during the process of maturation in the thymus and (b)

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H-2Kk expression in the virus-infected stimulator mice. If both conditions apply, little or no Db-restricted vaccinia virus-specific response is generated, whereas operation of either condition is insufficient to prevent generation of cytotoxic T-cell activity restricted to H-2Db. These findings seem to indicate that lack of response at H-2Db reflects the concurrent presence of a dominant or suppressive influence of H-2Kk and vaccinia antigens (Zinkernagel et al., 1978d). It is not clear whether such immunodominance reflects gradations in the strength of complexation of vaccinia virus antigen with Kk versus Db or Kq versus Db. Alternatively, whether variability in numbers of specific precursors may be influenced by tolerance or other unknown mechanisms is not clear (Zinkernagel, 1978b; Bennink and Doherty, 1979). C. CONCLUSION The studies on expression and differentiation of immune responsiveness of antigen-specific helper or cytotoxic T cells reveal that genes regulating responsiveness and genes coding for the restricting Self-MHC antigens map to the same MHC subregions. The T cells’ phenotype of restriction specificity and of responsiveness is determined by the thymic MHC, not by the T cells’ genome, and is selected in parallel during the thymic residence. These qualities are valid for classical Zr genes coded by H-21 regulating T-helper cells as well as for genes regulating responsiveness of cytotoxic T cells. Categorization of the latter genes as Zr genes therefore seems to be justified, and it does not seem too far fetched to speculate that these Zr gene products and the products of the genes coding for the Self-H-restriction antigens are identical. Obviously, this theory is far from established. A recombination in the H-2K or D region separating restriction from Zr influence would clearly negate this hypothesis. However, if this speculation is true, then such Zr gene phenomena regulating T-cell responsiveness operationally becomes a direct function of MHC restriction. VI. Interpreting MHC Restriction and Ir Regulation of T Cells

The MHC-restricted specificity of T cells has preoccupied cellular immunology for quite some time, and many models and hypotheses have been advanced over the past 5 years (Katz et al., 1973; Shevach and Rosenthal, 1973; Zinkernagel and Doherty, 1974a,b, 1976; Doherty and Zinkernagel, 1974,1975b; Shearer, 1974; Lennox, 1975; Bevan, 197513; Schrader and Edelman, 1975; Katz and Benacerraf, 1976; Zinkernagel, 1976; 1977; Doherty et al., 1976a,b; Janeway et al., 1976; Blanden et al., 1976a; Miller and Vadas, 1977; Matzineer and Bevan,

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1977a; Rosenthal, 1978; Schwartz, 1978; Zinkernagel et aE., 1978b; Blanden and Ada, 1978; Langman, 1978a; Cohn and Epstein, 1978; von Boehmer et al., 1978b). What must be explained?: (1)the antigenspecificity of T cells; (2) the allelic and differential specificity of T cells for MHC products; (3) Zr-gene phenomena and the possibility that restricting Self-H and Zr-gene product map to the same H - 2 regions; (4) that both selection of T cells’ specificity for Self and the Zr phenotype are dictated by the thymic MHC; (5) the special case of alloreactive T cells. The main questions deal with most of the unanswered problems in cellular immunology: What is the nature of the T-cell receptor (s)? How do T cells and their receptor repertoire differentiate, or mature? What is the role of thymus and lymphoreticular cells in selection of the restriction specificity and in generation of diversity of the receptor repertoire? What is the function of MHC products? How do Zr genes function? Is alloreactivity a special case, as must be argued for two-receptor site models, or does it simply reflect that alloantigens are seen as altered Self? Devising a “theory of the theories” has been undertaken by many researchers who want to explain their results and by others who find this area of immunology intellectually fascinating and fertile ground for theoretical speculation. Analysis of the various proposals in a completely logical fashion is an impossible task, since the number of conclusive experiments that would allow dismissal of certain alternatives is still very small indeed. However, the proposed models can be analyzed according to common or distinguishing assumptions and mechanisms. The following “bits” have been used in the various models:

1. Number of germline genes: small or great? 2. Number of separate receptors on T cells: one, two, or more? 3. Is the binding site (one or two) formed by products of one or of two distinct genetic elements? 4. Germline genes coding for T-cell receptors: identical to Ig V genes, different and involving the MHC, or both together? 5. Zr genes function on T cells, or on antigen-presenting cells? 6. Generation of diversity: driven by Self-MHC, or not a somatic process? 7. Alloreactivity : mediated via one receptor (a single T-cell receptor for Self-H plus X, one alone of the two receptors for Self-H, for X, or two receptors? As is obvious, many of the possible permutations have been proposed at one time or another and reflect the lack of conclusive data needed to exclude the possible null hypothesis. Some speculations

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have become rather unlikely. An example is the proposal by Rothenberg (1976,1978)and by Blanden, Hapel, and Jackson (Blanden et al., 1976a) that virally induced changes in H-2-determined glycosylation patterns may cause expression of altered Self antigens by infected cells. Similarly, the notion that Self-H recognition is mediated via like-like interactions has been rendered untenable by the chimera studies (Section IV). The question of whether the T-cell receptor(s) is made up of V genelike products is not the main topic of this review. Evidence for this notion has been summarized recently (Eichmann, 1978; Rajewski and Eichmann, 1977; Binz and Wigzell, 1977) and seems to become more and more convincing. If true, this finding will put great constraints on any T-cell receptor model. If considered in the context of MHC polymorphism having evolved together with T cells to guarantee an optimally great receptor repertoire for individuals and the species (Section VII,C), this would mean that both anti-Self-H and anti-X are coded by VH genes; it would, on the other hand, render a single receptor model unlikely. Presently, whether generation of diversity is driven by Self-MHC products is as difficult to answer as whether the number of germline genes is small or great. The present discussion centers around the organization of T-cell receptors, MHClinked Zr control, and the nature of alloreactivity. Why are T cells MHC-restricted, a fact that distinguishes them clearly from B cells or antibodies? The operation of two-receptor specificities has been proposed as an answer. One receptor may have evolved from a cell interaction or differentiation recognition system and conserved this original function in one of its physiological activities. The second receptor may have evolved to introduce immunological specificity to such cell interactions. Whether the receptors have evolved from the same or two different origins is unknown. Any argument linking MHC polymorphism with T-cell restriction and responsiveness must be based on the assumption that both draw from the same gene pool. We discuss the arguments in favor of this interpretation from the point of view that intracellular parasites were strong selective forces shaping T-cell immunity (see Section VII). Accordingly, it is the effector function of T cells that is determined by the restriction specificity: K and D-specific T cells are lytic, I-restricted T cells are nonlytic, helping, activating, proliferating, etc. Therefore, as a consequence, the T cells employ dual specificity to concentrate on intracellular parasites, and, incidentally, are not distracted by free, extracellular antigens. Thus, we see this latter view as an important consequence of the fist reasoning rather than as a primary cause.

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The single-receptor model for T-cell recognition has the attraction of being simple and not requiring special rules for alloreactivity, alloantigens resembling complex antigens formed between Self-H and X. This is why this model was originally proposed (Zinkernagel and Doherty, 1974a,b; Doherty and Zinkernagel, 1974, 197513; and Shearer, 1974). We proposed the altered-Self model with the idea of virus and H-2 being recognized together, i.e., altered Self was defined functionally. This idea could represent a neoantigen or a complex of virus and H-2 (a little of virus, a little of H-2) being recognized. The latter idea implies that there are two receptor sites or specificities which are complexed in some way and is, in fact, a two-receptor site model. The clear distinction between a single site versus two separate recognition sites was made only subsequent to findings that the specificity for Self-H was distributed clonally, and that T-cell reactivity to minor transplantation antigens was MHC-restricted (Bevan, 1975a,b; Zinkernagel and Doherty, 1975a).The unsolved fundamental problem with a single receptor site model in the strict sense is how the unique antigenic determinant (neoantigen) forms as a result of Self-H and X complexing. As pointed out by Langman (1978a) and Cohn and Epstein (1978), the neoantigen model is subject to the rules that the new antigenic determinant cannot equal either Self or any of the various possible foreign antigens X or neoantigens formed between allo-H and X. However, it may also be argued that the latter possibility has not been examined, since no function would be seen unless the T cell was focused on the K or D antigen (lytic receptor) of the target cell. The possibility that any new determinant could mimic the one formed between histoincompatible Self-H plus X, a case for which there may be some evidence (Wilson et al., 1977; Doherty and Bennink, 1973),does not discriminate between one- and two-receptor site models. How the differentiating immunocompetent T cells learn these rules may be explained by variations of the Jerne model (Jerne, 1971; von Boehmer et al., 1978b). The fact that most negative selection and. most chimera experiments indicate a rather limited overlap of specificity repertoires (see Section IV) is not explained satisfactorily by a singlereceptor model in the strict sense. Any form of two-receptor site model, whether conceived as two receptor sites on two molecules, two receptor sites on one molecule, or two closely situated receptor sites within a single antigen-reactive cavity formed by one or two molecules, solves the aspect of thymic selection or learning more successfully, but leaves us with a more complicated explanation for the special case of allorecognition than would a single-receptor model.

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At present, it is difficult to reconcile some of the negative selection and chimera data (Section IV). Similarly, some of the results obtained for either model seem to be contradictory in themselves. Obviously, technical difficulties may explain some of the discrepancies. Keeping this in mind, our present view is that both selection at the level of thymic maturation, as well as some sort of Z region-dependent expansion in a postthymic stage are real and not obviously caused by suppression. This view is supported by the fact that for some haplotype combinations negative selection experiments have yielded results compatible with a rather high degree of restriction specificity. However, for other haplotype combinations, there exists a certain overlap of restriction specificity; either at the level of presumed restricted T help or at the level of cytotoxic T cells, unexpected cross-reactivities may be seen under the selective test conditions employed. Attempts to systematically study these overlaps of restriction specificities are continuing. Extending the interpretation of these data to favor or discredit a general case for single or dual specificity models is somewhat arbitrary. Allogeneic effects may be implicated in these aberrant reactivities seen in some negative selection experiments. Also, similar criticisms may apply to adoptive sensitization of chimeric lymphocytes or to experiments in which negative selection has been shown not to cross MHC barriers. The evidence and consequences that in most F1--* P chimeras (Zinkemagel, 1978b; Bevan and Fink, 1978) and in some negative selection experiments (Bennink and Doherty, 1978a) the restriction specificity for the host H-2 is rather strict, in fact, as strict as that expressed by T cells from unmanipulated mice, has been debated repeatedly (Bevan and Fink, 1978; Zinkemagel, 1978b; Matzinger and Mirkwood, 1978; Bennink and Doherty, 1978b; Doherty and Bennink, 1979; Blanden and Andrew, 1979). It is clear that all immunological specificity is relative; therefore, restriction specificity of cytotoxic T cells cannot be absolute. In fact, it has been shown that lymphocytes from H-2 incompatible irradiation bone marrow chimeras may express cytotoxic activities restricted to the tolerated H-2, that was not expressed in the thymus, upon secondary or tertiary restimulation against minor histocompatibility antigens (Matzinger and Mirkwood, 1978). These and comparably highly selective experiments involving negative filtration of lymphocytes through irradiated allogeneic recipients to eliminate alloreactivity and subsequent sensitization against antigens presented together with the same alloantigens cannot be used to compare or quantitate relevant precursor cells (Wilson et al., 1977; Bennink and Doherty, 1978a,b). Results of this type, therefore, do not

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distinguish between the two models of T cell recognition: a single receptor, for a neoantigenic determinant formed between Self-H and foreign antigens versus two receptor sites, one for Self-H and one for foreign antigen. As shown previously in thymus or irradiation bone marrow chimeras and in negative selection experiments, it is remarkable that the degree of restriction in primary antiviral responses by thymic chimeras is comparable to that by normal mice. Some results are from chimeras in which antigen presentation is optimal in association with both H-2 haplotypes involved in a given chimera; they reflect restricted T-cell activity generated during an acute primary antiviral response in uiuo. In the absence of a reliable assay to estimate the relative frequency of precursor cells in a defined in uitro system, the relative activity found in these chimeras, where no selection should occur at the level of sensitization, gives the best estimate of relative precursor frequencies we can obtain. T-cell activity, restricted to the thymic H-2, is at least 30- to 50-fold greater than for the second parental H-2 type that is not expressed in the thymus. This does not, however, exclude the possible presence of rare precursor T cells that may be restricted (by cross-reactivity?) to the MHC type absent from the thymus and may be boosted under selective conditions to become measurable. We feel, therefore, that to understand the general principles of T-cell restriction and recognition it is more important to further analyze and understand the high frequency of T cells restricted to thymic MHC than to generalize from a rare exception. The biologically relevant question is the same as for cross-reactivity of antibodies, namely: Is the efficiency difference between a high-affinity antibody and a cross-reactive antibody, although intellectually and physicochemically only of relative magnitude, biologically absolute? From this point of view, it becomes a crucial matter whether an unmanipulated animal has 1000 precursor T cells to recover from a particular virus infection, or whether this repertoire is diminished 10- or 100fold and therefore insufficient for survival. MHC-linked Zr regulation is undoubtedly linked to T cells being MHC-restricted and is probably a direct consequence of this restriction. This basic idea seems to gain wide acceptance (see Benacerraf and Germain, 1978; Klein, 1979).Obviously, this can be argued from either a single or a double receptor site model. Within a single-site model, MHC-linked Zr genes could act in the two following ways: they could determine either antigenicity, i.e., by influencing complexation between Self and X, or, alternatively, the capacity of T cells to express the antigen-specific receptor. The question is, does the shoe (receptor) not fit the foot (antigen) or the foot (antigen) not fit the shoe

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(receptor)? These arguments were originally proposed for cytotoxic T cells to explain MHC polymorphism (Doherty and Zinkernagel, 1975b), but have also been extended to explain Zr genes regulating antibody responses (Zinkemagel, 1976a; Doherty et al., 1976a,b). Subsequently, Rosenthal(l978) and co-workers applied the idea in a more concretely stated way to antigen presentation by macrophages to explain Zr effects on proliferating T cells. These authors proposed that Zr gene products (Ia antigens?) bind antigens in a way that results in exposure, or binding, of certain determinants-determinant selection. More recently the somehow dormant implication of these ideas that the MHC products have a kind of receptor function for antigens has been stated in its extreme and explicit form by Benacerraf (1978).He proposed that about three amino acids may serve to form a triplet code for noncovalent binding of amino acids or the charged groups of the antigen. Another explanation is the suggestion that T cells restricted to a particular Self-H cannot express a receptor for a particular complex of Self plus X. This argument cannot be based easily on the assumption that tolerance prevents expression of a particular receptor, but rather implies restricted somatic diversification in the manner of Jerne (1971).The alternative proposal that a particular variable region cannot be expressed by T cells restricted to a particular allele is difficult to envisage in a single-receptor model. Except for the logical rules outlined earlier, there is no evidence for or against any of these proposals; thus they remain pure speculations. With respect to a two-receptor site model, the foregoing argument that Zr defects are determined by lack of appropriate complexation does not apply if we consider that there are two quite separate receptors. However, if Self-H and X are recognized by two proximate r e c e p tor sites, Self-H and X may need to be reasonably close to either trigger the two sites simultaneously or allow tight binding to occur. A tolerance argument can be made as follows: If a Self minor or major histocompatibility antigen resembles a viral antigen, then expression of this specificity is not permitted. This possibility has not been documented as yet, since, as far as tested, non-H-2 background genes do not seem to influence responsiveness in the virus models. In any case, the invoked tolerance cannot be a general one for vaccinia virus antigens, since all mice are phenotypically high responders when their overall capacity to generate cytotoxic T lymphocytes is assessed. Zr defects arise because there is a defect in the receptor repertoire that is linked to recognition of Self. Thus, expression of a receptor site for Self somehow prevents the expression of certain sec-

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ond receptor sites for X. Theoretically several mechanisms may be responsible for this occurrence. Langman (1978a) and Cohn and Epstein (1978) propose a “preclusion” model. Accordingly, selection of the anti-Self-H receptor before that for anti-X leaves a hole in the germline repertoire from which no anti-X can be selected. If by chance the receptor for X is usually derived from the germline used for the anti-Self-H receptor, low responsiveness or unresponsiveness may result. However, this proposal is a highly problematic explanation for why cytotoxic T lymphocytes’ unresponsiveness to completely (at least serologically) unrelated viruses should be regulated by the same MHC alleles, as is the case for Dk-associated nonresponsiveness for both vaccinia virus and Sendai virus. Also, it does not explain why there should be complete nonresponsiveness to all influenza A viruses presented in the context of H-2Kb (Doherty et al., 1978; Effros and Doherty, unpublished). Therefore, it is probably not a general explanation. An alternative concept has been proposed by von Boehmer et al. (1978b). They assume that maturing T cells first express two identical receptor sites, both with specificity for Self-H. One of these, probably on a different constant region, is free to mutate somatically away from its original specificity for anti-Self-H. Since the formation of the anti-X repertoire is, according to a modification of Jerne’s (1971) original concept, driven by Self-H, Zr defects may arise if a particular X does not lie within the range of feasible diversifications starting from a certain anti-Self-H receptor. Neither the Langman-Cohn nor the modified Jeme model can readily incorporate the concept that VH gene products may contribute at least part of the T-cell receptor for X. This is, however, envisaged in a third speculation that certain variable-type regions (e.g., VH products for a particular anti-X) may or may not combine with anti-Self-H receptors, This rule includes the assumption that anti-Self-H has a similar character on the T-cell receptor as allotypic markers on Ig, or that the phenomenon can be compared to the finding that certain hypervariable regions do not combine with certain framework residues in Ig. Based on some experimental evidence, O’Neill and Blanden (1979) have speculated that some responsiveness differences may be explained by the finding that certain K or D products are expressed in significantly different amounts on homozygous versus heterozygous cells, the implication being that the lesser amount of K or D expressed, the lower the response. At present, it is difficult to evaluate this proposal on the basis of the available data. In these models, one point might be overlooked in explainingzr gene

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phenomenology, i.e., intergenic Zr gene complementation as shown for I region-restricted T cells (Dorf and Benacerraf, 1975; Munro and Taussig, 1975; Warner et al., 1978; Schwartz et al., 1978). Most noncomplementing Ir phenomena involving the I-A region resemble the similar phenomena outlined for K and D, but some complementing examples suggest that the MHC may code either for several or more than only restricting Self-H. It is unclear whether Zr complementation is best explained by any of the following possibilities: (1) in these responses intercellular complementation occurs, each regulated or restricted to different H-21 subregions; (2) the restricting elements in I-EIC are partially coded in I-A (Jones et al., 1978; Silver and Russell, 1979; Cook et al., 1979), or (3) parts of the T-cell receptors are coded by the MHC [e.g., something analogous to a light chain, whereas the heavy chain is an Ig variable gene product-like structure (Cohn and Epstein, 1978)l. If it is true that Zr gene phenomenology is a mere reflection of T cells being MHC-restricted and that both the restricting element and Zr gene product are identical, then the linkage of T-cell repertoire to MHC (i-e.,Ir gene regulation) may be solely phenotypic. Biochemical analysis of T-cell receptors and MHC products should eventually provide some of these answers. Allorecognition can be viewed as a special case from many points of view. The generally accepted view is that K and D alloantigens are cell-surface structures that are receptors for the lytic message, possibly because these alloantigens are linked to a synapse-like mechanism. Similarly, I-region determinants are identical with or linked to receptors for proliferative signals, etc. Alloreactivity is readily explained by any of the single-receptor models, since from this point of view alloantigens are just a form of new antigenic determinants. Within two separate receptor models, alloreactivity can be mediated in three different ways: (1) via the anti-X receptor (Langman, 1978a; Cohn and Epstein, 1978); or (2) as anti-Self-H type receptors alone (Janeway et al., 1976; von Boehmer et al., 1978b); or (3)via anti-Self-H plus anti-X; the latter would actually follow a pattern-a modification of the Matzinger and Bevan (1977a) model-in which T cells recognize a public and a private specificity or two public specificities of a particular transplantation antigen or a determinant of the alloantigen plus a minor transplantation antigen. As pointed out in Section III,D, the finding by Shearer et al. (1975), and subsequently by Burakoff et al. (1976b) and by Lemmonier et al. (1977), that TNP-specific syngeneically restricted T cells may also be alloreactive accommodates the single-receptor model. Or, as shown by Bevan (1977), cytotoxic T cells that are minor transplantation antigen specific, and syngeneically restricted, can be selected so as to be

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highly reactive to a particular alloantigen, which has been taken as evidence in favor of a single-receptor model. For similar results with cloned anti-Self + H-Y-specific cytotoxic T cells, see von Boehmer et al. (1979a). However, this apparently pleasing interpretation is not really compelling or discriminatory, since, under the strongly selective pressure of restimulation or cloning procedures, it is readily envisaged that any of the other possible recognition mechanisms may apply. One of the major unresolved problems for proponents of the two separate receptors concerns the apparently high relative frequency of alloreactive T cells. Does this reflect the fact that the germline genes for the T-cell-receptor variable regions code for receptors for the species' alloantigens? Or alternatively, is the high frequency only a consequence of the special characteristics of MHC products? That is, does the usual presentation of alloantigen on immunocompetent cells provide potentiating and inducing signals from the stimulator cells back to the responding cell? and are alloreactive T cells much more readily detected than other antigen-reactive T cells because of the mechanics of MHC products? Although the first of these concepts is more widely accepted, the latter concept can explain why alloantigens or cells not derived from lymphohemopoietic cells do not induce alloreactive T cells despite their excellent antigenicity as evidenced in §'Cr release assays. It may well be that K- and D-like MHC products are ordinary antigens, and only these combinations with some as yet undefined stimulatory or inducing quality coded at H-2Z makes them unique (Lafferty and Woolnough, 1977). Therefore, from our present preoccupation with MHC restriction, an important question is whether alloreactivity is functionally mature and expressed before antigen reactivity or whether both develop simultaneously. The modified Jerne model (von Boehmer et al., 1978b)predicts that alloreactivity is mature before Self-restricted T cells; the LangmanCohn and a single-receptor model predict simultaneous emergence. Any additional two-receptor model that qualitatively separates antiMHC from anti-X receptors by assigning VH character to anti-X but not to anti-MHC would also predict that alloreactivity matures first and independently of diversification of anti-X. Attempts at analyzing this question in various chimeras are now in progress in several laboratories. However, technical problems with such chimeras, as elaborated in Section IV, or simple sensitivity differences of alloreactive versus syngeneic cytotoxic T cell induction and testing may render this task very difficult indeed. Most models, with the exception of a few, have so far survived, and opinions periodically shift support from one-receptor site models to two-receptor site models. In some cases, the packaging of the proposal

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with an appealing name has been more relevant than the (absent) facts. Such nebulous terms as physiological interaction, altered Self, and complexing have heated up discussions and minds alike and served at times to make the subject more etymological than immunological. At present, various arguments seem to favor a form of the two-receptor site model, but a single-receptor site model has not been formally excluded. However, attempting to argue one-site or two-site models in the absence of structural information about T cell receptors may ultimately prove as useful as much medieval theology. VII. In Vivo Relevance of MHC-Restricted Cytotoxic T Cells

As stated before, the fact that observations from in uitro experiments with MHC-restricted virus immune T cells are readily applicable to relevant in uiuo biological situations has been crucial in clarifying the biological role and importance of MHC restriction and of major transplantation antigens in general. Protection of the host against intracellular infectious agents is a very complex process (reviewed in Notkins, 1975). The potential for involvement of immune-specific mechanisms such as T cells (Blanden, 1970, 1971a,b; Mims and Blanden, 1972) and antibodies, possibly including antibody-dependent cell-mediated effector function (Perlmann et al., 1972; Steele et al., 1973; Shore et al., 1974, 1976; Rager-Zisman and Bloom, 1974; Ramshaw, 1975), is obvious. However, many nonspecific effector mechanisms, such as macrophage activation (Allison, 1974; Blanden, 1971b; Krahenbuhl and Remington, 1971; Blanden et al., 1976b), interferon and lymphokines (Isaacs and Lindenman, 1957; Merigan, 1964; Baron et al., 1964; Wheelock, 1965; Glasgow, 1966; Braun and Levy, 1972; Lindahl et al., 1972; Harfast et al., 1975; Rager-Zisman et ul., 1976; Welsh, 1978a,b; Gidlund et al., 1978; Trinchieri and Santoli, 1978; Trinchieri et al., 1978a,b) must be considered. These may be superimposed on genetic factors exemplified by natural resistance of macrophages (Bang and Wanvick, 1960; Allison, 1965; Goodman and Koprowski, 1960; Lindenmann, 1964; Lindenmann et al., 1978). In view of this complexity, it is difficult to assign in uiuo correlates to particular cells whose activities are assessed in uitro. Therefore, demonstration of in uiuo relevance must always be limited by the purity of the subpopulation under investigation and the number of parameters that can be compared. Several approaches have been used to demonstrate the in uiuo activities of defined cell populations. One approach is to use adoptive transfer of cells to reconstitute animals depleted of particular cellular

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subsets. Another method is the transfer of defined cell populations to recipients for acceleration of a particular immune phenomenon. The criteria used to establish whether effector cells operating in uitro are the same as those functioning in uiuo include typing by serological analysis of cell-surface markers, a process best done with lymphocytes recovered from the site of action in uiuo; comparison of dose-response relationships and specificities; and analysis of proliferative kinetics. Another approach is to remove the subpopulation of cells that is active in uitro and then to determine whether the in uiuo effects are modified in a comparable fashion. Most of these criteria have been met in analyzing the relevance of immune cytotoxic T cells in uitro and in v i m . This section summarizes the evidence suggestive that cytotoxic T cells specific for minor transplantation antigens may be involved in graft rejection and the extensive amount of data strongly indicating that virus-specific cytotoxic T cells generated during virus infection in uiuo are crucially involved in both antiviral protection and inflammation (for review see Allison, 1974; Blanden, 1974; Doherty and Zinkernagel, 1974; Blanden et d ,1976b, Notkins, 1975; Bloom and Rager-Zisman, 1975). These immune processes may also lead to T cell-mediated immunopathology (for reviews see Rowe, 1954; Mims, 1964; Hotchin, 1963, 1971; Lehmann-Grube, 1971; Doherty and Zinkernagel, 1974; Cole and Nathanson, 1975). A. IMMUNEPROTECTION

As stated in Section 11, from observations in murine systems we know that during an acute primary systemic virus infection there exists an intimate relationship between the kinetics of viral growth and elimination and the detection of virus-specific, MHC-restricted cytotoxic T cells (Marker and Volkert, 1973b; Blanden and Gardner, 1976). Adoptive transfer (Mitchison, 1954; Mackaness, 1964, 1969) of immune protection in both poxvirus and LCMV was achieved. Normal mice were infected with virus some 16-24 hours prior to cell transfer. The recipient mice were killed 24 hours later, and virus titers in spleen and/or liver were assessed quantitatively as plaque-forming units. The effector cells capable of suppression and elimination of virus in uiuo were characterized as &bearing (Blanden, 1971; Mims and Blanden, 1972; Blanden et d.,1975; Blanden and Kees, 1976; Zinkernagel and Welsh, 1976),Ig negative (Blanden et d.,1975), and nonadherent (Blanden, 1971b), and they were found in uiuo to follow the same kinetics of activity as cytotoxic T-cell activity measured in uitro (Blan-

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den and Gardner, 1976).This result is compatible with the interpretation that reconstitution of bone marrow-derived macrophages provides an accessory cell population for protection (Blanden, 1971b), as in protection against L. monocytogenes (Mackaness, 1964, 1969; Lane and Unanue, 1972; Blanden and Langman, 1972; Blanden et al., 1976b). Thus, it seems that in uitro cytotoxic T-cell activity and in uiuo protective effects have similar dose-response relationships. Some indirect evidence also suggests that these lymphocytes accumulate in specific splenic viral lesions (Blanden, 1974). Finally, it has been demonstrated that the in uiuo protective effect is virtually absolutely dependent on donor or immune T cells and the recipient host sharing at least the K or D regions of the H-2 gene complex; H-2Z regioncompatible T cells do not confer a significant level of protection (Kees and Blanden, 1976; Zinkernagel and Welsh, 1976; Ertl et al., 1977). Furthermore, the fine specificity of the MHC restriction of T cellmediated antiviral protection corresponds to that of cytotoxic T cells as demonstrated with H-2 mutant mice (Kees and Blanden, 1976). With controlled experimental conditions, antiviral protection in uiuo is a much more sensitive and biologically relevant assay than in uitro titration of cytotoxic T-cell activity, since a quantitative assessment can be made by using a scale of 4 logs of virus titer reduction. The lack of in uiuo cross-reactivity between virus-immune T cells operating in the context of wild-type H - 2 K b and mutant H-2Kbm1, and the absence of antiviral protection associated with transfer of Z region-compatible immune spleen cells, greatly strengthens the biological validity of the H-2 restriction phenomenon and the special role of K and D versus I-region products. More recently, antiviral effects of influenza-immune cytotoxic T cells have been demonstrated in the lungs of mice with virus pneumonia (Yap et al., 1978; Ennis et al., 1978). The existence of the cross-reactive T-cell response in influenza was correctly considered to be of little biological relevance for immune protection (Ennis et al., 1977a,b), and the long-term attention of influenza virologists has been directed mainly at the hemagglutinin (Kilbourne, 1975).This is an absolutely logical position when thinking of protection. However, T cells function essentially to promise recovery from virus infections (Blanden, 1974), not to protect against primary exposure. Cytotoxic T cells invade the lungs of mice with pneumonia caused by intranasal inhalation of an H2N2 virus after 6 days only if the mice have not previously encountered influenza A virus or if the mice are primed several weeks earlier with a serologically non-cross-reactive HONl virus (Bennink et al., 1978). Any protective effect of such prior exposure may be relatively marginal and could easily be confounded by

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many other factors operating in the clinical situation (reviewed in Doherty et al., 1977b). Nevertheless, evidence has existed for many years that mice primed with an HONl virus may be partially protected on subsequent challenge with an H2N2 virus (Schulman and Kilbourne, 1965; Werner, 1966; Floch and Werner, 1978). Furthermore, Webster and Hinshaw (1977) have shown that mice immunized with purified internal matrix protein are partially protected against subsequent respiratory exposure to a heterologous influenza A virus. However, the most compelling evidence comes from a series of elegant adoptive transfer and T-cell localization experiments performed by Yap and Ada (1978a-c; Yap et al., 1978). As with other virus models (see above), H-2K-D restricted Ly 2,3+T cells taken at the peak of the cytotoxic response from mice primed with either the same or a different influenza A virus greatly depress titers of influenza virus in the lungs. This is not to say that T cells cannot under some circumstances play an immunopathological role in influenza (Singer et al., 1972; Suzuki et al., 1974; Cate and Mold, 1975; Sullivan et al., 1976; Wyde et al., 1977). Any process that involves massive and synchronous elimination of functional cell populations may provide an acute physiological crisis (Doherty and Zinkernagel, 1974). However, the results of Yap and Ada establish beyond reasonable doubt that cell-mediated immunity, whether a function of virus-specific or cross-reactive T cell subsets, may be beneficial against influenza. The previously discussed in uiuo experiments can be criticized in various ways by postulating that allogeneic effects (reviewed in Katz, 1972) or allogeneic inhibition (reviewed in Hellstrom and Moller, 1965) operate in the recipient mice. Positive or negative allogeneic effects complicate many experimental approaches in cellular immunology (McCullagh, 1972; Katz, 1972). Their role in the aforementioned models has not been properly investigated. These effects seem unlikely to be a major factor in these experiments because a short-term (24 hours) adoptive transfer system is used and reciprocal F, ZSParent interactions function as well as syngeneic combinations. Even so, the possibilities of interference cannot be completely excluded. The direct involvement of cytotoxic T cells in antiviral protection in uiuo is also supported by the in uitro demonstration that virus-specific cytotoxic T cells can act antivirally, provided infected target cells are lysed before infectious virus has been assembled (Zinkernagel and Althage, 1977). Once most of the viral progeny are assembled, cytotoxic T cells may cause W r release and target cell death, but the cytotoxic T cells cannot suppress release of infectious particles. This evidence, in combination with indications that virus-specific antigens appear very early after infection [e.g., vaccinia virus (Ada et al., 1976;

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Jackson et al., 1976; Koszinowski and Ertl, 1976), VSV (Zinkernagel et al., 1977d)], strongly supports the concept that virus-specific cytotoxic T cells may stop viral replication by destroying the host cell during the eclipse phase of viral infection. All cells throughout the body are potentially susceptible to infection with some virus or other. Thus, the fact that K- and D-restricted T-cell recognition is lytic and that K and D determinants are present in variable amounts (Klein, 1975) on all cells of the vertebrate host fits the special requirements needed for elimination of viruses via a cell-surface surveillance mechanism (Doherty and Zinkemagel, 1975b). If true, this argument emphasizes that immune protection is mediated via host-cell destruction, which implies that, although disease may be directly caused by the cytopathic effect of the virus, disease may also be a consequence of the T cell-mediated destruction of infected host cells. However, one should not neglect the absolutely essential ancillary role of macrophages in “mopping-up” released infectious virus. This latter mechanism has been analyzed to some extent for the facultative intracellular bacterium Listeria monocytogenes. As elaborated by Mackaness (1964,1969) and co-workers, L. monocytogenes are eliminated by specific T cells (Lane and Unanue, 1972; Blanden and Langman, 1972) that activate macrophages to increased nonspecific bactericidal capacity. These T cells are apparently not cytotoxic and are MHC-restricted to H-21 (Zinkernagel et al., 1977a). This mechanism fits the concept that unlike viruses, intracellular bacteria do not undergo an eclipse phase, nor are they eliminated via host cell destruction, but rather by intracellular digestion in phagocytic cells. H2Z-coded structures that are expressed on selected cells (e.g., macrophages) may thus be regarded to function as cell-specific receptors for differentiation signals that induce enzymes in macrophages, Ig production in B cells, etc. (Zinkernagel, 1977). As presented here, similar arguments can be extended to toxins, parasites, and other antigens (e.g., bovine serum albumin). Thus, the relevance of these basic mechanisms to the pathogenesis of viral disease depends on the equilibrium between the cytopathic effect of the intracellular parasites and their virulence as opposed to the immunocompetence and immune responsiveness of the host. The practical clinical consequences of this concept in understanding the associations of MHC with disease will be discussed in Section VI1,D.

B. T CELL-MEDLATED IMMUNOPATHOLOGY The concepts developed in the preceding section seem also to explain the pathophysiology of several diseases, namely acute LCM

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(Traub, 1936, 1939; Rowe, 1954; Johnson and Mims, 1968; Hotchin, 1971; Doherty and Zinkemagel, 1974; Cole and Nathanson, 1975), Coxsackie B endocarditis (Woodruff and Woodruff, 1974; Wong et al., 1977a,b,c), and perhaps human hepatitis (Mackay, 1976). Originally, Traub (1936, 1939) found that intracerebral injection of LCMV induced death in adult mice, but not in neonates. The neonates became virus carriers, mimicking the normal maintenance pattern for LCMV in nature. This observation was one of the key experiments on which Burnet and Fenner (1949) built their concept of Self-non-Self discrimination central to the idea of immunological tolerance. The fact that immunoincompetence prevented death from LCMV injected i.c. was confirmed in classical studies by Rowe (1954) that established the immunopathological pathogenesis of this laboratory disease in mice. During the last 10 years, it has become increasingly obvious that the immune effector lymphocyte responsible for this disease is a T cell (Rowe, 1954; Rowe et al., 1963; Hirsch et al., 1967; Gledhill, 1967; Cole et al., 1971; Gilden et al., 1972a,b; Doherty et al., 1976c; Johnson et al., 1978). Virus injected i.c. tends either to distribute throughout the cerebrospinal fluid or to escape directly into the blood (Mims, 1960). This determines the distribution of both virus growth and accumulation of inflammatory cells in LCM, the disease process in the brain being localized predominantly to the meninges and cerebrospinal fluid. The meningeal inflammatory exudate may be isolated by tapping the cisterna magna (Carp et al., 1971; Doherty, 1973) and constitutes a very potent source of cytotoxic T-cell activity (Zinkernagel and Doherty, 1973; Doherty and Zinkernagel, 1974). The observation that cytotoxic T cells are localized in the actual lesion has been confirmed for poxvirus-induced meningitis (Hapel and Gardner, 1974; Morishima and Hayashi, 1978). Cole and co-workers first demonstrated that T cells could adoptively transfer acute LCM disease to mice that had become virus carriers as a result of immunosuppression with cyclophosphamide (Gilden et al., 1972a,b). Antibodies or B cells did not induce the disease. We modified this model in the following way: mice were infected i.c. with LCMV and immunosuppressed 2 days later with an appropriate dose of cyclophosphamide; the appropriate lymphocytes were then adoptively transferred to recipients after another 2 days (Doherty and Zinkernagel, 1975d). Normal lymphocytes did not cause disease, whereas T cells from LCMV-infected mice induced accelerated LCM. The interval between transfer and death (as little as 1$to 2 days) and the percentage of mortality directly correlated with the cytolytic capacity of the transferred lymphocytes. The effector cells were 6 positive, as

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already shown by Cole et al. (1972) and negative for Ig and Fc- receptors. Furthermore, the transferred T cells and the recipient mice had to be K or D compatible for disease to occur, i.e., accumulation of inflammatory cells in cerebrospinal fluid. Compatibility in the H-21 region caused little if any inflammation (Doherty et al., 1976a,c). As for antiviral protection in uiuo, T cells involved in induction of LCMV sharply discriminated between K b and the Kbml mutant specificities (Doherty et al., 1976~).It seems unlikely that host cells such as macrophages were recruited to collaborate with the transferred T cells in the induction of disease, since clinical LCM occurred rapidly in recipients treated with both cortisone and cyclophosphamide (Zinkernagel and Doherty, 1975~).Both drugs prevent generation and release of bone marrow-derived macrophages from the bone marrow and render such mice susceptible to L. monocytogenes, even when a source of effector T cells is provided. The presence of high concentrations of virus-specific cytotoxic T cells in the cerebrospinal fluid of mice with acute LCM and the striking similarities of effector T cells assayed in uitro and in this adoptive transfer model in uiuo are compelling evidence for the idea that cytotoxic T cells are involved rather directly in causing disease. Since after i.c. inoculation virus spreads preferentially in the meninges and choroid plexus, it seems probable that cytotoxic T cells destroy the blood liquor barrier that maintains the pressure disequilibrium between the cerebrospinal fluid and the blood stream. The consequence of this breakdown is acute brain edema, as has been documented very colorfully by showing that Evans' blue leaks into the interstitial brain tissue (Doherty and Zinkemagel, 1974). When large dose,s of viscerotropic LCMV (i.e,, a virus strain that grows widely throughout the body) are injected i.c., mice may well become clinically ill, but do not die (Hotchin, 1963, 1971). Th'1s socalled high-dose paralysis probably has nothing to do with conventional high-dose immunological paralysis. The phenomenon may best be explained by the following postulate: when there is a relatively high concentration of LCMV only in the meninges and the choroid plexus, T cells are recruited preferentially to the brain. This occurs when a neurotropic virus is used or mice are injected i.c. with a low dose of viscerotropic virus. When a high dose of viscerotropic virus is given, extensive replication occurs throughout the body, with the result that cytotoxic T cells are recruited to many other infected tissue sites. This dilution of the effector populations results in less damage to the brain-liquor bamer. Such an explanation is based on the assumptions that a host always makes a maximal cytotoxic T-cell response and

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that the relative damage depends on both the extent of lesions and the relative distribution of lesions. This also fits the fact that LCMV carriers do not die from acute LCMV when transfused with immune cells, as do the mice given virus i.c. as adults and are immunosuppressed prior to transfusion with immune lymphocytes. In fact, the immuriofluorescent studies of Mims (1964) have revealed that meninges are relatively weakly stained in carriers, but that many other organs or tissues are positive for LCMV antigens. High doses of LCMV injected into normal mice induce severe liver lesions that are completely absent in nude mice. Histologically, the picture resembles that of autoaggressive hepatitis in humans. Several points support this comparison with systemic LCM disease, particularly with respect to liver and human hepatitis: (1) immunosuppression is usually clinically beneficial; (2) virus-carrier status does not comprise liver pathology; (3) transfer of lymphocytes, but not of immune serum, from immune donors to virus carriers induces liver pathology of predominantly mononuclear infiltrates (Kohler et al., 1974; Mackay, 1976). An additional model illustrating T cell-mediated immunopathology has been studied by Bryere and Williams (1964), Svet-Moldavsky et al. (1964, 1968), and Holterman and Majde (1969, 1971). They observed that skin grafts taken from mice that carried viruses of low cytopathogenicity as Friend virus, MSV, or LCMV were rejected as rapidly as allografts by inbred syngeneic recipients. Viruses thus seemed to expose antigens that behaved as transplantation antigens. Retrospectively, these experiments were the first evidence indicating that T-cell monitoring in both the Self and alloreactive situation is comparable. These observations are of obvious practical consequence for human transplantation surgery. Virus carrier status may exist in humans for many more viruses than is currently suspected. Cytomegalovirus is one candidate that has been suspected to bring about rejection of transplants in recipients that may be optimally matched for HLA, but immune to this carrier virus. C. MHC POLYMORPHISM The polymorphism of MHC products has been a puzzle for a long time, and many speculations attempt to explain it (Snell, 1968; Benacerraf and McDevitt, 1972; Bodmer, 1972, 1973; Amos et al., 1972; Burnet, 1972; Klein, 1975,1976,1979; Doherty and Zinkemagel, 1975b; Snell, 1978; Langman, 1978; Zinkemagel, 1978b, 1979). These speculations include tolerance models in which infectious agents might mimic MHC products to escape immunosurveillance (reviewed in Snell, 1968; Bodmer, 1972; Amos et al., 1972), theories that MHC

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products function as receptors for intracellular parasites, such as viruses (e.g., Helenius et al., 1978), and the proposal that polymorphism is a mere accident because some linked genetic loci, such as the T loci, are polymorphic (Bodmer, 1972; Amos et al., 1973; Klein, 1975,1976). We strongly believe that evolution of T-cell restriction and Zr genes is intimately linked to that of MHC polymorphism. Since specificity for Self-H as well as responsiveness to X are determined by MHC products, polymorphism expands the receptor repertoire optimally to guarantee survival of the species by protection from intracellular parasites, and polymorphism superimposed on gene duplication maximizes the receptor repertoire of individuals (Doherty and Zinkernagel, 1975b; Langman, 1978a; Zinkernagel, 1978b, 1979; Klein, 1979). The discovery of MHC restriction and regulation, notably with respect to virus-specific cytotoxic T cells and cell-mediated immunity to intracellular bacteria indicates that the present role(s) of H-2 genes reflects the operation of strong evolutionary pressures. The obvious implication is that MHC polymorphism and T cells are both products of natural selection driven by infectious diseases. We must remember that, until a few decades ago, infectious diseases were the greatest causes of both morbidity and mortality, particularly during infancy and childhood. Hygiene, preventive immunization, and antibiotics have changed the picture drastically. In terms of human evolution, these are very recent achievements. Interestingly, the two highly polymorphic systems known in higher vertebrates are linked with immunology: Ig allotypes and MHC products. Allotype-linked Zr phenomena have been found to regulate mostly expression of antibodies directed against polysaccharides. However, MHC-linked Zr genes regulate T-cell activities, directed against foreign antigens expressed on cell surfaces. The similarities between polymorphism of the two systems as seen from an infectious disease standpoint are indeed convincing. Maybe the relevance of antibody-mediated protection against intracellular bacteria with their protective capsule, such as pneumo-, staphylo-, strepto-, meningo-, or gonococci, will become more obvious if active or passive immunization becomes the therapy of choice because of increasing resistance to antibiotics, thus reviving the intuitively correct choice of immunologists in the 1920s and 1930s (reviewed in Heidelberger, 1956).For the present discussion we shall restrict ourselves to T cells and their special relationship to MHC products and intracellular parasites. If we consider mainly infectious agents that cause rapidly progressing and often lethal disease, in contrast to many but more chronic

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parasitic diseases, such as filariasis, trypanosomiasis, or malaria, a simplistic scheme of disease contains two categories: extracellular agents, particularly bacteria with polysaccharide-rich capsules and walls that often produce toxins, and intracellular agents, such as viruses or intracellular bacteria. It is the latter category that seems to preoccupy T cells; the former is the main target of antibodies and the complement system. Let us, for the sake of the present argument, restate that MHC-linked Zr genes do not directly influence B cells but only T cells. Therefore, the two immune effector mechanisms and the polymorphism of involved allelic molecules are apparently linked as would be expected. Ig allotype-linked Zr genes regulate optimal responsiveness of antibodies to sugars and polysaccharides, whereas MHC-linked Zr genes deal with maximal responsiveness of T cells to intracellular parasites, for which proteinic antigens may serve as useful model systems. The studies of immunoresponsiveness of mice to virus, assessed as their potential to generate K- or D-restricted cytotoxic virus-specific T cells, show that immune protection depends on the H-2 alleles of the host. For example, mice of the H-2* haplotype infected with LCMV respond very well to Dk plus LCMV, but markedly less to Kk plus LCMV. In contrast, the same mice infected with vaccinia virus, a poxvirus, generate great cytotoxic activity against Kk plus vaccinia virus, but virtually none with specificity for Dk plus vaccinia virus (Zinkemagel et aZ., 1978~).Since LCMV and poxvirus (ectromelia) are among the most prevalent natural murine pathogens, this differential responsiveness is as revealing as the fact that every inbred strain of mice known has a high overall responder phenotype, i.e., all mice have strong K- or D-restricted responses to these viruses, and we have not yet found any strain that is a nonresponder at both loci. Our assumption is that the main task of immunity is to help the host overcome infectious diseases. The great selective pressure exerted by infectious agents has led to coevolution of T-cell immunity and MHC polymorphism. Protracted growth of intracellular parasites (viruses or intracellular bacteria, such as Listeria, BCG, etc.) is prevented mainly by T lymphocytes, whereas extracellular parasites are eliminated predominantly by antibodies and other neutralizing factors in association with complement and phagocytes (Notkins, 1975; Bloom and RagerZisman, 1976; Blanden, 1974, 1976b). As previously discussed, T cells seem to promote recovery of the host both by destroying acutely infected cells during the eclipse phase of virus infection and by recruiting macrophages to sites of pathology. In contrast, facultative intracellular bacteria and fungi are apparently not controlled by lytic T cells,

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but rather by nonlytic T cells that activate macrophages to increased bactericidal capacity (Mackaness, 1964, 1969; Blanden and Langman, 1972; Lane and Unanue, 1972). Thus, at least against some viruses, immune protection is mediated partially via host cell destruction. The clinical outcome of an infection is determined by the cell-destroying (cytopathic) capacity of the virus competing with cell destruction by T cells seeking to prevent the virus from spreading and with macrophages functioning to eliminate free virus. The termination of viremia by circulating antibody is also a major factor. This sequence immediately implies that the balance of beneficial versus harmful effects of cellular immunity depends on such factors as: cytopathogenicity of the virus, rapidity of spread, organ and/or cell tropism, antigenicity of the virus or parasite, immunocompetence, and the immune-response phenotype of the host. We have already summarized evidence from two virus infections that represent extremes and may illustrate these concepts. Poxvirus in humans (ectromelia in mice) is a highly cytopathic virus. Cellmediated immunity is essential for overcoming this infection, as documented by the fact that T cell-deficient children often develop general vaccinoses upon vaccination and that T cell-deficient mice rapidly die when infected with mouse poxvirus (Blanden, 1974). Hepatitis virus in humans and LCMV in mice provoke the other extreme response to infection. These viruses are not very cytopathic or their cytopathogenicity may have been modified, and immunodeficient hosts do not die of the virus infection. However, in immunocompetent virus-infected hosts, tissue damage seems to be caused by the T cell response of the host rather than by the virus per se (reviewed in Hotchin, 1971; Cole and Nathanson, 1975; Doherty and Zinkernagel, 1974). Thus, from the standpoint of cell-mediated immunity, the factors governing severity of disease can be viewed as (1) a balance of immune destructive effects of the response and (2) the predisposition to high versus low responsiveness, which is strongly influenced by H-2 -linked Zr genes. These findings offer a teleological explanation for MHC polymorphism and gene duplication within the MHC (Doherty and Zinkernagel, 1975b). T cells are MHC-restricted and recognition of Self-H limits recognition of foreign antigens by T cells. At the level of a species, polymorphism of Self-H-and for the individual gene duplication together with polymorphism-allows the receptor repertoire to increase optimally so as to minimize “holes” in the receptor repertoire and responsiveness. If mice possessed only one lytic MHC restriction structure, e.g., Dktype (a low-responder allele for vaccinia virus, a

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high-responder allele for LCMV), not only the individual, but also the entire species would be in jeopardy from, say, a poxvirus pandemic. Polymorphism diminishes the chance of extinction by (1) eliminating the possibility that viruses could adapt by mutation to mimic the species’ Self-H markers or could escape immune surveillance by other means, (2) restricting H-2-regulated low responsiveness or unresponsiveness to only a few members of a species, and (3) causing heterozygosity, which in combination with duplication of certain H-2 regions gives each individual maximal immune responsiveness and protection . D. MHC-ASSOCIATED DISEASES The performance of MHC-linked Zr genes in the virus models just described could relate to empirical associations found between MHC and susceptibility to disease (reviewed in McDevitt and Bodmer, 1974; Dausset and Svejgaard, 1977). It i s obvious that resistance to infection involves factors other than viruses and environmental influences. For instance, immunologically specific mechanisms, but also nonspecific and nonimmunologic host defense mechanisms, participate. Of course, one must also distinguish disease caused by the infectious agent (sometimes caused by the failure of immune protection) from disease caused not by the infectious agent itself but by the related immune responses. Here we speculate only on the course of disease in terms of the direct relationship between intracellular parasites and T-cell immunity. For the sake of the present argument, we disregard many other possible mechanisms that can directly or indirectly influence disease susceptibility in an MHC-dependent fashion; for example, complement deficiencies, autoantibody, and imune complex diseases are not considered in this proposal but may be explained similarly but less directly. These general concepts of T cell-mediated immunity are subject to Zr-gene regulation and involve recognition of modified cell surfaces followed by target cell destruction and inflammation via recruitment of various ancillary cells. In this context, the following combinations or theoretical categories of disease susceptibilities are logical. ( 1) High responsiveness results in relatively increased resistance to infectious cytopathic virus, but may decrease resistance to poorly cytopathic viruses, since high responsiveness tends to tip the balance of immune protection toward autoaggression via direct cell destruction or chronic inflammatory processes. (2) Low responsiveness results in relatively great susceptibility to acute cytopathic virus infection, a possibility

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that has been eliminated by natural selection by infectious agents. Low responsiveness could however increase the susceptibility to poorly cytopathic virus infection because the wider spread of virus allows immunological host cell destruction to become more extensive. Chronic virus infections have been suspected as the cause of so-called autoimmune diseases for some time. In fact, most of the associations between MHC and susceptibility to disease relate to autoaggressive or autoimmune diseases, not to acute infections. This may simply reflect the state of modem medicine. However, resistance or susceptibility to acute virus infections would tend to be encountered well before reproductive maturity is reached. Natural selection would tend to eliminate these types of low responders as may have occurred extensively with the great interchange among individuals on previously isolated land masses that has characterized the past 500 years of human history. In contrast, MHC-dependent immune responsiveness may vary considerably for poorly or noncytopathic viruses and therefore induce more or less detectable levels of immunologically mediated autoaggression. Since most of the diseases are chronic and rarely interfere with reproduction, such MHC-linked variations may become apparent as MHCassociated diseases (Zinkemagel, 1978a,b).

E. CONCLUSION MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells. In viuo MHC-restricted cytotoxic T cells are critically involved in early antiviral recovery, whereas nonlytic T cells act antivirally or antibacterially via I-mediated macrophage activation. Since MHC products define the effector function, but in parallel also influence the receptor repertoire that can be expressed by T cells, we consider MHC polymorphism and gene duplication to have evolved together with T cells under the selective pressure of intracellular parasites to expand the T-cell receptor repertoire optimally at the level of the population and the individual. It generally seems that the association between MHC and disease susceptibility is obvious for diseases characterized as autoimmune or autoaggressive and may reflect that noncytopathic viruses, which are not life-threatening, induce more or less extensive autoaggression dependent on MHC-linked immune responsiveness. Thus, immune responsiveness determined by limitations of the T-cell receptor repertoire, MHC polymorphism, and MHC-associated diseases are all con-

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sequences of T-cell functions being determined by MHC-coded cellsurface antigens, i.e., because T cells are restricted. VIII. Finale

Why are T cells restricted to the MHC products? We have attempted to deal with this question and analyze it from the point of view of cell-mediated immunity to intracellular parasites. Obviously, when examined from the viewpoint of individual homeostasis, MHC restriction makes sense only when brought into the broader context of possible evolutionary pathways, especially with respect to interactions between components of the “fluid” organ formed by lymphocytes. The sometimes complex and technically difficult experimental manipulations used for analysis are always subject to possible hidden artifacts. Nevertheless, these investigations have revealed fundamental fascinating links in the possible chain of events in lymphocyte differentiation, maturation, interaction, and effector-cell function that were unsuspected a few years ago. An understanding of the biological role of major transplantation antigens has probably been the main result of these efforts. Even at this early stage, only 5 years after the initial discovery of MHC restriction in the virus models, a clear biological basis has been established for the polymorphism and duplication found for H - 2 K and H - 2 D in the mouse, and H L A - A and H L A - B in man.

ACKNOWLEDGMENTS We would like to thank Drs. G. Ada, R. Blanden, B. Benacerraf, M. Bevan, E. Bubbers, S. Burakoff, J. Dausset, J. Klein, U. Koszinowski, R. Langman, J. P. Levy, A. McMichael, M. Rollinghoff, G. Shearer, E. Simpson, H. von Boehmer, and H. Wagner for their critical comments and help in completing this manuscript. We thank Ms. Phyllis Minick and Andrea Rothman for their excellent editorial assistance and Ms. Annette Parson, who typed this manuscript expertly and with great devotion. Part of the work reported was supported by U.S. Public Health Service Grants Al-13779, Al-07007, A1-00273, CA-20833, and Al-14162. This is Publication No. 1702 of the Immunopathology Department, Scripps Clinic and Research Foundation and was completed on March 15, 1979.

ABBREVIATIONSUSED IN T H E TEXT T cell MHC H-2 HLA lr genes B cell

Thymus-derived lymphocytes Major histocompatibility complex Murine major histocompatibility gene complex Human major histocompatibility gene complex Immune response genes Bone-marrow-derived lymphocyte

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Self-H X LCMV LCM TNP i.c. MSV SV40

vsv

DNP i.v. i.p. ts Ig ATXBM VH

.

Self major transplantation antigen Foreign antigen Lymphocytic choriomeningitis virus Lymphocytic choriomeningitis Trinitropheny 1 Intracerebral Murine sarcoma virus Simian virus 40 Vesicular stomatitis virus Dinitrophenyl Intravenous Intraperitoneal Temperature-sensitive Immunoglobulin Adult thymectomized, lethally irradiated, bone marrow reconstituted Hypervariable region of the heavy chain

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mediated immunity after exposure of mice to both live and inactivated rabies virus. Proc. Natl. Acad. Sci. U S A . 74,334-338. Wilson, D. B., Lindahl, K. F., Wilson, D. H., and Sprent, J. (1977). The generation of killer cells to trinitrophenyl-modified allogeneic targets by lymphocyte populations negatively selected to strong alloantigens. J. E x p . Med. 146,361-367. Woan, M. C., Yip, D.-M., and Tompkins, W. A. F. (1978). Autochthonous, allogeneic and xenogeneic cells as targets for vaccinia immune lymphocyte cytotoxicity. J . Immunol. 120,312-316. Wolfe, S . A., Tracey, D. E., and Henney, C. D. (1976). Induction of'hatural killer" cells by BCG. Nature (London)262,584-586. Wong, C. Y., Woodruff, J. J., and Woodruff, J. F. (1977a). Generation of cytotoxic T lymphocytes during coxsackie virus B-3 infection. I. Model and viral specificity. J . Immunol. 118,1159-1164. Wong, C . Y., Woodruff, J. J., and Woodruff, J. F. (197713). Generation of cytotoxic T lymphocytes during coxsackie virus B-3 infection. 11. Characteristics of effector cells and demonstration of cytotoxicity against viral infected my0fibers.J. Immunol. 118, 1165-1169. Wong, C. Y., Woodruff, J. J., and Woodruff, J. F. (1977~).Generation of cytotoxic T lymphocytes during coxsackie virus B-3 infection. 111. Role of sex.J. Immunol. 119, 591-597. Woodruff, J. F., and Woodruff, J. J. (1974). Involvement of T lymphocytes in the pathogenesis of coxsackie virus Bs heart disease. J . Immunol. 113, 1726-1734. Wrathmell, A. B., Gauci, C. L., and Alexander, P. (1976). Cross-reactivity of an alloantigen present on normal cells with the tumour-specific transplantation type antigen of the acute myeloid leukemia (SAL) of rats. Br. Cancer]. 33, 187-194. Wright, P. W., and Herberman, R. B. (1973).Immune response to gross virus-induced lymphoma: Comparison of two in vitro assays of cell-mediated immunity. J . Natl. Cancer Inst. 50,947-956. Wyde, P. R., Couch, R. B., Mackler, B. F., Cate, T. R., and Levy, B. M. (1977). Effects of low- and high-passage influenza virus infection in normal and nude mice. Infect. Immun. 15,221-229. Yap, K. L., and Ada, G. L. (1977). Cytotoxic T cells specific for influenza virus-infected target cells. Immunology 32, 151-160. Yap, K. L., and Ada, G. L. (1978a). Cytotoxic T cells in the lungs of mice infected with an influenza A virus. Scand. J . Immunol. 7,73-80. Yap, K. L., and Ada, G. L. (1978b). The recovery of mice from influenza virus infection: Adoptive transfer of immunity with immune T lymphocytes. Scand. J . Immunol. 7, 389-397. Yap, K. L., and Ada, G. L. (1978~). The recovery of mice from influenza A virus infec tion: Adaptive transfer of immunology with influenza virus-specific cytotoxic T lymphocytes recognizing a common virion antigen. Scand. J . Immunol. 8,413. Yap, K. L., and Ada, G. L., and McKenzie, I. F. C. (1978). Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature (London) 273,238. Zarling, J. M., Nowinski, R. C., and Bach, F. H. (1975). Lysis of leukemia cells by spleen cells of normal mice. Proc. Natl. Acad. Sci. U.S.A. 72,2780-2784. Zarling, D. A., Keshet, I., Watson, A., and Bach, F.(1978).Association of mouse major histocompatibility and Rauscher murine leukemia virus envelope glycoprotein antigens on leukemia cells and their recognition by syngeneic virus-immunecytotoxic lymphocytes. Scand. J . Immunol. 8,497-508.

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Zinkemagel, R. M., and Doherty, P. C. (1974b). Restriction of in uitro T cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature (London)248,701-702. Zinkemagel, R. M., and Doherty, P. C. (1974~).Immunological surveillance against altered self components by sensitized T lymphocytes in lymphocytic choriomeningitis. Nature (London) 251, 547-548. Zinkemagel, R. M., and Doherty, P. C. (1975a).H-2 compatibility requirement for T cell mediated lysis of targets infected with lymphocytic choriomeningitis virus. Different cytotoxic T cell specificities are associated with structures coded in H-2K or H-2D. J . E x p . Med. 141,1427-1436. Zinkemagel, R. M., and Doherty, P. C. (1975b). Peritoneal macrophages on targets for measuring virus-specific T cell-mediated cytotoxicity in uitro.J . Zmmunol. Methods 8,263-266. Zinkemagel, R. M., and Doherty, P. C. (1975~).Cortisone-resistant effector T cells in acute lymphocytic choriomeningitis and Listeria monocytogenes infection of mice. Austr. J . E x p . Biol. Med. Sci. 53,297-303. Zinkemagel, R. M., and Doherty, P. C. (1976a). Does the apparent H-2 compatibility requirement for virus-specific T cell mediated cytolysis reflect T cell specificity for “altered self’ or physiological interaction mechanisms. In “The Role of Products of the Histocompatibility Gene Complex in Immune Responses” (D. H. Katz and B. Benacerraf, eds.), pp. 203-211. Academic Press, New York. Zinkernagel, R. M., and Doherty, P. C. (1976b). The concept that surveillance of self is mediated via the same set of genes that determines recognition of allogenic cells. Cold Spring Harbor Lab. XLI, 505-510. Zinkernagel, R. M., and Doherty, P. C. (1977a). Major transplantation antigens virus and specificity of surveillance T cells. The “altered self’ hypothesis. Contemp. Top. Zmmunobiol. 7, 179-220. Zinkemagel, R. M., and Doherty, P. C. (197713). Possible mechanisms of disease susceptibility association with major transplantation antigens. In “HLA and Disease” (J. Dausset and A. Svejgard, eds.), 1st ed., pp. 256-268. Munksgaard, Copenhagen. Zinkernagel, R. M., and Klein, J. (1.977). H-2 associated specificity of virus-immune cytotoxic T cells from H-2 mutant and wild-type mice: M523 (H-2Kka)and M505 (H-2KW)do, M504 (H-2Dds) and M506 (H-2Kfa)do not cross-react with wild-type H-2K or H-2D. Zmmunogenetics 4,581-590. Zinkernagel, R. M., and Oldstone, M. B. A. (1976). Cells that express viral antigens but lack H-2 determinants are not lysed by immune T cells but are lysed by other anti-viral immune attack mechanisms. Proc. Natl. Acad. S c i . U.S.A. 73,3666-3670. Zinkemagel, R. M., and Welsh, R. M. (1976). H-2 compatibility requirement for virusspecific T cell-mediated effector functions in uioo. I. Specificity ofT cells conferring antiviral protection against lymphocytic choriomeningitis virus is associated with H-2K and H-2D.J Zmmunol. 117,1495-1502. Zinkernagel, R. M., Dunlop, M. B. C., and Doherty, P. C. (1975). Cytotoxic T cell activity is strain-specific in outbred mice infected with lymphocytic choriomeningitis virus. J. Zmmunol. 115, 1613-1616. Zinkemagel, R. M., Dunlop, M. B. C., Blanden, R. V., Doherty, P. C., and Shreffler, D. C. (1976). H-2 compatibility requirement for virus-specific T cell-mediated cytolysis. Evaluation of the role of H-21 region and non H-2 genes in regulating immune response. J . Erp. Med. 144, 519. Zinkemagel, R. M., Althage, A., Adler, B., Blanden, R. V., Davidson, W. F., Kees, U., Dunlop, M. B. C., and Shreffler, D. C. (1977a). H-2 restriction of cell-mediated

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immunity to an intracellular bacterium. Effector T cells are specific for Listeria antigen in association with H-21 region coded self-markers. J. Exp. Med. 145, 1353-1367. Zinkernagel, R. M., Callahan, G. N., Streilein, J. W., and Klein, J. (1977b). Neonatally tolerant mice fail to react against virus-infected tolerated cells. Nature (London) 266, 837. Zinkemagel, R. M.. Althage, A., and Jensen, F. C. (1977~).Cell-mediated immune response to lymphocytic choriomeningitis and vaccinia virus in rats. J. Zmmunol. 119,1242-1247. Zinkernagel, R. M., Adler, B., and Holland, J. (1977d). Cell-mediated immunity to vesicular stomatitis virus infections in mice. Exp. Cell Biol. 46, 53. Zinkernagel, R. M., Adler, B., and Althage, A. (1977e). The question of derepression of H-2 specificities in virus-infected cells: Failure to detect specific alloreactive T cells after systemic virus infection or alloantigens detectable by alloreactive T cells on virus infected target cells. Imrnunogenetics 5,367-378. Zinkemagel, R. M., Callahan, G. N., Klein, J., and Dennert, G. (1978a). CytotoxicT cells learn specificity for self H - 2 during differentiation in the thymus. Nature (London) 271,251-253. Zinkemagel, R. M., Callahan, G. N., Althage, A., Cooper, J., Klein, P. A., and Klein, J. (197813). On the thymus in the differentiation of “H-2 self-recognition” by T cells: Evidence for dual recognition?]. Exp. Med. 147, 882-896. Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Streilein, J. W., and Klein, J. (19784. The lymphoreticular system in triggering virus-plus-self-specific cytotoxic T cells: Evidence for T he1p.J. Exp. Med. 147, 897-911. Zinkemagel, R. M., Althage, A., Cooper, S., Kreeb, G., Klein, P. A., Sefion, B., Flaherty, L., Stimpfling, J., Shreffler, D., and Klein, J. (1978d).Zr genes in H - 2 regulate generation of antiviral cytotoxic T cells: Mapping to K or D and dominance of unresponsiveness. J . Exp. Med. 148, 592. Zinkemagel, R. M., Althage, A., Jensen, F., Streilein, J. W., and Duncan, W. R. (1978e). Cell-mediated immunity to viruses in hamsters. Fed. Proc. 37, 2078-2081. Zinkernagel, R. M., Althage, A., and Holland, J. J. (19780. Target antigens for H-2 restricted vesicular stomatitis virus-specific cytotoxic T cells. J. Zmmunol. 121, 744-748. Zinkemagel, R. M., Althage, A., Cooper, S., Callahan, G. N., and Klein, J. (19788). In irradiation chimeras, K or D regions of the chimeric host, not of the donor lymphocytes determine immune responsiveness of antiviral cytotoxic T cells.]. E x p . Med. 148, 805-810. Zinkemagel, R. M., Klein, P., and Klein J. (1978h). Host-determined T cell finespecificity for self-H-2 in radiation bone marrow chimeras of standard C57BU6 (H-zb),mutant Hzl (H-2ba),and F, mice. Zmmunogenetics 7, 73. Zinkemagel, R. M., Althage, A,, Waterfield, E. M., and Pincetl, P. (1979). Two stages of H-2 dependent T cell maturation. Proc. Symp. Cell Lineage Stem Cell Detennination, Seillac, France, May 20-24, 1979. (in press). Zweerink, H. J., Courtneidge, S. A., Skehel, J. J., Crumpton, M. J., and Askonas, B. A. (1977a). Cytotoxic T cells kill influence virus-infected cells but do not distinguish between serologically distinct Type A viruses. Nature (London) 267,354-356. Zweerink, H. J., Askonas, B. A., Millican, D., Courtneidge, S. A., and Skehel, J. J. (197%). Cytotoxic T cells to type A influenza virus; viral hemagglutinin induces A-strain specificity while infected cells confer cross-reactive cytotoxicity. Eur. J . Immunol. 7,630-635.

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ADVANCES IN IMMUNOLOGY. VOL 27

Murine lymphocyte Surface Antigens I A N F. C . MCKENZIE A N D TERRY POTTER Department of Medicine. University of Mefboume. Austin Hospital. Heidelberg. Victoria. Australia

I . Introduction .......................................................... I1. Classification of Alloantigenic Determinants ............................ I11. Production and Testing of Antisera ..................................... A. Production of Alloantisera .......................................... B . Methods of Detection .............................................. C . Contaminating Antibodies in Antisera ............................... IV. Characterization of Antisera ............................................ A . Genetic Analysis ....... ....................................... B . Tissue Distribution ..... ....................................... C . Functional Characterization ........................................ D . Further Characterization ........................................... V. Histocompatibility ( H )Loci-CMAD of General Distribution ............ A . H-2 CMAD (H-2K. H-2D. H-2G. H-2L) .............................. B . Non-H-2 Histocompatibility Loci ................................... C . Hh-1 .............................................................. VI. Lymphocyte Alloantigens . . ....................................... A . The Thy-1 L o c u s . , ..... ....................................... B. The Tla Locus .................................................... C . The QQ-1 Locus ................................................... D . The Qa-2 and Qa-3 Loci ........................................... E . The Ly-1 Locus (Lyt-1) ............................................ F. The Ly-2 and Ly-3 Loci ............................................ G. The Ly-4 Locus (Lyb-1) ........................................... H . The Ly-5 (Lyt-4) Locus ............................................ I . The Ly-6 Locus ................................................ J . TheALA-1 Locus .................... ......................... K. The Ly-7 Locus ................................................... L . The Ly-8 Locus ................................................... M . The LyM-1 Locus ................................................. N . Ly-b Specificities ................................................. 0. Qat-4. Qat-5 ...................................................... P. l a Loci .......................... ............................ Q . Other Specificities . . . . . . . . . . . . . . . ............................ R . Chemistry ofCMAD .............................................. VII . Erythrocyte Alloantigenic (EQ)Loci .................................... VIII . Miscellaneous Antigens .............. .............................. e-1) Locus .................. ................. B . F Antigen . . ..................................................

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D . The T Complex . . . ............. E . The H-Y Locus ................... ............. F. The N K Specificity ................................................ G . Ly-X Loci ........................ .......................

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IX. Xenoantisera Recognizing Lymphocyte Cell-Membrane Determinants .... A . Antilymphocyte Sera (ALS) ........................................ B. Mouse-Specific Lymphocyte Antigen (MSLA) ....................... C . Mouse Thymus-Derived Lymphocyte-Specific Surface Antigen (MTLA).................................................. D . Brain-Associated Theta (BAB) ...................................... E . Mouse-Specific Peripheral Lymphocyte Antigen (MPLA) ............ F. Xenoantisera-Detecting Determinants Present on Killer T Cells ........................................................... G. Antisera to Purified T-cell Populations ............................. H . Thymocyte-B Lymphocyte Antigen (Th-B) .......................... I . Mouse-Specific Bone Marrow-Derived Lymphocyte Antigen (MBLA) .................................................. J . Other Sera Detecting B-Cell Xenoantigenic Specificities ............. K. Mouse-Specific Plasma Cell Antigen (MSPCA) ...................... L . ML-2 Antigen ..................................................... M. Antibodies to the LPS Receptor on B Cells ......................... N . Xenogeneic Anti-Ia Serum ......................................... X. Relationship of Murine Leukemia Virus (MuLV) and CMAD ............ A . The Expression of Viral Antigens ................................... B. Classification of MuLV ............................................. C . Antigens Induced by Virus .......................................... D . Structural Viral Components as CMAD ............................. E . Virus-Related Antigens ............................................ F. Expression of Viral Antigens after Lymphocyte Activation ............ C. Conclusion ....................................................... XI . Functional Studies with Serological Markers ........................... A . Cytotoxic T Cells (TK)............................................. B. Phenotype of Helper T Cells (TH)for Antibody Production ........... C . Phenotype of T Cells Involved in Antibody Suppression (T,) ......... D . Phenotype of T Cells Involved in the Suppression of Cell-Mediated Responses .......................................... E . Phenotype of T Cells Involved in Delayed-Type Hypersensitivity (DTH)............................................ F. The Mixed-Lymphocyte Reaction (MLR) ............................ G . Host-versus-Graft and Graft-versus-Host (GvH) Reactions ............ H . Phenotype of Cells Undergoing Blast-Cell Transformation ............ I . Phenotype of Cells Involved in MIF Production ..................... J . The Ly Phenotype of T Cells in the Production of Eosinophilia ...................................................... K . Summary of Functional Data ....................................... XI1. CMAD in Studies of T-cell Ontogeny and Differentiation ............... A . Ontogeny ......................................................... B. The Prothymocyte ................................................. C . Differentiation in the Thymus ...................................... D . Differentiation in the Periphery .................................... E . Other CMAD in T-cell Differentiation .............................. F. Summary ......................................................... XI11. CMAD in B-Cell Differentiation and Ontogeny ......................... A . Stem Cells: B Stem Cell. the Immature B Cell ...................... B. CMAD of B Cells .................................................

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MURINE CELL-SURFACE ANTIGENS C. CMAD of Antibody-Forming Cells ................................. D. Ontogeny of CMAD and Receptors on B Cells . . . . . . . . . XIV. Expression of CMAD on Mouse Leukemias and Lymphomas A. Chemically Induced T Lymphomas.. ............................... B. In Vitro-Maintained T-cell Lines .................................. C. Other T-cell Lymphomas . .... .................... D . Radiation-Induced T Thym .... .................... E. Virus-Induced Leukemias and Lymphomas ......................... F. Phenotype of Abelson Virus-Induced B-Cell Tumors ................. G. Plasmacytomas and Other B-Cell Tumors ........................... H. Mastocytomas ..................................................... I. Other Tumors ..................................................... J. Conclusion ........................................................ XV. Conclusion ......................... .............................. Abbreviations Used in the Text ........................................ References ........................................................... Note Added in P r o o f . . ................................................

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I. Introduction

In the mouse, at least 40 different loci have been described that code for lymphocyte cell membrane alloantigenic determinants (CMAD) (Snell et al., 1976). There is, therefore, a relatively large degree of structural variation in an apparently homogeneous lymphocyte population, and one of the recent exciting advances in this field has been the demonstration that the CMAD may be used to distinguish between different functional subpopulations of lymphocytes. This was first noted when T cells and B cells could be distinguished on the basis of the presence of the Thy-1 specificity and of surface immunoglobulin (1g)-T cells being defined as Thy-1+, Ig-; and B cells, as Thy-1-, Ig+ (Reif and Allen, 1964; Raff, 1969). More recently, this distinction has been extended so that now subsets of both T cells and B cells can be classified and defined in terms of their function and their cell-surface phenotype. This subdivision has proved to be of value in studies of normal lymphocyte function, ontogeny, and differentiation and in the examination of lymphoid tumors. Although many loci have been described, it is likely that the genetic variations found thus far represent only a small proportion of the different cell membrane componentsfor there are more genetic polymorphisms to be unraveled, newer and more sensitive techniques to be used, and a wider range of functional assays to be applied. Furthermore, although immunologists are primarily concerned with lymphocytes, there are likely to be extensive polymorphisms of other cell types involved in immunological reactions such as macrophages, granulocytes, and other leukocytes. The potential for variation of the lymphocyte surface is extremely large, particularly if one considers the diverse functions of lymphocytes.

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The major aim of this review is to describe genetic polymorphisms that lead to structural variations in the cell surface. These variations lead to differences between strains which subsequently elicit the production of antibody or graft rejection, so that the CMAD may be detected. In addition lymphocyte heterogeneity will be described, i.e., variation determined by the presence or the absence of a particular CMAD within an individual, as detected serologically or by the ablation of a defined function. The review will comprehensively discuss most of the CMAD known in the mouse at this time. As many of these are related to histocompatibility antigens, these will be briefly discussed (Section V). As many antisera also contain antibodies to red cell alloantigens and other miscellaneous antigens, these are also briefly reviewed (Sections VII and VIII). Also, as some specificities detected by xenoantisera may reside on the same, or closely related, molecules as the alloantigenic determinants, a chapter on these has been included (Section IX). However, the major theme of the review deals with the description of the structure and function of the Ly antigens in mice (Sections VI, XI, and XIII). Similarly, we do not discuss in detail other lymphocyte surface molecules, such as immunoglobulin (Ig), Fc receptors, or the complement receptors-these have been extensively reviewed elsewhere (Moller, 1973; Warner, 1974; Nussenzweig, 1974). Cell-surface markers found in other species have recently been presented in detail (Gotze, 1977; Chess and Schlossman, 1977; Gasser, 1977). Other extensive reviews of CMAD are those of Boyse and Old (1969), Snell et al. (1976), Beverley (1977), Simpson and Beverley (1977), Snell (1978). II. Classification of Alloantigenic Determinants

A classification of CMAD is presented in Table I. Basically the classification follows traditional lines (Snell et al., 1976), the major subdivisions being as follows: (1) histocompatibility (H) loci, defined by histogenic methods; (2) the lymphocyte and other loci, the majority defined by serological methods (cytotoxicity or immunofluorescence); and (3) the red cell (Ea) loci, which have been defined by hemagglutination. However, we prefer a broader classification of CMAD into those of general distribution, i.e., CMAD found on all cells, and those of restricted distribution, i.e., whose distribution is restricted to one or two cell types. It should be pointed out that these groups are by no means exclusive, and that some overlap occurs. For example, the H loci were traditionally described by the rejection of skin grafts, which demonstrated that these particular CMAD were present on skin tis-

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sues. However, with the passage of time, the H designation has also implied that the specificities have a generalized distribution (with the possible exception of mature red cells), as measured by the ability of different tissues to immunize or tolerize for a subsequent skin graft (Snell et al., 1976). However, extensive tissue testing has not been done for all the H loci thus far described, and it is possible that some of these belong in the second group, where there is a restricted distribution of the CMAD. An example of this is the Sk-1 locus, which is present on skin tissues, but not on lymphoid tissues. Furthermore, to offer the label of H locus only to antigens of general distribution, characterized by skin graft rejection, is unnecessarily restrictive and excludes allograft rejection reactions directed at tissue-specific antigens, such as those confined to thymus, bone marrow, platelets, kidney, or other grafted tissue. For example, Thy-1.2 thymus cells injected into Thy-1.1 mice are ultimately rejected, the mode of rejection satisfying the criteria for the definition of an H locus with regard to specificity, heightened rejection of a second graft (memory), and the ability of the response to be adoptively transferred to syngeneic recipients by lymphocytes. We would therefore suggest that any CMAD can function as a histocompatibility antigen in the appropriate setting, and have accordingly set out the classification shown in Table I. This point is not merely of academic interest, for transplantation of thymus, bone marrow, and other tissues in man involves consideration not only of HLA and non-HLA histocompatibilities (i.e., Group A in Table I), but also of tissue-specific incompatibilities of the type listed under Group B, such as kidney-specific antigens and the ABO antigens. The determination of the nomenclature for new loci has recently been discussed in detail (Snell, 1977; Lyon, 1977). However, CMAD of restricted distribution are posing a nomenclature problem. The fist such locus described was 8, with two alleles and specificities: 8-C3H and 8-AKR(Reif and Allen, 1964). This was changed to Thy-l on the basis of the specificities being found only on the thymus. However, these specificities are now known to be present on extrathymic T cells, brain, and possibly skin, so that as a descriptive term Thy-L is inappropriate, but is accepted by current usage. Later, the Ly-L,-2,-3 loci were described and were designated for antigens with a distribution restricted to lymphocytes. As specificities of apparently lymphocyterestricted distribution are described, additional Ly numbers have been added-up to Ly-8 thus far. Boyse et al. (1977) attempted to clarify the Ly nomenclature by using “t” and “b” prefixes (e.g., Lyt-1, Lyb-1) indicating the T- or B-cell distribution of the specificities (Table 11), but this nomenclature has not yet received widespread accep-

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TABLE I CLASSIFICATION OF CELLMEMBRANEALLOANTIGENIC DETERMINANTS (CMAD) A. Loci Coding for CMAD of General Tissue Distribution ( H Loci) I. MHC: Major histocompatibility complex (H-2) a. Defined by recombination: H-SK, H-SZA, H-SIC, H-211, H - X , H-2D b. Defined by mutation: (i) C57BL/6H-2Kbmutants (21locus); (ii) H-2L; (iii) not mapped but defmed by mutants H-2h, H-BbD,H-2dc,H-2dd 11. Non-H-2 histocompatibility loci a. Defined by congenic lines: H-1, H-3, H 4 , H-7 to H-12, H-39 b. Defined by recombinant inbred lines (Bailey): H-1 to H-38 (excluding H-4, H-5, H-6, H-9-14, H-3133) c. Defined in non-H congenic strains H(Tla):H-31, H-32 H(Ly-l ),H(Ly-2-N8),H(Ly-2-N16),H(Ly-2, Ly-3) H (Ea-2 ) ? Hh-1 d. Defined by mutants (i) detected by skin grafts; (ii) associated with visible markers, e.g., H(Eh), H(ep),H k o ) e. Sex chromosome linked: H-X, H-Y B. Loci Coding f or CMAD of Restricted Tissue Distribution I. Loci coding for alloantigens present on lymphoid and related cells a. T cells: Tla, Thy-1, Ly-1 (Lyt-1),Ly-2, (Lyt-2)Ly-3, (Lyt-3),Ly-5, ( L y t 4 ) ,Q a - I , Qa-2, Qa-3, GIX, ZA (ICIE),ZJ, Qat-4, Qat-5 b. B cells: Lyb-1, ( L y 4 ) ,Lyb-2, Lyb-3, L yb4, L ybd , Lyb-6, Lyb-7, LyM-1, Pc-I, l a (ZA, ZCIE) c. Both T and B cells: Ly-6, Ly-7, L y 8 , ALA-1 d. Not assigned: Mph-1, DAG, N K , Pgm-1, Ly-X 11. Red cell Ea-1, Ea-2, Ea-3, E a 4 , Ea-5,Ea-6, Ea-7 111. Miscellaneous Sk-1, Sk-24 F, Hh-I, H-Y, Tlt IV. Virus-related antigens Pca-1, X-1,Gx,GRADAI. GERLD, MuLV-A a

Sk may change to Skn.

tance, as some of the Ly antigens do not fit into this classification, (e.g., Ly-7 and Ly-8) and some antigens initially considered to be restricted to T (Ly-5 = Lyt-4) or B (Ly-4 = Lyb-1) cells, may have a broader tissue distribution. However, it would be helpful to have a nomenclature giving some indication of the differences between the Ly specificities. In Table I, the CMAD of general distribution (Group A) have been divided into those found within the H-2 complex, the MHC of the mouse, and into “non-H-2” loci. Within this general definition, the loci coding for the Ia specificities fit into Group Byas these are of restricted

185

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TABLE I1 NOMENCLATUREIN Original description ~

USE FOR SOME OF THE

Currently used nomenclature

Suggested nomenclature'

Thy-1 Ly-1 Ly-2 Ly-3 LY4 Ly-5 Ly-6 Ly-7 LY-8

Thy-1 Lyt-1 L4t-2 Lyt-3 Lyb-1 Ly t-4

~~

0 Ly-A, mu Ly-B Ly-c

-

~ _ _ _ _ _ _ _ _ _

a

LY ALLOANTICENIC LOCI

-

Lyb-2, b-3, b-4, b-5, b-6, b-7

~

Boyse et al. (1977).

distribution. In Group A, the H loci, defined by graft rejection and/or serologically, can be divided into those defined by recombination (H-2K7H-2ZA, H-2ZJ, H-2ZC7H-2G7H-2D) and those defined by mutation. In all there may be up to 12 of these H loci within the H-2 complex, although some of the loci defined by mutation may be identical with those defined by recombination. The site of the H-2bm,H-2b, H-2dc, and H-2dd mutations have not yet been mapped with H-2 (McKenzie et al., 1977c; Klein, 1978a). The minor, non-H-2 loci have been defined by the use of conventional congenic lines, as well as the recombinant inbred strains of Bailey. Non-H-2 loci have also been defined by mutation (Melvold and Kohn, 1976). Of particular relevance to this review are the H loci that have been defined using the Ly congenic strains of mice, when it was found that the congenic strain and its partner reciprocally rejected skin grafts. It is not completely clear in these cases whether the H locus is identical, or closely linked, to the Ly locus in question, or whether it has been included, by chance, in the production of the congenic strain. Other H loci have been described in mice with dominant or recessive markers (Bailey, 1971), and there are also the sex chromosome H loci. In Group B, loci have been classified as being present on lymphoid or related cells, red cells, and other tissues (miscellaneous), and in addition there are loci that can be classified as being virus related. One area where the classification is incomplete involves tissue distribution, which has not been defined for all the specificities.

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TABLE I11 PROBLEMS ASSOCIATEDWITH ALLOANTISERUMPRODUCTION AND TESTING" Problems

Comments

1. Selection of immunization schedule a. Cell type:, whole cells, purified cells, membranes, subcellular fractions

a. Usually 5 x 108 to 5 x 108 whole cells ip weekly x 6 (3first injection sc), then bleed and immunize weekly b. Adjuvants: Freunds, BCG, Corynebac- b. Adjuvants of little value terium parvum, Bordetella pertussis c. Frequency of immunization and c. Some sera (Thy-1.2) are IgM and earlier bleeds possible, 1 or 2 injections bleeding also suitable for anti-Ia antibodies

2. Selection of donwhecipient combination a. Contaminating antibodies a. Use tables to select appropriate combinations and use congenic strains if possible b. Some genetic backgrounds give poor b. F1 hybrid female usually best responantibodies due to Ir gene effects and Ig ders. class of antibody c. Congenic pairs often give poor anti- c. Many trials may be needed for the best body response combination. 3. Individual variations Inbred mice and F1recipients vary greatly in the magnitude and specificity of antibody response to CMAD 4. Weak antisera Weak antibodies to well defined CMAD, especially in congenic lines 5. Handling mouse sera Bleeding, storage, stability, lyophilization

Individual bleeds with selection of good responders; transfer cells to irradiated recipients; manipulation to remove suppressor cells (3) Change combination, 7 use adjuvants, ? use helper effects of H-2 or TNP Sera usually very unstable at room temperature and 4"; store at -70"; avoid repeated freeze-thaw and storing in diluted form, some are mostly IgM and deteriorate rapidly with storage and lyophilization

6. Problems in testing

a. Autoantibodies b. Antiviral antibody (especially when testing on tumors)

a. Absorb with tissue of immunized recipient b. Absorb with tissues expressing viral determinants; with virus, or with tumors, antisera are mostly IgM, so use IgG-dependent assay, e.g., protein A-RFC

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TABLE 111 (Conttnued) Problems

Comments ~~

c. Aggregates and immune complexes in sera d. Multispecificity of sera

e. Noncomplement-fixing antibodies

f. Complement (C’) in cytotoxic tests i. Rabbit serum usually essential, but is toxic ii. Anticomplementary factors in mouse sera g. Target cell problems: Tissues vary in density and content of CMAD; i.e., strains differ in “expression’ of some CMAD,e.g., Ly-4, Ly-6, Ly-7, Ia h. Extrapolation: Small-scale experiments may not always be able to be scaled up for large-scale depletions

c. Absorb sera with platelets; ultracentrifuge or microaerofuge 15-45 minutes d. Absorb sera before testing; used selected target strain and tissue; used congenic strains for production testing or for absorption e. Use different technique, e.g., fluorescence, rosetting; use cells with Ig removed by capping i. Select rabbits for high C‘ and low toxicity, absorb with divalent cations removed ii. Lyophilize, use 2-3-stage tests if sera are anticomplementary g. Select best target (? tumor) and best target strain h. For large experiments, test runs are essential with sera used at “plateau” levels of lysis; repeat serum and C’ treatment may be required

7. Results a. False positive: Autoantibody, antiviral antibody; Fc binding of aggregates, toxic C‘ b. False negative: C’, weak, nonfixing antibody, weak antisera, loss due to repeated freeze-thaw, sera anticomplementary

-

See also Shen et al. (1975).

Furthermore, some of the specificities may well be virus related, as is the case for Pca-1. Other problems in the classification, such as duplication, have recently become apparent; for instance, it is likely that the Ly-6,ALA-1, DAG, Ren-1, and Ly-8 loci are identical (Section VI). 111. Production and Testing of Antisero

In this section the methods used in the production and serological testing of alloantisera and some of the problems encountered will be

188

IAN F. C. MCKENZIE AND TERRY POTTER

described. An extensive summary of these problems is given in Table 111, and other details have been given by Shen et al. (1975). The principal aim in production is to raise antisera that are of high titer and monospecific. However, in practice few of the currently produced alloantisera satisfy these aims. Before discussing the practical difficulties in the production and use of antisera, several concepts will be emphasized. First, the genes thus far described that code for CMAD have been described by either histogenic or serological methods, i.e., skin grafting, cytotoxicity, and hemagglutination. The use of newer, more sensitive, techniques, such as the fluorescence-activated cell sorter (FACS) and the production of monoclonal antibodies using hybridomas, may well identify many new systems. Second, antisera should be assumed to be complex unless certain criteria for monospecificity have been satisfied. In this context, it should not be forgotten that the number of specificities that can potentially be defined by any antiserum is a function of the number of strains tested (Snell and Stimpfling, 1966). Third, a basic point, the serological characterization of an antiserum involves both direct testing and absorption. Finally, where possible, congenic strains of mice should be used for direct testing, absorption, and functional tests.

A. PRODUCTION OF

ALLOANTISERA

In general, multiple immunizations (6-10) of lymphoid tissue are required to produce anti-CMAD antibody. It has been found that females give better responses than do males, and that an F1 hybrid usually gives a better response than does the homozygous recipient.

I. Selection of DonorlRecipient Combinations Ideally, antisera raised between Ly congenic pairs should be the most specific, but with the exceptions of Thy-1 and several Ly specificities, such immunizations have not been successful, probably because a single determinant is an insufficient stimulus for antibody formation. It has recently been found that additional antigenic differences at the cell surface induce greater T-cell help by the “associative recognition” phenomenon and lead to greater antibody formation (Lake and Douglas, 1978). A list of the currently available congenic lines is given in Table IV. The combinations used originally for Ly serum production and those selected as the best currently available for production of Ly antisera are presented in Table V. It should be emphasized that for many antisera it is advantageous to use an F1as the recipient, possibly to overcome the effects of Zr genes, which lead to nonresponsiveness (Lilly et al., 1973; McKenzie, 1975b; Shinohara et

al., 1978).

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TABLE IV CONGENIC C TRAINS AVAILABLE OR IN PRODUCTION FOR CMAD' Locus

Congenic line

Donor of allele

Stage of developmentv

Thy-1

A.Thy-1 a A.ALThy-1 B6.PL(74NS) B6.Tlaa A.Tlab B6.TlaC B6.Lyt-1.1 B6.Lyl a B6.Lyt-2.1 B6.Lyt-2.1, Lyt-3.1 B6.PL(75NS) BIO.LPa BIO.C-H-3' BlO.C(Z8NX) B6.Ly-Sb C3H.B6 B6.C-H-2 B6.Lyb-2.1 B6.Gx3 129.GxBALBlc.PcBG.Pc+ B6.Ea-2" BlO.Ea-2=,Ea-7' BIO.L C3H.EaAb C3HIBi.H-Ga B10.129 (5M)

AUN A.SW PUJ A C57BU6 BALBlc C3H Mixed C3H RF PL LP BALBlc BALBlc SJL C57BU6 BALBlc IlSt 129 C57BU6 C57BW6 BALBlc RFM RIII C57L C57BU10 C3HflAn 129

Available, Boy Available, Sf Available, Sn Available, Boy Available, Boy In preparation, Boy Available, Boy Available, Sn Available, Boy Available Boy Available, Sn Available, Sn Available, Sn Available Sn In preparation, (N13) Boy Available, Cherry In preparation, (N6) McK In preparation, (N7) Boy Available, Boy Available, Boy In preparation, (N10) Boy In preparation, Boy Available, Boy Available, Sn Available, Eg Available, Sf Available, Li Available, Sn

Tla Ly-1 Ly-213 LY-4 Ly-5 LY-6' Ly-7 Lyb-2 GV-12

Pc-1

Ea-2 Ea-3 Ea-4 Ea-6 Ea-7

Histocompatibility loci: congenic strains up to H-30, see Klein (1973); H-2 congenic stocks, see J. Klein (1975). Boy, Boyse; Eg, Egorov; Li, Lilly; McK, McKenzie; Sf, Shreffler; Sn, Snell. 'The congenic lines in preparation for DAG (C57BU6 allele onto a BALBlc background-J. Sachs) and Ren-1 (C57BW6 allele onto an A background-P. Halloran) will also be congenic at Ly-6, if these specificities prove to be the same.

2 . Immunization Protocols In general, the optimal immunization schedule for each specificity has not been individually determined but is usually based on regimens initially developed for raising antibodies to H-2 specificities. A commonly used schedule consists of an initial 6-10 intraperitoneal injections of lymphoid tissue (1 donor per 10-20 recipients) followed by bleeding and further immunization on alternate weeks for up to 3-6

TABLE V STRAIN COMBINATIONS FOR PRODUCTION OF

Antiserum

Additional loci at which donor and recipient differ

Specificity

Thy-1

Thy-1.1

C3Ht

AKR

NC

4000 T

A.AL.Th y-1

Thy-1.2

(BIO.A X A.AL)F, (C57BL/6 x A)F, AKRt

A.Thy-1 a C3H

C C NC

2000 T 2000 T 5000T

C57BL/6 ASL1*

Ly-1.1

(B6.PL(74NS)x RF)F, (A.Thy-la X AKR.H-2')Fl' C57BU6t

SL2*

C C NC

2000 T 2000 T 1qGPC)T

B6Ly-1 B6.Lyt-1.1 ELI*

C C NC

500T 1000 T

Ly- 1.2

(C57BL/10 x LP.R111)F1 (C57BL/6 x BALB/c)F, DBAI2f

Ly-2.1

C3WAn C57BU6t

CE K36*

NC NC

250 T 100(GPC)T

B1O.BR

CE

NC

500 T

(B1O.AKM x 129)F, (C3H X I)Flt

B6. PL(75NS ) ELI*

C NC

64T 50(GPC)T

(C3H x BDP)Fl

B1O.BR

NC

250 T

(C3WAn X B6.Ly-2.1)Fl

C57BW6 or ERLD* C58 C58

C

1000 T

Ly-1, Ly-3, Ly-6, Lyb-2, Lyb4, Lyb-5, Lyb-6, Lyb-7, ALA-I, LyM-1 Nil Nil Ly-1, Ly-3, Ly-6, Lyb-2, Lyb4, Lyb-5, Lyb-7, ALA-I, LyM-1 Nil Nil H-2, Tla, Ly-2, Ly-4, Ly-7, Lyb-2, Lyb-4, Lyb-5, Lyb-7 Nil Nil H-2, Tla,Ly-2, Ly4, Ly-7, Lyb-2, Lyb4, Lyb-5,Lyb-7 Lyb-2, Lyb-4 H-2, Thy-1, Ly-3, Ly-4, Ly-7, Lyb-2, LyM-1 Ly4, Ly-6, Ly-7, Lyb-2, Lyb-5, Lyb-7, LyM-1 Nil H-2, Ly-I, Ly4, Ly-6, Ly-7, Ly8, Lyb4, Lyb-5, Lyb-7, LyM-I, ALA-1 Ly4, LyS, Lyb-4, Lyb-5, Lyb-7, LyM-1 Nil

NC NC

128 T 128 T

Ly-7 Ly-7, Lyb-2

Ly-2

Ly-2.2

Ly-3

Ly-3.1

(CBA x SJL)F,I (C3H x SJL)FI

Donor"

Titer-' and tissue'

Locus

Ly-1

RecipienP

Congenic or not2

Ly ANTISEM=

100(GPC)T

CE A B10.D2 A.SW A.SW SJL SJL C3H CXBD CXBE AKR CXBK C3H

NC NC NC NC NC NC NC C NC NC NC NC NC

250 T 64s 128 S 250 T 250 T 500 T

Ly-6

Ly-6.1 Ly-6.2

Ly-7 LY-8

Ly-7.2 Ly-8.1

C58t B1O.At (BALBIc x SWR)F,t SJLt (DA x SJL)F, (B6H-2’ X A.SW)F,t (SWR x A.SW)F, (C3H.B6 x C57BL/6)Flt (BALBIc x A)F,t (CXBG x A)F, (CBA X A.Thy-1 ‘)Fi (B6.C-H-2dx CXBG)F,t AKRt

Ly-8.2

C3H1

AKR

NC

160 LN

Lyb-2.1

I.29* I.29* A.SW CE BALBIc L1210*

NC NC NC NC NC NC

640 s

LY-4 Ly-5

Ly-3.2 Ly-4.1 Ly-4.2 Ly-5.1 Ly-5.2

500T

32 LN 250 LN 250 LN 250 LN 250 s ? LN

Lyb-3 Lyb-4

Lyb-4.1

C3Ht (C3H.I x C57BU6)F1 (SJL X CE)Fif (C3HIAN x BALB/c)F,t (CBNN P x BALB/cd)F,d t C57BLIKst

Lyb5

Lyb-5.1

C57BL/6t

DBAI2

NC

12 s

Lyb-5.2

DBAI2t

C57BLI6

NC

n.t. S

Lyb-6 Lyb-7

Lyb-7.1

CBAINt C57BLI6t

CBA/J DBA/2

NC NC

n.t. S n.t. S

LyM-1

LyM-1.2

CBAIJ

NC

16 S

Lyb-2

Lyb-2.2 Lyb-2.3

640 S

640 s

640 S 40 S 960s

Ly-6, Ly-7, Lyb-2, ALA-1 Ly-6, Ly-7, Ly-8, LyM-I, ALA-1 Ly-7 Ly-6, Ly-8, Lyb-2, ALA-1 Ly-6, Ly-8, Lyb-2, ALA-1 Lyb-2 Lyb-2 Nil Ly-7 Nil Ly-3, Ly-8, ALA-1 Nil Thy-1, Ly-1, Ly-3, Ly-6, Lyb-2, Lyb-4, Lyb-5, Lyb-7, LyM-I, ALA-1 Thy-1, &-I, Ly-3, Ly-6, Lyb-2, Lyb-4, Lyb-5, Lyb-7, LyM-I, ALA-1 ? ? Nil Nil Nil Ly-I, Ly-2, Ly-4, Ly-7, Lyb-2, Lyb-5, Lyb-7 H-2, Tla, Ly-1, Ly-2, Ly-4, Ly-7, Lyb-2, Lyb-4, Lyb-7 H-2, Tla, Ly-1, Ly-2, Ly-4, Ly-7, Lyb-2, L.gb-4, Lyb-7 Nil H-2, Tla, Ly-1, Ly-2, Ly-4, Ly-7, Lyb-2, Lyb-9, Lyb-5, Lyb-7 Lyb-2, Lyb-5, Lyb-7

References are given in the text: in addition see Shen et al. (1975). Dagger (f ) indicates original combination. Asterisk (*) indicates tumors: ASLl-AIJ, SL2-DBAI2, EL/4C57BL/6, ERLD-C57BL/6, I. 29.-IISt, K36-AKR, L1210-DBAI2 C, congenic; NC, non-congenic; GPC, guinea pig complement; T, thymus; S, spleen; LN, lymph node; n.t., not tested.

192

IAN F. C. MCKENZIE AND TERRY POTTER

months. However, to raise antibodies to some specificities (e.g., Thy1.2) only 1or 2 injections are required. Although they have been quite successful for protein antigens, we have found that adjuvants such as Freund’s, BCG, or Corynebacterium paruum do not enhance the production of antibodies to CMAD. A further complication in the production of alloantisera is that individual mice vary greatly in the magnitude and specificity of their antibody response. As the pooling of nonreactive serum with the reactive serum pools considerably weakens the antisera, it is necessary to select and retain only those individuals with a high antibody response after a short immunization course. Transfer of spleen and lymph node cells from selected high responders into irradiated (500 rad) syngeneic recipients has been successfully used to enlarge the pool of productive recipients (Shen et al., 1978). For the identification of previously undescribed specificities, it may be advantageous to use purified cell populations as the immunizing dose. For example, the use of blast cells led to the definition of ALA-1; also tumors, which represent the monoclonal expansion of a single cell type, led to the definition of the Ly-1, Ly-2, Lyb-2, and Lyb-4 specificities. Attempts to use solubilized and purified alloantigens have not been successful for the production of antibodies to CMAD, as apparently the immunogenicity is lost during the extraction procedure (Graff et al., 1971; di Padua et al., 1973). B. METHODS OF DETECTION Although most of the specificities in this review were described by the use of the standard cytotoxic test, the recent introduction of new techniques, such as the FACS and rosetting methods, should lead to identification of new antibody specificities that do not mediate complement-dependent lysis. One advantage of the rosetting and FACS methods is that the separated populations may be subsequently tested in functional assays, whereas in cytotoxicity tests the antigenbearing cells are lysed. As well, there are some indications that these methods may be more sensitive than the cytotoxic assays in use.

1 . Cytotoxicity The lysis of cells by antibody in the presence of complement is the conventional method of defining CMAD (Gorer and O’Gorman, 1956). Cytotoxicity is determined by using dyes, such as trypan blue, eosin, or fluorescein diacetate. The 51Crrelease assay (Sanderson, 1964; Wigzell, 1965) has also been used extensively; however, it is of restricted use in determining the precise number of antigen-positive cells in a population. The disadvantages of the cytotoxicity assays are that they

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detect only antibodies that bind complement, and it is difficult to detect the lysis of only a small number (5-15%) of cells. As well, there are problems inherent in the selection of the complement source. Although rabbit serum is a more potent source of complement than guinea pig serum (Haughton and McGehee, 1969; Koene et al., 1973), it contains naturally occurring substances, probably antibodies, that are cytotoxic to mouse cells. Therefore it is necessary either to select serum from individual rabbits for low toxicity and high complement activity or to absorb rabbit serum with mouse lymphoid or tumor cells in the presence of EDTA (Boyse et al., 1970b). 2 . Other Assays The recent introduction of the FACS has greatly extended the usefulness of immunofluorescence assays (Moller, 1961) in the detection of CMAD. The principles of the FACS have been reviewed elsewhere (Herzenberg and Herzenberg, 1978),and because of its sensitivity and sorting capabilities the FACS has become an extremely valuable tool in the study of CMAD. The development of rosetting assays involving an indicator system in which either an anti-mouse globulin reagent (Parish and McKenzie, 1978) or staphylococcal protein A (Sandrin et al., 1978) is coated via CrC13 onto sheep red cells also offers several advantages over the conventional cytotoxicity assays, such as the detection of small numbers of reactive cells (Parish and McKenzie, 1977) and increased sensitivity for some alloantibodies; these assays also permit the separation and recovery of a mixed cell population. Assays involving 1251-labeledprotein A or antibody have recently become very valuable as an assay system to screen the supernatants from antibody-secreting hybridomas (Lemke et al., 1978). Recently, immunoprecipitation from NP-40 solubilized radiolabeled membranes, followed by analyses on SDS gels, has been used to separately identify CMAD, e.g., Lyb-6 (Kessler et al., 1978).It should be noted that when different techniques are used to detect a specificity, the identity of products detected cannot be assured and that both techniques should be performed in backcross mice-as was done for immunoprecipitation and cytotoxicity studies of the Lyb-2 molecule (Tung et al., 1977). ANTIBODIESIN ANTISERA C. CONTAMINATING Antisera to CMAD, in addition to specific antibody, are likely to be contaminated with autoantibodies, antiviral antibodies, and soluble immune complexes. Three different types of autoantibodies have been described.

194

IAN F. C. MCKENZIE AND TERRY POTTER

1. Heat-labile IgM autoantibodies present prior to immunization, particularly in strains 129 or C58 (Schlesinger, 1965; Raff, 1971b). 2. Heat-stable autoantibodies arising during immunization; their production differs markedly among different strains (Boyse et al., 1970a). 3. In strains such as NZB, BXSB, and MRL, there is production of autoantibodies accompanied by immune complexes, and autoimmune disease, in an analogous manner to human SLE (Andrews et al., 1978). These autoantibodies have been termed natural thymocytotoxic antibodies (NTA) (Shirai and Mellors, 1971, 1972; Auer et al., 1974) and appear to define a T-cell antigen that has a similar distribution to BA8 and co-caps with Thy-1.2 (Parker et al., 1974). Antibodies to murine viral determinants are present in most, if not all, alloantisera (Nowinski and Klein, 1975; P. Klein, 1975), particularly those that are raised against tumors, which usually express a high concentration of viral determinants. For a similar reason, antiviral antibodies are a particularly important problem in the typing of tumors and have to be absorbed either with purified virus, with tissue from strains such as NZB or DBN2, which carry more virus than others, or with tumor lines that lack the specificity defined by the antisera. As antiviral antibodies are predominantly IgM, the use of techniques involving protein A, which detects only IgG antibodies, can overcome this problem. Soluble immune complexes of alloantibodies and antigen may occur in the sera of hyperimmunized mice and can lead to false-positive reactions, particularly in the fluorescence or rosetting assays. Such complexes are usually removed by absorption (e.g., with human platelets) and/or by ultracentrifugation or microaerofugation. A further problem with many of these antisera is that they contain antibodies to known, but unwanted, specificities (Table V). At present the most appropriate means of overcoming these problems is to absorb with tissues of the congenic partner (if available) or test the antisera on strains that express only the specificity under study. Almost all the problems outlined in this section will be overcome by the use of monoclonal antibodies produced by the fusion of spleen cells from immunized mice with in vitro myeloma cell lines derived from MOPC-21. The methods and results have been reviewed elsewhere (Herzenberget al., 1978).In brief, recipient mice are immunized, and, after two injections, the spleen is removed and fused with the myeloma in the presence of polyethylene glycol (PEG). The cells are then grown in hypoxanthine aminopterin thymidine (HAT) medium, where only the growth of the hybridoma is supported, as the myeloma

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lacks hypoxanthine guanine phosphoribyl transferase (HGPRT) and so cannot synthesize nucleotides in the presence of aminopterin. The hybridomas are subsequently cloned and then set up either in uitro or in uiuo,and secreted antibody is collected. Thus far, antibodies to H-2, Ia, and Thy-1 alloantigens have been produced, and recently to Ly-1.1 (P. M. Hogarth, personal communication). In addition, two new specificities Qat-4, Qat-5 (G. Hammerling, personal communication), which are present exclusively on T cells and is H-2 linked, has been defined by antibodies secreted by a hybridoma. IV. Characterization of Antisera

The definition of a previously unrecognized CMAD by a new antibody must satisfy a number of criteria, and extensive testing must be performed before deciding that the antibody is directed against products of a new locus. As more CMAD loci are being defined, the methods and principles are summarized in Table VI. In the early stages, when the antiserum is undergoing the preliminary studies, it is wise to set up both a repeat immunization and a reciprocal immunization system in order to show that the antiserum is reproducible and to define the other allele. Also, at the earliest opportunity steps should be taken to establish a congenic line for the particular specificity. The characterization of a new and unique antiserum is usually done in several different phases, as shown in Table VI. Preliminary studies are usually aimed at determining (1) the presence of any autoantibody in the serum; (2) the most appropriate serological technique to detect the antibody; (3) the most appropriate tissue on which to perform the genetic characterization. A. GENETICANALYSIS The aims of genetic analysis are two-fold: first, to establish that the antibody defines a new alloantigenic system; and, second, to map the locus defining the new CMAD. The initial step is to determine which strains express the specificity, and three groups should be analyzed for the strain distribution pattern (SDP): (1)conventional inbred strains, (2) congenic strains, (3) recombinant inbred (RI) lines. The SDP for most of the known loci are shown in Tables VII, VIII, XI, XV, XIX, XX, and XXI. It is frequently found that some specificities are expressed in a lower density in some strains, leading to difficulties in testing; e.g., Ly-4 specificities are expressed weakly ir1H-2~strains, similarly Iak (Shreffler and David, 1975) and Lyb-2.2 (Sat0 and Boyse, 1976) in C3H, and Ly6.2 in 129. Although a unique SDP suggests that

196

IAN F. C. MCKENZIE AND TERRY POTTER

TABLE VI CHARTFOR THE CHARACTERIZATION OF ALLOANTISERA DETECTINGCMAD Produce antisera

5

Obtain pool of sufficient size for all tests and freeze in small aliquots

.1

Testing (decide most appropriate serological technique, e.g., cytotoxicity, fluorescence, rosetting) 1 . Preliminay studies

a. Tissue distribution to determine optimal target (e.g., thymus, lymph node, spleen); test on antiserum donor thymus for autoantibody b. Absorb with serum donor thymus to remove autoantibody and repeat test (a) c. Set up matings for a repeat antiserum, and for a reciprocal antiserum 2. Genetic characterization a. Strain distribution pattern (SDP) on inbred strains; compare with known loci that code for CMAD (Ly,H, Ea, etc.) and other loci (Table VII) b. Absorption studies with positive strains and test on panel for the presence of multiple specificities c. SDP on recombinant inbred lines:

No. of lines CXB: BALBk and C57BL/6 (Bailey) 7 21 AKL: AKR and C57L (Taylor) 13 BXH: C57BLI6 and CsWHeJ (Taylor) SWXL: SWRIT and C57L (Taylor) 7 BXD: C57BLi6 and DBAIi (Tayior) 24 d. Study segregating populations: Backcross (+ x -) x - -: (i) single gene (1: 1 segregation) (ii) separate from other loci F2cross (+ -)Fl x (+ -)F,: (i) single gene ( 3: 1 segregation) (ii) allelism (iii) separate from other loci e. Test available congenic lines f. Linkage studies g. “Shortcuts”: co-capping, chemistry h. Set up matings for congenic line 3. Tissue distribution a. Thymus, lymph node, spleen: “clearing” absorptions to determine presence or absence of antigen; quantitative absorptions for comparative amounts b. Other tissues (red cells, liver, kidney, brain): nonquantitative absorptions c. T-and B-cell distribution d. Tumors of known T- and B-cell type. 4. Functional characterization T helper, suppressor, MLR, etc. B: PFC (IgM and IgG), PFC precursor 5. Further characterization a. Analysis for antiviral antibody b. Skin grafts for H loci when congenic strains are available c. Use of congenic strain to produce and analyze antisera when available

TABLE VII STRAINDISTRIBUTION PATTERN(SDP) OF SOME COMMONLY USED CMAD AND Strain

H-2 Thy-1

C57BLl6 C57L C57BWcd BALWc A SWR C3HlHeJ DBAll DBAl2 CE C58 AKR PL

b b

RF

k b b k

129 LP CBNJ BDP SJL NZB a

k

d a b

k q d k k k u

p S

d

2 2 2 2 2 2 2 2 2 2 2 1 1 1 2 2 2 1 2 2

Tla

2 1,2,3,5 2 1,2, 3, 5 1, 2, 3, 5 -

-

2 1, 2, 3, 5, 1,2,3, (5?) 2 2

-

1,2, 3, (5?) 1, 2,3, 5, 1,2,3,(5?)

Ly-1 Ly-2 2 2 2 2 2 2 1 1 1 2 2 2 2 2 2 2 1 2 2 2

Ly-3 Ly-4 Ly-5 Ly-6 Ly-7

2 2 2 1 2 2 2 2 1 2 2 2 2 1 2 2 2 1 1 1 2 2 1 1 1 2 2 1 1 2 1 2 1 1 1 1 2 1 1 1 1 2 1 1 2 1 2 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 2 1 1 1 1 2 2 2 1 1 2 2 2 2 1 1 1 2 N 1 2 1 1 1 1 2 1 1 2 2 2 1 2 2 2 2 1 1 1

Further SDP for Qa, Lyb loci will be found in Tables XI and XV.

* ND, not determined.

1 2 2 2 2 2 2 2 2 2 1 2 2 2

MOUSE STRAINS'

Ly-8

Lyb-2

Lyb-4

LyM-1 ALA-1

2 2 2 1 1 2 1 1 2 ND ND 2

2 1 1 2 2 1 2 1 1 3

-

1

ND 3 2 N 1 ND 3 ND

2 2 NDb 2 ND 1 2(?) 21 ND 2 21 2 IJD 2 2 21 ND 2 3 2 ND ND 2ND 2 2 D N D 2 1 ND ND 22 ND ND

1 3

1 D

N D 2 2 2 2

INBRED

N

1 1 2 ND

D

ND

-

ND

+ +

ND ND ND D N

+

ND ND

K

2 3 m

0 M

r 7

2

z

bM $ 2 Z

(II

IAN F. C. MCKENZIE AND TERRY POTTER

198

TABLE VIII STRAIN DISTRIBUTIONPATTERNS OF DIFFERENTLOCI I N THE CXB RECOMBINANT INBRED LINES O F BAILEY"~~ CXB recombinant inbred strain

Locus

Chromosome

a Aal

2 9

b

4

Bfo Bge Bgt

4 9 9

C

7

Car-2 CPZ CS

Dag Ea-4 Ea-6 Es-1 Exa

Fu-2 Gpd-1 Gpi-1 H ba Hbb 161 If-2 lg-1 Lap-1 Ly-4 Ly-6 Ly-7 Mod-1 MUP

Pc-1 Pre sco Sep-1 SPl Tam-1 Tla H-1

H-2 H-3 H-7 H-8 H-15

3 2

2

a 4

9 4

7 11 7 -

9

9 4

17 9

7 17 7 17 2 9

4

D

E

G

H

I

J

K

C C B C C C B C B C B C C C C C C B C B C C B C C B B C B C B C C C C C B C C C C C

C B B C C B B C C B B B B B B C C C B B C B B C B B C C C B B B C B C B B B B C C B

C C C C C C C B C C C B C B B C C B B C B B C C B C B C C B

B B C C C C B B B B C C B C B B B C C B B C B B C C C B C B C C B B C C B C C B B C

B B C C B B C C C B B B B B C B C C B C C B B B B B B B B B B B B B C B C B B B C B

B B C B B B B C B B C B C C C C B C B B C B C B B C C B C B C B B B B B B B B C C C

B C B C C B B B C B B B B C B C B B C B B C B C B B C C B C C B C C B B B B B C C B

C

B C C C B C B B C B C

199

MURINE CELL-SURFACE ANTIGENS TABLE VIII (Continued)

CXB recombinant inbred strain Locus H-16 H-17 H-18 H-19 H-20 H-21 H-22 H-23 H-24 H-25 H-26 H-27 H-28 H-29 H-30 H-34 H-35 H-36 H-37 H-38

Chromosome

D

E

G

H

I

J

K

4

C B B C B B B C B C C B C C C B C C B B

B C B B B B C C C B B B C C B C B B C C

C C C B C C B C B C B C B B C C C B C C

C C B B B C C C C B C B B C C C B C R C

C C C B C C C C C B C C C B B C B B C C

B B B B B C C C C B C B C C C B B C B B

B C B B B B B B B C B C B B B C C B C C

4

8 4 4 7

7

5 8

-

-

a Published by permission of Dr. D. Bailey, Jackson Laboratory, Bar Harbor, Maine. b C = BALWc; B = C57BU6.

the antibody may define a new system, in order to be sure that it is not merely recognizing a new specificity, i.e., a new allele, at a previously defined locus, segregation analyses must be performed. A comparison of the SDP in the recombinant inbred lines of Bailey (1971)and Taylor (BXD, BXH, AKXL, SWXL) is often a very useful shortcut for establishing linkage of CMAD loci to other markers (e.g., Table VIII). B. TISSUEDISTRIBUTION Lymphoid tissues can be tested both directly and by absorption, the absorption studies being necessary to determine the relative amounts of the antigen in a particular tissue. The presence of the antigen in nonlymphoid tissue is usually identified by nonquantitative absorptions, and it is always necessary to include both positive and negative controls for these absorption studies, as antibodies can be removed nonspecifically. To determine the relative density of the CMAD on the reacting tissues, quantitative absorptions are performed that involve comparison of the absorptive capacity of known numbers of cells. If

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IAN F. C. MCKENZIE AND TERRY POTTER

the antibody reacts with lymphocytes, then obviously it is important to determine whether this reaction is preferentially with T cells, B cells, or both. Enriched T-cell populations include nylon wool nonadherent cells (Julius et al., 1973), or populations depleted of Ig+ cells, or lymphoid cells from T cell-depleted mice. To determine the relative concentration of the specificity, it may be necessary to do absorptions with purified T and/or B cells, then test the absorbed sera on both populations, In addition, tumors of known T-cell or B-cell type can be used for characterization of specificities. For example, there is a large variety of thymomas and T-cell leukemias (Thy-1+, Ig-), which are representative of T-cell types. Further, there are a number of B-cell tumors and myelomas, e.g., the Abelson tumors and mineral oilinduced plasmacytomas. However, it cannot be assumed that T-cell or B-cell tumors carry all the T-cell or B-cell specificities, for there appears to be a selective representation of T-cell specificities on thymomas of different origin (Section XIV). The ontogenetic appearance of the determinant may also be of crucial importance in determining the relationship between the tissue of origin and confinement to a particular lineage. C. FUNCTIONAL CHARACTERIZATION By this stage of testing, one has considerable knowledge of the antiserum, its appropriate target, content of autoantibody, strain distribution, and one knows that it is distinct from other loci. The sera are now available for the appropriate functional characterization, which may be of great immunological interest, but which also serves to characterize the antisera. Sera can be tested for the known T-cell and B-cell functions, and these will become apparent later in this review. D. FURTHER CHARACTERIZATION Many CMAD have been further characterized, such as to their relationship to MuLV and as to whether the CMAD is present on skin, which can only be adequately tested when the relevant congenic line is available. Further studies may be done to determine the physical or chemical relationship to other determinants by the co-capping method or by coprecipitation and chemical analyses. If two specificities do not co-cap they are presumably present on different molecules and therefore are the products of different genes. However, the converse is not necessarily true, as some nonidentical molecules co-cap. Biochemical characterization of the antigen is usually done by lactoperoxidasecatalyzed labeling of cell surface molecules with lZ5Ifollowed by solubilization, precipitation with the antisera, and analysis of the

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precipitate by SDS-PAGE. The details of these procedures are described in the appropriate sections. V. Histocompatibility (H) Loci-CMAD

of General Distribution

The histocompatibility loci, in general, code for specificities found on all tissues although this has not been extensively demonstrated for every tissue and for every H locus. However, as these specificities occur on lymphocytes and as there are some particular anomalies associated with their detection and distribution, especially with the non-H-2 loci, several brief comments are made on H-2 and non-H-2 loci in this review. A. H-2 CMAD @-2K, H-2DYH-2G, H-2L) Within the MHC of the mouse (the H-2 complex) are found many loci that code for CMAD. At least four (H-2K7H-2DYH-2G7H-2L) code for antigens of general distribution; i.e., they can be detected by graft rejection and elicit antibody production. As discussed in Section 11, other H loci also occur within the MHC and have arisen by mutation within the complex, so that the four mentioned here should be regarded as the minimum number of CMAD within H-2. The Ia specificities, which are of restricted distribution, are considered in Section VI. The H-2K and H-2D loci code for both private and public specificities. The private specificities are of restricted strain distribution, being found in only one haplotype or recombinants developed therefrom, as opposed to the public specificities, which have wide distribution in both inbred and wild mouse populations. Many public specificities occur on the same molecule as do the private specificities, as they coprecipitate with either public or private antibodies. The specificity H-2.7 is coded for by the H-2G locus. This specificity can be demonstrated directly on red cells and, by absorption, on lymphocytes. The histocompatibility effect of the H-2G locus is not clear, as the appropriate recombinants are not available to test H-2G in isolation. The H-2L locus has a histocompatibility effect-as demonstrated by the H-2dbmutant, and as well codes for specificities H-2.64, H-2.65, which are found on lymphocytes. Whether there is a private H2L-coded specificity is not clear at present. The H-2K and H-2D loci are probably equivalent to HLA-A,B in man. The H-2G locus coding for a red cell specificity may be equivalent to the Rodgers or Chido blood groups in man, so that the comparative structure of the MHC in man and mouse appears to be very close, although these blood groups have recently been linked to C4 polymorphism (O’Neill and Dupont,

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IAN F. C. MCKENZIE AND TERRY POTTER

1979). Of relevance to the lymphocyte CMAD, which have restricted distribution, is the finding that specificities are differentially distributed on different classes of lymphocytes. For example, thymus cells have very small amounts of H-2 compared with peripheral lymphocytes, though a comparison of H-2 density on peripheral T cells and B cells has not yet been reported. The structure of H-2 antigens is also of relevance to CMAD of restricted distribution in that /32-microglobulin has been associated with the H-2K and H-2D specificities and with the closely linked TL and Qa specificities. A similar molecule may also be associated with the T/t complex. These three genetic complexes occur on the same chromosome and may be considered to have arisen from the one complex, possibly T/t (Gluecksohn-Waelsch and Erickson, 1970) through gene duplication and recombination. Klein (1977) has summarized the evidence that this complex represents a precursor of H-2. The TZa complex possibly arose from H-2, and the two appear to be closely related not only genetically, but also structurally, with the common occurrence of the p2-microglobulin. \In addition, there is a close interrelationship of the concentration of TL specificities and H-2D specificities on the cell surface, for when TL is expressed there are smaller amounts of H-2D, and, moreover, 'H-2D and TL antisera reciprocally block for each other. It is not clear whether this represents more than a physical association on the cell surface, or the use of a common intracellular pathway in the synthesis of these molecules. Whether /32-microglobulinis associated with any of the CMAD of restricted distribution is unknown. Also within the MHC are found loci that code for specificities of restricted distribution-in the Z region (Section V1)-and also, two monoclonal antibodies have been found (G. Hammerling, personal communication) that code for a specificity found only on T cells (Qat-4, Qat-5) (Section VI). B. NON-H-2 HISTOCOMPATIBIL~Y LOCI As mentioned in Section 11, there are a whole series of non-H-2 H loci that have been detected and separated by the use of congenic lines. In general these lead to graft rejection effects of lesser magnitude than those found with the H-2 complex, e.g., graft rejection times ranging from 15 to over 300 days. However, it should be noted that these histocompatibility effects tend to summate, so that multiple non-H-2 antigens can cause graft rejection of similar rapidity as found with an H-2 difference, e.g., with C57BW6 and 129. The definition of most of the non-H-2 H loci has required the production of the appropriate congenic line, and almost all of these have been produced by Snell on the C57BL110 background. In addition to their weak his-

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tocompatibility effects, these non-H-2 loci are noted for their inability to elicit production of cytotoxic antibodies when grafts are rejected or when multiple immunizations are performed. The reason for this is not known but may be related to the problem of lack of antibody production when immunizing between congenic strains differing by certain Ly specificities (see Section 111).Of particular importance here may be the fact that most of these congenic lines are on a C57BU10 background, a genetic background that may not be conducive to the formation of cytotoxic antibodies. Perhaps if these non-H-2 lines were placed on a different genetic background, antibodies could be produced. The lack of appropriate serological tests has certainly inhibited the studies of non-H-2 loci, for the cumbersome F1 complementation tests have to be used to define polymorphisms at different loci. However, it appears in general that some of the non-H-2 loci may be as polymorphic as the MHC, for there are now 6 alleles of the H-3 locus (Gasser, 1976). Although in general antibodies have not been demonstrated by immunizing between the non-H-2 H congenic strains, several exceptions should be mentioned. First, by immunizing between strains differing at the H-1 locus (C3H.K and C3H) Winn found antibodies that were not detectable by hemagglutination or cytotoxicity, but did neutralize tumors in the Winn assay (Winn et al., 1958).In the same combination we have been unable to find cytotoxic antibody. Second, the Ly-4 sera described extensively in Section VI, may contain anti-H-3 antibodies. The Ly-4 locus has been demonstrated to be linked to the H 3 locus, as both have a similar SDP, especially in the Bailey RI lines, and the H-3 congenic lines have been found also to differ at the Ly-4 locus. WhetherH-3 and Ly-4 are the same or different is currently being studied. However, recently Zink and Hayner (1977, 1978) identified, by fluorescence and hemagglutination, antibodies that may recognize H-1, H-3, and H-13 specificities. In the studies of Zink and Hayner, the antiserum (BALB/c x DBA/2)Fl anti-B1O.DS was used and, though not considered by the authors, could have contained a contaminating anti-Ly-4 antibody. Similarly, many of their other antisera could have contained antibodies to Ly specificities. However, it is important to realize that many of the Ly sera currently produced could contain specificities to non-H-2 CMAD. Third, in some recent studies using W W anemic mice as recipients of bone marrow grafts, Harrison and Doubleday (1976) demonstrated that various non-H-2 loci have different effects depending on whether the locus is being tested by skin or bone marrow grafting. Two loci, H-17 and H-26, appeared to express antigens more strongly on skin than on bone marrow, whereas the H-12 product determines an antigen more strongly expressed on bone marrow than on skin.

204

IAN F. C. MCKENZIE AND TERRY POTTER

Although studies on the non-H-2 (H) loci have progressed slowly over the years, more recently a new develapment has occurred in addition to those described above. This is the development of the T-cell lysis assay for strains differing at various n0n-H-2 loci (Bevan, 1976). Provided that the donor and recipient share either of the H-2K or H-2D specificities, then T cell-mediated killing can occur in uitro after in uiuo sensitization of killer cells against the appropriate target. Also of relevance to the studies of non-H-2 (H) loci is the production of antibodies against H-2 mutants (M. Cherry, personal communication), a fmding that has eluded many attempts in different laboratories. In these particular studies, immunization was performed with broad antigenic differences, which sometimes included H-2 differences, and, by appropriate selection of target cells, specific antibodies directed against mutant targets were demonstrated. A similar approach may lead to the detection of antibodies for some of the non-H-2 loci. In conclusion, the difficulty in demonstrating antibodies to non-H-2 CMAD may be a technical problem and several new approaches may lead to antibodies. It is not unlikely that these antibodies may contaminate many of the anti-Ly antisera currently in use. C. Hh-1 Hybrid histocompatibility (Hh) occurs when F1 animals, heterozygous at the MHC, reject parental bone marrow grafts-an unexpected finding according to classical laws of transplantation. The phenomenon has been observed with bone marrow transplants, particularly when low doses of bone marrow are used (105 cells), and also with some leukemias (Cudkowicz and Bennett, 1971; Shearer et al., 1977). The effector cell in the rejection is radiation resistant, and genetic factors play a major part in the determination of this phenomenon. The major locus affecting this is Hh-1, which maps within or close to the D end of the H-2 complex, although other loci may also influence the phenomenon. Similarities of the cell mediating this phenomenon and the natural killer (NK) cell have been discussed (Kiesslinget al., 1976, 1977),as both cell types are not conventional T or B cells, nor radiation sensitive. As well, both arise in the bone marrow and both act without prior immunization. The nature and phenotype of the cell mediating either or both of these phenomena is obscure at present, although the NK cell carries a specificity found in anti-Ly-1.2 serum (see Section VIII). Similarly the antiserum C3H anti-B1O.BR which could contain antibodies to Ly-1.2, Ly-2.2, Ly-4.2, Ly-6.2, and Tla, as well as other specificities, has been shown to abrogate the Hh-1 marrow rejection effect (Gregory et al., 1972).

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VI. lymphocyte Alloantigenr

In this section will be reviewed most of the known alloantigenic specificities, which are expressed predominantly on lymphocytes, although in many systems exclusive representation on lymphocytes has not been demonstrated by exhaustive absorption with nonlymphoid tissues. The systems to be described are now of immense valueparticularly the Thy-1, Ly-1, Ly-2, Ly-3, and Ia specificities-as they identify functional lymphocyte subpopulations. The antisera can therefore be used to isolate and study the role of these cells in various aspects of the immune response. The lymphocyte alloantigens are classified in Table I; the combinations used for antiserum production are given in Table V; and the strain distributions are shown in Tables VII, XI, XV. Their distribution on T and B cells are shown in Table IX; and tissue distritution in Table XII. Linkage relationships are shown in Table XIII. A. THET~Y-1LOCUS The Thy-1 alloantigen is of historical importance, as it was the first serologically detected alloantigen to be described in the mouse, which had a restricted tissue distribution to thymocytes, as opposed to the H-2 specificities, which were known to be of general distribution (Reif and Allen, 1964).The Thy-1 antigen has been of great practical importance, as it is the commonly used marker to distinguish T cells (Thy1+)from B cells (Thy-1-) in mixed populations. In addition, Thy-1 specificities have an interesting tissue and species distribution, and the chemical nature of this antigen is also of some interest.

1. Genetics The Thy-1 locus is found on chromosome 9 and maps near dilute (d) and malic enzyme (Mod 1) (Itakuraet al., 1971,1972; Blankenhorn and Douglas, 1972); it is therefore separate from all other loci determining TABLE IX DISTRIBUTION OF CMAD ON MUIUNET AND B CELLS

T cells Thymocytes exclusively: Tla, Glx Extrathymic T cells: Ly-6, Qa-3, IJ Intra- and extrathymic T cells: Thy-1, Ly-1, Ly-2, Ly-3, Qa-1, Qa-2 B cells Ia, Ly-4, Lyb-2, Lyb-3, Lyb-4, Lyb-5, Lyb-6, Lyb-7, LyM-1, Pc-1 T and B cells H-2, Ly-5, Ly-7, Ly-8, ALA-1 (Ly-6 appears to be on some B cells)

206

IAN F. C. MCKENZIE AND TERRY POTTER

CMAD (Table XIII). The locus has two alleles, Thy-1 a and Thy-1 *, which code for the specificities Thy-1.1 and Thy-1.2, respectively. These specificities used to be defined as 8-AKR and 8-C3H, as the antisera were originally made as C3H anti-AKR and AKR anti-C3H (Reif and Allen, 1964).Although these strains are not congenic and the antisera contain other specificities (Table V), these particular combinations are still commonly used to produce an effective anti-Thy-1 serum. The Thy-1.1 specificity is found in a few unrelated strains, such as AKR, BDP, RF, and PL. All other inbred strain carry the Thy-1.2 specificity. An AKR subline, AKR/Cum carries the Thy-1 rather than the Thy-1 a allele (Acton et al., 1973). 2. Production of Antisera The classical combination used to produce Thy-1.2 serum (AKR anti-C3H thymus) also contains antibodies to Ly-1.1, Ly-3.2, and possibly Ly-6.1 and Ly-8.1 (see Table V). However, in the presence of guinea pig complement only T cells are lysed by this particular antiserum; but with rabbit complement, cells other than T cells are lysed (Greaves and Raff, 1971), possibly owing to B-cell antibodies such as those found in Ly-8 antisera. The AKR anti-C3H antiserum also contains appreciable amounts of autoantibody, which should be removed by absorption with AKR thymus tissue. Considerable amounts of antiviral antibody are also present, which would be partially removed by absorption with AKR thymus. Anti-Thy-1 sera can also be prepared using the three Thy-1 congenic strains (Table IV) available on C57BL16 and on A strain backgrounds. The best congenic anti-Thy-1 is produced using F, hybrids; however, these sera contain large amounts of autoantibody and may have little specific antibody reactive with peripheral T cells, so that, in general, anti-Thy-1 produced in congenic strains has been a disappointing reagent in functional tests. A stronger anti-Thy-1 response can be induced by immunizing with Thy-1 plus other antigenic differences-presumably the multiple differences inducing more T-cell h e l p a phenomenon called “associated recognition” (Lake and Douglas, 1978). There is a recent report (Sharav et al., 1977) on the production of anti-Thy-1.1 and Thy-1.2 sera by immunizing the mice of the appropriate strain with mouse brain; however, the sera were weak and the authors concluded that the Thy-1 antigen present on brain had a weak capacity to elicit an antibody response in allogenic recipients. A detailed genetic analysis has demonstrated that the immune response to Thy-1 specificities is partly controlled by Zr genes mapping within the H-2 complex (Zaleski and Klein, 1978).

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3. Tissue Distribution The quantity of the Thy-1 antigen on T lymphocytes and cortisoneresistant thymocytes is considerably less than that of the majority of thymocytes (Aoki et al., 1969). This is reflected in the titers of the congenic antisera, e.g., 1: 1,000 for thymus, compared with 1: 16-1 : 64 for spleen and lymph node. Thy-1 is also present on brain, epidermal cells (Scheid et al., 1972), fibroblasts (Stern, 1973), some mammary carcinoma cells (Gillette, 1977), and transitorily on fetal skeletal muscle (Lesley and Lennon, 1977). 4 . Presence of Thy-1 Specijicities on T Cells

The original studies (Reif and Allen, 1964)described the presence of theta (4) on thymus cells only. Two years later Reif and Allen (1966), in a study of AKR leukemias, also noted the presence of the Thy-1.1 specificity on thymus-derived leukemic lymphocytes. Later Raff (1969, 1970, 1971a) demonstrated the presence of Thy-1 antigens on Ig- cells by immunofluorescence and suggested that, because of the reciprocal distribution of Thy-1 and Ig, these were the differentiating surface markers for T and B cells. This finding was substantiated by the finding of decreased numbers of Thy-l+ cells in spleen and lymph node following thymectomy (Raff and Wortis, 1970; Schlesinger and Yron, 1970) or antilymphocyte serum treatment (Raff, 1969; Schlesinger and Yron, 1969) of mice, and furthermore by the demonstration that athymic mice (nude) carry very low numbers of Thy-l+ cells (Raff and Wortis, 1970). In a variety of assay systems, Thy-1 antisera inhibited T-cell, but not B-cell, functions (Cerottini et al., 1970; Raff, 1971a; Vischer and Jaquet, 1972; Rouse et al., 1973). In the last decade, numerous studies using anti-Thy-1 sera have confirmed the original findings and reaffirmed the usefulness of Thy-1 as a T-cell marker, furthermore, there does not appear to be any differential distribution of Thy-1 on functional T-cell subsets, although the Ly1+2+3+ subset appears to be resistant to anti-Thy-1 + C’ (Eardley et al., 1978). 5. Thy-1 Expression during Lymphocyte Diflerentiation The concentration of Thy-1 alters markedly during the differentiation of T lymphocytes (Raff, 1971a). In contrast to the early observations of the virtual absence of Thy-l+ cells from athymic mice, it is now apparent that about 20% of spleen cells in nude mice express a low density of the Thy-1 determinant (Roelants et al., 1975; Loor et al., 1976). It was suggested that these cells are the thymocyte precursors, although their relationship to the Thy-1- bone marrow cells that can

208

IAN F. C. MCKENZIE AND TERRY POTTER

be induced in uitro to express Thy-1 is unclear (Goldstein et al., 1975; Komuro et al., 1975a; Scheid et al., 1975a,b). As mentioned above, the level of Thy-1 falls during maturation within the thymus, therefore the overall scheme of differentiation can be represented: Prothymocyte + immature thymocyte + mature thymocyte + mature T cell Thy-l+ Thy-l++++ Thy-1++ Thy-1++

6. Rat Thy-1.1 and Chemistry of the Thy-1 Molecule Rats carry a specificity related to, or the same as, mouse Thy-1.1. Douglas (1972) and Michael et al. (1973) showed that rat brain and thymocytes expressed an antigen that reacted with mouse anti-Thy-1.1 serum (summarized by Williams et al., 1976). However, in the rat, Thy-1.1 is expressed on a very low number of lymph node cells (Acton et al., 1974), and, in contrast to the mouse, it is found on about 40% of bone marrow cells. On this basis, it was considered that Thy-1 was not a T-cell marker in the rat (Williams, 1976). A specificity analogous to Thy-1.2 has not been detected in rats. Xenoantisera raised in rabbits against rat or mouse Thy-1 bearing tissues have demonstrated the similarity of the Thy-1 specificity in these two species. Some interesting studies have recently emerged on the biochemical structure of the Thy-1 specificity, using alloantisera and xenoantisera and conducting comparative studies in rats and mice. Using both xenoantisera and mouse anti-Thy-1.1, it was found that there were approximately 600,000Thy-1 molecules on the surface of rat thymocytes (Acton et al., 1974; Morris and Williams, 1975; Williams et al., 1976), so that Thy-1 must be a major constituent of the thymic cell surface. Similar amounts were also found in rat brain (Barclay et al., 1976). Some studies (Barclay et al., 1976) suggested that Thy-1 of both brain and thymus was a 250,000-dalton glycoprotein containing about 30% carbohydrate. In these studies, the carbohydrate composition was different in the different tissues, although identical amino acid compositions were found. In other studies, it was suggested that the Thy1.2 antigen was glycolipid in nature, with the specificity residing in the ganglioside (GM,) fraction (Esselman and Miller, 1974; Miller and Esselman, 1975).However, it was subsequently shown that anti-GM, sera, which reacted with thymocytes and peripheral T cells, reacted independently of Thy-1 phenotype (Stein-Douglas et al., 1976; Milewicz et al., 1976). The relationship of GM, and Thy-1 is more apparent than real and is due to the greater accessibility of GM, on T cells (Stein et al., 1978) rather than to a chemical association of the two. Furthermore, other studies have failed to find any Thy-1 activity in the

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lipid fraction of thymus cell isolates (Kucich et al., 1975; Arndt et al., 1976). More recent attempts to isolate the Thy-1 specificity have involved the use of xenoantisera for purification using an immunoprecipitation step, as alloantisera to the Thy-1 specificity do not precipitate from NP-40 extracts, but require the freeze-thawing of labeled thymocytes (Vitetta et aZ., 1973; Trowbridge et al., 1975). However, Jones (1972) was able to precipitate Thy-1 from NP-40 lysates with a noncongenic anti-Thy-1 serum; with a similar antiserum, Atwell et al. (1973) identified a 60,000-dalton product from cell extracts prepared in urea. Vitetta et al. (1973) precipitated a 35,000-dalton molecule from thymocytes lysed by freeze thawing. The molecule precipitated from mouse or rat thymocytes by Thy-1 xenoantisera is T25, a 25,000-dalton glycoprotein (Letarte-Muirhead et al., 1974; Kucich et al., 1975; Trowbridge et al., 1975; Williams et al., 1976), although the true molecular weight is probably 19,000 (Kuchel et d . ,1978): By the use of the xenoantiserum and analysis by affinity chromatography, it is clear that three different specificities are present on the rat Thy-1 molecule, which include Thy-1.1, a rat-specific determinant, and a cross-reacting rat-mouse xenoantigenic determinant (Clagett et al., 1973; Thiele and Stark, 1974; Morris and Williams, 1975; Morris et al., 1975). Presumably, mouse Thy-1 has the same structure (Morris et at., 1975; Williams et al., 1976). In this 19,000 MW glycoprotein, the evidence is suggestive of the Thy-1 specificity being protein rather than carbohydrate in nature. As Thy-1 antigens are also present on nonlymphoid cells, it is unlikely that the antigen itself has a primary immunological function. However, Williams et al. (1976) have postulated that these molecules may be involved in cell-cell interactions in the brain and lymphoid system m such a manner that the protein integrates the molecule into the membrane to allow a display of carbohydrate structures that mediate the cell interactions.

7 . A Histocompatibility Locus Associated with the Thy-1 Locus It is apparent that there is a histocompatibility locus associated with Thy-], and there is compelling evidence that these two loci are the same. The studies necessarily require the use of congenic lines, and in the study by John et al. (1972),A strain mice received a skin graft from A-Thy-1a. One of eleven mice rejected two successive grafts from the same donor. The remaining ten mice rejected neither first nor second grafts. However, of the eleven recipients studied, eight were producing anti-Thy-1 antibody, some to a titer of greater than 1: 1000 against AKR thymocytes, and these are examples of the dissociation between

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IAN F. C. MCKENZIE AND TERRY POTTER

an antibody response and graft rejection. In the reciprocal direction, A skin grafted to A-Thy-l a, very little anti-Thy-1 antibody was produced, but five of eight recipients rejected one or more grafts. In a further study (Staines and O’Neill, 1975) similar results were found, except that A-Thy-la grafts were rejected by a higher proportion of mice. Using the C57BL16-Thy-1a congenic mice, skin graft rejection has been readily observed in immunized mice (Henning and McKenzie, unpublished results). These strain differences are probably due to the presence ofH-2-linked Zr genes that influence the anti-Thy-1 immune response. The H-Thy specificities, similar to those of the Sk and H-Y loci, provide examples of antibody production induced by a skin graft in the absence of graft rejection.

8. Thy-l Hybridomas Recently two hybridomas have been produced that secrete antibody to the Thy-1.1 and Thy-1.2 specificities (P. Lake, personal communication). These antisera have a high titer on thymus cells and are also effective in eliminating peripheral T cells. B. THETZULocus The Tla locus defines several specificities found on thymocytes and leukemia cells, but absent from normal peripheral lymphocytes or other tissues (Boyse and Old, 1969; Old and Stockert, 1977). The TL specificities are the only ones found exclusively on intrathymic T cells. The specificities were initially identified (Table X) using several antisera raised in C57BW6 mice against A strain leukemias (Old et al., 1963). The antisera were unusual in that they reacted with several C57BW6 and A strain leukemias and with A strain thymocytes, but not with C57BW6 thymocytes. The TL specificities are of special interest because of their genetic and structural association with the H-2 complex, as the two gene complexes are closely linked on chromosome 17 and both contain &-microglobulin. Furthermore, the H-2D and Tla genes interact in the expression of H-2D and Tla specificities. In addition, the TZa system was the first in which antibody-induced modulation of CMAD was observed. Although the TZa gene locus was initially defined by the antisera listed (Table X), the Tla region on chromosome 17 is now becoming more complex, with the description of the Qa-I, Qa-2, Qa-3, H-31, H-32 loci (Flaherty and Wachtel, 1975; Stanton and Boyse, 1976; Flaherty, 1976; Flaherty et aZ., 1978a) and the finding of an Zr gene affecting the immune response to ferritin in this same region (Young et al., 1976). As it is likely that the region is even more complex, the TZa locus should be considered as a gene complex or region (Flaherty, 1976).

211

MURINE CELL-SURFACE ANTIGENS TABLE X PRODUCTION OF TL AND Oa ANTISERA* Antisera (A.Tlabx C57BU6)F1 a ASLl

B6.Tlaa a ERLD D-35 (BlO.A(2R) x A.CA)Fl a B1O.Y B6.Kl a ERLD B6.Kl a C57BU6 thymus

+ lymph node

Absorbed with (TL phenotype)

Antigens detected

-

TL-1, 2,3, 5, Qa-1 TL-I, 3,5 TL-5 TL-3, 5 TL-4 TL-5

BALBIc (TL-2) A.CA (TL-1, 2, 3) ERLD (TL-1,2,4) B6 or BALBIc lymph node

C57BU6 thymus or EL4

Qa-2 Qa-3

* From Flaherty et al. (1977b, 1978). The production of antiserum to the other specificities is discussed in Old et al. (1968).

1 . Genetics The TZa locus is linked toH-2 on chromosome 17, being 1.5cM from the H-2D locus (Table XIII). Similar to the H-2K and H-2D loci, a number of specificities have been identified at the TZa locus, and it is likely that the TZa locus is highly polymorphic, 5 specificities being currently defined. Allele

Specificities

Type strains

Tla" Tlab Tlac Tlad

TL-1, 2, 3, 5 TL-0 TL-2 TL-1,2, 3

A C57BU6 BALBlc A.CA

Recently the TZad(A.CA)allele has been defined with the description of the TL-5 specificity (Flaherty et aZ., 197%). Although there are only four phenotypes found on normal thymocytes, tumor cells may also express the TL-4 specificity. Leukemias may also show additional phenotypes; e.g., BALB/c and DBM2 thymocytes express TL-2 only, yet some leukemias from such strains have been typed as TL-1,2 and TL-1,2,4, respectively (Boyse et aZ., 1968~). The strain distribution of the TZa specificities is shown in Table XI. 2. Production of Antisera In the original definition of the TZa locus, the antisera were raised in C57BW6 mice against several A strain leukemias such as ASL1. The

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IAN F. C. MCKENZIE AND TERRY POTTER

development of the A-TZa and C57BW6-TZaa congenic mice enabled the production of more specific antisera. Antisera to T L 1 , 2 , 3, and 5 are currently produced as (C57BW6 x A.TLab)F, anti-ASL1, and by appropriate absorption (Table X) it is possible to produce sera specific for most of the TL specificities (Old et al., 1968; Flaherty et aZ., 1977b). Anti-TL-4 sera can be produced in C57BL16 TL+ mice against ERLD, and antibodies to T L 5 were initially detected in an H-2 antiserum to the H-2.35 public specificity. Although spleen and lymph node cells are TL- (see Table XII), some antisera produced by immunization with these tissues may contain anti-TL antibodies (Stanton and Boyse, 1976). This finding was attributed to the induction of T L antigen expression on prothymocytes from the lymph node and spleen in TL+ strains, probably in the thymus of the recipient strain (Komuro et aZ., 1973). Subcellular fractionation studies have demonstrated that TL antigens are also expressed on the surface of mitochondria1 membranes of leukemic and thymic cells of TL+ strains, but not on the mitochondrial membranes of spleen cells (Smith et aZ., 1974, 1975; Jeng et al., 1978). 3. Tissue Distribution By direct testing and absorption analysis, TL antigens were found to be expressed only on thymocytes and some leukemias, but not on bone marrow or peripheral lymphocytes (Table XII). In TL+ strains the specificities are present on the majority of thymocytes with a notable exception of the cortisone-resistant population (Old et al., 1963). Strober (1979) has demonstrated mice with an altered distribution of TL antigens. In shielded mice receiving multiple doses of total body irradiation, Lyl-2- TL+ cells have been identified in the spleen. 4 . Properties of TL Specijicities adopted five criteria for the identification of a TL Boyse et al. (1968~) specificity: (i) linkage to the Tla locus; (ii) confined to thymus cells and leukemias; (iii) undergoes antigenic modulation; (iv) appearance in some leukemias of strains that are TL- (i.e., an example of gene activation); (v) present on the cell surface in the TL region. Most of the TL specificities satisfy these criteria, but there are some differences between them (Boyse et d., 1968~). For example, T L 1 and TL-2 share all five of these properties, but TL-2 is always present on TL+ cells, which suggests that it may have some structural relationship to other specificities. Anti-TL-2 antisera are the weakest of the T L antibodies, and antiserum to the TL-2 specificity has no modulating effects, although the specificity is modulated along with other specificities when an anti-TL-l,2,3 serum is used. Indeed, the TL-2 antibody may inter-

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MUFUNE CELL-SURFACE ANTIGENS

TABLE XI STRAIN DISTRIBUTION PATTERN(SDP) OF CMAD LOCI LINKEDON CHROMOSOME 17 (H-2, Tla, Qa-1, -2, -3)5*Y Strain

H-2

B6 B6.Tla" B6.K.2 B6.Kl B6-H-2 B1O.A B10.D2 B1O.Y A A-Tla A. BY A.TL A.TH A.SW AKR-H-2 AKR BALBIc BALBIc-H-2 C57L C57BR C58 DBN1 DBN2 C3HlAn C3HffBi CBA RF 129 SJL PL SWR

b b b b

-

k d

1, 2, 3, 5 2

Pa a

1, 2, 3, 5

U

-

+

b tl t2

2 1, 2, 3, 5

-

S

-

a

Tla 1, 2, 3, 5 -

-

b

-

d b b

2

k k

1, 2, 3, 5 1, 2, 3 , 5 2

k

9

d

k k k k b S U

b

-

-

2 1, 2, 3, 5 1, 2, 3, 5 1, 2, 3 , 5

Qa-1 -

+ + +

Qa-2

Qa-3

+ + +

+ + +

-

-

+ +

+ + + + + + + +

-

-

+ -

-

+ + -

-

+ +

+ + + + + + +

+I-=

+ + -

+I-=

+

-

+ +

+

+

+

+

-

-

-

Data of Frelingeret al. (1974a), Flaherty (1976), Flaherty et al. (1977), and Stanton and Boyse (1976). " Symbols: +, present; -, absent; ., not tested as yet. Sublines may vary (Flaherty, 1978a).

fere with the modulation induced by other sera. The relationship of TL-4 and TL-5 to these general criteria is unknown at present.

5. Znduction of T L Expression in TL- Strains One of the extraordinary findings about the TZa system is that strains whose thymus cells are TL( -) can develop TL(+) leukemias (Old et aZ., 1963).Furthermore some TL(+) strains may express an array of T L

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IAN F. C. MCKENZIE AND TERRY POTTER

specificities different from that of the thymus (see above). The TL system therefore provides a clear example of the variable expression of genes under different conditions. It is apparent that the mouse genome contains the structural genes that are capable of expressing T L under the appropriate circumstances, e.g., the appearance of TL-1, 2, 4 in C57BW6 leukemias when the thymus is TL( -). Such an observation demonstrates the presence of regulatory genes for cell-surface antigens that govern the expression of, and are probably closely linked (Boyse and Old, 1971) to, the structural genes. It has been suggested that the TL specificities are MuLV related. In favor of this suggestion is the similarity with the observation that expression of C;, can be induced in C& mice by Gross virus (MuLV-G). Exposure to MuLV-G occurs spontaneously in older mice of certain strains and may be accompanied by malignant transformation (Stockert et al., 1971; see Section X). It was therefore suggested that the TL- to TL+ conversion, which was accompanied by leukemic transformation, was induced by virus activation either by irradiation or environmental stimuli. Although C57BW6 leukemias rarely express the MuLV-G related surface antigens, GCSA and GX,expression of T L on thymocytes during the preleukemic phase has been demonstrated (Stocked and Old, 1977). However, there is no direct correlation between TL and MuLV expression on preleukemic thymocytes, and furthermore the leukemic transformation is not always accompanied b y the expression of T L specificities. Therefore it is unlikely that TL activation is a direct consequence of infection by MuLV, although this possibility cannot be completely ruled out.

6. Modulation of TL Antigens A unique property of T L antigens is that their expression on normal thymus and leukemic cells can be suppressed by T L antibody (Boyse et al., 1967), a phenomenon referred to as antigenic modulation. This process was demonstrated in uitro by the loss of sensitivity to cytolysis produced by specific antisera and guinea pig complement. In uitro antigenic modulation is an active process, as has also been shown for antibody-induced capping of Ig, H-2, and other determinants; however, in contrast to Ig capping, which requires the binding of divalent or whole antibody molecules, T L antigens can be capped by Fab antibody fragments (Lamm et al., 1968). When rabbit complement is used, the “modulated” cells can be lysed by antibody; this suggests that the antigen-antibody complexes remain on the surface and are not pinocytosed (Stackpole et al., 1974). The finding that the complement component C3 is necessary for mod-

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215

ulation has suggested that C3 intercalates into aggregated T L antigen-antibody complexes and disrupts steric interrelationships essential for activation of guinea pig complement (Stackpole et al., 1978). The modulation phenomenon can be demonstrated in vivo with the loss of demonstrable T L antigens on TL+ leukemias passaged through congenic TL- mice previously immunized against T L (Lamm et al., 1968).The process can also be demonstrated in vivo on normal thymus cells through the “loss” of TL antigens in TL+ strains after exposure to TL antibody received either by injection or by maternal transfer (Lamm et al., 1968). Absorption analysis has demonstrated both in vivo (Boyse et al., 1967) and in vitro (Old et al., 1968) that, during the modulation process, as the amount of T L decreases there is a comparable increase in the amount of detectable H-2D, but not H-2K, products on the cell surface. This has been interpreted as evidence for a shared structural or functional pathway for these antigens. As the modulation effects disappear, the amounts ofH-2D return to normal levels, i.e., to approximately 34% of the amount detected when there are no TL specificities present (Boyse et al., 1968d). However, as these results were obtained from absorption analyses, it may be that these changes represent merely an alteration in the accessibility of the antigens rather than a true difference in the quantitative amounts of antigen present. Another interesting finding arising from these studies was the demonstration of the spatial relationships of H-2K, H-2D, and T L specificities on the cell surface. Boyse et ul. (1968b) clearly demonstrated, in reciprocal blocking studies, that these specificities were arranged on the surface as K, D, and TL, i.e., in the same arrangement as the gene order on chromosome 17.

7 . Histocompatibility Loci Associated with the Tla Complex Earlier studies indicated that there were histocompatibility loci associated with the Tla locus. First, in congenic mice, differing only by TZu, grafts were rejected, but no anti-TL antibody formed (Boyse et al., 1972). Second, in studies of a wide range of H-2 recombinant mice, graft rejection occurred that could be attributed to Tla incompatibilities (Demant and Graff, 1973). However, direct serological examination of isolated epidermal cells failed to reveal the presence of TL specificities on these cells (Scheid et al., 1972). More recently, using recombinant mice where the crossing-over has occurred between TZu and H-2D, the histocompatibility genes associated with the Tlu locus have been defined. There are two loci, H-31 and H-32 [originally called H(TZu-1)and H(Tla-2)],closely linked to Tla, but separate

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IAN F. C. MCKENZIE AND TERRY POTTER

from this complex. The Tla locus itself apparently does not cause skin graft rejection (Flaherty and Wachtel, 1975).

8. Chemistry Analysis of the chemistry of T L antigens suggests that they are very similar to H-2K or D antigens. They exist in tetrameric form (MW -120,000), being comprised of two disulfide-linked heavy chains and two &-microglobulin subunits (Anundi et aZ., 1975; Vitetta et al., 197513, 1976; Peterson et aZ., 1976). Because of the close similarity in structure of H-2, TZa, Qa, and T/t products and their linkage on chromosome 17, it is likely that all arose by duplication of the one ancestral gene (Wlt) and that all are functionally related.

C. THEQu-I Locus The Qa loci map near the TZa locus and anti-Qa antibodies contaminate many anti-TL sera, and so are most appropriately described here. Although T L specificities were considered to be found only on intrathymic T cells, some antisera raised against products of the TZa region were found to contain reactivity against peripheral lymph node cells (Stanton and Boyse, 1976). This finding initially suggested that, in contrast to earlier studies, TL specificities could be expressed on peripheral T cells. An alternative explanation was that T L antisera contained antibodies to unrecognized antigens present on peripheral T lymphocytes and coded for by genes in the TZa region. Thus in the antiserum (C57BL16 x A.TZab)F, anti-ASLI (an A strain leukemia) in addition to the thymus-reactive anti-TL antibody there was also an antibody cytotoxic for 36% of lymph node cells, but not for bone marrow, brain, liver, or kidney (Table XII) (Stanton and Boyse, 1976).This specificity was called Qa-1, and it could be further distinguished from TL specificities by two recombinant strains, B6.Kl and B6.m in which the recombinations occurred between the H-2D and TZa loci. Strain B6.Kl is Qa-1+,Qa-2+,TL-. The recombinant strains also fix the gene order as H-2D, Qa-1, Qa-2, Tla (Stanton and Boyse, 1976). Thus far, only one Qa-1 specificity has been defined and identified in several strains (Table XI). It should be noted that the strain distribution pattern of Qa-1 is different from that of the T L specificities. Approximately two-thirds of T cell-enriched lymph node populations are Qa-1+,and by cytotoxicity it was found that Qa-1 is expressed on 60% of Lyl cells in the spleen and on a proportion of Ly123 cells; i.e., Qa-1 can distinguish subsets within these two populations (Stanton et d., 1978; Cantoret d., 1978b). That Qa-1 occurs predominantly on T cells was shown by the selective inhibition of mitogen respon-

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TABLE XI1 RELATIVEDISTRIBUTIONOF SOME CMAD ON THYMUS, LYMPHNODE,SPLEEN,AND BONE MARROW^ Antigen

Thymus

Lymph node

Spleen

Bone marrow

Thy-1 Tla Ly- 1 Ly-2

100 95 95 95

60-70 <5 60-70 2.1-35 2.2-65 30 30 40-55 70 70-80 65 30-40 35 30 35 65 35

30-40

10 5

Ly-3 Ly-4 Ly-5b Ly-6 Ly-7 Ly-8 Lyb-2 Lyb-4 LyM-1 Qa- 1 Qa-2 Qa-3

95 10 95 5-10 NT 15 5 5 NT

+

20

5

<5 35-45 2.1-35 2.2-35-45 20 60 30 25-30 70-80 40-50 60 45

70 20 35-40

+

5 5 5 35 5-10 15 NT NT 40 5 75 5 5-10 5

The figures represent the percentage of cells reacting in each tissue-usually determined as a cytotoxic index. The references to each specificity will be found in the appropriate part of Section VI. +, Antigen present and detected by absorption (percent positive cells not available); NT, not tested. Data of Komuro e t al. (197513). However, we have found that Ly-5 is expressed on most lymphoid cells (see text).

siveness by anti-Qa-1 serum + C' when the PHA response was ablated, the Con A response was reduced to 40%, but the LPS response was not affected (Stanton et al., 1978). Furthermore, although Qa-1 occurs on a large proportion of Lyl cells, it is not present on the cells proliferating in an MLR, nor were TKor their precursors found to be Qa-l+ (Stanton et al., 1978). By contrast, antibody responses to SRC were increased by treatment with anti-Qa-1 + C', suggesting that Ts are Qa-l+ (Stanton et al., 1978). Clearly the Qa-1 specificity will be a useful marker to distinguish T-cell subsets. It should also be noted that the feedback suppressor cell is Ly123Qa-1+ (Eardley et al., 1978; Cantor et al., 1978a), and this cell may be activated by a LylQa-l+ helper cell (Cantor et al., 197813) (this is discussed further in Section X1,D). In studies of CML, Klein and Chiang (1978) observed that A.TH lymphocytes stimulated by A.TL cross-reacted with other targets of unrelated H-2 haplotypes. Selection of appropriate targets eliminated the possibility that the H-2K, H-2D7 or Z regions participated in this

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IAN F. C. MCKENZIE AND TERRY POTTER

cross-reactivity. The participating locus was mapped to the right of H-2D. As the SDP of the CML reactivity correlated with the expression of Qa-1 antigens, it was postulated that Qa-1 antigens also function as targets in CML (Klein, 197813).

D. THEQa-2 AND Qa-3 LOCI The production of two new recombinant C57BW6 congenic strains (K1 and K2) located the H-31 and H-32 histocompatibility loci of the TZa region more precisely and simultaneously led to the definition of the Qa-2 locus (Flaherty, 1976). The antiserum B6,Kl anti-B6 reacted with lymph node, spleen, thymus-in order of decreasing reactivity-and also with liver (weakly by absorption), but not red blood cells, brain, kidney, or epidermal cells. The Qa-2 antigens thus seem to be lymphocyte specific. Absorption analysis of this serum defined two determinants: (a) the Qa-2 specificity, which was found predominantly on T cells of the thymus, bone marrow, lymph node, and spleen, as well as being present on a small population of IgM-bearing cells, and (b) the Qa-3 specificity, which was present only on a subpopulation of peripheral T lymphocytes (Flaherty et aZ., 1978a) (Table XII). Specific antiserum to the Qa-2 antigen was prepared against the ERLD leukemia, and the Qa-3 antiserum was raised against lymph node and thymus cells, and absorbed with the EL-4 tumor (Table X). It should be noted that, as for the T L specificities, mice of the same H-2 haplotype can differ at the Qa-2 locus. Furthermore sublines may vary; e.g., BALB/c Boy is Qa-2+whereas BALB/c By is Qa-2- (Flaherty et al., 1978a). Forman and Flaherty (1978) were able to generate cytotoxic CML reactive T cells by immunization and in uitro restimulation between these two sublines. As with Qa-1 locus differences, the effector cells generated cross-reacted with strains expressing the Qa-2 antigen, suggesting that Qa-2 may also act as a CML locus (Forman and Flaherty, 1978). As with Qa-1, only one specificity has so far been identified for the Qa-2 and Qa-3 loci (Table XI). Attempts to raise antisera against an alternate specificity of these loci have so far been unsuccessful (Flaherty et d.,1978a). The linkage relationships of Qa-1, Qa-2 to TZa and H-2 are mentioned above, but Qa-3 has not been mapped in relation to Qa-2. As can be seen in Table XI, many H-2 congenic lines differ at the Tla region. Thus many H-2 and Ia antisera may contain contaminant antibodies directed against specificities determined by this region; e.g., A.TL anti-A.TH antisera contain antibodies against a lymph node alloantigen(s), as well as against IaS specificities (Flaherty et al., 1977a)-presumably the Qa-1 antibody, The H-2 and Tla loci are both complex, in that they determine many specificities, and this may also

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be true for the Qa loci. It has been suggested that the MHC concept of H-2 be extended to include the TZa region (Flaherty, 1976), particularly as there are several histocompatibility loci (H-31 and H-32) mapping within the region between H-2D and Tla (Flaherty and Wachtel, 1975). Supporting evidence for this concept comes from the observation that all H-2k strains so far studied are Qa-2-, whereas most other strains are Qa-2+ (Table XI). In the few functional studies that have been done with Qa-2 and Qa-3 antisera, it was found that depletion with antisera and complement abolished the proliferative response to Con A and PHA mitogens, but had no effect on the LPS response (Flaherty et aZ., 197813). Qa-2 antigens have a similar molecular weight (43,000)to H-2 and T L specificities, and similarly they are associated with a Pzmicroglobulin subunit (Michaelson et al., 1977). However, as they cap independently of these antigens, they are apparently represented on distinct molecules (Michaelson et al., 1977). As only one specificity has been defined at each of the Qa loci, it is possible that these antigens are virus associated. Recently, Meruelo et al. (1978) showed that MuLV determinants are selectively incorporated into H-2D products, and, in conjunction with the linkage of Qa, H-2D, and TZa, it is tempting to postulate that these findings suggest that such molecules are susceptible to incorporation of MuLV determinants. Additional Qa loci have recently been defined by monoclonal antibodies (see Section V1,O).

E. THELy-1 Locus (Lyt-1) The Ly-1 and Ly-2 specificities were the first clearly defined lymphocyte-specific antigenic systems (Boyse et al., 1968a), although there were suggestions from other studies that such systems existed (Sanderson, 1965).Both Ly-1 and Ly-2 loci (originally called LyA and LyB) were initially defined by sera produced against leukemias (see Table V for the antiserum combinations). The specificity “mu” described by Cherry and Snell (1969) with the antiserum ( R F x RIII)F, anti-C3H was most likely to be Ly-1.1. The use of the Ly-1 and Ly-2 antisera in recent years for the serological distinction of helper cells from killer T lymphocytes has added great interest to the study of these interesting CMAD. 1 . Genetics The Ly-1 locus is situated on chromosome 19 linked to ruby-eye (rm) (Table XIII). The locus is clearly distinct and separate from all other loci determining CMAD. As with most of the other Ly loci, this locus consists of two alleles, Ly-1 a and Ly-l*, which code for specificities

220

IAN F. C. MCKENZIE AND TERRY POTTER TABLE XI11 LINKAGERELATIONSHIPSOF SOME CMAD

Locus

Chromosome

Tla Qa-1 Qa-2 Thy-1 Ly-1 Ly-2 Ly-3 Ly-4 Lyb-2 Lyb-4 Lyb-6 Mls LyM-I

17 17 17 9 19 6 6 2 4 4 4

1 1

(Linkage group)

Useful markers

H-2D H-2D H-2D Dilute ( d ) ,malic enzyme (Mod-I) Ruby-eye ( r u ) Ldr-1, mi, Ly-3 I B peptide, Ly-2

H-3 LPS, Lyb-4, Mup, Lyb-6 Mls, Dip-1, Lyb-2, Lyb-6, M U P , LPS Lyb-2, LPS, Lyb-6, MUP LyM-I, Dip-1 M l s , Dip-]

Ly-1.1 and Ly-1.2. As shown in Table VII, most mouse strains carry the Ly-1.2 specificity with the exception of CBA, C3H, DBM1, and DBM2. 2 . Production of Antisera Using the Ly-1 congenic lines, which were produced on C57BL16 background, specific antisera can be produced either by crossimmunization between the congenic line and the parent, or by absorption with the congenic line (Tables IV and V). The anti-Ly-1.1 serum is one of the easiest to produce in congenic lines, and either (C57BU 6 x BALB/c)Fl anti-B6Ly-la (Shen et al., 1975) or (C57BU 6 x LP.RIII)Fl anti-B6Ly-1 a are suitable combinations for the production of antisera. AAer absorption for autoantibody, the sera have high titers on thymus (in excess of 1/1000), and lymph node and spleen reactions are also strong. The anti-Ly-1.2 serum is generally raised as C3H anti-CE; however, this antiserum is contaminated with an antibody recognizing an antigenic determinant on the natural killer cell (Glimcher et al., 1977) (see Section VIII). Neither Shen et al. (1975) nor our group have been able to produce an anti-Ly-1.2 serum using the Ly-1 congenic lines. The Ly-1.2 serum therefore has to be tested in congenic strains, or absorbed with these prior to use (Section 111).The Ly-1.2 sera are usually not as strong as the Ly-1.1 sera (Table V), with titers on thymus rarely greater than 1:256.

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3. Tissue Distribution Direct testing and absorption analysis have demonstrated the Ly-1.1 and Ly-1.2 specificities to be present on all thymocytes, a proportion of Thy-l+ lymphocytes (Table XII),but absent from nonlymphoid tissues. The Ly-1, Ly-2, and Ly-3 specificities are expressed within the Thy-l+ population, and only 5% of Thy-l+ lymphocytes do not express Ly-1, Ly-2, or Ly-3 (Shikuet al., 1975).By cytotoxicity testing, T cells in the lymph nodes and spleen can be separated into three distinct populations: (i) Ly1+2+3+:50-55% of peripheral T cells; (ii) Ly1+2-3-: 3 0 4 5 % of peripheral T cells; (iii) Lyl-2+3+: 5 1 0 % of peripheral T cells. Therefore 80-90% of peripheral T lymphocytes express Ly-1, and 6 6 7 0 % express Ly-2 and Ly-3. This serological differentiation also have a functional counterpart, as the three defined populations have different functions (Section XI). The use of more-sensitive techniques, such as the FACS, has been unsuccessful in isolating an Lyl-2+3+ population, which suggests that all peripheral T cells carry the Ly-1 antigen (B. Mathieson, personal communication). The implication of such a finding is that the Ly1-2+3+ population identified by cytotoxicity expresses a low concentration of Ly-1, which cannot bind sufficient antibody to activate the complement system. Absorption analysis suggests that all thymocytes express similar amounts of the Ly-1, -2, and -3 specificities. However, separation of thymocytes with the FACS has identified a subpopulation within the cortisone-resistant pool that is Ly1+2/3-, constituting about 10% of normal thymus cells (Mathieson et al., 1979). Comparative absorption studies with peripheral lymphoid cells and thymocytes have demonstrated that Ly-l+ T cells in different tissues carry similar amounts of the Ly-1 antigen (Boyse et al., 1968a).This is in contrast to expression of Thy-1, which is very much decreased in peripheral tissues. 4 . Ly-1 Distribution on Tumors

Although the Ly-1 specificities were originally described by antisera directed against leukemias, not all T-cell leukemias are Ly-l+, and a restricted distribution of Ly specificities is found in leukemias as well as with normal cells (see Section XIV). Indeed most BALB/c lymphomas were found to be Lyl-2+ (Mathieson et al., 1978).There is no correlation between T L and Ly-1 on tumors. 5. Chemistry Recent analysis of Ly-1.1 specificity by cell-surface labeling, immunoprecipitation, and analysis on SDS-PAGE showed the Ly-1.1

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specificity to be a trypsin-resistant glycoprotein of molecular weight 67,000 (Durda et al., 1978). However, whether the antigenic specificity is determined by protein or carbohydrate has not been determined. 6. Functional Studies Although these results are discussed in detail later, cells bearing the Ly-1 determinant have important functional properties. These are two populations: Ly1+2+3+and Ly1+2-3-. The Ly1+2+3+cells are generally considered to be the precursor cell in many systems as well as having an amplifier or augmenting function in other systems. Killer cells in a syngeneic tumor system were also shown to be Ly1+2+3+. The Ly1+2-3- cell has T helper functions in many different systems and is involved in DTH reactions, both as an effector cell and as a suppressor cell. These functions are fully discussed in Section

XI. F. THELy-2 AND Ly-3 LOCI The Ly-2 locus (originally called Ly-B) (Boyse et al., 1968a) was described simultaneously with Ly-1 , whereas the Ly-3 (originally called Ly-C) locus was described later (Boyse et al., 1971). However, as the Ly-2 and Ly-3 loci are closely linked genetically and as these two Ly antigens are always present on the same tissues (with one possible exception-a radiation-induced thymoma), they will be described together in this section. 1 . Genetics Ly-2 (Lyt-2)and Ly-3 (Lyt-3)loci are closely linked on chromosome 6 and are linked to the lactic dehydrogenase regulatory (Ldr-1)and microphthalmia (mi) loci (Table XIII). These two Ly loci are closely linked and have not as yet been separated by recombination. The close genetic linkage of Ly-2, Ly-3 specificities is also reflected in the close proximity of the specificities on the cell surface as demonstrated by their mutual interference in blocking tests (Boyse et al., 1968b);this could indicate that these specificities are either on the same molecule or on separate, but closely associated, molecules. At one stage there was some evidence that the Ly-3 antigen was structurally related to immunoglobulin light chains, as the IB peptide, a marker present on the variable region of some light chains, had a similar strain distribution to the Ly-3 specificity (Gottlieb, 1974), and this SDP included the Ly-2, 3 congenic lines. It was therefore considered that the Ly-3.1 specificity and the peptide were identical structures. However, chemical analysis of the Ly-3.1 specificity isolated a glycoprotein of 35,000

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MW but no polypeptide of 22,500 MW corresponding to an immunoglobulin light chain (Durda and Gottlieb, 1976; Gottlieb and Durda, 1976). Therefore it is unlikely that a single genetic locus codes for the IB peptide and for the Ly-3.1 specificity, but that they are coded for by closely linked loci. More recently, another locus, Pc-8, which determines an allotypic marker on a kappa light chain, was found to map approximately 3 cM from the Ly-2,3 loci (Gottlieb, 1976; Claflin et al., 1978). The Pc-8 marker is found on the kappa chain of antiphosphocholine antibodies, which show the same functional and idiotypic characteristics as a Pc-8 binding myeloma protein, HOPC8. While it is tempting to suggest that there may be some functional relationship between the presence of these markers on immunoglobulin light chains and the presence of Ly-2, 3 specificities on functional T-cell populations, the evidence for an association other than by genetic linkage is not clear at present. However, the possible significance of this observation has been discussed extensively elsewhere (Gottlieb, 1976; Claflin et al., 1978). The finding that cytotoxic cells to Ly-2 and Ly-3 determinants could be induced in a secondary in vitro response, suggests that these alloantigens may function as targets in CML (Rollingho f f e t al., 1977). 2. Production of Antiserum The Ly-2,3 antisera are produced as shown in Table V. These have significantly lower titers on thymus and peripheral cells than do the Ly-1 antisera. With the exception of the Ly-2.2 specificity, antisera to Ly-2 and Ly-3 specificities cannot be produced in congenic lines (see Section 111)and are produced in other combinations (see Table V and Shen et al., 1975). In these particular cases, the antisera should be tested on congenic lines in experimental systems or absorbed with these lines prior to use. The comments made in relation to the production of antisera (Section 111)are of particular relevance to the Ly-2 and Ly-3 specificities. It should be noted that CE and C58 strains are used extensively for these immunizations because of their unique Ly phenotypes-CE is Ly-1.2, 2.1, and 3.2 and C58 is Ly-1.2, 2.1, 3.1. Both of these strains are very useful, but unfortunately neither one is very good breeding stock. To overcome this problem, Shen (1977) has produced the derived stocks (DS). These donor strains for immunizations have been crossed to their respective recipients, an intercross made, and the mice with the homozygous Ly phenotype of the donor are selected and maintained, but not inbred. These DS lines breed substantially better than the CE and C58 strains and are useful for antiserum production.

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3. Tissue Distribution The tissue distribution of the Ly-1, -2, -3 specificities have been referred to above and in Table XII. Whereas virtually all lymphocytes are sensitive to Ly-1, -2, -3 antisera, there are differences between the Ly-1 and Ly-2,3 systems with regard to sensitivity of peripheral T cells to lysis. Ly-1 antisera have approximately the same titers and the same absorptive capacity on peripheral T cells as on thymocytes (Boyse et al., 1968a). However, Ly-2, 3 antisera have considerably lower titers on peripheral T cells (Konda et al., 1973). Furthermore, lymph node cells of Ly-2.1+ strains have a low absorptive capacity, and the Ly-2.1 specificity is detectable by cytotoxicity on only 30% of lymph node cells and on 20% spleen cells in comparison with Ly-2.2 (Table XII). Schlesinger and Schoen (1971) showed that cytotoxic activity in Ly-2.1 antisera produced in C57BL against C3H thymocytes was confined to the IgM fraction. If this is the case for Ly-2.1 antibodies raised between other strain combinations, then the apparent low expression of Ly-2.1 on peripheral T lymphocytes may merely reflect differences in antibody class rather than being a unique feature of this specificity. With regard to leukemias, the comments made for Ly-1 above also apply to Ly-2, in that leukemias have been found to have a restricted distribution of the Ly-2, 3 specificities. We have recently identified several irradiation-induced tumors (Hogarth and McKenzie, unpublished) that are Ly2+3-, although the Ly-2 reaction may be due to a contaminant antibody. 4 . Chemistry Immunoprecipitation and SDS-PAGE analysis of Ly-2.1 and Ly-3.1 specificities indicated that they were glycoproteins of molecular weight 35,000 (Durda and Gottlieb, 1978). Sequential precipitation of the Ly-2.1 and Ly-3.1 antigens demonstrated that they precipitate independently, which clearly demonstrated that these antigenic determinants reside on different molecules. These antigens were complexed with each other in the NP-40 extract, but trypsin dissociated the two specificities, where Ly-3.1 was preferentially destroyed (Durda and Gottlieb, 1978).

5. Functional Studies Although the functional studies are discussed in detail later, the Lyl-,2+,3+ cell has been identified as the phenotype of a suppressor cell for antibody formation and also of the killer cell involved in cellmediated lysis reactions. The two cell populations are not the same, as the suppressor cell carries Ia (IJ) determinants that are absent from the killer cell.

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6. H Loci Associated with the Ly-l,2,3 Loci H(Ly) Histocompatibility loci have been found to be associated with some of the Ly loci, although there are some differences noted between the different congenic strains used. However, the significance of graft rejection in the Ly congenic strains should be discussed. Skin graft rejection between strains congenic for an Ly locus, i.e., selected on the basis of CMAD of lymphocytes, could have three different interpretations. First, the Ly and H locus could be identical, i.e., the same CMAD expressed on skin cells within a graft (epidermal or endothelial antigen presumably), e.g., for Ia and H-Y specificities (Sections VI,P and VIII). Second, the loci could be closely linked, and when the appropriate recombinants are available the loci will be separated, e.g., the former H(Tla) loci have now been found to be distinct from the Tla locus and are now called H-31 and H-32. Third, the two loci (H and Ly ) could be quite separate, not linked, the H locus being carried along by accident in the production of Ly congenic lines. In this particular case, further backcrossing should eliminate the extra locus, or alternatively, the two should segregate independently in a backcross. In general, the demonstration of H loci in association with Ly loci has been accompanied by some difficulties. The loci are often "weak," i.e., give slow rejection patterns, and it is usual to use nonimmune as well as immunized mice as recipients. Furthermore, mice may reject a first graft and accept a second, and "crisis" episodes occur whereby the graft undergoes partial rejection, but is not totally destroyed and later recovers. As well, mice may or may not form antibody after skin grafting, and this does not always correlate with graft rejection. a. H(Ly-1). In studies of the Boyse B6-Ly-I " (N8) congenic line, first grafts were rejected and second grafts were rejected in accelerated fashion, with some crisis episodes noted in the direction B6-Ly-I " + B6. In the reverse direction, grafts were also rejected (Flaherty and Bennett, 1973). When B6-Ly-I" mice were challenged with ERLD (Ly-1.2+),only immunized mice rejected the tumor. Skin-grafted mice failed to produce anti-Ly antibody. In our laboratory (Henning and McKenzie, unpublished results), the Cherry Ly-l congenic line does not reject C57BW6 grafts, nor are grafts rejected in the reciprocal direction. The two Ly-l congenic lines are currently being tested against each other, but at this stage it appears the H(Ly-I ) is adjacent to, but separate from Ly-l (Flaherty and Bennett, 1973). b. H(Ly-2, Ly-3). When the Boyse B 6 - L ~ - 2 ~(N7) , 3 ~ congenic line was used as a donor for grafting, first grafts were accepted by C57BW6, although a weak rejection was noted in the opposite direction (Flaherty and Bennett, 1973). In our studies, the Cherry B6-Ly-2",3" congenic strain was not rejected by C57BW6 nor was there graft rejection

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in the reciprocal direction. Presumably, there is an H locus, associated with Ly-2, Ly-3 but separate from these loci. c. H(Ly-2). When B6-Ly-2" (N8) received a C57BW6 first skin graft, slow rejection was observed, and this was also observed in the reverse direction (Flaherty and Bennett, 1973). Immunized mice rejected their grafts more rapidly. However, when the B6-Ly-2" (N16) mice were examined, first grafts were not rejected and rejections were observed only in immunized mice. This was interpreted as indicative of two H loci associated with the Ly-2 locus. Again, our own studies (Henning and McKenzie) have not found any graft rejection for Ly-2 incompatibilities with the Cherry B6-Ly-2-Ly-3 congenic line. Presumably the H(Ly-2-N16)locus is closely linked to Ly-2 but is missing from the Cherry congenic line. G. THEL y 4 Locus (Lyb-1) The L y 4 locus described by Snell et al. (1973) was the first Ly specificity to make extensive use of the recombinant inbred lines. Other loci defined using these lines are Ly-6 and Ly-7. The locus is of interest, as it was one of the first to be described that had a predominant tissue distribution to B cells. However, it now appears that specificities may also be present on some T cells.

1 . Genetics The L y 4 locus is situated on chromosome 2 closely linked to the H-3 locus and also linked to other markers, such as un, w e , and pa. The linkage has not been demonstrated formally, but presumed from the identical strain distribution pattern of the inbred and recombinant inbred strains, as well as the finding that several of the H-3 congenic lines such as BlO.LP, BlO.C(28NX) all carry the Ly-4.1 specificity compared with Ly-4.2 specificity of C57BUlOScSn. Appropriate backcross studies have demonstrated this specificity to be separate from the H-2, Ly-1, -2, -3 and from the Ea and H loci (Snell et al., 1973). 2. Production of Antisera Antiserum to the Ly-4.2 specificity is produced as (BALB/c x SWR)F1 anti-BlO.D2. This antiserum also contains several other specificities (Table V). Immunizing between the H-3 congenic lines or between F1 hybrids of the congenic lines has not led to specific Ly-4 antibody, and so the sera have to be either tested on congenic lines or absorbed with congenic lines prior to use.

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3. Tissue Distribution In the original description of these specificities, it was clear that anti-Ly-4.2 had reactivity restricted to B cells. There was no direct reaction and little absorptive capacity with thymus cells (McKenzie, 1975a; McKenzie and Snell, 1975). Furthermore, thymus cells did not appear to react with an anti-Ly-4.2 serum in a sensitive immunoelectron microscopic method (Aoki et al., 1974b). The antiserum reacted with a proportion of cells in lymph node and spleen suggestive of their B-cell distribution (60% and 30%, respectively: Table XII); furthermore, when used with an anti-Thy-1 serum, additive effects were apparent. Studies of T cell-depleted mice (nude mice, ATXBM, ALStreated mice) demonstrate increased numbers of cells reacting with Ly-4.2 antiserum. All these findings pointed to differential distribution of Ly-4.2 specificity on B cells. However, it was noted that some pools of antisera did contain an additional reactivity. More recently studies on isolated T cells, prepared by elution from nylon wool columns, show that Ly-4 specificities are present on a subpopulation of T cells and that these antigens are differentially expressed on splenic T cells and lymph node T cells. A recent study (Gani and Summerell, 1977) has thrown some doubt on the restriction of Ly-4.2 specificity to B cells, as in these studies almost equal distribution of Ly-4 was found on T cells as on B cells, except that the thymus reaction was also absent. Cross-absorption studies using purified T cells and B cells demonstrated what seemed to be the same specificity present on both cells. The differences between the two studies are most likely in the antisera produced, in sources of complement used, and possibly in the different sublines of mice used. However, it is difficult to reconcile the absorption studies of Gani and Summerell (1977) with our data, for if weaker pools of sera were used and the complement was different, one would expect cells with low expression of the antigen not to be lysed, which was not the case in their studies. 4 . H-2-Linked Gene Efecting the Expression of L y 4 Speci$cities In the original studies of Ly-4 specificities it was noticed that H-2k strains gave weak reactions with Ly-4.2 antisera (Snell et al., 1973). Furthermore in a backcross of (C57BW6 x C3H)F, backcrossed to C57BIJ6, H-2k heterozygotes (bk) had a much lower reactivity with Ly-4.2 than did the H-2b homozygous mice. In other studies we have found Ly-4.2 and Ly-4.1 to be expressed in amounts of one-fifth to one-tenth in H-2k strains as compared with other strains. For example, BlO.BR, C58, and C57BWcd are extremely difficult to type with Ly-4.2 reagents, as are other H-2k strains with the Ly-4.1 specificity. Studies

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in recombinant mice indicate that the gene responsible for this effect maps near the D end of the H-2 complex (McKenzie, unpublished observations). 5 . Functional Studies Extensive functional testing has been done with Ly-4.2 and to a lesser extent with Ly-4.1 antisera, using pools of sera with reactivity directed predominantly to B cells. All the studies indicate the Ly-4 specificity to be carried on B cells, particularly on early B cells, although much smaller amounts of Ly-4 are present on antibody-forming cells (McKenzie et al., 1975). The Ly-4 specificity is also carried on SRC antigen-binding cells and has been found on cells responding to pokeweed and lipopolysaccharide mitogens but absent from cells responding to Con A and PHA (McKenzie and Plate, 1974; McKenzie, 1975a). Certain functional T cells, such as MLR responder cells and killer T cells, lacked the Ly-4 specificities, which were found to be carried predominantly by an MLR stimulator B cell (Plate and McKenzie, 1973). These studies suggest that certain pools of antisera contain predominantly anti-B cell activity. H. THE Ly-5 (Lyt-4) Locus The Ly-5 locus was originally defined by sera produced in reciprocal immunizations between the H-28 strains ASW and SJL (Komuro et d,1975b). Although the serum was produced using spleen and lymph node cells for immunization, the antibody was found to react with virtually all thymocytes, and the initial report demonstrated a peripheral tissue distribution characteristic of T cells (Komuro et al., 1975b). When tested on thymus cells, the antiserum reacted in a backcross with 50% of the population, indicating the segregation of a single locus. Most strains carry the Ly-5.1 specificity with the exception of SJL, DA, and STS, which express the Ly-5.2 specificity (Table VII). Linkage studies have not mapped the Ly-5locus as yet, but it is not linked to H-2, Thy-], Ly-2, or Ly-3 loci. It should be noted (Table IV) that the congenic line for Ly-5 is well toward final preparation, which should sort out some of the unexpected reactions encountered with this antiserum. Of practical importance is the finding that the Ly-5 antisera contain considerable amounts of autoantibody and extensive absorptions are required to remove this antibody. Although the original description of Ly-5 demonstrated a T-cell distribution of this antigen, several complicating factors have come to light since the original description. First, both A.SW anti-SJL and SJL anti-A.SW sera contain additional antibodies, e.g., to Lyb-2 specificities (Table V). However, in our laboratory we have also found

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Ly-5 reactivity, by absorption, with several BALB/c myelomas and with spleen cells of nude mice. Nonetheless, in many circumstances we find the antiserum to react preferentially with T cells. According to some more recent data (F. W. Shen, personal communication), the tissue distribution in certain pools of sera may be broader than that originally defined and may possibly include kidney and other tissues. In our laboratory, we have also found the serum to be reactive with macrophages, neutrophils, and eosinophils. Functional testing in T-helper, T-killer, and mitogen stimulation studies show that Ly-5 is present on all functional cells (Woody et d.,1977; Michaelides and McKenzie, unpublished results). Other studies, with radiationinduced tumors, have suggested that prothymocytes express Ly-5, but not Ly-1, -2, or -3 antigens (see Section XIV). Biochemical analysis of biosynthetically labeled thymus cells following precipitation and lentil lectin chromatography has identified an Ly-5.1 molecule of MW 130,000 on SDS run under both reducing and nonreducing conditions (Ewald and McKenzie, in press). I. THE Ly-6 Locus The Ly-6 locus was initially defined by the Ly-6.2 specificity and has proved to be of interest, as it was the first specificity to be found in peripheral rather than thymic T cells, i.e., to be a differentiation marker of mature T cells, This observation has also been substantiated by functional testing. More recent studies have indicated that the Ly-6 specificities may be identical to several other specificities, and it is very likely that the antigenic specificity is virus related or even a viral determinant. Furthermore, the specificities may have a broader tissue distribution than to peripheral T cells.

1 . Genetics The Ly-6 specificities have an interesting strain distribution pattern (see Table VII); certain strains, such as C57BW6, are clearly reactive with the Ly-6.2 antiserum. However, some strains, e.g., DBN1, DBNS, SWR, and in particular 129, appear to have a poor representation of the Ly-6.2 specificity and in early studies gave rise to difficulties in the typing of these strains. Strain 129, for example, appears to express only one-tenth of the Ly-6.2 specificity as found on the C57BU6 lines. The production of the Ly-6 congenic line C3H.B6 enabled the Ly-6.1 specificity to be defined, and this has been confirmed by the strain distribution pattern and testing on the recombinant inbred and congenic lines (McKenzie, unpublished). Although the Ly-6 locus has not yet been mapped, it is not linked to H-2, Ly-1, -2, -3, -4, -5, and -7 loci, several of the E a loci as well as Thy-1 and TZa loci.

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2 . Production of Antisera The Ly-6.2 specificity was initially described by the serum (BALB/c x A)FI anti-CXBD (a Bailey recombinant inbred strain), but more recently a better serum has been produced using (CXBG x A)F, antiCXBE (McKenzie et al., 1977a).The serum has also been produced in other laboratories as (CBA x A.-Thy-1 ")F1anti-AKR thymocytes (Horton et al., 1978) and as (BALB/c x A)FI anti-BlO.D2 (Halloran et al., personal communication). This latter serum also contains anti-Ly-4.2, but was tested on Ly-4.2- strains. It was of interest to find that all descriptions of the production of the Ly-6.2 specificity have required the presence of A strain in the recipient, and preliminary studies have 'indicated that this is most likely to be due to a single gene that is not H-2 linked (unpublished observations). Recently, the Ly-6.1 specificity has also been defined using the serum (C3H.B6 x B6)F1anti-C3H.

3. Tissue Distribution Original studies of the Ly-6.2 specificity in C57BL16 mice demonstrated its presence on approximately 5% thymus cells, 25% spleen cells, 70% lymph node cells, and 15% bone marrow cells (Table XII). In addition, cortisone-resistant thymocytes were reactive, and absorption studies showed the relative absence of Ly-6.2 from the majority of thymus cells. More recently, several studies have indicated that strains AKR and DBN2 have approximately 30% of thymus cells reactive with the Ly-6.2 specificity (M. A. Horton et al., personal communication). Absorption studies indicated the Ly-6.2 specificity to be absent from red cells, liver, and brain but to be present on kidney tissue and on a T-cell tumor (McKenzie et al., 1977a). More recently, absorption studies have indicated extensive amounts of Ly-6.2 on kidney tissue in approximately the same amount as H-2 antigens (P. Halloran et al., personal communication). The evidence for the predominant T-cell distribution of the Ly-6.2 specificity was based on the distribution of this antigen in the lymphoid tissues as well as studies in T cell-depleted mice, e.g., by treatment with anti-Thy-1 serum and complement or, by using nude mice. Furthermore, there were no additive effects in serological tests with anti-Thy-1 serum, but additive effects did occur with an anti-Ia serum (McKenzie et al., 1977a). Furthermore, Ly-6.2 was shown to be present on the tumor EM, and absent from tumor BPC-1, a B-cell plasmacytoma of C57BW6 origin. The earlier studies failed to find any inhibition by anti-Ly-6.2 on the IgM plaque-forming response; however, more-recent studies from our laboratory and from several other centers have found IgM PFC to be Ly-6+, and in addition, Con A and LPS blast cells were found to be

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Ly-6+ (A. Feeney, M. Horton, M. Michaelides, personal communications). Furthermore, in studying the response to B-cell mitogens (LPS and pokeweed mitogen) identified Ly-6.2+ cells, presumably B cells, which were mitogen responsive (M. Michaelides, personal conimunication). At present it could be considered that Ly-6 is present predominantly on T cells and activated B cells. Using a different antiserum, P. Halloran (personal communication), has identified 30%of B cells as being Ly-6+ and a greater proportion of reactive bone marrow cells than reported above. Ly-6 specificities have also been identified on three different mastocytomas (P815, CXBGABMCT-1,CB6AMC4) and peritoneal exudate mast cells (P. M. Hogarth, P. Halloran, personal communications). 4 . Relationship of Ly-6 Specificities to Other Specificities It was noted that the strain distribution pattern of Ly-6.2 and ALA1.2 were similar with several exceptions, notably with strains DBN1, DBN2, SWR, and 129. Studies in the author’s laboratory and by Drs. A. Feeney and M. Horton have now clarified the situation, and it appears very likely that the Ly-6 and ALA loci are identical. First, the strain distribution patterns, particularly with Ly-6.2 and ALA- 1.2 are now identical in both conventional and recombinant inbred strains (BXH and CXB). This identity is also seen in the Ly-6 congenic line (C3H.B6) (Morgan and McKenzie, unpublished results). In a backcross study, Ly-6.2, ALA-1.2, and Ly-8.2 were found to segregate together (Horton et al., 1978). It is therefore likely that the three loci are identical. The antiserum BALB/c anti-DBN2, described some years ago as recognizing a specificity called DAG (Sachs et al., 1973), also has the same strain distribution pattern, including the CXB recombinant inbred lines. Similarly the locus identified as Ren-l (P. Halloranet al., personal communication), made by immunizing with kidney, is apparently the same as Ly-6; i.e., Ly-6, ALA-I, Ly-8, DAG, Ren-2 may all be identical loci, although the antiserum used to define each of these may well contain other specificities. This sort of duplication emphasizes some of the problems that may arise in studying antisera to CMAD. The ALA-1.2 specificity to be described below was produced b y immunizingwithblastcells,andthe combinationusedforantiserumproduction also led to production of other antibodies, which were removed b y absorption with donor thymus cells. Apparently blast cells express greater amounts of Ly-6 (ALA) than do peripheral cells, hence the suggestion that the specificity was confined to blast cells. However, this was probably a reflection of the strength of the antisera rather than

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a true indication of tissue distribution. In other sera produced more recently, Dr. Feeney (personal communication) has produced ALA- 1 antisera that react directly with peripheral lymphocytes.

5. Relationship of Ly-6 SpecGcities to Viral Determinants The possibility of antiviral antibodies contaminating or being important in the Ly-6 sera was first raised by M. Horton (personal communication), during investigation of antisera to Ly-6.2, which was produced with AKR, a strain heavily infected with MuLV, as the donor. Absorptions with both virus-infected and noninfected N3T3 tumor cells indicated that viral antibody was probably not involved, as both infected and noninfected tissues absorbed the serum equally well (M. A. Horton et al., personal communication). In our laboratory we have been using purified AKR virus for absorptions and can clear all Ly-6.2 activity completely from the serum by this absorption whereas Ly-6.1 remains untouched. It is therefore likely that in some, if not all, batches of anti-Ly-6.2 sera the serum is directed toward a viral component. 6. Functional Studies

A number of functional studies done with the Ly-6 specificities indicate that they are present on certain functional T cells. Woody (1977) has described the presence of the Ly-6 specificity on killer cells but its absence from their precursors, demonstrating conclusively the presence of this specificity on activated cells. In addition Woody et al. (1977)demonstrated that only 50-60% of T cells were Ly-6+.Horton et al. (personal communication) have extended the Ly-6 testing of functional killer T cells (TK), and their results are shown in Table XIV. In all systems, the effector cells are Ly-6+ and the precursor cells are mostly Ly-6-, although in the TNP-modified self and in the TABLE XIV LY-6 PHENOTYPE OF KILLER T CELLS" Reactivity Allogeneic TNP-modified self Sendai

H-Y Tumor (Anti P815) Xenogeneic (anti-Wistar rat)

Precursor

-

*

Memory cell

Effector cell

? ?

+ + + + + +

+ +

?

-

r

?

Published by permission of Dr. M. Horton.

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xenogeneic (anti-Wistar rat) system there was a suggestion that some of the precursor cells were Ly-6+. In experiments to detect memory cells in each of these systems, the memory cell was Ly-6+ in the Sendai virus and anti-P815 allogeneic Tk systems, but clearly Ly-6- in the H-Y system. The studies may be one of the earliest indications of different types of memory for the TKcells. In addition, it is likely that Ly-6 is present on helper and antibody suppressor cells, and we have also demonstrated the DTH suppressor cell to be Ly-6+ (Thompson and Potter, unpublished results). In spite of earlier experiments to the contrary, it is now clear that Ly-6.2 and 6.1 are present on IgM plaqueforming cells. As well, Ly-6 specificities are present on the cells responding to PHA, Con A, PWM, and LPS and present on Con A, PHA, and LPS blasts (Michaelides and McKenzie, unpublished; A. Feeney, personal communication), At this time it is not clear why these antigens, which appear to be MuLV associated, are expressed on activated cells. In conclusion, Ly-6.2 is an interesting antiserum, as it has a restricted tissue distribution, predominantly to T cells. The absorption studies indicate that antibody reactivity is directed against a virusassociated antigen although it is not clear how this fits into the allelic representation of the Ly-6 specificities and their presence on most functional T cells. J. THEALA-1 Locus ALA-1 (activated lymphocyte antigen-1) is an alloantigen that was originally described as being expressed on mitogen-stimulated T and B blast cells (Feeney and Hammerling, 1976). The early studies with this serum demonstrated the specificity to be present only on activated or blast cells of peripheral T and B cells, not of blast cells derived from thymocytes (Feeney and Hammerling, 1976). It was therefore considered that ALA-1 was a differentiation antigen, and its distribution was restricted to the later stages development of T and B cells.

1 . Genetics and Antiserum Production Anti-ALA sera were produced by immunizing with blast cells formed in tissue culture when lymph node and spleen cells were exposed to PHA for 24 hours. The sera were initially produced as C58 anti-C3H (ALA-1.1) and C3H anti-C58 (ALA-1.2) (Table V). Both sera contained contaminating anti-Ly and possibly other antibodies, and so the sera were absorbed with donor-strain thymocytes prior to testing and were also absorbed with Con A-stimulated blast cells of the recipient in order to remove autoantibody. The strain distribution of the antisera is shown in Table VII. As mentioned for Ly-6, there

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IAN F. C. MCKENZIE AND TERRY POTTER

were initially some difficulties in testing several strains, such as 129; however, it is now clear that strain 129 is ALA-1.2, not ALA-1.1. The reciprocal strain distribution pattern indicated that the two specificities were allelic, and this pattern has subsequently been confirmed by testing on the recombinant inbred lines(A. Feeney, personal communication) although no backcross studies have been published. The ALA antiserum produced by Kerbel (1977) by immunizing between the strains BALB/c and C57BL16 appears to be very similar to that of Feeney and Hammerling, and its properties also suggest a striking similarity to Ly-6 specificities. 2 . Tissue Distribution The ALA specificities were clearly present on blast cells when stimulated with Con A or LPS. In direct cytotoxicity testing, thymocytes, lymph node, and spleen cells were nonreactive. Quantitative absorption studies showed that the number of normal spleen and lymph node cells required to absorb an equivalent amount of antiALA-1.2 antibody was 5-10 times greater than the number of Con A-stimulated blast cells used for the absorption. Similar absorptions using thymus cells did not clear the antiserum or reduce the titer at all. Furthermore, when thymus cells were cultured with Con A to form blast cells, no absorption of the antisera were noted; i.e., the specificity was not expressed on all blast cells, only on those formed from peripheral T or B cells. Absorption studies demonstrated that, like Ly-6, ALA specificities were present on kidney cells but absent from red cells, liver, brain, and skin. In cross-absorption studies it was shown that both LPS and Con A blast cells carried the same specificity. The evidence demonstrating the similarity of Ly-6, ALA-l, and several other specificities has been summarized in the Ly-6 section. 3. Functional Studies By using antisera and complement to deplete different functional populations, it was shown that ALA-1 was present on in viuo-primed TK cell cytotoxic for allogeneic tumor cells (Feeney and Hammerling, 1977).In vivo SRBC-primed helper T cells were found to be ALA-l+. The specificity appeared to be expressed on effector cells, rather than their precursors, as the precursor of helper cells and of IgM PFCs to sheep red cells were ALA-1-.

K. THE Ly-7 LOCUS The description of the Ly-7 locus is another example of the use of the Bailey RI lines for the production of antisera with restricted specificity

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(McKenzie et al., 197%). So far only the Ly-7.2 specificity has been defined, though several attempts have been made to raise an anti-Ly7.1 antibody. 1 . Genetics The strain distribution pattern of the Ly-7.2 antiserum is of interest in that all strains, other than C57BL/10 and C58, are reactive with the antisera, and, although strains SJL and C3H give weaker reactions than do other strains, all these strains can clear the antibody by absorption. Analysis of the CXB RI lines (Table VIII) differentiates Ly-7 from Ly-4, Ly-6, H - 2 , Ly-1, Ly-2, l a , and Tla. This was confirmed b y backcross studies (M. Cherry et al., unpublished data). 2 . Production of Antisera The Ly-7.2 specificity is defined by an antibody present in (B6.CH - 2 d x CXBG)F, anti-CXBK or anti-CXBH (Table V). In attempts to make the antiserum in several different combinations, it is apparent that the Ly-7.2 antibody response is H - 2 restricted, in that H - 2 haplotypes b, d, r are good responders, whereas k, q, s are nonresponders. The gene conveying responsiveness probably maps to the IA subregion of the H - 2 complex (Potter and McKenzie, unpublished results). Anti-Ly-7.2 is a good cytotoxic antibody (Table V), reacting on a subpopulation of spleen and lymph node cells to a cytotoxic titer of 1: 128 or greater (Table XII).

3 . Tissue Distribution Population studies indicate that Ly-7 is present on a subpopulation of peripheral lymphoid cells (Table XII). On direct testing thymus is nonreactive, but it is positive by absorption and contains approximately one-tenth the antigen content of Ly-7.2 in spleen. On spleen and lymph node there is lysis of 60-80% of cells; on bone marrow, lo%, on peritoneal exudate cells, 30%, and on Peyer’s patches lymphocytes, about 60% (Table XII). Although the antisera reacts with few thymus cells, it reacts with 60% of cortisone-resistant thyrnocytes, 50% of Ig- spleen cells, and 80% of Thy-1- lymph node cells (McKenzie et al., 1977b). Clearly the serum is reactive with a subpopulation of T and B cells. Comparative absorption studies of separated T (Ig- lymph node cells) and B (Thy-l- spleen) lymphocytes suggests that there may actually be two antibodies present in this antiserum: one reactive with B cells only and the other reactive with both T and B lymphocytes. The serum is nonreactive, by absorption, with kidney, liver,

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brain, and red cells. Extensive functional studies have not been done for Ly-7.2; however, the antisera eliminates direct IgM plaqueforming cells (McKenzie et al., 1977b). L. T H E L Y -Locus ~ In the original serum used to define the Thy-1.1 specificity (C3H anti-AKR), Frelinger and Murphy (1976) identified an antibody that reacted with C57BL/6 (Thy-1.2) lymphocytes. The specificity recognized by this antibody was designated Ly-8.2. Although thymocytes were used for the immunization, the Ly-8.2 specificity was also found to be on peripheral T and B cells. According to the strain distribution pattern of the known antigen specificities (Table VII), the C3H antiAKR serum could contain antibodies to Thy-1.1, Ly-1.2, Ly-3.1, Ly6.2, and Ly-7.2. However, it was considered that Ly-8.2 was a unique specificity because (1)it showed a unique strain distribution pattern, although it Should be noted that for Thy-1.2+ strains the strain distribution patterns for Ly-8.2, Ly-6.2, and ALA-1.2 are identical; (2) the recipient strain C3H and the typing strain C57BU10 were Thy-1.2, Ly-3.2, and Ly-7.1 and therefore would not react with the Thy-1.1, Ly-3.1, or Ly-7.2 contaminant antibodies, but this observation would not exclude the possibility that Ly-8.2 represents expression of different alleles at these loci; (3) the weak reactivity of Ly-8.2 antiserum with thymocytes was in contrast to the expression of Ly-1.2 and Ly-3.1 specificities; (4) a backcross demonstrated that Ly-4 (Lyb-I), Ly-6, Thy-1, and H-2 segregated independently of the Ly-8 locus. More recently, however, Horton et al. (1978) have demonstrated that antiLy-8.2 segregated with Ly-6.2. Furthermore, both sera gave similar patterns in the recombinant inbred strains. It is therefore likely that the serum used to identify the Ly-8.2 specificity contains Ly-6.2 antibody, and it is not unlikely that other specificities are present as well. To date, the only functional studies that have been reported for Ly-8 specificities is the finding that direct PFCs in primed populations are Ly-8+.

M. THELYM-ILocus Although the strains CBNJ and C3H/HeJ are H-2 identical (H-2k) and they are related in origin, skin grafts between them are rapidly rejected, and there is also a mixed lymphocyte reaction between them. The skin graft rejection could be attributed to differences at a number of non-H-2 histocompatibility loci. The MLR is attributed to the products of the MZs locus (minor lymphocyte-stimulating locus), CBA bearing the M l s d allele and C3H/HeJ the Mls" allele (Festenstein, 1974).

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Antiserum produced as C3H/HeJ anti-CBNJ spleen and lymph node cells contains a low-titered cytotoxic antibody defining a new specificity: LyM-1.2 (Tonkonogy and Winn, 1976). The antibody was found to react with bone marrow cells (75%), spleen cells (70%), and lymph node cells (30%), suggesting that the antigen was confined to the B-cell lineage. This was also suggested by the apparent reciprocal cytotoxicity of LyM-1 and Thy-1 antisera as well as inhibition of B-cell but not of T-cell functions and mitogen responsiveness (Tonkonogy and Winn, 1976). The antiserum was also shown to be cytotoxic for 75% of peritoneal exudate cells. An unusual aspect of this antiserum was that cytotoxic activity was demonstrable only in the presence of extremely high concentrations of rabbit complement (Tonkonogy and Winn, 1976). On the basis of the differing number of spleen cells of different strains reacting by cytotoxicity, the LyM-1 antiserum defined three groups (Table XV): (1) 70% lysis found in strains carrying the MZs" or MZsd alleles; (2) 50% lysis found in strains with the M l s b allele; (3)background (25%) lysis for strains with the MZs" allele as well RF/J and CE/J mice (not Mls", but M W ) . In a separate study, the capacity of the different strains to absorb LyM-1.2 antibodies was studied and suggested the possibility of a new third allele for the AKWJ strain, LyM-1.3 (Tonkonogy and Winn, 1977), the prototype strains for LyM-1.1 being C3H/HeJ and for LyM-1.2, the CBNJ strain. However, the MZs locus is not as simple as originally defined, as serological and other studies have indicated the existence of crossreactivity between the M l s a and Mlsd products: (a).C3H/HeJ antiCBNJ (MZsd) serum is cytotoxic for AKWJ (Mls") lymphocytes (Tonkonogy and Winn, 1976); (b) AKWJ cells are as efficient as CBNJ cells in removing the cytotoxicity of anti-LyM-1.2 (anti-CBNJ) for AKWJ target cells; (c) lymphocytes from BALB/c (MZsd) mice immunized with BALB/c-MZsacongenic spleen cells inhibited [ 14C]uridineuptake b y CBNJ ( MISd) macrophages-i.e., cross-reactivity expressed at the cellular level (Matossian-Rogers and Festenstein, 1977); (d) when negative selection of MZs" bearing cells leads to their removal, MLC reactivity to MZsd determinants also disappears (Ryan et d.,1979). In a (CBNJ x C3H/HeJ)F, x C3H/HeJ backcross study of 42 mice, Mls (defined by the MLR reaction) and LyM-1 (defined by cytotoxicity) segregated together and no recombinants were found (Tonkonogy and Winn, 1976). Subsequent studies have demonstrated that the genes leading to the MLR and the antigenic specificity are not identical (Tonkonogy and Winn, 1977). For example, there was no MLR between CBA (LyM-1.2+)and RF or CE (LyM-1.2-); however, there

r

n g

TABLE XV STRAIN DISTRIBUTIONFOR B-CELL CMAD"

Locus

LY-4 (Lyb-1) Lyb-2

B

Defined by

Specificity

Antiserumb

Assay

Strain distribution'

Ly-4.1

B1O.A Q A

Cytotoxicity

Ly-4.2 Lyb-2.1

(BALWCX SWR)FI Q B10.D2 (C3H.I x C57BU6)F, Q 1.29

Cytotoxicity Cytotoxicity

Lyb-2.2

(SJL X CE)F, Q A.SW Abs. with HSFS/N (C3WAn x BALB/c)F, (I CE Abs. with HSFSlN + PL (CBNNP X BALB/cd)FId (I BALB/c

Cytotoxicity

NWySn, BALB/c, AKR, C3WHe, DBN1, DBN2. SWR, 129, PL C57BU6, C57L, C57BWcd, C58(?), WB/Re, MA CBNJ, C3HUBi, C57BWcd, C57L, C58, DBN1, DBN2, HSFS/N, YSt, SWR A, BALB/c, CBA/T6, CBNN, C3WAn,

Cytotoxicity

C3WHeJ, C57BU6, PL, 129 AKR, CE, RF, SJL

Lyb-2.3

Lyb-3

Fluorescence

+: CBNTG, BALB/c, C57BU6, N J , DBN1 -:

Lyb-4

Lyb-4.1

C57BUKs (I L1210

+: DBN2, C3H/HeJ, CBNJ, SWWJ

Cytotoxicity

C57BUKs, C57BU6, C57L, BALB/c, N J , AKR, SJL, RF, CE, 129 DBN1, DBN2, C3WHeJ, CE, SWR, A U N -:

Lyb-5

Lyb-5.1

C57BU6 Q DBN2 Abs. with (CBNNP x DBA/2d)F,d

CBNN

Cytotoxicity

z

Y

M

$

U cl

i! 4

8cl F1s

Lyb-5.2 Lyb-6

Lyb-6.1

DBN2 (I C57BU6 Abs. with (CBNNP x C57BU6d)F1d CBNN (I CBNJ

Cytotoxicity

C57BU6, CBNJ, BALB/c, SJL, A, AKR, RF

SDS-PAGE

+: CBNJ, DBN1,

DBN2, SWR CBNN, C3WHeJ, N J , AKR, BALB/c, C57BU6, CE, SJL +: DBN1, DBN2, CE, C3WHeJ -: C57BU6, CBNJ, BALB/c, SJL, AKR, RF, NJ, A U N +: CBNJ, DBN1, DBN2, BALB/c, C57BU6 -: C3H/HeJ, SJL, N J ,RF, CE +: C3H/HeJ, A, Am, BALB/c, NZB, SJL, SWR, RF, PL, CE -: CSHOBi, C57BU6, C58, CBA-T6, DBN2, 129, I/St, C57L, C57BWcd -:

Lyb-7

Lyb-7.1

C57BU6 a DBN2 Abs. with (CBNNP x DBN2d)F,d

LyM-1

LyM-1.2

C3WHeJ a CBNJ

Blocking of antibody responses Cytotoxicity

Pc-1

Pca- 1

DBN2 a MOPC-70A

Cytotoxicity

For references, see the text.

* Abs., absorbed. +, Antigen present;

-, antigen absent.

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were strong MLR responses between C3H, RF, and C E (all LyM1 Z ) ;i.e., there appeared to be a discordance between Mls typing and the cytotoxic pattern of anti-LyM-1.2. In another study of the same backcross, 29 mice were examined and two recombinants were found (6.8% recombination frequency), which demonstrated that ‘MZs and L y M - 1 were separate loci (Dickler et aZ., 1977). By the use of the AKXL recombinant inbred lines, the MZs locus has recently been mapped to chromosome 1 near Dip-1 and Ald (Festenstein et al., 1977). It should be noted that the MZs locus was defined by an MLR between nonprimed cells (Festenstein, 1974), and, in a backcross, 50% of mice stimulate and 50% do not-findings compatible with the segregation of a single MZs locus. However, when in uitro MLRprimed cells are used, spleen cells from up to 90% of the backcross mice stimulate a secondary response (A. Ahmed, personal communication) indicating the segregation of at least two, perhaps unlinked, loci: In addition to the specificities already mentioned, the C3H antiCBA serum also contains antibodies that inhibit the binding of aggregated immunoglobulin to the Fc receptor (Dickler et d.,1977). The locus controlling these determinants was also found to be linked to the MZs locus and could conceivably be the same as LyM-1. Dickler et al. (1977) have drawn attention to the similarity of this MZs-LyM-1 region to the H - 2 complex in that, (1) there are closely linked determinants for MLR stimulation (MZs locus) and for serologically detected polymorphic antigens expressed on B cells, ( L y M locus); (2) antisera to the products of this region are capable of blocking the Fc receptor; (3) there may be histocompatability genes in this region, although this cannot be demonstrated until MZs or L y M - 1 congenic lines are available. N. Ly-b SPECIFICITIES Over the last 3-4 years there has been a series of specificities defined that appear to have a restricted distribution and are found only on B lymphocytes. To separate these from other Ly specificities, they have received an “Ly-b” designation and thus far Lyb-1 to Lyb-7 have been described. There are several interesting features associated with these Lyb loci. For instance, the CBAINaid mutant, a strain that lacks a B-cell subpopulation (see Table XVI) has been used to define the L y b - 3 , b-5 b-6, and b-7 loci. Several other features of these loci are of interest in that some were defined not by cytotoxic reactions, but by chemical isolation (Lyb-6)or the blocking of in uitro antibody production (Lyb-7) (Table XV). It should be noted that as some of these loci have only recently been defined, not all have been genetically map-

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TABLE XVI SUMMARY OF THE ABNORMALITIES OF THE CBNN STRAIN,WHICHCARRIES THE MUTANT (xid) GENE 1. Respond to only some thymus-independent antigens

Type I1 (no response)

Type I (response) TNP-Bmcella abortus TNP-lipopolysaccharide TNP-Nocardia antigen

DNP, Ficoll Levan Dextran POlY(I), POlY(C) Pneumococcal type 111, polysaccharide

2. Type I responses are also less than normal 3. Defect carried in Ig+B lymphocytes and stem cells, but not in Ig- or thymus cells 4. Mutant gene xid is X-linked; in F, of crosses between CBNN female and normal male, male F, (xi&-) express defect but female (xi&+) do not. 5. CBNN spleen contains fewer lymphocytes than normal and only 30% of the normal number of Ig+ cells 6. Ig+ cells in adult CBNN carry neonatal amounts of IgM (i.e., increased amounts, and sIgM usually decreased in adult as compared to neonate) 7. Abnormal mIgM: mIgD ratio in CBNN (1: 3 normal) 8. CBNN do not carry B-cell population expressing the Lyb-3 and Lyb-5 specificities 9. CBNN fail to stimulate Mls-determined mixed-lymphocyte responses 10. No proliferative response to anti-p or anti-rc reagents 11. xid gene appears to control the development of a unique B-cell subpopulation (Mls+, Lyb-3+, Lyb-5+), which is absent in CBNN mice, rather than the development of CMAD expressing these specificities

ped, nor completely separated from all other Ly loci in backcross studies. Also for many of these loci the complete strain and tissue distributions are not as yet available. Several recent reviews of these loci have recently been published (Ahmed et al., 1978; Paul et al., 1978).These sera may prove to be of immense value in studies of B cell heterogeneity, ontogeny, differentiation, tolerance, and suppression.

1. Lyb-1 (Ly-4) The Lyb-l or Ly-4 locus has been described in detail above. The relationship of this locus to the other B-cell markers to be described below has not been determined.

2 . Lyb-2 When normal C3H.I (H-21)mice were immunized with an ascites tumor, 1.29 (H-29,an antiserum was produced, which, after absorption with thymus and spleen cells of C57BU6 or BALB/c, was found to contain an antibody with a predominant B lymphocyte reactivity (Sato

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IAN F. C. MCKENZIE AND TERRY POTTER

and Boyse, 1976). It was also found to react with about 40% of bone marrow cells, but not with thymocytes. As the reactivity of bone marrow was greater than that of Ig+ cells, this marker was considered to be present not only on mature B cells, but also on B cell precursors, and possibly on the surface of null cells (Sato and Boyse, 1976). In contrast to most of the other Ly- loci, the Lyb-2 locus appears to have at least three alleles (Shen et al., 1977) (Tables VII and XV), which show a unique strain distribution pattern. It should be noted that many sera used conventionally for anti-T cell antibodies could well contain this specificity (see Table V). Our own (unpublished) observations suggest that these antibodies are not necessarily present in antisera raised across an Lyb-2 incompatibility. Backcross studies have demonstrated the Lyb-2 locus to be separate from other CMAD loci and have mapped the locus to chromosome 4, approximately 6 map units from Mup-1 and adjacent to Pgm-2 (Sato et al., 1977). This linkage was confirmed by the recombinant inbred strain distribution pattern (Taylor and Shen, 1977). Recent studies have also demonstrated the linkage ofLyb-2, the LPS response gene, Lyb-4, Lyb-6, and M u p - 1 in this order on chromosome 4 with a distance of 4 c M for the Lyb-2-Lyb-6 interval (A. Ahmed and S. Kessler, personal communication). Functional studies have demonstrated that, for the IgG response, the majority of antibody-forming cells to sheep red cells are Lyb-2+; however, only a minor proportion of IgM PFCs are Lyb-2+. The tentative implication is that PFCs are mainly Lyb-2- and are derived from Lyb-2+ precursors (Shen et al., 1977). As Lyb-2 is present on bone marrow and the precursors of antibody-forming cells, it appears to be a B lymphocyte antigen that persists from the early to the later stages of B-cell differentiation. Immunoprecipitation studies of the Lyb-2.1 specificity on spleens from Lyb-2.1+ backcross mice have identified a cell surface polypeptide of molecular weight 40,000-45,000, and although the molecular weight was similar to that of H-2 and T L molecules, no &-microglobulin subunit was identified (Tung et al., 1977). The CBAIN Mouse and the xid Mutation. The CBNN strain arose as a mutation in the CBNH line at the National Institutes of Health in 1966 and was developed as a special subline (Berning et al., 1978) when it was found that CBNN was totally unresponsive to type I11 pneumococcal polysaccharide (Paul et al., 1978). This lack of responsiveness was inherited as an X-linked trait and resulted from the absence of the recessive xid gene in this strain (Amsbaugh et al., 1972). This mutation led to a defect in B-cell development and manifested a variety of defects (Table XVI) (Paul et al., 1978). Adult CBNN mice

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lack a proportion of splenic B cells and have excess numbers of immature B cells with increased amounts of surface IgM and a decreased concentration of surface IgD. The specific B-cell subpopulation that is lacking in the CBNN mutant also expresses the Lyb-3, Lyb-5, Lyb-7, and Mls determinants, so that this strain is crucial for the development of antisera recognizing these specificities. The extent of the defect is shown in Table XVI.

3. Lyb-3 The Lyb-3 specificity was first identified when (CBA/N x BALB/ c)F1defective male mice were immunized with BALB/c parental spleen cells (Huberetal., 1977).As thexid gene is X-linked, in the F, hybridonly hemizygous male mice carry the defect. Therefore when CBA/N female (XmXm)and normal male mice (XY) are crossed, the male F, hybrid mice (XmY)will manifest the defect (X" = X chromosome bearingxid mutant gene). The antiserum produced by Huberet al. (1977) was not cytotoxic for normal spleen cells, but was detected by fluorescence and reacted with a proportion of splenic B cells from all strains, with the exception of CBNN. The Lyb-3 specificity was not detected directly on bone marrow, thymocytes, or peripheral T cells, or by absorption on kidney or brain, but it was present on 30% of spleen cells and 46% of purified B cells (Huber et al., 1977). Using the FACS for further analysis, it was found that Lyb-3 was confined to small B lymphocytes (Huber et al., 1977). Of considerable interest was the finding that, when anti-Lyb-3 was injected together with purified B cells and a low dose of sheep red cells into lethally irradiated BALB/c mice, there was a marked increase in the anti-sheep red cell PFC response (Huber et al., 1977). It was considered that the Lyb-3 determinants were closely associated with, or on the same molecule as, the antigen receptor on the reactive population of B cells. In ontogenic studies, the Lyb-3 specificity was not detected on spleen cells of newborn mice, but could be found after 2 days and reached the adult levels after 15 weeks (Ahmed et al., 1978). Because all strains except CBNN are Lyb-3 positive, genetic mapping of the Lyb-3 locus by conventional approaches has not been possible. Recent chemical studies by lz5I cell-surface labeling, immunoprecipitation with anti-Lyb-3, and subsequent SDS-PAGE have identified a single-chain polypeptide (MW 68,000)(Cone et al., 1978). Considerable efforts were made to disprove identity with the heavy chains of cell-surface IgD. In addition, the isolated molecule did not bind to an Lens culinaris lectin column and therefore may not be glycoprotein or, at least, may not have exposed mannose residues. The

244

IAN F. C. MCKENZIE AND TERRY POTTER

Lyb-3 molecule was also unusual in that it retained antigenic activity after SDS-PAGE, although this is not a unique finding for cell-surface proteins and glycoproteins. Absorption of anti-Lyb-3 serum with the isolated 68,000 MW polypeptide fraction eliminated the SRC PFC enhancing effect (Cone et aZ., 1978). 4 . Lyb-4 When C57BWKs (H-29 mice were immunized with the L1210 ascites tumor cells (of DBN2 origin), an antiserum was produced that was cytotoxic for B cells in spleen, lymph nodes, and Peyer’s patches of adult DBN2 mice, but was nonreactive with thymocytes and peripheral T cells, and gave only low reactivity (-5%) with bone marrow cells (Freund et al., 1976). These findings were somewhat surprising as the L1210 tumor, which was induced with methylcholanthrene, had previously been regarded as a T-cell tumor. The antiserum has a unique strain distribution (Table XV), which was quite different from that of the H-2, Zg allotype, Mls, and Lyb-l loci (Freund et aZ., 1976). Backcross studies by Howe et al. confirmed these earlier studies and showed that Lyb-4 was separate from the H-2, I g allotype, A (Agouti), Lyb-1, and MZs loci. Additional backcross studies have now established the linkage of Lyb-4 with Mup-1 and Lyb-2 on chromosome 4 (Howe et al., 1979). An interesting aspect of the linkage studies was that although DBN2 (Lyb-4.1) showed such a pattern, no such linkage of Lyb-4 was observed for C3H/He either in a backcross or examination of the BXH-RI lines (Howe et aZ., 1979). This observation suggested that in C3H mice there has been a translocation of the Lyb-4 locus. An alternative explanation, that the anti-11210 serum contained multiple specificities, was excluded after cross-absorption studies with DBN2 and C3H demonstrated that both were Lyb-4.1+. To date, no Lyb-4.2 specificity has been described. Functional studies with the anti-Lyb-4.1 serum has mainly concentrated on its relationship to the Mls loci and to the blocking of the MLR with this serum. It has already been stated above that Lyb-4 and Mls loci have a different strain distribution and segregate independently in a backcross. However, antiLyb-4.1 serum can block an MLR (induced by either H-2 or MZs loci differences) provided that the stimulator cell is Lyb-4.1+ (Freund et aZ., 1977). The same specificity of blocking was shown in parent-F, MLR combinations, and it was postulated that the Lyb-4.1 determinant acts as a second signal augmenting lymphocyte activation in the MLR. Although this antiserum (C57BLIKs anti L1210) could potentially be multispecific, the cytotoxic activity and the MLR blocking activity segregated together. Recent studies of the ontogeny of the

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Lyb-4.1 specificity showed that it was absent on mice less than 4 days old, but reached adult levels b y 6 weeks of age (Ahmed et al., 1978). Immunoprecipitation studies with anti Lyb-4.1 serum on L1210 ascites tumor cells have resulted in the identification of a 44,000 MW polypeptide (Howe e t al., 1979). However these workers have not examined the segregation of the precipitated molecule and serologically defined Lyb-4.1 in a backcross. The similarity of this gene product to H-2K and D molecules, both in molecular weight and their role in the MLR, is clearly of interest. 5. Lyb-5 This antiserum, like anti-Lyb-3, was also produced using the CBNN mutant. In studies designed to repeat the Lyb-3 experiments, (CBNN 0 x DBN2 6)F, 6 mice were repeatedly immunized with F, hybrid 0 spleen cells, but no cytotoxic antibody was produced (Ahmed et al., 1977). By means of an alternative approach, C57BU6 mice were immunized with DBN2 cells, and the serum was then extensively absorbed with thymocytes and spleen cells from the abnormal (CBA/N 0 x DBN2 6)Fl CT mice. This protocol yielded a weak cytotoxic antiserum that reacted with a subpopulation of B cells in the adult and normal F, Q’s,but not with the mutant F, CT spleen cells (Ahmed et al., 1977). The specificity detected was called Lyb-5.1 and was present on Ig+, CR+ (complement receptor)-bearing B lymphocytes, which had a low to intermediate density of surface Ig; studies using the FACS showed that only 5% of sIg- cells were Lyb-5+. The strain distribution pattern of the Lyb-5.1 specificity is unique (Table XV), positive strains being DBNB, DBN1, CE/J, C3H/HeJ, SWWJ, and ALJN. The reaction with ALJN is important and serves to distinguish Lyb-5.1 from a contaminating Lyb-7.1 antibody also present in the serum (Ahmed et al., 1977; Subbarao et al., 1979a). The strain distribution and linkage studies demonstrated that Lyb-5.1 was separate from H-2, Zg allotype, M l s , other Lyb loci and from sex-linked loci. More recently, a DBN2 anti-C57BLJ6 reagent was prepared in the same way, and this recognizes the Lyb-5.2 determinant. Furthermore, although the antiserum is made using the CBNN mutant, Lyb-5 was considered to be separate from Lyb-3 for two reasons (Ahmed et al., 1977). First, all strains, with the exception of the mutant CBNNxid strain, are Lyb-3+, whereas a wider polymorphism is found for Lyb-5.1 (Table XV); and second, Lyb-3 antiserum is not cytotoxic. However, these differences could be due to other causes, such as a variation in density of Lyb-5 specificities among different strains. Also the different F, hybrids may make different classes of antibodies.

246

IAN F. C. MCKENZIE AND TERRY POTTER

Segregation analysis of Lyb-3 and Lyb-5 cannot be performed, so that examination of the Lyb-5 congenic line, which is now at the N6 generation (A. Ahmed, personal communication), should establish whether they are distinct loci. The Lyb-5.1 specificity is present on a subpopulation of B cells (approximately 50-60%). It was found that cells remaining after depletion by cytotoxicity of Lyb-5.1f cells, had a high s1gM:sIgD ratio. The Lyb-5.1 specificity was not detected until 14 days of age, and the level slowly increased until adult levels were expressed at 5 weeks of age. After depletion with anti-Lyb-5.1 and complement, spleen cells from other strains show similar defects to those seen in the CBNN mutant. Together, these observations indicate that Lyb-5.1 is present on a late-developing population of B lymphocytes-in fact, the same subpopulation which is absent in the CBNN&d mice and which also express the Lyb-3 and Lyb-7 specificities (Ahmed et al., 1978). 6. Lyb-6

The Lyb-6 specificity is unique in that it was initially defined b y biochemical analysis, not by conventional testing, such as cytotoxicity, fluorescence, or rosetting (Kessler et al., 1979). Antibody to this specificity is raised in CBA/N mutant mice, hyperimmunized with CBA/J spleen cells, and in conventional immunoprecipitation and SDS-PAGE analysis identifies a polypeptide of MW 45,000. The strain distribution pattern of this specificity is unique (Table XV) and partially serves to distinguish this locus from other Lyb loci and from the H-2, MZs, and l g allotype loci. However, there is a striking similarity to the strain distribution of Lyb-2.1 and Lyb-4.1. Studies in both recombinant inbred and backcross mice have established that the Lyb-6 locus is linked to, but distinct from, Lyb-2, Lyb-4, and M u p - 1 on chromosome 4 (Kessler et al., 1979).It is also of great interest that the gene for LPS (un)responsiveness in C3H/HeJ mice is also on this chromosome (Watson et al., 1978). The Lyb-6 specificity has been detected on lymphocytes in the lymph nodes and spleen and Peyer’s patches of adult mice, but not on thymocytes or bone marrow cells. Separation of spleen cells on the FACS showed that Lyb-6 was expressed only on Ig+ cells. Depletion by cytotoxicity indicated that only 50% of Lyb-6+ cells were also Lyb-5+; however, all Lyb-6+ cells also expressed Lyb-4. Antisera produced as C3H/HeJ anti-CBNJ, in addition to defining LyM-1.2 by cytotoxicity, also contains immunoprecipitating antibodies for Lyb-6.1. However, as the cytotoxic and immunoprecipitation reactions segregated independently in the (C3H/HeJ x CBNJ) x C3H/HeJ backcross, they apparently define distinct specificities. It is

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surprising that the cytotoxic antibody in this antiserum does not also give rise to an immunoprecipitation reaction. By radioiodination and immune precipitation, the Lyb-6 specificity appears to arise late in development. It is barely detectable on spleen cells of l-week-old mice and reaches adult levels at 4-5 weeks. Studies are in progress to confirm this scheme of expression on individual cells with a fluorescent anti-Lyb-6 reagent (S. Kessler, personal communication).

7 . Lyb-7 The Lyb-7 specificity was identified by a contaminant antibody in the Lyb-5 antiserum. In the backcross (C57BW6 x DBA/2)F, x C57BW6, the cytotoxic reactions of anti-Lyb-5 segregated (50% reactivity) as a single gene product. However, it was found that in 50% of these mice the in vitro response to TNP-Ficoll (an antigen to which CBNN is unresponsive) could be blocked b y the Lyb-5 antiserum. This blocking effect segregated independently of the cytotoxic effect and was attributed to the presence of another antibody defining a new specificity Lyb-7.1 (Subbarao et al., 1979a,b). The Lyb-5 and Lyb-7 specificities could also be separated on the basis of their distinct strain distribution pattern (Table XV). By absorption with strain AL/N (Lyb5.1, Lyb-7.2) it is possible to prepare a more specific anti-Lyb-7.1 reagent from the Lyb-5.1 antiserum (Subbarao et al., 1979a). The different strain distribution of these specificities among the Bailey and Taylor RI lines further distinguishes these specificities from each other and suggests linkage of the Lyb-7 locus to the ZgCH locus. Backcross analysis has shown linkage between the Lyb-7 and ZgCH loci with a separation of 15-21 cM; however, the blocking activity of anti-Lyb-7 is not due to contaminating anti-ti antibody (Paul et al., 1978). The observation that Lyb-7 is linked to an Ig marker has led to some interesting speculation on the role of this specificity in B-cell triggering (Paul et al., 1978; Subbarao et al., 1979a). Similar to Lyb-3 and Lyb-5, Lyb-7 specificities are also on the subpopulation of B cells absent in the CBNN mice. Although the relationship of these specificities to each other remains to be determined, it may be that the allogeneic determinants of the polymorphic Lyb-5 and Lyb-7 loci are expressed on a molecule that also carries Lyb-3 as a constant region marker (Subbarao et al., 1979a,b). 0. Vat-4, Vat-5 Recently G . Hammerling (personal communication) has identified the presence of two loci within or adjacent to the mouse MHC ( H - 2 ) which code for specificities found only on T cells. The antisera were produced by the production of a hybridoma antibody where immuniza-

IAN F. C. MCKENZIE AND TERRY POTTER

248

tion was performed across an H-2 difference. The antibodies produced were of restricted tissue distribution to T cells. The number of T cells reacting indicates that these specificities are separate from IJ and probably also separate from the IC/E Ia specificities described by Okuda and David (1978), which appear on T cells. The findings are of great interest and provide an example of the potential value of sera raised by the use of hybridomas (G. Hammerling and L. Flaherty, personal communication). P. Za LOCI Within the H-2 complex are several loci coding for specificities found primarily on lymphocytes. As these loci map in the same region as the H-2-linked immune response (Zr) genes, the specificities have been designated as Z region-associated (Ia) antigens. There are four named Ia loci (Zu-I, Za-3, Za-4, Za-5) coding for the serologically defined specificities and several other loci, defined by functional and serological studies, that have not yet been designated (Murphy, 1978; Tada et al., 1978a). Several subregions (designated Z-A, I-B, I-J, I-E, I-C)of the Z region, each having their own marker locus or loci, have been defined through recombination. As the Ia specificities have a restricted distribution, they are reviewed in this section; however, this TABLE XVII SUMMARY OF Ia SPECIFICITIESDEFINED BY SEROLOGICAL OR NONCONVENTIONAL MEANS~ Subregion I-A

I-B

1-1

I- E

I-c

a

Locus

Specificities

BY

Tissue/factor distribution*

Ia-1, -2, -3, etc.

B cells, M4, sperm, epidermal, Lad( B) ?la-I ? T blasts, Fc+ T cells, acceptor for TaF Not la-1 (?la-6) ? Helper and amplifier factors ?la-1 ? AE F No serological or factor-related determinants la-4 Unnamed, found in Ts, ?acceptor for Ts b, k , s haplotypes factor, ? Con A promoter cells, ? M4, ?Lad(T) TIa-4 ? Suppressor factors Not la# ? Helper T (Th2) la-5 7,22 B cells la-3 Ia-6 T,Lad07 ?la-3 ? T blasts, Fc+R T cells ? Not Za-3 MLR suppressor factors la-1

References are given in the text (Section VI).

* M4,macrophage; Lad, lymphocyte-activating determinant on B cell (B)or T cell (T); AEF, allogeneic effect factor; TaF, T cell amplifying factor.

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249

discussion will be brief, as the Ia systems have been extensively reviewed elsewhere (Shreffler and David, 1975; Meller, 1976; Snell et al., 1976; McDevitt, 1978; Murphy, 1978). Several features of the Ia specificities are distinct from those of other CMAD: (1) The specificities are highly polymorphic. Although this is also a feature of the H-2K and H-2D loci, it is in contrast with the Ly loci. (2) Ia specificities are strongly immunogenic, as high-titered antiserum is obtained after 1 or 2 injections; ( 3 ) some Ia specificities were defined b y functional testing before any serological analysis was performed. (4) Ia specificities show a unique association with the B-cell Fc receptor (Dickler and Sachs, 1974). 1 . The Z-A Subregion The Z-A subregion contains the Za-1 locus, which codes for specificities expressed predominantly on B cells, but also found on macrophages (Cowing et al., 1978), sperm, epidermis (Hammerling, 1976), and more recently on Langerhans cells in the skin (Sting1et al., 1978). The Z-A and ZCIE coded specificities are those commonly found in most “anti-Ia” antisera. Like the H - 2 K and D-coded specificities, the Za-1 B-cell specificities can also be classified as public and private. For example, Ia-2 is the “private” Ia“ specificity whereas Ia-1, -3, -15, -18 are public specificities, although there is not such a clear distinction between public and private Ia specificities as occurs with the H-2 specificities (Shreffler and David, 1975). Immunoprecipitation studies have demonstrated that Za-1 specificities are present on the same molecule and have identified the Ia structure as being a 58,000-60,000 MW molecule consisting of two chains: a (MW 33,000)and p (MW 27,000) (David, 1976; Cullen et al., 1976). It is not clear whether the allotypic specificities occur on one or both of these chains. Anti-Ia sera can inhibit the MLR reaction induced by the lymphocyte-activating determinants (Lad) on B cells, the Lad locus in the Z-A subregion giving rise to the strongest MLR of all (Shreffler and David, 1975). Whether the serologically determined Ia specificities are identical to the Lad specificities is uncertain. Anti-Ia sera can block antigen-induced T-cell proliferation; macrophage function in the in vitro PFC response; the in vitro response to LPS; and the response of T cells to syngeneic tumors in two different systems (Shreffler and David, 1975; Niederhuber et al., 1975, 1978; Schwartz et al., 1976; Okuda et aZ., 1978; Ponzio et al., 1977). In some circumstances, anti-Ia sera can also block the proliferative response to PHA, but whether this is due to Za-1 -coded determinants or other Z-A subregion products is not known (M. Michaelides, personal communication). Anti-Ia sera can also block several types of T-cell-B-cell collab-

250

IAN F. C. MCKENZIE AND TERRY POTTER

oration (Munro and Taussig, 1975), but again, which locus within the I-A subregion is responsible for this is not clear. Within the Z-A subregion, but almost certainly distinct from ZU-I, are loci involved in other immunological functions. Several studies have established that Ia specificities on T cells, or T cell-derived factors, are quite distinct from those on B cells (Tada et al., 1978a). This differentiation was based on various findings: (1) Helper augmenting (amplifying) and suppressor T-cell factors were found to be H - 2 haplotype specific. In contrast, the B-cell antigenic specificities are crossreactive and are widely distributed through different haplotypes. (2) T cells, but not B cells, can remove the functional anti-T cell activity from anti-Ia sera (i.e., helper and suppressor) and can also absorb the soluble factors produced by these cells (Okumura et al., 1976; Murphy, 1978). Anti-Ia sera absorbed with B cells can still react with an amplifying T-cell factor, whereas absorption with T cells removes the effect (Tokuhisa et al., 1978). ( 3 ) Ia antigens of the Z-J subregion have been found only on T cells (Murphy et al., 1976a; Okumura et al., 1977), and this may be the case for the Ia-6 specificity (Okuda and David, 1978) and possibly for some other T-cell specificities found within the Z-J and Z-E subregions (Hayes and Bach, 1978). Indeed, there have been a number of other studies demonstrating Ia specificities on both T cells and B cells-using either blast cells, chemical isolation, or FACS analysis (Frelinger et al., 1974 , 1976; Fathman et al., 1975; Goding et al., 1975; Gotze, 1975, 1976; Kramer et al., 1975; David et al., 1976; Niederhuber et al., 1976). All these points provide compelling indirect evidence for the separation ofall Ia specificities into those of either T-cell or B-cell distribution. The other specificities coded for by the Z-A subregion occur on T-cell blasts and on Fc+ p cells, and the same locus (?la-] ) codes for an acceptor site on T cells for an augmenting factor (Frelinger et al., 197413; Stout et al., 1977; Tada et al., 1978a). For reasons outlined above, another locus, separate from Za-1 and provisionally named Za-6, codes for the Ia specificities found on helper and amplifier factors, as anti-Ia sera can absorb these factors, Another factor, allogeneic effect factor (AEF), which acts on B cells, also bears determinants coded for by the Z-A subregion-possibly the Za-1 locus (Armerding et al., 1974). The Z-A subregion also contains a histocompatibility locus, H-21A (Klein et al., 1976).

2 . The Z-J Subregion The Z-J subregion was initially defined by alloantisera cytotoxic for T cell-regulating allotype and carrier-specific suppression (Murphy et

251

MUFUNE CELL-SURFACE ANTIGENS

al., 1976a; Okomura et al., 1976). Serological studies of the I-J subregion were considerably advanced by the use of two recombinant strains, B10.A(3R) and BlO.A(5R), which differ only in the I-J subregion. Cross-immunization of these strains produces anti-IJb and antiI J k sera, respectively; however, both antisera contain large amounts of anti-MuLV antibodies. These antisera are mostly characterized by their interference of suppressor activity, although the reaction of antiIJ sera with 8-10% of spleen cells can be detected by rosetting (Parish and McKenzie, 1978), b y a sensitive cytotoxic assay (Hayes and Bach, 1978), or by using T cells purified through antigen-coated columns (Okomura et al., 1977). Until recently it was considered that the Z-J subregion contained the one locus, Za-4, which coded for the specificities found on Ts (Murphy, 1978), on cells bearing the acceptor site for Ts factors, on Con A promotor cells (Frelinger et al., 1976), and on macrophages (Niederhuber, 1978). The presence of IJ determinants on macrophages is of interest and was .detected by the use of anti-IJ sera and complement to remove the PFC-potentiating role of macrophages. However, of all the Ia sera, anti-IJ is the only serum able to block this reaction in the absence of complement (J. E. Niederhuber and D. C. Shreffler, personal communication). In addition, IJ' T cells were found to carry Lad products as defined by the MLR (Okudaet al., 1977). As well, IJ determinants have been found on suppressor factors (reviewed by Murphy, 1978). Whether the la-4 or another locus coded for all these determinants is not apparent. More recently, definite evidence for a serological complexity of the Z-J subregion is apparent, and at least one other locus has been identified within the I-J region. Studies from Tada et al. (1978b) demonstrated that some helper cells (TH) were IJ', in addition to Ts being IJ'. In these studies two different types of helper cells, called T h l and Th2, were identified in a DNPKLH system, where T cells, purified on nylon wool, were obtained from KLH-primed animals and mixed with B cells from DNP-primed mice and cocultured. The T h l and Th2 cells were differentiated by the following characteristics:

Characteristic

Thl

Th2

Adherence to nylon wool Ly- 1

Pass

Adhere

11 Helper activity

+ +

+ + +

IAN F. C. MCKENZIE AND TERRY POTTER

252

These findings are in obvious contrast to the earlier findings, which have demonstrated only suppressor activity associated with the Z-J subregion products. It was therefore appropriate to determine whether the IJ determinants present on Th2 cells were identical with those present on Ts. Some elegant studies demonstrated these to be different, in that after absorption of a BlO.A(SR)anti-BlO.A(SR)serum with Ly23 cells, the activity for Ts was removed but remained for Th2. If the antiserum was absorbed with Lyl cells, the reactivity for Th2, but not Ts, was removed (Tada et al., 1978a,b). Reacts with Serum ~

~

BlO.A(3R)anti-5R sera = anti IJk

Absorbed with ~

~~

Nil Lyl+ Ly2+3+

TS ~~

+ + -

Th2 ~

+

-

+

These two different IJ specificities have not been named as yet. A similar splitting of the IJ sera have been observed with some T-cell hybridomas produced by Tada et al. (1978a). Recently an H-2ZJ locus was defined in two separate studies, one involving tolerance induction (Streilein, 1979) and the other by skin grafting (McKenzie and Henning, 1979).The use of anti-IJ serain vivo has also led to some exciting data in a syngeneic tumor graft system and in other systems. It was initially found that anti-IJ sera would potentiate the in vivo PFC response (Pierres et al., 1977), and later studies (Greene et al., 1977) showed that A/J mice receiving daily injections of IJksera were able to reject two A-strain tumors (SaI and SI5091a). In both these studies the anti-IJ sera presumably eliminate IJ' Ts, which augments the type of immunity studied. Confirmation of these studies have come from other laboratories using different systems, but we have been unable to effect immune responses by anti-IJ sera in several systems where Ts may be acting, such as in the production of anti-Ly sera, enhancement studies, or in augmenting the immune response to non-H-2 antigens. There have been no chemical studies on Z-J region products. 3 . The Z-E Subregion The Z-E subregion was initially defined by the specificity Ia-22 present on H - 2 k but absent in H - 2 d (Shreffler et al., 1976), and recently the Ia-23 specificity of H - 2 d has been identified and mapped to the I - E subregion. In immunoprecipitation studies, it was found that the Ia-7

MURINE CELL-SURFACE ANTIGENS

253

specificity coprecipitated with both H - 2 k (Ia-7, -22) and H - 2 d (Ia.7, -23) Z-E products (Okuda and David, 1978), so that Ia-7 is a public specificity and Ia-22 and -23 the private specificities of the Za-5 locus. These specificities occur on B cells and in distribution and function are analogous to the Za-l locus products. It is also likely that an Lad locus occurs within the Z-E subregion, although it is not clear whether this is identical with the Za-5 locus (Okuda and David, 1978). By cytotoxicity IE specificities, reacting with antisera raised against Con A-induced T-cell blasts, have been demonstrated on T cells (Hayes and Bach, 1978). I n contrast to other “anti-IE” antisera, these antisera did not react with B cells, suggesting that they identify a new locus within the Z-E subregion.

4 . The Z-C Subregion The specificity Ia-6 identifies the Z-Cd subregion and is the only specificity for this subregion (Shreffler and David, 1975). The specificity is difficult to detect, in that the cytotoxic activity is due to an IgM antibody and was originally found only in early bleeds of the immunized mice, and it has been difficult to reproduce the antibody (David, 1976). The serum reacts with 15-20% of lymph node cells, with some T-cell lymphomas, and is able to block an MLR reaction occurring between strains differing at the Z-C subregion. Furthermore, some of these antisera react with T-cell blasts and with Fc+ T cells (Frelinger et al., 1976; Stout et al., 1977). The activity in the antisera can be absorbed with T cells but not with B cells (David, 1976). I n addition, the Z-C subregion products have been found to inhibit the action of suppressor factors generated during an MLR reaction. Although anti-IC sera can react with the suppressor factors, it is unlikely that the Za-3 locus codes for the Ia specificities found on these factors (Rich and Rich, 1976a,b). Because of the difficulty in reproducibly making an anti-Ia-6 reagent and because both IE and IC products have only been identified by cytotoxicity or immunoprecipitation, in one or two haplotypes, many investigators consider Z-EIC to be one subregion. Chemical studies of the Z-EIC region have identified a similar product to the Z-A subregion, with both a and /3 chains (Cullen et al., 1976). An H - 2 I C histocompatibility locus has also been identified (McKenzie and Henning, 1976). Q. OTHERSPECIFICITIES

Other specificities have been noted in different sera. For instance, an antibody in the B6-H-2Kanti C E (Ly-2.1) sera is currently being studied (B. Mathieson, personal communication), although this may re-

254

IAN F. C. MCKENZIE AND TERRY POTTER

semble Ly-4.1 (Lyb-1.1). We have recently identified new specificities in a serum 129 anti-C57L and in the reciprocal immunization. The sera react predominantly with thymus cells (Potter, Thompson and McKenzie, unpublished results).

R. CHEMISTRY OF CMAD Many CMAD have been characterized biochemically for their molecular weight and composition. In general, it has been found that the antigenic determinants reside on glycoproteins; however, whether the antigenicity lies in the protein or the carbohydrate portion has not been determined for many of these CMAD. The assay that has been used most extensively is the radioimmunoprecipitation method (Marchalonis, 1977). In essence this technique involves the labeling of cell-surface proteins with 1251in the presence of lactoperoxidase. As the enzyme molecules are too large to penetrate the cell membrane, only cell-surface proteins are iodinated. After solubilization of the cell by detergents such as Nonidet-P40 (NP-40), the proteins bearing the antigen are separated from the other labeled molecules by immunoprecipitation with the specific antiserum. The soluble antigen-antibody complexes can be precipitated with either xenoantibodies to immunoglobulins or staphylococcal protein A. The molecular weight of these purified antigens can be determined by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). The molecular weights of various CMAD obtained by this, or a similar, technique are presented in Table XVIII. Further characterization of the antigenbearing proteins requires the determination of their amino acid composition and sequence, as well as peptide mapping studies. To date, only MHC antigens (H-2K, D, and Ia) have been studied by these methods (Vitetta and Capra, 1978); surprisingly, there is little information available on the other CMAD. Comments regarding the relationship between the gene products identified by immunoprecipitation and other techniques (e.g., cytotoxicity) were made earlier (Section 111,B). VII. Erythrocyte Alloantigenic (€a) Loci

The E a loci code for antigens expressed predominantly on red cells but are included in this review for two reasons. First, these antibodies contaminate many of the anti-Ly sera. Second, many of these anti-red cell sera react not only with red cells, but also with other tissues, including lymphocytes. Such reactions may lead to some confusion unless the appropriate target tissue is selected.

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255

TABLE XVIII THEMOLECULARWEIGHT OF SOME CMAD" Locus

MW of molecular bearing antigenic determinant

H-2K and H-2D TL Qa-2 IA, ICIE Thy-1 Ly-1 Ly-2 Ly-3 Ly-5 Lyb-2 Lyb-3 Lyb-4 Lyb-6 Pc-1

45,000' 45,000-50,000b 43,000' 60,000 19,000 67,000 35,000 35,000 130,000 45,000 68,000 44,000 45,000 105,000-110,000

" For references, see the text. Associated with prmicroglobulin subunits.

So far seven Ea antigenic systems and an additional locus (Earn) affecting antigen expression have been described. The strain and tissue distributions are presented in Table XIX. The Ea antigens apparently do not have any effect on skin graft rejection, although two of the loci were originally designated as histocompatibility ( H ) loci-Ea-5 (H-5) and Ea-6 (H-6), but the H designation has now been dropped. It is not unlikely that at least some of these specificities are present on erythroid precursors and could be important in bone marrow grafting studies. The Earn locus determines the electrophoretic mobility and agglutinability of erythrocytes (Rubinstein et al., 1974). At this locus there are two alleles Earnh (high) and Eaml (low), which show a unique strain distribution, and this is of importance in selecting the appropriate target cells to use for hemagglutination assays. A similar study (Lilly, 1974) indicated that hemagglutination of mouse red cells was also effected by a single dominant gene closely linked or identical to the Ea-4 locus. The relationship ofEarn to this locus is unknown. As indicated in Table XIX, most of the Ea loci have two alleles, but for , one specificity has been some of these loci (Ea-3, Ea-4, E u - ~ )only identified. Linkage analysis has mapped two of these loci: Ea-l on chromosome 8, and Ea-6 on chromosome 2. The controversy over the chromosomal location ofEa-2 has not yet been resolved, as Snell et al., (1967) and Popp (1967) reported independent segregation of H-2 and

E U LOCI AND

Locus Ea-1

Ea-2 (R, Z, rho, H-14)

TABLE XIX SPECIFICITIES EXPRESSEDON MOUSE RED CELLS, WHICH MAY BE EXPRESSEDALSO OR CONTAMINATE ANTI-Ly SERA

Alleles Ea-1 Ea-1 Ea-1' Ea-2'

Specificities

Antisera

Ea-l.l(A) Made between feral Only in feral mice mice Ea-1.2(B) Nil(?) Most inbred strains Ea-2.1(R) C3H a RIII; RIII, F/St, RF/J, RFM C3H a RFM

E ~ z - 2 ~Ea-2.2(Z) RIII a C3H Eu-3 Ea-3" Ea-3.1 C57BU10 a C57L (lambda, A) E u - ~ ND' ~ -

Ea-4 (BL, D) Eu-4"

Strain distribution

ND

E ~ z - 4 ~Ea-4.2

-

(C3H x DBA/2)F, C57BU10; NZB a B10.D2

Most inbred strains C57L C57BU10; BALB/c; A; C3H All inbred strains except C57BL C57BL

Tissue distribution

ON

LYMPHOCYTES

References

-

Singer, 1964

-

Foster et al., 1968

Liver, spleen, thymus, brain, lungs, testis

-

-

Snell, 1971; Hoecker and Pizarro, 1962; Popp, 1967, 1969; Hoecker et al., 1959

-

Egorov, 1965

-

Shreffler, 1966; Klein and Martinkova, 1968

?Thymus, ?spleen, kidney, lung

-

Ea-5 (alpha, 4

Ea-6 (delta, 6)

Ea-5"

Ea-5.1

E d b

ND

Ea-6"

Ea-6.1

Ea-Gb Ea-6.2

Ea-7 (T)

Ea-7"

Ea-7.1

E Q - ~Ea-7.2 ~

Earn

Earnh

-

Earn'

-

* ND, not determined.

C57BL a C3WSt lymphoma

(C3WSt a DBN2)F, a: E M ; C3HlSt a MCIM A a Meth A (BALBIc sarcoma) C57BFUcd a C3HlHe 129 a C57BU10

A, 129, YBR, I, FISt, C3WSt C57BU10, C3WHe, RF, C58, DBN1 A, C57BU10, 129, YBR, I, C3H/He, FISt, CBA, AKR, C58

Kidney, lung, lower levels on spleen and brain, testis

Amos et al., 1963

Testis, brain, lower levels on lung, liver, kidney

Amos 1958; Amos et al., 1963; Lilly, 1974

BALBIc, DBN2, DBN1, HTC, RF, C3WSt LP, 129, C3HIHe, CBA, C58, BUB A, C57BU10, C57L, BALBIc, DBN2, C57BWcd, DBN1, SWR, SJL, DA, CE, AKR, RF, HTG, I A, SJL, 129, DBN2, C3WHe, AKR C57BU10, C57L, BALBIc, RF

-

Amos, 1959; Stimpfling and Snell, 1968 -

Rubinstein et al., 1974

-

- 5

3 M

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Ea-2 loci, whereas Pizarro and Vergara (1973) mapped Ea-2 between the H-2 and T complex. Antibodies to the Ea specificities are generally identified by hemagglutination assays. For use in other systems it should be realized that these antisera are possibly contaminated with Ly antibodies; for example, in the Ea-2.1 antiserum leukoagglutinating and cytotoxic antibodies were detected, which could have been distinct from the Ea-2.1 antibodies, but cross-absorption studies indicated that the Ea-2.1 specificity was most likely to be present on both red cells and lymphocytes (Popp, 1967). The Ea-2.1 specificity also seems to be present on liver, spleen, thymus, brain, lungs, and testis (Popp, 1969). Ea-4.2 and Ea-5.1 specificities also appear to be expressed on other tissues, including thymus (Ea-4.2) and spleen (Ea-4.2 and Ea-5.1) (Table XIX). As some of these Ea antibodies react with lymphoid tissues, especially by absorption, the description of any putative new Ly locus should include hemagglutination and absorption studies with red cells, especially when techniques more sensitive than cytotoxicity are used. VIII. Miscellaneous Antigens

Cell membrane alloantigens have been detected on the surface of a number of other cell types apart from lymphocytes, such as epidermal cells, liver cells, macrophages, and sperm. In some cases the presence or the absence of these specificities on lymphocytes has not been excluded, and so these CMAD are briefly described herein. As well, there are some unusual findings with these specificities, which may be relevant to some of the Ly specificities or to lymphocyte alloantibodies. The SDP of these antigens are shown in Table XX. A. M p h - l ( MACROPHAGE-1)Locus An antibody reactive with macrophages has been described in an antiserum that was prepared against peritoneal exudate cells obtained either from normal mice or from mice injected with starch or bacteria (Archer and Davies, 1974). The antiserum was cytotoxic for about 60% of peritoneal exudate cells but gave virtually no reaction with lymph node cells. The antiserum was considered to detect an antigen present only on macrophages (hence the terminology Mph-1) although some reactivity with neutrophils could not b e excluded. Most strains were found to express the Mph-1.2 specificity, but the alternative specificity (Mph-l.l), was found in strains I/St and F/St. The M p h - 1 locus has been mapped to chromosome 7, linked to pink e y e dilution ( p ) and

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TABLE XX OF STRAIN DISTRIBUTION

SOME OF THE

MISCELLANEOUS ANTIGENS

Locus

Specificity

Strain

Mph-1

Mph-1.1 Mph-1.2 Type 1 Type 2 Sk-1.1 Sk-1.2 NK+ NKLyX-1.1 LyX-1" LyX-2.1 LyX-2.2 LyX-2" LyX-3.1 LyX-s

I/St, F/St C57BU6, BALB/c, N J , C3H, 129, D B N 2 , AKR CBA, C3H, AKR, D B N 2 C57BU6, BALB/c, A A, AKR, CBA, C3H C57BU6, BIO.A CE, C57BU6, NZB BALB/c, CBA BALB/c, C3H/He N J , C57BU6, D B N 2 , AKR, SJL NJ,BALB/c, D B N 2 DBN2 C3H/He, C57BLI6, AKR, SJL C57BU6, AKR, SJL N J , BALB/c, C3H/He, D B N 2

F Sk-1 NK Ly-x

chinchilla (cch)(Archer, 1975).More recent studies have indicated that the Mph-1.2 serum is cytotoxic for cultured monocytes, but not polymorphs, and that, by absorption, spleen, liver, and lymph node cells carry approximately one-fiftieth the amount of antigen as do normal peritoneal exudate cells (J. Archer, personal communication). Additional studies showed that peritoneal exudate cells obtained from normal or stimulated mice have the same amounts of Mph-1.2 on their surface, and it may be that activated macrophages do not have the Mph-1 antigen (J. Archer, personal communication). In other studies, anti-Mph-1.1 sera have been effective in vivo in an antitumor system (J. Archer, personal communication), but the anti-Mph-1 serum failed to show any effect in a passive skin graft enhancement study. Clearly, further studies are required with the Mph-1 sera to determine the precise cell that carries the specificity and the effect of these sera in vitro and in v i m . An alloantigenic antiserum reacting only with macrophages would naturally be of immense value for studying the role of this cell in many different cellular interactions.

B. F ANTIGEN The F antigen is described herein, as the conclusions obtained with this interesting antigen may have some bearing on the antibody response to lymphocyte CMAD and, in particular, to autoantibody formation. Unlike most of the other alloantigens, the F antigen is not

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identified directly, but rather by its induction of a liver-specific autoantibody response in an appropriate donorlrecipient combination (Iverson and Lindenmann, 1972). It is probably not a membrane component, but is detected only in liver extracts. Strains fall into two groups with regard to F antigens. Type 1 strains are CBA, C3H, AKR, and DBA/2; C57BL/6, BALB/c, and A are type 2 (Silver and Lane, 1975). Antibody is produced only by immunizing between the two types, not within the types, and is detected by precipitation of lz5Ilabeled liver extract. As the antibody reacts with liver extracts from both donor and recipient, it is an autoantibody. The unusual type of reactivity of the antibody has been interpreted as indicating that the F antigen has two reactive sites, one of which is considered to be reactive with autoantibody, is tissue specific, and is the same in all mice, i.e., a “constant” site. It is postulated that there is also a second site, on the same molecule, which exists in at least two allelic forms, i.e., a “variable” site, which leads to alloantigen expression and the production of the antibody. Presumably this variable site acts as a carrier and is recognized by helper T cells, which are tolerant to the constant form and respond only to the allogeneic variable form. Stimulation of the carrier response by the helper T cells is required for the B cells to mount a response to the invariant liver-specific site to which they are not tolerant, to form an anti-F autoantibody. The final complication, noted with anti-F antibody formation, is that not all strains have the ability to make the antibody, and the response depends on at least two genes: one maps to the I-A subregion within the H-2 complex; the other is a non-H-2-linked Ir gene (Silver and Lane, 1977; Long et aZ., 1978). The molecular weight of the antigen has been found to be 40,000 by Lane and Silver (1976), but in another study a larger molecule (MW 72,000-93,000) was observed (Mihas et aZ., 1976). C. Sk-1 AND Sk-2 LOCI(Sk = SKIN) The S k - l locus in the mouse codes for alloantigens found on skin and brain, but absent from lymphocytes. It was apparent that skin carried a tissue-specific alloantigen when it was demonstrated that neonatal mice, rendered tolerant by the injection of donor lymphocytes, subsequently rejected skin grafts but maintained a state of lymphocyte chimerism. In other mice, skin grafts survived; however, in both situations epidermal reactive alloantibodies were produced. The observations suggested that skin and lymphocytes were antigenically different and that the skin alloantigen (Sk) was expressed in several forms (Lance et al., 1971). Using antisera obtained from the skingrafted mice, the antigen was also detected on brain and on a neuro-

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blastoma. In the same study, the serological typing of epidermal cells showed them to be, H-2+, Thy-1+, Sk+, HY+, TL-, Ly-1-, Ly-2-, Ly-3-, Pca-. There are certain similarities between the Sk, H-Y, and Thy-1 specificities in that they occur on skin and brain and they elicit the production of antibodies when grafts are rejected or retained. Studies with the anti-Sk sera showed that strains carrying Sk-1.1 specificity are A/J (and congenics), AKR, C3H, and CBA, whereas the Sk-1.2 specificity is carried by C57BL/6 and BIO.A. No other strains have been tested. In addition, absorption or direct testing indicated that the Sk specificities were carried only by brain and skin, not by any of the lymphoid tissues (Scheid et al., 1972). The Sk-1 locus is not H - 2 linked (Scheid e t al., 1972). Recently a (C57BU6 x A)F, x C57BU6 backcross was studied (Wachtel et al., 1977); in this cross 71% of the backcross mice rejected their grafts, which suggested that two loci were segregating (Sk-1 and S k - 2 ) , but as yet there is no further information on the Sk-2 locus. However, Snell et a l . (1976) have suggested that either one of the S k loci may be identical to one of the H loci identified by Bailey in the recombinant inbred strains (Table VIII). The Sk loci are of importance in demonstrating that tissue-specific antigens other than those present on lymphocytes exist and may be responsible for some important transplantation reactions. (Note: Dr. M. C. Green has drawn attention to the use of Sk for Scaly and has suggested that Sk in the current context be altered to S k n . )

D. THET COMPLEX The T complex, which is linked to the H - 2 complex on chromosome 17, has been shown to exert a profound effect on embryonic differentiation, tail deformities, sperm differentiation and behavior, as well as having a suppressive effect on genetic recombination in this region (see extensive reviews by Bennett, 1975; Klein and Hammerberg, 1977). Although the early interest in this region was focused on its influence on differentiation and the description o f t alleles, more recently it has become possible to study T antigens serologically (Snell, 1979) wherein striking analogies have been found between T gene products and the H - 2 antigenic specificities. Anti-T alloantisera have been produced by the injection of sperm (Yanagisawa et al., 1974) or by using an undifferentiated teratocarcinoma (Artzt e t al., 1974; Stevens, 1967; Jacob, 1977). The antisera are cytotoxic by the dyeexclusion methods on sperm of particular t haplotypes and also react against the teratocarcinoma. The antigen detected by these antisera has been designated F9, and this is considered to be a T-gene product. In addition to its effect by cytotoxicity, it has also been shown by

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immunofluorescence to be present on the cells of early embryos, spermatogonia, and spermatocytes (Dubois et al., 1976). Extensive serological analysis (Artzt and Bennett, 1977), both by direct testing and by absorption to remove sperm autoantibodies, has demonstrated the presence of at least six specificities in the two strains used; this was the maximum number that could be detected. In addition, recombination has been demonstrated within the T complex, which must therefore contain at least two different loci whose products can be detected serologically. In this context, the T complex therefore resembles the H - 2 k and H - 2 D loci. Biochemical studies of the F9 antigen have demonstrated that it consists of two components of molecular weight 40,000 and 12,000. It was therefore considered that the small component was the same as µglobulin (or a similar molecule), as it appears in early embryos, which carry the T specificities and lack H-2 antigens (Dubois et al., 1976; Hakansson and Peterson, 1976). Current speculation as to the nature of the T gene product is that it may be the evolutionary precursor of H-2K, D molecules and function primarily in cell recognition during embryogenesis (Artzt and Bennett, 1975). In favor of this hypothesis is the sequential appearance of the T-gene product and H-2 in embryos and in the teratocarcinoma, MD-1, which is able to differentiate in vitro. In this tumor, the disappearance of F9 and appearance of H-2 at different times was shown (Forman and Vitetta, 1975; Dubois et al., 1976). In this particular study, Pzmicroglobulin was present only in association with H-2 specificities. More recently, the F9 antigen has been identified by a xenoantiserum (Kemler et al., 1977), which reacted with F9+ cells, and with 8-cell embryos by indirect fluorescence. The reaction could be blocked by using a mouse anti-F9 antibody. In addition, the xenogeneic antiserum was able to prevent cleavage of mouse embryos to form a morula and blastocyst although the effect was reversible by removing the antiserum. In addition to the structural and functional similarity to the H - 2 complex, a histocompatibility antigen ( H - 3 9 ) has now been identified near the Tlt complex (Artzt et al., 1977). Of relevance to studies of the MHC has been the finding of linkage disequilibrium between the T and H - 2 complexes, possibly due to the ability of different T alleles to suppress crossing-over in this region. The T complex is also associated with tail defects in the mouse, and it is of interest that in man there have been several reports of the recurrence of spina bifida controlled by a gene(s) linked to the HLA complex (Amos et al., 1975; Fellous et n l . , 1977).

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E. THEH-Y Locus There is a locus on the Y chromosome, H-Y, which codes for specificities present on sperm, lymphoid tissues, skin and other tissues, including germ cells of male mice (reviewed by Wachtel, 1977). The locus is mentioned in this review, as many anti-Ly sera probably contain anti-H-Y antibodies. The H-Y antigen was first described as a transplantation antigen when it was found that female mice of some strains rejected isogeneic male skin grafts (Eichwald and Silmser, 1955). It was initially thought that the expression of the H-Y antigen was dependent on the presence of testosterone, but it is now clear that this is not the case, for grafts from female donors do not express H-Y when carried by isogeneic males. In addition, male/female chimeras express H-Y only in the male cells. Also studies of the X-linked testicular feminization mutant (Tfm) show that H-Y expression is hormone independent, for in this mutant mouse there is a failure of target tissues to respond to testosterone, but male mice still express the H-Y specificity. However, it is not completely clear whether a structural or a regulatory gene for H-Y is present on the Y chromosome of mice. For example, mice bearing the S x r gene have sex reversal in that XX (female) karyotype mice, apparently lacking the Y chromosome, have male characteristics and are typed as H-Y+, possibly owing to the presence of a small piece of the Y chromosome in these mutants. However, in other experiments (Kralova and Demant, 1976) it was demonstrated that Y-linked male factors are regulatory and that the structural locus is in fact associated with the K end of the H - 2 complex. In addition, it is clear that the H - 2 complex is involved in several different ways in the rejection of H-Y incompatible grafts (Gasser and Silvers, 1971; Hurme et al., 1978a). In male mice, it has also been found that the H - 2 complex determines the rate of graft rejection due to an H-Y incompatibility. In addition, extensive studies indicated that the H-Y antigen is the same in mice of different haplotypes, and indeed is cross-reactive with the H-Y antigen of other mammals and birds (Wachtel et al., 1974). However, H-2* female mice reject male grafts more rapidly than mice of other H - 2 haplotypes. In addition, other genes, not MHC associated, also effect the expression of the H-Y antigen, as a C57BW10 background seems to give rise to more rapid graft rejection than does the C3H background. The MHC seems to be involved in the generation of cytotoxic cells against the H-Y antigen in vitro (Hurme et al., 1978b). The H-Y antigen occurs in many different species, and this has been demonstrated b y grafting experiments or by serological means. It is of

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interest that the H-Y antigen is limited to the heterogametic sex, whether it be male or female. For example, in birds the heterogametic sex is the female, the males are homogametic, and in this species the H-Y specificity is expressed in females (Wachtel et al., 1975). Studies of the H-Y locus have advanced more rapidly with the serological detection of H-Y. This was initially done b y a sensitive cytotoxic test (Goldberg et al., 1971) involving specially absorbed complement, but later by using a mixed hemabsorption hybrid antibody test, which led to the formation of rosettes (Wachtel et al., 1975). Recently, the H-Y antigen on human peripheral blood mononuclear cells has been detected by immunofluorescence using antisera raised in the mouse (Galbraith et al., 1978). Serological testing can also be done by absorption rather than by direct testing. These serological tests have confirmed the skin-grafting studies and have also demonstrated that cells prepared from thymus, spleen, lymph node, bone marrow, or peripheral blood of males, can react with anti-H-Y serum by absorption, but not usually directly in cytotoxic testing. Using a serological approach combined with skin grafting, Melvold et al. (1977) have demonstrated a dissociation between the ability to reject an H-Y incompatible graft and the serological H-Y type of the donor’s cells, suggesting a further complexity in the H-Y system. Among the progeny of an irradiated male mouse was a sterile male who received several skin grafts and was found to lack the H-Y antigen. Furthermore, karyotyping showed that this mouse lacked the Y chromosome. However, serological testing on brain or spleen cells demonstrated that H-Y may be present. It therefore appears that there may be two H-Y loci-H-Y-l determining the antigen present on skin, and H-Y-2 determining the antigen demonstrable with antisera-but the relationship between these two specificities is not as yet apparent.

F. THE NK SPECIFICITY The natural killer (NK) cell is found in spleen and bone marrow of some mouse strains and is identified by the in vitro lysis of selected tumor cell targets. The cell is unique in that it can cause killing without prior sensitization and acts on either isogeneic or allogeneic tumor-cell targets (Kiessling et al., 1976). Tumor cells seem to be a necessary target, and the target has to be infected with MuLV. The cell appears to be a lymphocyte, but of neither the conventional T- nor B-cell lineage as the cell-surface phenotype is Thy-1-, Ig-, Ly-1-, Ly-2-. Furthermore, the cell does not adhere to nylon wool (Kiessling et al., 1976). Herberman et al. (1978) have recently reported that NK cells express a low density of Thy-1 antigen and may be prethymic T

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cells. The NK cell has been identified as carrying a specific antigen (Glimcher et a l . , 1977). The antisera used were C3H anti-CE and (C3H x BALB/c) F1 anti-CE. In the first serum, anti-Ly-1.2 was also present; however, this was removed by absorbing with BALB/c, and the anti-NK antibody remained. The absorbed antiserum reacted with splenic NK cells from C57BL/6, NZB, but not with CBNT6 which also carries NK cells. As well, BALB/c and C3H must be negative, as these strains were used in the F1hybrid to produce the antiserum, although C3H spleen cells also have NK activity. It is therefore not clear whether the NK antigen is absent from some NK+ strains or whether it exists in allelic forms. There has recently been some attempt to correlate the properties of the cell involved in hybrid histocompatibility (Hh-1) with the NK cell or with another cell, called an M cell, which has similar properties to the NK cell (Kiessling et al., 1977). These three cell types share the properties of (1)being neither T cell nor B cell, although of bone marrow origin; (2) acting without prior immunization; (3)being radiation resistant, although irradiation removes the NK precursor cells from bone marrow; (4) being susceptible to the anti-bone marrow agent ?3r. However, although the properties of these cell types are similar, it remains to be demonstrated that all these effects can be mediated by the same cell type. G. Ly-X LOCI At least three loci have been described that are associated with the X chromosome, and these are designated Ly-X (Zeicher et al., 1977, 1979). The basis for the discovery of these systems was that several strains have been found to have X chromosome-linked regulation of immune responsiveness to several thymus-independent antigens. Strains BALB/c, DBAJ2, and SJL are low responders to type I11 pneumococcal polysaccharide, poly(I), poly(C), and denatured DNA, respectively. Three alloantisera (anti-X1,-Xz, and -XJ were raised in nonresponder male F1hybrids against the identical F1high-responder females, i.e., (XloWx Y)F1 anti-(Xlowx Xhish)F1female. Recipient and donor mice were F1males and F1 females, respectively (Zeicher et al., 1977). It was hypothesized that the high immune response was associated with a specific cell-surface antigen present on the lymphocyte membrane, and immunization in such cases led to the production of antisera reactive with the corresponding high-responder strains but not with low-responder strains. Four different specificities were detected-LyX-1.1, LyX-2.1, LyX2.2, and LyX-3.1-and these reacted against products of X chromo-

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somes of BALB/c, DBA/2, and SJL, respectively (Table XX). The sera reacted with approximately 10% of thymus cells, spleen cells and lymph node cells and with approximately 25% of nylon wool nonadherent T cells obtained from the spleen. The anti-LyX-2 serum reacted with DBA/2, which suggested the presence of another antibody: LyX2.2. For each of the three loci, the reactivity of the antisera was confined to strains with a high response to the three different antigens, and the low responders were nonreactive. In the subsequent study, Zeicher et al. (1979) found that the antiLyX-2 reagent consisted of two different antibodies: those reacting with T cells (called Lyt-X) and those reacting with B cells (Lyb-X), and these were regarded as being separate, but with the same strain distribution pattern. On examination of the additive effects with several different antisera to Ly-X, Thy-1.2, Ly-1.1, or Ly-2.1, no additive effects were noted with the Ly-1.1 antisera, which suggested that Ly-2/3 and Ly-X were present on the same lymphocyte population whereas Ly-1 and Ly-X were present on different populations. These studies were confirmed by using peanut agglutinin to separate immature and mature thymocytes. It therefore appears that there may be several specificities present on T lymphocytes and possibly on B lymphocytes, which may be coded for by X-linkedzr genes. However, formal proof of the multiplicity of this system must await the appropriate backcross studies. IX. Xenoantisera Recognizing Lymphocyte Cell-Membrane Determinants

A whole series of xenoantisera have been prepared, usually in rabbits, by immunizing with mouse tissues, such as thymus, B cells, brain. More recently, fractionated cells, purified cell populations, and cell membranes have been used. These sera have potential usefulness, as they are usually of high titer and appear to react with all strains; however, they suffer from the disadvantage that extensive absorption may be required to demonstrate specificity. Initially almost all rabbit anti-mouse sera contain antibodies that appear to react with all tissues of all mice, and this so-called “species-specific” antibody requires removal b y absorption with liver, red cells, platelets, etc., before tissue specificity can be demonstrated. (To our knowledge this antibody has not been shown to be mouse specific, and “species specific” is therefore a misnomer.) The production of xenoantisera is now receiving considerable attention in different studies in man, where the production and use of alloantisera directed to non-MHC products (Chess and Schlossman, 1977) is quite limited and the production suffers from

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certain ethical considerations. Furthermore, the use of xenoantisera i n vivo has been found, with one or two exceptions, to be far superior to the use of alloantisera in inhibiting specific reactions. Studies in mice therefore are of great relevance in the practical production of such antisera for use in man. A. ANTILYMPHOCYTESERA (ALS)

ALS has been extensively studied in the last decade and is the most effective agent for the prolongation of skin allografts in mice. ALS is also used in man for immunosuppression for renal allografts, although the graft prolongation in man is not as obvious as in mice, presumably because much lower doses are given. The production and use of ALS has been covered extensively in earlier reviews (Wolstenholme and O’Connor, 1967), and only several comments need to be made. First, ALS is usually made against mouse thymus cells [antithymus sera (ATS)] and after appropriate absorption appears to b e T-cell specific. The specific component is probably the same as mouse specific lymphocyte antigen, described below. Second, ALS has also been made in many different ways, using tissues other than mouse thymus cells. I n these circumstances a whole range of other antibodies would also be produced, i.e., anti-B cell antibodies, anti-plasma cell antibodies so that ALS made against a tissue containing a broad range of cells, e.g., spleen, would therefore contain some of the more specific components described below. Third, the structure of the molecule, detected by ALS, is probably the same or related to the structures described by Williams et a l . (1976) (see Section VI).

B. MOUSE-SPECIFICLYMPHOCYTE ANTIGEN(MSLA) Antisera to MSLA (Shigeno e t al., 1968) was prepared from rabbit antimouse thymus sera after in vivo absorption. The absorbed antiserum reacted with an antigen present on thymocytes, lymphocytes, T-cell leukemias but not sarcomas. The sera also had in vivo immunosuppressive effects, and tissue specificity, but no strain specificity, was demonstrated. Furthermore, the antisera were found to contain small amounts of anti-H-2 antibodies, but no antibodies recognizing TL, Ly-1, Ly-2, or Thy-1 alloantigenic specificities. LYMPHOCYTE-SPECIFIC SURFACE C. MOUSE THYMUS-DERIVED ANTIGEN (MTLA) MTLA antisera have been produced by immunizing rabbits with either mouse thymus, boiled thymus, brain homogenate, and tumors including lymphosarcomas (Bron and Sauser, 1973; Rabinowitz et al.,

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1974; Ochiai et al., 1975) or thymocyte membranes purified on Sephadex or by polyacrylamide gel electrophoresis (Sauser et al., 1974). All these antisera have the same T-cell reactivity as described for MSLA antisera and lack any strain specificity. It is likely that MSLA and MTLA specificities are the same. However, in contrast to other sera, the antisera produced by Rabinowitz et a l . (1974) seem to contain reactivity for alloantigens such as Thy-1.2 and possibly Ly-1.2, Ly-2.2. However, for the specific demonstration of these antibodies exhaustive absorptions were not performed, and the authors noted that the detection of the alloantigenic specificity was always weak. A comparison of anti-Thy-1.2 and anti-MTLA sera in reciprocal blocking studies demonstrated that both antisera react with specificities that are probably carried on the same molecule (Sauser et al., 1974) (see Section VI, A).

D. BRAIN-ASSOCIATEDTHETA(BA8) A T-cell antigen recognized by rabbit antisera to homogenized mouse brain has been described b y a number of investigators (Asakuma and Reif, 1968; Golub, 1971; Sauser et al;, 1974; Rabinowitz et al., 1974). The antisera appear to satisfy the requirements for a T cell-specific marker, as after little absorption there appears to be no activity for B cells, although the antiserum is immunosuppresive in viuo for a subsequent in uitro plaque assay, presumably by interacting with helper T cells. The antiserum is a useful in uitro reagent for the detection and elimination of T cells by cytotoxicity methods, although by using the rosetting methods we have found that the reactivity is not confined to T cells (Owen and McKenzie, unpublished results). Recently it has been shown that rabbit anti-mouse brain antiserum reacts with the pluripotential hematopoietic stem cells (CFU-s) of bone marrow (Filppi et al., 1978). The relationship between the specificities detected by BAO, MSLA, MTLA, and Thy-1 sera are not clear, but it is possible that all react with determinants present on the same molecule (see Section VI, A).

E. MOUSE-SPECIFIC PERIPHERALLYMPHOCYTE ANTIGEN (MPLA) During an attempt to raise an anti-MBLA serum against lymph node cells from thymectomized, irradiated, and liver-reconstituted mice (Raff and Cantor, 1971), an antiserum was produced that reacted with 100% of lymph node cells and only 4% of thymocytes. The antigen defined was called MPLA, as it apparently recognized a peripheral lymphocyte antigen. No further studies have been reported with this serum.

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F. XENOANTISERA-DETECTING DETERMINANTS PRESENT ON KILLER T CELLS Antisera raised against inocula enriched for killer cells have been described by several investigators. A rat antiserum against mouse immune peritoneal exudate lymphocytes was found to react with an antigen, Ka, present on T cells cytotoxic for a number of different tumors (Sullivan et al. 1973). Absorption studies showed that this antigen was not present on thymus or bone marrow cells and was confined to mature functional T cells, but apart from cytotoxicity no other T cell functions were studied with this serum. The population identified by the antiserum was also shown to be Thy-l+ Ly-l-2+; i.e., this was the first demonstration that killer cells were Ly-1-2+ (Sullivan et al., 1973) (see also Section XI). A similar antiserum, raised in guinea pigs against BALB/c killer cells, reacted with about 20% of Ig- spleen cells and inhibited killer-cell activity (Rothstein et a1., 1978). Helper-cell function and mitogen responsiveness were not impaired by this antiserum. In other studies, a xenoantiserum made against nude mouse splenocytes was reported to react with a determinant [mouse killer T-cell antigen (MKTCA)] present on cytotoxic T cells, IgM plaque-forming cells, but not on helper T cells (Kisielow et al., 1976).

G. ANTISERA TO PURIFIED T-CELL POPULATIONS A number of studies have indicated that T-cell or B-cell specific antisera can be produced by immunization and subsequent reciprocal absorption with purified T or B lymphocytes. For example, T and B cells separated by electrophoresis have been used as the inocula and the absorbing cells in order to prepare specific T-cell and B-cell antisera (Zeiller and Pascher, 1973). These antisera recognized separate and distinct determinants on B cells, peripheral T cells, and functional T cells (Zeiller and Pascher, 1973). In another study, xenoantisera made against cortisone-resistant thymocytes and absorbed with cortisone-sensitive thymocytes reacted only with cortisone-resistant thymocytes, lymph node cells, and spleen cells (Papiernik and Bach, 1977).

H. THYMOCYTE-B LYMPHOCYTE ANTIGEN (Th-B) Immunization of rabbits and goats with the BALB/c myeloma MOPC-104E gave rise to an antiserum that recognized an antigen present on both thymocytes and B cells but not on peripheral T cells (hence designated Th-B: Yutoku et a1 ., 1974,1975,1976). The absence of the Th-B antigen from peripheral T cells suggested that it appeared relatively early during differentiation, but was lost during T-cell mat-

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uration (Yutoku et al., 1975). It was also suggested that the Th-B antigen was present on immature thymocytes and is lost as subcapsular cells in the thymus migrate toward the medulla (Stout et al., 197513). The Th-B specificity may also be present on prothymocytes, as it is present on lymphoid tissues of nude mice (Stout et a1 ., 1975a).

I. MOUSE-SPECIFICBONEMARROW-DERIVED LYMPHOCYTE ANTIGEN (MBLA) An antiserum was made in rabbits to lymph node cells of thymectomized, irradiated, fetal liver-reconstituted CBA mice; after extensive absorption with red cells, liver, and thymocytes, it reacted with B cells in lymph node, spleen, and approximately 40% of bone marrow cells (Raff et d.,1971). This antiserum showed reciprocal cytotoxicity to anti-Thy-1 antisera (Raff et a1 ., 1971) and also reacted with MOPC-315 myeloma cells, direct IgM+ plaque-forming cells (Niederhuber and Moller, 1972), and antigen-binding B cells (Niederhuber and Moller, 1972). The serum therefore appeared to react with B, not T, cells. Further evidence supporting the restriction of the MBLA specificity to B cells were the findings on immunofluorescence testing with antiMBLA and MSLA sera, where reciprocal patterns were noted (Lame1972). The MBLA specificity was lin et d.,1972; Niederhuber et d., also found on some Ig- bone marrow small lymphocytes (Ryser and Vasalli, 1974) and may therefore be one of the earliest serologically detected markers of B-lymphocyte differentiation.

J. OTHERSERADETECTINGB-Cell XENOANTIGENIC SPECIFICITIES A B-cell antigen (given no specific name) was identified by a rabbit antiserum made against lymph node cells obtained from nude mice (Kakiuchi et a1 ., 1976). The antiserum was found to react with B cells only and could decrease the number of antibody-forming cells in irradiated mice reconstituted with bone marrow. However, in contrast to MBLA, the effect of this anti-B serum on direct plaque-forming cells was minimal, which suggested that this antigen was present on B cells but absent from antibody-forming cells. The relationship of this antigen to the MKTCA is not clear. Gorczynski (1977a,b) prepared a series of anti-B cell antisera by immunizing with spleen, lymph node, bone marrow, as well as fractionated bone marrow, cells. After appropriate absorption, the antisera reacted with different B-cell subsets, such as stem cells or more mature B cells, but no further studies of these sera have been reported as yet.

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271

K. MOUSE-SPECIFICPLASMACELL ANTIGEN (MSPCA) Immunization of rabbits with plasma cell tumors, such as MOPC104E or MPC-67, produced antisera that, after absorption, reacted with plasma cell tumors and a small percentage of spleen cells in plaqueforming assays (Takahashiet al., 1971; Watanabe et al., 1971a,b; Harris et al., 1972). The antigen appeared to be reactive with the same cell population as the Pca-1 alloantigen. In addition the antisera reacted with other plasmacytomas, with the notable exception of MOPC-70A, which differs from some of the others in the class of immunoglobulin secreted. The plasma cell, kidney, liver, and brain (PKLB) antigen (Yutoku et d., 1974, 1977) appears to be almost identical to MSPCA, although it was made against the plasma cell tumor AJD-PC-22A and reacts, by absorption, with the nonlymphoid tissues mentioned. Similar to antiMSPCA, it does not react with MOPC-70A. L. ML-2 ANTIGEN A second determinant recognized by the xenogeneic anti-MOPC104E serum was detected after separating cells on the FACS (Stout e t al., 197513). The antigen, ML-2, was found on splenic B cells but was absent from thymocytes and splenic T cells. This antigen could be identical to MBLA.

M. ANTIBODIESTO THE L P s RECEPTORON B CELLS An interesting antibody has recentlv been prepared taking advantage of the finding that C3H/HeJ is a nonresponder to the mitogenic effect of the lipid A moiety of LPS. The antiserum was prepared b y immunizing rabbits with C3HITif (LPS high responder) and the resultant antiserum was absorbed with the nonresponder C3WHeJ (Forni and Coutinho, 1978). The antiserum reacted with a subfraction of B cells from LPS high-responder strains and did not react with the two LPS nonresponder strains (C3H/HeJ, C57BWlOScCr). It was also found that the cellular reactivity of this antiserum, and the ability of cells to bind LPS, segregated together in individual backcross mice. In addition, LPS (especially the lipid A fraction) and the anti-LPS receptor antisera competed for binding to the same site on the B-cell membrane. It was therefore suggested that the xenogeneic antiserum reacted with the LPS triggering receptor on B lymphocytes (Forni and Coutinho, 1978).

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N. XENOCENEIC ANT1-k SERUM A number of studies have indicated that Ia antigens can be recognized by xenogeneic antisera. This was first shown using rat antimouse antisera (Sachs and Cone, 1973) and has now been shown in a number of studies by immunizing with B cells, B-cell tumors, or purified membrane components (Billinget al., 1976). In both man and mouse these sera apparently react with Ia antigens present on the surface of B cells. The precise relationship of these antibodies to alloantibodies is not clear, but it is likely that both are present on the same molecule, although it may be that the xenogeneic serum reacts with specificities present on one chain, and the alloantibodies with the other chain, as has been suggested for the DRW reactions in man (Snary et al., 1976). As with other xenoantisera, no strain specificity has been demonstrated. An interesting anti-Ia-like antibody has recently been described (Parish et al., 1976a). It was produced by immunizing rabbits with mouse whole sera and absorbing with dialyzed sera. The antiserum reacts with B cells, with some T cells, and with macrophages, but is not cytotoxic, and a rosetting assay has to be used for its detection (Parish et al., 1976b). The characterization of these sera have been described in detail elsewhere; however, it appears that these antisera recognize carbohydrate-defined Ia antigens in contrast to alloantibodies, which recognize both protein- and carbohydratedefined alloantigens (McKenzie et al., 1977d). An interesting recent observation is the finding that a xenogeneic anti-DRW serum reacts with Ia-like molecules in a number of species, including the mouse (Kvist et al., 1978). X. Relationship of Murine Leukemia Virus (MuLV) and CMAD

There have recently been a number of different examples wherein CMAD and MuLV appear to be associated-either directly or indirectly. For example, as discussed in Section 111, viral antibodies contaminating alloantisera are providing considerable problems in the analysis of CMAD. This has proved to be particularly troublesome when tumors are examined with anti-Ly sera (Mathieson et al., 1978; Wettstein et al., 1976; Hogarth and McKenzie, unpublished). In addition, it has recently been established that normal lymphocytes and pluripotent hemopoietic stem cells express viral structural proteins, and the expression of MuLV is related to the stage of cell differentiation (Lonai et al., 1974; Elder et al., 1977; Staber et al., 1978). Furthermore, some CMAD, e.g., GIx, Pca-l, and X-l, appear to be of viral origin or closely related to viral determinants. Other possible relation-

273

MURINE CELL-SURFACE ANTIGENS

ships between CMAD and MuLV have also been suggested, such as between H-2 and endogenous virus (see discussion by Snell et al., 1976). We therefore considered it appropriate to include a condensed and simplified review of MuLV and their relationships to CMAD. Detailed reviews of MuLV have recently been published (Aaronson and Stephenson, 1976; Old and Stockert, 1977; Gardner, 1978; Levy,

1978). The first suggestions of the etiological role of virus in murine leukemia were the classical studies that demonstrated that cell-free preparations of lymphoid tissue from leukemic mice were leukemogenic (Gross, 1951). Later, isolation of the virus and induction of leukemia with purified virus confirmed the viral etiology of the particular murine leukemia under investigation (Gross, 1970). It now appears that a viral genome is incorporated into the cellular genome of all mouse cells (Aaronson et al., 1971; Chattopadhyay et al., 1974). The integrated viral genome (or provirus) is regarded as being “endogenous,’’ in that it is inherited through the germ line and behaves essentially as a host gene under the same regulatory influences as other cellular genes (Aaronson and Stephenson, 1976).

A. THE EXPRESSION OF VIRAL ANTIGENS The structure of a typical murine RNA virus is represented in Fig. 1. The virus particle is composed of a bilayer lipid envelope derived from the host cell (Fig. 2), and this has viral proteins inserted into it. This envelope surrounds an icosahedral capsid, which encloses RNA and RNA-dependent DNA-polymerase. Several viral polypeptides

lipid bilayer

acquired from host c e l l

1

major envelope glycoprotein minor envelope protein

P 10

::: P 30

envelope

s t r u c t u r a l proteins forming capsomer~. some of t h e s e proteins-capsid are a s s o c i a t e d with ribonucleoprotein.

FIG. 1. Structure ofa type C murine virus. Numbers indicate molecular weight of glycoprotein (gp)or polypeptide (p).

X

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IAN F. C. MCKENZIE AND TERRY POTTER

Membrane pinches off to give envelope SUrrOUIIding mature virion

t Membrane extruded, surrounds capsid. Host membrane components almost completely replaced

viral envelope components inserted into cell membranes Host cell surface components displaced

Host cell membrane components

--bilayer

Host cell lipid membrane Viral envelope (glyco) proteins

I

\

,

1

Icosahedral capsid surrounding ribonucleoprotein core

I

I

\

,

\

I \

I

\

I

I

‘0’

Productively infected cell

FIG.2. Budding of RNA virus from host cell. In the case shown, host components have not been incorporated into the virus. Bubbers et al. (1978) have shown that Friend virus incorporates H-2D products.

have been identified by standard biochemical techniques, and by the use of recombinant strains of viral mutants, these proteins have been characterized as products of regions of the viral genome designated gag, p o l (polymerase), and env (envelope) (Baltimore, 1975). The p o l gene codes for the RNA-directed DNA polymerase, the env gene for an

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275

85,000 MW polyprotein, which is cleaved to a 15,000 MW [p15 (E)] and a 70,000 MW polyprotein. The gag gene produces a 65,000-68,000 MW protein, which gives rise to several internal polypeptides (p10, p12, p15, and p30) associated with the core of the virion. In many laboratories, the 70,000 MW glycoprotein of the env gene has been resolved into components of69,OOO MW and 71,000 MW (gp69, gp71). Until 1974 several different nomenclatures were used to describe the virion proteins identified during purification procedures, and consequently much of the literature was very difficult to correlate. The nomenclature suggested by August e t al. (1974) has received widespread acceptance. According to this system, proteins are designated by their biochemical nature and molecular weight: g p = glycoprotein, p = protein, followed by the molecular weight times e.g., gp70 is a glycoprotein of molecular weight 70,000. The structural role or properties of the protein may be indicated by a capital letter: e.g., C, core; E, envelope; N, nuclear. This has made it possible to designate the differences between the 15,000MW proteins expressed on the envelope (p15E) or the core (p15C). Each of these viral components carries antigenic determinants described as interspecies, group- and type-specific antigens, but the relative proportions of these antigens vary. Thus p30 mainly expresses group specificities, and p12 bears predominantly type specificities. Interspecies determinants are common to RNA leukemia viruses found in all mammalian species; group specificities are common to viruses associated with a given species: e.g., the group-specific antigens of murine leukemia viruses differ from those of the corresponding feline viruses. Type-specific determinants are the property of individual viruses. The major structural MuLV products so far identified are gp70 (gp69/71), p30, p15 (including p15E), p12, and p10. These proteins may be isolated and used in the preparation of antisera in rabbits or goats, and these antisera have demonstrated that each of the proteins is antigenically different. Furthermore, protein products induced by xenotropic viruses are antigenically different from those induced by ecotropic viruses. Structural comparison of the gp70 glycoproteins from different anatomical sites (e.g., lymphoid organs, kidney, or serum) has revealed that there are difference between the gp70 molecules expressed in different tissues within an individual (Elder et d., 1977).

B. CLASSIFICATION OF MuLV Isolation of viruses from a variety of leukemic and normal cells has led to the realization that there are many murine leukemia viruses,

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IAN F. C. MCKENZIE AND TERRY POTTER

each with a characteristic host range, nucleotide sequence, interference pattern, as well the biochemical and serological differences in the viral products described above. These properties have led to MuLV being classified in several different ways. Although the relationships between the different classifications are becoming clearer, at present there is no integrated classification available.

1 . Host Range Endogenous viruses may be classified into ecotropic, xenotropic, and amphotropic on the basis of their host-range restriction: ecotropic viruses grow only in mouse cells, xenotropic viruses grow only in cells from other species, and amphotropic viruses are isolated from wild mice and grow in cells from many species, including mouse (Hartley and Rowe, 1976). The ecotropic viruses may be further classified on the basis of restricted growth in cells from different mouse strains. N-tropic viruses grow best in NIH Swiss mice, B-tropic in BALB/c, and NB-tropic grow in both strains. The murine host-range restriction is determined by the Fu-l locus (Lilly and Pincus, 1973). It is important to note that host range patterns may not be stable; for example, N-tropic viruses grown in BALB/c mice may, after several suboptimal passages, recover and grow in this strain, resulting in an NB-tropic virus (Hartley et al., 1970). Nucleic acid hybridization studies have demonstrated that N-tropic and B-tropic viruses are closely related (Callahan et al., 1975). Xenotropic viruses were originally isolated from NZB mice and have since been isolated from many strains including C57BW6, NIH/Swiss, and nude mice (Levy, 1978). Isolation procedures suggest that strains such as NZB and C57L carry only xenotropic viruses, whereas other strains such as AKR, DBA, C57BL/6, BALB/c, and CBA carry both ecotropic and xenotropic viruses. Within the xenotropic viruses are found further differences in the species in which such viruses grow optimally, suggesting the existence of several distinct xenotropic virus subtypes, even within one mouse strain (Levy, 1978).

2. Nucleic Acid Homology Comparison of the homologies of the nucleic acids isolated from different xenotropic viruses with a complementary DNA probe, synthesized from BALB/c and NZB xenotropic viral RNA, showed that the genome of all the xenotropic viruses, excepting NZB and NIWSwiss, were relatively homologous (Callahan et al., 1975). On this basis, xenotropic viruses have been classified as either MuLV-X" or MuLV-XB, which includes virus iso-

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277

lated from AKR, BALB/c, and other strains that also carry ecotropic viruses and replicate best in rat and rabbit cells. By contrast, MuLV-XP viruses are restricted to strains from which ecotropic viruses have not been isolated, such as NZB and NIH/Swiss, and these grow best in human and other primate cells.

3. Antigenic Diferences Radioimmunoprecipitation assays for the core protein p12 and the envelope glycoprotein gp70 have led to the separation of the endogenous MuLV into three subclasses (Stephenson et al., 1975). Class I is composed of ecotropic MuLV, class I1 contains most of the known xenotropic viruses, and class I11 is represented by NZB and NIH/Swiss xenotropic viruses. Both class I and I1 viruses are inducible by halogenated pyrimidines in vitro (Aaronson and Stephenson, 1976). C. ANTIGENS INDUCEDBY VIRUS The induction of endogenous virus, or infection of the cell with exogenous virus, can lead to several alternative modes of expression of viral or virus-related antigenic determinants. The simplest outcome of such an infective process is that there are no additional antigenic determinants expressed on the cell surface. At the other extreme, there could be active virus production accompanied by an expression of many new determinants, such as found with the GCSA system. There are other possibilities, such as the virus-controlled elaboration of the expression of a host gene, e.g., Pca, Abelson antigen, or the expression of viral determinants without an accompanying production of virus, as found with the Glxsystem. In productive viral infection, viral products are synthesized and assembled into intact virus at the budding site on the cell surface (see Fig. 2), and at this stage viral internal components may also be expressed on the cell surface, but separate from the viral budding site (Tung et al., 197613). As the viral envelope is acquired from the cell membrane during the budding process, it is possible that some CMAD are included on the viral envelope. Recently Bubbers et al. (1978) have shown selective incorporation of some H-2D specificities into the Friend virus, and we have observed a similar phenomenon with the AKR virus, as anti-Ly-6.2 activity is removed by AKR virus (see also Section VI).

D. STRUCTURAL VIRALCOMPONENTS AS CMAD The reactivity of antitumor sera with several virus-induced tumors was shown to be a consequence of recognition of CMAD of viral origin or association (Gross, 1970). As such reactions were encountered origi-

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IAN F. C. MCKENZIE AND TERRY POTTER

nally with Gross virus-induced tumors, the systems are prefixed with the symbol G, e.g., GIx,GCSA. These antigens may also be expressed on normal cells as well as leukemic cells, and some show selective expression in lymphocyte differentiation, e.g., confinement to T-cell lineage, The expression of many of these antigens on normal tissue varies among different strains; the strain distribution pattern for such antigens is presented in Table XXI.

1 . GCSA All leukemias induced by Gross-AKR virus have a common surface antigen, the Gross cell-surface antigen (GCSA). GCSA was identified after absorption of antiserum produced in either C3H or C57BW6 mice against Gross virus-induced leukemias, and was shown by cytotoxicity to be present on the surface of leukemias and normal lymphoid tissues of strains with a high incidence of spontaneous leukemias, such as AKR and C58 (Old et al., 1965). Further analysis showed that GCSA could also be found in normal and malignant tissues of low-incidence strains (Nowinski e t al., 1968). The antigen was never found in the absence of replicating MuLV and is now considered to be an invariable marker of productive MuLV infection (Stockert et al., 1971). As some GCSA- strains, such as NZB, can become GCSA+ with increasing age (the so-called conversion strains), the classification of strains as GCSA+ or GCSA- is usually made on the basis of anti-GCSA antibody absorption by normal spleen from young mice. It appears that antisera to GCSA recognize determinants expressed on the viral internal components p30, p15, p12, and p10 (Tung et al., 1976b; Snyder et al., 1977), and so the antigen can be detected by immunoelectron microsTABLE XXI STRAINDISTRIBUTION OF MuLV-RELATED CMADa.b Strain

C57BI46 C58 A BALBIC C3H/Figge AKR NZB

129 a

GRADAUGERLD)

GCSA

GlX

-

-

-

+

+ +

+

+ + + +

+ +

-

+ + +c -

-

-

-

References are given in text. antigen present; -, antigen absent. NZB is a “conversion strain” in expression of GCSA.

+,

+ + + + + +

x-1

Pca- 1

-

-

+ -

-

+ + + +

+ + + + + -

MURINE CELL-SURFACE ANTIGENS

279

copy of the cell surface, but not of the viral envelope. GCSA may also be expressed on the cell surface away from the budding site; however, whether it is expressed exclusively on virion core components is uncertain, as some pools of GCSA react against viral envelope proteins gp70 and p15(E) (Ledbetter and Nowinski, 1977). The expression of GCSA in F, hybrids made between GCSA+ and GCSA- strains is under the control of the Fu-1 locus, which has two alleles, Fu-1 and Fv-1 b, that determine the susceptibility of the particular strain to infection by N-tropic or B-tropic MuLV (reviewed by Old and Stockert, 1977).

2. GSA An antigen related to the Gross-virus was found in the serum of normal NZB mice, as well as virus-infected or tumor-bearing mice, and was designated the Gross soluble antigen (GSA) (Aoki et al., 1968). It is likely that GSA is GCSA that has been released into the serum. The antigenic determinants defining this system are also located on p30 and p15.

3. GLK A further MuLV-Gross associated determinant is the Glx antigen, which was initially detected, by a cytotoxic antibody, on the lymphocytes of some strains (Stockert et al., 1971, 1972) (Table XXI). In GCSA- strains, only the thymus is Glx+; however, in the GCSA+ strains, the Glx antigen is found in spleen. Recent evidence suggests that serum, sperm, and seminal vesicles of Glx+strains also possess the antigen (Obata et al., 1976).As the Gross AKR virus has a widespread occurrence in inbred strains, it is difficult to produce alloantisera to the Glxdeterminants. Therefore antiserum to the Glx antigen was initially produced in rats, which are highly susceptible to MuLV infection as they are not naturally exposed to these viruses. The serum is usually produced against a Gross-MuLV induced rat leukemia, such as C58NT in (W/Fu x BN)Fl hybrids. However, mouse alloantibody to Glx, with the same specificity as the rat Glx antisera occurs spontaneously in (B6.Glx+x 129)F1 mice (Obata et al., 1976). Spontaneous production of this antibody in mice is accompanied by decreased expression of Glx on thymocytes of these mice, an observation similar to the antigenic modulation phenomenon described in the T L system (Obata et al., 1976). The Glx antigen occurs in four different cell-surface concentrations in different strains and has the quantitative ratios of 3 : 2 : 1: 0. The expression of the antigen, in the prototype strain, 129, is dependent on

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IAN F. C. MCKENZIE AND TERRY POTTER

two unlinked genes: Gu-1, which has been mapped to chromosome 17 (linkage group IX, hence the designation Glx),and Gu-2, which has been located on chromosome 1 (Stockert et al., 1972). Both Gu-l and Gu-2 positive alleles must be present for the expression of the Glx antigen, In contrast to strain 129, in AKR mice the Gu-I locus maps to chromosome 4 (Ikeda et al., 1973), an observation that has altered concepts about the genetics of this system (reviewed by Boyse, 1977) and led to the discussion of quasi-linkage. The GIx specificity differs from GCSA in that Glxcan be expressed in the absence of productive viral infection, as in strain 129 (Glx+, GCSA-). However, infection by most ecotropic viruses, particularly by N-tropic virus generally leads to expression of the GIxantigen, even in strains classified as being Glx-. In contrast to the earlier observations that only ecotropic viruses could induce expression of GIx (O’Donnell and Stockert, 1976),mapping studies of immunoprecipitates and tryptic digests has suggested that Glx- gp70 is coded for by a xenotropic viral genome (Elder et al., 1977; Tung et al., 1978a). Several observations have demonstrated that GIx is a type-specific antigenic determinant carried by the gp70 molecule, and these include: (a) removal of serum Glx by antiserum to MuLV gp70; (b) molecular weight analysis of the anti-GIx immunoprecipitate; (c) steric inhibition of goat anti-gp7O attachment by anti-Glx serum; (d) absorption of Glxantibodies by whole Gross virus; (e) mouse thymocytes may express gp70 as detected by cytotoxicity of anti-MuLV gp70 (Obata et al., 1975; Tung et al., 1975b; Villano et al., 1975; Elder et al., 1977). With the exception of BALB/c and the Glxcongenic strain 129, GIx-, all strains may express gp7O-like molecules (Strand et al., 1974). Therefore it is apparent that there are at least two antigenically different forms of gp70, i.e., Glx+ or Glx- (Tung et al., 1975a): G ~ g p 7 0 expressed on 129 thymocytes; 0-gp70 expressed on C57BL/6 thymocytes. A further serologically distinguishable form of gp70 is X-gp70, which occurs only on leukemias or cells from strains that produce large amounts of virus (Tung et al., 1976a). Antibody to X-gp70 is present in anti-X.l sera (see below). Similar to Glx-gp70, X-gp70 is expressed on some radiation leukemias of X-gp70- strains; however, unlike the GIx system, expression of X-gp70 is dependent on productive viral infection. The presence of additional antigenic determinants on gp70 seems likely (e.g., Gupta et al., 1977: see GRADAl below). In summary, GIx is an antigenic determinant on the gp70 molecule expressed on the thymocytes, sperm, and other tissues from several strains, even in the absence of productive viral infection.

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281

4 . G R A DGERLD A~~ Two more antigens related to MuLV-Gross have recently been identified. These have been designated GRADAl (Obata et al., 1978) and GERLD (Old and Stockert, 1977) because of their presence on the leukemias RADA-1 and ERLD. Antibodies to these specificities are found in the serum of nonimmunized mice, anti-GRADAl being found in NIH/Swiss and anti-GERLD in the sera of (C57BL x 129)F1mice. Both these antigens are detected by cytotoxicity on leukemias and normal lymphoid tissues of strains (such as C58, AKR, C3H/Figge) with a high leukemia incidence. Although GIx is expressed on the gp70 molecule, its independence from Glx and the other Gross related antigens is indicated by a unique strain distribution pattern. The fact that under certain circumstances mice can recognize the GIX, GCSA, GRADA1, and GERLD antigenic determinants as foreign, with an accompanying antibody production, raises the possibility that these specificities have a role in the spread of virus in the host and in the emergence of transformed cells (Obata et al., 1978).

E. VIRUS-RELATEDANTIGENS Several antigenic determinants have been described, such as X-1, Pca, that are not part of the virion but are associated with virusinduced transformation. These are collectively referred to as virusrelated antigens. Originally GCSA and GIxwere thought to be virus related, but they have since been shown to be on virion components. This may also prove to be the case for some of the antigens described below.

1 . x-1 The observation that BALB/c radiation-induced leukemias could be rejected by BALB/c F1 hybrids, with an accompanying production of cytotoxic antibodies to the leukemia, suggested the existence of an antigen X-1, present on the leukemia and to which the F1hybrids, but not homozygous BALB/c mice, could respond (Sato et al., 1973).The standard anti-X-1 typing sera are obtained from BALB/c F1 hybrid mice that have rejected the radiation-induced leukemia RL c3 1 (Sato et al., 1973), and the sera are cytotoxic for this tumor. The X-1 antigen is present on normal tissues, but the concentration of the antigen is so low on normal lymphocytes that it cannot be detected by a direct technique. Therefore strains are typed for X-1 by their capacity to absorb anti-X-1 cytotoxic antibody, and the sera are subsequently tested on RL c3 1.

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IAN F. C. MCKENZIE AND TERRY POTTER

Thus far, C58, AKR, NZB, and 129 have been typed as X-l+ and other strains as X-1- (Table XXI). The X-1 antigens appear to be unrelated to GIXand GCSA antigens as the standard Glx+/GCSA+leukemia, E 6 G2, is X-1- (Old and Stockert, 1977). The relationship, if any, of the X-1 antigen to MuLV has not been established as yet; however, its expression on leukemias of X-1- strains (Old and Stockert, 1977) is characteristic of other MuLV-related antigens. In relation to the radiation-induced leukemias, it should be noted that the resistance of (BALB/c x C57BL/6)F1hybrids to the radiation-induced leukemias is conferred by a C57BL/6 gene in the H-2K region. This may be the Rgv-1 (resistance to Gross virus) gene of Lilly (1970) conferring the ability to mount an antibody response to the X-1 and possibly other antigenic determinants on such leukemias. 2 . Plasma Cell Locus (PC-1) Pea-1 is an antigen present on plasma cells but absent from thymocytes and T lymphocytes and is defined by the reaction of the antiserum DBA/2 anti-MOPC-70A (a BALB/c myeloma) (Takahashi et al., 1970a,b, 1971). The serum reacts with plasma cells of many strains (Table XXI) and has a unique strain distribution pattern. Of relevance to MuLV is the finding that the Pca typing of certain relevant strains is C57BL/6-, C58-, BALB/c+, NZB+, 129-. Transfer experiments and plaque assays have demonstrated that the antigen is present only on plasma cells (both IgG and IgM), but not on the precursor B cells, suggesting that Pea-1 is a differentiation antigen acquired late in the process of maturation of B cells to plasma cells. The antigen is also present on the surface of most plasmacytoma cells. The Pea-1 antigen has also been detected, by absorption, on cells obtained from the liver, kidney, brain, and lymph nodes, a distribution not unlike that of the xenogeneic PKLB antigen (see Section IX). Because of the occurrence of natural antibodies to the Pea-1 antigen in the sera of neonatal mice (Herberman and Aoki, 1972), it has been suggested that the Pea-1 antigen may represent expresssion of products of a virus present in latent form in some normal tissues of Pea-1 positive strains (Aoki et al., 1974a). Furthermore, so far there has been no report of an allelic product in Pca-1- strains-an observation consistent with viral induction of antigen expression (see Old and Stockert, 1977, for a more detailed discussion). Recent biochemical analysis has demonstrated that the Pca-1 alloantigen is expressed as a cell-surface membrane component (MW 105,000-110,000) that is different from any other CMAD or MuLV related antigen so far characterized (Tung et al., 1978b). The antigen should be of value in B-cell functional and differentiation studies together with the other B-cell specificities described in Section VI.

MUMNE CELL-SURFACE ANTIGENS

283

3. Abelson Antigen The Abelson antigen has been found on all cells transformed into tumor cells by the Abelson murine leukemia virus (MuLV-A) and is also found on uninfected cells from BALB/c fetal liver, bone marrow, and spleen, but not other lymphoid tissue (Risser et al., 1978). The Abelson antigen is expressed on normal cells only from BALB/c mice and, as MuLV-A was originally isolated following infection of a BALB/c mouse with Moloney virus, the authors suggest that the antigen is a serological marker of a BALB/c gene normally encoding a cell-surface molecule incorporated into the Moloney virus genome during the generation of Abelson virus.

F. EXPRESSION OF VIRAL ANTIGENS ACTIVATION

AFTER

LYMPHOCYTE

Viral antigens have been demonstrated on lymphocytes after activation by either mitogen or antigens such as LPS, lipid A, and PPD (Phillips et al., 1976; Moroni and Schumann, 1975; Lee and Ihle, 1976; Schumann and Moroni, 1976, 1978), by sheep red cells (Moroni and Schumann, 1977; Wecker et al., 1977; Schumann and Moroni, 1978), and by allogeneic cells during a GVH reaction (Datta and Schwartz, 1976). These observations suggest that the expression of the MuLV genome may be linked to the differentiation process. The finding of viral determinants on activated lymphocytes is of importance in the analysis of lymphocyte subpopulations by mouse alloantisera. Results should be interpreted with caution, as many of these functional studies (Section XI) are performed with antisera that are potentially contaminated with antibodies to viral determinants. G. CONCLUSION MuLV antigens are important in serological studies of CMAD because many alloantisera such as anti-Ly sera contain contaminating antiviral antibodies (induced by Gross-AKR type and related MuLV), which are primarily directed against p30 and gp70 (Stephenson e t aZ., 1976). Because of the possible association of C-type viruses with differentiation pathways, a current problem is to assess whether other alloantigens, in the Ly series, are virus associated. XI. Functional Studies with Serological Markers

As the T-cell population manifests a variety of different immunological functions, and as there also exists heterogeneity of T cells with regard to the expression of particular L y specificities, there have been extensive investigations to determine whether the functional T-cell

TABLE XXII SURFACE

ANTIGENIC PHENOTYPE OF FUNCTIONAL

T-CELL SUBPoPULATIONS

Surface antigenic phenotypea Function Cytotoxic cells (stimulus) H-2K andlor H - 2 D disparityb

I-region disparityb H-Ye Chemically (TNP) modified selfc Virus-associated antigensb Tumor-associated antigensb Helper cells 1. Antibody responses Antigen-specific secondary response Hapten-carrier systems in uitro Adoptive transfer in uiuo Non-antigen specific, Con A-induced

Ia

Ly-1

Ly-2J3

Other

References

r

n Y

-

-

NT

NT NT NT NT -

+ + NT

-Ly1.2 +Lyl.l -

-

+

-

+ + + + +

+

LyS+LyG+ALA-l,Fc+

Cantor and Boyse (1975b); Shikuet al. (1975); Stout et al. (1976); Feeney and Hammerling (1977); Woody (1977), Woody et al. (1977); Meliefet al. (1978) Simpson (personal communication) Simpson (personal communication) Cantor and Boyse (1976) Pang et al. (1976); Yap et al. (1978) Shiku et al. (1976) Stutman et al. (1977) Cantor and Boyse (1975a) Feldmann et al. (1975a, 197%) Tada et al. (1978b) Murphy et al. (1976b); Okumura et al. (1976) Jandinski et al. (1976)

B 2

E

i? U cl

Bv.e 0

3

2

Non-antigen specific, MHC-induced: Whole H - 2 , M l s , or I region alone H - 2 K or H - 2 D alone 2. For enhanced generation of killer cells in CML Suppressor cells 1. Antibody responses Antigen specific

TNP-self Allotype suppression Con A-induced IgE production Arising during GvH Neonatal virus-infected thymocytes Memory 2. Cell-mediated responses Con A stimulated suppression of TK Delayed hypersensitivity

NT NT

+ +

NT

+

+ +

IJ+

-

+

+

+ + +

NT IJ' NT NT

-

-

NT

+ + +

+

-

NT

-

NT -

-

+

+

-

Swain and Panifilli (1979)

Cantor and Boyse (1975b)

-

+ + + + + -

TL

Feldmann et al. (1975a); Beverley et al. (1976); Cantor et al. (1976); Weitzmann et al. (1976); Heuer et al. (1977); Ramshaw et al. (1977); Zan-bar et al. (1978); Bottomly et al. (1978) Cantor and Boyse (1976) Herzenberg et al. (1976); Okumura et al. (1976) Jandinski et al. (1976) Watanabe et al. (1977) Pickel and Hoffman (1977); Shand (1977) Mosier et al. (1977) Loblay et al. (1978) Jandinski et al. (1976)

Ly5+Ly6+

Huber et al. (1976b) Thompson, Potter, and McKenzie (submitted)

(Continued)

TABLE XXII

(Continued)

Surface antigenic phenotype" Function

Ia

Ly-1

Ly-2/3

In aioo tumor growth MLR proliferationd (stimulus) Whole H-2 or I region alone

IJ+

NT

NT

-

H-2K or H-2D alone DTH effector Secretion of soluble factors Antigen-specific supressor factors Nonspecific helper factors Proliferatiue responses Mitogens: PHA Con A LPS PWM Soluble antigens Tumor antigens MZF production During MLR Stimulation with antigen (DNBSOJ

NT -

+ + +

IJ+

-

NT

+ + + + + + + +

+/-

+ + + + -

NT -

Other

References Greene et al. (1977)

-

+

Cantor and Boyse (1975a); Michaelides and McKenzie, unpublished observations. Wettstein et 01. (1978) Huber et al. (1976b), Vadas et al. (1976)

+

Tada et al. (1977)

-

Ly5+Ly6+

Pickel et al. (1976)

+

Michaelides and McKenzie, unpublished Stanton et al. (1978), Flaherty et al. (1978b)

+

Ly5+LyG+Qal+Qa2+Qa3+ Ly5+Ly6+Qa2+Qa3+ Ly4+Ly5+LyG+LyM-l Ly4+Ly5+Ly6+ Ly6+Ly7+

+

Ly5+LyG+Ly7+

Newman et al. (1978) Basten, personal communication

+

-

-

Basten, personal communication Lerman et al. (1978)

+, Antigen present; -, antigen absent; NT, not tested. * Generated from Lyl-2+ precursors. This response is generally enhanced by Lyl cells. Precursor cells are Ly1+2+. Identified by uptake of [3H]thymidine.

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subsets bear a unique cell-surface phenotype, particularly of the Ly-1, -2, -3 specificities. A summary of the findings is presented in Table XXII. The underlying principle of most of these investigations is to lyse cells with anti-Ly sera and complement and to determine the capacity of the remaining cells to mediate a defined function, and compare this with the nondepleted population. If the effect is abrogated by antiserum treatment, then the assumption is made that the cells responsible for such a function carry the antigen defined by the antiserum. The major reservations in evaluating these functional studies concern the assay systems and specificity of the antiserum used. The assays in most cases depend on in vitro analysis; cytotoxicity is generally assessed by 51Crrelease, antibody production by a PFC response, and proliferation by [3H]thymidine incorporation. Although these represent perhaps the best of currently available techniques, sensitivity varies among different laboratories. Also the antisera used in many studies vary in strength; for example, anti-Ly-2.2 sera are usually weaker than anti-Ly-1.1 sera (Table V) (Shen et al., 1975). The problems of raising congenic antisera were discussed earlier (Section 111), and the possible contamination of antisera with extra antibodies against known or unknown specificities should also be kept in mind. The antisera could conceivably also contain antibodies directed against “immunoregulatory factors” produced in vivo. Should they exist, such antibodies would drastically affect systems such as the Mishell-Dutton cultures used in many of the investigations. In addition, contamination of antisera with antiviral antibodies (Section X) may also be a problem in the interpretation of functional studies, particularly as MuLV determinants have been identified on both helper T cells and on plaque-forming cells (Section X) (Wecker et al., 1977). To reduce the likelihood of such unknown factors affecting the interpretation of results, several approaches have been adopted: (1)testing the direct effects of antisera on both pairs of the congenic strains, or (2) using antisera that have been absorbed with both partners of the congenic strains before functional testing with the noncongenic strain (Shen et al., 1975). A further complication could arise should there exist further subpopulations in respect to the density of the surface antigens under investigation. For example, should there in fact actually be two subpopulations of Ly123 cells, one having a high Ly-1 but low Ly-2/3 density and the other having a low Ly-1 but high Ly-2/3, then, because of the varying strength of the antisera used, marginal results may lead to misinterpretations. A similar effect may account for the observation that killer cells strains with the Ly-la allele are

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IAN F. C. MCKENZIE AND TERRY POTTER

Lyl-2+3+ whereas those with the Ly-l* allele are Ly1+2+3+,although whether this reflects the difference in titer of the two antisera has not been determined. Although the above example is only speculation, it does demonstrate the need to interpret observations with caution. So far most of the experiments described have used functional testing of the cell population remaining after treatment with antisera and complement. Thus the antigen-positive cells eliminated by the treatment are indirectly examined on the basis of a loss of function in the surviving population. Another approach that has been used recently relies on separation methods not based on cytotoxicity (e.g., FACS, rosetting), so that the antigen-positive cells are retained. In particular, the use of cytotoxicity has meant that Ly123 cells cannot be studied directly, as treatment with either anti-Ly-1, or anti-Ly-2/3 antisera leads to their removal. For this reason, their contribution to the overall response is generally indirectly assessed by a comparison of the effect of treatment with each antiserum, and the relative effect of mixing the two residual populations; i.e., treatment with anti-Ly-1 and complement leaves the Ly23 population, treatment with anti-Ly-2/3 and complement leaves the Ly123 as the deleted population: Antisera used

Cells deleted

Cells remaining

Anti-Ly-1 Anti-Ly-2 or -3 Mixture of A and B

1+2-3-, 1+2+3+ 1-2+3+,1+2+3+ 1+2+3+

1+2-3-(B)

1-2+3+(A)

1+2-3-, 1-2+3+

A further point in many of the ensuing studies to be described, is that it is not always clear whether the Ly phenotype described is that of the effector cell, its precursor, or a secondary cell involved in the modification (amplification or suppression) of another pathway. However, where known this is indicated (Table XXII). A. CYTOTOXIC T CELLS(TK) 1 . Allogeneic Killer Cells Killer cells specific for H-2K and H-2D products of the allogeneic stimulator cells may be induced either in uivo or in uitro in the MLR. By selective depletion of primed populations, Cantor and Boyse (1975a,b) showed that the killer cells generated had a unique surface phenotype. In strain C57BL/6, T K belong to the Ly23 subclass, i.e., are Ly-1.2-. However, in strains bearing the Ly-1.1 specificity (e.g., B6Ly-la), the effector cell appears to be Ly1+2+3+(Shiku et al., 1975).

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289

Killer cells can also be generated in vitro against Z-region determinants, although the response is of a lower magnitude. With this restricted difference the killer effector cells also have the Ly23 phenotype in the C57BU6 strain, i.e., the same as those activated by a whole H-2 incompatibility (Simpson and Beverley, 1977). An amplification role of Lyl cells for the generation of Ly23 killer effector cells has also been established by using stimulation between strains with restricted H-2 differences (Cantor and Boyse, 197513).In support of this finding, Melief et al. (1979) showed that with stimulation between C57BU6 and the mutant H-2Kba,i.e., an H-2K difference only, maximal generation of Ly23 killer effectors from Ly23 precursors was dependent on T-cell help provided by Lyl cells. In this study the helper Lyl cells did not contribute directly to the killer cell pool and were apparently not required for cultures involving both a K and Z-A region disparity. By contrast, using H-2 recombinant (rather than the K b mutant referred to above), Bach and Alter (1978) have demonstrated t w o alternative pathways used by T lymphocytes in the generation of TK: (a) with an entire H-2. difference, Lyl helper T cells respond to Z-region specificities and help the Ly2,3 cells to respond to K or D products (as found by Cantor and Boyse, 1975b); (b) with K - or D-region differences (Z-region similarity), the Ly123 cells provide an essential role in the generation of TK. A similar observation was made for the proliferative response (Wettstein et d., 1978, and see below). Other models have shown that with restricted differences or with a non-H-2 target TK have a unique phenotype. For instance, the secondary cytotoxic response of female cells stimulated in vitro by spleen cells from males of the same strain was found to be directed against the H-Y antigenic product which associates with H-2K or H-2D antigens (Gordon et al., 1975). In this system the effector phase was mediated by an Ly23 cell derived from an Ly123 precursor cell (E. Simpson, personal communication). Other surface markers have also been shown to be present on killer T cells generated by in vitro allogeneic stimulation. These are Ly-5 (Lyt-4) and Ly-6 (Woody et al., 1977; Woody, 1977), ALA-1 (Feeney and Hammerling, 1977),and the Fc receptor is also present on TK(Stout et al., 1976). Unlike the Ly-1,2,3 antigens, the Ly-5 and Ly-6 specificities appear to be on nearly all peripheral T lymphocytes, at least on effector T lymphocytes, and thus have so far been of little value in distinguishing between the different functional groups. However, there are some data to suggest that Ly-6, although present on killer cell effectors, may be absent from the precursors (Woody, 1977).

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IAN F. C. MCKENZIE AND TERRY POTTER

A similar effect was observed with ALA-1 (Feeney and Hamerling, 1977)and the Fc receptor (Stoutet al., 1976). These latter specificities, when used in conjunction with the Ly-1,2,3 specificities may therefore serve to distinguish precursor from effector cells. 2. Syngeneic Killer Cells In the systems examined, the cytotoxic response was directed against an antigenically modified syngeneic target, the modification being produced by either chemical treatment, viral infection, or the expression of tumor-specific antigens. a . Chemically Modi$ed Self-Antigens. TK may be induced by stimulating with autologous cells conjugated with TNP, and these TK effector cells are Ly23 cells (Cantor and Boyse, 1976). In contrast to the allogeneic system, the generation of killer cells in this system was dependent on the presence of Ly123 cells, possibly as precursor or amplifier cells, and the effector cells were generated predominantly from Ly23 cells. b. Virus-Associated Antigens. It is now well established that the killing of virus-infected target cells by sensitized T cells is restricted to those systems where there is an H-2K or H-2D compatibility between the stimulator and the target (Zinkernagel and Doherty, 1974). The primary in vivo and the secondary in vitro responses to ectromelia are inhibited by anti-Ly-2, but not by anti-Ly-1, serum (Pang et al., 1976). Although the in vitro response of memory immune cells was abrogated by both Ly-1 and Ly-2 antisera, mixing experiments were not done in these studies to ascertain whether the effect was due to Ly123 cells or an interaction between Lyl and Ly23 cells. In another study it was found that the in vivo response to influenza virus infection was dependent on the presence of Ly23 killer cells (Yap et al., 1978). c. Tumor-Associated Antigens. Cytotoxic effector cells obtained from C57BU6 mice immunized with the Meth 4 sarcoma and assayed against radiolabeled tumor cells belong to the Ly123 population (Shiku et al., 1976). By contrast, in C3H mice an anti-mammary tumor cytotoxicity was due to Ly23 cells, but for maximal response both Ly123 and Lyl cells were also required, particularly for the later peak of in vitro activity (Stutman et al., 1977; Stutman and Shen, 1978) B. PHENOTYPE OF HELPERT CELLS(TH) FOR ANTIBODY PRODUCTION Helper T cells ( T H ) also appear to have a distinct cell-surface phenotype and in most studies have been found to consist of Lyl cells.

MUFUNE CELL-SURFACE ANTIGENS

291

Cantor and Boyse (1975a) observed an enhancement of the SRC plaque-forming responses in mice irradiated and reconstituted with L y l cells, but not those given Ly23 cells. Thus T H were found to belong to a different T-cell subset to suppressor cells (T,) and TK,with regard to surface antigenic phenotype. Other systems have since been extensively examined, and similar findings have resulted. In hapten-carrier systems in which T cells primed to the carrier cooperate with hapten-primed B cells for antibody production, it was found that carrier-specific helper cells, defined by the augmentation of the antihapten response following in vitro induction with DNP-KLH, belong to the Lyl population (Feldmann et al., 1975a). In this study the helper effector cells were generated from L y l precursor cells (Feldmann et al., 1977%).T H cells in the Feldmann system were found to be Ia-, but other studies using an adoptive transfer system have shown helper T cells to be Ia+ (Okumura et al., 1976). In these studies DNP-KLH primed spleen cells from (BALB/c x SJL)Fl were transferred to irradiated BALB/c recipients, and the recipients acquired the capacity to mount an antiDNP PFC response. By selective depletion of the transferred cells, it was shown that the helper T cells involved were Ia+ (Okumura et al., 1976). Further examination of this finding revealed that the anti-Ia serum that abrogated helper activity had no effect on suppressive activity (Murphy et al., 1976b). Similarly, the anti-Ia serum that abrogated suppression did not interfere with helper activity, establishing that the Ia determinants of helper and suppressor T lymphocytes are different. Studies by Tada et al. (197%) on in vitro induction of carrier-specific helper cells demonstrated that two types of helper cells distinguishable by their passage through (TH1),or adherence to (TH2),nylon wool. Treatment of the two T-cell subpopulations with anti-IJ sera and complement eliminated helper activity from the TH2, but not the THl,population. Thus the serological studies (reviewed by Murphy, 1978) indicate that there are distinct loci mapping in the I-J subregion coding for separate determinants expressed on either helper or suppressor T cells. Under appropriate circumstances, Con A-activated T cells can perform helper, suppressor, and killer functions (Dutton, 1975; Pierce and Kapp, 1976), The Con A-induced antibody helper function for the anti-SRBC response in vitro is also a function of the L y l subclass. It therefore appears from a number of different types of experiments that the Lyl cell functions as a T Hcell, although this phenotype has been associated with the DTH effector cell and the DTH suppressor cell (see below). Using culture conditions that usually give rise to Ts,

292

IAN F. C. MCKENZIE AND TERRY POTTER

and in fact which did so in unfractionated T cells, it was found that Lyl cells could only function as THcells (Eardleyet al., 1978). The conclusion reached was that the Ly1+2-3- phenotype is a stable and invariant marker of T cells, which are capable of only helper activity. However, the target of this helper activity can be either a B cell, leading to increased antibody production, or Ly123 Qal cells resulting in the generaion of Ts (Cantor et al., 1978a; Eardley et al., 1978). Recently it has been shown that both LylQal+ and LylQal- cells are required for optimal antibody formation, and it has been suggested that LylQal+ cells can act on both B cells and regulatory T cells, whereas LylQal- cells act only on B cells (Cantor et a2 ., 1978b). However, optimal antibody production required the presence of both LylQal+ and LylQal- populations.

c. PHENOTYPE OF T CELLS lNVOLVED I N ANTIBODY SUPPRESSION (T,) Suppressor T cells of antibody production have been described in a variety of experimental systems and display differences with respect to their antigen dependence and specificity. In general, these have mostly been found to have the Ly23Ia+ phenotype and can therefore be distinguished from the Ly231a-TK (Table XXII). 1 . Hapten and Protein Antigens The T suppressor cell generated in the in vitro primary response to a high dose of an antigen such as DNP-KLH can inhibit the antihapten antibody response. The active suppressor cells are Ly23Ia+ (Feldmann et al., 1975a; Beverley et al., 1976) and are derived from a Ly23Ia- precursor cells (Feldmann et al., 1977b). Suppression can be adoptively transferred so that antibody production in vivo is reduced. For example, in a hapten carrier system (DNP-BSA) the suppressor effector cell has been characterized as Lyl-2+IJ+ (Zan-bar et al., 1978). Using a similar transfer system, Bottomly et al. (1978) were able to show that the suppressor cells to phosphocholine, generated by anti-T15 antibodies, had suppressor specificity for both idiotype (hapten) and carrier. The Ly phenotype of these suppressor T cells was found to be Lyl-2+. Thus in all studies, the Ts is Ly23Ia+ (IJ'). As noted elsewhere in this review, the Ia determinants present on Ts are those coded for by the Z-J region, in particular by the Za-4 locus (Murphy, 1978; Tada et al., 1978a).

MURINE CELL-SURFACE ANTIGENS

293

2. Cellular Antigens Suppressor cells generated in vivo by high doses of sheep erythrocytes, and assayed by their effect on the plaque response in vitro, also belong to the Ly23 population (Cantor et al., 1976; Weitzmann et al., 1976). In cyclophosphamide-treated recipients, the suppressor cell of antibody formation to horse erythrocytes was shown to be Ly1-2+Iaf (Ramshaw et al., 1977). In another system Ly23 cells partially suppressed the antibody (PFC) response to TNP-conjugated syngeneic spleen cells, but Ly123 cells were found to be the major effector cells in the suppression (Cantor and Boyse, 1976). 3 . Allotype Suppressor Cells (BALB/c x SJL)F, mice usually produce immunoglobulins bearing either one or the other of the parental allotypic markers. However, chronic suppression of a particular allotype can be induced by the neonatal injection of antibody to a particular allotype (Herzenberg et a1 ., 1976).The phenomenon is due to the induction of Ts, and suppression is effected b y an Ly 23Ia+ cell, and the target cell may be an Lyl helper cell (Herzenberget a1., 1976; Murphy et a1., 1976a; Okumura et al., 1976). 4 . Con A-Znduced Suppressor T Cells

In addition to inducing cell proliferation, in vitra stimulation by Con A induces the appearance of Ly23 cells capable of the nonspecific suppression of antibody and of cell-mediated responses (Jandinski et al., 1976). The suppressor cell in this system is phenotypically identical to the antigen-specific suppressor cell (i.e., Ly23), and it has therefore been concluded that the development of suppressor function by this separate lineage of T cells does not necessarily require overt antigen stimulation (Jandinski et a1., 1976).

5. Other Antigens Heuer et al. (1977) investigated a low zone tolerance system in which suppressor cells generated by low doses of antigen (bacteriophage, fd) could be transferred to normal mice. The transferable suppressor cell had the characteristic Lyl-2+Ia+phenotype. I n several other systems the surface phenotype of the T suppressor cell for antibody formation has been found to differ from the Ly23IJ+ cell described above. For example, L y l spleen cells from nonimmune mice can nonspecifically suppress IgE antibody responses in irradiated recipients (Watanabe et al., 1977). However, as stated by the authors, it

294

IAN F. C. MCKENZIE AND TERRY POTTER

is not clear whether the suppression in this system is direct, or whether it reflects interaction of the transferred cells with a radioresistant cell in the recipient. Similarly, the antigen nonspecific suppressor cells generated during a graft-versus-host reaction, and capable of inhibiting the in vitro response to sheep erythrocytes, belong to the Ly123 population (Pickel and Hoffman, 1977; Shand, 1977). Infection of newborn mice with mouse thymic virus enriches for T-cell suppressor activity, whereas other T-cell functions, such as helper or killer activity, MLR proliferation, and mitogen responsiveness, are depleted (Cohen et al., 1975).Thymocytes generated in such infected mice are capable of suppressing antibody formation by adult B cells and carry not only Ly-1 and Ly-2 determinants, but T L as well, i.e., are TL+ Ly123 (Mosier et al., 1977).The developmental relationship between these neonatal suppressor cells and the cells mediating suppression in the adult is uncertain. The differences in these systems probably reflect the different antigens used, but clearly point to the fact that several different types of suppressor T cell exist. Whether they are functionally related and merely represent the development or differentiation of the effector Ts, or whether they represent totally separate pathways of differentiation, is not clear.

6. The Surface Phenotype of the Memory-Suppressor T Cell Basten and colleagues (Loblay et al., 1978) have recently described a Ts memory cell for a response to DNP-HGG. Suppressor T cells induced b y immunizing with alum-precipitated HGG and B . pertussis organisms were capable of abrogating antigen-specific antibody responses in mice reconstituted with primed cells following irradiation. The suppression was long lived, and even greater suppression, attributed to a “memory suppressor” cell, was observed 4 weeks after its initial induction. This memory cell belonged to the Ly23Ia+ population.

D. PHENOTYPE O F T CELLS INVOLVED IN THE SUPPRESSION O F CELL-MEDIATED RESPONSES Suppression of cell-mediated responses has been investigated in two systems: (1) development of allogeneic killer T cells during a mixed lymphocyte reaction; (2) delayed-type hypersensitivity (DTH) responses in vivo. 1 . T s for Killer Cell Generation As mentioned above, Con A-induced suppressor cells were shown to be capable of nonspecific suppression of both antibody-mediated and

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295

cell-mediated responses in the in uitro induction of cell-mediated cytotoxicity to H-2 targets (Jandinski et al., 1976). In both cases the Ly23 cells were responsible for the suppression.

2 . DTH Suppression Suppression of DTH has been ascribed to the same Ly subclass as the antibody suppressor, i.e., Ly23 (Huber et al., 1976b). However, Ramshaw et al. (1977) have reported that the DTH suppressor cell in their study had the Ly1+2-3- phenotype. The differences are probably explicable in terms of the different assay systems used. In the Ramshaw study, suppression was induced by high doses of SRC, and the putative Ts was transferred together with antigen to cyclophosphamide-treated recipients. Suppression was assessed after further antigenic challenge by measuring the degree of footpad swelling. Huber et al. (1976b) used a different system, involving the transfer of nonimmune spleen cells together with antigen into thymectomized irradiated recipients. In addition, Huber et al. used C57BU6 mice and Ramshcaw et al. used CBA, and these strains have different alleles for the Ly-1 and Ly-2 loci. Alternatively, the studies also suggest that there are two different types of Ts involved in the suppression of DTH. However, it is likely that the Huber study examined suppression of the effector cell of DTH, whereas the Ramshaw study examined the suppression of the DTH inducer cell. In this case, it appears that suppressor cells can act in at least two different stages of the DTH response. Further investigations of the suppressor cell in the Ramshaw system have shown the phenotype to be Lylf2-3-4-5+6+7-Ia-IJ(Thompson, Potter and McKenzie, unpublished results).

3. The Role of T s in Tumor Growth Some interesting results on the role of Ts in tumor graft rejection have recently been demonstrated with the in uiuo use of anti-IJ sera (Greene et al., 1977) (see Section V1,P). Briefly, the anti-IJk sera were able to hasten the rejection of a syngeneic tumor graft-presumably by reacting with and inhibiting Ts for the induction of TKfor the tumor. The study is important, for it is one of the few examples of the in uiuo use of alloantisera. 4 . The Feedback Suppressor T Cell In several recent in uitro and in uiuo studies, the Ly123Qal+ cell has been shown to have potent suppressive activity. In studies designed to convert Lyl cells into Ts by stimulating in uitro with large doses of SRC, it was found that Lyl cells could give rise to TH, but not Ts (Eardley et al., 1978).

296

IAN F. C . MCKENZIE AND TERRY POTTER

However, when immune Lyl cells were added to cultures of T cells, SRC, and nonimmune Lyl cells, potent suppressive effects were generated, It was concluded that the immune Lyl cells induced cells bearing Ly2 to become functional Ts, and that the level of suppression generated was directly proportional to the number of immune Lyl cells added. Further experiments, including i n uiuo SRC immunization, indicated that the target cell for the Lyl cell was an Ly1+2+3+Qal+cell, termed a “feedback suppressor cell” (Eardley et al., 1978). It was also suggested from these studies that the target of the feedback suppressor cell was the initial reactive L y l TH cell. In a second series of experiments, the in uiuo counterpart of the in uitro studies were examined (Cantor et al., 1978a). It was found that mice lacking Ly123 cells did not show the feedback suppressor phenomenon; however, this effect was restored by the addition of Ly123 cells. Of clinical relevance to the development of autoimmune disease was the finding that NZB mice, which spontaneously develop autoia:mine disease, had few Ly123 feedback suppressor cells. Recent experiillents indicate that L y l Q a l + cells are responsible for inducing the Ly123Qalf feedback suppressor cell (Cantor et al., 197813)

5 . Ly Phenutype of Cells Secreting Soluble Factors in Help and Suppression It is apparent that induction of the immune response both in uitro and i n uiuo is dependent on both specific and nonspecific soluble factors, which in most studies have been found to carry Ia determinants (reviewed by Tada et al., 1976, 1977, 1978a; Feldmann et al., 1977a). Whether this is a general mechanism of helper- and suppressor-cell action is not clear as yet. However, Lyl cells secrete molecules bearing Ia antigenic determinants as detected by inhibition of rosetting by Ia antisera (McKenzie and Parish, 1976). In addition, cells undergoing allogeneic stimulation in uitro, and which produce “T cell replacing factor” (as defined by restoration of the sheep red cell antibody response in T cell-depleted cultures), also belong to the Lyl (TH) population (Pickel et al., 1976). Further studies have indicated that cells that secrete suppressor factors are phenotypically identical to those that have been described as suppressor cells in other systems, i.e., Ly23IJ+ (Tada et al., 1977). E. PHENOTYPE OF T CELLS INVOLVED IN DELAYED-TYPE HYPERSENSITIVITY (DTH) The DTH response is initiated b y a T cell and involves many different cells, including monocytes and macrophages. The DTH effector

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cell to SRC has been characterized as Lylf2-Ia- (Huber et al., 1976b). Similarly Lyl cells were found to be the effector cells in DTH to fowl y-globulin (Vadaset al., 1976). However, foradifferentantigen (DNFB), the DTH cells were not solely L y l or Ly23, and an interaction between L y l and Ly23 (or Ly123) cells has been suggested (M. Vadas et al., personal communication). The DTH effector cell (TDTH) therefore belongs to the same population as the TH(Ia L y l cells). Whether these cells are the same (which would seem unlikely) or different is not clear on the basis of antigenic phenotype; however, the use of additional cell markers, such as Qa, Ly-5, Ly-6, may sort out this overlap.

F. THE MIXED-LYMPHOCYTE REACTION (MLR) It is generally accepted that there are two populations involved in the MLR response-one that proliferates in response to Z-region determinants, and another that generates killer cells specific for H-2K and D products (Bach e t aZ., 1973). In general (but not exclusively) T cells respond to determinants found predominantly on B cells. The strongest MLR reaction occurs where there are H-2 differences, and within the H - 2 complex the Z-A subregion gives rise to the strongest stimulation of all (Shreffler and David, 1975). In addition, by the use of H-2 recombinant and mutant mice, Lad (lymphocyte-activating determinants) have also been found associated with (? identical to) the H-2K, H - 2 D , H-2L, ZJ, ZEIC loci or subregions of the H - 2 complex. There is no L a d locus associated with the S region or with the TZa locus. Another locus, called MZs (minor lymphocyte stimulating), fonnerlyM locus, has been defined by Festenstein and colleagues (reviewed by Festenstein, 1974). This locus is located on chromosome 1, has four alleles, and is linked to the L y M - 1 locus. The definition of other L a d loci (if any) must await the production of congenic strains, but there appear to b e no L a d loci associated with the Ly-l to L y - 5 loci. It has not been determined whether the stimulating determinants for the major and minor L a d s are the same as those detected serologically or different. Blocking studies show that all H-2 antisera can inhibit the proliferative response when there are only restricted differences, e.g., anti-H-2K sera can block for an H-2K difference and so on. In addition anti-Ia sera can block an MLR for a difference of the whole H - 2 complex. Anti-Ly-4 ( b l ) and Lyb-4.1 sera can also block an allogeneic response (Plate and McKenzie, 1973; Freund et aZ., 1977), suggesting the possibility that these determinants are involved in the delivery of a second signal in lymphocyte activation (Ahmed, personal communication). Antisera to Ly-1, -2, -3 and Ly-6 have no effect on the stimulating cell in an MLR, either by re-

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moval of a stimulator or in blocking studies (Michaelides and McKenzie, unpublished results). The proliferative response to H-2 determinants is accompanied by the production of TKcells. For incompatibilities of the whole H-2 complex, Lyl cells constitute the majority of the proliferating cells (Cantor and Boyse, 1975a). The strongest proliferation occurred in response to I-region determinants. As described earlier, the TK cells generated were Ly23 cells and their target specificities were the H-2K or H-2D antigens (Cantor and Boyse, 1975a).These two T-cell subsets, Lyl and Ly23, arose from different precursor cells, and the Lyl cells did not give rise to Ly23 TK cells, although the proliferating Lyl cells did enhance the generation of TK cells (Cantor and Boyse, 1975b). The model arising from these studies was that Lyl cells proliferated in response to I-region products and maximized the generation of killer Ly23 cells, which were stimulated by H-2K and H-2D products. Supporting evidence for this scheme also came from Nagy et al. (1976), who showed that the proliferating Lyl blasts bound I-region antigens and the cytotoxic Ly23 cells bound H-2K- and H-2D-region products released from the stimulator cells. By depletion experiments we have established that the secondary proliferative response following restimulation of these cells is confined to the Lyl population (Elliott, Potter and McKenzie, unpublished observations). In the absence of an I-regian incompatibility, such as with a H-2K or H-2D incompatibility, MLR proliferation is reduced but still detectable. With the H-2 mutant H-2 the proliferative response in this restricted difference is mediated by Lyl and Ly123 cells (Wettstein et al., 1978). Using a similar mutant, Melief et al. (1979), also observed approximately 50% inhibition of the proliferative response by Ly-2 antisera treatment. A similar variation of Ly phenotypes of the cells responding to different MHC antigens has also been observed in the “allogeneic effect” (Swain and Panfilli, 1979). In this system cells responding in an MLR are tested for their capacity to help (or amplify) the in vitro response of splenic B cells to SRC. It was found that the helper cells generated in response to incompatibilities at the whole haplotype, the I region, or the Mls locus, are Lyl cells. However, when the difference is restricted to K (B6+B6-H-2ba) or D (BIO.T(GR)+BIO.G), the helper cell is Lyl+2+ (Swain and Panfilli, 1979). Clearly the role of the Ly123 cells in the MLR has not been definitely established and may vary with the type of stimulation encountered.

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G. HOST-VERSUS-GRAFT AND GRAFT-VERSUS-HOST (GvH)

REACTIONS There have been few published data on the phenotypes of cells mediating graft rejection, or the GvH reaction. Recently, B. Huber and H. Cantor (personal communication) have shown that in reconstituted “B” mice unprimed Ly23 cells alone are incapable of graft rejection, and that Ly123 cells are required to induce graft rejection. Furthermore, in this system immune Ly23 cells in combination with immune Lyl cells can cause rapid graft rejection. In another study (Loveland and McKenzie, unpublished) “B” mice receiving sensitized spleen cells were able to reject skin grafts. The cell population responsible for graft rejection was Thy-1+, Ly-1+, Ly-2’, Ia-, i.e., Lyl cells from primed mice appear to be able to cause graft rejection (these could be Lyl or Ly123 cells). Both of these studies are in keeping with the in vitro studies, i.e., a role of TH(Ly-l+)cells interacting with Ly23 cells for the generation of TI(. As with other systems, the major problem appears to be the difficulty of obtaining pure populations of cells for reconstitution. Although this is particularly relevant for Ly123 cells (see Section XIII), it should be noted that for all in vivo experiments incomplete depletions may lead to expansion of such a pool following transfer. This problem is compounded by the relatively longer time scale involved in in vivo experiments, which may allow the effects of the contaminating cells to be expressed. GvH effector cells may belong to the Ly123 population; however, as pointed out by the investigators, an interaction between Lyl and Ly23 cells could also be responsible for this effect (Huber and Cantor, 1976). H. PHENOTYPE OF CELLSUNDERGOING BLAST-CELL TRANSFORMATION

1 . Cells Responding to T-cell and B-Cell Mitogens There have been few studies reported on the phenotype of cells responding to mitogens other than the observation that Con A and LPS responder cells are Ia+ (Niederhuber et al., 1975, 1976). One study (Hirst et aZ., 1975) demonstrated that Ly123 cells respond to PHA, whereas Lyl cells respond to Con A. Treatment of SJL lymph node cells with antisera and complement before stimulation suggests that the Con A response of Ly23 cells was suppressed by Lyl or Ly123 cells (S. Lerman et al., personal communication). The effect of Con A is strain and concentration dependent, as in another study (Rabinowitz

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et al., 1978) it was found that, in the C3H strain, L y l cells were responsive to Con A. In our own studies (Michaelides and McKenzie, unpublished) it has been found that both Ly-1 and Ly-2 antisera can abrogate the PHA or Con A response, suggesting that either all T cells were responding to these mitogens or that the major response was due to Ly123 cells. The appropriate cell-mixing studies to determine which of these alternatives is the case have not been performed. Some of the other anti-Ly reagents have also been tested, and it was found that anti-Ly-5 and Ly-6 antisera abrogated the response to all four mitogens (PHA, Con A, LPS, and PWM). The finding of Ly-6 specificities on cells responding to the B-cell mitogens indicates that these B cells are Ly-6+ or, alternatively, that Ly-6+ cells are involved as accessory cells in this B-cell response. Ly-4 studies have been reported previously (McKenzie and Plate, 1974), where it was found that the antigen was on cells responsive to B-cell, but not T-cell, mitogens. Anti-Ly-7 sera inhibited the LPS response but were virtually without effect on the response induced by any other mitogens. The results with anti-Ia sera are of interest. Initially it was found that cells responsive to Con A, LPS, and PWM were Ia+ (Niederhuber et al., 1975, 1976; Michaelides and McKenzie, unpublished observations) and that PHA-responsive cells were Ia-. However, we have recently found that anti-Ia and complement has some inhibitory effect on the PHA response. Many of these results are difficult to interpret and give variable effects in repeated experiments. This may be related to the observation that treatment with anti-Ly sera and complement has different effects depending on the concentration of the lectin used (Rabinowitz et al., 1978). There have been surprisingly few reports on the surface phenotype of the blast cell itself; in some preliminary studies, Con A blast cells are predominantly Ly1+2-3-4-5+6+7-Ia+ (Michaelides and McKenzie, unpublished observations). 2 . Cells Responding to Antigen-Induced Blastogenesis Preliminary studies of the phenotype of the cells involved in the in vitro blastogenic response to DNBS03 indicate that the responding cells are Ly1+2+3+6+7+Ia+.Whether all cells in the cultures are responding, or whether the response requires an interaction between T cells and B cells prior to blastogenesis, has not been determined (A. Basten, personal communication).

3. Cells Proliferating in Response to S yngeneic Tumors Two studies have examined the Ly phenotype of cells undergoingin vitro proliferation to a syngeneic tumor. In the first study, SJL spleen

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30 1

cells cultured with irradiated reticulosarcoma cells of the same strain, undergo a considerable degree of proliferation as measured by L3HIthymidine uptake (S. Lerman et al., personal communication). The proliferation was abrogated by cytotoxic depletion with anti-Ly-1 but not with anti-Ly-2 serum + C‘. Furthermore, the anti-Ly-2 and complement treatment, in some experiments, markedly increased the cell proliferation (Lerman et al., 1979). It was also found that although the responding cells were Ia- and the SJL tumor Ia+, anti-IaS serum in the medium (SJL is H-2s) inhibited the proliferation. This observation suggested that the cell surface Ia antigen was the stimulating component, or alternatively, that it forms a complex with the stimulatory antigen (Ponzio et al., 1977). I n the second study (Okuda e t aZ., 1978), GRSJJ14 leukemia cells stimulated GRS spleen cells to proliferate. Several GRSL lines were tested, but stimulation was observed only with Ia+ lines, and this proliferation could be inhibited by anti-Ia sera. Both sets of results suggest that Ia antigens on tumor cells may be important in inducing blast-cell proliferation and therefore be involved in one type of immune surveillance (Okuda et al., 1978). IN M I F PRODUCTION I. PHENOTYPEOF CELLSINVOLVED

In two different systems the phenotype of the lymphoid cells involved in MIF production has been studied. In the first, the M I F was produced b y T-cell activation during an MLC reaction (Newmanet al., 1978). It was found that both Lyl and Ly23 cells produced M I F and that these same cells were involved in the production of an “inducer of plasminogen activation” (IPA). It was not clear from these studies whether the Ly123 cell was involved as well as the two subsets mentioned. In the second study (Basten et d.,unpublished results), M I F production by cells activated in culture by DNBSOI was attributed to the Lyl population. The cell was also found to be Ia-Ly5+6+7+. It is not clear whether these two different results represent a real difference in that different T-cell subsets produce MIF, or whether the results are merely a reflection of the different systems used.

J. THE LY PHENOTYPE OF T CELLS I N THE PRODUCTION OF EOSINOPHILIA A novel recent finding has been that certain parasites induce an inflammatory peritonitis containing large numbers of eosinophils, and that the eosinophilia is T-cell dependent. The eosinophilia does not occur in nude mice but can be restored by injecting T cells or thymocytes. Depletion experiments suggest that the L y l cells are

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LAN F. C. MCKENZIE AND TERRY POTTER

more efficient in promoting an eosinophilia than Ly23 cells (Johnson et al., 1979). K. SUMMARY OF FUNCTIONAL DATA The use of anti-Ly sera has been of great value in assigning particular immune functions to distinct cell types. The use of Ly antisera, however, should not be restricted to defining the antigenic phenotype of immune-reactive cells. These sera potentially offer a means by which the feasibility of many models invoked to explain various immune phenomena (e.g., antibody suppression) may be investigated. An example of this is the recent work of Eardley et al. (1978) and Cantor et al. (1978a,b) in the identification of the “feedback suppressor” cell (Lyl+ Ly2+ Qal+). The overall findings with the 3 classes of cells best characterized can be summarized (Table XXIII) as follows. Class 1: Lyl cells (30-35% of peripheral T cells). These enhance the functional activity of other cells (B cells, killer T cells as well as the monocytes and macrophages mediating DTH) after antigenic stimulation. Lyl cells appear to selectively recognize I-region determinants, as shown by their activity in helping antibody production and in the DTH phenomenon, both of which are under I-region restriction (Feldmann et al., 1977a; Tadaet al., 1976; Miller et al., 1976). Such an TABLE XXIII SUMMARY OF I M M U N E FUNCTIONS OF

THREECELL TYPES

Lyl cells Enhance antibody production by B cells Initiate DTH responses Induce pre-TK to differentiate to TK Stimulated by I-region determinants in a mixed lymphocyte reaction Secrete helper factors Proliferate in uitro in response to tumor antigens Suppressor role in IgE production and possibly delayed-type hypersensitivity responses Secrete migration inhibition factor

Ly23 cells

Killer cells to allogeneic, viral, and H-Ydeterminants Suppressor cells in antigen specific, Con A-induced, and allotype suppression systems Secrete antigen-specific suppressor factors

Ly123 cells Possible precursor role in many systems Killer cells to tumor antigens Nonantigen-specific helper cells induced by H-2K or H-2D determinants Suppressor cells in antibody responses to TNP-self, and nonspecific suppression arising during GvH or in neonatal cells I n uitro proliferation in response to PHA, Con A, soluble antigens, H-2K or H-2D incompatibilities.

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association is clearly demonstrated by the proliferative response to Z-region determinants in the MLR. Further evidence associating the generation of helper activity and the I region comes from the studies of Gershon and Cantor (1977). Their studies demonstrated that even across an H-2 barrier, Lyl cells could generate substantial help in a primary i n uitro PFC response to sheep red cells. As association between Lyl cells and the I region has also been demonstrated in the i n uiuo opsonization of alloreactive Ly 1cells binding complexes bearing IA determinants (Hutchinson and Bonavida, 1979). Class 2: Ly23 cells (10% of peripheral T cells). This population includes the Ia+ (IJ) suppressor cells and the Ia- allogeneic killer cells. The association of killer cells with H - 2 K and H-2D region products, as earlier suggested by Bach et al. (1973), has been confirmed for the Ly23-bearing killer cells. Class 3: Ly123 cells (50-5570 of peripheral T cells). As discussed, functions of these cells have been difficult to establish accurately. It was generally accepted that these cells were the precursors of the Lyl and Ly23 cells, but did not directly mediate any immune reactivity themselves. More recently, however, it has been established that in several systems these cells may contribute directly to cytotoxicity, suppression, and MLR proliferation. XII. C U D in Studies of T-cell Ontogeny and Differentiation

Studies of T-cell differentiation and ontogeny have been enhanced by the use of antisera to the alloantigens Thy-1, TL, Ly-1,2,3. In combination with organ culture, cell-transfer systems, the use of nude (athymic) mice, the T6 chromosomal marker, and thymic hormones, as well as assays of lymphocyte function, the serological techniques have enabled a composite picture of T-cell development and differentiation to be deduced. We have not attempted to review this extensive topic, as several excellent reviews of T-cell differentiation have recently been published (Cantor and Weissman, 1976; Cantor and Boyse, 1977a,b; Owen, 1977; Stutman, 1977, 1978). Rather, we have summarized some of the conclusions reached in these reviews and discuss the implications of recent data obtained with the FACS (Mathieson et al., 1979). Several models incorporating the current theories of differentiation with respect to the expression of CMAD are presented in Fig. 3. A. ONTOGENY The appearance of the T-cell CMAD have been investigated both i n uiuo, by studying the acquisition of these markers in neonates, and i n

w

B

Thy-1 Ly123 TL+

Thy-1 TL+-Thy-l

\'Thy-1 Other markers: Ly5+

Ly5+

-e::k;:i

$i:tLThy-1 Ly123 TL-

Ly123 TL+

Thy-1 Ly123 TL-

Lyl TL+

Thy-1 Lyl TLLy5+ Ly6+

Thy-1 Lyl Thy-1 Ly23 Thy-1 Ly123

I

(2, 3)

Thy-1 Lyl Ly5+ Ly6+

FIG.3. Expression of cell-membrane alloantigenic determinants. h: The cortisone resistant population may not be derived from the cortisone-sensitivepopulation and may never have expressed TL. b: At least some peripheral T cells may be derived from the cortisonesensitive pool, e.g., the PTP of Stutman and Shen (1979).References: (1)Cantor and Boise (197%); (2) Mathiesonet al., 1979);(3)Scollay e t al. (1978).

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vitro with organ (thymus) culture systems. Stem cells migrate into the thymic epithelial remnant about day 11 of gestation in the mouse embryo, and by day 13 Thy-l+ cells can be identified by fluorescence or by cytotoxicity (Owen and Ritter, 1969). In studies of neonatal T cells bearing Ly antigens, Ly123 cells, but not Lyl or Ly23 cells, were detected in the spleen of 7-day-old mice. The numbers of Lyl and Ly23 cells gradually increased to the tenth week, when they accounted for 20% of the total number of spleen cells (Cantor and Boyse, 1975~).Adult thymectomy led to a 50% decrease in Ly123 cells accompanied by a relative increase in Lyl and Ly23 cells (Cantor and Boyse, 1975~).Taken together, these studies suggested that splenic Ly123 cells are derived directly from the thymus and differentiate in the periphery to Lyl and Ly23 cells. Organ cultures of fetal thymus commencing at day 13-14 contain T h y - l T L - blastlike cells, which rapidly give rise to Thy-1+TL+ small lymphocytes (Owen and Raff, 1970; Owen 1977; Mandel and Kennedy, 1978). A second wave of cell proliferation that gives rise to Thy-l+TL- small lymphocytes was seen at about the tenth day of culture and could occur in the absence of the first, suggesting the existence of independent pathways of T-cell differentiatibn (Mandel and Kennedy, 1978). In a similar organ culture system and a related in vivo study, it was evident that the Ly123 specificities were not detected by cytotoxicity until day 14 and reached adult levels b y day 19 of fetal life (Kamarck and Gottlieb, 1977). On these cells Ly-1,2,3 antigens appeared simultaneously, suggesting that Ly123 cells are derived before Lyl and Ly23 cells.

B. THE PROTHYMOCYTE It is generally accepted that hemopoietic cells migrate from the marrow to the thymus, and that cells from the yolk sac also migrate to marrow after an additional maturation step (reviewed by Owen, 1977; Stutman, 1978). The presence of a specific T-cell progenitor separate from the pluripotential stem cell and from a myeloid precursor has also been identified (Phillips et al., 1977).This cell presumably has none of the T-cell markers, as a Thy-l-Ly-TL- cell isolated from bone marrow on a density gradient can be induced in vitro to differentiate, by exposure to thymopoietin and other hormones or chemicals (Komuro et al., 1973, 1975a). The cell thus induced expresses Thy-1, TL, and Ly markers. It should be noted in these studies that, although a variety of substances that increase intracellular CAMP can also induce this differentiation step, they also lead to B-cell differentiation and only thymopoietin appears to be T-cell specific (Scheid et al., 197513; Goldstein et al., 1975, 1976). These studies thus define a prothymo-

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cyte (Thy-l-TL-Lyl23-) + thymocyte (Thy-l+TL+Lyl23+) differentiation step. The studies must be reconciled with the finding of a low-density Thy-l+ cell in nude mice (Loor and Kindred, 1973; Scheid et al., 1975a; Loor et al., 1976; Roelants et aZ., 1976). This cell is presumably a prothymocyte, as the injection of nude mice with thymic humoral factors results in the appearance of Thy-1+TL+ cells in the spleen (Scheid et aZ., 1975b). Consistent with this finding is the observation that the cell in nude mice identified by Roelants et al. (1976) is also Thy-l+TL+. A similar cell, bearing a low density of BA8 and designated a “pre-T-cell,” is also found in nude mice and can be induced to differentiate into TL+ Con A-responsive cells by incubation with thymic epithelium. The finding of Thy+Ly123- thymomas (P. M. Hogarth and I. F. C. McKenzie, unpublished observations) (see Section XIV) that are also Ly5+ suggests that Ly5 may be acquired during early T-cell development and possibly expressed on prothymocytes, particularly as we find that nude mice have Ly-5+ cells. The relationship (identity) of the low Thy-l-bearing cell of nude mice to the prothymocyte, defined by its induction in uitro, is not yet established.

c. DIFFERENTIATION IN THE THYMUS Within the thymus are the cortisone-sensitive (CS) cortical cells, which are Thy-l+TL+Lyl23+ and a small proportion (-5%) of cortisone-resistant (CR) medullary Thy-l+TL-Ly123+ cells. The CR population is characterized by a lower density of Thy-1 and an increased density of H-2 and is considered to be immunologically competent. Therefore, both by surface antigenic phenotype and functional capabilities the CR thymocytes are analogous to peripheral T lymphocytes, and it has been suggested that they constitute the export cell of the thymus, i.e., the immunocompetent T cell (Konda et al., 1973). In many of the classical models of T-cell differentiation, the CS cortical cells are thought to give rise to the CR population that constitute the pool from which all peripheral T cells arise. However, it is not definitely established whether the CR population is derived from the Thylhlgh,TL+cells or whether they represent a separate line of differentiation (Shortman et al., 1975; Weissman et aZ., 1975). Furthermore, it is not established whether or not peripheral T cells are solely derived from the CR population, or whether there is more than one pathway from prothymocyte to T lymphocyte, as would be expected in view of the heterogeneity in expression of Thy-1 on peripheral T cells (Cantor et d.,1975). The postthymic precursor (PTP) cell described by Stutman and Shen (1979) is cortisone sensitive, suggesting that at least

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some peripheral T cells are derived from cells other than those of the CR population. The early data obtained on cytolysis of thymocytes with Ly antisera indicated that each antiserum (Ly-1.2, Ly-2.2, Ly-3.2) lysed 90% of C57BW6 thymocytes but only 35-60% of the CR population (Cantor and Boyse, 1975a). This observation was consistent with there being some degree of differentiation within the thymus and has been further substantiated by data obtained with the FACS (Mathieson et aZ., 1979). Analysis on the FACS has identified a Ly1+23- population representing about 10% of normal thymus cells and 50-70% of the CR population. The intrathymic differentiation of Lyl cells has recently been demonstrated through observations of thymus cells labeled in situ with fluorescein isothiocyanate (Scollay et a1., 1978). Cells emigrating from the thymus to the periphery could be identified by the presence of the fluorescein label. Analysis of the fluorescent thymocytes and peripheral T cells with rhodamine-conjugated Ly antisera suggested that of the cells emigrating from the thymus, 67% were Lyl cells, 31% Ly123, and only 1-2% Ly23. As there were so few Ly23 cells identified in this way, it raises the possibility that Ly 123+Ly23 differentiation may occur in the periphery. Studies of the antigenic phenotype of thymic tumors also supports this contention, as there is differential antigen expression, presumably correlated with differentiation of cells within the thymus (Section XIV) (Mathieson et al., 1978; Hogarth and McKenzie, unpublished observations).

D. DIFFERENTIATION IN THE PERIPHERY Among peripheral T cells, the three subsets characterized and their relative frequencies are: Ly123 (50-55%), Lyl (30-35%), and Ly23 (10%). The studies of Cantor and Boyse (1975b) clearly demonstrated the different functional roles of Lyl (helper) and Ly23 (killer, suppressor) cells. Later studies by Jandinski et aZ., (1976) established that isolated Lyl cells from nonimmune mice were programmed for helper-amplifier function before any extrinsic antigenic stimulus. Similarly, isolated Ly23 cells activated by Con A could generate killer function and were therefore committed to a restricted range of functions before overt stimulation. However, it was not clear from these studies whether any further differentiation could occur, i.e., whether Lyl cells could give rise to Ly123 or Ly23 cells, and similarly for the Ly23 cells; or whether the Lyl and Ly23 cells were “irreversibly” committed to the prescribed function. Several definitive studies support this latter contention. Huber et al. (1976a) prepared ATXBM “B” mice, by adult thymectomy, lethal irradiation, and reconstitution with

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Thy-l-depleted bone marrow cells. By the addition of selected subclasses of T cells, these mice could be made “B-Lyl” mice (i.e., those given T cells depleted with Ly2 and complement) or “B-Ly23” mice (Lyl-depleted T cells). As treatment with either Lyl or Ly2 antisera depletes the Ly123 population, it was not possible to produce any B-Ly 123” mice. When these mice were examined for their immunological capabilities, it was found that “B-Lyl” mice could express helper, but not killer, activity, whereas the “B-Ly23” mice expressed the converse. These studies concluded that the two lines of differentiation leading to Lyl and Ly23 cells were divergent and that there was no differentiation of Lyl+Ly23 or Ly23-Lyl. The common precursor of the Lyl and Ly23 cell lineages has not been established, although Huber et al. (1976a) suggested that likely candidates were either the TL-Ly123 subclass or the TL+Ly123 thymocyte. As stimulation with chemically altered syngeneic cells induces an Ly123-Ly23 differentiation (Cantor and Boyse, 1976), and polyclonal activation of purified TL-Ly123 cells gives rise to Lyl helper cells, it has been postulated that TL-Ly123 cells are the precursors of Ly23 and Lyl cells (Cantor and Boyse, 1977a,b). Stutman (1978; Stutman and Shen, 1979),has provided direct evidence that there are immunocompetent postthymic precursor cells that give rise to Lyl and Ly23 cells. These studies have identified in the periphery immature T cells that undergo maturation under the influence of thymic hormones. This postthymic precursor (PTP) cell is (a) nonrecirculating and found mainly in spleen and bone marrow; (b) rapidly dividing and sensitive to thymectomy; (c) resistant to ALS, but sensitive to hydrocortisone; (d) adherent to nylon wool; (e) immunologically incompetent, as determined by nonresponsiveness in an MLR or to mitogens and by the failure to confer helper activity or initiate a GvH response; (f) Thyl+TL-Ly123+ (Stutman, 1978; Stutman and Shen, 1979). Injection of these cells from the spleens of neonatal CBNT6 mice into thymectomized recipients gave rise to both Lyl and Ly23 cells carrying the T6 chromosome marker. It has been proposed that this maturation occurs after direct contact of the Ly123 PTP cell with Ia+ “inducer” cells (Stutman, 1978). “

E. OTHERCMAD IN T-CELL DIFFERENTIATION Some of the more recently described specificities may prove to be useful in the study of T-cell differentiation. In particular, the Qa-2 and Ly-6 antigens, although not present on the majority of the thymocytes, .are expressed on the CR thymocyte population (see Section VI).

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The ALA-1 specificities are expressed primarily on activated T cells (Section VI). Other CMAD expressed on functionally distinct T-lymphocyte subpopulations include IJ and Qa-1 determinants (see Section XI). The potential usefulness of these markers to the study of T-cell differentiation has not been investigated.

F. SUMMARY An overall view encompassing all these studies is presented in Fig. 3. It can be seen that there are two fundamental questions still unanswered: (1) Is the cortisone-resistant population derived from the cortisone-sensitive pool, or does it represent a separate lineage? (2) Is it this CR population that gives rise to peripheral T cells? From the studies described here it appears that there are two lines of differentiation, or rather, a continuing differentiation of Ly123+Lyl Ly23. One occurs in the thymus (Mathieson et al., 1979; Scollay et al., 1978), the other in the periphery but under a thymic influence (Stutman, 1978; Stutman and Shen, 1979). The failure to detect any Lyl- thymocyte or peripheral T cells on the FACS (B. Mathieson, personal communication) indicates that the Ly23 cells defined by cytotoxicity carry a low amount of Lyl. This could be interpreted as a decrease in, but not a total absence of, expression of Lyl accompanying the differentiation of Ly123+Ly23. As the Lyl cells defined by cytotoxicity are also characterized as Ly-2/3- on the FACS, either a total disappearance of the Ly-2/3 antigens accompanies the Ly123-Lyl differentiation or alternatively Lyl cells may not be derived from Ly123+ (TL+or TL-) cells. The data of Cantor and Boyse (1977a,b) and Stutman (1978) reported above, would conflict with the latter possibility.

+

XIII. CMAD in B-Cell Differentiation a n d Ontogeny

The use of the TL, Thy-1, Ly-1,2,3 CMAD to study T-cell differentiation, ontogeny, and functional heterogeneity provides an elegant example of the potential usefulness of serological techniques in studying cellular events. However, such extensive studies have not been performed for B-cell differentiation and ontogeny, for until recently the number of CMAD detected on B cells was limited. In particular, there has been no CMAD detected on early, immature B cells, analogous to TL in the T-cell differentiation line. Consequently, the early events in B-cell differentiation are obscure, and we rely on the evidence obtained from investigations of the CFU-S and CFU-C (Phillips et aZ., 1977), adoptive transfer studies to enumerate pre-B and B cells

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IAN F. C. MCKENZIE AND TERRY POTTER

(Lafleur et al., 1972), and a variety of other in vivo and in vitro assay systems (see Ahmed et al., 1978). Overall these studies suggest a differentiation line (Phillips et aZ., 1977). Pluripotential stem cell + B stem cell + pre-B cell + B cell

antibody-forming cell

It is evident that there are B-cell subsets and that there is a heterogeneity of B cells with respect to mitogen responsiveness (Janossy and Greaves, 1975), cell size (Gorczynski and Feldman, 1975), adhesiveness and charge (Schlegel et aZ., 1975),recirculating capacity (Strober, 1975). A considerable amount of data on B cell differentiation has accumulated through studies of several surface components, such as immunoglobulin class and density (Strober, 1975; Scher et al., 1976; Vitettaet al., 1977; Abneyet al., 1978), Fc and C3 receptors, as well as the Ia, Ly-4, and Pca-1 alloantigenic specificities. The more recent identification of the Lyb-2, Lyb-3, Lyb-4, Lyb-5, Lyb-6, Lyb-7, and LyM-1 alloantigens should add considerably to our knowledge of the B-cell lineage. In this section we review the use of CMAD in B-cell differentiation and, because they have been reviewed elsewhere (Nussenzweig, 1974; Warner, 1974; Parish, 1975; Vitetta et al., 1977; Moller, 1978), refer to sIg, Fc, and C3 only for comparative purposes. A flow chart of B-cell differentiation, adapted from Ahmed et al. (1978), is shown in Fig. 4. A. STEMCELLS:B STEMCELL, THE IMMATURE B CELL Although a pluripotential stem cell has been identified in fetal liver, bone marrow, and neonatal spleen, a B-lymphocyte stem cell analogous to those found for T lymphocytes and the myeloid series, has not yet been defined (Phillips et al., 1977). The earliest cells committed to the B-cell lineage express an Fc receptor and intracellular (cytoplasmic) IgM. These cells give rise to immature Fc+ sIg+, B cells that respond to B cell polyclonal activators, class 1 thymus-independent antigens (e.g., TNP-Brucella abortus) (see Section VI) and are capable of an IgM response to thymus-dependent antigens. It is also possible to induce tolerance in these cells (Nossal and Pike, 1975; Cambier et al., 1976). To date, no CMAD of restricted distribution has been detected on these immature B cells; however, these cells have not been examined for the LyM-1 and the Lyb series of antigens, which are expressed during the later stages of development.

B. CMAD OF B CELLS B cells at this stage are found in the bone marrow and show varying degrees of maturation. These cells are distinguished from the immature B cells by the expression of Ia antigens (i.e,, Fc+, sIgM+, Ia+)and

Cell

CMAD. Receptors

Antigen Responses

Tolerance Induction

Pluripotent stem cell

?

-

?

B stem cell

?

-

I I 1 I

?

Pre-B cell

Fc+, cytoplasmic IgM

Immature B cell

Fc+, surface IgM, Lyb-2+

B cell

Fc+, la+, IgM+

B cell

Fc+, Ia+, IgM+, IgD+, C3+, Ly-4+, Lyb-2+

PCA, TI Class 1, TD IgM, IgG; memory IgM, IgG

B cell

Fc+, IgM+, IgD+, Ia+, C3+, Ly-4+, Lyb-2+, Lyb-3+, Lyb-4?, Lyb-5+

PCA, TI Class 1, TD IgM, IgG; memory IgM, IgG

Fc+, Ia?, IgD+, C3+, Ly-4+, Lyb-2+, Lyb-5+, LyM-1+

FCA, TI Class 1, 2; IgG PFCs only

Fc+, Ia+, IgM+,IgD+, C3+,Ly-4+,Lyb-2+,Lyb-3+, LyM-1+, Pca+, (?Lyb-4, 5, 6)

PCA, TI Class 1, 2; IgM and IgG PFCs

Ly-4+, ALA-1+, Pca+

IgM PFC response

Ly-4+, ALA-1+, Pca+

IgG PFC response

IgG+

Memory

I 1

1

B cell

B cell

B cell

PCA, TI Class 1, T D IgM

FIG.4. Flow chart of B-cell differentiation. PCA, polyclonal (B cell) activator; PFC, plaque-forming cell; TD, thymus dependent; TI, thymus independent (classes 1 and %see text). Adapted from Ahmed et al. (1978).

0

c c

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the capacity to mount both IgM and IgG responses to T-dependent antigens. The next stage of differentiation is a commitment to a particular isotype, reflected in the expression of sIgG, sIgA, and/or sIgD, which appears to precede the expression of the C3 receptor (i.e., Fc+, sIgM+, sIgD+, Ia+, C3+). These cells are found predominantly in the spleen and bone marrow and also carry the Ly-4, LyM-1, and Lyb-2 antigenic determinants. It is possible to induce tolerance and memory in cells at this stage of development, and by analogy it is likely that this cell is the last stage of differentiation reached in CBNN mice. The expression of Ia antigens before sIgD suggests the sequence p- + p+ (IgM response) + p+Ia+ w+Ia+tS+(IgM and IgG responses) (Ahmed et al., 1978). The differentiation of the Fc+ sIgM+ sIgD+ Ia+ C3+ cell along two separate lines (Ahmed et al., 1978) is suggested by (1)the stimulation of different B-cell clones by Class 1 T-independent antigens, by Class 2 T-independent antigens and by T-dependent antigens (Lewis and Goodman, 1977); (2) the absence in CBNN mice of a T-dependent responsive B cell producing IgG antibody after primary immunization (Ahmed et al., 1977); (3)the absence in CBNN mice of a T-dependent B cell that is resistant to tolerization by Class 1 T-independent antigens and soluble T-dependent antigens (Scottet al., 1977; Merchant et al., 1978); (4) the differential distribution of Lyb-1, Lyb-2, and Ia antigens on PFCs and their precursors. Ahmed et al. (1978) suggested that one line of differentiation leads to a sIgM+, sIgD+, Ia+, MLS+, C3+, Lyb-3+ cell, capable of memory induction but resistant to tolerance, whereas the alternative line leads to cells that are Lyb-5+ and are not capable of memory, are resistant to tolerance induction by soluble T-dependent antigens, and generate IgG PFCs. The relationship between these two possible lines of differentiation, the PFC precursors, and the different types of PFCs is not clear at present. C. CMAD OF ANTIBODY-FORMING CELLS Takahashi et al, (1970a,b, 1971) were the first to describe the presence of antigenic determinants that were confined to antibody-forming or plasma cells. The Pca-1 specificity that they identified was also expressed on plasma cell tumors (Sections X and XIV). Since then, the only other alloantigen identified on plasma cells, but not on B cells, is ALA-1 (Feeney and Hammerling, 1977). Recent studies by Shen et al. (1977) suggest that the converse is true for Lyb-2 specificities, which are found on PFC precursors but only on a minor portion of IgG PFCs. Alloantigens present on both B lymphocytes and PFCs include Ly-4 (McKenzie and Snell, 1975), Ly-7 (McKenzie and Potter, unpublished observations), Ly-8 (Frelinger and Murphy, 1976), and LyM-1 (Ton-

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konogy and Winn, 1976). It was of interest to find that antibodyforming cells formed in response to priming with low doses of SRC were Ly-4-, whereas cells generated by priming with high doses of SRC consisted of two populations-Ly-4+ (70%) and Ly-4- (30%) (McKenzie et al., 1975). This finding suggested that there are two separate lines of B-cell differentiation to IgM PFCs. A differential expression of Ia determinants on PFCs has been observed by Press et al. (1975) and McDevitt (1976), who found IgG, but not IgM, precursors to be Ia+. Plasma cells have also been shown to lack surface Fc and C3 receptors (Basten et al., 1972; Dukor et aZ., 1972) as well as having a reduced expression of surface immunoglobulin (Takahashi et al., 1971). D.

ONTOGENY OF

CMAD AND RECEPTORS ON B CELLS

Although Ig- Fc+ cells have been identified in fetal liver (Cline and Moore, 1972; Rosenberg and Parish, 1977), there is some controversy over whether adult levels of Fc+ cells in the spleen are attained in the neonate (Forni and Pernis, 1975) or not until 4-6 weeks of age (Rosenberg and Parish, 1977). The C3 receptor appears late in ontogeny and is barely detected before 2 weeks of age, adult levels appearing in the next 2-4 weeks. The frequency of C3+ and C3- B cells appears to be under H-2 control (Gelfand et al., 1974a,b; Ferreira and Nussenzweig, 1976). Induction of precursor cells with LPS indicated that the appearance of the C3 receptor was preceded by at least two distinct differentiation steps and that Ia and sIg were expressed before the C3 receptor (Hammerling et al., 1976). Fc+ Ig- Ia- C 3 - - , Fc+ Ig+ Ia- C3-+ Fc+ Ig+ Ia+ C3-+ Fc+ Ig+ Ia+ C3+

The ontogeny of different CMAD is shown in Table XXIV, which encompasses the data from Ahmed e t al. (1978), in addition to data presented in Section VI and our own unpublished observations. The appearance of CMAD in fetal mice has not been investigated, but at birth a small number of Lyb-2+ Ia+ cells are detected in the spleen and rise, with the total number of B cells, to adult levels by 2 4 weeks. The Lyb-3, Lyb-5, and Lyb-6 specificities appear shortly after birth and reach adult levels by approximately 4 weeks of age. XIV. Expression of CMAD on Mouse leukemias a n d lymphomas

Studies of CMAD on lymphoid tumors in the mouse have played a key role in the definition and characterization of many different alloantigenic specificities. The original descriptions of the Ly-1, -2, -3, TZa, Pc-1, and Lyb-2 loci all involved tumors as immunogen or targets, and

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TABLE XXIV APPROXIMATE TIMEOF FIRST APPEARANCE OF CMAD, Fc AND '23 SURFACE Ig' Fetal life: 10 Days 13 Days 15 Days Birth Postnatal: 3 Days 1 Week 2 Weeks

RECEPTORS, AND

Fc IgM, Thy-1 TL, Ly123 Ia, Ly-4, Lyb-2 Lyb-3 IgD, Ly-7, Lyb-4, Lyb-6 C3, Ly-I, Ly-2/3, Lyb-5

For discussion, see text and Ahmed et al. (1978).

many T-cell and B-cell tumors have a different cell-surface phenotype (Shevach et al., 1972a,b; Warner et al., 1978; and Mathieson et al., 1978; and reviewed by Potter, 1976). The study of the phenotype of lymphoid tumors is of importance, as tumors can provide large numbers of relatively homogeneous cells that can be used for pathophysiological studies of lymphocyte function. In this context it should be noted that functional tumors have been found in both man and mouse (Stocker et al., 1974; Feldmann et al., 1975; Proffitt et al., 1975a,b; Roman and Golub, 1976). The importance of plasmacytomas and their monoclonal antibodies provides strong evidence of the productive value of tumor studies (Potter, 1976). Furthermore, studies of tumors may be of particular importance in the study of new cell types, present in such small numbers that they cannot normally be detected; e.g., the phenotype of the Ia+, Ig-, Thy-l- bone marrow cell that we have recently identified (McKenzie, unpublished) has also been described in several SJL tumors and in some Friend virus-induced tumors (Chesebro et al., 1976, 1978; Ponzio et al., 1977). Before describing the studies defining the CMAD of tumors in detail, certain problems in the use of antisera with tumors will be discussed (see also Section 111). First, many murine tumors express large amounts of MuLV determinants, and, as mouse alloantibodies contain anti-MuLV antibodies, false-positive reactions can occur (Sections I V and X). Second, tumor cells are often asynchronous, and the expression of certain CMAD has been shown to vary with the cell cycle (Pellegrino et al., 1974).Finally, tumors maintained in uivo usually contain a number of infiltrating normal cells, which are a particular problem in absorption studies. For example, it has recently been shown that contamination of an Ia- tumor-cell population with 1% of normal Ia+ cells could give rise to a positive absorption of an anti-Ia antiserum (Wettstein et al., 1976).This can be overcome by passaging the line in uitro; however, under these circumstances, greater amounts of MuLV are

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expressed on the cell surface. Also, tumor cell lines undergo changes in CMAD expression in culture. Some of the earliest studies performed on the CMAD of leukemias and lymphomas were those demonstrating that AKR lymphomas and leukemias were Thy-l+ and, by their mode of origin and morphology, were clearly T-cell tumors (Asakuma and Reif, 1968). In later studies a number of tumors were typed for Thy- 1and Ig markers and thus were easily characterized as being of T or B cell origin (Shevach et al., 1972a,b). An enormous amount of work has been done with mineral oil-induced plasmacytomas with regard to their properties and the structure of the monoclonal antibodies that they secrete. The use of these plasmacytomas also led to the definition of the Pca-1 specificity as well as several other specificities detected by xenoantibodies (Section VIII). These studies are reviewed elsewhere (Potter, 1976), and this section therefore concentrates mainly on the CMAD of T-cell tumors, which have been extensively typed with Thy-1 and Ly reagents. The presence of the TL antigen in leukemias of TL- strains was discussed earlier (Section VI). A. CHEMICALLY INDUCEDT LYMPHOMAS A number of BALB/c lymphomas induced b y l-ethyl-l-nitrosourea ( E N U ) have been typed for Thy-1, TL, and the Ly-1,2,3 specificities (Mathieson et al., 1978). The tumors were mostly of recent origin-to avoid the changes that occur when cells have been extensively passaged. The tumors were tested directly by cytotoxicity, by immunofluorescence, and by absorption. Several patterns, which were stable over many generations, were noted: (1)most tumors (11 of 13 Thy-l+ or TL+ tumors) showed a different expression of the Ly-1 and Ly-2 specificities and were typed as either Ly1+2- or Lyl-2+; (2) two cell lines (BALENTL 9 and P 1798) were Ly1+2+; (3)all the tumors were Thy-l+; (4) most tumors were TL+, but one was Lyl+TL(Mathieson et al., 1978; results confirmed in our laboratory). These studies are of importance, as they clearly document the occurrence of TL+ Lyl+2- and TL+Lyl-2+ cells, i.e., the retention of T L specificities by cells that could have been partially differentiated from the TL+Ly1+2+3+cell (Mathieson et al., 1978). If T L specificities are considered as differentiation antigens for leukemias, the presence of those cells suggest that some degree of T-cell differentiation occurs within the thymus (Section XIII).

B. In Vitro-MAINTAINED T-CELL LINES Some of the tumors described above were also grown as in vitro cell lines and were found to express the same phenotype in vitro as in

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uiuo: BALENTL 13was Thy-1+, Ly1+2-; seven other BALENTL lines were Thy-1+, TL+, Ly-1-2+, and two other lines were Thy-1+, Ly1+2+ (Kim et al., 1978). Terminal transferase (TdT), an enzyme found in thymic, but not in peripheral, T cells and considered as a marker for T-cell differentiation (Baltimore, 1974; Coleman et al., 1974; Kung et al., 1975) was also measured in these cell lines. All tumors showed some TdT activity, although the level of activity varied among the different lines, and this did not correlate with the Ly phenotype; e.g., in the Lyl-2+ group, there was a 6-fold variation of TdT levels (Kim et

al., 1978). C. OTHERT-CELL LYMPHOMAS A number of naturally occurring T-cell leukemias and lymphomas have been studied for Thy-1 and Ig, although only a few have been typed with Ly and Ia reagents (Shevach et al., 1972a,b; Krammer et aZ., 1976; Mathieson et al., 1978). These “spontaneous” tumors arise in AKR, C58, and several other strains and are associated with MuLV infection (Section X). Two AKR tumors (LS13 and LS34) were typed as Thy-1+, TL-, Ly1+2- by direct testing, and was subsequently found that many of the AKR tumors are TL- (Old et al., 1965). Another tumor, P1798, occurred in an estrogen-treated BALB/c mouse and was typed as Thy-l+, TL+, Ly1+2+;another, L4946, was Ly1+2- (Potter, 1976). Tumors involved in the original description of Ly-1 and Ly-2 specificities have been Ly typed as E 8 G2: Thy-1+, TL-, Ly1+2-; ERLD: Thy-1+, TL+, Ly1+2+;and EL4: Thy-1+, TL-, Ly1+2+(Old and Stockert, 1977). Although the Thy-1+, Ig- surface phenotype has been used to identify tumors as being of T-cell origin (Shevach et al., 1972b; Mathieson et al., 1978), a number of Thy-1+, Ig+ leukemias and lymphomas have been identified (Boylston and Mowbray, 1974; Harris et al., 1973; Ramasamy and Munro, 1974; Greenberg and Zatz, 1975; Haustein et ul., 1975; Warner et al., 1975; Krammer et al., 1976; Greenberg et al., 1977). Several unusual AKR tumors have been more fully characterized, and they appear to be unique in that (1)they arose in the periphery of old AKR mice (rather than in the thymus in younger mice); (2) the mice had been thymectomized or had thymic atrophy; (3)the AKTB-2 tumor typed as Thy-1+, Ly-1+, Ly-2-, Ia+, Ig+, Fc+; and the AKTB-3 tumor typed as Thy-1+, Ly-1+, Ly-2-, Ig+, Fc+. These typing results were obtained when the tumors were obtained from the spleen, but when the AKTB-1 line was obtained from lymph nodes, it typed as Thy-l+, Ig-, Fc-, Ia-. In these three tumors, the B-cell markers, Zg, l a , and Fc, were all present or absent together (Greenberg et al., 1977). In another series, fifteen AKR tumors were

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typed with a variety of T- and B-cell markers. All tumors were Thy-1+, MSLA', MBLA-, C3-, a proportion of the tumors were TL+, several were Fc+, and one was Ig+ (Krammer et d.,1976). Recently, several Ia+tumors have been identified in A.TL and A.TH mice (Goding and Warner, 1975). These tumors occurred with an incidence of 2% and involved lymph nodes, spleen, and thymus. The tumors were Thy-1+, Ig- and, as the A.TL tumors were rejected b y A.TH mice and vice versa, it was concluded that they probably carried Ia antigens. In our laboratory, we have typed only one of these tumors (A.TH-SLl), which had the unique surface phenotype Thy-l+ Ly 1+2+3+4+5-6+7-TL+PCA-Ia+.

D. RADIATION-INDUCED T THYMOMAS We have induced a number of different thymomas in C57BW6 or C57BU10 mice, or their congenic lines, by multiple-dose irradiation. The cells were typed by direct cytotoxicity, rosetting, and absorption, using a wide range of CMAD antisera (P. M. Hogarth and I. F. C. McKenzie, unpublished observations). Several different groups of tumors occurred: (a) Thy-l+ Ly1+2+3+4-5+6-Ia-; (b) Thy-l+ Lyl-2+3+4-5+6-Ia+; (c) Thy-l+ Lyl-2+3+4+5+6+Ia+;(d) Thy-lLyl-2-3-4-5+6-Ia-; in addition, some tumors typed as Ly-2+3--an unusual phenotype. These different phenotypes are of interest for several reasons. First, the finding that all tumors were Thy-1+, Ig- and carried T-cell rather than B-cell markers indicates that these were clearly of T-cell origin. Second, the phenotypes examined correlated reasonably with the phenotype of known functional T cells of their precursors. For example, group a could be the Ly1+2+3+precursor cell; group b has the phenotype of a suppressor cell (as it is Ly2+3+Ia+);group c is similar, but also expressed Ly-4 and Ly-6, but as yet no functional studies have been done with these tumors. Third, several new phenotypes are identified. In group d the Lyl-2-3- cell may be a prothymocyte, and in this context it is of interest that it is also Ly5+. In the final group, the tumors were identified as being Ly2+3-; if these results can be confirmed, this is the first description of an Ly2+3- cell. Fourth, the low incidence of the Ly-6.2 specificity, being expressed on only one of the Ly thymomas (group c), supports the contention that it is a peripheral T-cell marker. E. VIRUS-INDUCED LEUKEMIAS AND LYMPHOMAS In addition to the AKR tumors, which are closely associated with MuLV (Gross), other MuLV can cause tumors; those induced by the F M R group have been most extensively studied. Tumors induced by

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the Friend virus appear to be of either of two types. First, those that are probably erythroid tumors and do not react with Thy-1, Ly, or Ia antisera and are Ig-(Hogarth and McKenzie, unpublished results). The second group type as Thy-1-, Ia+, Ig- (Cheseboro et al., 1976, 1978). They are also Fc+, and as mentioned above, we have identified a normal bone marrow cell with the same phenotype, but whose function is unknown (McKenzie, Owen, and Parish, unpublished observations). The two Rauscher virus-induced tumors that were studied gave different phenotypes: RBL-3 was Thy-l-Ig+ and RBL-5 was Thy-l-Ig(Shevach et al., 197213; P. M. Hogarth, personal communication). The Moloney viral tumor MBL-2 was Thy-l+Ig-, YAAC-1 is Thy-l+Ig(Shevach et al., 1972b). We have typed YAAC-1 and found the surface phenotype to be Ly1+2-3-4-5-6-7-TL+Ia+Pca-.

F. PHENOTYPE OF ABELSON VIRUS-INDUCEDB-CELLTUMORS The MuLV-A, or Abelson, virus arose in studies of Moloney leukemia virus (Section X).In contrast to the Moloney virus, the Abelson virus produces tumors in B cells and gives rise to lymphosarcomas (Abelson and Rabstein, 1970a,b) and plasmacytomas (Potter, 1976). In most studies the tumors are Thy-1- Ig- (Sklaret al., 1975a,b), although surface Ig has been found in some (Warner et al., 1978). In a study of six different MuLV-A-induced tumors, three groups were studied, (a) lymphosarcomas (ABLS tumors) typed as TL-, Thy-1-, Ly1-2-3-4+5+6-7+, Ia-, Pca+ (1 of 2 was positive); (b) two plamacytomas typed the same except that they were Ly4+,7+, Pea+, Ia+; (c) two tumors considered to have characteristics of both group a and group b were also Lyl-, 2-3-4+5+'-, 7+/-, and Ia+/- (Hogarth and McKenzie, unpublished results). On the basis of these and other studies, it has been suggested that MuLV-A acts on early B cells and that the plasma cell is also a site of infection with MuLV-A. G . PLASMACYTOMAS AND OTHER B-CELLTUMORS The characteristics of these cells have been extensively reviewed elsewhere (Potter, 1976) although surface characterization has mostly involved the use of the Pca, Ly-4, Ly-7, and Ig markers and they have not been extensively studied with the newer Lyb markers. A number of MOPC plasmacytomas have been typed as Ly-4+,7+and most (but not all) are Pea+ (Warner et al., 1978; Hogarth and McKenzie, unpublished results). Some of the MOPC tumors are also Ly1-2-3-4-5-TLpThy-l-Ia+ (Hogarth and McKenzie, unpublished results). On the basis of the expected absence of the TL, Thy-1, and

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Ly-1,2,3, markers, and the differential distribution of Ly-4, Thy-1, Ia, and Pca, a differentiation pathway of B cells has been designed (Warner et al., 1978): from pre-B (Ly4-7+Ia+Pca-) through B cells to antibody-forming B cells (Ly4+7+Ia+Pca+).Another series of B-cell tumors have also been typed with Ly-4,7 antisera (Warner et al., 1978): WEHI 231, 279, and PK3 are Ly4+7+Ia+,whereas the T lymphomas WEHI-22 and 7 were Ly4-7-Ia-; and S49 (T lymphoma) was Ly4-7+; and a myeloid leukemia, WEHI 265, was Ly4-7- (Warner et al., 1978). Recently, five spontaneously derived BALB/c tumors and one chemically induced (BALENLM 17) were examined for Fc, C3, Ia, and sIg. With one exception, all the tumors were Thy-1-, Ia+, Fc+, Ig+, C3-. The one exception, M12, was Ia-Fc+, and clearly demonstrates that Ia specificities and Fc receptor are separate structures (Kim et al., 1979). H. MASTOCYTOMAS The mastocytoma P815 is often used as a target in CML assays and is L ~ 4 - 7 ~Recently . we have typed three mastocytomas: P815, CXBGAMCT-1, CBGABMC-4. For all three tumors, the phenotype was Ly1-2-3-4-5-6+7-Ia-Pca-Tla-H-2+ (Hogarth and McKenzie, unpublished), and P815 has also been typed as Ly6+ by P. Halloran (personal communication). The Ly-6 specificities (Ly-6.2 or Ly-6.1) were the only Ly markers present on these mastocytomas. It has been suggested that mast cells could be an end-stage functional T cell (M. F. Burnet, personal communication), and it is intriguing to note that Ly-6 has been found, predominantly, on peripheral functional T cells (Section VI). It has not been determined whether normal mast cells are Ly-6+. I. OTHERTUMORS A number of other tumors have been studied with one or more of the Ly markers. The SJL strain produces several interesting tumors. First, spontaneously occurring reticulosarcomas have recently been typed as Thy-1-, Ig-, Ia+ (Ponzio et al., 1977). The origin of this cell is unknown, but several Friend virus-induced tumors described recently (Chesebro et al., 1978) have the same phenotype (Thy-l-Ig-Ia+) (see above). Another group of SJL tumors were those induced b y giving young, thymectomized mice regular feedings of DMBA (Haran-Ghera and Peled, 1973; Linker-Israeli and Haran-Ghera, 1975). Some of the tumors were weakly Thy-l+ but were also Ig+, Fc+, and C3+. It is most likely that these are B-cell tumors.

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J. CONCLUSION As stated in the introduction to this section, murine lymphoid tumors are of interest because they represent an expansion of a normal cell type and are therefore useful in providing large numbers of cells for study. Second, they may lead to the identification of new cell types. However, do tumors represent the expansion of normal cell types? Are they functionally of value to study? Or are there technical problems, especially associated with MuLV, so prominent that the results are of no real value? The technical problems can be overcome, especially if tumors are tested by absorption or by techniques that appear not to detect MuLV (Section 111). Furthermore, in general lymphomas and leukemias fall mostly into T(Thy-1+, Ig-) or B(Thy-1-, Ig+) cell types. There are well defined exceptions to this, and some tumors, studied in detail, are Thy-1+, Ig+, Fc+, Ia+-i.e., they have the markers of both T cells and B cells. In these cases, the rigid definition of T cells and B cells can be questioned, and the tumor under examination could represent expansion of a Thy-1+Ig+ cell not normally detected in the mouse. Alternatively, the expression of new or unusual markers may be a characteristic of the malignant transformation, with gene derepression as the explanation for the simultaneous occurrence of CMAD. Arguing against derepression as a general phenomenon to explain the occurrence of the different phenotypes is the finding that both T-cell and B-cell markers tend to fit the concepts of differentiation, in that there are Lyl+2+,Ly1+2-, and Lyl-2+Thy-l+ cells and that these markers have not been found on Ig+ cells. The second question of the functional relevance of the tumors can be answered by citing some of the data. Several BALENTL tumors (Lyl-2+) were found to suppress the MLR and the generation of killer T cells (Kim et al., 1978), whereas a Ly1+2- tumor had no such suppressor activity. These functions correlate with the known functions of the Lyl-2+ cell. However, these studies need to be extended and Ly typing performed on other functional T-cell tumors. XV. Conclusion

Most of the known antigenic determinants found on the surface of murine lymphocytes have been summarized in this review. Several points will be reemphasized. 1. Although the detection of CMAD by antisera involves many technical difficulties, the recent introduction of monoclonal cell hybrids for antibody production should overcome many ofthese problems,

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32 1

and investigators can look forward to an abundant supply of hightitered, specific reagents. 2. The polymorphisms revealed to date will undoubtedly be expanded b y using different cell types for immunization, with different donor-recipient combinations and the use of more sensitive methods to detect such antibodies. 3. A surface molecule must exist in polymorphic forms within the available inbred strains to be detected with alloantisera. It is likely that there are molecules on the lymphocyte surface that are antigenically uniform within all strains of mice. The identification of such systems will require highly specific xenoantisera, and so far studies in this area have received far less attention than the alloantigenic s y stem s. 4. In spite of the problems, the studies of CMAD have yielded generous rewards, particularly in the identification of the relationship between T-cell function and surface antigenic phenotype. The study of CMAD in T-cell and B-cell differentiation and ontogeny will undoubtedly continue to expand and will provide a very valuable tool. 5. With a few exceptions, the primary function of the CMAD remains unknown, although the role of Ia, Lyb-3, and Lyb-7 in the regulation of B-cell responses has suggested that these molecules are involved in antigen-induced cell activation. Further chemical characterization of many CMAD should provide a better insight into their particular function. 6. Although the study of murine CMAD has uncovered many interesting findings, the ultimate test of such studies will be their applications for man. By analogy it would be expected that similar systems exist in man. However, for ethical reasons, antisera to these determinants cannot be raised b y planned alloimmunization and must therefore be prepared as xenoantisera or by monoclonal antibodies from cell hybrids. The identification and characterization of such systems may ultimately lead to immunotherapy in human disease states, e.g., in the elimination or administration of selected lymphocyte subpopulations.

ACKNOWLEDGMENTS The authors gratefully acknowledge the help in the preparation of this manuscript of Dr. Aftab Ahmed, Naval Medical Research Institute; Dr. Geoff Shellam, University of Western Australia; and Mark Hogarth and Gillian Morgan from the University of Melbourne. For secretarial assistance, we are indebted to Miss J. Wong, Mrs. J. Hanni, and Miss K.Hanni. The original work reported herein was supported by grants obtained from the N.H.M.R.C.,Tobacco Research Foundation, Anti-Cancer Council of Victoria (Australia), and the National Cancer Institute, N.I.H., Bethesda, Maryland (CA-22080).

322

IAN F. C. MCKENZIE AND TERRY POTTER ABBREVIATIONS

ATXBM, adult thymectomized irradiated, bone marrow reconstituted BCG, Bacille Calmette-Gubrin BLyl, ATXBM mice, given Lyl T cells BLy23, ATXBM mice, given Ly23 T cells BSA, bovine serum albumin C‘, Complement C3, (receptor for) third component of complement CFU-s, colony-forming unit stem cell cM, centimorgan CML, cell-mediated lympholysis Con A, concanavalin A CR, cortisone resistant CS, cortisone sensitive DNFB, 2,4-dinitrofluorobenzene DNP, 2,4-dinitrophenyl DMBA, 2,4-dimethylbenzathracene DTH, delayed-type hypetsensitivity EDTA, ethylenediaminetetraacetic acid Fab, antigen-binding fragment of antibody FACS, fluorescence-activated cell sorter Fc, (receptor for) crystallizable fragment of Ig GvH, graft versus host HGG, human y-globulin Ig, immunoglobulin (sIg, surface immunoglobulin) KLH, keyhole limpet hemocyanin

USED IN

THE TEXT LPS, lipopolysaccharide Lyl cell, Ly1+2-3- T cell Ly23 cell, Lyl-2+3+ T cell Ly123 cell, Ly1+2+3+T cell MHC, major histocompatibility complex MIF, migration inhibition factor MLR (MLC), mixed lymphocyte reaction MuLV, murine leukemia virus MuLV-A, MuLV-Abelson MuLV-G, MuLV-Gross MW, molecular weight NP-40, Nonidet P-40 PACE, polyacrylamide gel electrophoresis PFC, plaque-forming cell PHA, phytohemagglutinin PPD, purified protein derivative PTP, postthymic precursor PWM, pokeweed mitogen RI, recombinant inbred SDP, strain distribution pattern SDS, sodium dodecyl sulfate SRBC, (SRC) sheep red blood cell TDTH, T cell effecting DTH TH, helper T cell TK,killer T cell Ts, suppressor T cell TD, T-cell dependent TI, T-cell independent TNP, trinitrophenyl

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ADDED IN PROOF Several new loci and specificities have been described recently: (a) T100. Identified by a contaminant antibody in Ly-2.1 serum which reacts with thymocytes and lymph node cells of most strains including BALB/c but not with C57BL16, C58, C57L, and several CXB lines. The CXB pattern suggested linkage to the H-25 locus, which was confirmed by direct testing on the H-25 congenic strain. The antibody is weakly cytotoxic but precipitates with a 100,000 MW glycoprotein [Durda et al. (1979). J . Zmmunol. 122, 1451. A similar antibody has apparently been described by Ledbetter et al. (manuscript in preparation) with a rat monoclonal antibody. This glycoprotein is now referred to as Lgp-100. (b) H-2T, Qed-1. These may be identical or closely linked to the Tla locus (K. Fischer-Lindahl, personal communication). (c)Mac-1 defines a new specificity which is apparently only present on macrophage-like cell lines and absent from other tissues. It is identified by a rat monocolonal antibody [Springer (1979). Eur. J . Zmmunol. 19, 3011. (d) Thy-1. In addition to the monoclonal Thy-1 anZmmunol. tibodies described in the text, Marshak-Rothstein et al. (1979). 122,24911 have produced similar hybridomas. NOTE

u.

ADVANCES IN IMMUNOLOGY, VOL. 27

The Regulatory and Effector Roles of Eosinophils PETER F. WELLER AND EDWARD J. GOETZL The Howord Hughes Medical Indifvto Labomtory ot HarvardMedical School, and Departments of Medicine, Haward Medical School and the Robert B . Brigham Hospital Division of the Affiliated Hospitals Center, Inc., Boston, Massachusetts

I. Introduction ........................................................... 339 11. Eosinophil Production and Distribution ................................. 340 340 A. Production of Eosinophils in the Bone Marrow ....................... 341 B. Immunological Control of Eosinophil Production ..................... C. Release, Tissue Distribution, and Fate of Eosinophils ................. 345 111. Cellular Properties of Eosinophils ...................................... 347 IV. General Functions of the Eosinophil .................................... 349 A. Modulation of Eosinophil Migration .................................. 349 B. Endocytosis and Associated Events .................................. 353 V. Involvement of Eosinophils in Immunological Responses . . . . . . . . . . . . . . . . . 354 A. Association with Antibody Production and Delayed Hypersensitivity 354 Reactions .......................................................... B. Eosinophil Receptors for Immunoglobulins and Complement Components ....................................................... 355 C. Modulation of Immediate Hypersensitivity Reactions ................. 356 D. Association with Human Immunodeficiency Diseases . . . . . . . . . . . . . . . . . 360 VI. The Role of the Eosinophil in the Host Response to Helminthic Infections ............................................................ 360 364 VII. Concluding Remarks ................................................... 365 References ................................... ......................

I. Introduction

The eosinophilic polymorphonuclear (PMN) leukocyte, or eosinophil, was initially recognized and defined by the characteristic staining of its cytoplasmic granules with aniline dyes, such as eosin. Myriad studies subsequently have documented that increased numbers of eosinophils are found in the blood and affected tissues in association with most allergic reactions and parasitic helminth infections, as well as in the course of some neoplastic, inflammatory, and immunodeficiency diseases (Beeson and Bass, 1977). While the molecular mechanisms underlying the development of eosinophilia remain to be elucidated, immunological pathways were implicated in view of the temporal association of the rises in antibody titer and eosinophil levels following the administration of some immunogens, the accelerated and more pronounced eosinophilia produced by rechallenge with antigen, and the passive transfer of eosinophilia by T 339 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-022427-5

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lymphocytes from animals with peripheral blood eosinophilia. Specific immunological principles capable of augmenting the production of eosinophils have been recognized recently by the application of in vitro bone marrow culture techniques. The striking local tissue eosinophilia that was observed following immunological stimulation of mast cells or lymphocytes also may be attributable, in part, to the array of specific mediators released by these cells. Contemporary investigations of the functions of eosinophils have been facilitated by the development of improved approaches for obtaining purified populations of eosinophils and of more sensitive biochemical and immunochemical methods to characterize cellular constituents and receptors. Enzymes contained preferentially in eosinophils as compared to other leukocytes have been identified and, in some instances, have been shown to rapidly degrade mast cellderived mediators. A tentative integration of this evolving body of facts has led to the concept that the normal eosinophil is a tissue cell with a unique capacity to contain and terminate most immediate-type hypersensitivity reactions. Recent in vitro studies of the antibody and complement-dependent helminthicidal activities of purified eosinophils and analyses of the in vivo effects of the depletion of eosinophils b y the administration of specific antisera have suggested the importance of eosinophils in host resistance to helminthic infections. The eosinophil thus may serve both regulatory and effector roles in a variety of immunological responses. II. Eosinophil Production and Distribution

A. PRODUCTION OF EOSINOPHILSIN THE BONE MARROW During the fetal and neonatal development of animals, eosinophil production may occur in extramedullary sites including the liver, spleen, thymus, and lymph nodes, as well as in the bone marrow (Foot, 1963; Yoffey and Bhurgan, 1964; Sin and Sainte-Marie, 1965). By adulthood, most eosinophils are produced in the bone marrow, where the total number of eosinophils equals or exceeds the combined number in all other tissues and is substantially greater than that in the blood by a ratio of 200 : 1 in the rat (Rytomaa, 1960) and 400 : 1 in the guinea pig (Hudson, 1968) (Fig. 1). Although a distinct bone marrow progenitor of eosinophils has not been identified, several lines of evidence suggest that eosinophilic and neutrophilic leukocytes may originate from different stem cells. The circulating level of eosinophils is not diminished in patients with congenital agranulocytosis (Gilman

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Genemlm

Emergence

Meon circulating

Mean life

time = 34hr Bone morrow

time L. 10-80hr Morrow r e w v e

time 9 12hr Circulation

lime days Tissues

number of eOSinODhtls

Marginal pool

300-400

FIG.1. Generation and maturation ofthe eosinophil. N, nucleus; 0,primary lysosomal granule; ID, crystalloid granule; 0 , smaller homogeneously dense granule; invaginations in the plasma membrane represent microendocytic vesicles.

et al., 1970) or drug-induced neutropenia (Connell, 1969). Human eosinophils lack lysozyme (West et al., 1975), which is found in high concentrations in neutrophils. Further, eosinophil peroxidase differs from neutrophil peroxidase in terms of ultraviolet absorption spectra, substrate preferences, susceptibility to various inhibitors, and antigenicity (Archer and Broome, 1963; Archer et al., 1965; Salmon et al., 1970; Migler and DeChatelet, 1978). Moreover, the level of eosinophil peroxidase is normal in the face of genetic deficiencies of neutrophil and monocyte peroxidase (Lehrer and Cline, 1964; Salmon et al., 1970), and, conversely, the concentration of neutrophil peroxidase is unaffected by the genetic absence of eosinophil peroxidase (Presentey and Szapiro, 1969). Although such quantitative and qualitative differences in the mature cells could reflect disparate differentiation of eosinophils and neutrophils after the early promyelocyte stage, data obtained from in vitro studies of bone marrow-derived clones have supported the existence of unique stem cells for eosinophils. In human marrow cultures eosinophil colonies develop more slowly than neutrophil and macrophage colonies (Johnson et al., 1977). The application of a cluster-transplantation technique to the culture of human marrow cells demonstrated that the development of colonies of eosinophils was independent of neutrophil and macrophage colonies, which contained no eosinophils (Dao et al., 1977). By employing velocity sedimentation prior to culture, the eosinophil colony-forming cells of mouse bone marrow could be separated from stem cells that generated neutrophil or macrophage colonies (Metcalf et al., 1974). B. IMMUNOLOGICAL CONTROLOF EOSINOPHILPRODUCTION While the mechanisms underlying the stimulation and regulation of eosinophil production are incompletely understood, specific eosinophil colony-stimulating factors (CSFs) have been identified in

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uitro and the possibility of their immunological derivation has been confirmed (Fig. 2). Mouse bone marrow, spleen, and fetal liver cells were stimulated to produce eosinophil-like granulocytes in in vitro cultures by the introduction of a 50,000 MW factor that had been elaborated by mouse lymphoid cells stimulated by pokeweed mitogen (Metcalf et al., 1974). This eosinophil CSF was electrophoretically separated from neutrophil and macrophage CSF activities that had been generated concomitantly. Ruscetti et al. (1976) demonstrated that the development of eosinophils in in uitro cultures of marrow cells was promoted by the addition of medium obtained after specific antigen challenge of lymphocytes from mice sensitized to Trichinella, whereas medium obtained after the lymphocytes were challenged with heterologous antigen was without effect. The potentially restricted specificity of the immune response to Trichinella was suggested by the finding that splenic lymphocytes from mice sensitized to Bacille Calmette-Gu6rin (BCG), when incubated with either purified protein derivative (PPD) or Trichinella antigen, failed to elaborate CSF activity. Confirmation of the lymphocytic origin of some soluble factors that mediate eosinophilopoiesis has been obtained in viuo. Trichinel la antigen-stimulated lymphocytes, obtained from rats sensitized to Trichinella, when implanted intraperitoneally in cell-tight diffusion chambers, induced peripheral blood eosinophilia in the recipient rats (Basten and Beeson, 1970). Conversely, when normal mouse bone

Circulation

1

IT ,lymphocytes1 T lymphocytes

RG.2. Regulation of the production and distribution of eosinophils -, Generation inhibition. CSF,colony-stimulating factor. or release; ---->, stimulation; -,

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marrow cells in similar chambers were implanted intraperitoneally into mice experiencing an eosinophilia induced by rechallenge with antigen, the compartmentalized marrow cells developed greater numbers of eosinophils than those implanted in noneosinophilic rats (McGarry and Miller, 1974). With marrow cells and primed spleen cells in adjoining compartments separated by cell-impermeable membranes, increased intracompartmental marrow eosinophilopoiesis was produced by antigen-specific stimulation of the spleen cells (Miller and McGarry, 1976; Miller et al., 1976). That cell-free medium, conditioned by the prior incubation of spleen cells with specific antigen, stimulated eosinophilopoiesis when injected into mice with corticosteroid-induced eosinopenia further suggested a critical in vivo role for lymphocyte-derived eosinophilopoietic factors (Miller et al.,

1976). In experimental animals, considerable evidence has been collected in support of the participation of T lymphocytes in the induction of augmented eosinophilia by immunogens (Fig. 2), and some of the functional determinants of the immunogens have been defined. Intravascular-but not subcutaneous, intramuscular, or intraperitoneal-injection of Trichinella spiralis larvae elicited a prolonged eosinophilia that was augmented by a secondary challenge with larvae (Basten et al., 1970). This augmented eosinophilic response could be abolished by neonatal thymectomy, repeated administration of antilymphocyte serum, or chronic thoracic duct drainage (Basten and Beeson, 1970). In previously irradiated rats the development of eosinophilia on administration of larvae required prior reconstitution with both lymphocytes and bone marrow cells. The secondary eosinophilia that was characteristic of the reaction to rechallenge with antigen was seen only when the reconstituting lymphocytes were derived from animals sensitized to Trichinella larvae. The augmented eosinophilic response could be transferred adoptively with thoracic duct or peripheral blood lymphocytes in suspensions or in cell-tight peritoneal chambers, but not by lymph or plasma. That the eosinophilic response to injected Trichinella larvae was dependent on sensitized T lymphocytes was established by utilizing T lymphocytedeficient mice which manifested normal neutrophilic responses to bacterial infection, but required thymic reconstitution to develop an eosinophilic response to larvae (Walls et al., 1971). Similarly in irradiated mice, the eosinophil response normally observed after rechallenge with antigen (Speirs et al., 1974) required reconstitution with both normal stem cells and specifically sensitized lymphocytes (McGarry et al., 1971). As in rats, the eosinophilic response of mice

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was a function of sensitized T lymphocytes and was independent of B lymphocytes and of antibody formation (Jenkins et al., 1972; Speirs et al., 1973; Ponzio and Speirs, 1973, 1975; Walls, 1976). The failure of congenitally athymic (nude) mice to develop the usually increased eosinophilic responses associated with helminthic infections, such as Schistosomiasis mansoni (Fine et al., 1973; Hsu et al., 1976; Phillips et al., 1977), Ascaris suum (Nielsen et al., 1974), and Trichinella spiralis (Ruitenberg et al., 1977), provides additional confirmation of T-lymphocyte involvement in the augmentation of eosinophilia. The observations that the administration of Trichinella larvae by routes that resulted in pulmonary or peripheral embolization yielded augmented eosinophilia (Basten et al., 1970) suggested a need for tissue sequestration of particulate immunogens in mediating T lymphocyte-dependent eosinophilia. Thus antigen-containing homogenates and extracts of Trichinella larvae failed to induce augmented eosinophilia when utilized for rechallenge, but were able to prime rats for the eosinophilia induced by embolized larvae (Walls and Beeson, 1972a). The antigens involved need not be helminthic in origin, since other immunogenic particles, including dextran beads (Walls and Beeson, 1972b) and latex beads coated with human y-globulin (Schriber and Zucker-Franklin, 1975), elicited eosinophilia after embolization into the pulmonary vascular bed (Fig. 2). The tissue immobilization of particulate antigens requisite for the T lymphocyte-mediated eosinophil response might function by providing high local concentrations of antigen-antibody complexes (Butterworth, 1977). Local eosinophil accumulations, which may be associated with blood eosinophilia (Litt, 1968; Parish and Coombs, 1968; Parish, 1972), have been detected in relation to antigen-antibody complexes in the peritoneal cavity (Litt, 1961; Speirs and Osada, 1962), in draining lymph nodes (Cohen et al., 1964), and in the skin (Cohen and Sapp, 1965). T lymphocyte-dependent stimulation of eosinophil production has not been related causally to either regional concentrations of immune complexes or to other T lymphocyte products, such as eosinophil stimulation promotor (ESP) (Colley, 1973; Greene and Colley, 1974) and eosinophil chemotactic factor precursor (ECF,) (Cohen and Ward, 1971). That eosinophilia is not totally ablated in T lymphocyte-deficient animals (Fine et al., 1973; Phillips et al., 1977; Rothwell and Love, 1975) suggests the existence of T lymphocyte-independent mechanisms of eosinophilopoietic stimulation. Mahmoud et al. (1977, 1978a) have demonstrated a factor termed an “eosinophilopoietin” in the serum of mice rendered eosinopenic with specific antieosinophil serum (Fig. 2). This principle stimulated intramedullary eosin-

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ophilopoiesis and elicited blood eosinophilia in normal mice, exhibited a molecular weight of between 186 and 1375 by gel filtration, and was digestible by Pronase. A similar factor, which also stimulated eosinophilopoiesis in normal mice, has been identified in the sera of patients with schistosomiasis, but not of patients with hypereosinophilia of unknown etiology (Mahmoud et al., 197813). Neither the cellular origins nor the relationship to other eosinophilopoietins is known for these factors. Both IgE-producing B lymphocytes and mast cells have been hypothesized as additional sources of eosinophilopoietins (Parish e t al., 1977).

c. RELEASE, TISSUEDISTRIBUTION,AND FATEOF EOSINOPHILS Autoradiographic studies have indicated that rat eosinophils normally exhibit an emergence time from bone marrow to blood of 60-73 hours, a half-life in the circulation of 8-12 hours, and a tissue half-life of approximately 22 hours (Foot, 1965) (Fig. 1).The elimination of rat eosinophils from the blood was independent of their age, or random (Foot, 1965; Spry, 1971a). In Trichinella-infected rats, the respective times of the eosinophil cycle were significantly shortened, so that the generation time fell from 30 hours to 9 hours and the emergence time from marrow to blood was reduced from 41 hours to 18 hours (Spry, 1971a,b). Neutrophil emergence times were not shortened, indicating that separate mechanisms regulate the maturation and release of the two types of granulocytes. In a study of three normal humans given a pulse of tritiated thymidine, the mean eosinophil generation time was 34 hours (Fig. 1) and eosinophils were removed from the circulation randomly with a half-life of about 2 hours (Parwaresch et al., 1976). The latter study also suggested the existence of two populations of eosinophil precursors: one divided slowly, but yielded eosinophils that left the marrow rapidly and circulated for a long time, whereas the other precursor divided more rapidly and produced eosinophils that remained in the marrow longer and subsequently circulated only briefly (Parwaresch et al., 1976). Studies in patients with hypereosinophilia have indicated more complex patterns of eosinophil release and distribution, including the possibility of the cycling of a fraction of tissue eosinophils back into the circulation (Herion et al., 1970; Greenberg and Chikkappa, 1971; Dale et al., 1976). Plasma from Trichinella-infected rats has been shown to contain an “eosinophil releasing factor,” which can double the number of circulating eosinophils within 6 hours in recipient normal rats (Spry, 1971a), implying the existence of a mobilizable pool of eosinophils, analogous to a marrow reserve or a “marginated” population. The factors that govern the normal distribution of eosinophils from

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PETER F. WELLER AND EDWARD J. GOETZL

the circulating blood pool into the tissues have not been elucidated. It has been suggested that the peripheral blood eosinophilia, which may develop in lymphomatous rats, was a consequence of a blockade of the normal patterns of eosinophil emigration from the blood (Spry, 1972). The eosinopenic effects of such agents as P-adrenergic agonists (Koch-Weser, 1968; Srivastava et al., 1977), prostaglandins (Kurosawa, et al., 1978), and corticosteroids (Beeson and Bass, 1977) are of uncertain etiologies (Fig. 2). The eosinopenia that frequently ac,companies infection and inflammation (Weiner and Markovin, 1952) has been studied in Trichinella-infected mice with eosinophilia (Fig. 2). Bacterial and viral infection and noninfectious inflammation resulted in rapid blood eosinopenia, the appearance of eosinophils at the periphery of the inflammatory lesions, an inhibition of eosinophil egress from the marrow, and, with chronic infections, an impairment of eosinophilopoiesis (Bass, 1975a). These effects were not mediated by either endotoxin or increased endogenous levels of corticosteroids and could be reproduced by a partially characterized “eosinopenic factor” isolated from inflammatory exudates (Bass, 1975b, 1977). In normal adult humans, the peripheral blood level of eosinophils ranges from 0 to 450 per microliter, exhibits diurnal variation, and may fluctuate considerably within minutes (Discombe, 1946; Rud, 1947; Best et al., 1953; Horn et al., 1975). In children, eosinophil counts tend to be somewhat higher, with discrete peaks discernible at about a month after birth (Matheson et al., 1957) and in the period from 4 to 8 years of age (Cunningham, 1975). Blood eosinophil counts are elevated in atopic individuals, even those without symptoms (Felarca and Lowell, 1967). Quantitative studies of the distribution of eosinophils have established that the eosinophil is primarily a tissue cell (Fig. 1). The ratio of the number of eosinophils residing in the tissues to those in the blood has been estimated to be 100 : 1 in man (Stryckmans et al., 1968), 200 : 1 in the rat (Rytomaa, 1960), and 300 : 1 in the guinea pig (Hudson, 1968). Few eosinophils are found in the heart, kidneys, or liver, but they are abundant in organ systems with epithelial surfaces exposed to the environment. Thus, outside of the bone marrow, the bulk of tissue eosinophils is localized i n the gastrointestinal tract, the skin, the lungs, the lower urinary tract, and the uterus during estrus (Rytomaa, 1960). The uterine localization (Ross and Klebanoff, 1966) may be related to estrogen receptors on the eosinophil (Tchernitchin et al., 1975).The ultimate fate of the eosinophil after localization in the tissues is not certain, but possibilities include simple dissolution, shedding into the respiratory or gastrointestinal tracts (Teir et al., 1963), and consumption by macrophages (Speirs and Osada, 1962; Ross and Klebanoff, 1966).

THE REGULATORY AND EFFECTOR ROLES OF EOSINOPHILS

347

Ill. Cellular Properties of Eosinophils

The mature human eosinophil shares with the neutrophil and basophil an overall polymorphic shape, but the nucleus of the eosinophil is distinctly bilobed and lacks a nucleolus (Gross, 1962; Zucker-Franklin, 1974). The most characteristic microscopic feature of the eosinophil is a class of large ellipsoidal cytoplasmic granules, which contain an electron-dense crystalloid core enclosed in a less dense matrix (Miller et al., 1966). Large spherical primary lysosomal granules proliferate during the early development of eosinophils (Fig. 1) and mature into the crystalloid granules subsequent to the myelocyte stage (Spicer and Hardin, 1969; Hardin and Spicer, 1970; Bainton and Farquhar, 1970; Scott and Horn, 1970). Another class of eosinophil cytoplasmic granules are small and homogeneously dense and develop during the metamyelocyte stage, progressively increase in numbers with cellular maturation, and become more abundant in tissuelocalized eosinophils (Fig. 1) (Parmley and Spicer, 1974). This apparently adaptive production of small granules by mature eosinophils is not a characteristic of neutrophils, and, thus, may relate to the functional significance of the greater density of endoplasmic reticulum and Golgi lamellae in eosinophils than in neutrophils (Tchernitchin, 1973; Parmley and Spicer, 1974). Eosinophil granules contain an array of enzymes generally comparable to those in neutrophil lysosomes, but the human eosinophil lacks lysozyme and the content of peroxidase, P-glucuronidase, and acid phosphatase exceeds that of neutrophils by 2- to 3-fold (West et al., 1975). Physicochemical and functional polymorphism of lysosomal enzymes is as common in eosinophils as in other leukocytes. For example, the acid phosphatase activity from the crystalloid granules of horse and guinea pig eosinophils is electrophoretically separable into two discrete enzymes (Gessner et al., 1973; Heyneman et al., 1976). The horse eosinophil acid phosphatases differ in terms of affinities for the granule, thermolability, substrate preferences, and inhibitor specificities (Heyneman et al., 1976). Enzymes of apparent similarity may differ substantially in function in eosinophils as compared to neutrophils. The bactericidal role and the contributions to oxidative metabolism of peroxidases vary for each of the classes of leukocytes. The failure of the eosinophil peroxidase to participate in bactericidal reactions initially was demonstrated by inhibiting peroxidase activity in intact leukocytes, which slightly enhanced eosinophil bactericidal activity while inhibiting that of neutrophils b y nearly 50% (Bujak and Root, 1974). Although the peroxidases of both human eosinophils and neutrophils catalyzed the iodination of proteins in the presence of H 2 0 2(Migler et al., 1978), a biochemical basis for the eosinophil defi-

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PETER F. WELLER AND EDWARD J. GOETZL

ciency was suggested by studies documenting that only the neutrophil peroxidase utilized chloride for this reaction and catalyzed the efficient generation of bactericidal products from amino acids (DeChatelet et al., 1978). That eosinophil peroxidase may have other roles has been indicated by studies of superoxide production. The ingestion of opsonized zymosan by eosinophils from eosinophilic subjects stimulated the rate of production of superoxide to a constantly high rate for over 3 hours, whereas the rate of production by phagocytosing neutrophils fell after 1 hour (Tauber et al., 1979). Inhibition of peroxidase activity by sodium azide significantly suppressed superoxide production by eosinophils, which suggested that eosinophil peroxidase contributed to superoxide generation. Enzymes that are preferentially contained in human eosinophils as compared to other leukocytes are present in the granules and in membrane structures. Arylsulfatase is localized predominantly in the small granules of eosinophils from several species, with lesser amounts in the crystalloid granules (Archer and Hirsch, 1963; Parmley and Spicer, 1974; Wasserman et al., 1975a), and exhibits type B characteristics (Wasserman et al., 1976). Type B arylsulfatase has a higher isoelectric point and lower molecular weight than type A, demonstrates normal first-order kinetics, and is uniquely susceptible to inhibition by pyrophosphate and sodium chloride. Phospholipase D is also present at higher levels in human eosinophils than in neutrophils and mononuclear leukocytes (Kater et al., 1976). Eosinophil phospholipase D has a molecular weight of 60,000 and an isoelectric point of 5.8-6.2 and cleaved choline from L-a-phosphatidylcholine with a pH optimum of 4.5-6.0. The lysophospholipase activity, which is selectively concentrated in eosinophils, is of approximately 32,000 MW by gel filtration and appears to be localized predominantly in the plasma membranes (Weller et al., 1978a). Some granule-associated proteins without apparent enzymic activity have been found exclusively or predominantly in eosinophils. A major basic protein of the core of crystalloid granules contains approximately 13%arginyl residues and has a molecular weight of 9200-11,000, depending on the species examined (Gleich et al., 1974; Gleich et al., 1976; Lewis et al., 1978). The abilities of major basic protein to precipitate with DNA, neutralize heparin, and activate papain appear to be based largely on its strong positive charge (Gleich, 1977). Other distinct cationic proteins (Olsson et al., 1977) and the Charcot-Leyden crystal protein (Gleich et al., 1976) have been extracted from eosinophil granules and purified. Consistent alterations in the serum levels of such eosinophil-derived proteins have been noted in some human diseases (Venge et al.,

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349

1977a,b, 1978), but these observations have not clarified their biological role. IV. General Functions of the Eosinophil

A. MODULATIONO F EOSINOPHILMIGRATION Although eosinophils are positioned predominantly in the tissues, their unique effector capabilities can be manifested fully only after directional influx and local accumulation at sites of specific reactions. Eosinophil chemotaxis, initiated by the presentation of a concentration gradient of a stimulus, and chemokinesis, evoked as a function of stimulus concentration irrespective of a gradient, are regulated by several immunological pathways (Goetzl, 1976) (Table I). Factors that are preferentially chemotactic and chemokinetic for eosinophils, as compared to other types of leukocytes, were elaborated when lymphocytes from animals and human subjects exhibiting delayed hypersensitivity were challenged in vitro with the homologous antigen. One such factor, termed the eosinophil stimulation promoter (ESP), was analogous to other lymphokines and was secreted in fully active form (Colley, 1973; Weller et al., 1978b). Specific antigen challenge of lymphocytes from sensitized guinea pigs resulted in the carrier-specific elaboration of an antigen-containing precursor macromolecule (ECF,) that becomes chemotactic for eosinophils when mixed with homologous IgGcontaining immune complexes (Torisu et al., 1973). Activation by immune complexes of the classical complement pathway and by microbial polysaccharides of the alternative complement pathway leads to the elaboration of fragments such as C5a (Kay et al., 1973) and complexes such as C567 (Lachmann et al., 1970), which attract eosinophils as well as other leukocytes with no apparent preference. In contrast, C3bBb of the alternative pathway is predominantly chemotactic for neutrophils (Ruddy et al., 1975). Although subacute and delayed immunological pathways can contribute some principles capable of enhancing eosinophil migration, immediate-type hypersensitivity reactions produce the broadest spectrum of factors that selectively influence the traffic of tissue eosinophils (Table I). IgE-dependent activation of fragments of guinea pig and human lung tissue releases from preformed stores an array of eosinophil chemotactic stimuli including histamine (Clark et al., 1975), low molecular weight peptide eosinophil chemotactic factors of anaphylaxis (ECF-A) (Kay et al., 1971; Kay and Austen, 1971), and intermediate molecular weight eosinophil chemotactic factors of

w

m 0

3r

TABLE I EOSINOPHILCHEMOTACTIC AND CHEMOKINETIC FACTORS GENERATED BY IMMUNOLOGICAL REACTIONS

Type of hypersensitivity response A. Immediate

Factor"

Structure or source

E C F-A tetrapeptides Intermediate MW ECF 12-HETE and 11-HETE

350-400 MW acidic tetrapeptides 1500-2500 MW acidic peptides Mast cell lipoxygenase products of arachidonic acid Mast cell cyclooxygenase product of arachidonic acid

LCF

Chemotactic activity

Ratio of optimal concentrations for chemotactic Chemodeactivation kinetic and chemotactic activity stimulation

+ +

0

10-3

0

lo-'

+

0

1

+

0

lo-'

r

M

9

i5U Other eosinophildirected activities Increase in number of C3b receptors

-

1

M

U

-b

Q

-

3r

-

Histamine

PGD,

B. Subacute

C5a

C. Delayed

C567 ESP ECF,

111 MW amine

Mast cell cyclooxygenase product of arachidonic acid 17,000 MW basic polypeptide 25,000 MW protein 65,000-70,000 MW antigen-con taining protein complex

+

0

+ + + +

0

+ 0

-

+

-

-

Inhibition of migration (HJ; enhancement of migration (HJ; increase in number of C3b receptors Inhibition of migration at high concentrations

iQz C

r

;Pr

lo-' -

-

ECF,, eosinophil chemotactic factor precursor; ECF-A, E C F of anaphylaxis; 11-and lBHETE, 11-and 12-~-hydroxyeicosatetraenoic acid; LCF, lipid chemotactic factor; PGD2, prostaglandin Dz; ESP, eosinophil stimulation promoter. Dash (-) = No data are available.

P B

v)

352

PETER F. WELLER AND EDWARD J . GOETZL

1500-2500 mw by gel filtration (Boswell et d., 1978). Human lung ECF-A is comprised in part of two acidic tetrapeptides (Goetzl and Austen, 1975), and the intermediate molecular weight factors consist of a family of peptides of varying acidity and hydrophobicity (Boswell et al., 1978). The challenge of mast cells also leads to the production and release of the lipoxygenase metabolites of arachidonic acid, 11hydr0xy-5,8,12,14-eicosatetraenoic acid ( 11-HETE), and 12-Lhydroxy-5,8,10,14-eicosatetraenoicacid (12-HETE), and diverse cyclooxygenase metabolites of arachidonic acid, of which prostaglandin D2 (PGD2)is the quantitatively predominant product (Roberts et al., 1978). PGD2 is a highly potent chemokinetic factor for eosinophils and, to a lesser extent, neutrophils (Goetzl et al., 1979). 12-HETE is chemotactic for PMN leukocytes, with a preference for eosinophils, and exhibits chemokinetic activity at concentrations below the peak for chemotaxis (Goetzl 1977; Goetzl and Gorman, 1978). IgG,directed stimulation of the mast cell-rich rat peritoneal cavity results in the elaboration of lipid chemotactic and chemokinetic factors that act on eosinophils and neutrophils, while IgE-specific stimulation yields only the lipid chemotactic activity (LCF) (Valone and Goetzl, 1978). The generation of this chemotactic activity is inhibited by the prior introduction of indomethacin, which suggests that arachidonic acid or a related fatty acid serves as the biosynthetic precursor. The principles that regulate eosinophil chemotaxis by altering the activity of the stimuli or the responsiveness of the eosinophils have been reviewed recently (Goetzl, 1976; Goetzl and Austen, 1976a). Of special interest is the phenomenon termed deactivation, by which chemotactic factors induce a state of unresponsiveness to subsequent chemotactic stimulation (Ward and Becker, 1970; Goetzl et al., 1974). The ratio of the concentrations required for peak deactivating and for chemotactic effects for each chemotactic stimulus varies from the ECF-A tetrapeptides to approximately 1 for 12-HETE and related lipid factors (Table I) (Goetzl and Austen, 1977). While the ability of the ECF-A tetrapeptides and histamine to elicit the selective accumulation of eosinophils has been confirmed in vivo (Bryant et al., 1977), the mechanism may not be chemotactic in nature. ECF-A induces predominantly chemotactic deactivation, rather than activation, i n uitro (Goetzl and Austen, 1976b), and histamine also exerts a major inhibitory effect in vitro by way of an H2 receptor-coupled increase in the eosinophil concentration of cyclic AMP (Clark et al., 1977). Recent evidence indicates that the inhibitory role of histamine may be amplified by a pathway involving histamine stimulation of mononuclear leukocytes, which results in the release of an eosinophil-

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353

immobilizing factor (Kownatzski et al., 1977). Thus, the predominant natural function of the ECF-A tetrapeptides and histamine may be to trap eosinophils attracted by other stimuli to mast cell-related inflammatory foci, while HETE and PGDz would continue to stimulate eosinophil random migration chemokinetically after dissipation of the concentration gradients. It is likely that functions of the eosinophil other than migration also will be altered by chemotactic factors, as it has been shown that C5a and formylmethionyl peptides alter neutrophil adherence in vitro and intravascular sequestration in vivo and result in neutrophil aggregation (O’Flaherty et al., 1978). Studies to the present have indicated that eosinophil-selective chemotactic factors affect the expression of some plasma membrane receptors, as will be discussed, and markedly stimulate the rate of production of lipoxygenase metabolites of arachidonic acid.

B. ENDOCYTOSIS AND ASSOCIATEDEVENTS As for other leukocytes of the PMN series, eosinophils engulf particles, form phagolysosomes, and undergo lysosomal degranulation (Archer and Hirsch, 1963; Zucker-Franklin et al., 1966; Cline et al., 1968; Cotran and Litt, 1969; Zeiger and Colten, 1977). Both phagocytosis and bactericidal reactions are less efficient for eosinophils than for neutrophils (Cline, 1972; Bujak and Root, 1974), and elevated levels of eosinophils offer little protection against bacterial infection in children with congenital neutropenia (Gilman et al., 1970). In some respects, endocytosis and related functions of eosinophils resemble more the comparable events in macrophages (Cohn, 1975) than in other PMN leukocytes. The development of increased numbers of vesiculotubular and other membranous structures within rat peritoneal eosinophils following an in vitro exposure to colloidal gold or fetal calf serum is indicative of prominent microendocytic activity (Fig. 1) (Simson and Spicer, 1973; Komiyama and Spicer, 1975). Progressive increases in the number of acid phosphatase- and arylsulfatase-positive small granules are seen late in the development and maturation of tissue eosinophils (Parmley and Spicer, 1974). The acquired flexibility of some eosinophil plasma membrane receptors, to be discussed subsequently, combined with the enhanced microendocytic activity and lysosomal adaptability of tissue eosinophils, provides a functional profile that is clearly distinct from that of neutrophils. Some aspects of the degranulation reactions of eosinophils exhibit unique characteristics as well. The phagocytosis of opsonized zymosan by eosinophils results in the release of histaminase, arylsulfatase

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PETER F. WELLER AND EDWARD J. GOETZL

B, and P-glucuronidase, while the calcium ionophore A23187 induces the selective release of histaminase alone (Zeiger and Colten, 1977). Cytochalasin B inhibits the release of histaminase from eosinophils, but not from neutrophils. Of the immune complexes that elicit the release of granule-associated enzymes from eosinophils, those containing IgE have the highest potency (Takenaka et al., 1976). The interactions of anti-IgE or a specific allergen with eosinophils results in the apparently preferential synthesis and release of prostaglandins E l and Ez (PGE, and PGEJ (Hubscher, 1975a,b), which implies that eosinophils possess receptors for IgE as well as the enzymes required for the release and metabolism of arachidonic acid (Samuelsson et al., 1978). V. Involvement of Eosinophils in Immunological Responses

A. ASSOCIATIONWITH

ANTIBODY PRODUCTION AND DELAYED HYPERSENSITIVITY REACTIONS Marked eosinophilia was noted in the cortical areas of regional lymph nodes that drained the sites of primary immunization of animals with protein or carbohydrate antigens (Cohen and Sapp, 1963; Litt, 1963; Litt, 1964a). The accumulation of eosinophils in the lymph nodes reached a peak level within 24 hours of the injection of antigen, lasted for 5-7 days after a single dose, and increased substantially with repeated doses of antigen. These and similar observations and the demonstration of small quantities of antigen within eosinophils (Roberts, 1966) initially suggested that eosinophils participate in the processing of antigen for presentation to macrophages and lymphocytes (Speirs, 1970). Despite early controversy, it now appears that the eosinophils localized in lymph nodes after immunization are engaged in the endocytosis of antigen-antibody complexes (Sabesin, 1963; Litt, 1964b), but are not required for an immune response (Mahmoud, 1977). While eosinophils may be intimately associated with macrophages and lymphocytes in a variety of delayed hypersensitivity reactions, antibodies frequently are responsible for the observed phenomena. The repeated injection of antigen into the same skin site of guinea pigs that previously had been immunized to establish delayed hypersensitivity led to accelerated reactions with increased numbers of eosinophils (Arnason and Waksman, 1963). An azobenzene arsenate conjugate of acetyltyrosine, which induced delayed hypersensitivity without antibody production, elicited similar accelerated cutaneous reactions that lacked significant eosinophilia (Leber, et al,, 1973).

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Marked peritoneal eosinophilia followed the primary and subsequent injections of Trichinella spiralis larvae in rats and, in both instances, achieved a peak level within 5 days (Walls et al., 1974). While the formation of eosinophil rosettes around macrophages within 48 hours of challenge suggested a possible role for macrophage-derived factors, the course of rosette formation coincided with the appearance of immunoglobulin on the surface of the macrophages, and such rosettes could be produced in uitro by adding eosinophils to normal macrophages that had been preincubated with immune complexes. Factors that are apparently selectively chemotactic for eosinophils have been generated in uiuo and in uitro in several lymphocytedependent systems (Torisu e t al., 1973; Kay e t al., 1975), but only the eosinophil stimulation promoter (ESP) (Colley, 1973) has been established to be a chemotactic and chemokinetic stimulus in the absence of antigen or antibody (Lewis et al., 1977). ESP is elaborated predominantly by T lymphocytes in mice, although macrophages are required for antigen-induced production (Greene and Colley, 1976); the lymphocyte source has not been established in other species. ESP is analogous to other lymphokines with respect to its molecular weight of approximately 25,000, susceptibility to inactivation by heating or proteolysis, stimulation of release by mitogens, and suppression of generation by inhibitors of protein synthesis (Greene and Colley, 1974; Weller et al., 197813). While any primary function for eosinophils in delayed-type hypersensitivity reactions remains to be elucidated, the recent demonstration that isolated schistosome egg granulomas produce ESP (James and Colley, 1975) supports the possibility that this and other immunologically mediated responses in part serve to recruit eosinophils, which augment the local capacity for defense.

B. EOSINOPHILRECEPTORSFOR IMMUNOGLOBULINS AND COMPLEMENT COMPONENTS As for other types of leukocytes, eosinophils possess plasma membrane recognition units for immunological reactants that facilitate the recognition and endocytosis of opsonized particles and cells by eosinophils and enhance the associated degranulation and cytotoxic activity. While generally present at lower levels than on neutrophils, receptors for IgG, C3b, C3d, and C4 have been demonstrated by rosetting techniques on eosinophils from normal and hypereosinophilic subjects (Gupta et al., 1976; Tai and Spry, 1976; Spry and Tai, 1976, 1977; Anwar and Kay, 1977a; Parillo and Fauci, 1978). As for monocytes and lymphocytes, C4 and C3b shared the same receptor on eosinophils, while C3d had a discrete receptor (Gupta et al., 1976). In

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PETER F. W L L E R AND EDWARD J. GOETZL

contrast to the C4-C3b receptor, the C3d receptor was expressed more strongly on eosinophils than on neutrophils. The release of lysosomal enzymes and the production of PGE by human eosinophils that have been incubated with immune complexes both attained the highest levels when complexes composed of IgE and anti-human IgE were employed (Hubscher, 1975a,b; Takenaka et al., 1977), but IgE receptors have not been demonstrated on eosinophils by direct techniques. The relative functional role of IgG and C3b receptors has been assessed in terms of the adherence of human eosinophils to schistosomula (Ottesen et al., 1977). In serum, the schistosomula activated the complement cascade predominantly through the alternative pathway and the C3b that was generated accounted for over 80% of the observed adherence of eosinophils, while IgG antibody mediated the remainder of the specific effect. A role for IgE receptors in this reaction could not be detected. Both complement receptors and receptors for homologous and heterologous IgG were increased in number on eosinophils of some patients with hypereosinophilic syndromes, and the extent of the increase was related generally to the level and duration of the eosinophilia (Spry and Tai, 1977; Parillo and Fauci, 1978). Such plasma membrane receptors of human eosinophils possessed an adaptive flexibility that was also demonstrable in uitro. Histamine and the ECF-A tetrapeptides, but not serotonin or bradykinin, increased by over 100% the density of C3b receptors on eosinophils without affecting those on neutrophils (Anwar and Kay, 197%). As serum IgE levels were elevated in a substantial number of hypereosinophilic subjects irrespective of the specific diagnosis (Parillo et al., 1979), mast cell- or basophil-mediated pathways may be involved in the modulation of expression of eosinophil receptors in a variety of human diseases in addition to allergic reactions.

c. MODULATIONOF IMMEDIATE HYPERSENSITIVITY REACTIONS Mast cell-mediated (Lowell, 1967; Felarca and Lowell, 1967) and parasitic diseases (Conrad, 1971) may be characterized by eosinophilic tissue infiltrates; factors that selectively attract eosinophils have been identified in some of these entities, including allergic rhinitis with nasal polyps (Kaliner et al., 1973), cold urticaria (Center et al., 1979), and invasive helminthic diseases, such as filariasis (Rubin et al., 1975) and schistosomiasis (James and Colley, 1975). Eosinophils generally appear soon after the onset of mast cell-mediated reactions. Occasionally a second wave of lesional eosinophils is appreciated several hours after challenge with antigen or anti-IgE, which is associated with a

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357

recurrence of other manifestations of immediate-type hypersensitivity responses, and lacks apparent involvement of complement proteins or lymphocytes (Pepys et al., 1968; Dolovich et al., 1972). The diverse mast cell-generated eosinophil chemotactic factors that elicit the early eosinophil response have not been detected in later lesions. However, a specific eosinophil chemotactic factor has been extracted from some delayed anaphylactic reactions and shown to be a protein of approximately 70,000 mw (Hirashima et al., 1976). The peak tissue concentrations of this factor occurred at the time of both the maximal delayed eosinophil response and the macrophage response, but the eosinophil-specific factor was separable chromatographically from the macrophage chemotactic activity. Irrespective of the specific time course of the hypersensitivity reaction, the eosinophils arrive after the humoral phase has been established. The immobilized eosinophils retain other cellular and metabolic functions, in addition to a full complement of lysosomal and other enzymes, which permit them to modulate the ongoing mast cell reaction b y inhibiting the release of chemical mediators and by inactivating some of the mediators that have been released (Austen et al., 1976) (Table 11).The incubation of human eosinophils with anti-human IgE serum stimulated the synthesis and release of both PGEl and PGE2, in quantities exceeding those released by stimulated neutrophils (Hubscher, 1975a,b). The mixture of PGEl and PGE2 inhibits the release of histamine from human basophils, and presumably from mast cells, by increasing the intracellular levels of cyclic AMP. As a further cellular mechanism for the inhibition of release or expression of TABLE I1 MODULATION OF IMMEDIATE-TYPE HYPERSENSITIVITY REACTIONS BY EOSINOPHILS~ Effect on mast cell mediators

Mechanism or specific mediator

Eosinophil function or constituent

Increase of CAMP in mast cell

Elaboration of PGE, and PGEz Phagocytosis

~~

1. Inhibition of release or

expression of mediators

2. Inactivation of mediators by nonenzymic factors 3. Enzymic degradation of mediators

Removal of extruded mast cell granules Heparin Histamine SRS-A PLF Lysophospholipids

Major basic protein His taminase Arylsulfatase B Phospholipase D Lysophospholipase

a PGE, and PGE,, prostaglandins E, and E,; PLF, platelet-lytic factor; SRS-A, slowreacting substance of anaphylaxis.

358

PETER F. WELLER AND EDWARD J. GOETZL

mediators of immediate-type hypersensitivity reactions, eosinophils apparently preferentially phagocytosed extruded mast cell granules (Mann, 1969), which avidly retain both macromolecular heparin and a cationic chymotrypsin-like protease that is active while still in the granule (Yurt and Austen, 1979). Nonenzymic factors and specific enzymes contained preferentially in the eosinophil are capable of inactivating some mast cell-derived mediators (Table 11).The major basic protein inactivated commercial heparin and the heparin in the mast cell extracts by a nonenzymic mechanism that is presumably related to a charge interaction (Gleich, 1977). Eosinophil major basic protein also increased the uptake and desulfation of heparin by macrophages (Fabian et al., 1978). Eosinophil histaminase, like that of the neutrophil, deaminated histamine with an extracellular pH optimum of 6-8 (Zeiger et al., 1976). A role for eosinophil arylsulfatase B in the modulation of mast cell reactions was suggested by the demonstration that the purified enzyme inactivates SRS-A from human and rat sources in a time- and dose-dependent manner (Wasserman et al., 1975b). That the arylsulfatase B activity was responsible for the inactivation of SRS-A was supported by the copurification of p-nitrocatechol sulfate-cleaving and SRS-A-inactivating activities and by the finding of an identical pH optimum for the two activities of the purified enzyme. Finally, doserelated inhibition of SRS-A inactivation by p-nitrocatechol sulfate and reciprocal inhibition of the cleavage of the preferred synthetic substrate by SRS-A confirmed that the two substrates were competing for the same active site (Wasserman et al., 1975b). Phospholipase D from eosinophil and cabbage sources substantially inactivated a crude platelet-active material, obtained by IgG,dependent stimulation of the mast cell-rich rat peritoneal cavity, as assessed by a decrease in its ability to release ['Clserotonin from prelabeled platelets (Kater et al., 1976). Two distinct platelet-active factors were subsequently resolved and freed of the SRS-A and lipid chemotactic factors present in the crude material by chromatography on diethylaminoethyl cellulose in organic solvents (Valone et al., 1979).A platelet-lytic factor, recognized by its ability to concomitantly release lactic acid dehydrogenase and ['4C]serotonin from platelets, was found in the chloroform :methanol (7 : 1,v :v) eluate from diethylaminoethyl cellulose, while the methanol : aqueous 1.0 M ammonium carbonate (1: 1, v : v) eluate contained a noncytotoxic plateletactivating factor (PAF). Purified eosinophil phospholipase D inactivated the platelet-lytic factor in a time-dependent reaction at pH 5.5, without affecting the activity of the PAF. As the activation of phos-

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pholipase AP generates lysolecithins as well as the polyunsaturated fatty acid precursors of prostaglandins and thromboxanes, it is not surprising that immunologically challenged mast cells release lysolecithin (Keller, 1962; Strandberg et al., 1975). Lysolecithin possesses a wide range of stimulatory and inhibitory cellular activities, including effects on adenylate and guanylate cyclase (Shier et al., 1976; Anderson and Jawarski, 1977) and prostaglandin synthetase (Shier, 1977), facilitation of cellular membrane fusion (Poole et al., 1970), and cytotoxicity at high concentrations (Reman et al., 1969). While the influence of eosinophil lysophospholipase on the biological effects of mast cell-derived lysolecithin remains to be elucidated, preliminary studies of the inhibition of inflammation by a bacterial lysosphospholipase (Modollel and Munder, 1972) project a significant involvement for this enzyme in the immunoregulatory profile of the eosinophil. Although human and other eosinophils thus possess the appropriate enzymic constituents to exercise a regulatory role in immediate-type hypersensitivity reactions, the cellular characteristics of their contribution are only presently being elucidated. The granular enzyme histaminase was released from eosinophils engaged in phagocytosis or exposed to the calcium ionophore A23187 (Zeiger and Colten, 1977). In addition to the fact that phagocytosing eosinophils released only a relatively small proportion of the total cellular content of arylsulfatase B (Goetzl et al., 1974; Zeiger and Colten, 1977), other evidence has suggested that SRS-A inactivation may be an intracellular event, as contrasted with the fluid phase inactivation of histamine b y histaminase. The inactivation of SRS-A by intact eosinophils achieved peak rates at pH levels of 7.2-7.4, occurred in the absence of release of arylsulfatase B, was more rapid and complete than that attained with the arylsulfatase B extracted from the same number of eosinophils, and was suppressed by metabolic inhibitors that did not affect the isolated enzymes (Wasserman e t al., 197513). While the intracellular localization of eosinophil phospholipase D and its mechanism of action in intact eosinophils are not known, a phospholipase D-like activity of comparable size has been found in sera of normal and eosinophilic patients, which may be identical to the previously described acid labile-plasma enzyme that inactivated a lipid PAF (Farr et al., 1978). Finally, the suppression of blood and tissue levels of eosinophils in guinea pigs b y prior treatment with specific antieosinophil sera markedly retarded the reaccumulation of histamine in skin sites following IgG,-mediated passive cutaneous anaphylaxis reactions (Jones and Kay, 1976). Thus, the eosinophil may also fulfill a role in the restora-

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tion of the integrity of the mast cell population following immediatetype hypersensitivity reactions.

D. ASSOCIATION WITH HUMANIMMUNODEFICIENCY DISEASES Peripheral blood and tissue eosinophilia are associated with some congenital and acquired alterations in T-lymphocyte function or immunoglobulin levels. Eosinophilia is found in patients with Swisstype (Hitzig et al., 1971) and sex-linked (Miller and Schieken, 1967) combined immunodeficiency, the Nezelof syndrome (Lawlor et al., 1974), and the Wiskott-Aldrich syndrome (Bach et al., 1968). Patients experiencing graft-versus-host reactions often develop tissue and blood eosinophilia (Kersey et al., 1971). Striking increases in the eosinophilia may be manifested by patients with either lymphocyte or immunoglobulin deficiencies in the course of Pneumocystis carinii infections (Burke and Good, 1973). Similarly, a patient with agammaglobulinemia developed eosinophilia with visceral larva migrans (Huntley and Costas, 1965). The eosinophilia observed in patients with the syndrome of hyperimmunoglobulinemia E and recurrent infections may represent an abnormal recruitment of mast cell pathways (Hill and Quie, 1974). In apparent contrast to other immunodeficiencies, spindle cell thymomas associated with hypogammaglobulinemia may be associated with eosinopenia or the complete absence of eosinophils (Waldmann et al., 1967). An absence of eosinophils and basophils and low serum levels of IgA and IgE in a single patient has been associated with repeated infections, asthma, hemolytic anemia, vasomotor rhinitis, alopecia, scabies, and warts (Juhlin and Michaelson, 1977). VI. The Role of the Eorinophil in the Host Response to Helminthic Infections

With the exception of the protozoan parasite Pneumocystis carinii, which may be associated with eosinophilia in immunodeficient patients (Burke and Good, 1973), it is metazoan parasites (nematodes, trematodes, and cestodes) that characteristically produce tissue and peripheral blood eosinophilia in the host. The most striking eosinophilia is generally found during the tissue-invasive or tissuemigratory phases of helminthic infections (Conrad, 1971), although increased levels of eosinophilia may be provoked by courses of antihelminthic chemotherapy (Ottesen and Weller, 1979). Eosinophil chemoattractants, presumably involved in evoking local accumulations of tissue eosinophils, may be released directly from helminths (Campbell, 1942; Tanaka and Torisu, 1978; Tanaka et al., 1979). The

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bulk of the eosinophil chemotactic activity generated in association with helminthic lesions, however, is believed to be a function of the host response that is mediated by immediate, subacute, and delayed hypersensitivity pathways. As documented in animal models of helminthic infections, specificallly sensitized T lymphocytes are critical to the stimulation of eosinophilopoiesis by helminthic antigens (Basten and Beeson, 1970) as well as to the production of an eosinophil chemokinetic and chemotactic lymphokine (ESP) (Colley, 1973; Lewis et al., 1977). Although some evidence suggests that the eosinophil plays a prominent protective role in the host response to a variety of helminths, as reviewed by Butterworth (1977), most of the data have been developed in relation to the trematode Schistosoma mansoni (Butterworth, 1977; Phillips and Colley, 1978; Colley and James, 1979). Two lines of evidence have emerged from in vivo studies of schistosomiasis that support the concept of a specific effector function for eosinophils (Fig. 3). First, schistosomula migrating in immune animals were damaged as they penetrated the skin and the histopathology of the infiltrates around these schistosomula demonstrated that eosinophils were much more prominent in hosts exhibiting specific immunity to schistosomes (Hsiiet al., 1971, 1974; von Lichtenberget al., 1976). Similarly, when schistosomula were introduced into the lungs without penetrating the skin, the rate of intrapulmonary death of the schistosomula and the intensity of the local eosinophil infiltration were greater in immune rats than in those not sensitized previously (von Lichtenberg et al., 1977). Second, the administration of apparently monospecific antieosinophil sera (Mahmoud et d . , 1973) to immune mice prior to reinfection with S. mansoni abolished the protective effect of active antischistosomal immunity. Similarly, the administration of an-

--

---

--c--

FIG. 3. Eosinophil-mediated cytotoxicity for helminthic parasites. IgC, immunoglobulin C; C3b, a fragment of the third component of complement; MBP, eosinophil major basic protein; Enz., enzymes; 02-, superoxide.

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tieosinophil serum to noninfected mice prior to initial infection abolished the passive immunity transferable with mouse antischistosomal serum (Mahmoud et al., 1975). In contrast, antineutrophil, antilymphocyte, and antimacrophage sera failed to alter the state of immunity. Thus eosinophils were selectively localized at the principal sites of schistosomal invasion, and the abolition of eosinophils resulted in abrogation of the heightened host resistance bestowed by both active and passive immunity. Additional data substantiating an effector role for eosinophils in immunity against helminths have come from in vitro studies. Butterworth and his colleagues (1974, 1976, 1977a,b) utilized the release of chromium-51 from prelabeled schistosomula as a measure of cytotoxicity to demonstrate the schistosomulicidal activity of human and baboon peripheral blood leukocytes. Leukocyte-mediated release of radiolabel was complement independent but required noncytophilic IgG opsonizing antibody. The cytotoxic damage was mediated by populations of eosinophils of up to 90% purity (Butterworth et al., 1977a), could be ablated by antieosinophil serum but not antineutrophil serum (Butterworth et al., 1975),and was not dependent on the presence of mononuclear leukocytes (Butterworth, 1977). The cytotoxic effect was inhibitable by the addition of immune complexes (Butterworth et al., 1977b), supporting the thesis that eosinophil adherence to schistosomula was mediated by Fc receptors (Mackenzie et al., 1977). More recent work (Vadas et al., 1979) has demonstrated that neutrophils induce release of %r at as high a level as eosinophils, but that eosinophils exhibited greater antibody-dependent adherence to schistosomula and mediated much more prominent morphological damage to the schistosomula than did neutrophils. Other studies have also demonstrated killing of schistosomula by neutrophils (Dean et al., 1974; Hsii et al., 1977) and by macrophages (Capron et al., 1975; Mahmoud et al., 1 9 7 8 ~ )The . nature of the respective contributions of these cells remains to be defined. Phase-contrast cinemicrographic (Densen et al., 1978) and electron microscopic studies of the interactions of human (Glauert and Butterworth, 1977) and rat (McLaren et aZ., 1977) eosinophils with schistosomula have indicated that intimate contact between eosinophils and schistosomula was followed by eosinophil degranulation (Fig. 3). The deposition of peroxidase-positive granule material onto the surface ofthe schistosomulawas followed by development ofmicroscopically apparent lesions. The prior addition of inhibitors of microfilament function, glycolysis, or esteratic activity impaired eosinophilmediated release of slCr from schistosomula (David et al., 1977). More

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recently, eosinophil major basic protein, which is released from eosinophil granules during the antibody-dependent interaction between human eosinophils and schistosomula, has been shown to damage schistosomula ofS. mansoni, an effect shared by other polycationic factors (Butterworth et al., 1979). In addition to the antibody-dependent schistosomulicidal activity of eosinophils, activation of the alternative pathway of complement on the surface of the schistosomula appeared to contribute to the eosinophil-mediated damage in infected rats. The deposition of C3b on schistosomula led to the adherence of rat eosinophils through specific C3 receptors, and damage to the subjacent surface of the schistosomula by the attached eosinophils (Ramalho-Pinto et aZ., 1978). In infected rats, eosinophils also exerted cytotoxic effects on schistosomula by way of a mast cell-dependent pathway initiated by specific IgG,, antibody and the mast cell potentiation was reproduced by the tetrapeptides of ECF-A (Capron et al., 1978a; Capron et al., 1978b). In the same model, IgE antibody-antigen complexes and macrophages manifested cytotoxicity for schistosomula at a time later than that of the peak eosinophil effect (Capron et al., 1975, 197813). Eosinophils not only have been implicated as effector cells in the immune defense against reinfection with schistosomula, but also may fulfill a similar role in resistance to other helminthic infections. For example, eosinophils were cytotoxic to larval stages of Trichinella spiraZis (Grove et al., 1977; Kazura and Grove, 1978; Perrudet-Badoux et al., 1978). As neutrophils express similar cytotoxic activity for Trichinella larvae in vitro (Bass and Szejda, 1979), the uniqueness of the in vivo contribution of the eosinophils requires further definition. Eosinophil-mediated cytotoxicity has also been demonstrated in vitro for epimastigotes of the protozoan Trypanosoma cruzi (Sanderson et al., 1977) and for avian and mammalian cells (Sanderson and Thomas, 1978; Parillo and Fauci, 1978; Van Epps and Bankhurst, 1978). In addition to the cytotoxic actions of eosinophils that are directed against larval schistosomula, the abundance of eosinophils in schistosome egg-induced hepatic granulomas have suggested an additional role with respect to mediating the immunopathogenetic responses to the eggs (Colley and James, 1979). Eosinophils from animals immune to schistosomiasis and normal eosinophils activated b y incubation with immune serum or with ESP containing medium were capable of destroying S . mansoni eggs (James and Colley, 1976, 1978a,b,c). Isolated egg-induced hepatic granulomas secreted chemoattractant ESP (James and Colley, 1975), and at times eosinophils constituted 25-50% of the cells in the egg-induced granulomas (Mahmoud et al.,

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1975). The administration of antieosinophil serum resulted in reduction both of the size of the granulomas and of their eosinophil content (Mahmoudet al., 1975). Since hepatic granulomas are the source ofthe most severe and irreversible pathology in schistosomiasis (Warren, 1972), appropriate modulation of the function of the eosinophils may be a central determinant in the response that either destroys the parasite or contributes to host-tissue injury and fibrosis. VII. Concluding Remarks

Eosinophils differ from other leukocytes in their genesis and maturation, their morphology and biochemistry, and their functional abilities. Eosinophils possess apparently unique capabilities to modulate immediate-type hypersensitivity reactions by virtue of specific biochemical constituents that degrade mast cell-derived mediators. Specific receptors and cytotoxic mechanisms may endow eosinophils with the capacity to serve as effector cells in immunologically mediated host resistance to infections with helminths. Of special interest is the evidence that points to the existence of functionally discrete subpopulations of eosinophils, a subject worthy of further investigative effort. The most intriguing evidence for two developmentally distinct subpopulations of eosinophils derives from the finding in bone marrow cultures of two types of progenitor cells for human eosinophils that were separable by velocity sedimentation (Johnson et al., 1977) and from the demonstration of the existence of separate short- and long-lived cohorts of circulating human eosinophils (Parwaresch et al., 1976). The distribution of eosinophils migrating through micropore filters in response to chemotactic agents in vitro is far more heterogeneous than that of neutrophils, a finding that is compatible also with the presence of distinct subpopulations of eosinophils exhibiting differential chemotactic responses to the same chemoattractants (Clark et

al., 1975). The major unsolved question that is raised by these findings, as well as by the heterogeneity of enzyme content (Parmley and Spicer, 1974) and receptor expression among eosinophils (Tai and Spry, 1976), is whether the observed subpopulations are a consequence of inherent developmental differences or are acquired as a result of subsequent differential maturation, or occur secondary to stimulation and activation of the eosinophils. Eosinophils are distributed principally in tissues where they contain more small granules rich in acid phosphatase and arylsulfatase than do blood eosinophils (Parmley and Spicer, 1974). Although normal tissue eosinophils may differ biochemically

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and functionally from the less mature blood eosinophils, the relationship of such differences to the involvement in tissue responses has not been elucidated. The genesis of grossly hypogranulated and vacuolated eosinophils observed in allergic (Connell, 1968), parasitic (Saran, 1973), and idiopathic (Spry and Tai, 1976) hypereosinophilic states and possibly representing functionally “spent” cells is similarly unclear. Further studies of the cellular biology, immunology, and clinical abnormalities of eosinophils will be required to unravel the factors that regulate their protective functions and potential for host-tissue damage.

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Subject Index Antisera characterization of functional, 200 further characterization, 200-2 10 genetic analysis, 195-199 tissue distribution, 199-200 production and testing of, 187-188 contaminating antibodies, 193-195 methods of detection, 192-193 production of alloantisera, 188-192 Autoimmune diseases, anti-receptor, other, 42-43

A

Acetylcholine receptor amount effect of antigenic modulation on,

26-27 effect of complement on, 25-26 autoimmune response to, in human myasthenia gravis antibodies to AChR, 33-36 cause of response, 3 9 4 2 pathological mechanisms impairing transmission, 36-39 function, effect of bound antibody on,

B

24-25 immunization with, 14-17 molecular properties biochemical, 6-9 electrophysiological, 5-6 synthesis and destruction, 9-11 Alloantigenic determinants, classification of, 182-187 Alloantigenic loci, erythrocyte, 254-258 Antibody bound, AChR function and, 24-25 production, eosinophils and, 354-355 Antigens miscellaneous F antigen, 259-260 H-Y locus, 263-264 Ly-X loci, 265-266 Mph-1 (macrophage-1) locus, 258-259 NK specificity, 264-265 Sk-l andSk-2 loci (Sk = skin), 260-261 T complex, 261-262 target conclusion, 95-96 evidence for interaction of self-H and foreign antigens, 91-93 nature of restricting self-MHC determinants, 74-80 nature of virally induced antigens, 80-9 1 special case of alloantigens, 93-95 Antigenic modulation, amount of AChR and, 2 6 2 7

Bone marrow, eosinophil production in, 340-34 1 c

Cell membrane alloantigenic determinants B-cell differentiation and ontogeny,

373

309-3 10 CMAD of antibody-forming cells, 3 12-3 13 CMAD of B cells, 310-312 ontogeny of CMAD and receptors on B cells, 313 stem cells: B stem cell, the immature B cell, 310 expression on mouse leukemias and lymphomas, 313-315 chemically induced T lymphomas,

315 conclusion, 320 in oitro-maintained T-cell lines,

315-316 mastocytomas, 319 other tumors, 319 other T-cell lymphomas, 316-317 phenotype of Abelson virus-induced B-cell tumors, 318 plasmacytomas and other B-cell tumors, 318-319 radiation-induced T thymomas, 317

374

SUBJECT INDEX

virus-induced leukemias and lymphomas, 317-318 T-cell ontogeny and differentiation differentiation in periphery, 307-308 differentiation in thymus, 306-307 ontogeny, 303-305 other CMAD in, 308-309 prothymocyte and, 305-306 summary, 309 Chimeras, MHC incompatible, 105-106 Complement amount of AchR and, 25-26 eosinophil receptors for, 355-356 Cytotoxic T-cells MHC-restricted, in uioo relevance, 128-129 conclusion, 140-141 immune protection, 129-132 MHC-associated diseases, 139-140 MHC polymorphism, 135-139 T-cell-mediated immunopathology, 132- 135 virus-specific characterization of effector cells, 67-69 evidence for MHC restriction in other species and outbred populations, 69-72 generation of effector cells and assay, 58-67 models for recognition by effector T cells, 72-74 D

Delayed hypersensitivity, eosinophils and, 354-355 E

Effector cells ontogeny, 96-97 conclusion, 109 differentiation of T-cell restrictionspecificity, 97-104 MHC incompatible chimeras, 105106 negative selection experiments, 106-109

role of lymphohemopoietic cells in T-cell maturation and antigen presentation, 104-105 Endocytosis, eosinophils and, 353-354 Eosinophil cellular properties, 347-349 general functions endocytosis and associated events, 353-354 modulation of migration, 349-353 involvement in immunological responses association with antibody production and delayed hypersensitivity reactions 354-355 human immunodeficiency disease and, 360 immediate hypersensitivity reactions and, 356-360 receptors for immunoglobulins and complement components, 355-356 production in bone marrow, 340-341 immunological control, 341-345 release, tissue distribution and fate, 345-346 role in host response to helminthic infections, 360-364 Erythrocyte, alloantigenic (EA) loci, 254258 H

Helminthic infections, host response, role of eosinophils in, 360-364 Histocompatibility (H) loci cell membrane alloantigenic determinants of general distribution H-2 CMAD (H3K, H-2D, H-2G, H-2L), 201-202 Hh-1, 204 non-H-2 loci, 202-204 Human, antibodies to AChR in, 33-36 I

Immediate hypersensitivity, eosinophils and, 356-360 Immunodeficiency disease, human, eosinophils and, 360

SUBJECT INDEX Immunoglobulins, eosinophil receptors for, 355-356 Immunopathology, T cell-mediated, 132-135 Ir genes, evidence for MHC-coded, regulating expression of cytotoxic T-cells, 110-115 1

Lymphocyte alloantigens cell membrane alloantigenic determinants of general distribution ALA-1 IOCUS, 233-234 chemistry of CMAD, 254 H-2t, 247-248 la loci, 248-253 Ly-1 locus (Lyt-l),219-222 Ly-2 and Ly-3 loci, 222-226 Ly-4 ~ O C U S(Lyb-I),226-228 LY-5 locus ( L Y - ~ ) 228-229 , Ly-6 IOCUS, 229-233 Ly-7 IOCUS, 234-236 Ly-8 locus, 236 Ly-b specificities, 240-247 LyM-1 ~OCUS,236-240 other specificities, 253-254 Qa-1 IOCUS, 216-218 Qa-2 and Qa-3 loci, 218-219 Thy-1 IOCUS, 205-210 Tla locus, 210-216 Lymphohemopoietic cells, role in T-cell maturation and antigen presentation, 104-105 M

Major histocompatibility gene complex associated diseases, 139-140 polymorphism, 135-139 restriction, interpretation and It- regulation of T-cells, 118-128 role in defining T-cell specificity and responsiveness druing ontogeny, 96-97 conclusion, 109 differentiation of T-cell restrictionspecificity, 97-104 MHC incompatible chimeras, 105106

375

negative selection experiments, 106-109 role of lymphohemopoietic cells in T-cell maturation and antigen presentation, 104-105 role in determining T-cell responsiveness, 109-110 conclusion, 118 evidence for MHC-coded Zr genes regulating expression of cytotoxic T-cells, 110-115 influence of thymic selection of T-cell restriction specificities on responsiveness during T-cell ontogeny, 115-118 Murine leukemia virus, relationship to cell membrane alloantigenic determinants, 272-273 antigens induced by virus, 277 classification of MuLV, 275-277 conclusion, 283 expression of viral antigens, 273-275 expression of viral antigens after lymphocyte activation, 283 structural viral components as CMAD, 277-281 virus-related antigens, 281-283 Myasthenia gravis clinical features of, 11-13 experimental autoimmune in other species mice, guinea pigs, goats, monkeys and frogs, 31-33 rabbits, 27-31 Myasthenia gravis experimental autoimmune in rats acute and passive, 17-22 chronic, 22-24 effect of antigenic modulation on amount of AChR, 26-27 effect of bound antibody on AChR function, 24-25 effect of complement on amount of AChR, 25-26 immunization with AChR, 14-17 human, autoimmune response to AChR antibodies to AChR, 33-36 cause of response, 3 9 4 2 pathological mechanisms impairing transmission, 36-39

376

SUBJECT INDEX 5

Serological markers functional studies with, 283-288 cytotoxic T cells, 288-290 host-versus-graft and graft-versus-host reactions, 299 Ly phenotype o f T cells in the production of eosinophilia, 301-302 mixed-lymphocyte reaction, 297-298 phenotype of cells involved in MIF production, 301 phenotype of cells undergoing blast cell transformation, 299-301 phenotype of helper T cells for antibody production, 290-292 phenotype of T cells involved in antibody suppression, 292-294 phenotype of T cells involved in suppression of cell-mediated responses, 294-296 phenotype of T cells involved in delayed-type hypersensitivity,

296297 summary, 302-303 1

T-cell, restriction-specificity, differentiation of, 97-104 Thymic selection, of T-cell restriction specificities, influence on responsiveness during T-cell ontogeny,

115-118

Transmission impairment, pathological mechanisms of, 3 6 3 9 neuromuscular, 3-5 X

Xenoantisera recognizing lymphocyte cell-membrane determinants, 266267 antibodies to LPS receptor on B cells,

27 1 antilymphocyte sera (ALS), 267 antisera to purified T cell preparations, 269 brain-associated theta (BAB), 268 ML-2 antigen, 271 mouse-specific bone marrow-derived lymphocyte antigen, 270 mouse-specific lymphocyte antigen,

267 mouse-specific peripheral lymphocyte antigen, 268 mouse-specific plasma cell antigen,

271 mouse thymus-derived lymphocytespecific surface antigen, 267-268 other sera detecting B-cell xenoantigenic specificities, 270 thymocyte-B lymphocyte antigen,

269-270 xenoantisera-detecting determinant present on killer T cells, 269 xenogeneic anti-Ia serum, 272

CONTENTS OF PREVIOUS VOLUMES

Volume 1

Antibody Production by Transferred Cells

CHARLES C . COCHRANE AND FRANK J. D ~ X O N

Transplantation Immunity a n d Tolerance

M . HASEK, A. T. HRABA

LENCEROVA,

AND

Phagocytosis

DERRICK ROWLEY

Immunological Tolerance o f Nonliving Antigens

Antigen-Antibody Reactions i n Helminth Infections

RICHARDT. SMITH

E. J. L . SOULSBY

Functions o f the Complement System

ABRAHAMG. OSLER

Embryological Development of Antigens

REED A. FLICKINGER

In Vifro Studies of the Antibody Response

ABRAM B. STAVITSKY

AUTHOR INDEX-SUBJECT INDEX

Duration o f Immunity i n Virus Diseases

J. H . HALE Volume

Fate a n d Biological Action o f Antigen-Antibody Complexes

In Vitro Studies o f the Mechanism o f Anaphylaxis

WILLIAM 0. WEICLE Delayed Hypersensitivity to Simple Protein Antigens

P. G . H .

C E L L AND

3

B. BENACERRAF

The Antigenic Structure o f Tumors

K. FRANK AUSTEN AND JOHN H . HUMPHREY The Role o f Humoral Antibody i n the Homograft Reaction

CHANDLER A. STETSON

P. A. CORER

Immune Adherence

AUTHOR INDEX-SUBJECT INDEX

D. S. NELSON Reaginic Antibodies Volume 2

D. R. STANWORTH Nature of Retained Antigen a n d i t s Role i n Immune Mechanisms

Immunologic Specificity a n d Molecular Structure

DANH . CAMPBELL A N D JUSTINE S. GARVEY

FREDKARUSH Heterogeneity o f y-Globulins

JOHN L. FAHEY

Blood Groups i n Animals Other Than M a n

W. H. STONEA N D M . R. IRWIN

The Immunological Significance o f the Thymus

Heterophile Antigens a n d Their Significance i n the Host-Parasite Relationship

J . F. A. P. MILLER, A. H. E. MARSHALL.A N D R. c. WHITE

C. R . JENKIN

Cellular Genetics of Immune Responses

AUTHOR INDEX-SUBJECT INDEX

C.. J . V. NOSSAL

377

378

C O N T E N T S OF PREVIOUS V O L U M E S

Volume 4

Volume 6

Ontogeny a n d Phylogeny of Adaptive Immunity

Experimental Glomerulonephritis: Immunological Events a n d Pathogenetic Mechanisms

ROBERT A. GOOD AND BEN W. PAPERMASTER

EMILR. UNANUE AND FRANK J. DIXON

Cellular Reactions i n Infection

EMANUEL SUTER AND HANSRUEDYRAMSEIER Ultrastructure of Immunologic Processes

Chemical Suppression of Adaptive Immunity

ANN E . GABRIELSON ROBERT A. GOOD

AND

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

MACLYNMCCARTYAND STEPHEN I. MORSE Structure and Biological Activity of Immunoglobulins

SYDNEYCOHENA N D RODNEYR. PORTER Autoantibodies a n d Disease

H . G . KUNKELAND E. M. TAN Effect of Bacteria a n d Bacterial Products on Antibody Response

J. MUNOZ AUTHORINDEX-SUBJECT INDEX

Nucleic Acids a s Antigens OTTO J. PLESClA A N D

WERNER BRAUN In Vitro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DUTTON Developmental Aspects of Immunity

JAROSLAVSTERZL AND ARTHUR M. SILVERSTEIN Anti-anti bodies

PHILIPG . H . C E L L AND ANDREW S. KELUS Conglutinin a n d lmmunoconglutinins

P. J. LACHMANN AUTHOR INDEX-SUBJECT INDEX

Volume 5 Natural Antibodies a n d the Immune Response

STEPHEN V. BOYDEN lmmunological Studies with Synthetic Polypeptides

MICHAELSELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIP Y. PATERSON The Immunology of Insulin G . POPE

c.

Tissue-Specific Antigens

D . C. DUMONDE AUTHORINDEX-SUBJECT INDEX

Volume 7 Structure a n d Biological Properties of Immunoglobulins

SYDNEYCOHEN A N D CESAR MILSTEIN Genetics of Immunoglobulins i n the Mouse

MICHAEL POTTER AND ROSE LIEBERMAN Mimetic Relationships between Group A Streptococci a n d Mammalian Tissues

JOHN B. ZABRISKIE Lymphocytes a n d Transplantation Immunity

DARCYB. WILSON AND R. E . BILLINCHAM

379

CONTENTS OF PREVIOUS VOLUMES Human Tissue Transplantation

Phylogeny of Immunoglobulins

JOHN P. M E R R ~ L L

HOWARD M. GREY

AUTHOR INDEX-SUBJECTINDEX

Slow Reacting Substance o f Anaphylaxis ROBERT P. ORANGE AND

K. FRANK AUSTEN Volume 8 Chemistry a n d Reaction Mechanisms o f Complement

HANSJ. MULLER-EBERHARD Regulatory Effect o f Antibody on the Immune Response JONATHAN UHR AND

w.

GORAN MOLLER The Mechanism of Immunological Paralysis

D. w.DRESSERAND N. A. MITCHISON

OSCARD. RATNOFF Antigens o f Virus-Induced Tumors

KARL HABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS AUTHOR INDEX-SUBJECT

In Vitro Studies o f Human Reaginic Allergy

ABRAHAMC . OSLER, LAWRENCEM. LICHTENSTEIN, A N D DAVIDA. LEVY AUTHOR INDEX-SUBJECT

Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, a n d the Inflammatory Response

INDEX

INDEX

Volume 11 Electron Microscopy of the lmmunog lobul ins

N . MICHAEL GREEN Genetic Control o f Specific Immune Responses HUGH 0. MCDEVITT AND

Volume 9 Secretory lmmunag l o b u l i ns

THOMAS B. TOMASI, JR., AND JOHN BIENENSTOCK Immunologic Tissue Injury M e d i a t e d by Neutrophilic leukocytes

CHARLES G . COCHRANE The Structure and Function of Monocyter a n d Macrophages

ZANVILA. COHN The Immunology a n d Pathology o f N Z B Mice

J. B. HOWIE A N D B. J . HELYER AUTHOR INDEX-SUBJECTINDEX

BARUJBENACERRAF The lesions i n C e l l Membranes Caused by Complement JOHN H. HUMPHREY AND

ROBERT R. DOURMASHKIN Cytotoxic Effects of lymphoid Cells I n Vitro

PETERPERLMANNAND GORAN HOLM Transfer Factor

H. S. LAWRENCE Immunological Aspects o f Malaria Infection

IVOR N. BROWN AUTHOR INDEX-SUBJECTINDEX

V o l u m e 10 Volume 12 Cell Selection by Antigen i n the Immune Response GREGORY SlSKlND AND

w.

BARUJBENACERRAF

The Search for Antibodies with Molecular Uniformity

RICHARDM.

&MUSE

380

CONTENTS OF PREVIOUS VOLUMES

Structure a n d Function o f yM Macroglobulins

HENRYM ETZCER Transplantation Antigens

R. A. REISFELTAND B. D. KAHAN The Role o f Bone Marrow in the Immune Response

NABIHI. A B ~ AND U MAXWELLRICHTER Cell Interaction i n Antibody Synthesis

D. W. TALMACE, J. h D O V I C H , A N D H. HEMMINCSEN The Role o f lysosomes in Immune Responses AND GERALDWEISSMANN PETER DUKOR

Molecular Size a n d Conformation of Immunoglobulins

KEITH J. DORRINCTON AND CHARLES TANFORD

Volume 14 lmmunobiology o f Mammalian Reproduction

ALAN E. BEER AND

R. E. BILLINCHAM Thyroid Antigens and Autoimmunib

SIDNEYSHULMAN Immunological Aspects o f Burkitt's lymphoma

GEORGEKLEIN Genetic Aspects o f the Complement System

CHESTER A. ALPER FREDS. ROSEN

AND

The Immune System: A Model for Differentiation i n Higher Organisms

L. HOOD AND J. PRAHL AUTHOR INDEX-SUBJECTINDEX

AUTHOR INDEX-SUBJECTINDEX Volume 15 Volume 13 Structure a n d Function of Human Immunoglobulin E

HANSBENNICHA N D S. GUNNAR 0.JOHANSSON Individual Antigenic Specificity of Immunoglobulins

JOHNE. HOPPER AND ALFRED NISONOFF In Vitro Approaches to the Mechanism o f Cell-Mediated Immune Reactions

The Regulatory Influence of Activated T Cells on B Cell Responses t o Antigen

DAVIDH. KATZ A N D BARUJBENACERRAF The Regulatory Role o f Macrophages i n Antigenic Stimulation

E. R. UNANUE Immunological Enhancement: A Study of Blocking Antibodies

JOSEPHD. FELDMAN

BARRYR. BLOOM Immunological Phenomena i n leprosy and Related Diseases

J. L. TURKAND A. D. M. BRYCESON Nature a n d Classification o f Immediate-Type Allergic Reactions

ELMERL. BECKER AUTHOR INDEX-SUBJECTINDEX

Genetics a n d Immunology of Sex-linked Antigens

DAVIDL. GASSERAND WILLYSK. SILVERS Current Concepts of Amyloid

EDWARDC. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN AUTHOR INDEX-SUBJECTINDEX

381

C O N T E N T S OF PREVIOUS V O L U M E S Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, a n d ldiotypes

J. B. NATVIGAND H . G . KUNKEL Immunol o g i c a I Un responsiven ess

JEAN-CHARLESCEROTTINI K. THEODORE BRUNNER

AND

Antigenic Competition: A Review o f Nonspecific Antigen-Induced Suppression

HUGH F.P ~ o s AND s DAVIDEIDINGER

WILLIAM0. WEIGLE Participation of lymphocytes i n Viral Infections

E. FREDERICK WHEELOCK STEPHEN T. TOY

Cell-Mediated Cytotoxicity, Allograft Rejection, and Tumor Immunity

AND

Immune Complex Diseases in Experimental Animals a n d Man

C. G. COCHRANE AND D . KOFFLER The lmmunopathology o f Joint Inflammation i n Rheumatoid Arthritis

Effect of Antigen Binding on the Properties o f Anti body

HENRYMETZCER lymphocyte-Mediated Cytotoxicity a n d Blocking Serum Activity to Tumor Antigens

ERIK H E L L S T R ~ M AND INGEGERDH E L L S T R ~ M

KARL

AUTHOR INDEX-SUBJECTINDEX

NATHANJ. ZVAIF,LER AUTHOR INDEX-SUBJECTINDEX Volume 19 Molecular Biology o f Cellular Membranes with Applications t o Immunology

Volume 17 Anti Iym phocyte Seru m

S. J. SINGER EUGENE M. LANCE, P. B. MEDAWAR, Membrane Immunoglobulins a n d Antigen A N D ROBERT N. TAUB Receptors on B a n d T lymphocytes

In Vitro Studies of Immunologically Induced Secretion o f Mediators from Cells a n d Related Phenomena

ELMERL. BECKER A N D PETER M. HENSON

Receptors for Immune Complexes on lymp hocytes

VICTOR NUSSENZWEIG

Antibody Response t o Viral Antigens

KEITH M . COWAN

Biological Activities af Immunoglobulins of Different Classes and Subclasses

HANS L. SPIEGELBERG

Antibodies t o Small Molecules: Biological a n d Clinical Applications

VINCENT P. BUTLER, JR., SAM M. BEISER

NOEL L. WARNER

SUBJECTINDEX

AND

AUTHOR INDEX-SUBJECTINDEX

V o l u m e 20

Volume 18

Hypervariable Regions, Idiotypy, a n d Antibody-Combining Site

Genetic Determinants o f Immunological Responsiveness

DAVIDL. GASSER A N D WILLYS K. SILVERS

J. DONALD CAPRA AND J. MICHAEL KEHOE Structure a n d Function o f the J Chain

MARIAN ELLIOTT KOSHLAND

382

CONTENTS O F PREVIOUS VOLUMES

Amino Acid Substitution a n d the Antigenicity of Globular Proteins

MORRISREICHLIN The H-2 Major Histocompatibility Complex a n d the I Immune Response Region: Genetic Variation, Function, a n d Organization

DONALDc. SHREFFLER A N D CHELLAS . DAVID

Delayed Hypersensitivty i n the Mouse

ALFRED J . CROWLE

Cellular Aspects of Immunoglobulin A

MICHAELE. LAMM Secretory Anti-Influenza Immunity YA.

s. SHVARTSMAN AND

M. P. ZYKOV SUBJECTINDEX Volume 23 Cellular Events i n the IgE Antibody Response

KIMISHICE ISHIZAKA

SUBJECTINDEX

Chemical a n d Biological Properties of Some Atopic Allergens

T. P.

Volume 21 X-Ray Diffraction Studies of Immunoglobulins

ROBERTOJ. POLJAK Rabbit Immunoglobulin Allotypes: Structure, Immunology, a n d Genetics

THOMAS J. ~

N D T

Cyclical Production of Antibody a s a Regulatory Mechanism i n the Immune Response

WILLIAM0. WIECLE Thymus-Independent B-Cell Induction and Pa ralysis ANTONIO COUTINHO AND GdRAN MBLLER

SUBJECTINDEX

Volume 22 The Role of Antibodies in the Rejection a n d Enhancement of Organ Allografts

CHARLESB. CARPENTER, ANTHONY J. F.D'APICE, AND ABUL K. ABBAS Biosynthesis of Complement

HARVEY R. COLTEN Graft-versus-Host Reactions: A Review

STEPHENC. GREBEAND J. WAYNESTHEILEIN

KING

Human Mixed-Lymphocyte Culture Reaction: Genetics, Specificity, a n d Biological Implications

B o DUPONT,JOHN A. HANSEN, EDMONDJ. YUNIS

AND

lmmunochemical Properties of Glycolipids a n d Phospholipids

DONALDM. MARCUSAND GERALDA. SCHWARTING SUBJECTINDEX Volume 24 The Alternative Pathway of Complement Activation

0. G ~ T Z E AND H. J . MULLER-EBERHARD Membrane a n d Cytoplasmic Changes i n B lymphocytes Induced by LigandtSutface Immunoglobulin Interaction

GEORGE F. SCHREINER EMIL R. UNANUE

AND

Lymphocyte Receptors for Immunoglobulin

HOWARDB. DICKLER Ionizing Radiation and the Immune Response

ROBERTE.ANDERSON A N D NOEL L. WARNER SUBJECTINDEX

CONTENTS OF PREVIOUS VOLUMES Volume 25

Volume 26

Immunologically Privileged Sites

Anaphylatoxins: C3a a n d C5a

F. BARKER AND R. E. BILLINGHAM

CLYDE

Major Hirtocompotibility Complex Restricted Cell-Mediated Immunity

M. SHEARER AND ANNE-MARIE SCHMITT-VERHULST GENE

Current Status of Rat lmmunogeneticr

DAVIDL. GASSER Antigen-Binding Myeloma Proteins of Mice

MICHAEL POTTER Human lymphocyte Subpopulations

L. CHESSAND S. F. SCHLOSSMAN SUBJECTINDEX

383

TONYE. HUGLIAND HANSJ. M~LLER-EBERHARD H-2 Mutations: Their Genetics a n d Effect on Immune Functions

JAN KLEIN The Protein Products of the Murine 17th Chromosome: Genetics a n d Structure

ELLENS. VITETTA AND J. DONALD CAPRA

Expression and Function of Idiotypes an lymphocytes

K. EICHMA" The B-Cell Clonotype Repertoire NOLAN H. SIGNALAND NORMANR.

KLINMAN SUBJECT INDEX

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