Midwinter Conference of Immunologists

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Midwinter

Conference

A. MALLEY, Oregon

Regional

14, 143-156

Primate

of Immunologists

N. WARNER, Research Beaverton. Received

(1979)

AND A. NISONOFF

Center, Oregon May

505 Northn,est 97005

185th Avenue.

2. 1979

The Eighteenth Midwinter Conference of Immunologists was held January 20 through 23, 1979, at the Asilomar Conference Grounds, Pacific Grove, California, with Noel Warner and Al Nisonoff as cochairs. The subject of the conference, “Cell Structure, Signals, and Communications,” was discussed during five halfday sessions by invited speakers. In addition, there was a poster session involving 20 participants, who discussed a variety of topics related to the main theme of the conference. The Fifth Annual Dan H. Campbell Memorial Lecture was given by S. J. Singer (University of California at San Diego). His lecture was preceded by a few introductory remarks by George Feigen (Stanford University), a close associate and friend of Dr. Campbell for many years. A. Nisonoff (Brandeis University) introduced Dr. Singer, whose topic for the opening talk was “Molecular Interactions in Membranes.” Dr. Singer pointed out that the activation of different types of cells by the binding of ligands such as antigens, mitogens, hormones, and growth factors to specific receptors in the cell membrane is an important phenomenon in cell biology, and in cell immunology in particular. Little is known, however, about the molecular events that occur after this binding and that ultimately result in activation. This is no doubt in part due to our present incomplete understanding of molecular interactions in membranes and their consequences. For example, there are many indications that after the binding of ligands a clustering of the specific receptors in the membrane is induced. What does such a clustering of receptors achieve? In studies that have been carried out in his laboratory and those of others it appears that the antibody- or lectin-induced clustering of specific receptors generally leads to a linkage of the clusters across the membrane to cytoskeletal structures containing actin, myosin, a-actinin, and perhaps other proteins. This transmembrane linkage may be an important event in the activation process, perhaps as the event that is required for endocytosis of the ligand-receptor complex. This event apparently is also responsible for triggering the process of capping. Another type of molecular interaction in the fluid membrane may be an important factor in the mechanism of immune T-cell cytotoxicity. The fact that such cytotoxicity is histocompatibility restricted has led to the notion that certain antigens form a stable molecular complex with histocompatibility antigens to form a

143 OWO-1229179/090143-14$01.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved

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new recognition structure (altered self). On the other hand, complexes may form in a different manner. The relevant antigen, having been itself clustered, may bind histocompatibility antigens, and this type of association may be an important means of concentrating the histocompatibility antigen into the region of contact between the immune T cell and the target cell. Evidence for such a cluster-induced association with H-2 antigens was presented. The first session of the conference, convened by Al Nisonoff (Brandeis University), dealt with supermolecular aspects of membrane structure. Dr. Thomas P. Stossel (Massachusetts General Hospital), the first speaker, talked about mechanisms of action of contractile proteins during phagocytosis. Actin, myosin, and actin-binding protein (ABP) have been purified from macrophages and characterized. The fact that these proteins are concentrated in the cell periphery and move into pseudopods supports a role for contractile proteins in the mechanism of phagocytosis by macrophages. As in other movements, phagocytosis requires (1) force generation, (2) orientation of force, and (3) control mechanisms. As in muscle, cyclic actin - myosin cross-bridging explains force generation because macrophage actin and myosin filaments interact to hydrolyze ATP and contract in the presence of Mg”+ (macrophage myosin may require phosphorylation for this activity). However, unlike myosin and actin filaments in sarcomeres, those in macrophages are randomly oriented. The ABP crosslinks cortical actin into a three-dimensional gel. Cytochalasin B or a new 180,000-dalton macrophage protein plus micromolar calcium reversibly dissolves ABP-actin gels by limited severing of actin filaments. According to classical network theory, abrupt gel-sol transformations accompany small changes in crosslink:polymer ratios near a critical point. The ABP-actin interaction conforms to this theory and hence is a powerful system for control of cytoplasmic consistency. Myosin dispersed within the network draws filaments from less crosslinked to more crosslinked regions. Therefore, direction of movement and its control can be established by cytoplasmic calcium gradients. Dr. Leroy Hood (California Institute of Technology) next discussed the structure and evolution of major histocompatibility gene products. The major histocompatibility complex (MHC) of mammals plays a central role in the functioning of the immune system. The MHC encodes three classes of gene functions: (1) transplantation antigens, (2) immune responsiveness, and (3) control of certain complement components. The fact that the MHC gene complex in mammals encompasses a large segment of DNA raises the question of how many genes are included in the MHC. Moreover, an evolutionary question is raised: Why have these three classes of genes in the MHC remained linked for a long period of time, presumably throughout vertebrate evolution? Dr. Hood first discussed the advent of new microsequencing techniques and how they, along with monoclonal antibodies, will permit us in the future to characterize and isolate the transplantation antigens of I, antigens. Next he reviewed the amino acid sequence data on transplantation antigens and the evolutionary and genetic conclusions and paradoxes posed by these data. Finally, he discussed the structural data on I, antigens and the implications these data have for the putative functions of these cells.

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The first session concluded with a paper by Ian Trowbridge (Salk Institute of Biological Studies) on the biochemistry and genetics of lymphocyte surface molecules. The biosynthesis of Thy-l glycoprotein in Thy-l- mutant lymphoma cell lines was studied as a model system in the investigation of the ways in which the expression of specific molecules on the lymphocyte cell surface may be regulated. Thy-l- mutants fall into five complementation classes, and synthesis of Thy-l glycoprotein can be detected by metabolic labeling in mutants from four of the five classes. The oligosaccharides found on the Thy-l glycoproteins made by the mutant cells differ from those of the wild-type glycoprotein, an indication that abnormal glycosylation of the Thy- 1 molecule may interfere with its transport to the cell surface and integration into the plasma membrane. The fact that a Thy-l- mutant was isolated by cytotoxic selection with concanavalin A (Con A) provides strong evidence in support of this possibility. The precise biochemical lesion in class E Thy-l- mutant cells has been identified. In these mutants there is a block in the synthesis of the lipid-linked oligosaccharide intermediate of asparagine-linked oligosaccharides of cellular glycoproteins; this leads to the accumulation of two lipid-linked oligosaccharides that are smaller than the major lipid-linked oligosaccharide found in wild-type cells. These oligosaccharides are then transferred to nascent polypeptides, and as a result the high-mannose oligosaccharides of glycoprotein of class E mutant cells contain fewer a-linked mannose residues than the glycoproteins of wild-type lymphoma cells. This defect in glycosylation selectively interferes with the expression of Thy-l glycoprotein on the surface of mutant cells. Thy-l glycoprotein is degraded more rapidly in all classes of Thy-l- mutants than in wild-type cells. However, the increased rate of degradation does not seem sufficient to account for the deficit of Thy-l molecules on the surfaces of mutant cells. Substantial amounts of Thy-l glycoprotein can be demonstrated within Thy-l mutant cells by immunofluorescence and immunoelectron microscopy. It seems likely, therefore, that the abnormal glycosylation of Thy-l glycoprotein blocks the transport of Thy-l molecules to the cell surface. The nature of this block is unknown but cannot involve the loss of a general signal for transport of the cell surface provided by normal oligosaccharides since other glycoproteins with aberrant oligosaccharides are found in normal amounts on the cell surface. Monoclonal antibodies have been obtained against several surface molecules of mouse lymphoid cells that can be detected biochemically. These molecules include Thy- 1 and T200 glycoproteins. With the exception of Thy-l glycoprotein, the molecules are not detectable on nonlymphoid tissues. The potential use of such monoclonal antibody reagents for studying the mechanism of hematopoietic differentiation was illustrated by the quantitative changes in the expression of T200 glycoprotein that were detected during the differentiation of Friend-virusinduced erythroleukemia cells exposed to dimethyl sulfoxide. Although T200 glycoprotein is found on many types of hematopoietic cells, in cytotoxic ablation experiments monoclonal antibody against T200 discriminates against thymusderived lymphocytes and may be useful in studies of lymphoid differentiation. The second session, on biochemical, pharmacologic, and cellular aspects of cell regulation, was chaired by Kenneth Melmon (Stanford University). It opened with

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John David (Harvard University), who discussed the biochemical characterization of a putative glycolipid for migration-inhibitory factor (MIF) and the existence and properties of two distinct MIFs. A lymphocyte mediator of cellular immunity MIF alters the behavior of macrophages. Studies so far indicate that MIF is indistinguishable from macrophageactivating factor (MAF), a lymphocyte mediator that modulates the structure, biochemistry, and function of macrophages and enhances their microbicidal and tumoricidal capabilities. Thus, one important mechanism for the activation of macrophage function occurs through the lymphocyte mediators MIF/MAF. He reviewed several studies by members of his associates, including those by H. Remold, T. Higgins, D. Liu, W. H. Churchill, and W. Piessens, that focused on the interaction of guinea pig MIF with macrophages. These studies led to three conclusions. First, a macrophage glycolipid appears to act as a receptor MIFi MAF. This glycolipid requires both fucose and sialic acid for its action. Second, there are two distinct MIF molecular species, pHS-MIF and pH3-MIF, that can be separated from each other by isoelectrophoresis; the two MIFs have different properties (see the discussion below). Third, macrophage-associated esterases control the mediator-cell interaction. The blocking of the macrophage esterase by esterase inhibitors results in a macrophage that is more responsive to MIF/MAF. This is partly due to the fact that pHS-MIF is destroyed by the macrophageassociated esterase (as well as by trypsin) whereas pH3-MIF, the larger species of the two, is resistant. These principles also are applicable to human MIF/MAF and monocytes. The second speaker, Thomas C. Merigan (Stanford University), spoke on a regulatory role for interferon as a lymphokine. Recently, a role for interferon in resistance to viral infection has been clearly demonstrated with anti-interferon antibody. In man some evidence of efficacy has been seen in trials with interferon in infections ranging from the common cold to herpesviruses and hepatitis B. It is now recognized to have a number of cell regulatory effects. Interferon is under study as a normal regulator of immune and other differentiated cell functions, and as a component in resistance to tumors. The interferon produced by the immune system appears more active as an immunoregulator than that produced by nonlymphoid cells. Antitumor effects have been observed with murine interferon in spontaneous and virus- and carcinogeninduced tumors. There are several possible mechanisms for this antitumor effect. For example, interferon inhibits the proliferation of transformed (and normal) cells in vitro. In addition, interferon enhances the cytotoxic action of macrophages and both T and natural killer lymphocytes. Preliminary trials suggest an antitumor action in man, and it is not surprising that bone marrow suppressive effects are the major factor limiting dosage. These effects are readily reversible and occur at serum levels active in inhibiting marrow growth in \vitr’o. Thus, the wide array of biological activities exhibited by interferon offer increasingly interesting avenues for both fundamental biological and clinical investigations I The next speaker, Kenneth Melmon (Stanford University), discussed the possible roles for histamine as a modulator of immunity. Dr. Melmon opened his discussion by indicating that mediators of inflammation may be modulators of the immune process. He emphasized the fact that histamine as a mediator of inflam-

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mation can feed back and inhibit the inflammatory process as it pertains to histamine release or lysosomal enzyme release from phagocytosing neutrophils. He then presented evidence that histamine is capable of modifying the function of T suppressor cells, which in unprimed animals can respond to histamine, and that in the process of commitment to an effector function, both B cells and T cells develop histamine receptors. Stimulation of the T-cell population interferes with the release of lymphokines, T effector cell cytolysis, and many other functions testable on these cells. He also described the preparation of histamine columns by insolubilizing conjugates of histamine to rabbit serum albumin and their usefulness. These affinity adsorbents were able to separate cells possessing receptors to histamine. Splenic leukocytes were separated chromatographically across these adsorbents. The nonadherent cells lacked T suppressor function, T effector function, and B-cell capacity for humoral antibody production. He concluded that the receptors for histamine are nonrandomly distributed and that they may modulate a number of different immune functions. This session concluded with a talk by Charles W. Parker (Washington University School of Medicine) on the biochemical events of lymphocyte activation. When lymphocytes are subjected to a variety of immunologic and other stimuli, most of which apparently act at the cell surface, blast transformation and division or differentiation may be induced. Despite much effort, the mechanism by which these processes are initiated and coordinated is still not completely understood. It has been argued that cyclic GMP (cGMP) is a major intracellular messenger for cell proliferation, but direct evidence is very limited. Although cyclic AMP (CAMP) clearly can inhibit mitogenesis and promote differentiation, its action may be more complex. Dr. Parker and his associates have suggested that there may be more than one functional pool of CAMP in human lymphocytes and that a localized pool of CAMP acting in or near the plasma membrane may play an early stimulatory role in mitogenesis. Since CAMP normally acts by promoting the phosphorylation of proteins, as an approach to the evaluation of this hypothesis his laboratory has studied protein phosphorylation in lymphocytes prelabeled with 32POi-. As noted previously by his group, three mitogens, phytohemagglutinin (PHA), Con A, and A-23187, produced rapid generalized increases in labeling. In addition, by a modification of previous methods for breaking cells, we can now show increases in at least four proteins in the cytosol, two of which separate in the 60,000 to 70,000 molecular-weight range in sodium dodecyl sulfate-polyacrylamide gels. Since a phosphorylated protein with a similar molecular weight has recently been shown in mammalian cells transformed by avian sarcoma viruses and is apparently responsible for the phenotypic expression of transformation in these cells, these observations are of considerable interest. Whether the phosphorylation is mediated by CAMP or is independent of CAMP remains to be established. The subject of the third session, with Arthur Malley (Oregon Regional Primate Research Center) as chairman, was the regulation of cell proliferation and differentiation, and the opening paper was given by Martin Cline (University of California at Los Angeles). He spoke on the regulation of hemopoiesis by humoral and other factors. A variety of cellular interactions control the production of blood cells in

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hematopoietic organs. The interactions include (a) the involvement of T lymphocytes in granulopoiesis, monocytopoiesis, and erythropoiesis and (b) the involvement of mononuclear phagocytes in granulopoiesis, T lymphocyte differentiation, and perhaps erythropoiesis. The stromal cells of hematopoietic organs provide a unique microenvironment that influences the development of stem cells. The rich diversity of hematopoietic cellular interactions may serve as a model for interactions in other tissues and organs comprising multiple cell types. Blood-cell interactions have been elucidated in vivo in animals and in \~itro in recently developed culture systems applicable to human studies. For several years cellular interactions affecting hematopoietic cell proliferation and differentiation have been described in terms familiar to cell biologists: for example, the activation of macrophages by soluble products of lymphoid cells is well established. More recently, permanent lines of hematopoietic cells have provided tools for studying induced differentiation of eukaryotic cells at the molecular level. It is likely that these model systems will provide the ultimate explanation of the influence of one cell upon the proliferation and differentiation of its neighbor. It is also possible that the techniques used to examine these interactions may have unanticipated applications in various therapies for human diseases. For example, it has recently been reported that mixtures of hematopoietic stem cells cultured for long periods in vitro can change their antigenic characteristics and subsequently proliferate in host animals that would ordinarily reject their growth. This observation may have important biological and technological implications for human bone marrow transplantations for aplastic anemia and malignant disease: currently, transplantation is restricted to donors and recipients possessing hematopoietic cells closely similar in surface antigenic structure. The possibility that one could artificially alter that structure is challenging. Margritt Scheid (Sloan-Kettering Institute) talked about differentiative induction of lymphocyte populations in vitro. The results from a dual assay for the induction of Thy-l ’ T cells and of CR t B cells from marker-negative precursors confirm that thymopoietin is at present the only known selective inducer of prothymocytes. In contrast, various inducers. including ubiquitin, are active in both assays. Pharmacologic evidence indicates that there are different cellular receptors for ubiquitin and thymopoietin. Prothymocytes and pro-CR+ B cells constitute two distinct populations in bone marrow and spleen; their distribution in density gradients is different, and elimination of either population enriches the other proportionately. There are no noteworthy induction differences in these two populations with regard to kinetics, dependence on temperature and protein synthesis, activation by CAMP, or inhibition by cGMP. The opposite inductive effects of CAMP and cGMP have been corroborated by the use of pharmacologic agents that raise or lower the levels of intracellular cyclic nucleotides. In contrast, a third induction assay, which monitors acquisition of the PC’ surface phenotype, indicates that this differentiative step, the last known for B cells, is initiated by cGMP and inhibited by CAMP. Induction of PC is also inhibited by thymopoietin, an indication that the inductive selectivity of thymopoietin is not due to restriction of its receptors to the ‘I lineage ceils. Rather, it seems that receptors for thymopoietin also occur on PC-

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inducible and other B cells, although in this case they are geared biochemically to inhibition rather than expression of the succeeding gene program. This fact suggests a role for thymopoietin in the coordinated interregulation of thymocyte classes, in addition to its better-known function as a thymic inducer of prothymocytes. Present data conform to the scheme in which the cyclic nucleotides CAMP and cGMP, and agents that affect intracellular levels of these mediators, influence reciprocally the early and late (functional) phases of lymphocyte differentiation as a whole and in which thymopoietin reciprocally influences the differentiation of the B and T classes of lymphocytes. Kendall A. Smith (Dartmouth Medical School) next spoke on the immunologic significance of T-cell growth factor. T-cell growth factor (TCGF), derived from T-cell mitogen- or antigenstimulated mononuclear cells, selects for and supports the continuous exponential proliferation of both human and murine cytotoxic T lymphocytes. Studies on the molecular and functional characteristics of TCGF have revealed that its activity resides in a 14,000- to 15,000-dalton protein with an isoelectric point of pH 5.65. Production of TCGF was found to be T-cell specific in that only T-cell mitogens or antigens elicited TCGF production and mature peripheral T cells were required. Removal of Thy- l-positive cells from spleen cell populations markedly decreased TCGF production in response to T-cell mitogens. Immature T-cell populations produced little TCGF; thymocytes released only 1 to 2% of the TCGF activity produced by spleen cells. Spleen, lymph node, and bone marrow cells from nude mice produced no measurable TCGF. In contrast, cortisol-resistant thymocytes produced TCGF in amounts equivalent to spleen cells. Adherent splenic cells were also found to be required for TCGF production. Spleen cells purified on nylon columns were deficient in TCGF production; however, reconstruction with 1 to 5% adherent cells resulted in normal TCGF production. The response to TCGF was also found to be T-cell specific. Spleen and thymus cells activated by T-cell mitogens or antigens adsorbed TCGF activity, whereas unstimulated cells or spleen cells stimulated by B-cell mitogens did not. Absorption occurred rapidly (within 2 hr at 37°C) and was dependent on cell concentration. Similarly, TCGF supported the continuous proliferation of cells activated by T-cell mitogens or antigens but did not initiate or sustain proliferation of unactivated cells. It was observed that although thymocytes produced minimal amounts of TCGF and exhibited weak proliferative responses to T-cell mitogens, stimulation with mitogens in the presence of TCGF resulted in strong proliferative responses. These results prompted an investigation of the effect of TCGF on prothymocytes in nude mice. The addition of TCGF to nude spleen, lymph node, and bone marrow cells together with Con A resulted in normal proliferative responses. Furthermore, the addition of TCGF to nude spleen cells in the presence of alloantigen resulted in proliferation and the generation of alloantigen-specific cytolytic nude lymphocytes. Such cells were placed in continuous culture in the presence of TCGF. A nude cytolytic cell line has been maintained in culture for more than 3 months. The cells, which have retained their cytolytic activity and specificity, contain Thy- 1 antigen.

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The results of this experimentation indicate (1) that TCGF is the proliferative signal in the T-cell immune response, (2) that T-cell activation requires mitogen or antigen but that proliferation is mediated solely by TCGF, (3) that TCGF probably interacts with activated T cells through a TCGF-specific receptor, (4) that the macrophage requirement for T-cell proliferation may be explained by the requirement of adherent T cells for TCGF production, (5) that a primary function of the thymus is to effect the maturation of those cells necessary for TCGF production. and (6) that thymic immunodeficiency may be attributable to a paucity of cells capable of TCGF production. Biochemical mechanisms involved in antigen- and mitogen-induced lymphocyte differentiation were discussed by Jim Watson (University of California at Irvine). Helper-T-cell-replacing factors (TRF) secreted by Con A-treated mouse spleen cells have been purified to homogeneity by salt precipitation, gel filtration, ionexchange chromatography, and isoelectric focusing (IEF). Three assay systems have been used to quantify purified TRF activity: (1) the stimulation of antibody responses to erythrocyte antigens in T-cell-depleted spleen cultures, (2) the ability to synergize with the T-cell mitogens Con A and PHA to stimulate strong proliferative responses in murine thymocyte cultures in which neither is active alone, and (3) the amplified production of cytotoxic T lymphocytes in culture. The TRF activity is found in protein-containing molecules of 35,000 daltons; there is considerable heterogeneity in isoelectric points ranging from 4.0 to 4.8, and TRF is active in each culture assay in concentrations of less than lo-!’ M. Current studies involve the iodination of IEF-purified TRF and analysis by gel electrophoresis for the determination of polypeptide chain composition. Production of these molecules requires the presence of T cells, but it has not been formally shown that they are secreted by T cells. A limiting dilution analysis has revealed that about 1 in 20,000 spleen cells produces TRF. The biological activity of TRF can be clearly distinguished from that of lower molecular-weight factors such as LAF by a dependence on macrophages in the mode of action. Current work involves the preparation of an antiserum to TRF and the development of a radioimmunoassay to quantify TRF with ‘2”I-labeled TRF. The radioimmunoassay will be used to compare identity to other T-cell-replacing activities, known as specific helper factors, allogeneic factors, and LAF. The fourth session dealt with the biochemical structure and biological functions of lymphocyte membrane components. This session was chaired by Noel Warner (University of New Mexico School of Medicine). The first speaker, Paul D. Gottlieb (Massachusetts Institute of Technology), spoke on the structure and genetics of Lyt-2 and Lyt-3 antigens. The Lyt-2 and Lyt-3 thymocyte alloantigens are present on nearly all murine thymocytes and on approximately 50% of peripheral T lymphocytes. In particular, these alloantigens have been shown to be present on subsets of T cells that have killer and suppressor activity, but not on T helper cells. The genetic loci governing expression of the Lyt-2 and Lyt-3 alloantigens are closely linked to each other on chromosome 6 of the mouse, and there is evidence that the Lyt-2 and Lyt-3 antigenic determinants are topographically adjacent on the cell surface. He has shown that a genetic locus governing expression of a variety of immuno-

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globulin light-chain polymorphisms is very closely linked to the Lyt-2 and Lyt-3 loci. He has been studying the biochemical nature of the Lyt-2 and Lyt-3 molecules because they are present on particular functional subsets of T cells and may be involved in mediating their functions, and because he wishes to determine whether they might be structurally related to light chains. The approach taken by Dr. Gottlieb involved radiolabeling the thymocyte surface with lzsI and lactoperoxidase or NaB3H, and galactose oxidase, solubilizing the plasma membrane with NP-40 detergent, precipitating the relevant antigens with anti-Lyt alloantisera, and displaying the precipitated products on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. To control for non-Lyt differences between strains of mice, he performed parallel experiments on pairs of B6-congenic strains, which differ only at the locus of interest. Results suggest that the Lyt-2 and Lyt-3 antigenic determinants reside on glycoprotein molecules with an apparent molecular weight of 35,000. That they are present on different molecules is indicated by their behavior in sequential precipitations with one antiserum and then another, and by their differential sensitivity to trypsin. Their similarity in overall structure and close linkage raises the possibility that the Lyt-2 and Lyt-3 molecules may be related in structure and evolution. In attempting to produce a congenic antiserum specific for the Lyt-3.1 antigenic specificity, he observed that the B6-Lyt-2a strain was a responder whereas B6 (which differs from B6-Lyt-2” only at the Lyt-2 locus) was a nonresponder. He has shown that the F, hybrid of B6 x B6-Lyt-2a is a nonresponder (or very weak responder), and that in back-crosses to the responder, response is segregated with the Lyt-2 locus. Thus, a locus closely linked to Lyt-2 appears to govern the immune response to Lyt-3.1, and he has suggested several mechanisms to explain this surprising result. The next speaker, Brigitte T. Huber (Tufts University School of Medicine), spoke on the structure and functions of Lyb-3 cell surface antigen. The aim of her research is to identify differentiation markers on murine B cells, which are expressed selectively at different stages of B-cell development, or on independent sublines of B cells. Using the CBA/N mutant mouse, which has a well-characterized B-cell defect, she has been able to raise an antiserum that defines Lyb-3, a marker exclusively expressed on 50% of splenic B cells and 100% of lymph node B cells in adult mice of all strains tested, except CBA/N. Lyb-3 is absent on neonatal spleen cells and adult bone marrow cells. In addition to delining a mature subset of B cells, Lyb-3 serves as a functional receptor: Lyb-3 antibodies can trigger a T-cell-dependent response either if the signal given by the antigen or the T-cell help is suboptimal. Her laboratory is now in the process of analyzing the exact functional character of the Lyb-3 receptor molecule. Using a similar approach, they are currently defining a new H-2 linked marker that is expressed on the same subset of B cells as Lyb-3. Lenore A. Herzenberg (Stanford University) spoke on T-cell regulation of Bcell differentiation. The first of the memory cells to appear after antigenic stimulation carries surface IgD and remains in primed animals for varying periods of time; the length of time depends on the strength of the priming stimulus and the amount of T-cell help

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available to support further differentiation. This IgD+ memory cell gives rise to a second memory cell that does not carry surface IgD and that represents the bulk of the memory pool in well-stimulated animals. Fluorescence-activated cell sorter (FACS) “double transfer” experiments directly showed that IgD+ memory cells give rise to IgD- memory cells in adoptive recipients when carrier-primed T cells and antigen are supplied. In these experiments, FACS-isolated IgD+ cells from the original primed donor were transferred to “first recipients” and allowed to expand and differentiate. Splenic B cells from these recipients were then FACS-sorted into IgD+ and IgD- populations. These populations, when transferred to second recipients, showed that the original IgDl cells had generated an exclusively IgD- memory population in the first recipients. Similar “double transfer” experiments showed that IgD- memory cells give rise to more IgD- memory cells but not to IgD+ memory. Two kinds of T helper cells are required for the differentiation and expansion of mature IgD- memory populations. The double transfer experiments showed that the expansion of IgD- memory populations requires help from carrier-primed T helper cells (CTh). Experiments with mice whose ailotypes had been suppressed showed that IgD+ to IgD- differentiation requires help from a second “Igspecific” T helper population. This population, which is depleted by allotype suppressor T cells, is also required for expression of IgD- memory and for production of the suppressed allotype. Data are insufficient to establish whether CTh are also required for memory expansion. Finally, she presented data that showed the IgD’ memory pool gives rise to predominantly low-affinity responses even when taken from donors boosted more than once. Afftnity maturation of the response is correlated with the emergence of IgD- memory and appears to proceed entirely within this population. These findings are summarized in the diagram below of the B-cell differentiation sequence. CTh (IgTh?) No T Virgin

------+

,--. J exp:, * : Early I Memory'-Cells

IgTh (CTh?) y

~'EXp:\ IgTh ' : (CTh?) Mature 1 Memory :Le.mm. CellZ?

IgD+

IgDf

Igf

IgM+

(IgM?)

I@-

(IgG?)

(I&?)

>

tgc aft

+ I >;c;

The final speaker of the fourth session was Rolf Zinkernagel (Scripps Clinic and Research Foundation), who spoke on the role of MHC gene products in T-cell recognition and responsiveness. The MHC codes for major transplantation antigens (H) on the cell surface that are involved in cell-mediated immunity as follows: (1) T cells express dud Specificity for foreign antigens and for self-H, (2) self-H determines the T-cell function (T cells specific for self-K and D are lytic; those specific for self-1 are nonlytic and induce differentiation of target B cells or macrophages), and (3) MHC genes that seem to regulate the capacity of T cells to respond (Zu genes) map to the same MHC regions as the genes coding for the restricting self-H. By means of chimeras,

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it was found that neither T-cell specificity for self-H nor responsiveness was determined by the genome of the T cells but rather by the MHC of the thymus and some lymphohemopoietic ceils; thymic selection of restriction specificity and responsiveness were coupled. It therefore appears that Zr gene products and restricting self-H are identical and Zr gene phenomena are merely a consequence of T cells being MHC restricted. Polymorphism and gene duplication of MHC products can thus be viewed as mechanisms that maximize the T-cell repertoire and responsiveness at both population and individual levels. This view of the MHC restriction of T cells has practical implications for attempts to reconstitute immunodeficient patients and for better understanding of MHC disease association. Eli Benjamini (University of California at Davis School of Medicine) convened the fifth session on the interactions of components of lymphocyte membranes. The first speaker was Henry Metzger (National Institutes of Health), who discussed the structure of Fc receptors. All cells implicated as either direct or indirect participants in immune responses bind one or more types of immunoglobulins (Ig) to their surface membranes. In all instances such exogenous Ig is bound through its Fc region. In view of the variety of cells, the variety of Ig isotypes that are bound, and the various functions that have been ascribed to these (Fc receptors), it is not surprising that considerable structural differences between different receptors have been noted. To date the most detailed information is available on the protein that binds IgE to the surface of mast cells. The properties of this Fc receptor, in sit/r as well as in its soluble form, was described and the progress toward its purification and structural elucidation was reviewed. The mechanism by which this protein acts as a sensor for surface antigen-antibody reactions was discussed (the molecular details are largely unknown). Comparative aspects with other Fc receptors were also discussed. Endotoxin-induced platelet membrane perturbation as a model system for transmembrane signaling was the subject of the next talk, which was presented by David C. Morrison (Scripps Clinic and Research Foundation). Bacterial endotoxins (lipopolysaccharides [LPS]) have been used extensively to probe the mechanism(s) of activation by murine B lymphocytes. Such activation has been demonstrated to result in induction of DNA synthesis, initiation of polyclonal and T-cell-independent specific antibody synthesis, and induction and/or shedding of surface membrane markers. The active component of the LPS molecule responsible for lymphocyte activation has been demonstrated to be the lipid A region. Lipid A has been shown to bind to the lymphocyte membrane. It is clear that the binding interaction is not, by itself, sufficient to initiate lymphocyte triggering and that certain events occuring after binding are required. However, the precise nature of these other biochemical events remains poorly understood. His approach to these questions has as its basis the observation that LPS (lipid A) can bind to and/or perturb, in addition to B lymphocytes, virtually every cell (erythrocytes, platelets, granulocytes, macrophages, fibroblasts, endothelial cells, liver cells, nerve cells, and muscle cells) with which it comes in contact. Therefore, there may be at least one fundamental biochemical parameter of lipid A and/or mammalian cell membranes that governs this interaction and/or capacity to

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be perturbed. In order to gain potential insight into the interactions between lipid A and cell membranes, Dr. Morrison and his associates examined the capacity of a lipid-A-rich preparation of LPS from Salmonella minnrsota Re 595 to perturb rabbit platelet membranes. They chose this system because of the relatively simple nature of the platelet structure, the ease in obtaining pure cell populations, and the facility with which cell responses (secretion) can be ascertained. In their initial experiments LPS added to purified preparations of platelets in buffer for short periods of time at 37°C resulted in profound, time-dependent morphological changes in the platelets. As ascertained by both transmission and scanning electron microscopy, the LPS initiated the appearance of numerous pseudopodia and “lingerlike” projections, yet the integrity of the intracellular constituents (granule-localized serotonin and cytoplasmic lactate dehydrogenase [LDH]) remained intact. The subsequent addition of Ca2+ (but not Mg2+ or Ba2+) resulted in the selective secretion of granule-localized serotonin with no loss of cytoplasmic LDH. Tenfold greater concentrations of Mg”+ or Ba2’ were ineffective in blocking the Ca2+-mediated response. Binding studies with radiolabeled 133Ba2Sand 4sCa2+ have indicated a selective association of Ca2’ with the platelets. These data provide evidence that LPS can selectively mediate the binding and/or translocation of Ca’+ in the triggering of a platelet response. Considerable evidence suggests that the platelet must somehow “process“ the LPS and thus allow the Ca”+-dependent response. Binding of LPS to the platelet is independent of time and temperature, but the secretory response is critically dependent upon both of these variables. Inhibition of binding by purified platelet membranes suggests that the LPS binds primarily to membrane constituents. Van? Hoff plots (log rate versus l/Tabs) indicate a partial linear dependence with a rather pronounced biphasic inflection point between 35 and 39”C, an indication that membrane fluidity may contribute to the effective processing of the LPS, perhaps by regulating LPS insertion into the lipid region of the membrane. Additional evidence for processing has been provided by experiments with polymyxin B (PB), a cationic antibiotic that binds tightly to LPS. If added to platelets at zero time, PB has no demonstrable effect independent of the presence or absence of LPS. If added after 90 min of preincubation, however, PB causes lysis (release of serotonin and LDH) of LPS-treated but not control platelets, an indication that the PB recognizes a different form of the LPS associated with the platelet (the amount of LPS associated with the platelet during this time is constant). These accumulated data indicate that, after binding, a time- and temperaturedependent alteration of the LPS occurs, perhaps within the platelet membrane. This alteration then allows the LPS to recognize and/or specifically translocate calcium and to trigger a platelet secretory response. A delineation of this LPS processing mechanism may provide fundamental information on mechanisms of LPS-lymphocyte interactions at the cell membrane as well as information on LPS-mammalian cell membrane interactions in general. Immunochemical studies on lymphocyte surface antigens in which monoclonal xenogeneic antibodies were used were discussed by Jeffrey Ledbetter (Stanford University). He talked about several monoclonal antibodies that were produced by immunization of rats with mouse spleen or thymus cells and subsequent fusion

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of the rat spleen with NSI mouse parental myeloma. He also gave a general introduction on the monoclonal antibody approach to the analysis of cell surface antigens and then discussed in detail several monoclonal antibodies recognizing lymphocyte cell surface antigens. The monoclonal antibody H12 recognizes an antigen present on all thymocytes and 35% of spleen ceils. The spleen cells bearing this antigen were shown to be non-Ig-bearing cells by two-color FACS staining. Further analysis with AKIUJ and AKR/Cu mice showed that H12 recognizes the Thy 1.2 antigenic determinant. Immunoprecipitation and two-dimensional gel analysis revealed that the molecular species bearing Thy 1.2 antigenic determinants is a family of related glycoproteins (molecular weight of 25,000 to 30,000) that vary in their extent of glycosylation. The monoclonal antibody C7 recognizes an antigen on all thymocytes, splenic T and B cells, and approximately 20% of bone marrow cells. The C7 antibody must recognize a new cell surface polymorphism since cells from C57B116 and C57Bl/lO mice do not express this antigen. Typing of F, and back-crossed mice with C7 showed that the antigen is controlled by a single codominant gene. Immunoprecipitation and two-dimensional gel analysis revealed that the molecular species bearing C7 antigenic determinants from spleen is a glycoprotein with a molecular weight of 100,000. Immunoprecipitation from labeled thymocytes with C7 antibody showed the lOO,OOO-molecular-weight component plus a larger species of 150,000 daltons. Preliminary evidence suggests that the 150,000molecular-weight species from thymocytes is a highly glycosylated form of the smaller species; neuraminidase digestions of the C7 antigen from spleen and thymus are identical on two-dimensional gels. The third monoclonal antibody Hll is expressed on all thymocytes, 50% of spleen cells, and 50% of bone marrow cells. The Hl 1 antigen is expressed on the non-Ig-bearing cells in the spleen and is restricted to the large bone marrow cells. Two lines of evidence suggest that the Hl 1 staining in the spleen is not restricted to Thy-l-bearing cells: Hll consistently stains 10 to 15% more cells in spleen than anti-Thy 1, and Hl 1 stains 20% of nude spleen cells whereas monoclonal anti-Thy 1 stains less than 5% of nude spleen cells. The conference ended with a presentation by Emil Unanue (Harvard University Medical School), who spoke on the mobility of lymphocyte membrane components and their interaction with submembrane structures. One of the first effects that takes place upon interaction of B lymphocytes with antigen is the reorganization of plasma membrane components. Studies on B lymphocytes exposed to anti-Ig have shown that capping of the complexes is accompanied by a reorganization of cytoplasmic actin and myosin and subsequent marked stimulation of translatory motion. The membrane interaction stimulates the activation of the microfilament network. The activation of the contractile function is accompanied by a marked efflux of intracellular calcium ion and a loss of about one-fourth of the exchangeable intracellular calcium ion pool. These changes require a crosslinked ligand. Interaction of ligands with other plasma proteins such as H-2 proteins also leads to capping, but this capping is slow and is not accompanied by activation of the contractile proteins nor by a change in

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calcium ion fluxes. Drugs that change calcium ion metabolism affect capping of Ig but not of H-2. The overall data suggest that molecules on lymphocyte membranes can be separated into two sets according to their capacity to interact with the contractile proteins. Surface Ig can signal metabolic changes that rapidly induce activation of contractile elements of the cell. This activation of locomotion may be crucial during the early stages of the reorganization of the lymph node architecture that follow the entrance of antigen. The metabolic processes that lead to the activation of locomotion are not sufficient to induce proliferation or differentiation of the B cell. ACKNOWLEDGMENT This conference was supported in part by the Office of Naval Research, Biochemistry under Contract NOOO14-77-GOO59, NR202-092.

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