- Email: [email protected]
Midwinter Conference of Immunologists
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
MEETING Midwinter
25,283-294
(1982)
REPORT
Conference
of Immunologists1
ESTHER F. HAYS, PATRICIA JONES, C.GARRISON AND EDGARG.ENGLEMAN Laboratory
of Biomedical
FATHMAN,
and Environmental Sciences, 900 Veteran Avenue, University of California, Los Angeles, California 90024
The Twenty-first Midwinter Conference of Immunologists was held January 23-26, 1982, at the Asilomar Conference Grounds, Pacific Grove, California. The chairpersons were Patricia Jones, C. Garrison Fathman, and Edgar Engleman. The conference topic was “Cell Recognition and the MHC.” It included five half-day sessions by invited speakers and a poster session. In addition to the scientific sessions, a sense motion was proposed as follows: “The following propositions indicate policies which we believe should be promoted by universities, public granting agencies, journals, and professional societies concerned with the standards and integrity of academic research in the biological sciences. They are not conceived as a formal set of rules and regulations, but as desirable objectives to be facilitated in ways appropriate to a given institution and with emphasis on voluntary cooperation within the research community: “ 1. Faculty members who receive research support from any agency, public or private, should be willing to disclose the source and amount of personal income derived from private enterprises in areas related to their research. “2. Granting agencies and journals, whose peer review processes require access to confidential grant applications or the refereeing of manuscripts, should carefully consider restricting participation of individuals who have a financial or commercial interest in the research areas under review. “3. Acceptance by a university of support from any source for a faculty member’s research should always be contingent on the assurance of adequate provisions for peer review and the absence of conflicts of interest that compromise educational standards and commitment. A standing faculty committee should verify that acceptable standards of review have been met and should, where there is doubt, initiate an appropriate ad hoc review procedure.” A panel consisting of Leon Wofsy, Ray Owen, and Hugh McDevitt discussed the motion. There was a lively discussion from the floor after which each part of the motion was offered for a show-of-hands vote. There was approximately 90% approval of all three parts of the motion by the persons attending the conference. ’ This work relates to Department of the Navy Grant N 00014-82-G-0025 issued by the Office of Naval Research. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 283 0090-1229/82/l 10283-12$01.00/O Copyright AI1 rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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The meeting began with the Dan H. Campbell memorial lecture entitled. “Cell Recognition and the MHC.” This was given by Dr. Rolf Zinkernagel. University of Zurich, Switzerland. Dr. Zinkernagel reviewed his pioneering studies and the work of others in this field and presented recent findings of his laboratory regarding thymic education of bone marrow progenitors in H-2 restriction. The first scientific session was entitled, “Cell Recognition and the Origin of the MHC.” Dr. Charles H. Bigger presented work carried out by himself and Dr. William Hildemann (University of California, Los Angeles). The topic of his talk was “Major Immunogene Complexes (MIC) in Phylogenetic Perspective.” He stated that an MIC has been identified in some 13 mammalian species thus far studied. In the best studied species, i.e., mice, rats. and humans, three groups of MIC genes are characteristic: (a) polymorphic genes governing strong alloincompatibility, (b) multiple Ir loci regulating T- and B-cell responses, (c) complement genes. The uniqueness of the MIC apparently resides in the clustering of these genes determining immunorecognition and immunoregulation, not in the individual effects of constituent genes. Self-recognition has been emphasized as the most critical function of the cell membrane structures determined by the MIC by some, while others have focused on regulation of cell interactions and development. In addition, attention has been called to nonimmunological effects including mating behavior, plasma testosterone levels, cyclic AMP concentrations, and cleft palate susceptibility in mice. Probable MICs have been identified in certain birds. anuran amphibians, and advanced bony fishes. An ancestral MIC may also be present in reptiles, urodele amphibians, primitive fishes, and even invertebrates. A precursor to the MIC may already have evolved in certain sponges and hydrozoans among the lower invertebrates. Certain species, but not all, of sponges consistently exhibit strong alloincompatibility associated with extensive genetic polymorphism. This acute cytotoxic reactivity is caused by cell-mediated immunity with specific memory. Higher invertebrates vary substantially in the timing and intensity of allogeneic incompatibilities, though impressive genetic polymorphism remains the rule. Multiple histocompatibility loci and Ir loci, both independent of the MIC, apparently evolved early. However, self-nonself discrimination in volves cell-surface markers or receptors specified by far more than a single gene complex. Dr. Bigger concluded that his phylogenetic studies suggest that criteria for the MIC and its multifarious functions now invite more skeptical evaluation. The next paper was presented by Dr. Richard A. Lerner (Scripps Clinic and Research Foundation, La Jolla, Calif.). He discussed the molecular basis of cell-cell recognition in the cellular slime mold, ” Dictyostelium discoidrum ,’ * Recent biochemical experiments have defined a lectin ligand system which is involved in cohesion and aggregation of Dictyostelium discoideum cells. The two molecules involved are a tetrameric carbohydrate binding protein (CBP) which consists of four subunits with approximate molecular sizes of 26K and a glycoprotein receptor with an approximate molecular size of 80K. His laboratory isolated a mutant in which CBP is present but has lost the ability to bind to galactose. This mutant both provided definitive evidence for the role of CBP in development and gave a point of departure for further studies. Dr. Lerner stated that if one imagines that cell-cell interaction occurs via a lock and key bond then the mutant
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can be thought of in terms of an altered key and should be suppressible by selecting for cells where an altered lock now fits the altered key. A second-site suppressor mutant was isolated which developed normally but still had a mutation in CBP. The prediction was that these cells had an altered 80K receptor which bound to the mutated CBP and allowed development to proceed. Two predictions were reported to follow from this hypothesis: (a) Since the strain with the outside suppressor has both an altered CBP and an 80K receptor, its cells should interact (synergize) with themselves but no other cells. In other words, the manipulation of mutating a cell surface binding molecule and suppressing the mutation via a second-site lesion in the receptor should create a new strain with private recognitive specificity. (b) In two strains which do not synergize with each other, the homologous CBPs should bind to the homologous receptors, but not vice versa. Data were presented which suggested that both of these predictions hold, therefore, he has been able to construct in the laboratory strains of Dictyostelium discoideum with private cell-cell recognition. The general point is that where molecules involved in cellular recognition are concerned, extragenic suppression of mutants can lead to constraints in the abilities of the cells to recognize anybody but themselves. Dr. Lerner concluded that these strains of slime mole should provide a point of departure for understanding the molecular basis of cell-cell interaction and species-specific recognition. Dr. Virginia Scofield (Hopkins Marine Station, Stanford, Calif.) presented her data describing protochordate allorecognition controlled by an MHC-like gene system. She stated that the tunicate Botryllus shows the capacity for allogeneic recognition by colony fusion or rejection. These responses are controlled by genes which are similar to loci of the vertebrate major histocompatibility complex (MHC). Colonies are comprised of clones of individuals enclosed in a single tunic and connected by a common circulation. Anastamosis between blood vessels of new buds and the colonial vascular system, or between blood vessel termini (ampullae) of growing edges of the same colony, are normal events in colony growth. Intraspecific parasitism by fusion between colonies of unlike genotype is prevented by interruption of the anastamosis sequence after a limited exchange of blood between the incompatible colonies. Allogeneic recognition between blood cells is followed by cell-mediated effector responses which isolate the involved ampullae . This colony specificity is controlled by a single, highly polymorphic gene locus which also prevents self-fertilization. The extreme polymorphism of the fusibility locus and its linkage to genetic elements which affect fertilization resemble properties of the linked H-2 and t regions of the mouse, whose loci control histocompatibility and sperm function, respectively. These similarities between the Botryllus and vertebrate systems, and the apparent restriction of allogeneic recognitions in both cases to certain blood cells, suggest that Botryllus colony specificity is controlled by genes of a primordial MHC. Dr. Scofield concluded that the identity between fusion and self-sterility genes also suggests that the gene families which participate in vertebrate adaptive immunity evolved from loci which prevented self-fertilization in hermaphroditic ancestors to the vertebrates. “Genetic Organization and Functional Relationships of T/t-Complex Mutations
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in the Mouse” was the title of a presentation by Dr. Dorothea Bennett. The coauthor was Dr. Karen Artzt. A summary of their data follows. Variants of mouse chromosome 17, called t haplotypes, occur in wild populations as frequent polymorphisms. They have a perplexing variety of properties, which include effects on tail length, early embryonic differentiation, the maturation and function of male germ cells, and suppression of meiotic recombination. Eight different categories of t haplotypes have been defined on the basis of their differing homozygous effects on embryonic development; these various lethal factors all appear to be functionally related to one another, and to operate by interfering with specific cell interactions. Serological and biochemical evidence suggests that t haplotypes govern abnormal glycosylation of cell surface molecules. Until recently, the recombination suppression in t-i + heterozygotes prevented attempts to analyze whether the relationship between t lethal factors was one of allelism, or of a broader type, possibly that of membership in a common multigene family. Now it has been shown that recombination occurs freely between two different complementing t haplotypes, and Drs. Artzt and Bennett have been able to map five lethal t factors relative to one another. They are all clearly not allelic. and in fact are distributed over more than 20 CM of chromosome 17. The H-2 complex has long been known to be included in the region of crossover suppression in t haplotypes; thus, the two sets of loci are inherited as a supergene complex. Furthermore, strong linkage disequilibrium exists between t haplotypes and H-2 haplotypes. Since their high transmission from males drives up the frequency oft haplotypes, this situation must serve as an important regulator of H-2 polymorphisms in wild mouse populations. Their recombination experiments have shown that the H-2 complex maps to an unexpected location with t haplotypes; its position is between the loci of T and tf, and is closely flanked by t lethal factors. Dr. Bennett stated in conclusion that the physical proximity of the two sets of complex loci may be fortuitous, but can also be used as a base for speculation on some functional or genetic relationship between them. In the second session, the papers dealt with MHC Genetics and Gene Products. Dr. Donald C. ShreMer (Washington University, St. Louis. MO.) spoke about the genetic organization and functional aspects of the S region of the MHC. He pointed out that although the structural, functional. and/or evolutionary relationships of the S-region (Class III) products to those of Class I (H-2) and Class IT (I) products remain obscure, significant advances have been made in the structural, functional, and genetic definition of the Class III products. The initial S region marker, the Ss protein, has been clearly divided into two structurally distinct, but homologous, products of duplicate S-region structural genes. One of these products is the classical C4 complement component: the other, sex-limited protein (Sip), has about 80 to 90% amino acid sequence homology with C4, but has no detectable activity in the classical complement assays. By radiolabeling in macrophage cultures, followed by immunoprecipitation. it has been possible to show that C4 and Slp are three-subunit molecules. They are both synthesized at 200K intracellular precursors, which are processed at the time of glycosylation and secretion into the medium into subunits (C4a = 98K, p = 74K, y = 34K; Slpcv =:
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105K, p = 72K, y = 32K). Subunit order in both precursors is /?-a-y. These properties were established by pulse-chase, peptide mapping and partial microsequencing. Multiple structural variants of C4 and Slp have mapped the structural genes to the S region. One C4 variant of particular interest has reduced functional activity, about 25% of normal, in a hemolytic assay. This defect is associated with a 4K dalton reduction in size of the CYchain (which must be proteolytically cleaved for activation). This reduction is due to the absence of an oligosaccharide group from the C-terminal half of the (Y chain, probably due to amino acid substitution. Other variants of C4 and Sip are being examined. Three variant modes of expression of the Slp molecule are also of particular interest: (1) sex limited (testosterone induced); (2) permissive (genetic background induced); (3) constitutive. The human and guinea pig MHCs carry structural genes for C2 and Bf of the complement system, as well as C4. Dr. Shreffler’s laboratory has detected S-region-localized quantitative functional variations in murine C2 (3fold) and Bf (40-fold). The Bf functional activity differences were found to be associated with constant Bf levels by immunochemical assay, strongly suggesting structural differences among Bf allelic products. Dr. Thomas Stanton (University of Washington, Seattle, Wash.), discussed the expression of Qa- 1 antigens. He pointed out that the Qa- 1 cell surface glycoprotein is a Class I molecule similar to H-2K, D, and L molecules in molecular weight, associated with P,-microglobulin and similar in amino acid sequence. The Qa-1 locus has been mapped to a position on mouse chromosome 17 telomeric to the Tla locus. He has been able to identify three alleles, Qa-la, Qa-lb, and Qa-ld by cytotoxic testing, and recently has identified three additional alleles by absorption analysis with one anti-Qa-1 antiserum. Thus, the Qa-1 locus appears to have greater polymorphism than previously thought. In addition, sequential immunoprecipitation of biosynthetically labeled B 10.M lymphocytes revealed that Qa- 1 is complex consisting of at least two loci encoding cell surface molecules. Expression of Qa-1 is controlled by a dominant locus which maps to the region between H-2S and Tla. All strains which are H-2Dk are low expressors of Qa-1. Low expression is changed to high expression after cell activation by mitogens. Data concerning the serological and functional analysis of the I region of the MHC were presented by Dr. Donald B. Murphy (Yale University School of Medicine, New Haven, Conn.). Loci mapping in the I region of the murine major histocompatibility complex (MHC) regulate immune responses (Ir loci) and control cell surface glycoprotein antigens (Ia loci). A central question is whether Ia glycoprotein molecules are products of Ir loci. Utilizing monoclonal anti-Ia antibody, produced by hybridoma Y-17, T-cell-proliferative responses to foreign antigens under Ir gene control were specifically blocked. It was shown that the quantitative levels of Ia molecules on the cell surface influence immune response potential. Studies by Dr. Murphy and colleagues have provided evidence for preferential association of Ia chains in Fl hybrids, resulting in aberrant (reduced) expression of certain Ia complexes on the cell surface. Their study, coupled with studies by others utilizing monoclonal anti-Ia antibody and I-region mutant strains, provides overwhelming evidence that Ia glycoprotein molecules are Ir gene products. In addition, Dr. Murphy’s group showed that quantitative (i.e.,
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amount of antigen expressed) as well as qualitative (i.e., allelic product expressed) expression of Ia molecules on the cell surface is critical in determining the Ir phenotype of the organism. The final paper in this session was presented by Dr. Benjamin D. Schwartz (Washington University, St. Louis, MO.). The title was “Molecular Relationships of the Human DR, MB, MT, and Te Alloantigens,” He said that the HLA-linked Class II alloantigens include the HLA-DR, MB, MT, and Te determinants, and that recent reports have demonstrated that most DR-homozygous B-lymphoblastoid cell lines express two structurally distinct Class II molecules. Dr. Schwartz’s group has used alloantisera carefully screened by absorption and immunochemical studies to exclude the presence of serologically undetected anti-DR, anti-MB, anti-MT, and anti-Te antibodies, to explore the molecular relationships of the human Class II alloantigens. Radiolabeled Class II molecules were immunoprecipitated with appropriate alloantisera and analyzed by a series of sequential immunoprecipitations and by two-dimensional gel electrophoresis. It was found that two structurally distinct sets of Class II molecules could be isolated from DRS-positive cells. One set (H,L,) bears DRS and MT2, while the second set (H,L,) bears DR5, MT2, MB3, and MT4. Thus, on DR 5 cells, MB3 and MT4 are on the same molecule (H,L,), and DRS and MT2 are on both sets of molecules. Preliminary data indicate the existence of a third set of Class Ii molecules (H1L3 or H,L,). In contrast, on DR4 cells, anti-MB3 precipitates only H,L, molecules, anti-MT4 precipitates both H,L, and H,L, molecules, and antiTe22 immunoprecipitates H,L, molecules. The results suggest random association of heavy and light chains of the human Class II molecules. The structure and organization of MHC genes were the topic of the third session. Dr. Stanley G. Nathenson (Albert Einstein College of Medicine, N.Y.) presented his data regarding structural studies on Class I H-2 products from mouse MHC mutant strains and their implications for molecular recognition. It was pointed out that the genes which comprise the major histocompatibility complex are intimately involved in a number of phases of the immune response defense mechanism. The classical transplantation antigen molecules (Class I), controlled by the mouse MHC (H-2), H-2K, H-2D, and H-2L, are highly polymorphic products of interest to immunologists because of their role at the cell surface as molecules which, in association with foreign antigen, serve as targets for immune T-cell recognition. Dr. Nathenson summarized some of his recent findings on the biochemical properties of the K, D, and L histocompatibility molecules with special emphasis on their overall organization and on the structural alterations present in these molecules from mouse MHC mutant strains. The study of mutants provides a method for analysis of MHC gene structure as well as an approach for probing the structural basis of K. D, and L recognition by the T-cell receptor. The most complete data obtained so far have been from the H-2K” series of mutants for which limited discrete structural changes were found differentiating the mutant and parent H-2Kh molecules. These findings suggest that alterations as small as a single amino acid exchange apparently are sufficient to alter recognition by the T-cell receptor. Studies were also presented on the mapping of polymorphic Class I DNA sequences within the MHC using Southern blot analysis of congenic and recombinant inbred mouse strains.
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A molecular dissection of the mouse MHC was given by Dr. Michael Steinmetz (California Institute of Technology, Pasadena, Calif.). His laboratory has isolated and sequenced cDNA clones encoding Class I molecules (transplantation antigens) of the mouse MHC. Southern blot analyses of germline or liver DNA from several inbred strains of mice using these cDNA clones as probes have demonstrated that there are 10 to 15 bands cross-reacting with the Class I probes. These cDNA clones have also been used to isolate 30-40 different Class I genes from a BALBlc sperm DNA library constructed in phage A. Two of these Class I genes have been characterized by DNA sequencing, and their origin, with respect to the genetic map of the MHC, has been determined. One, gene 27.1, appears to be a nonfunctional Class I gene and is located within the Qa-2,3 region to the right of the D marker locus. The second, gene 27.5, has been shown by transformation experiments and DNA sequencing studies to encode the Ld molecule. Both of these genes are split into eight exons separated by seven introns. The exons correlate precisely with structurally and functionally defined protein domains. The first exon encodes the signal or leader peptide; three exons are found for the three external domains of the molecule; a separate exon encodes the transmembrane domain and; surprisingly, three exons encode the small cytoplasmic domain. More recently a cosmid library of BALB/c sperm DNA has been screened with the Class I cDNA probes with the aim to obtain a complete map of the mouse MHC at the molecular level. Fifty-four cosmid clones which were isolated with the cDNA probes could be ordered into 13 clusters by restriction mapping. These 13 clusters encompass 837 kb of DNA and contain 36 Class I genes. One cluster, 191 kb in length with seven Class I genes, has been shown to contain the pseudogene 27.1 and therefore maps to the Qa-2,3 region. A second cluster with two Class I genes contain the Ld gene. A comparison of the 36 Class I genes shows that the exon encoding the third external domain is far more highly conserved than the two exons for the first and second external domains. This finding supports the hypothesis that the conserved, immunoglobulin-like third external domain interacts with p,-microglobulin whereas the variable domains are involved with antigen recognition by the T-cell receptor. Dr. Sherman Weissman (Yale University School of Medicine, New Haven, Conn.) presented data describing cloning and structure of human HLA genes. He described his isolation of an HLA cDNA clone which turned out to be HLA-B7. The HLA structure is a large extracellular protein with four regions with a carbohydrate moiety, a transmembrane portion, and a small cytoplasmic portion. The portion just outside the cell membrane links to B2 microglobulin. Dr. Weissman also presented data in which he used his cloned HLA DNA to transfect mouse cells. The result was a mouse cell that expressed the human HLA antigens. Dr. Jack Strominger (Harvard University, Cambridge, Mass.) presented a discussion of progress in several areas of research regarding human HLA-DR antigens. First the structure was described; two chains (~29 and ~34) both pierce the membrane. The heavy chain has two extracellular domains each defined by a disulfide loop. The N-terminal region is highly polymorphic. The C-terminal region is highly conserved and has very strong sequence homology to the (~3 region of the HLA-A, HLA-B, HLA-C antigens and to C, domains of immunoglobulins.
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The heavy chain appears to have an extracellular region without a disulfide loop and a second region with a disulfide loop. He next stated that there is a separation of subsets of HLA-DR antigens. Three subsets on consanguinous homozygous HLA-DR6 cells were defined by immunoprecipitations with alloantisera and monoclonal antibodies. One of these is the DC-l antigen. These subsets were separated on a preparative scale using monoclonal antibodies. These subsets contain at least three different B chains and two or possibly three IX chains. Data were presented showing that two cDNA clones and several genomic DNA clones corresponding to the HLA-DR CYchain gene have been obtained. First, a novel method involving immunopurification of HLA-DR mRNA-containing polysomes was employed; the specificity of an crchain-specific monoclonal antibody coupled with the use of a Stuphy1ococcu.s aureus Protein A-Sepharose column were the basis of the immunopurification. The mRNA was purified nearly to homogeneity and it was then relatively easy to use to prepare ds DNA which was cloned into pBR322. The cDNA clone has been sequenced and used to show that it detects a single BamHl band in genomic DNA digests. It was also used to obtain a genomic clone from a human genomic DNA library and the structure of the a-chain gene within the clone is being elucidated in Dr. Strominger’s laboratory. The fourth session dealt with HLA genes in immune recognition and regulation. Dr. William E. Biddison (National Cancer Institute, Bethesda, Md.) discussed the role of HLA gene products in the control of cytotoxic T-lymphocyte (CTL) responses to influenza virus. He presented an analysis of cytotoxic T-lymphocyte restriction antigens in man by the use of HLA-A variants. It was pointed out that the self-specificity of human (CTL) that respond to non-MHC foreign antigens has been analyzed in a limited number of experimental models. The principal CTL restriction antigens for the responses to the male H-Y antigen, influenza virus, measles virus, herpes simplex virus, and cytomegalovirus have been reported to be highly associated with the serologically defined HLA-A and HLA-B specificities. Studies of CTL recognition of influenza virus in conjunction with selfHLA-A2-associated antigens have demonstrated that a small proportion of donors possess HLA-A2 antigens which are serologically indistinguishable but which can be readily discriminated by CTL as well as by isoelectric focusing and peptide mapping. A related set of observations has been made for CTL recognition of HLA-A3. Influenza-immune CTL obtained from selected HLA-A3-positive donors could distinguish between the virus-infected target cells of unrelated HLA-A3-positive donors. The patterns of recognition of HLA-A3-related CTL restriction antigens differed for the responses to two non-cross-reacting viruses (types A and B influenza). The results of these studies suggest that (1) there is a strong but incomplete association between the self-antigens recognized by influenza-immune CTL and the serologically defined HLA-A2 and HLA-A3 antigens; (2) each HLA-A and HLA-B molecule may possess multiple CTL, restriction antigens, each of which may function as self-recognition structures for CTL that respond to different foreign antigens; and, (3) the HLA-A region may be considerably more polymorphic than current serological analyses have revealed.
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Dr. John Stobo (University of California, San Francisco, Calif.) discussed the immune response gene control of collagen reactivity in man. He stated that previous studies from his laboratory had indicated that human T-dependent reactivity to collagen, as measured by the production of leukocyte inhibition factor (LIF), is controlled by immune response genes closely linked to those coding for HLADR4. All HLA-DR4 + individuals tested are collagen responders. Observations, which indicate that absence of detectable collagen reactivity in the peripheral blood mononuclear cells (PBMC) of HLA-DR4 individuals does not reflect an absence of collagen reactive T cells, were presented. First, irradiation (1000 rad) of the PBMC of 20 HLA-DR4 collagen nonresponders results in the appearance of collagen reactivity. The specificity of this reactivity to collagen and collagenrelated peptides is identical to that seen in HLA-DR4+ collagen responders. Second, fractionation of T cells from HLA-DR4collagen nonresponders on a live-step discontinuous bovine serum albumin gradient yields a population of high-density, collagen-reactive T cells. Finally, cytolytic treatment of nonresponder T cells with a monoclonal antibody (OK-T@ specific for suppressor T cells results in the appearance of collagen reactivity. Addition of unresponsive (i.e., nonirradiated or low-density T cells) to autologous responsive (i.e., irradiated or high-density T cells) results in specific suppression of collagen reactivity. Radiation-sensitive, collagen-specific suppressive influences cannot be detected in the PBMC of HLA-DR4+ collagen responders. These studies indicate that the absence of collagen reactivity in HLA-DR4nonresponders reflects a predominance of collagen-specific suppressive influences rather than an absence of collagen-reactive T cells. The ability to detect collagen reactivity in HLA-DR4+ individuals reflects an absence of collagen-reactive suppressive influences. Human immune suppression genes were the subject of a talk by Dr. Takehiko Sasazuki (Tokyo Medical and Dental University, Tokyo, Japan). Immune responsiveness of 84 members from 18 healthy families to streptococcal cell wall (SCW) antigen was tested by antigen-specific, monocyte-dependent T-cell proliferation in virro. The SCW antigen was extracted from the streptococcal cell wall by pepsin digestion, and was purified by ammonium sulfate fractionation, gel filtration through Sephadex G- 100, and DEAE- Sepharose column chromatography. The maximum likelihood scoring method revealed that low responsiveness to SCW antigen was controlled by a single dominant gene. Log score for linkage between the gene controlling low responsiveness to SCW antigen and HLA was 3.709 at 8 (recombinant fraction) = 0.00 indicating a close linkage between the HLA and the gene controlling the low responsiveness to SCW antigen in vitro. The coculture of T cells from high responders with low-responder monocytes showed vigorous response to SCW antigen whereas T cells from low responders did not respond to this antigen even with HLA-haploidentical high-responder monocytes. Low responsiveness to SCW antigen was thus expressed on the T-cell level but not on the monocyte level. Furthermore, whole peripheral lymphocytes from low responders suppressed the immune response of HLA-D haploidentical high responders. The nylon-wool column-passed T cells from low-responder peripheral blood lymphocytes treated with anti-HLA-DR monoclonal antibody with complement completely abolished the immune response of the HLA-D
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haploidentical high responder. By using the monoclonal antibodies against various T-cell markers, it appeared that the suppressive activity was found on the T cell which was positive for Leu 2a but negative for Leu 3. The low responder did respond to SCW antigen after the Leu 2a-positive T-cell fraction was depleted from the peripheral lymphocytes by panning using anti-Leu 2a monoclonal antibody. All these observations clearly document the existence in man of an HLA-linked immune suppression gene which controls the low responsiveness to SCW antigen via the suppressor T-cell fraction. Dr. David G. Marsh (Johns Hopkins University School of Medicine, Baltimore, Md.) presented his studies of the genetics of the human immune response to pollen allergens. He said that studies of response toward inhaled allergens (e.g., pollens) provide a good model for understanding the genetics of human immune response because of the immunogenetically limiting, low-dose antigenic exposure and the wide array of highly purified allergens available. Two of the important genetic controls appear to be a non-HLA-linked IgE-regulating gene and HLA-linked genes. Immunoglobulin E responses toward allergens of molecular weight ca. 10,000 daltons, such as ragweed Ra3, appear to be controlled by both the IgEregulating gene and the HLA-linked Ir and Is genes; but response to the structurally less complex molecule, ragweed Ra5 (molecular weight 5000 daltons), appears to be confined to HLA-linked genetic control. Using extremely pure Ra5 (299.9% pure), Dr. Marsh has recently found that 36/38 (95%) of persons with detectable IgE antibody to Ra5 have Dw2, versus 30/139 (22%) of persons who have Dw2 in the RaS-negative (although ragweedallergic) category. This striking difference is significant (P < 0.0001). 1gG antibody responses to Ra5 were studied in 61 of the study subjects who had been treated for their allergy by injections of ragweed antigens. All 22 treated subjects who possessed Dw2 made good IgG responses, including 9 who were not allergic to Ra5. On the other hand, only 1l/39 Dw2-negative subjects made IgG responses to Ra5 following treatment. This difference was significant (P < 0.0001) and the level of the responses in DwZnegative subjects was significantly lower than that in Dw?positive subjects. These data provide perhaps the best evidence yet for HLAlinked immune response (Ir) genes in man. Thus, HLA-Dw2 provides an excellent marker for human immune response to Ra5. Probably the main reason why the association is so strong is because the structure of Ra5 is much less complex than those of most other allergens studied. Immune recognition of Ra5 may be restricted primarily to a single site on the molecule. Using model allergens of simple well-defined chemical structure like Ra5, Dr. Marsh stated that it should be possible to build up a “genetic fingerprint” of human immune responses relating antigenic structure to genetic loci of the HLA system. The final session was a panel discussion of the roles of H-2 gene products in immune recognition and regulation. The discussion was moderated by C. Garrison Fathman (Stanford University) and the participants were Dr. Eli Sercarz (University of California, Los Angeles), Dr. John Kappler (National Jewish Hospital,
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Denver), Dr. William E. Paul (National Institutes of Health) and Dr. Harvey Cantor (Harvard Medical School). There were three major areas of discussion during the panel session; one on the nature of antigen recognized by T cells, the second on the nature of the T-cell receptor, and the final being the nature of MHC restricting elements. Regarding the nature of antigen as recognized by T cells, the following points were made: (1) T cells and B cells recognize different epitopes. Actually, even different T-cell subpopulations recognize unique epitopes; (2) native and denatured antigens often cross-react totally; (3) peptide fragments are often just as (or more) effective than intact antigens; (4) antibodies against antigens do not block T-cell recognition; (5) T-cell recognition of antigen/MHC is not blocked by free antigen; (6) antigens taken up by antigen-presenting cells are not effective for approximately 1 hr. Are the antigens “processed” by these cells? In the discussion of the nature of T-cell receptor, it was pointed out that (1) recognition of antigen and MHC gene product is physically linked; (2) MHCrestricting element (A,B) and antigen (X, Y) must be on the same cell: (3) the interaction of A and X can be recognized by a specific T-cell receptor to yield a tight, ternary complex. Some clones which are specific for A + X also can recognize B + Y; however, such a clone would not recognize A + Y or B + X. Fusion of a T-cell specific for A + X to a T cell specific for B + Y yields a hybrid reactive to A +XandB + YbutnottoA + YorB +X. The following outline reflects the discussion on the nature of MHC restricting elements: Restricting elements = Ir gene products = serologically defined structures, K, D, L; IA, IE, (I-J?) (1) Relevant MHC expression is in the antigen-presenting cell, not the responding T cell; (2) gene complementation in expression of the IE structural molecule correlates with IE-linked Ir genes; (3) anti-MHC monoclonal antibody which detects structural products can also block T-cell recognition; (4) mutants which affect structural products also affect expression of the Ir gene and restricting element; (5) transfection with a cloned structural gene transfects the restriction element. Specific immune response (Ir) genes generally determine the ability or lack of ability of individuals to respond to distinct antigens. It is now clear that the products of Ir genes are I-region-associated (Ia) antigens. Ia antigens are found on antigen-presenting cells (APC), B lymphocytes, and some T cells. A critical aspect of their function is that they are corecognized, with antigen, by specific “histocompatibility-restricted” T lymphocytes and this corecognition is necessary for T-cell activation: thus, the absence of a suitable Ia molecule needed for the presentation of a particular antigen can underlie Ir gene control. Alternatively, Ir genes may affect the existence of regulatory suppressor cells which mask a clear potential for response to the antigen. Nonetheless, the reason that certain Ia molecules fail to allow responsiveness to a specific antigen has not been resolved. Two major possibilities exist. First, Ia molecules on APC may engage in an active interaction with antigen which is
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critical to the recognition of the antigen (or an antigen-la complex) by T cells. Such “antigen-la pairs” have been experimentally demonstrated. la molecules may be incapable of interaction with antigens for which they express a nonresponder phenotype or may interact in an essentially unrecognizable way. This may be referred to as the “failure of antigen-processing theory.” Alternatively, the reason that given Ia molecules are associated with unresponsiveness to specific antigen is that no T cells exist which are capable of recognizing the particular antigen-la pair. This “absence of T cells” may be due either to the lack of representation of genes encoding the relevant receptor(s) in the genome, from a failure to positively select T cells expressing responsiveness to that antigen- la pair in the course of intrathymic maturation and selection, or from the deletion of such T cells during the process of establishing self-tolerance. Data presented in the panel discussion supported the concept that there were two distinct types of restricting elements recognized by T cells in the context of MHC-restricted recognition. First, the lr gene products were synonymous with la, Class II molecules. Second, the restricting elements for cytolytic effector T cells were shown conclusively to be Class I molecules. This demonstration relied in part upon studies presented by Jeffrey Frelinger (University of Southern California, Los Angeles, Calif.), which showed that transfection of L cells with genes encoding a Class I molecule not expressed on the L cells would allow them to become effective targets of cytolytic effector cells whose restriction specificity was encoded within the gene transfected into the L cell. The question of the form of antigen recognized by T cells was addressed in part by preliminary data from Dr. Harvey Cantor’s laboratory. These data suggested that certain nominal antigens might be found in covalent linkage with l-region molecules, thus forming a binary complex of la and antigen in the absence of a T-cell receptor for antigen. That this cannot be a universal mechanism for l-region restriction of recognition of nominal antigen was agreed to by all participants. Dr. Sercarz presented evidence that T cells and B cells recognized different nonoverlapping epitopes on a multideterminant antigen. Surprisingly, very few epitopes make an imprint on the entire T-cell system, although it was pointed out that a characteristic hierarchy of utilization of epitopes exists so that even each potential epitope is not always presented. It was suggested that each set of antigenpresenting cells, addressing different subpopulations of T cells, might possess a limited and unique “recognition chemistry.” Finally, the consensus of the panel was that there are no hard data which would allow a concrete suggestion about the molecular nature of the T-cell receptor for antigen. It is anticipated that in the succeeding Midwinter Conferences, the molecular nature of the T-cell receptor will be described. Received
April
29, 1982; accepted
June 4, 1982.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
MEETING Midwinter
25,283-294
(1982)
REPORT
Conference
of Immunologists1
ESTHER F. HAYS, PATRICIA JONES, C.GARRISON AND EDGARG.ENGLEMAN Laboratory
of Biomedical
FATHMAN,
and Environmental Sciences, 900 Veteran Avenue, University of California, Los Angeles, California 90024
The Twenty-first Midwinter Conference of Immunologists was held January 23-26, 1982, at the Asilomar Conference Grounds, Pacific Grove, California. The chairpersons were Patricia Jones, C. Garrison Fathman, and Edgar Engleman. The conference topic was “Cell Recognition and the MHC.” It included five half-day sessions by invited speakers and a poster session. In addition to the scientific sessions, a sense motion was proposed as follows: “The following propositions indicate policies which we believe should be promoted by universities, public granting agencies, journals, and professional societies concerned with the standards and integrity of academic research in the biological sciences. They are not conceived as a formal set of rules and regulations, but as desirable objectives to be facilitated in ways appropriate to a given institution and with emphasis on voluntary cooperation within the research community: “ 1. Faculty members who receive research support from any agency, public or private, should be willing to disclose the source and amount of personal income derived from private enterprises in areas related to their research. “2. Granting agencies and journals, whose peer review processes require access to confidential grant applications or the refereeing of manuscripts, should carefully consider restricting participation of individuals who have a financial or commercial interest in the research areas under review. “3. Acceptance by a university of support from any source for a faculty member’s research should always be contingent on the assurance of adequate provisions for peer review and the absence of conflicts of interest that compromise educational standards and commitment. A standing faculty committee should verify that acceptable standards of review have been met and should, where there is doubt, initiate an appropriate ad hoc review procedure.” A panel consisting of Leon Wofsy, Ray Owen, and Hugh McDevitt discussed the motion. There was a lively discussion from the floor after which each part of the motion was offered for a show-of-hands vote. There was approximately 90% approval of all three parts of the motion by the persons attending the conference. ’ This work relates to Department of the Navy Grant N 00014-82-G-0025 issued by the Office of Naval Research. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 283 0090-1229/82/l 10283-12$01.00/O Copyright AI1 rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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The meeting began with the Dan H. Campbell memorial lecture entitled. “Cell Recognition and the MHC.” This was given by Dr. Rolf Zinkernagel. University of Zurich, Switzerland. Dr. Zinkernagel reviewed his pioneering studies and the work of others in this field and presented recent findings of his laboratory regarding thymic education of bone marrow progenitors in H-2 restriction. The first scientific session was entitled, “Cell Recognition and the Origin of the MHC.” Dr. Charles H. Bigger presented work carried out by himself and Dr. William Hildemann (University of California, Los Angeles). The topic of his talk was “Major Immunogene Complexes (MIC) in Phylogenetic Perspective.” He stated that an MIC has been identified in some 13 mammalian species thus far studied. In the best studied species, i.e., mice, rats. and humans, three groups of MIC genes are characteristic: (a) polymorphic genes governing strong alloincompatibility, (b) multiple Ir loci regulating T- and B-cell responses, (c) complement genes. The uniqueness of the MIC apparently resides in the clustering of these genes determining immunorecognition and immunoregulation, not in the individual effects of constituent genes. Self-recognition has been emphasized as the most critical function of the cell membrane structures determined by the MIC by some, while others have focused on regulation of cell interactions and development. In addition, attention has been called to nonimmunological effects including mating behavior, plasma testosterone levels, cyclic AMP concentrations, and cleft palate susceptibility in mice. Probable MICs have been identified in certain birds. anuran amphibians, and advanced bony fishes. An ancestral MIC may also be present in reptiles, urodele amphibians, primitive fishes, and even invertebrates. A precursor to the MIC may already have evolved in certain sponges and hydrozoans among the lower invertebrates. Certain species, but not all, of sponges consistently exhibit strong alloincompatibility associated with extensive genetic polymorphism. This acute cytotoxic reactivity is caused by cell-mediated immunity with specific memory. Higher invertebrates vary substantially in the timing and intensity of allogeneic incompatibilities, though impressive genetic polymorphism remains the rule. Multiple histocompatibility loci and Ir loci, both independent of the MIC, apparently evolved early. However, self-nonself discrimination in volves cell-surface markers or receptors specified by far more than a single gene complex. Dr. Bigger concluded that his phylogenetic studies suggest that criteria for the MIC and its multifarious functions now invite more skeptical evaluation. The next paper was presented by Dr. Richard A. Lerner (Scripps Clinic and Research Foundation, La Jolla, Calif.). He discussed the molecular basis of cell-cell recognition in the cellular slime mold, ” Dictyostelium discoidrum ,’ * Recent biochemical experiments have defined a lectin ligand system which is involved in cohesion and aggregation of Dictyostelium discoideum cells. The two molecules involved are a tetrameric carbohydrate binding protein (CBP) which consists of four subunits with approximate molecular sizes of 26K and a glycoprotein receptor with an approximate molecular size of 80K. His laboratory isolated a mutant in which CBP is present but has lost the ability to bind to galactose. This mutant both provided definitive evidence for the role of CBP in development and gave a point of departure for further studies. Dr. Lerner stated that if one imagines that cell-cell interaction occurs via a lock and key bond then the mutant
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can be thought of in terms of an altered key and should be suppressible by selecting for cells where an altered lock now fits the altered key. A second-site suppressor mutant was isolated which developed normally but still had a mutation in CBP. The prediction was that these cells had an altered 80K receptor which bound to the mutated CBP and allowed development to proceed. Two predictions were reported to follow from this hypothesis: (a) Since the strain with the outside suppressor has both an altered CBP and an 80K receptor, its cells should interact (synergize) with themselves but no other cells. In other words, the manipulation of mutating a cell surface binding molecule and suppressing the mutation via a second-site lesion in the receptor should create a new strain with private recognitive specificity. (b) In two strains which do not synergize with each other, the homologous CBPs should bind to the homologous receptors, but not vice versa. Data were presented which suggested that both of these predictions hold, therefore, he has been able to construct in the laboratory strains of Dictyostelium discoideum with private cell-cell recognition. The general point is that where molecules involved in cellular recognition are concerned, extragenic suppression of mutants can lead to constraints in the abilities of the cells to recognize anybody but themselves. Dr. Lerner concluded that these strains of slime mole should provide a point of departure for understanding the molecular basis of cell-cell interaction and species-specific recognition. Dr. Virginia Scofield (Hopkins Marine Station, Stanford, Calif.) presented her data describing protochordate allorecognition controlled by an MHC-like gene system. She stated that the tunicate Botryllus shows the capacity for allogeneic recognition by colony fusion or rejection. These responses are controlled by genes which are similar to loci of the vertebrate major histocompatibility complex (MHC). Colonies are comprised of clones of individuals enclosed in a single tunic and connected by a common circulation. Anastamosis between blood vessels of new buds and the colonial vascular system, or between blood vessel termini (ampullae) of growing edges of the same colony, are normal events in colony growth. Intraspecific parasitism by fusion between colonies of unlike genotype is prevented by interruption of the anastamosis sequence after a limited exchange of blood between the incompatible colonies. Allogeneic recognition between blood cells is followed by cell-mediated effector responses which isolate the involved ampullae . This colony specificity is controlled by a single, highly polymorphic gene locus which also prevents self-fertilization. The extreme polymorphism of the fusibility locus and its linkage to genetic elements which affect fertilization resemble properties of the linked H-2 and t regions of the mouse, whose loci control histocompatibility and sperm function, respectively. These similarities between the Botryllus and vertebrate systems, and the apparent restriction of allogeneic recognitions in both cases to certain blood cells, suggest that Botryllus colony specificity is controlled by genes of a primordial MHC. Dr. Scofield concluded that the identity between fusion and self-sterility genes also suggests that the gene families which participate in vertebrate adaptive immunity evolved from loci which prevented self-fertilization in hermaphroditic ancestors to the vertebrates. “Genetic Organization and Functional Relationships of T/t-Complex Mutations
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in the Mouse” was the title of a presentation by Dr. Dorothea Bennett. The coauthor was Dr. Karen Artzt. A summary of their data follows. Variants of mouse chromosome 17, called t haplotypes, occur in wild populations as frequent polymorphisms. They have a perplexing variety of properties, which include effects on tail length, early embryonic differentiation, the maturation and function of male germ cells, and suppression of meiotic recombination. Eight different categories of t haplotypes have been defined on the basis of their differing homozygous effects on embryonic development; these various lethal factors all appear to be functionally related to one another, and to operate by interfering with specific cell interactions. Serological and biochemical evidence suggests that t haplotypes govern abnormal glycosylation of cell surface molecules. Until recently, the recombination suppression in t-i + heterozygotes prevented attempts to analyze whether the relationship between t lethal factors was one of allelism, or of a broader type, possibly that of membership in a common multigene family. Now it has been shown that recombination occurs freely between two different complementing t haplotypes, and Drs. Artzt and Bennett have been able to map five lethal t factors relative to one another. They are all clearly not allelic. and in fact are distributed over more than 20 CM of chromosome 17. The H-2 complex has long been known to be included in the region of crossover suppression in t haplotypes; thus, the two sets of loci are inherited as a supergene complex. Furthermore, strong linkage disequilibrium exists between t haplotypes and H-2 haplotypes. Since their high transmission from males drives up the frequency oft haplotypes, this situation must serve as an important regulator of H-2 polymorphisms in wild mouse populations. Their recombination experiments have shown that the H-2 complex maps to an unexpected location with t haplotypes; its position is between the loci of T and tf, and is closely flanked by t lethal factors. Dr. Bennett stated in conclusion that the physical proximity of the two sets of complex loci may be fortuitous, but can also be used as a base for speculation on some functional or genetic relationship between them. In the second session, the papers dealt with MHC Genetics and Gene Products. Dr. Donald C. ShreMer (Washington University, St. Louis. MO.) spoke about the genetic organization and functional aspects of the S region of the MHC. He pointed out that although the structural, functional. and/or evolutionary relationships of the S-region (Class III) products to those of Class I (H-2) and Class IT (I) products remain obscure, significant advances have been made in the structural, functional, and genetic definition of the Class III products. The initial S region marker, the Ss protein, has been clearly divided into two structurally distinct, but homologous, products of duplicate S-region structural genes. One of these products is the classical C4 complement component: the other, sex-limited protein (Sip), has about 80 to 90% amino acid sequence homology with C4, but has no detectable activity in the classical complement assays. By radiolabeling in macrophage cultures, followed by immunoprecipitation. it has been possible to show that C4 and Slp are three-subunit molecules. They are both synthesized at 200K intracellular precursors, which are processed at the time of glycosylation and secretion into the medium into subunits (C4a = 98K, p = 74K, y = 34K; Slpcv =:
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105K, p = 72K, y = 32K). Subunit order in both precursors is /?-a-y. These properties were established by pulse-chase, peptide mapping and partial microsequencing. Multiple structural variants of C4 and Slp have mapped the structural genes to the S region. One C4 variant of particular interest has reduced functional activity, about 25% of normal, in a hemolytic assay. This defect is associated with a 4K dalton reduction in size of the CYchain (which must be proteolytically cleaved for activation). This reduction is due to the absence of an oligosaccharide group from the C-terminal half of the (Y chain, probably due to amino acid substitution. Other variants of C4 and Sip are being examined. Three variant modes of expression of the Slp molecule are also of particular interest: (1) sex limited (testosterone induced); (2) permissive (genetic background induced); (3) constitutive. The human and guinea pig MHCs carry structural genes for C2 and Bf of the complement system, as well as C4. Dr. Shreffler’s laboratory has detected S-region-localized quantitative functional variations in murine C2 (3fold) and Bf (40-fold). The Bf functional activity differences were found to be associated with constant Bf levels by immunochemical assay, strongly suggesting structural differences among Bf allelic products. Dr. Thomas Stanton (University of Washington, Seattle, Wash.), discussed the expression of Qa- 1 antigens. He pointed out that the Qa- 1 cell surface glycoprotein is a Class I molecule similar to H-2K, D, and L molecules in molecular weight, associated with P,-microglobulin and similar in amino acid sequence. The Qa-1 locus has been mapped to a position on mouse chromosome 17 telomeric to the Tla locus. He has been able to identify three alleles, Qa-la, Qa-lb, and Qa-ld by cytotoxic testing, and recently has identified three additional alleles by absorption analysis with one anti-Qa-1 antiserum. Thus, the Qa-1 locus appears to have greater polymorphism than previously thought. In addition, sequential immunoprecipitation of biosynthetically labeled B 10.M lymphocytes revealed that Qa- 1 is complex consisting of at least two loci encoding cell surface molecules. Expression of Qa-1 is controlled by a dominant locus which maps to the region between H-2S and Tla. All strains which are H-2Dk are low expressors of Qa-1. Low expression is changed to high expression after cell activation by mitogens. Data concerning the serological and functional analysis of the I region of the MHC were presented by Dr. Donald B. Murphy (Yale University School of Medicine, New Haven, Conn.). Loci mapping in the I region of the murine major histocompatibility complex (MHC) regulate immune responses (Ir loci) and control cell surface glycoprotein antigens (Ia loci). A central question is whether Ia glycoprotein molecules are products of Ir loci. Utilizing monoclonal anti-Ia antibody, produced by hybridoma Y-17, T-cell-proliferative responses to foreign antigens under Ir gene control were specifically blocked. It was shown that the quantitative levels of Ia molecules on the cell surface influence immune response potential. Studies by Dr. Murphy and colleagues have provided evidence for preferential association of Ia chains in Fl hybrids, resulting in aberrant (reduced) expression of certain Ia complexes on the cell surface. Their study, coupled with studies by others utilizing monoclonal anti-Ia antibody and I-region mutant strains, provides overwhelming evidence that Ia glycoprotein molecules are Ir gene products. In addition, Dr. Murphy’s group showed that quantitative (i.e.,
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amount of antigen expressed) as well as qualitative (i.e., allelic product expressed) expression of Ia molecules on the cell surface is critical in determining the Ir phenotype of the organism. The final paper in this session was presented by Dr. Benjamin D. Schwartz (Washington University, St. Louis, MO.). The title was “Molecular Relationships of the Human DR, MB, MT, and Te Alloantigens,” He said that the HLA-linked Class II alloantigens include the HLA-DR, MB, MT, and Te determinants, and that recent reports have demonstrated that most DR-homozygous B-lymphoblastoid cell lines express two structurally distinct Class II molecules. Dr. Schwartz’s group has used alloantisera carefully screened by absorption and immunochemical studies to exclude the presence of serologically undetected anti-DR, anti-MB, anti-MT, and anti-Te antibodies, to explore the molecular relationships of the human Class II alloantigens. Radiolabeled Class II molecules were immunoprecipitated with appropriate alloantisera and analyzed by a series of sequential immunoprecipitations and by two-dimensional gel electrophoresis. It was found that two structurally distinct sets of Class II molecules could be isolated from DRS-positive cells. One set (H,L,) bears DRS and MT2, while the second set (H,L,) bears DR5, MT2, MB3, and MT4. Thus, on DR 5 cells, MB3 and MT4 are on the same molecule (H,L,), and DRS and MT2 are on both sets of molecules. Preliminary data indicate the existence of a third set of Class Ii molecules (H1L3 or H,L,). In contrast, on DR4 cells, anti-MB3 precipitates only H,L, molecules, anti-MT4 precipitates both H,L, and H,L, molecules, and antiTe22 immunoprecipitates H,L, molecules. The results suggest random association of heavy and light chains of the human Class II molecules. The structure and organization of MHC genes were the topic of the third session. Dr. Stanley G. Nathenson (Albert Einstein College of Medicine, N.Y.) presented his data regarding structural studies on Class I H-2 products from mouse MHC mutant strains and their implications for molecular recognition. It was pointed out that the genes which comprise the major histocompatibility complex are intimately involved in a number of phases of the immune response defense mechanism. The classical transplantation antigen molecules (Class I), controlled by the mouse MHC (H-2), H-2K, H-2D, and H-2L, are highly polymorphic products of interest to immunologists because of their role at the cell surface as molecules which, in association with foreign antigen, serve as targets for immune T-cell recognition. Dr. Nathenson summarized some of his recent findings on the biochemical properties of the K, D, and L histocompatibility molecules with special emphasis on their overall organization and on the structural alterations present in these molecules from mouse MHC mutant strains. The study of mutants provides a method for analysis of MHC gene structure as well as an approach for probing the structural basis of K. D, and L recognition by the T-cell receptor. The most complete data obtained so far have been from the H-2K” series of mutants for which limited discrete structural changes were found differentiating the mutant and parent H-2Kh molecules. These findings suggest that alterations as small as a single amino acid exchange apparently are sufficient to alter recognition by the T-cell receptor. Studies were also presented on the mapping of polymorphic Class I DNA sequences within the MHC using Southern blot analysis of congenic and recombinant inbred mouse strains.
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A molecular dissection of the mouse MHC was given by Dr. Michael Steinmetz (California Institute of Technology, Pasadena, Calif.). His laboratory has isolated and sequenced cDNA clones encoding Class I molecules (transplantation antigens) of the mouse MHC. Southern blot analyses of germline or liver DNA from several inbred strains of mice using these cDNA clones as probes have demonstrated that there are 10 to 15 bands cross-reacting with the Class I probes. These cDNA clones have also been used to isolate 30-40 different Class I genes from a BALBlc sperm DNA library constructed in phage A. Two of these Class I genes have been characterized by DNA sequencing, and their origin, with respect to the genetic map of the MHC, has been determined. One, gene 27.1, appears to be a nonfunctional Class I gene and is located within the Qa-2,3 region to the right of the D marker locus. The second, gene 27.5, has been shown by transformation experiments and DNA sequencing studies to encode the Ld molecule. Both of these genes are split into eight exons separated by seven introns. The exons correlate precisely with structurally and functionally defined protein domains. The first exon encodes the signal or leader peptide; three exons are found for the three external domains of the molecule; a separate exon encodes the transmembrane domain and; surprisingly, three exons encode the small cytoplasmic domain. More recently a cosmid library of BALB/c sperm DNA has been screened with the Class I cDNA probes with the aim to obtain a complete map of the mouse MHC at the molecular level. Fifty-four cosmid clones which were isolated with the cDNA probes could be ordered into 13 clusters by restriction mapping. These 13 clusters encompass 837 kb of DNA and contain 36 Class I genes. One cluster, 191 kb in length with seven Class I genes, has been shown to contain the pseudogene 27.1 and therefore maps to the Qa-2,3 region. A second cluster with two Class I genes contain the Ld gene. A comparison of the 36 Class I genes shows that the exon encoding the third external domain is far more highly conserved than the two exons for the first and second external domains. This finding supports the hypothesis that the conserved, immunoglobulin-like third external domain interacts with p,-microglobulin whereas the variable domains are involved with antigen recognition by the T-cell receptor. Dr. Sherman Weissman (Yale University School of Medicine, New Haven, Conn.) presented data describing cloning and structure of human HLA genes. He described his isolation of an HLA cDNA clone which turned out to be HLA-B7. The HLA structure is a large extracellular protein with four regions with a carbohydrate moiety, a transmembrane portion, and a small cytoplasmic portion. The portion just outside the cell membrane links to B2 microglobulin. Dr. Weissman also presented data in which he used his cloned HLA DNA to transfect mouse cells. The result was a mouse cell that expressed the human HLA antigens. Dr. Jack Strominger (Harvard University, Cambridge, Mass.) presented a discussion of progress in several areas of research regarding human HLA-DR antigens. First the structure was described; two chains (~29 and ~34) both pierce the membrane. The heavy chain has two extracellular domains each defined by a disulfide loop. The N-terminal region is highly polymorphic. The C-terminal region is highly conserved and has very strong sequence homology to the (~3 region of the HLA-A, HLA-B, HLA-C antigens and to C, domains of immunoglobulins.
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The heavy chain appears to have an extracellular region without a disulfide loop and a second region with a disulfide loop. He next stated that there is a separation of subsets of HLA-DR antigens. Three subsets on consanguinous homozygous HLA-DR6 cells were defined by immunoprecipitations with alloantisera and monoclonal antibodies. One of these is the DC-l antigen. These subsets were separated on a preparative scale using monoclonal antibodies. These subsets contain at least three different B chains and two or possibly three IX chains. Data were presented showing that two cDNA clones and several genomic DNA clones corresponding to the HLA-DR CYchain gene have been obtained. First, a novel method involving immunopurification of HLA-DR mRNA-containing polysomes was employed; the specificity of an crchain-specific monoclonal antibody coupled with the use of a Stuphy1ococcu.s aureus Protein A-Sepharose column were the basis of the immunopurification. The mRNA was purified nearly to homogeneity and it was then relatively easy to use to prepare ds DNA which was cloned into pBR322. The cDNA clone has been sequenced and used to show that it detects a single BamHl band in genomic DNA digests. It was also used to obtain a genomic clone from a human genomic DNA library and the structure of the a-chain gene within the clone is being elucidated in Dr. Strominger’s laboratory. The fourth session dealt with HLA genes in immune recognition and regulation. Dr. William E. Biddison (National Cancer Institute, Bethesda, Md.) discussed the role of HLA gene products in the control of cytotoxic T-lymphocyte (CTL) responses to influenza virus. He presented an analysis of cytotoxic T-lymphocyte restriction antigens in man by the use of HLA-A variants. It was pointed out that the self-specificity of human (CTL) that respond to non-MHC foreign antigens has been analyzed in a limited number of experimental models. The principal CTL restriction antigens for the responses to the male H-Y antigen, influenza virus, measles virus, herpes simplex virus, and cytomegalovirus have been reported to be highly associated with the serologically defined HLA-A and HLA-B specificities. Studies of CTL recognition of influenza virus in conjunction with selfHLA-A2-associated antigens have demonstrated that a small proportion of donors possess HLA-A2 antigens which are serologically indistinguishable but which can be readily discriminated by CTL as well as by isoelectric focusing and peptide mapping. A related set of observations has been made for CTL recognition of HLA-A3. Influenza-immune CTL obtained from selected HLA-A3-positive donors could distinguish between the virus-infected target cells of unrelated HLA-A3-positive donors. The patterns of recognition of HLA-A3-related CTL restriction antigens differed for the responses to two non-cross-reacting viruses (types A and B influenza). The results of these studies suggest that (1) there is a strong but incomplete association between the self-antigens recognized by influenza-immune CTL and the serologically defined HLA-A2 and HLA-A3 antigens; (2) each HLA-A and HLA-B molecule may possess multiple CTL, restriction antigens, each of which may function as self-recognition structures for CTL that respond to different foreign antigens; and, (3) the HLA-A region may be considerably more polymorphic than current serological analyses have revealed.
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Dr. John Stobo (University of California, San Francisco, Calif.) discussed the immune response gene control of collagen reactivity in man. He stated that previous studies from his laboratory had indicated that human T-dependent reactivity to collagen, as measured by the production of leukocyte inhibition factor (LIF), is controlled by immune response genes closely linked to those coding for HLADR4. All HLA-DR4 + individuals tested are collagen responders. Observations, which indicate that absence of detectable collagen reactivity in the peripheral blood mononuclear cells (PBMC) of HLA-DR4 individuals does not reflect an absence of collagen reactive T cells, were presented. First, irradiation (1000 rad) of the PBMC of 20 HLA-DR4 collagen nonresponders results in the appearance of collagen reactivity. The specificity of this reactivity to collagen and collagenrelated peptides is identical to that seen in HLA-DR4+ collagen responders. Second, fractionation of T cells from HLA-DR4collagen nonresponders on a live-step discontinuous bovine serum albumin gradient yields a population of high-density, collagen-reactive T cells. Finally, cytolytic treatment of nonresponder T cells with a monoclonal antibody (OK-T@ specific for suppressor T cells results in the appearance of collagen reactivity. Addition of unresponsive (i.e., nonirradiated or low-density T cells) to autologous responsive (i.e., irradiated or high-density T cells) results in specific suppression of collagen reactivity. Radiation-sensitive, collagen-specific suppressive influences cannot be detected in the PBMC of HLA-DR4+ collagen responders. These studies indicate that the absence of collagen reactivity in HLA-DR4nonresponders reflects a predominance of collagen-specific suppressive influences rather than an absence of collagen-reactive T cells. The ability to detect collagen reactivity in HLA-DR4+ individuals reflects an absence of collagen-reactive suppressive influences. Human immune suppression genes were the subject of a talk by Dr. Takehiko Sasazuki (Tokyo Medical and Dental University, Tokyo, Japan). Immune responsiveness of 84 members from 18 healthy families to streptococcal cell wall (SCW) antigen was tested by antigen-specific, monocyte-dependent T-cell proliferation in virro. The SCW antigen was extracted from the streptococcal cell wall by pepsin digestion, and was purified by ammonium sulfate fractionation, gel filtration through Sephadex G- 100, and DEAE- Sepharose column chromatography. The maximum likelihood scoring method revealed that low responsiveness to SCW antigen was controlled by a single dominant gene. Log score for linkage between the gene controlling low responsiveness to SCW antigen and HLA was 3.709 at 8 (recombinant fraction) = 0.00 indicating a close linkage between the HLA and the gene controlling the low responsiveness to SCW antigen in vitro. The coculture of T cells from high responders with low-responder monocytes showed vigorous response to SCW antigen whereas T cells from low responders did not respond to this antigen even with HLA-haploidentical high-responder monocytes. Low responsiveness to SCW antigen was thus expressed on the T-cell level but not on the monocyte level. Furthermore, whole peripheral lymphocytes from low responders suppressed the immune response of HLA-D haploidentical high responders. The nylon-wool column-passed T cells from low-responder peripheral blood lymphocytes treated with anti-HLA-DR monoclonal antibody with complement completely abolished the immune response of the HLA-D
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haploidentical high responder. By using the monoclonal antibodies against various T-cell markers, it appeared that the suppressive activity was found on the T cell which was positive for Leu 2a but negative for Leu 3. The low responder did respond to SCW antigen after the Leu 2a-positive T-cell fraction was depleted from the peripheral lymphocytes by panning using anti-Leu 2a monoclonal antibody. All these observations clearly document the existence in man of an HLA-linked immune suppression gene which controls the low responsiveness to SCW antigen via the suppressor T-cell fraction. Dr. David G. Marsh (Johns Hopkins University School of Medicine, Baltimore, Md.) presented his studies of the genetics of the human immune response to pollen allergens. He said that studies of response toward inhaled allergens (e.g., pollens) provide a good model for understanding the genetics of human immune response because of the immunogenetically limiting, low-dose antigenic exposure and the wide array of highly purified allergens available. Two of the important genetic controls appear to be a non-HLA-linked IgE-regulating gene and HLA-linked genes. Immunoglobulin E responses toward allergens of molecular weight ca. 10,000 daltons, such as ragweed Ra3, appear to be controlled by both the IgEregulating gene and the HLA-linked Ir and Is genes; but response to the structurally less complex molecule, ragweed Ra5 (molecular weight 5000 daltons), appears to be confined to HLA-linked genetic control. Using extremely pure Ra5 (299.9% pure), Dr. Marsh has recently found that 36/38 (95%) of persons with detectable IgE antibody to Ra5 have Dw2, versus 30/139 (22%) of persons who have Dw2 in the RaS-negative (although ragweedallergic) category. This striking difference is significant (P < 0.0001). 1gG antibody responses to Ra5 were studied in 61 of the study subjects who had been treated for their allergy by injections of ragweed antigens. All 22 treated subjects who possessed Dw2 made good IgG responses, including 9 who were not allergic to Ra5. On the other hand, only 1l/39 Dw2-negative subjects made IgG responses to Ra5 following treatment. This difference was significant (P < 0.0001) and the level of the responses in DwZnegative subjects was significantly lower than that in Dw?positive subjects. These data provide perhaps the best evidence yet for HLAlinked immune response (Ir) genes in man. Thus, HLA-Dw2 provides an excellent marker for human immune response to Ra5. Probably the main reason why the association is so strong is because the structure of Ra5 is much less complex than those of most other allergens studied. Immune recognition of Ra5 may be restricted primarily to a single site on the molecule. Using model allergens of simple well-defined chemical structure like Ra5, Dr. Marsh stated that it should be possible to build up a “genetic fingerprint” of human immune responses relating antigenic structure to genetic loci of the HLA system. The final session was a panel discussion of the roles of H-2 gene products in immune recognition and regulation. The discussion was moderated by C. Garrison Fathman (Stanford University) and the participants were Dr. Eli Sercarz (University of California, Los Angeles), Dr. John Kappler (National Jewish Hospital,
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Denver), Dr. William E. Paul (National Institutes of Health) and Dr. Harvey Cantor (Harvard Medical School). There were three major areas of discussion during the panel session; one on the nature of antigen recognized by T cells, the second on the nature of the T-cell receptor, and the final being the nature of MHC restricting elements. Regarding the nature of antigen as recognized by T cells, the following points were made: (1) T cells and B cells recognize different epitopes. Actually, even different T-cell subpopulations recognize unique epitopes; (2) native and denatured antigens often cross-react totally; (3) peptide fragments are often just as (or more) effective than intact antigens; (4) antibodies against antigens do not block T-cell recognition; (5) T-cell recognition of antigen/MHC is not blocked by free antigen; (6) antigens taken up by antigen-presenting cells are not effective for approximately 1 hr. Are the antigens “processed” by these cells? In the discussion of the nature of T-cell receptor, it was pointed out that (1) recognition of antigen and MHC gene product is physically linked; (2) MHCrestricting element (A,B) and antigen (X, Y) must be on the same cell: (3) the interaction of A and X can be recognized by a specific T-cell receptor to yield a tight, ternary complex. Some clones which are specific for A + X also can recognize B + Y; however, such a clone would not recognize A + Y or B + X. Fusion of a T-cell specific for A + X to a T cell specific for B + Y yields a hybrid reactive to A +XandB + YbutnottoA + YorB +X. The following outline reflects the discussion on the nature of MHC restricting elements: Restricting elements = Ir gene products = serologically defined structures, K, D, L; IA, IE, (I-J?) (1) Relevant MHC expression is in the antigen-presenting cell, not the responding T cell; (2) gene complementation in expression of the IE structural molecule correlates with IE-linked Ir genes; (3) anti-MHC monoclonal antibody which detects structural products can also block T-cell recognition; (4) mutants which affect structural products also affect expression of the Ir gene and restricting element; (5) transfection with a cloned structural gene transfects the restriction element. Specific immune response (Ir) genes generally determine the ability or lack of ability of individuals to respond to distinct antigens. It is now clear that the products of Ir genes are I-region-associated (Ia) antigens. Ia antigens are found on antigen-presenting cells (APC), B lymphocytes, and some T cells. A critical aspect of their function is that they are corecognized, with antigen, by specific “histocompatibility-restricted” T lymphocytes and this corecognition is necessary for T-cell activation: thus, the absence of a suitable Ia molecule needed for the presentation of a particular antigen can underlie Ir gene control. Alternatively, Ir genes may affect the existence of regulatory suppressor cells which mask a clear potential for response to the antigen. Nonetheless, the reason that certain Ia molecules fail to allow responsiveness to a specific antigen has not been resolved. Two major possibilities exist. First, Ia molecules on APC may engage in an active interaction with antigen which is
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ET AL.
critical to the recognition of the antigen (or an antigen-la complex) by T cells. Such “antigen-la pairs” have been experimentally demonstrated. la molecules may be incapable of interaction with antigens for which they express a nonresponder phenotype or may interact in an essentially unrecognizable way. This may be referred to as the “failure of antigen-processing theory.” Alternatively, the reason that given Ia molecules are associated with unresponsiveness to specific antigen is that no T cells exist which are capable of recognizing the particular antigen-la pair. This “absence of T cells” may be due either to the lack of representation of genes encoding the relevant receptor(s) in the genome, from a failure to positively select T cells expressing responsiveness to that antigen- la pair in the course of intrathymic maturation and selection, or from the deletion of such T cells during the process of establishing self-tolerance. Data presented in the panel discussion supported the concept that there were two distinct types of restricting elements recognized by T cells in the context of MHC-restricted recognition. First, the lr gene products were synonymous with la, Class II molecules. Second, the restricting elements for cytolytic effector T cells were shown conclusively to be Class I molecules. This demonstration relied in part upon studies presented by Jeffrey Frelinger (University of Southern California, Los Angeles, Calif.), which showed that transfection of L cells with genes encoding a Class I molecule not expressed on the L cells would allow them to become effective targets of cytolytic effector cells whose restriction specificity was encoded within the gene transfected into the L cell. The question of the form of antigen recognized by T cells was addressed in part by preliminary data from Dr. Harvey Cantor’s laboratory. These data suggested that certain nominal antigens might be found in covalent linkage with l-region molecules, thus forming a binary complex of la and antigen in the absence of a T-cell receptor for antigen. That this cannot be a universal mechanism for l-region restriction of recognition of nominal antigen was agreed to by all participants. Dr. Sercarz presented evidence that T cells and B cells recognized different nonoverlapping epitopes on a multideterminant antigen. Surprisingly, very few epitopes make an imprint on the entire T-cell system, although it was pointed out that a characteristic hierarchy of utilization of epitopes exists so that even each potential epitope is not always presented. It was suggested that each set of antigenpresenting cells, addressing different subpopulations of T cells, might possess a limited and unique “recognition chemistry.” Finally, the consensus of the panel was that there are no hard data which would allow a concrete suggestion about the molecular nature of the T-cell receptor for antigen. It is anticipated that in the succeeding Midwinter Conferences, the molecular nature of the T-cell receptor will be described. Received
April
29, 1982; accepted
June 4, 1982.