Immunology Today, vol. 8, No. 11. 1987

It was fitting that the opening session of a symposium to mark the 50th anniversary of the discovery of the H-2 complex* was held at the Jackson Laboratory in Bar Harbor, Maine. Here, Peter Gofer and George Snell collaborated to demonstrate that mouse blood group antigen-2, described by Gofer in 1936, was the strongest of the histocompatibility antigens then under study by Snell. Snell officially opened the conference and tributes to Gofer and Snell were given by B. Amos (Duke University, Durham, USA) and J. Stimpfling (McLaughlin Institute, Great Falls), themselves pioneers in the study of the major histocompatibility complex (MHC). G. Hoecker (Chile), who worked with both Gofer and Snell, aptly compared the importance of Snell's introduction of congenic resistant strains to the analysis of histocompatibility with the use of haptens by Landsteiner to unravel the specificity of the humoral immune response. In his role as H-2's Virgil, J. Klein (Max Planck Institute, T0bingen, FRG) gave an elegant history of the 'golden age' of the MHC. Beginning with the origins of inbred mice at Miss Lathrop's farm and ending his history before the discovery of H-2 restriction, Klein lamented the introduction of modern science's 'wildwest ethics' during the expansion of interest in the MHC, which he said lessened the enjoyment and excitement of the earlier era. It is clear that the study of H-2 has become a field of scientific endeavor strikingly different from the pioneering immunogenetic studies that were done on a gene family with no known function other than the encoding of transplantation antigens and immune response genes. But in Klein's view the major mysteries of H-2 have already been unraveled and only the details of ils function remain for further investigation. Yet, given the central role of the MHC in immune function, its potential for revealing complex structure-function relationships, and the importance of the MHC supergene family in investigations on fundamental genetic processes in mammalian cells, it seems likely that H-2 will continue to surprise and confound us. Nonetheless, this 50th anniversary

Fifty years of//--2 research fr0mGe0rgeLCarbm meeting may be one of the last devoted to H-2 as a whole rather than to specialized aspects of its organization and function, and it was gratifying that such a high proportion of the investigators resDonsible for the development of our understanding of H-2 were present at the celebration. The presentations and posters which followed the historical perspective conveyed the excitement of post-golden age H-2 research. Although the tools of molecular biology are answering questions unapproachable even ten years ago, it remains clear that earlier 'tools' of H-2 research such as congenic and intra-H-2 recombinant strains persist as the foundation for much current effort. The historical perspective also was maintained throughout the conference by introductory 'overviews' from S. Nathenson (Albert Einstein College of Medicine, New York), P. Jones (Stanford), P. Doherty (John Curtin School of Medical Research, Canberra), H. McDevitt (Stanford), H. Festenstein (London Hospital), and L. Hood (Caltech).

Geneticorganization One possible reason for the impression that the H-2 enigma is essentially 'solved' is the remarkable progress that has been made in understanding the genetic organization of the MHC. So far, 50 genes have been isolated from the H-2 region of the BALB/c mouse; 34 of these are class I (including the histocompatibility antigens defined by Gorer and Snell), 7 are class II (originally defined as immune response genes) and 8 are unrelated or 'entrapped' loci (M. Steinmetz, Hoffman-La Roche, Basel; Hood T. Meo, Pasteur Institute). The K and / regions have been physically linked by cloning, chromosomal walking and pulsed-field electrophoresis into a cluster of 600 kilobases (kb). The D region has been physically linked to the Qa region in a 500 kb gene cluster and defines the location of the tumor necrosis factor c~ and 13 genes centromeric to D. Other clusters encompass the S region and Tia * 'H-2 antigens,genes,molecules,function'took regions. Clones linking the S region to either the K-I cluster or the Dplaceon 5-9 June1987.

Lt~) 1987. ElsevierPublications,Cambridge 0167 4919/87/50200

region cluster have not yet been isolated and large stretches of DNA intervene. Undoubtedly, interesting results will be forthcoming when the physical linkage map is complete and the uncloned stretches become available. For example, M. Bennett (Southwestern Medica! Center, Dallas) reported that the elusive hybrid histocompatibility (Hh) genes and possibly trans-acting regulators are located between S and D; these conclusions were based on his assessment of natural resistance to bone marrow transplantation in intra-H-2 recombinant mice. Although the Hh genes appear to encode 'recessive transplantation antigens', SClD (severe combined immunodeficiency) mice, which have an impairment in the ability to rearrange successfully immunoglobulin and T-cell receptor genes, are able to recognize Hh-incompatib!e cells (Bennett; G. Carlson, Jackson Lab.). The advances in the molecular biology of the MHC have also helped to reveal and analyse fundamental mechanisms in mammalian genetics. For example, sites for meiotic recombination are not distributed randomly throughout the MHC - 'hotspots' for recombination have been identified between the K and i regions, and within the Ec~ and El3 genes (Steinmetz; W. Lafuse, Ohio State Univ.; C. David, Mayo Clinic, Rochester). Repetitive DNA sequences similar to those of chi sequences have been found in the vicinity of some of these hotspots, but their involvement in crossover enhancement has not yet been demonstrated directly. Classical analysis of H-2 mutants suggested the occurrence of unusual processes, and thc~c have been confirmed by molecular analyses. Microrecombination among MHC class I genes appears to be responsible for the multiple nucleotide substitutions seen in several spontaneous Kb mutants; in at least some cases these gene conversion-like events occurred pre-meiotically during mitotic amplification of the germ line (J. Gelibter et al., Albert Einstein College of Medicine, New York). The role of gene conversion-like processes during evolution in generating the high

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degree of H-2 polymorphism remains controversial. Based on extensive analysis of wild mouse populations, F. Figueroa (Max Planck Institute. T0bingen, FRG) maintained that MHC polymorphism evolved over millions of years and was generated mainly by point mutations with little or no contribution of gene conversion-like mechanisms. In contrast, using oligonucleotide probes specific for alleles of H-2K, L Pease and co-workers (Mayo Clinic, Rochester) found that some sequences of H-2K were shared by genes in the Oa and T/a regions, consistent with the hypothesis that diversity can be generated by transfer of coding sequences among class I genes. Other polymorphic sequences were unique to H-2K and probably resulted from point mutation. The largest number of class I genes are in the Oa and T/a regions, but their funct=~n remains a mystery. C. Warner (Iowa State Univ.) described a Q-region gene, Ped, that influences the rate of preimplantation embryo development. The expression of the allele giving fast development was found to concord with the expression of Qa-2 on preimplantation embryos, consistent with the possibility that the Ped gene product may be a Qa-2 antigen. Probes specific for the transmembrahe r_~j=ion~of i..ndiv~'~! Q-reg~n genes (Hood) are being used to examine the tissue-specific and temporal expression of these genes, and should prove valuable in determining whether they are developmentally important. Some Q-Tla region products may also serve as restriction elements. The maternally transmitted antigen Hmt may be a class I antigen that presents the mitochondrial Mtf gene product to cytotoxic T cells (S. Richards and K. Fischer-Lindahl, Howard Hughes Medical Institute, Dallas). D. Murphy (Yale) described a novel Q-regioncontrolled determinant on B cells whose expression is also controlled by an I-region gene and raised the possibility that the Q-region product may be serving as a restriction element.

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H-2 restriction As expected, much time was devoted to the mechanism of/-/-2 restriction. The variety of approaches to the problem taken by many different labs have led to the consensus

that the peptide antigen must be bound by both the T-cell receptor and the class I or class II restricting element. Rather than outline the individual contributions that were presented, I will try to give a neneral picture of H-2 restri~ion anc[ of the problems that remain. The ability of class I and class II MHC molecules to interact selectively with different antigenic peptide fragments has been repeatedly demonstrated. This interaction has been assessed directly and through the use of monoclonal antibodies to block the binding of peptides. The use of both natural and engineered H-2 mutants is proving valuable in dissecting the nature of the antigen recognition by H-2 molecules. (A particularly attractive approach for in-vitro mutagenesis has been developed by Jeff Frelin~er and coworkers (Univ. North Carolina, Chapel Hill) that allowed the rapid creation of a library of all possible single base substitutions in the alpha-1 domain of I-I-2Dp.) The peptide bound to the MHC molecule is then recognized by the T-cell receptor. It is still not clear whether the binding site of the T-cell receptor is specific for the antigenic peptide alone, as presented by the class I or class II molecule, or whether the antigenic determinant includes selfMHC structures that must be recogIIIC.t~.,

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genic peptide. Related to this question is the nature of alloreactivity. R. Blanden (John Curtin School of Medical Research, Canberra) proposed that alloreactive T cells have a lower affinity than MHC-restricted T cells because the cell-surface concentration of H-2 is higher than that of H-2-viral antigen complexes on stimulatory cells. Another interesting aspect of H-2 restriction is the difference between class II-restricted recognition by T helper cells and the class I restriction generally seen in cytotoxic T cells. Several mechanisms that can account for MHC class restriction were presented. One obvious mechanism is the selective binding of 'accessory molecules' involved in the MHC/antigen/T-cell receptor complex; for example, C. Janeway (Yale) presented evidence that CD4 molecules dictate MHC class restriction by their simultaneous interaction with the T-cell receptor and class II molecules. Recycling of H-2 molecules from the cell surface

through endosomes and back to the surface can also be involved in class restriction. For example, B cells, which present antigen in association with class II molecules, do not recycle their class I molecules but do so extensively with their class II antigens (B. Pernis, Columbia University, New York). It is assumed that H-2-restricted antigen presentation involves internalization and processing of antigen and this has been visualized via electron microscopy (T. Delovitch, Univ. Toronto). However, the possibility that association (or even processing) occurs at the cell surface has not been eliminated. Although it is clear that selective binding of peptides by class II molecules can account in most cases for 'immune response gene' phenomena, it is still not known whether the T-cell receptor complex can recognize only peptide determinants bound to the 'antigenselective site' of MHC molecules or whether conformational determinants on unprocessed antigens that are physically associated with H-2 can also be recognized. M. Edidin (Johns Hopkins) presented physicochemical evidence for H-2 haplotype-selective close association between class I molecules and foreign antigen, gained by measuring fluorescence resonance energy transfer between fluoresceinated 132-microglobulin and Texas red-conjugated Sendal virus glycoproteins on fibroblasts. Regardless of whether this sort of interaction between non~,rocessed antigen and class I molecules is sufficient for H-2-restricted recognition by the T-cell receptor, such interactions could be important for inducing internalization and processing of antigen and might also be involved in the nonimrnunological functions of class I molecules such as the regulation of the functional expression of the insulin receptor (R. Goodenow, Univ. California, Berkeley). H-2 antigensand disease In his review on the discovery of H-2 restriction, Doherty emphasized the fact that the finding arose from studies on the mechanisms and genetics of resistance to disease. The role of H-2 in disease is, and is likely to remain for some time, a major focus of studies on the MHC. Festenstein reviewed the controversial early work on the possibility that a!tered H-2 antigens could serve as tumor-

, lmrnunology Today, vol. 8, No. 11, 1987

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specific antigens. H. Stauss (Univ. Chicago) presented recent work on the only tumor-specific antigens cloned to date and which have been shown to be novel class I MHC genes. Expression of the antigen encoded by one of these genes is essential for the rejection of the ultra-violet light-induced tumor from which it was isolated. The influence of I-I-2 expression on tumor rejection, progression and metastasis was emphasized by studies ~uch as those of G. Hammerling (Heidelberg) and associates in demonstrating that transfection of t-l-2 genes can lead to tumor rejection, probably by serving as restriction elements for tumorassociated antigens. Trans-acting deregulation of MHC expression by radiation-induced leukemia virus is one means by which the leukeinia cells can avoid the ho~t immune response (D. Meruelo, New York Univ.). While it is clear that increasing H-2 expression can lead to tumor rejection in some cases, results from John Frelinger's group (Univ. Rochester) emphasized that inducing H-2 alone may not be sufficient to induce rejection of all tumors, even those that are deficient in H-2 expression. Tissue-specific and locus-specific regulation of H-2 expression are importan, factors in susceptibility to pathological conditions such as the demyeiination caused by Theiler's virus (R. Melvold, Northwestern Univ., Chicago; Rodriguez, Pease and David). Elucidation of the molecular mechanisms responsible for regulating H-2 expression is progressing rapidly- for example, in identifying the distinct sequences responsible for responsiveness to the interferons - but much remains to be learned. Particularly relevant to disease is expression of MHC antigens by discrete cellular subpopulations within the central nervous system. Tissue-specific post-translational modification of expression could also turn out to be very important, and an exciting approach for analysing intracellular transport and processing is provided by the panel of class II variant cell lines developed by L. Glimcher and co-workers (Harvard) and the class I-variant somatic cell lines described by Nathenson and colleagues. The efforts of the transplantation immunogeneticists in the 'golden age of H-2' have had far-reaching efl.:ts. The dired medical benefits

of organ transplantation are of obvious importance, but they are minor compared with the impact that H-2 immunogenetics continues to have on immunology and mammalian genetics. Although some may feel that the major secrets of the MHC have been revealed, the 'master trickster' (to quote Klein) may have

some surprises still to disclose, perhaps enough to warrant a symposium commemorating the 75th anniversary of the discovery of the major histocompatibility complex. George Carlson is a Staff Scientist at The Jackson Laboratory,Bar Harbor, MA 04609, USA.

Mapping the autoimmunizingepitopeson acetylcholinereceptors tramGili In the autoimmune disease myasthenia gravis (MG), patients experience muscle weakness and fatiguability owing to loss of functional acetylcholine receptors (AChR) at the neuromuscular junction. The loss rn,~ybe causedby modulation of the receptors by anti-AChR autoantibody and~or by complement-mediated focal lysis at the post-synaptic membrane. The structure of the AChR has been studied primarily because of its function as a neurotransmitter receptor. The~enefit of these studies to immunologists investigating MG was illustrated at a recent meeting* in which the use of the products of DNA technology to study the ,,,,,,~u,~u,~y~atmechanisms of MG was among the topics discussed. More than 85% of patients with MG have autoantibodies to conformational determinants on the human AChR, a pentameric structure in which the subunits ((x2133'8) are arranged to form a central ion channel. Approximately 60% of autoantibodies from these patients are directed towards an area on the ec-subunit which Tzartos and Lindstrom 1 have named the main immunogenic region (MIR; Fig. 1). The position of the MIR on the two (x-subunits is of significance in the pathogenesis c~:i ~he disease, since modulation occurs via cross-llnking of receptors by antibody. More accurate definition of the antibodybinding sites within the MIR and also of the T-ceU recognition site(s) in the AChR could lead to the development of immunotherapeutic strategies. The recent publication of the amino *First EuropeanConferenceon Myasthenia Gravis, Maastricht,The Netherlandsheld on 15-16June1987. 1987. ElsevierPubhcat,ons.Cambridge 0167

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acid sequence of all four subunits of the AChR from the electric fish (Torpedo) 2-4 and the eL- and -ysubunits of human AChR has facilitated studies into the localization of the immunodominant sites within the AChR. The use of synthetic peptides was a recurring theme at the conference.

B-cell epitopes Two approaches have been used to define the MIR on the (x-subunit: antibody mapping by direct binding to expressed DNA products or synthetic peptides; and competition between patients' sera and monoclonal antibodies (mAbs)in binding to native AChRs. From a full length cDNA for the (x-subunit of mouse AChR, T.Barkas (Lausanne)was able to construct fusion proteins for several parts of the extracellular domain. Probing immunoblots with (x-bungarotoxin or mAbs confirmed that the major (x-bungarotoxin binding site was within the sequence cx179-216 and restricted the MIR to a region between residues 37-85. Some anti-MIR mAbs required the presence of (x6-37 to stabilize their binding, suggesting that a subsidiary loop of the MIR exists within this region. A study has also been made of the binding of anti-MIR and anti-AChR antibodie~ found in MG to an overlapping series of synthetic 18-mer peptides collectively representing 83% of the human (~-subunit. Several, but not all, anti-MIR mAbs bound preferentially to a peptide correspondin~j to residues 65-78 (S. Tzartos, Attains~,and sera from patients with genera{ized ,MG also bound to this fragment in enzyme-linked immunosorbent assays (B. Conti-

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Fifty years of H-2 research.

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