Workshop I MHC and Transplantation

Immunogenetics Laboratory, LMU Munchen, Pettenkoferstr. Sa, SOOO Munchen 2, 'fMPI for Neurology, Josef-Schneider-Str. 10, S700 Wurzburg, FRG

1.1 Immunogenetics of Myasthenia gravis

A. ANDREAS-ZIETZ, E. KELLER, V. ARBARA, B. SCHALKE", H. WEKERLE'f, S. SCHOLZ, H. ZANDER, and E. D. ALBERT

It appears that the clinical syndrome of Myasthenia gravis must be heterogeneous and that the association with HLA is restricted to only a subgroup of these patients. The HLA associations observed in Caucasoid patients are with HLA-Al, B8 and DR3, and it remains unclear as to whether the disease is more strongly associated with HLA-BS or with DR3. In an attempt to better characterize the various subtypes of Myasthenia gravis with immunogenetic markers and in order to more closely identify the localisation of the Myasthenia gravis disease susceptibility gene within the HLA complex, we have begun to test a large number of patients from throughout Germany. A total of 215 patients were typed for HLA-A, B, C, and DR antigens using conventional serology. A preliminary analysis of these data identifies the following factors as being highly associated with HLA-BS and DR3: Myasthenia gravis per se, Myasthenia gravis and female sex, age of onset below 40 years in both sexes, thymic hyperplasia in both sexes, high antibody titers (>60) against acetylcholin receptor. Investigat­ing the controversial issue of the association with BS or with DR3, we have found the following distribution: BS+/DR3+: 56 (26 %) p=O.OOOl; BS+/DR3-: 12 (5.6 %) p= 0.016; BS-/DR3+: 25 (11.6%) p=0.0003. In the controls, these frequencies are 7.3%, 2.4% and 4.S % respectively. The most likely explanation is, that the disease susceptibility gene for Myasthenia gravis must be localized between HLA-B and -DR, because both BS+/DR3-, and BS-/DR3+ cases are marginally increased. In an attempt to utilize the high power of resolution of DRlDQ typing by RFLP, we have investigated a total of 96 patients and have found that all DR3 positive patients carry one subtype of DR3 (DR«3a»: 27, DR<<3b»: 0), while in the controls we found 5 individuals positive for DR<<3a» and 5 individuals positive for DR<<3h». We conclude from these data that the disease susceptibility gene for Myasthenia gravis could be localized in the DR region.

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Institut fUr Immunologie, Goethestr. 31, 8000 Miinchen 2, FRG

1.2 Characterization of a murine Qa Class 1 gene which is only expressed in tumor cells

D. BEVEC and E. H. WEISS

The Qa region of mice carrying the H-2k haplotype encodes only a reduced number of the Qa region genes found in other mouse strains, and no H-2k Qa gene products have been identified so far. We isolated a class I sequence from an AKR cosmid library linked to a gene homologous to the Q4 gene in the C57BL strain. The AKR Q5 gene is completely different by restriction enzyme mapping from any other Qa region gene described so far. We determined the entire DNA sequence of this gene and used an AKR Q5 specific oligomer derived from an unique sequence in exon 4 to map the gene in recombinant mouse strains to the Qa region. Only mice of the H-2k haplotype contain AKR Q5 homologous sequences. This gene, in contrast to the other Qa genes, codes for an intact transmembrane domain which is similar to

H-2Dd. The DNA and protein sequence of AKR Q5 differ significantly from other class I sequences. Using the oligomer as probe the AKR Q5 gene was shown to be transcribed in transformed cell lines only. A 1,6 kb Q5 mRNA is present in several AKR thymomas and the fibroblast cell line L929. The gene is not transcribed upon transfection into different target cells.

Weare currently replacing the putative AKR Q5 promoter region with an equivalent fragment of the H-2Db gene in order to obtain transcription of the gene and expression of the gene product.

This work was supported by the Deutsche Forschungsgemeinschaft (We 1067-2) and by the Genzentrum Miinchen.

Biogen Research Corp., Cambridge, MA, U.S.A.

1.3 Regulation of MHC class II genes. Differential expression of la in a macrophage cell line

E. C. BOTTGER, M. A. BLANAR, and R. A. FLAVELL

Treatment of the murine macrophage cell line P388D.1 with interferon-y(IFN-y) leads to a disparate Ia gene expression pattern. IFN induced the accumulation of Ea and Au mRNA's, both of which were not expressed in untreated cells. In contrast, Ap and Ep mRNA's were constitutively expressed at a low level and showed a delayed time-course of induction. Blocking protein synthesis with cycloheximide inhibits the IFN-y-mediated En mRNA accumulation in P388 cells. Unexpectedly, cycloheximide alone induces Aa, but not Ap, Ea, or Ep mRNA. Nuclear run-off assays revealed that IFN-y induces Ia mRNA at the transcription level. These results establish a role for transcriptional activation in the IFN-y-mediated expression of Ia genes and indicate that induction of Ea mRNA in P388 D.1 cells by IFN-y requires de novo synthesis of a protein intermediate. Our findings furthermore suggest that the regulation of expression at the mRNA level may be different for the distinct class II genes.

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Biogen Research Corp., Cambridge, MA, U.S.A.

1.4 Transcriptional activation of HLA-DRa by interferon-y requires a novel trans-acting protein

E. C. BOTTGER, M. A. BLANAR, and R. A. FLAVELL

Stimulation of the human epithelial-like cell-line, HeLa, with y-interferon (IFN-y) results in the induction of steady-state levels of HLA-DRa mRNA. Using a sensitive RNase-mapping procedure, we can detect induced DRa mRNA as early as 8 h following treatment with IFN-y; maximal accumulation occurs by 48 h. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-y-induced accumulation of DRa mRNA, indicating that de novo synthesis of a transacting protein factor is required for major histocompatibility complex class II gene induction. Nuclear run-off transcription assays revealed that IFN-y acts by directly stimulating the rate of transcription of the HLA-DRa class II gene. Similarly, IFN-y increased the transcription rate of the HLA-A2 class I gene as well as that of the human invariant chain gene. IFN-y-induced transcription of HLA-DRa and the invariant chain gene was blocked by treatment with cycloheximide but IFN-induced transcription of HLA-A2 was unaffected. Our findings indicate that transcriptional induction of HLA-DRa and the invariant chain gene by IFN-y requires the action of a novel trans-acting protein.

Institute of Immunology, University of Munich, Goethestr. 31, D-8000 Munich 2, FRG

1.5 Differential expression of class II transcripts and surface expression in activated T cells

M. DIEDRICHS and D. J. SCHENDEL

HLA-class II molecules are highly polymorphic transmembrane glycoproteins each consist­ing of an a heavy chain and a noncovalently associated ~ light chain. These molecules are encoded in man by genes located in the HLA-D region on chromosome 6. Three major subregions, DR, DQ and DP, each containing several a and ~ chain gene sequences, have been identified. Class II molecules show distinct tissue distributions:: they are constitutively expressed on many cells of the immune system such as mature B cells, monocytes, dendritic cells but only on T cells following antigen activation. We were interested to compare class II expression between a B cell-derived lymphoblastoid cell line (LCL) and activated T lympho­cytes from the same donor as well as among T cells following allogeneic stimulation, activation with soluble antigen (purified protein derivative of tuberculin (PPD», or mitogen activation with phytohemagglutinin (PHA). The class II surface expression on T cell lines or clones was determined using locus-specific monoclonal antibodies and the presence of class II mRNA was analyzed by Northern blot hybridization with DR, DQ and DP ~-chain specific eDNA probes. Nearly all activated T cells showed expression of DR molecules, DP expression was variable and only T cells belonging to the CD4+ subpopulation expressed DQ. No DQ expression was seen on CD8+ cell lines. The detection of DR, DQ and DP mRNA transcripts correlated with the patterns of surface expression, indicating that regulation of gene expression accounts for the differences in surface expression among the various activated T cells.

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llnstitute of Immunology and Genetics, German Cancer Research Center, D-6900 Heidel­berg, FRG, 2Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands

1.6 HLA-C transgenic mice: immunological competence and reactivity with HLA alleles

O. DILLI, T. HUNTI, s. KocHI, F. KIEVITS2, P. IVANYI2, and G. J. HAMMERLING1

The human major histocompatibility complex encodes three classical class I antigens, HLA­A, Band C. Of these HLA-A and B are well known to act as strong transplantation antigens and as restriction molecules for recognition of foreign antigen by cytotoxic T lymphocytes. In contrast, little is known about HLA-C, and it is not clear whether HLA-C has the same functional properties as HLA-A and B. Transgenic C57Bl/6 mice expressing the HLA-Cw3 gene were established. Functional studies demonstrated that transgenic skin was rapidly rejected by normal C57Bl/6 mice and that cytotoxic T lymphocytes generated by immuniza­tion of the Cw3 transgenic mice with influenza and Sendai virus were restricted by the Cw3 molecule. These data suggest that HLA-Cw3 has immunological functions comparable to those of HLA-A and B. Next, the reactivity against other HLA alleles was investigated. After immunization of Cw3 transgenics with B6 fibroblasts transfected with HLA-A2 both H-2 restricted and unrestricted A2 specific CTL were observed by using appropriate targets. Normal and Cw3 transgenic B6 mice contained these CTL in comparable frequencies (1/ 20,000 unrestricted A2 specific CTL and 1/4000 H-2b restricted A2 specific CTL). These data show that the expression of an HLA antigen in the thymus does not skew the T cell repertoire towards a more effective recognition of HLA antigens.

Dept. of Immunology, University of Kiel, D-2300 Kiel, FRG

1.7 Immunogenicity of T -depleted bone marrow is strongly reduced by additional depletion of accessory cells: implications for prevention of graft rejection

P. DREGER,]. TREUMER,]. HARPPRECHT, J. STEINMANN, and W. MULLER-RuCHHOLTZ

In clinical bone marrow (BM) transplantation, prevention of graft-versus-host reaction can be achieved by T-depletion of the graft. Unfortunately, T-depletion is complicated by a strongly increased incidence of graft rejection. Therefore, we studied the possibility of preventing rejection of a BM graft by reducing its immunogenicity, rather than unduly increasing the immunosuppressive conditioning of the host. Considering the fact that a graft's own accessory cells (AC) playa crucial role in its immunogenicity, we have studied our newly developed monoclonal antibody (MoAb) K31 for its effect on the accessory capacity and the immunogenicity of human BM, and compared it with clinically established T -depleting MoAbs including anti-lymphocyte/monocyte MoAbs, such as CAMPATH-l. K31 recognizes a p180 glycoprotein expressed on nearly all cells of the human lymphoid and monocytic lineages but not on stem cells or any other hemopoietic cell. Results: 1) Pretreatment with K31 + rabbit complement (C) completely eliminated the capacity of irradiated human BM cells to support mitogen-induced proliferation of purified autologous T cells, indicating the absence of AC. On the other hand, the accessory function of

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the BM was not affected after similar pretreatment with the established MoAbs. 2) When tested in a MLC-like assay where MoAb+C treated BM cells served as stimulators, K31 treated BM was unable to induce proliferation of allogenic T cells, whereas the allostimulatory capacity of BM treated with die established MoAbs was not reduced. 3) Addition of autologous peripheral blood adherent cells to K31 + C treated BM restored its accessory function as well as its allostimulatory capacity, whereas autologous T cells had no effect.

Conclusions: 1) Depletion of AC in addition to T cells strongly reduces the immunogenicity of a BM graft and thus may prevent its rejection. 2) Because passenger cells with accessory function are known to contribute largely to the immunogenicity of solid organ grafts as well, reduction of immunogenicity of solid organ grafts may be similarily achieved by ex vivo perfusion with K31.

llnstitute of Immunology and Genetics, German Cancer Research Center, D-6900 Heidel­berg, FRG, and 2Memorial Sloan Kettering Institute, NY, U.S.A.

1.8 HLA transgenic mice as a novel tool for the generation of allele specific HLA antibodies

G. J. HAMMERLING\ O. DILLl, S. YOUNG YANd,and U. HAMMERLINd

So far, conventional HLA typing depends mainly on the use of human alloantisera. Attempts to replace these antisera by murine monoclonal antibodies have not been very successful, because upon immunization with HLA the mice produce predominantly antibodies against monomorphic HLA determinants (determinants present on most HLA alleles). In contrast to normal mice, HLA transgenic mice should be tolerant for most HLA determinants and therefore be suitable for production of allele specific antibodies. We have established HLA class I transgenic mice, expressing HLA antigens on the same tissues as H-2. Immunization of an HLA-B7 transgenic mouse with splenocytes from an HLA-Cw3 transgenic mouse resulted in the establishment of several Cw3 specific monoclonal antibodies. With some of these mAb the Cw3 antigen could be even split into 2 subgroups. These findings show that HLA transgenic mice are a useful tool for the generation of allele specific HLA mAb and for the detection of new HLA specificities. It is anticipated that such mAb will refine HLA typing in the future.

lThe Charing Cross Sunley Research Centre, Lurgan Avenue, London, W6 8LW, U.K. 2Philipps-Universitat, Marburg, FRG, 31mperial Cancer Research Fund, London, U.K.

1.9 Effect of Iymphokines on MHC class II gene expression in a murine B cell transfectant carrying a DR gene construct

U. HARTWIGl.2, A. VENKITARAMANl, H. lKEDA3, J. TROWSDALE3, and M. FELDMANNl

Little is known about the molecular mechanisms by which the lymphokines interferon y(IFNy), interleukin 4 (IL4) and tumor necrosis factor a (TNFa) regulate MHC Class II gene expression. We analysed the effect of these lymphokines on MH C Class II gene expression in a murine B cell transfectant carrying a DR gene construct in an attempt to distinguish their

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actions at the transcriptional and posttranscriptional levels. A cosmid vector containing 8 tandem copies each of the complete DRa and DR~ cDNA sequences individually driven by SV40 promoters was transfected into the murine B cell lymphoma, A20, by electroporation and one stably expressing transfectant cell line was used for further study. Recombinant murine IFNy, IL4 and TNFa enhanced expression of endogenous murine MHC Class II genes in the transfectant cells both at the mRNA and protein level, with distinct kinetics and degrees of enhancement. However, these lymphokines had differential effects on the degree and kinetics of expression of DR mRNA and protein. These results suggest that multiple mechanisms may be involved in the effect of lymphokines on MHC Class II expression. Inferences regarding transcriptional and post-transcriptional effects of the lymphokines will be discussed.

Abteilung Innere Medizin III, Vniversitat Vim, Steinhovelstr. 9, D-7900 Ulm/Donau, FRG

1.10 Primary in vitro induction of cytotoxic T cells in human HLA­identical donor-host pairs

T. HOFFMANN, M. THEOBALD, D. BVNJES, and W. HEIT

The immunological mechanisms underlying clinical tolerance, graft versus host disease (GvHD) and graft rejection (GR), respectively, following human HLA identical MLC negative bone marrow transplantation (BMT) are not clearly defined so far. While minor histocompatibility antigens (MiHA) in mice could be defined by the use of inbred strains, corresponding information is not available in man. Apart from serological investigations, much interest lies on the in vitro generation of effector cell populations directed against non MHC determinants. We present here data from five BMT patients and their HLA identical MLC negative donors. Primary in vitro stimulation with donor and host cells prior to BMT was performed under limiting dilution (LD) conditions and cultures were tested for cytotoxic­ity subsequently. In all cases we found high frequent reactivities against the stimulating HLA identical target cell with frequencies ranging from 1/570-1/3800. They equal frequencies established by HLA different alloantigeneic stimulation of the same responder cells (1/1000-11 4000). In all five pairs, a decrease of specific lyses at higher responder cell numbers was observed; in 2/5 cases a decline of positive wells at high responder cell numbers allowed frequency estimation of suppressor cells, which were found to be 10-fold lower than the concerning frequencies of cytotoxic cells. Split well assays and clonal analyses confirmed minor reactive cultures not to be reactive against 3rd party-, K 562- or autologous targets. Besides, phenotyping of individual clones showed the pattern of classical cytotoxic T lym­phocytes. None of the clones tested showed humoral activity for interferon, tumor necrosis factor or IL 2.

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Institute of Microbiology and Immunology, and '·Dept. of Medicine, University of Ulm, D-7900 Ulm, FRG

1.11 Frequency of donor-reactive helper T lymphocytes in renal transplant recipients

J. Jooss, B. ZANKER*, H. WAGNER, and D. KABELITZ

The mechanism(s) of graft tolerance and graft rejection are still poorly understood. Humoral (antiidiotypic antibodies) as well as cellular components (suppressor, cytotoxic, helper T cells) have been supposed to contribute to the overall outcome of «tolerance» or «rejection». We have previously described that a selective reduction of donor-reactive cytotoxic T lymphocyte precursors (CTL-p) occurs in certain recipients of a well-functioning kidney allograft. Since a substantial number of patients with well-functioning kidney graft did not show a reduction of donor-specific CTL-p, we asked in the present study if changes occurred in these patients at the level of circulating donor-reactive helper T lymphocytes. To this end we have established a limiting dilution (LD) culture system to measure frequencies of alloreactive interleukin-2 (IL2) producing helper T lymphocyte precursors (HTL-p). Following this protocol, graded numbers of T cells are cocultured with irradiated (donor or third party) EBV-transformed stimulator cells in the absence of additional «factors». After 3-4 days, supernatants are screened for IL2 on IL2-dependent CTLL test cells, and frequencies of alloreactive HTL-p are determined based on the distribution of positive and negative wells at a given responder cell concentration. Typically, one out of 300-800 unseparated (E+) T cells produces IL2 in this system. Data will be presented on the frequency of donor-reactive HTL-p in renal allograft recipients. Our preliminary results indicate that no changes of circulating donor-specific HTL­p are seen in patients with a selective reduction of donor-specific CTL-p. On the other hand, out of ten patients with stable graft function and unchanged frequency of donor-specific CTL­p, one patient could be identified with no detectable donor-reactive HTL-p. These results indicate that nonreactivity at the level of donor-reactive IL 2 producing helper cells may possibly contribute to the clinical state of «tolerance» in kidney transplant recipients.

Institut flir Immunologie und Genetik, Deutsches Krebsforschungszentrum, 1m Neuenheimer Feld 280, 6900 Heidelberg, FRG

1.12 The role of the a 3 domain of MHC class I antigens in cytotoxic T lymphocyte responses

U. KALINKE, G. J. HAMMERLING, and B. ARNOLD

Class I MHC antigens which are important for immune reactions are composed of three distinct domains, a 1,2 and 3, of which a3 is proximal to the plasma membrane. Work with mutant and hybrid class I antigens led to the conclusion that most determinants which are recognized by cytotoxic T lymphocytes (CTL) are generated by the a 1 and a2 domains. The a 3 domain does not affect the fine specificity. Since the xeno immune reaction is much weaker than the allo immune reaction although the difference between xeno and self is much more greater than the difference between allo and self, we asked whether we can localize structures on class I antigens which are involved in species specific interactions. Using targets expressing hybrid antigens between mouse H-2kk and human HLA-A2, we found that the species of the a3 domain is of importance for CTL-Iysis. For instance, primary anti H-2Kk CTL could not lyse target cells expressing hybrid class I antigens consisting of a 1 and a2 of H -2K k and a 3 of

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HLA-A2. In addition, preliminary experiments showed that after sensitization of mice with syngeneic tumor cells expressing a hybrid antigen consisting of a 1 and a 2 of HLA -A2 and a 3 of H-2Kk CTLs could be obtained, which lysed targets of another MHC haplotype expressing the hybrid antigen. This is remarkable, since sensitization with cells expressing the whole HLA-A2 resulted in CTLs which recognized the HLA-A2 only on syngeneic but not on allogeneic transfectants. Obviously the replacement of a 3 of HLA-A2 by a 3 of H-2Kk led to an H-2 unrestricted recognition of the target. The question, which protein on CTL can be responsible for a species specific interaction will be discussed.

Immunogenetics Laboratory, LMU Miinchen, Pettenkoferstr. 8a, FRG;'MPI for Psychiatry, Kraepelinstr. 10, 8000 Miinchen 2, FRG; 2Neurological Hospital, Karls University, Katharinska 30, 120000 Prague, Czechoslovakia

1.13 DNA typing for HLA-DR and -DQ in 126 patients with narcolepsy

E. KELLER, A. ANDREAS-ZIETZ, B. ROTH2, H. SCHULZ" S. SCHOLZ, and E. D. ALBERT

A total of 126 patients with the full-blown clinical picture of narcolepsy (including cataplexy) was investigated using a system of DR/DQ typing with Taq I and EcoRI RFLP analysis. All but two patients were positive for HLA-DR2. The DR2 negative patients are clinically indistinguishable from the DR2 positive cases. All DR2 positive patients carry the same subtype of DR2 (DR«2a», which corresponds to the serologically defined DRw15). All DR2 positive patients also carry the same split of DQwl (DQw«la», which corresponds to the serological definition of DQw6).The DR2 negative cases are also negative for this split of DQwl. We conclude from these results that the association between narcolepsy and DR2 is not absolute, a fact which is borne out also by the recent report of NEELY 1987, a substantial percentage of DR2 negative cases among Negroid patients with narcolepsy. Our data do not allow to distinguish whether the susceptibility gene for narcolepsy is located in the DR region or in the DQ region. The results however are compatible with the assumption of a narcolepsy gene in the DQ region. We have also analysed the second DR haplotype of the 124 DR2 positive narcolepsy patients. To our surprise, we found that only two patients, possess DRw6, while we would have expected the presence of DRw6 in 15 % (18 patients). This very low frequency of DR w6 is also found but never discussed in the literature. On the other hand, we find the alleles DR2 and DRS overrepresented on the second haplotype of DR2 positive patients. We conclude from these findings that the presence of DRw6 has a protective effect against the development of narcolepsy and that in general the second HLA haplotype in the patients is not neutral with respect to disease susceptibility.

Supported by SFB217, project A2.

Institut fiir Immunologie und Genetik, Deutsches Krebsforschungszentrum, D-6900 Heidel­berg, FRG

1.14 IFN-y induces assembly of MHC class 1 heavy chains with ~2microglobulin

D. KLAR and G. J. HAMMERLING

Assembly of histocompatibility class I heavy chains with ~2microglobulin (~2m) has been shown to be necessary for cell surface expression. Studies on the H-2 class I deficient but

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interferon-y (IFN-y) inducible fibrosarcoma BC2 and the lung carcinoma CMT 64.5 showed that transfection of allogeneic H -2 class I genes leads to expression of the class I proteins, but they are found only intracellularly and not on the cell surface. Inspite of large amounts of ~2m in the cells, no association of the transfected class I chain with ~2m was observed. However, stimulation with IFN-y induced assembly and surface expression. These findings show that the assembly of class I heavy chains with ~2m is not a spontaneous event but appears to be a process which needs regulation. The defect of these cells may be the lack of factors necessary for assembly which can be induced by IFN-y. In other systems, proteins have been described which may be involved in the assembly of multichain complexes like immunoglobulins and influenza hemagglutinin. In case of nonassembly these proteins are associated with the free chains (1, 2). However, we have not yet found proteins in BC2 and CMT 64.5 which are associated with free class I heavy chains. Investigations which clarify this defect are under way.

1. GETHING, M-]., K. McCAMMON, and]. SAMBROOK 1986. Cell 46: 939. 2. HAAS,]. G., and M. WABL. 1983. Nature 306:387.

Hospital for Joint Diseases, Department of Rheumatic Diseases, New York University School of Medicine, 301 E. 17th St., New York, NY 10003, U.S.A.

1.15 Mapping the MHC class II epitope dependency of a polymorphic monoclonal antibody by site directed mutagenesis

B. LANG, P. F. MERRYMAN, R. M. CRAPPER, J. P. DALTON, J. SILVER, and R.WINCHESTER

Using site directed mutagenesis and cDNA mediated gene transfer, we studied the binding characteristics of the polymorphic monoclonal antibody (MoAb) 109d6, which detects an epitope that is associated with susceptibility to rheumatoid arthritis. This epitope is encoded by both, DRw53~r and DRw10~1-chain genes as shown by immunoprecipitation and transfection. As these ~-chains are identical in their third diversity region (amino acid 67 to 73) only, which include the distinctive sequence of three consecutive Arginine residues at positions 70 to 72, we postulated that this region is important in the formation of the epitope. Therefore we mutagenized the DRw10B1 gene at position 70, converting an Arginine residue to Glutamine and the DRw53B4 gene at position 71, converting an Arginine to Threonine. Each of the native and mutagenized cDNAs were transfected together with DRA cDNA into mouse fibroblasts using the calcium phosphate method. Transfectants expressing the highest levels of membrane Ia were selected by fluorescence activated cell sorting, cloned and studied for reactivity with a panel of monoclonal anti-DR antibodies and MoAb 109d6. Cells transfected with native or mutagenized DRw10B1 or DRw53B4 cDNAs showed comparable expression of monomorphic DR-antigens recognized by DR-specific MoAb SG157. However, the cells transfected with the mutagenized DRw10B1 gene were greatly reduced in their reactivity with MoAb 109d6 as compared to the unmutagenized transfectants. In contrast, there was no significant difference in reactivity with MoAb 109d6 of cells transfected with the DRw53B4 gene mutagenized at codon 71. From these results we conclude that the Arginine residue at position 70 is crucial in the formation of the epitope recognized by MoAb 109d6 in DRw10~1-chains. On the other hand, the Arginine at position 71 does not appear to be critical for the MoAb 109d6 binding in DRw53~rchains.

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Dept. of Immunology, Brunswiker Str. 4, D-2300 Kiel, FRG

1.16 T cell depletion of bone marrow transplants (BMT) by magnetic immunomicrospheres (MIMS) in a MHC fully allogeneic mouse model

G. LEYHAUSEN, S. WINOTO-MORBACH, M. BLANK, and W. MULLER-RuCHHOLTZ

In MHC fully allogeneic BMT virtually all donor marrow cells that are irreversibly determined for MHC alloreactivity must be eliminated. An established approach in mouse models is T cell depletion by incubation of BM with Thy-1 antibody and complement. However, the application of this principle is restricted to situations in which complement­activating antibodies and adequate sources of complement are available. We here provide the first evidence that magnetic cell separation with MIMS, which is not limited to complement­activating antibodies, is as effective as T-cell depletion by cytotoxic measures. Magnetic microspheres were coupled to rabbit-a-mouse IgM and in a second step to monoclonal Thy-1.2 antibody. C57 (H-2b) BM cells were incubated with these MIMS and separated by a single run through several magnetic fields. Ten to twelve week old lethally irradiated Balb/c (H-2d)

mice received 5X107 C57 BM cells. Results: Purity of the separated BM cells, tested by microscopic evaluation, was over 99 %.

Vitality of these cells was 94-98 %. In 17/18 recipients of MIMs-treated BM cells permanent survival and chimerism were achieved. Restitution of immunocompetence and induction of specific allograft tolerance were shown by normal rejection of third party skin and permanent acceptance of BM donor strain skin. In contrast, 21121 control animals died within 25-38 days.

Conclusions: 1) MIMS provide high purity of cells by magnetic separation. 2) MIMS do not affect the vitality of BM cells. 3) MIMS separation is nontoxic for hemopoetic stem cells. 4) MIMS separation can be performed in a model system to completely prevent GVHR in MHC fully allogeneic BMT. 5) MIMS allow specific cell depletion independent of complement.

Institut fiir Immunologie, Universitat Miinchen, Goethestr. 31, D-8000 Munchen 2, *Institut fur Experimentelle Immunologie, Deutschhausstr. 1, D-3550 Marburg, FRG

1.17 Identification of a functional HLA-class I gene, closely linked to HLA-A

G. MESSER, J. RAGOUSSIS*, A. ZIEGLER*, G. RrETHMULLER, and E. H. WEISS

Distinct from the human leucocyte antigens HLA-A, -B, and -C of the major histocompati­bility complex, we isolated a complete, genomic class I gene (coscda12) from a human cosmid library. A 1.5 kb intragenic low copy probe used for long distance mapping, revealed a close neighbourhood to the HLA-A locus. A 50 kb Sadl restriction fragment hybridized both to the HLA-A2 and the coscda12 probe. In order to characterize a possible gene product, we transfected the cosmid into murine P815 and L929 cells and selected stable transfectant clones. Of the HLA reagents tested, only the monomorphic antibodies W6/32 and B9.12.1. reacted specifically with the coscda12 transfectants. In order to determine the molecular structure, the cda12 gene was sequenced. It shows a similar exonlintron organization as the known class I genes. But it has a high divergence from the classical class I sequences. In order to study the

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expression of this functional gene, linked to HLA-A, a cda12 specific oligonucleotide was synthesized, to investigate various tissues and cell lines. We will use the single copy probes of cda12 for chromosomal walking experiments to physically link HLA-A with cda12 and study this region in more detail.

This work was supported by Deutsche Forschungsgemeinschaft (SFB 217).

Ilnstitute of Pathology, Heidelberg University, 21nstitute of Immunology and Genetics, German Cancer Research Center, Heidelberg, FRG

1.18 Abrogation of ~rmicroglobulin but not of HLA-A, B, C heavy chain expression on the transcriptional level in human colon carcinoma cells

F. MOMBURG1 and S. KOCH2

Immunohistological studies have shown that in a considerable proportion of colon car­cinomas, HLA-A, B, C antigens are lacking in all tumor cells or in a subpopulation. These studies have been performed with monoclonal antibodies, such as W 6/32, against framework determinants of class I antigens, the formation of which is dependent on the association of HLA-A, B, C heavy chains with ~Tmicroglobulin (~2m). Using HC-to, a monoclonal antibody reacting with free HLA-B and C heavy chains, and a rabbit antiserum to free HLA­A, B, and C heavy chains, we have re-examined 15 colon carcinoma tissue samples that were completely (11) or partially (4) negative with HLA-A, B, C:~2m antibodies and ~2m antibodies in the tumor cell population, but exhibited a strong staining of non-neoplastic stromal cells. In serial frozen sections, these class I antigen/~2m-negative tumor cell (sub )populations showed a mostly strong expression of free heavy chains, with the exception of one tumor which contained W6132-/HC-I0+ and W613Z-IHC-I0- subsets. In situ hybridizations were per­formed with three of these tumors in order to address the question whether the expression of ~2m is blocked on the transcriptional or translational level. 35S-labeled RNA probes for ~2m (HindIII-Sacl fragment from pS4.11 cloned into pGEM7) and HLA-B8 (Pstl-Sacl fragment from pMF28 cloned into pGEM3) strongly hybridized to stromal cells, whereas only the HLA-B8 probe hybridized to the neoplastic cells indicating the absence of detectable amounts of ~2m mRNA.

Institute of Immunology, University of Munich, Goethestr. 31, D-8000 Munich 2, FRG

1.19 RFLP and biochemical typing of HLA-B27 variants

E. NOESSNER, E. LEDERER, and D. J. SCHENDEL

The addition of RFLP analyses to serological, cellular and biochemical methods enables HLA class I variants to be easily distinguished. Studies with cytotoxic T lymphocytes (CTL) provided the first indication that HLA-B27 could be split into various subtypes (1). Subse­quent analysis with I-dimensional isoelectric-focussing (IEF) revealed that several of the CTL­defined subtypes could be biochemically distinguished as well (2). While the HLA-B27W, K, and D variants are easily identified by their IEF patterns, the B27C and B27W subtypes are

XXth Meeting of the Society of Immunology . 129

difficult to discriminate by IEF. We isolated genomic DNA from B27 cells, including local and B27 subtype reference cells (kindly provided by B. Breur-Vriesendorp and P. Ivanyi, Amster­dam). Following digestion with BglII and hybridization with an HLA-B locus specific cDNA probe (kindly provided by E. WeiB of our institute) two RFLP patterns associated with B27 could be identified. Fortunately the B27C and B27W variants have different RFLP, thus the problem of distinguishing between them in IEF can be clearly resolved by RFLP analysis.

1. BREUNING, M. H., C. J. LUCAS, B. S. BREUR, M. Y. ENGELSMA, G. G. De LANGE, A. J. DEKKER, B. BIDDISON, and P. IVANYI. 1982. Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and the role in self-recognition. Hum. Immunol. 5: 259.

2. NEEFJES, J. J., B.S. BREUR-VRIESENDORP, G. A. van SEVENTER, P. IVANYI, and H. P. PWEGH. 1986. An improved biochemical method for the analysis of polymorphism of HLA class-I antigens. Definition of new HLA class-I subtypes. Hum. Immuno1.16: 169.

Institut ftir Immunologie und Genetik, Deutsches Krebsforschungszentrum, 1m Neuenheimer Feld 280, und *Pathologisches Institut, Universitat Heidelberg, 6900 Heidelberg, FRG

1.20 Regulation of the MHC class II-associated invariant chain

U. PESSARA, F. MOMBURG*, and N. KOCH

The regulation of the MHC class II antigen associated invariant chain (Ii) was studied in stimulation experiments. The expression of the human Ii gene was examined in the human carcinoma cell line HT-29, whereas the regulation of the murine Ii gene was studied in rat fibroblasts transfected with the murine Ii gene and by in vivo experiments. In HT-29, which is constitutively negative for Ii and MHC class II antigens, Ii-expression was not induced by IFNy-treatment alone. Only the combination of TNFa together with IFNy led to a strong induction of Ii, whereas TNFa alone did not show any stimulatory effect. A different pattern of regulation was observed for the transfected murine Ii gene: It showed a low constitutive expression, which could be enhanced by IFNy. But again, a combination of IFNy and TNFa had the most pronounced effects on Ii-expression; TNFa alone had only some effect on expression of murine Ii. To investigate Ii stimulation in vivo, mice were treated i.v. with IFNy, TNFa, or a combination of both. The most striking effects were observed in renal tubular epithelium: Although IFNy alone showed a stimulatory effect, a combination of IFNy and TNFa strongly enhanced the expression of Ii, suggesting that this synergistic effect potentially occurs under physiological conditions.

Institut flir Immunologie der Joh.-Gutenberg-Universitat Mainz, FRG

1.21 SV40 transformed bone marrow macrophage clone Bl exhibits a distinct pattern of MHC class II molecule synthesis and antigen presentation function

S. QUERNER, F. J. SCHNEIDER, A. B. RESKE-KUNZ, and K. RESKE

In contrast to B cells, dendritic cells and a number of T cell clones unstimulated murine bone marrow macrophages (BMMcIl), derived from in vitro cultures of BM cells in the presence of

130 . XXth Meeting of the Society of Immunology

M-C~F are constitutively la-. Treatment of this BMM<I> population with rIFN-y induced high levels of mRNA for the polymorphic n and ~ proteins including the non MHC encoded invariant y chain. This de novo appearance of n, ~ and y transcripts was not paralleled at the protein level, because metabolic labeling in conjunction with specific immunoprecipitation and twodimensional gel electrophoresis revealed n, ~-heterodimers without associated proteins of the invariant y chain group (F. J. SCHNEIDER et al. 1987. Eur. J. Immunol. 17: 1235). In the course of establishing cloned accessory cells of BMM<I> origin by immortalization through SV40 transformation, the BMM<I> clone B1 was obtained. Based on cytochemical and functional criteria B 1 cells appear arrested in the promonocyte state of BMM <I> differentiation. In contrast to BMM<I>, untreated B1 cells produced significant message levels for n, ~ and y that were increased to maximal values by IFN-y stimulation. In addition, low levels of synthesis of n, ~ and y were observed in naive B1 cells. The synthesis of all three chains was substantially augmented in response to IFN-y. As in the case of nonnal BMM<I>, the influence of IFN-y on the class II molecule synthesis in B1 cells was strictly dose- and time-dependent. Unstimulated as well as IFN-y treated B1 cells were active in cooperating with T clone cells inducing antigen-specific T cell proliferation. Thus, B 1 cells appear to be instrumental in elucidating the steps involved in the differential translation of the polymorphic and the invariant chains in BMM<I>.

Supported by the Deutsche Forschungsgemeinschaft, SFB 311, Mainz.

Institut fiir Imm~nologie und Genetik, Deutsches Krebsforschungszentrum, 1m Neuenheimer Feld 280, 6900 Heidelberg, FRG

1.22 Deletion subclones of murine invariant chain: investigations of interaction between MHC class II antigens and invariant chain

K. SCHENCK and N. KOCH

The invariant chain (Ii) is a glycosylated membrane-bound protein that associates intracellu­larly with MHC class II antigens (Ia). The genes for human and murine Ii have been cloned, and their gene organization, nucleotide and deduced amino acid sequences have been deter­mined' previously.

Although it is tempting to speculate that Ii plays a role in la-mediated antigen presentation and/or processing, no such function has been clearly demonstrated to date. To determine the site in Ii that is responsible for interaction with la, a series of controlled deletions were introduced into the gene for murine Ii. The resultant constructs were transfected into la­positive/Ii-negative cells, and the transfectants are being tested for expression of the truncated Ii gene and for association of la and Ii.

Institute of Immunology, University of Munich, GoethestraBe 31, D-8000 Munich 2, FRG

1.23 Monoclonal antibodies recognize antigens on activated cells

B. SCHWEIGHOFER and R. WANK

By immunisation of mice with activated cells, two monoclonal antibodies were found which recognize antigens on EBV transfonned lymphoblastoid cell lines, leucemic cells and PHA-

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blasts. The recognized determinants segregate with the HLA-A, B segment and are strongly associated with the antigen HLA-A24. The antibodies were analysed in the single cell ELISA (1) and FACS as well as in blocking studies. The binding patterns were very similar but not identical with the reaction pattern of the T-cell clone 234 (2) which recognizes an HLA-A24 associated determinant on activated lymphocytes of the immunizing cell. Further characterisa­tion of the epitopes recognized by these monoclonal antibodies using isoelectric focussing and precipitation experiments will be undertaken

1. HOLZMANN, B., J. P. JOHNSON. 1983. A beta-galactosidase linked immunoassay for the analysis of antigens on individual cells. J. Immun. Methods 60: 359.

2. WANK, R. 1988. A human T-cell clone recognizes a Qa- or Tla-like antigen in man. In: Histocompatibility Testing 1988. Springer-Verlag, Berlin (in press).

German Cancer Research Center, D-6900 Heidelberg, FRG

1.24 Correlation between H-2 class-I-antigen-expression and susceptibility to natural killer (NK) cells

K. STURMHOFEL and G. J. HAMMERLING

NK cells are assumed to play an important role in the primary defence against malignant cells. For several tumor cells an inverse correlation between expression of H-2 class I-antigens on the one hand and susceptibility to NK cells and in vivo growth ability on the other hand have been reported. Thus, low H-2 tumor cells grow and metastasize more vigorously in vivo than H-2 positive cells, probably because they escape destruction by NK cells. In order to investigate the influence of H-2 class I on NK susceptibility, we selected from the strongly H-2b positive El4lymphoma several variants which lack H-2 expression. These class I negative clones are significantly better lysed by NK cells than the parenteral E14, and they also cannot grow in syngeneic hosts when inoculated i.v. or i.p. To determine whether this inverse correlation between H-2 expression and NK lysis is fortuitous, we restored H-2 expression by transfection with cloned H-2 genes. The de novo expression of transfected Db and Kb antigens rendered the El4 clones resistant to NK lysis. El4-null clones transfected with the neoR gene, still negative for class I, did not become resistant to NK lysis. These findings clearly demonstrate that the H-2 class I molecules themselves can influence NK susceptibility. However, the mechanism is not yet clear. Since MHC antigens can be viewed as primordial structures it is possible that they bind to and modify the as yet unknown target structures for NK cells.

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Institut fUr Immunologie und *Pathologisches Institut der Joh.-Gutenberg-Universitat Mainz, FRG

1.25 Stimulation of allogeneic T -lymphocyte responses by large unilamellar vesicles bearing conformation isomers of Lewis rat RT1.B specific class II molecules

J. SYHA, R. WEITZEL, H.-P. DIENES*, and K. RESKE

By use of the noncrossreactive mAb Mrc-OX3 and MRC-OX6, we identified two serologi­cally discrete a,~-heterodimers that were shown to represent stable conformation isomers of RT1.B (I-A equivalent) class II molecules residing at the cell surface of LEW rat spleen cells (K. RESKE, R. WEITZEL. 1985. Eur. J. Immunol. 15: 1299). The two isomers were suggested to arise within the trans Golgi compartment from an MRC-OX6 reactive biosynthetic intermedi­ate made up of terminally glycosylated a, ~ and invariant chains by release of the mature chain prior to membrane insertion. We developed an artificial lipid bilayer system as useful tool in assessing functional activities of reconstituted MRC-OX3 and MRC-OX6 specific a,~­heterodimers. Because small sized vesicles proved unsuitable in this respect, large unilamellar liposomes (LUL) were prepared by a detergent dialysis procedure and were morphologically characterized by electron microscopy. Affinity chromatography of nonionic LEW rat spleen cell extracts over monoclonal immunoadsorbents yielded highly purified a,~-heterodimers that were still recognized by the respective mAb. Coculture in the presence of allogeneic (LEW. A VN) T lymphocyte enriched spleen cells with LUL that contained the purified class II antigens led to a significant activation of the T cells as measured by 3H-TdR incorporation. Activation was induced by both MRC-OX3 and MRC-OX6 reconstituted liposomes demon­strating that two cell surface forms of the RT1.B class II molecule are functionally competent.

Supported by the Deutsche Forschungsgemeinschaft, SFB 311, Mainz.

Inst. Med. Immunology, Charite, Clinic for Internal Medicine, Charite, Humboldt-Univer­sitat Berlin, and Institute of Immunology, Klinikum Steglitz, Freie Universitat Berlin

1.26 Expansion of a CD 3+4-8- TCR alpha/beta- T cell subset in long­term kidney allograft recipients

H. D. YOLK, P. REINKE, R. VON BAEHR, and T. DIAMANTSTEIN

In 35 out of 90 long- term kidney allograft recipients an unusual high proportion of T cells bearing immature phenotype CD 3+4-8- TCR alpha/beta- recirculates in their peripheral blood. These cells are able to produce interleukin-2 and to proliferate in response to interleukin-2 following stimulation by different T cell mitogens. It seems to be probable that this subpopulation is identical with one observed in the thymus, in fetal blood, in the epidermis and in a minority in the peripheral blood and which show a rearrangement of TCR gamma/delta complexes. Although we found a great number of such T cells in two cases with an acute rejection crisis in the patient's urine sediment, we are not able to bring the detection of immature T cells in the peripheral blood in a safe relation to acute or chronic rejection episodes or to other complications (infections, glomerulonephritis etc.). Since many patients show reproducible either increased or normal values of such T cells, sometimes over periods of several months independent on the clinical course, a genetic component is probable. The observations might be an expression of an individual sensitivity of T lymphocyte maturation on immunosuppressive therapy.

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Dept. of Immunology, Brunswiker Str. 4, D-2300 Kiel, FRG

1.27 The electromagnetic separation principle: a basis for human pancreatic islet transplantation

S. WINOTO-MoRBACH, G. LEYHAUSEN, K. ULRICHS, and W. MULLER-RuCHHOLTZ

Successful human islet transplantation requires large amounts of viable islets which must be depleted of the strongly immunogenic exocrine tissue. The well established method of hand­picking in rodent models is far too laborious and time-consuming for the number of islets needed in human transplantation. In earlier studies, we adapted our magnetic microspheres (MMS) to large scale purification of collagenase-digested rat islets by construction of a specific MMS-lectin complex which binds selectively to rat exocrine tissue and showed that the separated islets remained in vivo functionally intact. We investigated lectins for their binding specificity to human exocrine tissue.

Results: 1) Ulex europaeus agglutinin I (UEA-I), which proved to be highly selective for rat exocrine tissue, is unsuitable in the human as it also binds strongly to endocrine cells. 2) Of the other 18lectins tested in immunofluorescence studies, 8/18 do not react with either exocrine or endocrine tissue, 9/18 show weak reactivity with exocrine tissue and only one reacts strongly and selectively with exocrine tissue. 3) Only the strongly selective lectin can be coupled to MMS without loss of function. 4) This particularly useful MMS-lectin complex appears to be similarly effective at electromagnetic separation in the human as UEA-I is in the rat 5) The viability of islets after separation is over 90 %.

Conclusions: 1) Our experience with lectins in rat and human pancreas studies show that each islet donor species requires a thorough search for a selectively binding lectin. 2) The electromagnetic separation principle is effective, simple, and fast and may solve the problems of quantity and purity in human islet transplantation.

Abteilung Immungenetik der Universitat, Gottingen, FRG

1.28 Sequence analysis of class I cDNA clones of the rat major histocompatibility complex

W. WURST, J. SCHMIDT, E. ROTHERMEL, and E. GUNTHER

Two groups of class I genes can be distinguished in the rat major histocompatibility complex (RTt), which are separated from each other by the class II gene cluster encompassing DP, A, and E-like genes and the class III region with the C4 and Bf genes. The RT1.A region determines classical class I antigens and the RT1.C region H-2Qa-like molecules. For further characterization of class I genes, six different class I cDNA clones have been isolated from an expression library constructed from poly(A +) mRNA of mitogen-stimulated T lymphocytes. Clone-specific sequences have been subcloned from the 3' end of the clones and four of them could be mapped to the RT1.C region. Sequence comparison reveals strong homology to class I genes of other species. One of these clones (11/3) carries a long insert - 2.4 kb instead of 1.0 to 1.3 kb found for the other inserts. Clone 11/3 has an open reading frame of 1.3 kb, which is followed by a 3' untranslated region of 1.1 kb. The coding region shows a mosaic-like

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structure. Two blocks of about 340 nucleotides each are homologous to class I sequences, namely to parts of exon 4 and to exons 5 to 8, which code for the transmembrane and cytoplasmatic parts of class I molecules, and to exon 2, respectively. The 5' part of about 340 kb, however, fails to show homology to class I sequences. With a 5' end gene probe clone 11/3 could be mapped to the RTl.C region, and by Northern blot analysis transcripts of 3.1 kb could be detected in lymphocytes, kidney, liver, testis and brain. By Southern blot analysis, this probe can be shown to detect three cross-hybridizing fragments in mouse and human genomic DNA. Clone 11/3 might code for a novel cell surface MHC molecule.