Workshop E: Ig, TCR, MHC Genetics

WORKSHOP E

19, TCR, MHC Genetics

'paul-Ehrlich-Institut, Langen, Germany; 2Amgen Center, Thousand Oaks, CA, USA; Institute, Ontario Cancer Institute, Toronto, Ontario, Canada

3 Amgen

E.l Official nomenclature for Tcell receptor variable gene segment families

Multiple DNA and protein sequence alignments have been constructed for the mouse and human T cell receptor alb, ~ and y (TCRA/D, B and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence a name was derived, which complies with the standard nomenclature (WHO Bull 71: 113-115, 1993). The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. This system is applicable to all species and all TCR loci characterized at any level from the first cDNA sequence to full mapping. By discriminating between alleles at the same locus versus genes from different loci, based on the characterization of true alleles and on restriction mapping combined with PCR analysis, we were able to reduce the number of greater than 150 different human TCRBV sequences in the database to a repertoire of only 65 unique TCRBV gene segments. An extension of this analysis to the over 100 TCRA V sequences results in a predicted repertoire of 44 different TCRAV gene segments. Our general observation that a member of a certain mouse subfamily is closer to a human V gene segment than to a member of another mouse subfamily supports the hypothesis to trans-species evolution. A novel alignment program revealed that despite their extreme diversity, the variable peptides consist of a relatively large number of functionally conserved amino acid residues. "·B. Arden and T. W. Mak sit on the WHO-lUIS standing committee on TCR designation.

142 . 25th Meeting of the Society of Immunology Institut fur Virologie und Immunbiologie and 'Neurologische Klinik der Universitat Wurzburg, Wurzburg, Germany

E.2 Analysis of superantigen (SAg) reactivity of alleles of rat TCRBV8.2 segments supports the crucial role of the CDR4 as superantigen contact point A. ASMUSS, T. HOCHGREBE, G. GIEGERICH l , T. HONIG, and T. HERRMANN Analysis of the superantigen (SAg) reactivity of TCRBV alleles may help to understand SAg-TCR interaction. Here was describe strain-specific differences of the response of TCRBV8.2 rat cells to the bacterial SAg SEB and the viral SAg of mtv-7. RT-PCR products of mRNA of eight TCRBV8.2 positive hybridomas obtained from four SAg non-responder strains were directly sequenced and compared to the SAg responsive TCRBV8.2 of LEW. Both differ by 7 aa substitutions at position 14, 51 and 52 (CDR2) and 70-73 (CDR4 - the proposed SAg binding site). The aa sequence of the new TCRBV8.2 was confirmed in 4 of 9 clones containing genomic PCR products obtained with TCRBV8.2 specific primers. The remaining 5 clones contained an additional TCRBV8.2 like element which has the responder-type CDR4 sequence. However, its pseudogenic nature (deletion of one nucleotide) argues against previously made speculations on TCR-SAg interaction based on its presumed expression OEM [1994] 179: 63). Finally, the SAg responsive TCRBV8.5 and the SAg unresponsive TCRBV8.2 allele differ in their CDR4 only at position 71, suggesting that CDR4 residue 71 is important for binding of SEB and mtv-7 SAg to TCRBV8.2.

Institute of Exp. Immunology, University Marburg, Marburg, Germany

E.3 Isolation of a putative MHC class I a chain gene fragment in skates M. BRANDL, B.JASPERT, and K.-U. HARTMANN In a number of fish species, for example in carp, rainbow trout, zebrafish and shark, MHC class I and II genes have been isolated. In this study a gene fragment of the skate, which is capable of belonging to a MHC class I a chain gene, has been isolated by polymerase chain reaction analysis (PCR). PCR was carried out with the genomic DNA of a skate liver (raya montagni, male). For amplification by PCR two highly degenerated oligonucleotide-primers were used. These primers correspond to the regions around the two conserved cysteines in the third domain of MHC class I chain of vertebrates (i.e. human, mouse, chicken, carp, shark) (1). Restriction sites were added at the 5'ends of each primer. The PCR products were analyzed by 1 % agarose gel electrophoresis. The obtained product had a length of about 200 nucleotides. The amplified DNA fragments were cloned into Bluescript vectors (Stratagene) and the nucleotide sequence of two clones, RI and RII, was determined by the chain termination method (Sanger). Compared with the membrane-proximal domains of the MHC class I molecules of other vertebrates the predicted amino acid sequence of the examined clones showed homology in more than 15

25th Meeting of the Society of Immunology . 143 residues. In summary, our first results indicate that expression of MHC-like molecules also exist in skates. 1. HASHIMOTO et aI., PNAS 1992, vol. 89, pp. 2209-2212.

IT echnische U niversitat, Institut fur Virologie und Immunologie, Dresden; 2Arztliche Gutachtergemeinschaft; Niederdorf; 3BGF A, Abt. Mol. Genetik, Bochum, Germany

E.4 Associations between anticentromere and antitopoisomerase I responses and HLA class II alleles in silica-associated systemic scleroderma K. CONRADI, H.-P. RIHS 3,J. MEHLHORN", K.-H. FRANK I, and X. BAUR 3 Anticentromere (ACA) and antitopoisomerase I antibodies (anti-Topo I) are markers for progressive systemic sclerosis (PSS) and can be detected years before disease manifestation. Oligotyping of MMC-II alleles has revealed a strong association of these autoantibody responses to sequences of MHC-II ~-chain molecules. To look for a possible genetic influence of autoantibody responses and PSS development in uranic miners with intensive quartz dust exposure we performed non-radioactive HLA-typing for DRB1, 3, 4,5, DQB1, and DPB1 with 73 DIG-ll-ddUTD labelled SSOs in 80 uranic miners, 29 silica-associated (sPSS) and 49 idiopathic PSS (iPSS) patients. In anti-To po I positive sPSS patients DRBl"'0301 and DQBl""0201 and in ACA positive sPSS patients DRBl""0800 and DQBl""0402 were significantly increased compared with unrelated controls and the studied iPSS group. Looking for «susceptibility epitopes» for the ACA response we found that 92.3 °lc, of sPSS and 83.3 % of iPSS in contrast to 100 'Yo described by REVEILLF et al G. Clin. Invest. 89 (1992), 1208) have DQB1 alleles with a polar glycine or tyrosine at position 26. In the case of the anti-Topo I response we couldn't find any association to previously described candidate epitopes and DQB1 alleles (DQBl""tyr 30, "TRAELDT 71-77). It can be suggested from this study that more than one shared polymorphic sequence in HLA class II alleles may be important for the specific anticentromere and anti-Topo I autoimmune response.

Rheumaklinik und Rheumaforschungsinstitut, Aachen, Germany

E.S Comparison of microcytotoxicity test system and ELISA for the detection of HLA-B27 T. DICK, R. MIERAU, and E. GENTH The determination of HLA-B27 is frequently used in the diagnosis of seronegative spondarthropathies, especially ankylosing spondylitis (AS). About 90 % of patients with AS carry the B27 allele. We compared the determination of cell surface B27 using a standard microcytotoxicity assay (MA) and the determination of soluble B27 by ELISA using patient sera. We tested 125 patients: a) 40 without B7 or B27, b) 19 with B7 without

144 . 25th Meeting of the Society of Immunology B27, c) 9 with B7 and B27, d) 5S with B27 without B7, according to the results of the MA. In 4 cases (all group b) the ELISA system was not able to type for B27. In the other cases the results of the groups a, c, d completely agreed in the two test systems. In group b) 13 patients were equally typed in both tests, two patients were B7 pos., B27 neg. in the MA and B27 pos. in the ELISA. In both cases borderline aD were recognized in the ELISA system which were misinterpreted by the software algorithm of the evaluation program. In 9S.6 % the results of the two test systems were identical. All tested B27 neg. AS patients were negative in both test systems. These data suggest that typing of patients with seronegative spondarthropathies for B27 can be performed using a standard MA or an ELISA system with nearly identical results.

11. Dept. ofInternal Medicine, University of Mainz, Mainz, Germany; 2Mucosal Immunity Section, Laboratory of Clinical Investigations, and 30ffice of the Director for Intramural Research, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA

E.6 TCR-VB gene usage by C04+ and C08+ T cells in patients with common variable immunodeficiency

Differential TCR-Va and V~ gene family expression occurs in CD4+ and CDS+ T cell subsets. We now used CDS+ and CD4+ T cell subpopulations of 5 patients from a recently defined subgroup of CD8high, CD57high patients with common variable immunodeficiency (CDShi CVI) (Blood S2:192, 1993) and 5 normal volunteers as a model to test the hypothesis that differences between CD4+ and CD8+ T cell V~ expression are strongly influenced by environmental factors such as antigen or superantigen. PBMC were separated by density gradient centrifugation, CD2+ cells obtained by a rosetting technique and CD4+ and CDS+ T cells purified by negative selection using immuno-magnetic beads. Total cellular RNA was isolated by phenol-chloroform extraction and reverse-transcribed using oligo dt. TCR-C~ mRNA content of CD4+ and CDS+ samples was determined using a quantitative RT-PCR technique as described (DNA and Cell Biology 12: 217, 1993) and adjusted to the same basis. Patient matched CD4+ and CDS+ T cell cDNA was then amplified within the same PCR run using primers specific for TCR-V~ families 1-20. Analysis of mean control CD4+ and CDS+ T Cell V~ gene usage frequencies shows that variability of CD4/CDS subset differences between CD4+ and CDS+ V~ values are 20-fold less than among individual V~ families (0.537 vs 0.026). Variability in TCR-V~ gene family usage frequencies is similar in controls and CVI patients but interestingly the mean absolute CD4/CDS difference is significantly greater in CVI patients (p < 0.0004). These increased differences in CD41 CDS TCR-V~ usage frequencies were equally distributed and not significantly different from one another. Only V~12 was a significant outlier with a CD4/CDS difference significantly greater than the other differences (p < 0.001). These findings demonstrate that environmental antigens can alter TCR-V~ family gene usage frequencies on mature CD4+ vs CDS+ T cell subsets in humans. They further support the concept that CDS hi CVI patients are both exposed to antigens which address T cells in the context of MHC (giving rise to a variety of increased CD4+ICDS+ T cell V~ familiy differences) and some (or one) antigens which address T cells bearing V~ families without regard to MHC context (giving rise to the V~12 difference).

25th Meeting of the Society of Immunology . 145 Institute of Transfusion Medicine, University of Kiel, Kiel, Germany

E.7 Fc parts of FcyR monoclonal antibodies do not functionally inhibit FcyR I and II B. FLESCH and]. NEPPFRT Functional inhibition of Fcy receptors (FcyR) by antibodies binding with their Fab part at or nearby the IgG binding site is reported. But it may also be speculated that there is a blocking of FcyRs by the Fc part of FcyR monoclonal antibodies via a three-molecular complex in that way that one FCyR antibody molecule binds specifically with its Fab part to the corresponding FcyR and with the Fc part to another FcyR. Corresponding observations had been made by us using HLA class I monoclonal antibodies inhibiting FcyR I and II specific phagocytosis. We tested this hypothesis using the monocyte immune phagocytosis inhibition assay with FcyRI and FcyRII specific targets. Phagocytosis of red blood cells (rbc) sensitized with anti D of the human isotype IgG 1 (hlgG 1) is mediated by FcyRI specifically, whereas FcyRII mediates phagocytosis of targets sensitized with anti glycophorin A of the mouse isotype IgG 1 (mlgG 1). Monocytes of at least 3 FcyRIl high responder (HR) and 3 low responder (LR) individuals were used in each assay. We tested the following mouse monoclonal antibodies: 22.2 and 32.2 (FcyRI specific) and IV.3, CIKM5, KB6I, FLIS.2, FLIS.26, 2EI and 4IHI6 (all specific for FcyRII, 41H16 recognizing only the HR phenotype on human monocytes). Monocytes from HR individuals ingested hlgG 1 as well as mlgG 1 sensitized targets (phagocytosis rates 69.0 % and 61.7 %) whereas LR monocytes selectively ingested the FcyRI specific hlgG1 sensitized rbc (71.3 'X,). Both FcyRI antibodies did neither inhibit FcyRI specific phagocytosis of hlgG 1 sensitized targets (55.0 'Yo and 46.7 % for LR; 73.S 0;\, and 47.1 % for HR) nor FcyRII mediated phagocytosis via the Fc part of the antibodies (62.4 % and 60.3 % for HR). All but one FcyRIl antibodies totally inhibited FcyRII specific phagocytosis of mlgG 1 sensitized rbc (rates between 0.0% and 2.7 % for HR) but did not significantly inhibit FcyRI specific phagocytosis in any case (rates between 46.0 % and SO.2 % for LR and HR). KB6I partially inhibited FcyRII phagocytosis of HR monocytes (21.7°1<). There is no functional inhibition of FcyRI or II by the Fc parts of the noncorresponding FcyR monoclonal antibodies.

IInstitut fur Virologie und Immunologie der TU Dresden, Dresden, Germany; lLudwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland; ) Arztliche Gutachtergemeinschaft Niederdorf, Niederdorf, Germany

E.8 Association of TNFa microsatellite alleles with progressive systemic scleroderma (PSS) K.-H. FRANKl, M. FOSSEI 1 , C. V.]ONGENEEL1 , K. CONRADI, A. SCHWAIl I, and]. MEHLHORN) Previously we could show a prevalence of the HLA-alleles DRBP0301 and DQBP0201 in anti scl-70-positive PSS patients, associated with quartz dust exposure. These DR3, DQ2 alleles are known to be in a linkage disequilibrium with the TNFa2 microsatellite

146 . 25th Meeting of the Society of Immunology allele and correspond with high TNFa production (POICOT et al. 1993. Eur. J. Immunol. 23: 224-231). Therefore we studied the TNFa microsatellite allele distribution in idiopathic and silica-associated PSS (iPSS, sPSS).

n iPSS

37

sPSS

24

PSS

61

Danish controls

150

TNFa2 + (TNFa212 or 2/0 +)

TNFa2 -

19(51.4%) (2 [5.4 %]) 19(79.2%) (5 [20.8 %]) 38 (62.3 %) (7 [9.8 %])

18

P = O.ot8

5

P < 0.001

23

P < 0.001

46 (30.7%)

104 (69.3 %)

The statistical significance is further increased in DR3, DQ2 patients. The data support the notion that TNFa may playa role in the pathogenesis of sPSS. Supported by the BMFT (07 NBL 03).

I. Medizinische Klinik, Homburg, Germany; 1 Rheumaklinik Aachen, Aachen, Germany

E.9 Expression of a human IgA rheumatoid factor in a phage expression system 1 A. GAUSE, G. CARBON, W. lUNG, R. MIFRAU , and M. PFREUNDSCHUH Heavy and light chain fragments of an IgAx rheumatoid factor (RF) were expressed as recombinant proteins in E. coli in order to test whether this recombinant antibody expression system is useful for analysis of human RF. A RF producing hybridoma which was established from a patient with rheumatoid arthritis (RA) and of which the nucleotide sequences of the V -genes encoding heavy and light chains are known (MIERAU et al. 1992) was used to clone the heavy and light chain from cDNA by specific PCR. The individual heavy and light chain fragments were cloned into the Immunozap Expression Vector (Stratagene) which in second step are combined into one vector. Identity of the heavy and light chain genes with the original sequences was shown by DNA sequencing. The expressed proteins were purified and compared to the binding properties of the original protein from the hybridoma supernatant. Therefore, a functional RF-enzyme immuno assay has been established which allows for the determination of the RF activity of the individual heavy and light chain fragments expressed as recombinant proteins in E. coli. By means of this test the RF-activity of the monomeric IgAVx protein could be demonstrated. The comparison of the binding properties of the recombinant protein and of the protein from the hybridoma will show whether this expression system will facilitate the analysis of human RF, especially of small nubers of RF producing cells and cells infiltrating synovial tissue.

25th Meeting of the Society of Immunology . 147 1Institut fur Klinische Immunologie und T ransfusionsmedizin, J ustus- Liebig-U niversitat, Gie/~en; 2 Abteilung fur Hamostaseologie und T ransfusionsmedizin, KerckhoffKlinik, Max-Planck-Institut, Bad Nauheim, Germany; 3 Serbio Research-Laboratories, Gennevilliers, france

E.10 Heparin-associated thrombocytopenia (HAT): isolation of the antibody and characterization of a multi molecular PF4heparin complex as the major antigen A. GREINACHER 1, B. POTZSCH 2, J. AMlRAI 3 , v. DUMMEL 1, A. EICHNER 1, and C. MUELLER-EcKHARDT 1 Sera of 35 patients with HAT, strongly reactive in the serotonin release assay (SRA), were assessed in a platelet factor 4 (PH )/heparin ELISA. Both tests correlated closely (p = 8.44 x 10- 5 ). We have isolated the HAT antibody by absorption and elution of HAT sera using human endothelial cells. Eluates gave similar results as the sera in the ELISA (p = 3.04 x 10-6 ), which also correlated very closely with the SRA (p = 1.45 x 10- 7). This demonstrates that HAT antibodies bind to the same epitope on platelets and on endothelial cells. High heparin concentrations released PF4 in a dose-dependent manner from microtiter plates if PF4/heparin, but not if PF4 alone, was covalently linked. Concomitant to the release of PF4, binding of HAT antibodies to PF4/heparin decreased, as indicated by median optical density (OD) OD 0.947 in the presence of buffer to OD 0.181 in the presence of 100 IU/ml heparin. Binding of 3 eluates was not inhibited by high heparin concentrations and they reacted also with PF4 alone. We conclude that multimolecular PF4/hcparin complexes represent the major antigen in HAT, which are present on platelets and endothelial cells. This multimolecular complexes form immune complexes after HAT antibody binding which concomitantly alterate platelets and endothelial cells. This may explain the thromboembolic complications in HAT.

lChildren's hospital of the friedrich-Alexander University, Erlangen, Germany; 2 Dept. of Genetics, Med. Institute of Bioregulation, K yushu University, fukuoda, Japan; 3Rheumakinderklinik, Garmisch-Partenkirchen; 4Labor fur Immungenetik, Kinderpoliklinik der LMU, Munich, Germany

E.ll DQA1 upstream regulatory region: polymorphism of the Y-Box in early onset pauciarticular-JCA

J. P. HAASl, A. KIMURA2, H. TRUCKENBRODT3, and E. D. ALBERT4 The upstream regulatory region (URR) of DQA1 was found to be polymorphic recently, with a least ten defined variants (QAP: 1.1,1.2,1.3,1.4,1.5,2.1,3.1,3.2,4.1,4.2). Most of these variants are characterized through sequence variations in the region between -210 to -240 bp upstream of the CAP site of DQA1-Exon 1. Besides this hypervariable region the promoter region of DQA1 includes cis-acting elements, known from other MHC-class II promotors, like the NFK-B binding site, Z-, X- and Y-Boxes. The Y-Box in the URR of DQA1 differs from the consensus sequence (bp -74 A instead of G) found

148 . 25th Meeting of the Society of Immunology in all other Y-Boxes of class II URRs. In addition to that, two of the QAP variants QAP 4.1 and 4.2 have been shown to carry a second substitution in their Y-Box (bp - 70 A instead of G), which causes a significantly decreased transcription of the DQAI-alleles (DQAP040I, "'0501, "'0601) carrying these QAP variants. The alleles DQAP040I, "'0501, "'0601 have been found to carry a sequence motif, which is highly associated with susceptibility to EOPA-JCA. We have studied the QAP polymorphism including the Y-Box variants in the URR of DQAl in 200 patients suffering from EOPA-JCA and in 224 unrelated healthy controls. Besides the association with DQAP0401, "'0501, "'0601, we have observed a strong association of susceptibility for EOP A -J CA with the less effective Q AP variants Q AP4.1 (Chi2 = 14.81, P < 10-4 ) and QAP4.2 (Chi2 = 50.18, P < 10- 9 ). In view of the pathogenesis of EOPA-JCA this fact draws attention (in addition to presentation of an «arthritogenic peptide» by peripheral cells) to a possible role of the thymus, where HLADQ molecules seem to have a special role in shaping the T cell repertoire.

Institute of Immunology and 1Institute of Medical Biochemistry, University Rostock, Rostock, Germany

E.12 Cell surface biotinylation as a tool for nonradioactive monitoring of membrane protein shedding S. HAUSMANN, R. CLAUS, R. KALUS, H. W ALZEL 1, and H. KOHLER A large number of membrane proteins are known to exist also in soluble form. These molecules may be generated either by alternative mRNA splicing or by release of the membrane-bound proteins from the cell surface. To monitor the latter mechanism we labeled cell surface proteins by biotinylation with D-biotin-N-Hydroxysuccinimide ester, cultivated cells up to five days, and separated cells from culture supernatant. Soluble proteins from supernatant as well as detergent solubilized ones from cell membranes were characterized by immunoprecipitation, SDS-PAGE, Western blotting with peroxidase-coupled streptavidine, and detection by enhanced chemiluminescence (ECL). By this way we studied shedding of HLA class I antigen from Balm I cells and found that the 44 kD membrane bound form disappeared from cell surface during culture, whereas a 36 kD probably cleaved form appeared in culture supernatant. Similar experiments were performed to investigate the fate of HLA class II antigens. Moreover, we could show that limited surface biotinylation had no influence on the viability of cell lines (Balm 1, Daudi) or peripheral blood mononuclear cells (PBMC). Even functional integrity of PBMC was only slightly affected as measured by LPS-induced cytokine release (IL-I, IL-6, TNFu) and PHA-induced 3H-thymidine incorporation. Our data reveal that surface biotinylation combined with highly sensitive ECL detection can be successfully used for characterization of shed membrane proteins without the disadvantages of 1251-radiolabeling. Supported by the Deutsche Forschungsgemeinschaft (DFG CI 110/1-2).

25th Meeting of the Society of Immunology . 149 Institut fur Anthropologie und Humangenetik, Munich, Germany; IUniversity of Innsbruck, Dept. of Internal Medicine, Innsbruck, Austria

E.13 LST-1, a novel gene in the human TNF region 1. HOLZINGER I, A. DE BAEY, and E. H. WEISS Extensive studies of the central MHC (class III) have revealed a remarkably high density of genes, several of which encode proteins essential for immune functions. The mouse B 1144 gene, identified as partial cDNA, is located 10 kb centromeric to TNFA and represents a B cell and macrophage specific transcript in mice. We describe the exact genomic localization of the human homologue and give a structural analysis of this leukocyte-specific transcript-l (LST -1) gene, which is strongly induced in U937 cells upon stimulation with IFN -yo We characterized the LST -I gene by sequencing the corresponding region of the HLA genome and by Northern blot and cDNA analyses. The previously described microsatellite markers TNF d and TNF e are located in the last intron of LST -1. Several eDNA clones were isolated coding for three long open reading frames (ORFs) two of which only partially overlap with the peptide sequence of the mouse cDNA. We located a polymorphic Pvu II restriction site about 260 bp downstream of the postulated polyadenylation signal of LST-1. Elucidation of the biological function of the LST -1 gene product awaits knowledge of the translated ORF. No clear homology was found to polypeptide sequences in the data banks. The close linkage to the TNF genes and the stimulation by IFN -y might indicate that LST -1 could be involved in the regulation of the immune response.

Transplantationslabor, Klinik fur Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany

E.14 After organ transplantation V~ gene usage is predominantly altered in the COB T cell subset S. HUNDRIESER and K. WONIGEIT Alterations of T cell receptor (TCR) V~ gene Jage can occur after clinical organ transplantation due to exposition of the T cell system to incompatible alloantigens and to immunosuppressive drugs. We have studied this question by comparing the V~ gene usage before and after transplantation in 3 patients with liver grafts and one patient with a kidney graft. Vf:l gene usage was analyzed in separated CD4 and CD8 subsets by PCR using oligonucleotide primers for defined V~ families. One month after organ transplantation significant alterations in the TCR Vf:l gene usage were observed in all patients. Interestingly, however, these changes were confined to the CD8 subset. Marked increases were observed for V~3, 4, 12, and 13, decreases were seen for V~6 and 24. The patterns differed from patient to patient. In a control person no alterations were observed during a similar interval. In one liver grafted patient TCR VB gene usage was characterized by an extreme increase of Vf:l12 after one month and reduction to the pretransplant value 4 months later. Additionally performed fine analysis of his TCR VB gene products in sequencing gels revealed changes in the clonal heterogeneity for Vf:l5a, 6, 7,

150 . 25th Meeting of the Society of Immunology 11, 12, 13, 20, 21, and 24 that persisted for more than 5 months after transplantation. These results indicate that alterations of TCR V~ gene usage after organ transplantation predominantly affect the CDS subpopulation. The mechanism underlying this subset specific effect remains to be clarified.

Transplantationslabor der Klinik fur Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany

E.1S Non IFN inducible MHC class I genes in the rat D. LAMBRACHT and K. WONIGEIT In order to analyze the number and organization of class I genes in the rat MHC (RTI system) we have established a cosmid library of the LEW rat strain (RTll haplotype). From this library a class I gene encoded by a 5.4 kb BamHI fragment was isolated on cosmid 121. This clone mapped in a cluster of overlapping cosmids located in the direct neighborhood of the deletion in the mutant haplotype Iml. Comparison of the restriction fragment length polymorphism pattern demonstrated that the BamHI fragment is present in the I and in the u haplotype. Transfection of clone 121 in mouse fibroblasts led to the expression of a rat class I antigen detectable with the common mouse anti-rat class I mab Oxl S. In contrast to other isolated and transfected rat class I genes this antigen is not inducible neither by IFN Y nor by IFN a although the complete 5' region is present. Hybridization with a probe containing the whole promotor region of the classical R Tl.AI gene (enhancer B, Interferon response element, enhancer A) demonstrated that the cosmid fails to hybridize suggesting major differences in the 5' regulatory sequences. Another typical feature of this antigen is a high trypsine sensitivity suggesting also characteristic differences in the a3-domain. A second class I gene with the same features (non inducible by IFN, high trypsin sensitivity) was isolated on a different cosmid mapping to the same class I region. The described genes belong to a previously undetected family of class I genes in the rat.

First Dept. of Medicine, Johannes Gutenberg University of Mainz, Mainz, Germany

E.16 T cell receptor (TCR) Bchain rearrangement in HLA-B27restricted synovial C08+ cytotoxic T cells E. MAY, R. DUCHMANN, B. ACKERMANN, B. GOERGEN, K.-H. MEYERZUM BDsCHENfELDE, and E. HERMANN The association of HLA-B27 with susceptibility to the spondylarthropathies may be related to the peptide presenting properties of this MHC class I molecule. The presence of HLA-B27-restricted cytotoxic T lymphocytes (CTL) with specificity for bacterial (Yersinia, Salmonella) or self antigens has recently been demonstrated by our group (Lancet 342: 646, 1993). To gain further insight into the molecular basis of antigen

25th Meeting of the Society of Immunology . 151 recognition, we have now sequenced the TCR-~ chains of HLA-B27-restricted CTL clones and lines « 3 clonal populations) and controls. HLA-B27-restricted CTL clones (all ar1-TCR+CD8+) and non-B27-restricted control CTL were derived from the SF of 3 HLA-B27+ patients with reactive arthritis (ReA) and from PBL of one B27+ healthy donor. Total cellular mRNA was isolated from each clone and reverse-transcribed. The eDNA was amplified by PCR using primers specific for 20 different TCRV~ families. PCR products of individual families were sequenced using a solid phase sequencing technique. - Results: Sequence analysis of 32 HLA-B27-restricted autoreactive or Y. enterocolitica specific CD8+ CTL clones and lines showed that V~ gene usage was skewed to families V~13, VrH4 and V~17, thus representing 68 % of all CTL. 15 control CTL expressed V02, 3, 6, 13, 14, and 16. In marc detail, Yersinia-specific B27-restricted CTL preferentially used V~13.3 and 14, auto reactive B27-restricted CTL preferred Vr1 13.2 and 17. Interestingly, V~ gene segments 13, 14 and 17 show high sequence homologies; these TCR V~ families have recently been shown to be expressed in HLA-B27-alloreactive CTL (L6I'EZ DE CASTRO et al.). In our study, we could also demonstrate nonrandom VDJ region usage in the HLA-B27-restricted CTL. In summary, these data may further our understanding of the structural requirements for the trimolecular complex of TCR, HLA-B27 and «arthritogenic» bacteria in the spondylarthropathies.

Med. Universitatsklinik u. Poliklinik, Abt. II, Tiibingen, Germany

E.17 HLA-class I Bantigen typing by DNA sequencing C. A. MOLI.ER, F. METZGER, C. FISCHER, I. STEIERT, E. KELLERl\IANN-KEGREISS, and H. SCHMIDT One of the main problems after bone marrow transplantation (BMT) remains severe acute graft versus host disease (GvHD) (> grade II) occurring in about 50 'Yo after BMT with marrow from unrelated donors or not completely HLA-identical family donors. Besides intensification of the immunosuppressive therapy during BMT further improving HLA matching of donor and recipient in matched unrelated BMT might diminish the incidence of GvHD. While HLA class II is meanwhile done by analysis of the DNA HLA class I typing is still performed by serology. To improve HLA class I matching before BMT from unrelated donors we started typing of the most polymorphic part of the second exon of HLA B antigens by sequencing. Total DNA was prepared from standard cell lines but also from mononuclear cells of patients with known HLA class I phenotype. Using a mixture of 3 oligonucleotides at the 5' end and HLA-Bw4 or HLABw6 specific at the 3' end a PCR reaction was performed which resulted in HLA-Bw4 or HLA-Bw6 specific DNA sequences from position 83 to 244 of the 2 nd exon. Sequencing of the amplified DNA was performed by cycle sequencing using Taq polymerase. The reaction mixture was analyzed during gel electrophoresis by a DNA sequencer (373A DNA Sequencer Applied Biosystems®). So far we were able to amplify and sequence genomic DNA segments from homozygous cell lines but also from cells of patients expressing HLA-Bw4 and HLA-Bw6 antigens. Preliminary results indicate that within the sequenced region HLA-B63 is identical with HLA-B':·S7011""5702. For the moment we are extending this method for sequencing the yd exon as well. Using slightly other sequencing conditions it seems possible that the method can also be used to sequence DNA from patients carrying two HLA-Bw4 or HLA-Bw6 antigens.

152 . 25th Meeting of the Society of Immunology Dept. of Immunology, Hannover Medical School, Hannover, Germany

E.1S Defective glycosylphosphatidylinositol (GPI)-anchor-biosynthesis in paroxysmal nocturnal hemoglobinuria (PNH) is caused by heterogeneous mutations of the PIG-A gene T. OSTENDORF, C. NISCHAN, J. SCHUBERT, and R. E. SCHMIDT GPI-anchored cell surface molecules play an important role in cell adhesion, signal transduction and regulation of the complement system. Both structure and biosynthetic pathway of the anchor have been elucidated. Recently some of the genes involved in biosynthesis have been cloned. In PNH GPI deficient peripheral blood cells fail to synthesize N-Acetyl-D-Glucosaminyl-Phosphatidylinositol (GlcNac-PI) which has been shown to be the first step in GPI-anchor biosynthesis. The recently cloned X-linked PIGA-gene appears to be one of at least three genes which participate in this early step. We now characterized the genetic defect in PI G- A in 7 different PNH patients from which we have generated GPI -positive and -negative cell lines each. Using reverse transcriptase polymerase chain reaction at least three different splicing variants of PI G-A were detected for each cell line. Cloning and sequencing of the patients full length PIG-A-coding region revealed heterogeneous mutations in transcripts of GPI -negative cell lines ranging from transitions, point deletions, greater deletions and splicing defects to instable full length transcripts. In one patient two different presumably lineage-specific defects were detected. Supported by the DFG (Schu 71312-2; Grad. Kol!. No. 120/6-1).

Dept. of Internal Medicine 1 and Dept. of Transfusion Medicine, University of Ulm, Ulm, Germany

E.19 Immunoglobulin gene usage in diabetes-associated human monoclonal autoantibodies recognizing glutamate decarboxylase W. RICHTER, K. JURY, D. LOFFLER, B. O. BOHM, and T. H. EIERMANN Islet cell antibodies directed towards the GAB A synthesizing enzyme glutamate decarboxylase (GAD) provide an early marker for islet cell destruction in individuals developing type 1 diabetes. Such human autoantibodies have not been accessible for analyses of kappa, lambda or heavy chain variable gene usage due to the lack of stable B cell lines. We obtained 6 human monoclonal IgG antibodies (MICA 1-6) directed towards GAD from an individual with newly diagnosed type 1 diabetes and analyzed the variable gene segment usage and somatic mutations observed in these GAD-reactive human monoclonal autoantibodies. After mRNA isolation, cDNA synthesis and amplification of immunoglobulin variable regions by polymerase chain reaction (PCR) sequences of heavy and light chain PCR products were determined by solid phase DNA sequencing of both strands. Sequence comparison demonstrated that all six MICAs originated from different B cell clones. MICA 1,2,5 and 6 showed high homology to published germ line V segments of the VH4 family in their heavy chains. MICA 3 was 95 % identical to a published VH1 germline V-region and MICA 4 showed a 94 % identity to a VH5 germline gene. The two

25th Meeting of the Society of Immunology . 153 pairs MICA 1 and 3 and MICA 4 and 6, which were able to block each others binding to GAD and therefore may bind an identical epitope in GAD used different variable genes in their heavy and light chains indicating that these epitopes in GAD may be highly immunogenic. Somatic mutations especially in CDR2 of the VH genes suggest an antigen driven immune response to GAD in type 1 diabetes.

Clinical Research Unit for Rheumatology and Dept. of Rheumatology and Clinical Immunology, University of Freiburg, Freiburg, Germany

E.20 T cell receptor usage in arthritis patients C. SCHATZLEIN, P. FIEDLER, E. v. SEYDUTZ, H. H. PETER, and H. EIBEL In rheumatoid arthritis (RA) auto reactive T cells are thought to contribute to the initiation and to the progression of the disease. Initially, upon activation T cells would invade synovial tissues. Once there, they could start an autoimmune response against synovial antigens that are not found at those sites where negative selection of T cells occurs. Analyzing the T cell receptor repertoire of synovial T cells might help to elucidate this process. Recently it was found by us and by others by PCR based methodology that synovial T cells of RA patients preferentially express specific TCR Va and V~ families. We extended our analyses by using a set of TCR Vf-l specific antibodies specific for 16 TCR V~ families. Synovial and peripheral T cells from RA patients were analyzed for the expression of these TCR V~ members among the CD4 and CDS subsets. In 9 RA patients we found TCR V~19, in 5 VI32 and in 3 V(:l6 to be preferentially expressed in synovial T cells. The other TCR V(:I subsets were highly variable. In two cases of chronic lyme arthritis we observed the preferential usage of the TCR V~3 and the TCR V~8 subsets. In particular, the synovial CD4 positive T cells used 10 x more frequently the VI:!3 (3.4 01<,) than peripheral T cells. Furthermore, 30 % of the synovial CD8+T cells expressed TCR V(:\8 receptors whereas only 8 'Yo of the peripheral CDS positive T cells were V~8+. To investigate whether these V~S+T cells expanded oligo- or polyclonally we purified the V(:l8+CD4 and CD8 subsets from peripheral blood and from the synovial fluid and determined the DNA sequence of 37 independent TCR VI:l8+ cDNA clones. By comparing the TCR V~ CDR3 regions of the eDNA clones we found them to be highly divergent. This result suggests that in lyme arthritis synovial TCR V~S+ cells are activated polyclonally and potentially by superantigen stimulation.

Med. Universitatsklinik u. Poliklinik, Abt. II, Tubingen, Germany

E.21 Regulation of HLA-class I genes by interferon H. SCHMIDT, I. STEIERT, E. KELLERMANN-KEGRFISS,]. WALZ, R. ZINSER, and C. A. MOU.ER Expression of HLA class I antigens can be modulated in vitro and in vivo by interferon (IFN) a but also by IFN y. A strong induction of HLA-class I antigens was found on both hematopoietic progenitors and normal peripheral blood mononuclear cells after one

154 . 25th Meeting of the Society of Immunology month of IFN treatment in 1S patients with myeloproliferative syndrome. By daily injections of IFN in the first month of therapy stimulation was continuously increasing suggesting a major effect of IFN a on hematopoietic progenitors with sustained enhanced expression of HLA-class I antigens during differentiation of myelomonocytic cells. Differential in vivo regulation of HLA-class I antigens by IFN was shown by comparison of HLA-A2 with HLA-B antigen expression. In vitro expression of the HLA-B7 and -Bw64 genes was significantly more inducible by IFN than the genes coding for the HLA-B27, HLA-B51, HLA-B3S, HLA-B39, HLA-Cw3, and HLA-A2 antigens after transfection into mouse L cells. Modification of the 5' ends of the HLA-B7 and HLA-B27 genes before transfection in mouse L cells revealed the presence of enhancer sequences responding to interferon treatment in the 5' untranslated region of the HLA-B7, but not of the HLA-B27 gene and suggested further independently acting enhancer elements downstream of the transcription initiation site. When different fragments of HLA B7 or B3S, including introns, 3' and 5' untranslated regions, were cloned in front of CAT genes IFN responding enhancers were only detected at the 5' end of the HLA-B7 gene. Further IFN independent enhancers could be detected within introns and at the 3' end of HLA class I genes. These findings may indicate specific regulatory mechanisms of HLA class I antigen expression possibly influencing T cell recognition in immune response.

, Clinical Research Unit for Multiple Sclerosis and Dept. of Neurology, 2 Institute for Virology and Immunology, University ofWiirzburg, Wiirzburg, Germany

E.22 Molecular analysis of rat T cell-receptor Va4 and VaS families M. STANGEL', N. E. TORRES-NAGEl2 , H.-P. HARTUNG!, K. V. TOYKA!, T. HONIG 2 , and G. GIEGERICH' We recently described novel rat T cell receptor (TCR) Va-families employing two monoclonal antibodies, G99 and G177, directed against these Va-elements. These chains were assigned to the TCR Va4 and VaS family because of high homologies to the respective mouse DNA sequences. Since there are multiple members of both Va-families described in the mouse, we used TCR Va4 and VaS-specific primers for PCRamplification from genomic DNA to clone further rat TCR Va-genes. Applying this method, we found several novel members of both the rat TCR Va4 and VaS family. Preliminary data from sequence analysis of TCR cDNA from T cell hybridomas and sorted T cells recognized by the G99 and G 177 antibodies showed that both antibodies recognize at least two different members of the respective TCR Va-family. Interestingly, the TCR Va4 chain is used by encephalitogenic T cells in PLlJ-mice in an autoimmune disease model, and both, TCR Va4 and VaS chains, have been found in Lewis rat experimental allergic neuritis (GIEGERICH et aI., unpublished results). The availability of novel anti-TCR antibodies and a broader knowledge of the TCR Va-gene repertoire might be helpful in the analysis of experimental autoimmune disease.

25th Meeting of the Society of Immunology . 155 Institut fur Immunologie, Universitat Munchen, Munchen, Germany

E.23 Long-term in vivo expansion of HLA-B35 alloreactive T cells with homologous TCRs suggests cross-stimulation via a persistent peptide/self MHC complex A. STEINLE, C. REINHARDT, P. JANTZER, and D. J. SCHFNDEL We generated HLA-B35 specific, alloreactive T cell clones that distinguish HLA-B35 variants differing by one amino acid position located in the peptide-binding cleft. Their postulated peptide dependency was confirmed by testing with TAP deficient mutant cell lines. Sequence analysis revealed that three of seven HLA-B35 specific T cells had homologous T cell receptors (TCRs) correlating with their similar fine specificities. They expressed Va2.5/Ja62.119 and V~4/J~2.7 rearranged TCRa and ~ chains and utilized highly related CDR3 sequences. This suggests that the specificity of at least some alloreactive T cells is determined by the interaction between CDR3 and the allopeptide. To study the abundance of this particular TCR specificity in vivo, we analyzed the Va2.5/Ja62.119 repertoire of unstimulated PBL of the responding cell donor who was never sensitized with HLA-B35. Sequencing of the cloned Va2.5/Ja62.119 junctional regions obtained from two PBL isolates separated by a nine year interval revealed that about 75 'Yo of the clones contained only a few transcripts identical or homologous to those of the three HLA-B35 specific T cells. This suggests that an immune response to a persistent antigen (pathogen?) has led to the expansion and maintenance of a set of T cells bearing homologous TCRs that through their crossreactivity dominate the alloresponse against HLA-B35. These findings reveal a new aspect of all ore activity and could have major implication in particular transplantation settings.

Dept. of Immunology, Hannover Medical School, Hannover, Germany; 1 Dana Farber Cancer Institute, Boston, MA, USA

E.24 IgG binding site of FcyRIII is located on the second immunoglobulin-like domain of the receptor A. TAMM, A. KISTER I, and R. E. SCHMIDT FcyRIll (CD16) is a low affinity receptor for IgG that binds only immune complexes with the Ka < 10 7 • PcyRIll belongs to the immunoglobulin superfamily and its extracellular part reveals 40 °It, of homology in the amino acid level to the high affinity receptor for IgE, FCERI. To define the conformational epitopes on human FcyRIll for the binding of IgG, chimeric FcyRIn/pcERI receptors were constructed replacing two to four amino acids (aa) on CD16 with the corresponding ones on PCERI a-chain in order to construct receptors with retained structure of CD16, but with decreased IgG-binding ability. Comparison of the ligand binding capacities of the chimeric receptors expressed on the 293 human embryonic fibroblasts to that of the wild-type CD16, revealed three chimeras with decreased binding affinities to IgG. These mutations involve three aas on the loop between C and C' [:I-strands, four on the F-G loop and four entirely on the P strand on the second immunoglobulin-like domain. According to the computerized three dimen-

156 . 25th Meeting of the Society of Immunology sional structure of the receptor, the aas substituted in the chimeric receptors are exposed on the surface of the molecule constituting one intact binding site for the ligand. The direct interaction of the single aas with IgG will be characterized with the single aa replacements in the regions identified to be involved in binding. Supported by SFB 244/ A09.