Workshop C: Antigen Processing and Presentation

1Department of Molecular Immunology, German Cancer Research Center, Heidelberg, 3DRK-Blucspendezentrale Ulm, Universitat Ulm, Ulm, Germany, and 2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic

C. 1 Differential association of HLA·DR molecules with tetraspans on the cell surface and in endosomal/lysosomalloading compartments N.O. ANDERS 1, H. KROPSHOFER 1, V. HOREJSI 2, A. WOLPL', G. MOLDENHAUER 1, G.]. 1 1 HAMMERLING , and A.B. VOGT Tetraspan molecules are highly glycosylated proteins with four transmembrane domains known to be expressed on Band T cells and to be involved in T cell activation. In B cells the tetraspan molecules CD82, CD81, CDG3 and CD53 are reported to be associated with the major histocompatibility complex (MHC) class II molecules forming multi-component clusters, however the function of these clusters is not known. In order to investigate which compartments harbor which type of MHC class II - tetraspan complexes subcellular fractions of EBV-transformed B cells were immunoprecipitated with antibodies recognizing HLA-DR-peptide complexes and analyzed for co-precipitating tetraspan molecules. We found that in endosomalllysosomal compartments, where peptide loading is thought to occur, a considerable subset of HLA-DR molecules are engaged in complexes with CD82 and CDG3. However, on the cell surface HLA-DR molecules are mainly associated with CD81, CD53 and CD82. Thus, maturation of class II molecules, which is reflected by loading with appropriate peptide and transport to the cell surface, appears to be paralleled by incorporation into tetraspan clusters of different composition. This might be important for the regulation ofHLA-DR trafficking to and from the cell surface and in particular for the quality of antigen presentation to T cells.

1Department of Molecular Immunology, German Cancer Research Center, Heidelberg, 3DRK-Blutspendezentrale Vim, Universitat Vim, Ulm, Germany, and 2Neuroimmunology Branch, NINDS, National Institutes of Health, Bethesda, USA

C. 2 HLA·DM is expressed on the cell surface of 8 cells where it controls peptide exchange and immunodominance S.O. ARNDT 1, A.B. VOGT 1, S. MARKOVIC-PLESE 2, K. POTZIES 1, G. MOLDENHAUER l , A. WOLPL 3, R. MARTIN 2, G.]. HAMMERLING 1, and H. KROPSHOFER 1 HLA-DM (OM) is a key player of antigen presenting cells (APCs) as it has been shown to act as a chaperone and editor in loading of antigenic peptides onto major histocompatibility complex (MHC) class II molecules in late endosomal and lysosomal compartments. However, the

342 . 30th Annual Meeting 1999 existence or a role of OM on the cell surface has remained elusive so far. Here we show that a subset of 0 M molecules resides indeed on the cell surface of B cells and catalyzes exogenous peptide loading. In addition, OM reveals to select for high-stability MHC class II-peptide complexes by removal of low-stability peptides. Accordingly, loading and presentation of an immunodominant epitope of myelin basic protein, a candidate autoantigen in multiple sclerosis, typically binding with low stability to its MHC class II restriction element, is counteracted by the editing activity of OM. This is first evidence that OM may co-control presentation of auto-epitopes implicated in autoimmunity.

Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany

c. 3

HLA-OO/ H2-0 in antigen presentation

P. BROCKE, EA ARMANDOLA, and G.]. HAMMERLING Loading of major histocompatibility complex class II (MHC II) molecules with antigenic peptides is a prerequisite for specific immune recognition by CD4+ T cells. This MHC lI:peptide complex formation is influenced by non-classical MHC II molecules like HLA-DM (mouse: H2-M) and HLA-DO (mouse: H2-0). HLA-DM facilitates antigen processing by catalyzing the exchange of invariant chain derived peptides (CLIP) from the MHC II binding groove for antigenic peptides, thereby functioning as a peptide editor favoring the presentation of stably binding peptides. HLA-DO tightly interacts and co-localizes with HLA-DM in lysosome-like vesicles of antigen presenting cells and seems to regulate HLA-DM function. We investigated the role of H2-0 in antigen presentation by reducing H20b protein expression in the LB27.4 mouse B lymphoma cell line using transfection with a ribozyme containing antisense construct which is complementary to 500 bp at the 3' end of the H2-0b chain mRNA. Antigen presentation of these antisense transfectants to three different T cell hybridomas (I-Ad :HEL8-29, I-Ad :Ova323-339, or I-Ab :Ova323-339 specific) was found to be enhanced in comparison to control tiansfectants. We also produced transgenic mice expressing H2-0a and b chains driven by the H2-Kb class I promoter resulting in 1,5 to 2 fold higher H2-0 expression as compared to wild type mice. Preliminary experiments with spleen cells from these H2-0 overexpressing transgenic mice showed reduced antigen presentation capacity. Taken together, in these cellular and in vivo studies H2-0 appears to downmodulate antigen processing/presentation of the protein antigens investigated here - presumably by downregulating H2-M in its function.

30th Annual Meeting 1999 . 343 1Department of Internal Medicine III and Institute of Clinical Immunology, FriedrichAlexander-University, Erlangen, Germany, 2Department of Nephrology, and 3Wieslab AB, Lund University, Lund, Sweden

C. 4 Goodpasture syndrome: Molecular characterization of the immunodominant B cell epitope H. BURKHARDT l , T. HELLMARK 2, C. UNGER 1, and]. WIESLANDER 3 Goodpasture syndrome is a prototype autoimmune disease characterized by formation of pathogenic autoantibodies against the basement membrane collagen type IV, which cause rapidly progressive glomerulonephritis often accompanied by lung hemorrhage. The non-collagenous domain of the a3 chain of type IV collagen (a3(IV)NC1) but not the homologous region of the al chain (al (IV)NCl), is the target of the pathogenic autoimmune response. The pathogenic autoantibodies bind in a conformation-dependent manner thereby limiting the application of linear synthetic peptides for epitope-mapping strategies. In the present study the identification of crirical target structures of the autoantibody response was pursued by the expression of the antigen as a recombinant protein in a human embryonic kidney cell line (HEK-293). This strategy enabled the construction of a variety of properly folded chimeric molecules, in which the wild-type a3(IV) NC 1 sequence was progressively replaced by the corresponding sequence of the homologous non-reactive al (IV)NCI. The introduction of single replacement mutations in certain positions of rhe amino-terminal third of a3(IV)NCl destroyed autoantibody binding completely. Based on this knowledge about critical amino acid residues wild type al (IV)NCI was selectively substituted in 9 discontinuous positions with amino acid residues from the a3(IV)NCl resulting in a recombinant construct that was recognized by all patients' sera (n=20). These 9 amino acid residues define a single conformational B cell epitope as the critical target of the nephritogenic antibody response in Goodpasture syndrome.

Division of Immunobiology, Institute of Zoology, University of Bonn, Bonn, Germany

C. 5 Presentation of antigenic sequences inserted into invariant chain C. CARSTENS, E. SIEVERS, A. K6NIG, and N. KOCH Invariant chain (Ii) associates early in biosynthesis to MHC II molecules. A segment of Ii binds to the peptide groove ofMHC II and prevents premature loading with peptides or polypeptides. We replaced the MHC II binding segment of Ii by an antigenic sequence and tested the recombinant Ii (rIi) in antigen presentation. These constructs were capable to elicit an IL-2 T cell response that was not obtained with peptide-loaded APC. Antigenic sequences were inserted in variable positions ofIi. The aim was to generate APC that were predominantly loaded with a single peptide. The efficiency of the rIi antigen fusion proteins to present antigen was tested by competition with exogenously provided peptide or protein antigens. Antigens provided from the endogenous source by rIi were dominantly presented and out-compete the presentation of other antigens.

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1Junior Research Group of Cell Biology and 2Department ofImmunology, Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Dilsseldorf, Germany

C. 6 Mercury-induced recruihnent of nucleolar autoantigen fibrillarin to antigen processing

In H-2' mice, the administration of mercury ions elicits an anti-nucleolar autoantibody (ANoA) response that targets a conserved epitope of fibrillarin, similar to the anti-fibrillarin response diagnostic of a subset of human scleroderma patients. To investigate the biological events underlying this B cell response, we treated eukaryotic cells with mercuric chloride (HgCI 2). By means of indirect immunofluorescence (IIF) and confocal microscopy we found, in interphase cells, a time and cell cycle dependent redistribution of fibrillarin from the nucleoli to distinct nucleoplasmic clusters and a colocalization of nucleoplasmic fibrillarin with the proteasomes. Relocalization of fibrillarin was also induced by other compounds such as transcription inhibitors. However, none of them induced colocalization of fibrillarin with proteasomes in the nucleoplasm. We therefore suggest the following cell-based disease model: mercury ions penetrate into cell, accumulate in the nucleolus and inhibit nucleolar transcription. In consequence, nucleolar structure is altered and the molecular context of fibrillarin is changed. Fibrillarin recruits to proteasomes in the nucleoplasm thus permitting processing and presentation of previously cryptic determinants and breaking tolerance of the immune system to self determinants.

I Department of Immunology, Max-Planck-Institut filr Infektionsbiologie, Berlin, Germany and 2Department of Microbiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, USA

C. 7 Modification of mycobacterial glycolipids by macrophages: Relevance for CDl-mediated immune responses? K. FISCHER 1, R. HURWITZ 1, D. CHATTERJEE2, H.L. COLLINS 1, K. HAGENS l , S.H.E. KAUFMANN 1, and U .E. SCHAIBLE 1

Human group I CD 1 molecules are able to present mycobacterial lipids to a subset ofT cells. In the case of group II COl molecules only a galactosylceramide and glycosylphosphatidyl-inositol but not bacterial glycolipids have been reported to bind to mouse and human CD 1d. Human CD 1b, c and d, and murine CD 1d traffic through late endosomal/lysosomal compartments in antigen presenting cells (APC) and all three molecules contain a tyrosine based endosomal targeting sequence. This raises the questions i) where in the APC are mycobacterial glycolipids loaded onto COl molecules and ii) if the lysosomal environment is important for processing of relevant antigens. We found that fluorescent labeled mycobacterial surface glycolipids are transferred from the early endosome like phagosome to late endosomes/lysosomes. When we analyzed subcellular fractions of macrophages infected with 14C-palmitic acid labeled Mycobacterium bovis

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BCG by thin layer chromatography (TLC) we found two mycobacterial glycolipid species independent of the phagosome. One of the two is similar to a glycolipid found in mycobacteria alone but the second one is not present in the mycobacterial glycolipid pattern. To determine if this is a modified mycobacterial glycolipid we isolated a number of mycobacterial glycolipids from TLC plates and integrated them into phosphatidylinositol liposomes which were incubated together with murine macrophages. Using this approach we found that all isolated mycobacterial glycolipids were metabolized by macrophages. In a second approach the same glycolipid preparations were incubated with lysosomes purified from macrophages which contain the enzyme subset that appears relevant for processing. One out of six glycolipids analyzed was found to be altered by lysosomal enzymes and the resulting product had a similar Rf value as the one observed in vesicle preparations from infected macrophages. This glycolipid and its enzymatically altered product are currently identified. Our data suggest that the enzymatic repertoire of the APC can process mycobacterial glycolipids. This may be a prerequisite to achieve glycolipid antigens for CD 1 mediated T cell recognition. Further studies aim to analyze if these glycolipids can bind the various CD 1 molecules and can induce specific T cells.

I Institute of Immunology and Transfusion Medicine, University of Lubeck, Lubeck, 2Department of Biochemistry, Rheinisch-Westfalische Technische Hochschule, Aachen, 4Bernhard-Nocht-Institute of Tropical Medicine, Hamburg, 5Institute of Experimental Microbiology, Friedrich-Schiller-University, lena, Germany, and 3UT Southwestern Medical Center, Dallas, USA

c. 8

Superantigenic activity of the Mycoplasma arthritic/is-derived superantigen (MAS) is mediated by a novel structure

A. GALUSCHKA',]. GROTZINGER2, U. FAGIN 1, H. KIRCHNER l , B. HOSCHLER 1, D.R. KAR p3, B. FLEISCHER 4, W. REICHARDT s , A. WOLLMER2, and L. RINK l Superantigens stimulate the T cell system as intact proteins by cross-linking major histocompatibility complex (MHC) class II molecules with variable parts ofT cell receptors (TCR) outside the usual antigen binding groove. In order to exert superantigenic effects, they apparently require a common structural fold, named the beta-grasp motif, that is present even in very unrelated superantigens such as staphylococcal enterotoxin A and toxic-shock syndrome toxin-I. The Mycoplasma arthritidis-derived superantigen (MAS, alternatively MAM) is the only known superantigen produced by a Mycoplasma, a cell wall-free prokaryote. Here we show that MAS has a completely different structure from all other superantigens. From its structural characteristics MAS is the prototype of a new group of superantigens. Despite its unique structure, MAS uses exactly the same molecular mechanism as other superantigens. It binds to the highly conserved residue H81 in the beta-chain of MHC class II molecules, which seems to be a common binding site of superantigens.

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Hans-Spemann-Labotatories, Max-Planck-Institute of Immunology, Freiburg, Germany

C. 9 Structure/function analysis of CIITA, the MHC class II transactivator

s. HAKE, C. KAMMERBAUER, and V. STEIMLE The human class II major histocompatibility complex (MHC) molecules playa critical role in immune responses by presenting processed exo-genous antigens to CD4+ T lymphocytes. MHC class II molecules (MHC-II) show a complex expression pattern with constitutive expression only on few specialized cell types and a widespread inducible expression. Specific cis-acting sequences have been recognized in the proximal promoters of the MHC-II genes to which different transcription factors bind to. While these transcription factors, RFX, NF-Y and X2bp are ubiquitous expressed and present also in MHC-II negative cells, a strictly concordant expression berween CIITA, the class II transactivator, and MHC-II RNA has been observed in multiple cell lines and tissues CIITA turned out to constitute the master regulator of MHC-II gene expression, because its expression is both necessary and sufficiant to induce expression of all MHC-II promoter containing genes. In our study we are concentrated on a structure/function analysis of CIITA. CIITA is probably not itself a DNA binding protein and is believed to act in a co-activator like fashion through protein-protein interactions with MHC-II promoter binding proteins. In the C-terminal part of CIITA we have found four Leucine-Rich Repeats (LRRs). LRRs have been identified in many different classes of proteins and have been shown to mediate protein-protein interactions. With Alanine-scanning mutagenesis of this region we identified a number of amino acid residues in which single and multiple alanine exchanges abolish CIITA function. Protein expression levels are not affected by these exchanges. These alanine mutants show no dominat negative effect, but abolish the dominant effect of a mutant of CIITA, where the first 335 amino acids are deleted. Additionally we could directly show the interaction of CIITA with rwo subunits of the RFX complex, RFX5 and RFX-ANK, by co-immuno-precipitation. These interactions are not affected by LRR-alanine mutagenesis. The third subunit of the RFX complex, RFX-AP show no binding to CIITA in co-immunoprecipitation assays. Preliminary data show that the interaction of CIITA with an yet unidentified 33kD protein is affected by LRR-alanine mutagenesis. We conclude that the LRRs ofCIITA are involved in protein-protein interactions, but not in the binding berween CIITA and the rwo subunits of the RFX complex, RFX5 and RFX-ANK.

lInstitut ftir Medizinische Biochemie und Molekularbiologie, Universitat Rostock, Rostock, Germany and 2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, USA

C. 10 Impact of the TCR CDR3 regions on the recognition of microbial peptides by MBP-specific T cell clones S. HAUSMANN1.2, M. MARTIN 2, L. GAUTHIER 2, and K.W WUCHERPFENNIG 2 T cells recognize antigenic peptides bound to an MHC molecule in a specific manner. However, a certain degree of degeneracy, i.e. crossreactivity towards a panel of structurally related peptide

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sequences, is also an essential feature of this recognition event. This has major implications in a variety of immune processes as for example during positive selection or the initiation of an autoimmune response by microbial peptides via «molecular mimicry». We studied structural aspects of degenerate T cell recognition using two human T cell clones specific for a peptide from myelin basic protein (MBP) presented by HLA-OR2. These clones have identical T cell receptors (TCR) except for a three amino acid difference in the a and ~ CDR} regions. The fine specificity of these clones, as determined by single amino acid-substituted analog peptides of MBP(85-101), differs only in the recognition of lysine at position 93. In the recently solved crystal strucrure of the HLA-0R2/MBP(85-99) complex this position has determined to be the central TCR-accessible side chain of the peptide (relative position P5). Therefore, the differences in the TCR COR3 regions between both clones determine the differential recognition of the central peptide side chain. This suggests a similar topology in peptide recognition as seen in the crystal structures of MHC class I-restricted TCRs bound to their corresponding MHC/peptide complexes, in which the COR3 regions of the TCR a and ~ chains form a central pocket which accommodates the side chain of the central amino acid. Furthermore, we used the T cell recognition motifs to search protein data bases for microbial peptide sequences, which may stimulate the MBP-specific clones. Out of 70 peptides synthesized five were able to stimulate one of the clones. The other, more specific T cell clone, only recognized three of them with a conservative lysine to arginine change at P5. The importance of the P5 side chain for the differential recognition of these microbial peptides was confirmed since a serine to arginine substitution at P5 in one of the peptides restored its ability to activate both clones. Thus, the degree of specificity/degeneracy in recognition of the central amino acid side chain of an antigenic peptide by MHC class II-restricted MBP-specific T cells is determined by the COR3 region of the TCR.

I Division of Immunobiology, Institute of Zoology, University of Bonn, Bonn, Germany and 2Human Immunogenetics Laboratory, Imperial Cancer Research Fund, London, UK

c.

11 High molecular weigt «super dimer» bands are artificially formed complexes of DR molecules and antibody

C. HITZEL 1, N. KOCH', U. GRONEBERG 2, M.

VAN

HAM 2, and]. TRowsDALE 2

C04+ T cells are activated by antigen presenting cells expressing MHC II molecules. The detection of dimers of dimers in MHC class II crystals has excited speculation about their possible functions in T cell antigen recognition. High molecular weight complexes isolated with mAbs have been described. These data have been suggested to be biochemical evidence for the existence of MHC II dimers. We investigated the composition of these high molecular weight DR bands. Under conditions, in which ORa~ heterodimers were readily detected, no DR complexes with an (aB)2chain composition could be identified. This result suggests that DR superdimers do not exist in B cells under normal physiological conditions. Two monoclonal antibodies (L243 and 01-12) immunoprecipitated high molecular weight DR complexes suspected to be superdimers. However, biochemical analysis revealed that, rather than superdimers, these were 50S-stable complexes of DR in combination with the antibodies. These findings question the existence of DR superdimers in vivo.

348 . 30th Annual Meeting 1999 Institut fur Immunologie, Universitat Heidelberg, Germany

C. 12 Antigen presentation by polymorphonuclear neutrophils !PMN): Induction of MHC class II expression and of MHC class II-restricted T cell proliferation C. IKING-KONERT, M. RADSAK, S. STEGMAIER, and G.M. HANSCH PMN of healthy donors do not express MHC class II antigens. In patients with chronic inflammatory diseases, however, MHC class II is found on up to 20% of the peripheral PMN. By cultivating PMN with gamma interferon (IFN-y) or supernatants of activated T cells MHC class II expression could be induced by de novo protein synthesis. In addition, also expression of the costimulatory molecules CDSO and CDS6 was induced. To test whether thus activated PMN might also be able to present peptide antigen, tetanus toxin C fragment (TT) specific T cell lines were established from a healthy, recently vaccinated individual. With PMN of that donor, but not of unrelated donors, T cell proliferation could be induced when TT was present. Only the CD4+ T cell proliferated, but not the CDS+. Proliferation could be inhibited by antibodies to MHC class II, CDS6 or ICAM-I, but not by antibodies to MHC class I or other PMN-surface antigens. PMN that had not been pretreated with IFN-y or T cell supernatants were also able to induce T cell proliferation. We found a drastic up-regulation of MHC class II and the co-stimulatory molecules during the co-culture. By Wortmannin, a phosphatidylinositol 3-kinase inhibitor, known to block the assembly of MHC class II-peptide complexes, T cell proliferation was greatly reduced, suggesting that PMN also process peptide antigens. Taken together, our data indicate that PMN are not only effector cells, but may also participate in the afferent limb of the immune response.

Division of Immunology and Allergology, Medical Institute of Environmental Hygiene, Heinrich Heine University, Dusseldorf, Germany

C. 13 Production of C04+ Tcell hybridomas against procainamide, a drug inducing lupus L.E. LAYLAND, M. WULFERINK, and E. GLEICHMANN Introduction: Drug-induced lupus erythematosus is associated with over SO different drugs. Procainamide (PA) , an anti-arrhythmic drug, belongs to the class of drugs well established for their role in inducing this autoimmune disease. PA is an arylamine compound and can be considered a prohapten. The consensus surrounding the complex pathogenesis ofPA-induced lupus regards that the first, pre-immunologic step is the N-oxidation of PA to the reactive metabolites, or haptens, hydroxylamine-PA (HAPA) and nitroso-PA. We propose that the second step involves T cell sensitization to neoantigens formed by these metabolires and presented by APCs, such as macrophages. To test this hypothesis we have established murine T cell hybridomas, within the BALBlc strain, that are sensitized, to as yet unidentified, neoanrigen induced by the metabolism of procainamide.

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Methods: As a metabolizing system we isolated white bone marrow cells (WBMC) , as a source of phagocyte precursor cells. To produce the HAPA-induced neoantigen, these cells were pulsed with procainamide, in vitro, for 24 h in serum-free medium. Popliteal lymph node (PLN) assays were performed, using different cell numbers of the antigenic material injected s.c into the hind foot pad. Increased PLN indices indicated that an immune response had occurred. T cells from lymph nodes immunized with the optimal concentration of antigen in IFA were fused with the CD4+ BW cancer cell line. Hybridoma specificity was identified through lL-2 bioassays. Results: Triplicate PLN assay results displayed a dose response only against PA-pulsed WBMC and nor towards naive WBMC. These results were statistically significant (p<0.05), with a dose of 5x I 06 WBMC/footpad. Three fusions generated CD4+ hybridomas which were specific to PA-pulsed WBMC to a stimulation index of up to 6 compared to naive WBMC. After subcloning, preliminary results from various hybridomas exhibited individual I_Ed and I_Ad MHC II dependency. By Western blot analysis, a protein adduct of a PA metabolite formed by WBMC has been detected. Whether this adduct corresponds ro the neoantigen recognized by the hybridomas is under investigation. Conclusion: Results indicate that phagocytes pulsed with PA, a prohapten, generate a neoantigen recognized by CD4+ T cells in vivo and in vitro. Whether the neoantigen confirms HAPA, the reactive metabolite, as the hapten is now being analyzed.

Institute fur lVirologie und Immunbiologie und 2Hygiene und Mikrobiologie, Universitat Wurzburg, Wurzburg, Germany

C. 14 Measles virus targets proteins into the exogenous processing pathway C. NEUMEISTER l , C. LODER2, V TER MEULEN 1, and S. NIEWIESK 1 Usually, after viral infection virus is processed and presented via the endogenous pathway for cytoroxic T cells (CTL). Proteins are degraded into epirope peptides, transported via the transporters of antigen presentation (TAP) into the endoplasmic reticulum (ER), bind ro an MHC class I molecule and are transported ro the cell surface. Here, we demonstrate that the TAP-deficient T2 cells present measles virus (MY) after transfection with the mouse MHC class I Ld molecule ro mouse CTL. Recognition depends on live virus as inactivated virus is not recognized. After infection with MV and canine distemper virus (CDV) the epirope peptide aa 280/289 present in the nucleocapsid proteins of both viruses is recognized by CTL whereas a vaccinia virus expressing MV-nucleocapsid protein (N) is not recognized. However, vaccinia per se does not inhibit presentation because a recombinant vaccinia virus expressing the epirope fused ro a peptide ensuring the transport into the endoplasmic reticulum in a TAP-independent fashion is well recognized. The TAP independent processing and presentation is not specific for MV-N, as beta-galactosidase expressed by MV is recognized by CTL, roo. The presentation of MV can be inhibited via chloroquine indicating that the exogenous pathway is used for presentation of MV by Ld. Subsequent studies by confocal microscopy have shown that Ld co-localizes with the endollysosomal marker LAMP-I. In addition, the nucleocapsid and the matrix protein, bur not the hemagglutinin protein co-localize with LAMP-I. As the matrix protein plays a prominent role in connecting the internal proteins (like N) to the envelope we assume that MV is responsible for transport of N into the endo/lysosome. This is supported by the fact that not only MV, bur also the related viruses CDV and Parainfluenza virus type I (Sendai) are recog-

350 . 30th Annual Meeting 1999 nized in a similar fashion. It might also explain, why MV is known to stimulate such a vigorous CD4+ T cell response besides a CD8+ T cell response.

lClinical Research Unit for Rheumatology, 3Institute of Biochemistry and Molecular Biology, Albert-Ludwigs-University Freiburg, Germany, and 2Neurosciences Group, Institute of Molecular Medicine, University of Oxford, Oxford, UK

c.

15 Effects of a chaperone of the HSP70 family on antigen presentation to a specific T cell clone

The primary function of MHC molecules is to present fragments of antigens to specific T cells. The processing of proteins into peptides - which are then presented by class II molecules and recognized by T cells - requires the interaction of numerous molecules, including proteases and chaperones. Alleles of class II molecules may differ not only in their binding capacity for specific peptides but also in their interaction potential with the antigen processing and peptide loading machinery. We study one example for the latter, i.e. the response of the autoreactive T cell clone PM-A, recognizing an epitope derived from the a-subunit of the human acetylcholine receptor (AChR), presented by antigen-presenting cells (APC) expressing either DRBI *0401 (71 Lys) or DRB 1*0408 (71Arg). If APC present the peptide epitope, both alleles have the same capacity to stimulate the T cells. However, using either a preparation of the human AChR or a recombinant a-chain (rl-437), 0401 presents much better than 0408 (Nicolle et af. (1995), Eur.j. Immunol., 25, 2119). The same phenomenon is observed if murine P388.Dl cells transfected with genes coding for DRBI *0401 or DRBI *0408 (TP0401lTP0408) are used. We now show preliminary evidence suggesting that processing of rl-437 is only possible in the presence of a chaperone of the HSP70 family in the antigen preparation. Supported by a grant of DAAD.

Max-Planck-Institute of Immunobiology, Freiburg, Germany

C. 16 Albumin-nickel complexes induce and activate nickel-specific T cells H.-J. THIERSE, H.U. WELTZIEN,]. VOLLMER, and C. MOULON T cell interactions with non-peptide antigens, particularly with metal ions such as Nt+, have only recently gained major attention. However, the molecular nature of e.g. Ni-induced antigenic determinants which eventually trigger the corresponding T cell receptors (TCR) has not yet been elucidated. We have previously observed that a large percentage of human T cell clones that were induced from peripheral blood lymphocytes (PBL) of Ni-allergic donors by NiS0 4 could also be driven into proliferation by addition ofNt+ complexed to human serum albumin (HSANi). Moreover, Ni-reactive T cell clones could also be obtained from the same donors by in vitro

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primary stimulation of PBL with HSA-Ni. In this case all clones were also triggered by NiS0 4. The resulting T cells were CD4+ and class II HLA-restricted, as indicated by a requirement for APC expressing autologous HLA class II alleles. Activation was also achieved by Ni-complexes with serum albumin of other species such as cattle, mouse, rat, chicken or pig, but not of dog. The effective activation by such albumin-Ni complexes did not require antigen processing, since T cell activation by HSA-Ni was also effective in the presence of glutaraldehyde fixed APC. In addition, commercially available horse radish peroxidase complexed to Ni via a synthetic Nibinding site also activated our T cell clones in presence of the respective APC. This suggests that Ni-carriers such as HSA transfer Ni++ ions to Ni-binding structures on MHC molecules on the APC which subsequently engage and activate the relevant TCR. We assume that such processes also playa role in vivo during sensitization for and/or elicitation of Ni-specific skin reactions. Supported by the BMBF (Klinische Forschergruppe «Pathomechanismen der allergischen Entziindung» FKZ 0IGC970l/7) and the BIOMED 2 program ofthe EU, project PL963713.

1Max-Planck-Institute of Immunobiology, Freiburg, Germany and zNovartis, Basel, Switzerland

c.

17 T cell-mediated nickel reactivity in HLA-DR1 transgenic mice

Nickel allergy represents the most common form of human contact dermatitis, and its correlation to Ni-reactive T cells is well established. However, although in vitro activation of Ni-specific CD4+ as well as CD8+ human T cells has recently been intensively studied, a relevant animal model to investigate Ni-reactivity in vivo is yet missing. Mice are usually not or only poorly sensitized by NiCl z or NiS0 4 for typical contact sensitivity, and in vitro activation of mouse T cells by Ni salts has proven difficult. One possible explanation for this phenomenon might be a greater potential of human versus mouse MHC molecules to complex and present Ni-ions to reactive TCR. We therefore tried to sensitize C57BL/6 mice, transgenic for the human HLA-DRl molecule. An aqueous solution ofNiCl z was applied to the abdomen and mice were tested for hyperreactivity on successive days by application ofNiCl z in DMSO to the ear. So far, neither the wild type C57BL/6 mice, nor the DRl transgenic animals exhibited any signs of contact sensitivity as evaluated by ear swelling measurement. In parallel experiments, we attempted to stimulate naive splenic T cells from either wild type or DRI transgenic mice in vitro by NiCl z. Unlike for wild type cells, DRl transgenic T cells proliferated (though poorly) in response to NiCl z and could be fused to TCR-deficient BW5l47 thymoma cells. Two Ni-responsive hybridomas were obtained, both of which turned out to be restricted to HLA-DRl. These data raise hope that improved regiments of sensitization might eventually lead to an animal model for nickel contact dermatitis. Supported by the BMBF (Klinische Forschergruppe «Pathomechanismen der allergischen Entziindung» FKZ OlGC970l!7) and the BlaMED 2 program of the EU, project PL963713

352 . 30th Annual Meeting 1999 JInstitute of Medical Microbiology, 2Department of Gynecology, and 3IIIrd Medical Department, Johannes Gutenberg University, Mainz, Germany

C. 18 Functional analysis of the MHC class II antigen presentation pathway . in cervical cancer W WALTER l , H. HOHN J, H. PILCH 2, S. GUNZEL2, B. SELIGER3, K. FREITAG l , C. NEUKIRCH 1, and M.J. MAEURER 1 HLA-DR-expression by tumor cells has been associated with enhanced infiltration of tumor infiltrating lymphocytes (TIL) in patients with cervical cancer. Increased CD4+ T cell reactivity directed against human papilloma virus (HPV)-associated T cell epitopes is correlated with viral persistence and disease progression. Presentation of tumor associated antigens by MHC class II molecules to CD4+ T cells may be instrumental in cellular immune responses directed against cervical cancer. CD4+ T cell activation is quantitatively controlled by the level of MHC class II expression on antigen presenting cells, but also requires additional factors including HLA-DM (DM), which catalyze peptide loading onto MHC class II molecules. We analyzed expression of CIITA, the MHC class II isotypes HLA-DR, -DQ and -Dp, the DM subunits DMA/DMB and DOA/DOB in cervical cancer cell lines in response to IFN-y by flow cytometry and RT-PCR. Expression of HLA-DR and -DP was coordinately induced by IFN-y in all cell lines examined. Induction of HLA-DR and -DP expression appeared to be quantitatively controlled by the level of IFN-y-induced CIITA expression. In contrast, HLA-DQ expression was not induced in response to IFN-y, implicating isotype-specific regulatory mechanisms. IFN-y induced coordinate expression of DMA and DMB, required for optimal MHC class II peptide loading. Additionally, IFN-y induced DOAIDOB expression in the cervical cancer cell line Me180. Exclusively IFN-y treatment of the HPV68+, HLA-AI,A32, B8, B44, C5, C7, DR*0401, DR9, DQY Mel 80 cell line resulted in significantTNF-a secretion in a HLA-DR4 -restricted CD4+ T cell line which defines a peptide epitope provided by HPV68. Coordinate expression ofMHC class II and DM under the control ofCIITA renders cervical cells into immune-competent antigen presenting cells as defined by activation of MHC class II-restricted and antigen-specific CD4+ T cells.

Departments of J Medical Microbiology and 2Immunology, Johannes Gutenberg-University, Mainz, Germany

C. 19 H2-M and H2-0 are differentially expressed in professional and nonprofessional antigen presenting cells

H2-M is a nonclassical major histocompatibility class II (MHC-II) molecule that catalyzes peptide loading of MHC class II molecules. Recent studies revealed the conserved MHC-II molecule H2-0 to be a modulator ofH2-M function. H2-0 binds H2-M and thereby influences the peptide repenoire, which is ultimately loaded onto MHC-II molecules. Here, we examined the regulation ofMHC-II, invariant chain (Ii), H2-M, H2-0 gene expression by IL-4 and IL-IO in

30th Annual Meeting 1999 . 353 different APC types by flow cytometry and semiquantitative RT-PCR. In detail: Stimulation of nonprofessional APCs from mesenchymal and epithelial origin by IFN-y markedly induced MHC class II, H2-Ma, -Mb and -Oa, but not H2-0b expression in the presence of the MHCII transactivator CIITA. In contrast, in B16 melanoma cells, IFN-y-induction of CIITA is associated with the coordinate expression ofMHC class II, H2-Ma, -Mb, -Oa and -Ob. Professional APCs, such as the macrophage cell line P388Dl exhibits constitutive H2-0a and -Ob expression, which is not inducible by IFN-y as compared to CIITA, MHC class II, H2-Ma and -Mb expression. Moreover, differential expression of CIITA, MHC class II, H2-Ma, -Mb relating to H2-0a and -Ob was observed in IL-4-, IL-I0- or IFN-y-treated A20 B cells. Finally, differential expression of H2-M and H2-0 apparently impacts on MHC class II restricted antigen presentation as demonstrated by the ability of P288Dl macrophages and A20 B cells to present native protein antigen to H2-Ad- and -Ed-restricted CD4+T cells. Differential expression ofH2and H2-M indicates that H2-M-mediated MHC class II peptide loading is modulated by H2-0 in a cell type specific manner.

o

I Pediatric Stem Cell Program, Children's Hospital and 2Medical and Natural Sciences Research Center (MNF), University ofTiibingen, Tiibingen, Germany

c. 20

First evidence for inhibition of staphylococcal superantigen-induced T cell proliferation by the copolymer GLAT

J.T. WESSELS I , K. SCHILBACH I , M. EYRICH\ S. LAL I , H. KALBACHER2, D. NIETHAMMER I , and P.G. SCHLEGEL I

The synthetic copolymer GLAT (L-Glu,L-Lys,L-Ala,L-Tyr) has previously been demonstrated to prevent lethal GVHD in the Bl 0.02 - BALB/c model (both H-2D)across minor histocompatibility barriers. Because of its promiscuous binding to a variety of murine MHC-II molecules, we decided to investigate the effect of GLAT on the oligoclonal stimulation of murine T cells (obtained from BI0.D2 mice in which there is no deletion of the respective V~- families) induced by the staphylococcal enterotoxin A (SEA) and B (SEB). In bulk lymphocyte cultures, GLAT inhibited superantigen-induced T cell proliferation in a dose dependent manner. Highly purified populations of CD4+ lymphocytes (depletion column) as responder cells and of dendritic cells as APC's (generated from spleen cells in the presence ofTNF-a and IL-4) were used for «defined lymphocyte culture" assays. Proliferation was measured by 3H thymidine incorporation in the presence of SEA, SEB and/or GLAT or controls. Specificity controls included HEL and nonbinding copolymer TGA. Results from these defined cultures with highly purified subsets showed strong inhibition of the T cell proliferation by SEA (If..Lg/ml) and by SEB (lOf..Lg/ml) in the presence of G LAT. The concentrations of SEA, SEB had previously been titrated to result in optimal T cell proliferation. To further demonstrate, that SEA and GLAT were able to compete in terms of their binding ability to the MHC-TCR complex, we designed a triple color fluorescence competition assay by coupling AMCA (as UV fluorochrome) to the N-terminus of GLAT. Control tests after coupling showed no loss in function of GLAT. Biotinylated SEA (Streptavidin-PE) and CDllc-FITC labeled APC's, generated as described above, were used to demonstrate that GLAT-AMCA was able to almost completely displace SEA from the MHCTCR complex. Resulrs reported from rhese studies represent the first evidence that the copolymer GLAT is capable of inhibiting oligoclonal T cell responses induced by SEA and SEB.