Presentation

WORKSHOP F

Antigen Processing/Presentation

IMax-Planck-Institut fur Immunbiologie, freiburg, G ermany; 2Center of Cytopharmacology, University of Milan, Milan, Italy

F.1 Establishment of an in vitro system to study primary and secondary T cell responses to Borrelia burgdorleri U. AlTENSCHMIDTI, P. Rl cClAR DI-C ASTAGNOU 2, and M. M. SIMON I Lyme disease is the most common tick-born illness in Europe and th e United States. Recent studi es ha ve shown that infecti on with the causati ve bacteria, Borrelia burgdorferi (B. burgdorferi), results in the induction of humoral and cellular immune responses . Investigations indicate that T cells are involved both in th e generation of optimal protective antibody responses and in the development of chronic arthritis and carditis. However little is known about the quality and quanti ty of spirochete -specific T cells induced in th e course of infection. This is mainly due to th e fact that the attempt to propagate B. burgdorferi reactive T cells in vitro are discouragi ng so far. In order to establish a suitable system to analyze spirochete specific T cell res ponses in vitro, various cell populations including transformed macrophage and dendritic cell lines were analyzed fo r surface expression of MHC class II molecules and their ability to present antigen to T cells. MHC class II structures were differentially up regulated by different antigen preparations of B. bllrgdorferi and among all antigen- presenting cells tested, dendritic cells were the most potent ones to ind uce spirochete-specific T cell responses. Proliferative responses of presensiti zed T cells were about 100 to lOOO- times higher compared to th ose of naive T cells. It is to be expected that this system will allow to cha racteri ze B. bllrgdorferi associated T cell epitopes relevant for protection and pathogenesis in Lyme disease.

Institut fur Immunologie, Mainz, G ermany

F.2 Establishment of different rat invariant chain isoforms and analysis of their effects on class II biosynthesis, transport and class II-restricted antigen presentation K. DEMLEITNER, s.

II.l. Y,

and K. RESKE

During biosynthesis, MHC class II molecules are tightly associated wi th the invariant y chain (Ii). This association has been implicated to control intracellular sorting of class II

158 . 25th Meeting of the Society of Immunology and to bear upon their peptide loading. The major y chain form (p33) is a glycoprotein of 33 kD. A second form results from alternative splicing of an additional exon (exon 6b). The resulting protein p41 carrying a 64 amino acid insertion has been proposed to play an important role in antigen presentation. We established a full-length eDNA clone (ypLRI 11.2) from a eDNA-library prepared from IFNy-stimulated Lew.AVN rat bone marrow macrophages. Clone ypLR/11.2 was found to contain exon 6b as well as an additional in frame start codon. The latter has been described in human but not in mouse giving rise to y protein species p35 and p43. Because recent studies demonstrated that the cytosolic tail of the invariant chain plays an important role in class II targeting, we constructed rat eDNA-clones corresponding to four y proteins namely p43, p41, p35 and p33. Moreover using PCR-techniques the second start codon present in p43 and p35 has been functionally deleted. To investigate the efficacy of the different y forms in transport and intracellular targeting of class II heterodimers and to assess their potential in antigen presentation COS cells were transfected transiently with these constructs with or without cotransfection of rat class II genes. COS cells transfected with the individual invariant chain eDNA clones express all four y chain forms which seem to follow common intracellular transport routes. Moreover immunofluorescence staining of saponin permeabilized a,~, invariant-chain transfectants revealed that similar patterns of intracellular distribution of class II molecules were detectable irrespective of the class II accompanying y chain species. Immunochemical and functional analyses of the individual a~y-transfectants are in progress.

Dept. of Virology, University of Heidelberg, Heidelberg; 1Dept. of Virology, University of Ulm, Ulm, Germany

F.3 Influence of flanking amino acids on antigenic peptide processing and presentation is independent of protein conformation M. EGGERS, T. RUPPERT, and U. H. KOSZINOWSKI

B. BOES,

M. DEL VAL, H.-J.

1

SCHLICHT ,

De novo synthesized proteins underly cytosolic degradation in cells. Processed antigenic

pep tides bind selectively to MHC class I molecules and are presented to cytotoxic T lymphocytes. Previous studies have shown that the MCMV pp89 epitope 168YPHFMPTN176 can also be correctly processed from a foreign carrier protein (DEL VAL, M. et al. 1991. Cell 66: 1145-1153). The amount of antigenic peptide depends on the insertion site - after insertion in the HBe-protein the amino terminal insertion results in lower quantities and also a diminished presentation. This unfavorable presentation could be rescued by substituting the flanking amino acids with a single alanine residue and inserting various oligo-alanine spacers. The observed effect could be caused by a negative influence of local flanking amino acids or a change in the conformation of the carrier protein. To test an local influence minigenes coding for a 17- and a 19-mer were constructed and expressed by recombinant vaccinia viruses. From these experiments we learned that the effect of the flanking amino acid observed in chimeric proteins is also true for small polypeptides. This local effect on antigen presentation could be due to a change in the proteolytic cleavage or to different transport properties of the arising pep tides into the endoplasmatic reticulum. Proteasome cleavage data of synthetic peptides representing sequences for efficient and poor antigen presentation are reported.

25th Meeting of the Society of Immunology . 159 Clinical Research Group on Allergy, Dept. of Dermatology, University of Mainz, Mainz, Germany

F.4 The failure of la+ T clone cells 81/04.1 to induce proliferation of TH1 cells correlates with a lack of expression of 87-2 S. FROSCH, U. BONIFAS,]. SCHWING, and A. B. Rr.SKE-KuNZ Optimal stimulation of T cell proliferation and IL-2 production requires not only signaling via the T cell receptor (TCR)/CD3 complex, but also costimulatory signals provided by a variety of adhesion molecules on the antigen-presenting cells (APC). After stimulation via the TCR/CD3 complex in the absence of costimulatory molecules T cells fail to produce IL-2 and to proliferate and develop a long-lived state of unresponsiveness. Here, we compared the accessory function of various APC to antigen-specifically stimulate the murine THI cell clone KIllS. Spleen cells and epidermal Langerhans cells (LC) induced KIllS T cells to produce IL-2 and to proliferate. In contrast, the T clone cells BII04.1, expressing MHC class II molecules constitutively, although inducing IL-2 secretion, failed to activate the KIllS T cells to proliferation. KIllS T cells were not rendered anergic by BII04.1. This correlates with the high expression of B7-1 molecules on BII04.I, responsible to prevent T cells from unresponsiveness. Other accessory molecules like LFA-I, ICAM-l and the ligands for CD2 were expressed on BII04.I at high levels and were functionally active. HSA was not expressed on BI/04.1, but does not seem to be involved in induction of proliferation of KIllS T cells because HSAtransfected CHO cells did not costimulate T cell activation. B7-2 molecules, recently shown to playa critical role in the initiation of T cell proliferation, were absent from BII 04.1. Therefore the lack of B7-2 on BII04.1 alone or together with a lack of a so far not defined costimulatory molecule(s) might be responsible for the failure to induce proliferation of KIllS T cells. LC on the other hand, express this costimulatory molecule(s) and are thus effective stimulators.

Abteilung fur Medizinische Virologie, Universitat Heidelberg, Heidelberg, Germany

F.S Ineffective processing of antigenic cytomegalovirus peptides in immunodepleted mice G. G. H. HENGEL, T. RUPPERT, and U. KOSZINOWSKI Control of murine cytomegalovirus (MCMV) disease is mediated by virus specific CD8+, CD4- T lymphocytes. T cells of the MCMV infected BALB/c mice recognize the nonapeptide YPHFMPTNL of the MCMV lEI protein pp89 presented by the MHC class I molecule Ld. It has been shown that MCMV can escape the CTL control in vitro by downregulation of class I molecules from the cell surface. Viral escape could be prevented by a counteracting upregulation of class I molecules mediated by interferon-y (HENGEL et ai.,]. Virol. 68: 289). We compared the efficiency of antigen processing in MCMV infected normal and full body gamma irradiated mice. The efficiency of antigen processing was determined as ratio between naturally processed antigenic peptide and virus productivity in individual organs. As measured by the peptide/virus ratio antigen

160 . 25th Meeting of the Society of Immunology processing in immunocompetent mice was 100- to 10000-fold more effective than in gamma-irradiated mice. This discrepancy might be either due to the depletion of antigen processing cells, or it may be caused by a lack factors (e.g. interferon-y) physiologically involved in antigen processing and presentation.

IInfectious Disease Unit, Mass. General Hospital, Harvard Medical School, Boston; 2Dept. of Public Health, San Francisco; 3NCI, Frederick; 4University of California, Davis, USA; 5Current address: Dept. of Internal Medicine III, University of ErlangenNurnberg, Erlangen, Germany

F.6 Fine analysis of epitopes recognized from HIV-l infected longterm survivors T. HARRER I, 5, E. HARRER!, S. BUCHBINDER2, D. MANN 3, T. YILMA\ R. P.jOHNSON\ and B. D. WALKERI Objective: Analysis of CTL epitopes recognized in a cohort of 10 healthy nonprogressing individuals with documented HIV -1 infection for 10 to 15 years. - Materials and methods: CTL clones or lines were obtained either by stimulation of PBMC under limiting dilution conditions with the help of unspecific mitogens or by stimulation of PBMC with autologous HIV -3B superinfected CD4-cells, and further characterized by epitope mapping. - Results: We could define several highly conserved epitopes located in areas of the viral genome with functional importance. An epitope in gp 120 is overlapping with the CD4-binding site. An epitope in p17 contains a putative matrix localization signal. An epitope in RT is targeting the sequence YMDD, the active site of the RNA-dependent DNA-polymerase. - Conclusions: Analysis of CTL epitopes recognized by healthy long-term non-progressing HIV -1 infected individuals is important for the rational design of both a preventive and a protective vaccine.

University of Darmstadt, Dept. of Microbiology, Darmstadt, Germany

F.7 Enhancement of uptake of lipopolysaccharide in macrophages by the OmpA protein of Gram-negative bacteria A. KORN,

z. RA]ABI, B. WASSUM, and K. NIXDORFI'

Monoclonal antibodies (mAb) to lipopolysaccharide (LPS) and to the major outer membrane protein OmpA from Proteus mirabilis were generated and used to follow the kinetics of uptake in macrophages of LPS as well as LPS bound to OmpA using a modified enzyme-linked immunosorbent assay in a microtiter plate culture system. In this assay, differentiation between antigen on the cell surface and antigen which had been internalized could be made. The mAb were of various IgG subclasses and showed strong reactivities with the respective antigens. Uptake of LPS by macrophages showed a relatively rapid increase during the first 4 h of culture and then progressed more slowly

25th Meeting of the Society of Immunology . 161 over the remaining 24 h observation period. When macrophages were pulsed with LPS for 30 min, the amount of LPS detectable on the macrophage surface decreased progressively thereafter over a 3 h interval, which indicated internalization of the antigen, and rose subsequently to a new level on the surface at 24 h. Uptake and internalization of LPS were more efficient when it was in complex with OmpA. In contrast, there was no augmentation of uptake when LPS was combined with bovine serum albumin. The level of detection of LPS in this assay system was approximately 10 ng/ml. When the fate of the same complexes was followed using the mAb to OmpA, the pattern of internalization was similar to that delineated by the mAb to LPS, but the protein did not reappear on the cell surface in a form detectable with mAb. The OmpA-LPS complex was taken up more efficiently than the protein alone in the early phases of culture (l-4h). This was not simply due to more rapid sedimentation of larger aggregates onto the macrophage surface; these differences were not seen when the cells were fixed prior to addition of the antigens. Macrophages were apparently stimulated to more active uptake of the OmpALPS complexes.

Institute of Medical Microbiology and Hygiene, Technical University of Munich, Munich, Germany

F.8 Oenaturated ovalbumin enters class I-restricted pathway of antigen presentation and allows in vivo sensitization of cytolytic C08+ T lymphocytes B. MARTINEZ-KINADER, H. WACNER, and K. HEEC Soluble proteins like ovalbumin (Ova) in native form fail to enter the endogenous pathway of antigen presentation and fail to sensitize class I -restricted cytolytic T lymphocytes in vivo. To target these antigens into class I-restricted pathway of antigen presentation and to allow effective T cell vaccination soluble proteins have to be entrapped into vehicles like ISCOMs or liposomes. We show here that denaturation of Ova by temperature or SDS allows to immunize in vivo class I-restricted CDS+ cytolytic T lymphocytes without the usc of a vehicle or adjuvant. The induced CTL recognize the immunodominant peptide from Ova (257-264) in the context of H2-Kb. In vitro, denaturated but not native Ova sensitizes EL4 target cells for Ova-specific recognition by CTL. Target cell sensitization can neither be prevented by cycloheximide nor by brefeldin A, indicating that de novo class I synthesis and transport is not required. In addition, transporter-defective cell line RMA-S is efficiently sensitized by denaturated Ova. In conclusion our data suggest that denaturated proteins enter class I -restricted route of antigen presentation by a so far unrecognized pathway.

162 . 25th Meeting of the Society of Immunology Institut fur Immunologie, and tpathologisches Institut, Joh.-Gutenberg-Universitat Mainz, Mainz, Germany

F.9 Dendritic cells (DC) develop from phagocytosis positive and class II negative Pro DC by in vitro differentiation of rat bone marrow in the presence of GM-CSF M. MEHLIG, C. SCHEICHER, H.-P. DIENES t, and K. RESKE We established an in vitro culture system to raise DC from bone marrow cells of LEWIS rats by the use of recombinant mouse GM-CSF. Following 10 days of continuous in vitro culture approximately 30 % of the BM-cells collected express excessive amounts of class II molecules as evaluated by FACscan analysis and biosynthetic labelling. The capacity of the BM-DC to phagocytose particulate antigen in conjunction with the cell's class II expression was explored and correlated with the maturational state of in vitro differentiating DC. Electron microscopic measurements demonstrated the presence of particles of varying size ranges in strongly class II positive cells with dendritic morphology. Adjacent to these cells, phagocytosis positive cells were detected that exhibited characteristic dendritic morphology but were totally class II negative as revealed by mAb OX6 staining. This pattern suggested that the two cell types might be interrelated and that both represent descendents from a common proliferation cluster. Since matured BM-DC confronted with polystyrol particles to not take up the beads it is concluded that phagocytosis positive, class II deficient Pro DC represent differentiating progenitors of the class II expressing mature DC population.

Institut fur Immunologie, Universitat Mainz, Mainz; 'Zentrum Biochemie, Biochemie II, Universitat Gottingen, Gottingen, Germany

F.10 Intracellular location of events involved in antigen processing is different in B cells and macrophages (Mph) S. MILBRADT, K. SCHLINK,]. HAMPL, T. BRAULKEt, K. YON FIGURAt, E. RODE, and G. GRADEHANDT Exogenous protein antigens (Ag) are processed after uptake by antigen-presenting cells (APC) prior to presentation to CD4+-T cells in combination with MHC class II molecules. The intracellular site of the proteolytical cleavage of the antigens is still uncertain as it is also the case for location of peptide binding to MHC class II. We could co-localize a number of molecules believed to be involved in antigen processing (Cathepsin B, invariant chain, C-form of MHC class II and a new MHC II-associated, peptide binding complex with an apparent mw of 90 kDa) in endosomes or pre-Iysosomes, irrespective of the type of APC investigated. Interestingly Mph, in contrast to B cells, contain the C-form and the peptide binding complex within lysosomes, indicating efficient loading of MHC class II-molecules in this compartment. To investigate the putative role of endosomes in proteolytical antigen processing we used approaches which allow selective depletion of endo/lysosomal enzymes like cathepsin Band D within the endosomes. For this purpose B cells and Mph were incubated with antisera directed

25th Meeting of the Society of Immunology . 163 against the 300 kDa mannose-6-phosphate receptor (MPR) to prevent guidance of man6-P-bearing enzymes to endosomes and subsequently lysosomes. When B cells were cultured under conditions selectively depleting endosomes from such enzymes the presentation of antigens needing proteolytic degradation to specific T cells was blocked. In contrast, processing of these antigens by anti-MPR-treated Mph was not influenced. As the presentation of processing-independent pep tides and the stimulation of alloreactive T cells was not affected possible, overall toxic effects of the antisera could be excluded. On the other hand, if APC were treated with cystamin which only affects protein degradation in lysosomes but not in endosomes only presentation of antigens by Mph was diminished. These data indicate that the proteolytic step of antigen processing as well as peptide loading to MHC class II-molecules in B cells occurs primarily in endosomes, whereas in Mph the lysosomes did also participate in both events.

Institut fur Immunologie, Mainz, Germany

F.ll Chondroitin sulfate derivatized invariant chain is synthesized by Langerhans cells U. NEISS, A. MARX, K. DEMUITNER, and K. RESKE The invariant chain, a nonpolymorphic glycoprotein which associates noncovalently with MHC class II molecules has been described to occur in a proteoglycan form (li-CS) that bears a chondroitin sulfate glucosaminoglycan. Recent data suggested that li-CS can function as an accessory molecule involved in APC-T cell interactions in allogeneic T cell responses. Epidermal Langerhans cells (LC) represent highly efficient antigen-presenting cells whose capacity to stimulate primary immune responses increases upon short term in vitro culture. In previous work we described the noncoordinate synthesis of class II and invariant chains by rat LC following short term culture. Thus LC cultured for 3 days synthesized abundant levels of invariant proteins in the absence of class II biosynthesis. The aim of our present study was to investigate the relationship of li-CS synthesis in fresh and cultured LC in the light of the functional characteristics of the two cell populations. Cells were grown in the presence of CSSJ-suifate for 4 h and were subjected to NP40 extraction with subsequent immunoprecipitation using class II and invariant chain specific mAb. In fresh and cultured LC distinct li-CS synthesis was detectable. However, in contrast to the biosynthesis of the invariant chain, synthesis of li-CS was not upregulated during LC culture. Thus quantitative differences in li-CS synthesis between fresh and cultured LC cannot be responsible for the increase of the LC's stimulatory capacity observed upon culture. Nevertheless qualitative changes in li-CS between fresh and cultured LC were observed. The molecular weight of li-CS derived from fresh LC exhibited size heterogeneity (40-80 kd) and was shifted to higher values in cultured LC. Further experiments using COS cells transfected with eDNA-clones of invariant chain forms p31 and p41 suggested that the differences in molecular weight of Ji-CS might result from chondroitin sulfate modification of different invariant chain core proteins.

164 . 25th Meeting of the Society of Immunology Abt. Neuroimmunologie, MPI fur Psychiatrie, Martinsried, Germany

F.12 Unusual regulation of MHC class I antigens in single, electrically active neurons of rat brain cultures H. NEUMANN, A. CAVALIE, D.JENNE, and H. WEKERLE In striking contrast to most other cells, functionally active neurons fail to express MHC class I antigens. The molecular mechanisms underlying this deficiency and its functional significance are unknown at present. We studied MHC class I gene expression in neurons of embryonic Lewis rat hippocampal cultures. We used a new technology combining whole-cell patch clamp analysis with RT-PCR to characterize neurons electrophysiologically and to amplify mRNA isolated from these particular single cells. Furthermore membrane expression of MHC class I products was analyzed by immunofluorescence staining. Hippocampal neurons identified by spontaneous or induced action potential firing failed to express MHC class I products on their membranes. Nevertheless, mRNA for class I was detected in 9/13 neurons investigated so far. In contrast, mRNA for P2micro globulin was only present in a minority of these neurons (2/13). Some neurons (4/ 13) expressed neither mRNA for class I nor for p2-microglobulin. However, we always detected mRNA for class I and ~2-microglobulin in cultured astrocytes. After repeated activation by interferon-y neurons became stainable with anti-class I mAbs, and under the same treatment, mRNA for class I and ~2-microglobulin became apparent. These preliminary data suggest, that in neurons availability of p2-microglobulin may be a limiting factor for membrane expression of MHC class I antigens.

I. Medizinische Klinik und Poliklinik der Johannes-Gutenberg-Universitat Mainz,

Mainz, Germany

F.13 Anti-neutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis, primary sclerosing cholangitis and autoimmune hepatitis T. ORTH, A. SCHWARTING, G. GERKEN, K.-H. MEYER ZUM BOSCHENFELDE, and W.-J. MAYET Antibodies against cytoplasmic components of neutrophil granulocytes have been described as markers for systemic vasculitis, especially for Wegener's granulomatosis. Furthermore, in recent studies ANCA have also been detected in ulcerative colitis (UC), primary sclerosing cholangitis (PC) and autoimmune hepatitis (AIH). Two types of ANCA can be distinguished performing indirect immunofluorescence: cytoplasmic or C-ANCA and perinuclear or P-ANCA. In this study sera of patients with UC (n=50), PSC (n = 20) and AIH (n = 50) have been investigated to determine the type of ANCA and to determine further the antigen specificities in human' granulocytes. Granulocytes were isolated from heparinized blood of healthy blood donors. Cytocentrifuged slides were fixed with ethanol and analyzed by immunofluorescence technique (1FT). 36 % of our patients with UC, 50 % of our patients with PSC and 54 % of our patients with AIH

25th Meeting of the Society of Immunology . 165 showed a cytoplasmic fluorescence pattern. In ELISA with coated total cytoplasmic extract from granulocytes we found elevated optical densities in comparison to normal human sera in UC: 15 %, PSC: 30 % and AIH: 80 'Yo. By Western blot analysis using total cytoplasmic neutrophil extract from granulocytes we observed positive reactivity with sera from patients with PSC and AIH against polypeptides ranging between 30 kD and 80 kD. Further work is in progress to characterize the target antigens human granulocytes in different diseases. Our data suggest that C-ANCA at level of 1FT are not specific for one disease, but are present in diverse conditions characterized by autoimmune chronic inflammation.

Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA

F.14 Peptide transporters form complexes with MHC class I ~rmicroglobulin dimers in the endoplasmic recticulum B. ORTMANN, M. J. ANDROLEWICZ, and P. CRESSWELl. Class I molecules present pep tides derived from endogenous, mainly cytoplasmic proteins to cytotoxic T cells. Most probably those pep tides are generated by the action of a cytoplasmic multi catalytic proteinase complex, the so-called proteasome, and are transported into the endoplasmic reticulum (ER) by a heterodimeric transmembrane protein complex formed by the transporters associated with antigen processing (TAP.1 and TAP.2). In the mouse class I heavy chains and to some extent class I-Brmicroglobulin(B2m) dimers are bound to the ER resident chaperone calnexin (IP90 in human, p88 in mouse). Here we show that class I heavy chain-B2m dimers of all alleles tested are retained in the ER of human lymphoblastoid cell lines in association with TAP proteins, and thus coprecipitate with TAP from cells solubilized in digitonin. In the r3 2 m negative cell line Daudi class I heavy chain remains bound to calnexin and does not associate with TAP, indicating that B2m is a necessary prerequisite for dissociation of heavy chain from calnexin and association with TAP. Isolated TAP-associated class I-r:l2m dimers can be loaded with class I binding peptides and are stable after Triton XIOO induced release from TAP in vitro. Class I binding peptides added to streptolysin O-permeabilized cells mediate specifically the dissociation of class I molecules from TAP in vivo. These data suggest that class I -B2m dimers after dissociation from calnexin and association with TAP bind peptides transported by TAP and dissociate from TAP ready for the transport to the cell surface. The determination of the size and the subunit composition of TAP-class I complexes is currently under investigation.

166 . 25th Meeting of the Society of Immunology 1Max-Planck-Institut fur Biologie, Abteilung Infektionsbiologie, Tubingen; 2Medizinisches Naturwissenschaftliches Forschungsinstitut, Tubingen; 3Naturwissenschaftliches und Medizinisches Institut an der Universitat Tubingen, Reutlingen, Germany

F.15 Peptides derived from a secreted neisserial antigen interact with purified human MHC class II molecules K. OTZELBERGERI, H. KALBACHER2, H. MAX 2, K. H. WIESMOLLER 3, and T. F. MEYER 1 Pathogenic Neisseria (N. meningitidis and N. gonorrhoeae) secrete IgA-proteases, highly specific bacterial enzymes which cleave human IgA1 antibodies. IgA protease activity has been demonstrated in cerebrospinal fluids of meningitidis patients and in genital gonococcal infections. This potential virulence factor induces an antibody response during clinical disease or after nasopharyngeal carriage. We were interested in the MHC class II restricted antigen recognition of this exogenous antigen by helper T cells and therefore examined the binding specificity to a panel of MHC class II molecules to identify candidate T cell epitopes. For this purpose we synthesized overlapping pentadecamer peptides and performed in vitro peptide binding assays (KROPSHOFER, H., J. Exp. Med. 1992). EBV-transformed homozygous cell lines were used to purify MHC molecules. The solubilized heterodimers were incubated with a fluorescence-labelled peptide representing a naturally occurring epitope and analyzed by high performance gel filtration using fluorescence detection to calculate the amount of bound labelled peptide. Unlabelled synthetic neisserial peptides were then added in excess. We found that several peptides compete with the binding of the reference peptide in the MHC binding groove. Interestingly even pep tides lacking prominent hydrophobic anchor residues show binding affinity to DR1w1, DR4w4, and DR4w14.

Institut fur Immunologie, Joh.-Gutenberg-Universitat Mainz, Mainz, Germany

F.16 In vitro grown bone marrow DC activated by uptake of bead-adsorbed antigen express ILl and IL12 mRNA and exhibit augmented T cell stimulatory capacity C. SCHEICHER, M. MEHLIG, R. ZECHER, and K. RESKE

Immature precursors of BM-DC in contrast to fully matured BM-DC exhibit pronounced phagocytic capabilities. Because in progressing GM-CSF supplemented BMcultures a fraction of approximately 4 % of DC precursors was found to arise daily we employed this culture system to measure the DC's capacity to process and present antigen adsorbed to polystyrol particles versus antigen applied in soluble form. Antigen coated polystyrol beads were added to DC cultures set up in parallel within intervals of 24 h starting at day 1. Twenty-four hours following particle administration to each culture dish sizable numbers of beads were observed having been internalized by DC precursors while the majority of the beads was found engulfed by codeveloping macrophages. By day 7 the percentage of phagocytosis-positive DC in all parallel cultures was evaluated by FACscan analysis using double-labelling. The processing and presentation of particle-bound as compared to soluble antigen was monitored by antigen

25th Meeting of the Society of Immunology . 167 specific MHC class II restricted T cell triggering. It was found that uptake of antigen by phagocytosis as compared to fluid phase endocytosis yielded significantly enhanced stimulatory values. Furthermore the proportion of transcripts of various cytokines expressed by DC which had engulfed antiugen coated beads and by untreated control DC were assessed by Northern blot hybridization. Significant differences in the amount of ILlu- and ILl2(p35, p40)-transcripts between the two cell preparations were noticed. This pattern of expression was seen reproducibly in phagocytosis positive DC in contrast to untreated DC and corresponding control BM-Mph.

Institut fur Immunologie, Universitat Mainz, Mainz; I Abt. Biochemie II, Zentrum Biochemie, Universitat Gottingen, Gottingen, Germany

F.17 MPR46 deficient macrophages (Mph) have a reduced capacity to present exogenous protein antigens K. SCHLINK, S. MILBRADT, E. RUDE, K. VON FIGURAl, A. KOSTER" U. MATZNER" R. POHLMANN!, and G. GRADFHANDT Tubulo-vesicular lysosomes of macrophages (Mph) appear to playa central role for the processing of antigens to peptides that subsequently bind to class II MHC-molecules. Lysomal enzymes are guided to their destination by two specific transporting receptors: the 46 kDa and the 300 kDa mannose-6-phosphate receptor (MPR46 and MPR300 respectively). The MPR46 was found to be extraordinary expressed especially in hematopoietic tissues (fetal liver, spleen, thymus, bone marrow), freshly isolated ex vivo T and B lymphocytes and in cultured bone-marrow derived Mph. MPR46-deficient mice have been obtained by «knocking-out» the respective gene in murine embryonic stem (ES) cells, followed by injection of MPR46-gene deficient ES-cells into the blastocysts of B16-mice. The mice resulting from this procedure appear to have a normal phenotype under specific pathogen free conditions and do not show any sign for lysosomal storage diseases. The serum levels of lysosomal enzymes are within the normal range. To analyze the role of MPR46 in antigen processing by Mph, in which lysosomes are shown by us to playa central role in this process, Mph were cultured from their bone-marrow precursor cells derived from MPR46-deficient mice and wild type (WT) controls. The amount and structure of the class II MHC-molecules, especially with regard to the C-form, which is formed after binding of antigenic peptides, was found to be identical in both populations. This was confirmed by biochemical and FACS-analysis. Nevertheless the capacity for presentation of antigens whose processing depend on proteolytic degradation was strongly reduced in MPR46-deficient Mph compared to WT control cells. No reduction of presentation of antigens whose processing does not involve proteolysis could be observed. Therefore we conclude, that only the proteolytic degradation of antigens depends on the presence of MPR46. To further confirm this hypothesis, the amount of the M6P-marked lysosomal enzyme Cathepsin B in WT and MPR46-deficient Mph was detected. It was found that Mph lacking MPR46 contained only 50 % of the Cathepsin B-level found in WT cells. These data suggest an exceptional function of MPR46 in the guided transport of lysosomal hydrolases in Mph. This was further confirmed by the finding that IFNy-stimulated P388.Dl Mph which have a mutation-based deficiency in MPR300-expression are comparable to bone-marrow derived Mph in their potency to present all types of antigens and the intracellular amount of lysosomal hydrolases.

168 . 25th Meeting of the Society of Immunology Institute of Immunology, University of Kiel, Kiel, Germany

F.1S Indirect recognition of HLA-A2 derived peptides S. STEGMANN, N. ZAVAZAVA, A. FREESE, and W. MOLLER-RuCHHOLTZ The crystal structure of the HLA-A2 molecule previously described by Wiley's group has opened up new possibilities for studying the regions of the HLA molecule that are of immunologic interest. In this study we looked at overlapping peptides of the HLA-A2 molecule (56-113) and examined the response of peripheral blood lymphocytes (PBL) obtained from patients who had received HLA-A2 mismatched renal allografts and from healthy persons (non-HLA-A2). Peptides were 5-15 residues long. Two of the peptides examined had a stimulatory effect on PBL in a 7 day proliferation assay. Both were from the a1 region. Parallel to these experiments, we measured the release of interleukin 2 in the supernatants. The same two pep tides from the a1 region which were good stimulators for PBL were able to induce the release of interleukin 2. The other peptides failed to induce measurable interleukin 2 production. Both experiments showed only little differences between the cultures of the renal patients and the healthy persons. These data suggest that the frequency of cytotoxic T cells that respond to indirect presentation of allo-MHC antigen is low. Using this assay we can now identify the immunogeneic CTL epitopes on MHC molecules and hopefully allow intelligent mismatches in clinical transplantation.

1 Institute for Anthropology and Human Genetics, Munich; 2Institutefor Microbiology, Regensburg, Germany; 3Dept. of Immunology, The Scripps Research Institute, LaJolla, CA,USA

F.19 Nonamer peptides binding to HLA-E in vitro M. UUlRECHT\ s. MODROW2, R. SHRIVASTAVA3, P. PETERSON 3, and E. H. WEISS 1 MHC class Ib molecules in the mouse have been shown to present antigenic peptides, whereas in humans such a task has only been described for CD1b. A function of the nonclassical HLA class I molecules, namely HLA-E, -F and -G, is still in dispute. In contrast to HLA-G and -F, HLA-E is strongly transcribed in all human tissues investigated to date. None the less, cell surface expression of HLA-E in vivo has not been demonstrated yet. We described a mouse myeloma cell (P3X63Ag8.653) cotransfected with the genes for HLA-E and human ~2m that showed low cell surface expression of HLA-E most likely due to impaired binding of endogenous peptides. We used these transfectants to define synthetic peptides capable of binding to HLA-E by means of their ability to enhance cell surface expression of HLA-E. Binding of peptides was confirmed by testing their effect on the thermostability of soluble empty HLA-E/human ~2m dimers expressed by Schneider cells. Two viral peptides binding to HLA-E were thus identified. As found for the classical MHC class I molecules, the minimal length of the peptides required for binding was nine amino acids. Data obtained from the mouse myeloma HLA-E transfectants indicated that in spite of the high constitutive mRNA expression, the HLA-E antigen might be only present on the cell surface in selected tissues. Therefore we are currently establishing HLA-E specific monoclonal antibodies to analyze tissue distribution of the HLA-E antigen and its subcellular localization in vivo. Supported by the Deutsche Forschungsgemeinschaft (SFB 217).

25th Meeting of the Society of Immunology . 169 1 Institut fur Anthropologie und Humangenetik, Munchen; 2Dermatologische Klinik und Poliklinik der Universitat Munchen, Munchen; 3Universitatsklinik GroJlhadern, GroJlhadern, Germany

F.20 HLA-G expression in human epithelial tissue

The HLA-G molecule belongs to the group of non-polymorphic MHC class I molecules which consists in man of HLA-E, -F and -G, the function of which has not been identified so far. A very restricted expression is characteristic for HLA-G in that it is mainly found in fetal tissues, especially in trophoblast, suggesting that HLA-G might playa role in the induction of maternal tolerance to the fetus. Moreover HLA-G is expressed in three alternatively spliced mRNA variants. The function of the shortened HLA-G heavy chains lacking either the a2 domain (HLA-GL'1a2) or both the (X2 and u3 domains (HLA-GL'1a2/a3) is not known. Interestingly, in cultured keratinocytes a strong induction of HLA-G mRNA was observed. HLA-G mRNA was also detected in biopsies taken from patients with different skin diseases including tumors and inflammatory lesions. Remarkably, the different samples varied in the expression pattern with regard to the different splice variants. In addition, we identified a new, alternatively spliced HLA-G mRNA coding for a soluble form of the molecule, explaining the secreted HLA-G antigen described previously. A possible functional involvement of HLA-G in the pathogenesis of skin diseases has to be discussed. We also investigated whether other epithelia might express HLA-G mRNA and analyzed different samples of normal uterine cervix, cervix displasias and carcinomas. No HLA-G mRNA could be detected in cervix tissue. Supported by the Deutsche Porschungsgemeinschaft (SFB 217).

Dept. of Immunology, University of Ulm, Ulm; lDept. of Virology, University of Heidelberg, Heidelberg, Germany

F.21 Contribution of the cellular hsp60 to presentation of stress induced self-epitope cross-recognized by a CDS T cell clone with specificity for a mycobacterial hsp60 peptide U. ZOGFL, B. SCHOEL, H. HENGEL 1, and S. H. E. KAUFMANN A cytolytic TCR a/~ CD8 T cell clone with specificity for the mycobacterial hsp60 peptidesoo-SOg was derived from C57BL!6 mice immunized with mycobacterial hsp60 in ISCOM. This T cell clone in addition, recognized IFNy-stressed syngeneic macrophages. By treatment of stressed target cells with hsp60-specific anti -sense oligonucleotides autoreactive lysis by this T cell clone was specifically inhibited, leaving the presentation of an unrelated peptide unaffected. For identification of the stress-induced epitope, peptides of 15 amino acids length, spanning the entire self-hsp60 with an overlap of 5 amino acids, were synthesized. However, none of these 48 peptides was able to prime target cells for recognition by the crossreactive T cell clone. Although the self-hsp60 peptideSlO_SlS shares partial homology with the mycobacterial hsp60 peptidesoo_sos, this

170 . 25th Meeting of the Society of Immunology peptide was not recognized by the auto reactive T cell clone. Sensitization of target cells for lysis by this T cell clone was made possible by replacing Asp in the self-hsp60 peptideSlO-S18 with Asn, which is known to be crucial for binding of peptides to the H2Db binding groove. We conclude that the known self-hsp60 is involved in the processing/presentation of the stress-induced self-epitope to the auto reactive T cell clone with specificity for mycobacterial hsp60, but itself is not the direct source of this self-epitope.