Workshop E: Antigen Processing and Presentation

Workshop E Antigen Processing and Presentation

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Division of Immunology, The Walter and Eliza Hall Institute of Medical Research and Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia

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E. 1 Cross-presentation of cellular antigens by lymph node dendritic cells G.M.N. BEHRENS1, M. LI1, C.M. SMITH1, G.T. BELZ1, F.R. CARBONE2, K. SHORTMAN1, and W.R. HEATH1 Dendritic cells (DC) can be divided into at least three subsets in the spleen, and five subsets in the lymph nodes. Splenic DC, are either CD4+CD8–DEC-205–, CD4–CD8+DEC-205+ or CD4–CD8–DEC-205–, whereas lymph node DC contain an additional two subpopulations: CD4–CD8–DEC-205+ and CD4–CD8intermediateDEC-205+. It has previously been reported that after in vivo immunization with cell-associated OVA, the CD8+ subset of splenic dendritic cells are the only subset capable of cross-presenting this antigen to CD8 T cells. This report demonstrates that CD8+ DC are also the only splenic DC subset capable of cross-presentation in an in vitro assay where DC are mixed with OVA-coated irradiated spleen cells. We used this same in vitro assay to determine which lymph node derived DC subsets were capable of cross-presentation of cell-associated antigen. OVA-coated bm1 splenocytes were only effectively cross-presented to OVA-specific CD8+ T cells by the CD4–CD8+DEC-205+ subset, corresponding to the same subset that cross-presents this antigen in the spleen. These data suggest that the ability to crosspresent cell-associated antigens is largely limited to a single subset of dendritic cells.

Departments of 1Internal Medicine II, 2Internal Medicine IV, 3Neurology, 4Immunology, Institute of Cell Biology, and 5Medical and Natural Sciences Research Center (MNF), Eberhard-Karls-University, Tübingen, Germany

E. 2 Cathepsin G is the functionally dominant protease for processing of myelin basic protein in lysosomal fractions of primary human B cells T. BURSTER1, A. BECK2, E. TOLOSA3, S. STEVANOVIC4, G. SCHWARZ5, H. KALBACHER5, and C. DRIESSEN1 Recent data demonstrated that the asparagine endoprotease AEP and cathepsin (Cat) S dominate proteolytic processing of the potential autoantigen Myelin Basic Protein (MBP) by B-lymphoblastoid cells (BLC) in vitro. However, there is little knowledge about the selection of proteases that are active in the MHC class II antigen processing compartment in primary human APC. To this end we exposed MBP and a mutant version that lacked the AEP processing site (MBP*) to lysosomal fractions purified from BLC and primary CD22+ peripheral human B cells, respectively. The emerging proteolytic fragments were identified by HPLC and mass spectrome-

68 · 33rd Annual Meeting of the German Society of Immunology try. Although MBP* was largely resistant to purified AEP, it was processed into fragments similar to those obtained with the wild type protein by BLC, albeit with slower kinetics. Processing by BLC was stopped by total inhibition of cysteine proteases. Lysosomes from primary B cells, in contrast, produced an entirely different fragmentation pattern from both MBP and MBP*, where a serine protease activity was functionally dominant. We identified this activity as CatG, a serine protease that is expressed by myeloid cells, but not by primary B cells on the RNA level. Exogenous CatG is bound to the surface of peripheral B cells and colocalizes with LAMP-1 in late endocytic compartments. Not unlike AEP, it destroys one immunodominant epitope of MBP while directly generating the N-terminus of a second immunodominant region. We suggest that CatG represents a major proteolytic activity in the endocytic compartment of peripheral human B cells that might play an important role in antigen degradation.

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Department of Molecular Cell Biology, Institut für umweltmedizinische Forschung, Düsseldorf, 2Department of Molecular Biology, Institute of Molecular Biotechnology, Jena, and 3 Computing Center, Technical University, Aachen, Germany

E. 3 Subnuclear recruitment of fibrillarin to nucleoplasmic proteasomes: Implications for altered processing of a nucleolar autoantigen M. CHEN1, T.D. ROCKEL1, G. STEINWEGER1, P. HEMMERICH2, J. RISCH3, and A. VON MIKECZ1 Although it is generally accepted that proteasomes occur in both, the cytoplasm and the nucleus, research has been focussing on cytoplasmic protein breakdown and antigen processing, respectively. Thus, little is known on the functional organization of the proteasome in the nucleus. Here we report that within the nucleus 20S and 26S proteasomes are normally segregated from nucleolar proteins such as fibrillarin, B23 and C23, since proteasomes occur in the nucleoplasm of the cells where a substantial fraction shows colocalization with speckles containing splicing factors. The significance of nucleoplasmic proteasome localization was analysed using a recruitment system: Sub-toxic concentrations of mercuric chloride (HgCl2) induced subcellular redistribution of fibrillarin and partial colocalization with nucleoplasmic proteasomes in different cell lines, and in dendritic cells isolated from mice treated with mercury (Chen et al. Mol Biol Cell in press). Immunoprecipitation of fibrillarin with antibodies to ubiquitin, as well as accumulation of fibrillarin in cells treated with specific inhibitor of proteasomes suggests that proteasome-dependent processing of this autoantigen occurs upon mercury induction and thus constitute the cell biological basis of autoimmune responses against fibrillarin which occur in mouse models by mercury and scleroderma.

33rd Annual Meeting of the German Society of Immunology · 69 Department of Cellular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany

E. 4 Specificity differences of thymus-selected T cells and corresponding hybridomas: Evidences for differences in abT cell receptor complex formation S. HELLWIG, B. GERLICH, U. PFLUGFELDER, and H.U. WELTZIEN Involvement of one or multiple abT cell receptors (abTCR) in TCR/CD3 T cell antigen receptor complex formation is a long-standing controversy in immunology. We have characterized two CD8+ murine CTL clones, one expressing two productively rearranged TCR a-chains. Both clones were hapten-specific and H-2Kb restricted. They were induced with the Kb -binding octapeptides* M4L-TNP (clone E6) or M4L-DNP (clone D1), and crossreacted with O4-TNP or with O4-DNP and M4L-TNP, respectively, i.e. either with different carriers or with a different hapten on the same carrier. Hybridomas produced from both clones by fusion with CD8a expressing BW5147 cells lost reactivity to the inducing antigens, but each kept one of the crossreactive specificities (hybridoma Hy-E6 reacted to O4-TNP, but not to M4L-TNP, Hy-D1 to M4L-TNP, but not to M4L-DNP or O4-DNP). This was in contrast to other hybridomas produced from T cell clones which always reflected the fine specificity of the clones of origin. Antibody stainings revealed a marked reduction of surface expression of one of the two TCR achains in hybridoma Hy-E6, but comparable levels of mRNA for both. Experiments expressing the individual TCRs of clone E6 by gene transfection identified one of them as selectively responding to Kb/O4-TNP, but not to M4L-TNP. Surface expression of the second TCR failed. In contrast antibody staining showed intracellular expression of this particular TCR in surface negative transfectants and hybridomas. Preliminary experiments with blue native gel electrophoresis suggest a different TCR complex formation on the CTL clone and hybridoma. Current studies aim at identifying the role of multiple abTCR complexes in defining antigen specificity and differences in abTCR/CD3 complex formation in T cell clones versus hybridomas. * M4L=SMQKFGEL, O4=SIIKFEKL. Supported by the Deutsche Forschungsgemeinschaft, SFB 388

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Department of Internal Medicine III, Johannes Gutenberg-University, Mainz and 2Institut für Molekulare Virologie, GSF-National Research Center for Environment and Health, Neuherberg, Germany

E. 5 Functional mechanisms of TAP and tapasin downregulation by HER-2/neu overexpression F. HERRMANN1, U. WOLLSCHEID1, I. DREXLER2, C. HUBER1, and B. SELIGER1 High levels of MHC class I surface expression depends on the generation of antigenic peptides derived from intracellular synthesized proteins and their transport from the cytosol into the endoplasmic reticulum (ER) by the transporter associated of antigen processing (TAP). In the

70 · 33rd Annual Meeting of the German Society of Immunology ER, the synthesized MHC class I heterodimer is loaded with peptides which is facilitated by the ER-resident chaperone tapasin (tpn). Several studies demonstrated a deficient expression of the transporter subunits TAP1 and TAP2 and/or tpn in human and murine tumors of distinct origin resulting in an immune escape of these malignant cells. In most cases, the impaired TAP and tpn expression can be induced by IFN-g suggesting dysregulation rather than structural alterations of these components as the underlying molecular mechanism. This study focuses on the molecular mechanism of an immune escape phenotype induced by HER-2/neu overexpression in NIH3T3 cells. We found that HER-2/neu significantly reduces MHC class I surface expression which is attributable to downregulation of TAP1, TAP2 and tpn mRNA and protein levels. This is further associated with deficient peptide transport function and decreased sensitivity to CTL- mediated lysis. Since this immune escape phenotype could be reverted by IFN-g treatment, we analyzed the downregulation of TAP and tpn in the HER-2/neu transfectants on the transcriptional level. The 5’ regions of TAP1, TAP2 and tpn genes were cloned in front of a luciferase (luc) gene and the promoter activity was measured. In accordance to the RNA data, all three promoters showed less activity in HER-2/neu+ cells. A set of promoter deletion mutants demonstrate that one specific transcription factor binding site may not solely responsible for the impaired promoter activity. Thus, HER-2/neu overexpression results in transcriptional downregulation of various antigen processing components which appear to be due a complex altered binding of transcription factors and/or repressors.

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Department Biology II, Ludwig-Maximilians-University and 2Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany

E. 6 Expression and processing of the HLA-G isoforms V. HOFMEISTER1, S. MAIER1, J.W. ELLWART2, and E.H. WEISS1 HLA-G is a HLA class Ib molecule mainly expressed in extravillous trophoblasts. Alternative splicing results in seven different membrane bound and soluble HLA-G variants with still debated function. Transport of MHC class I heavy chains to the cell surface is dependent on correct folding and assembly with b2-microglobulin and a bound peptide-ligand in the ER. Association of MHC class I heavy chains with b2-microglobulin and TAP can hint to correct folding and loading with peptide. To study the association of different HLA-G heavy chains with b2-microglobulin and TAP, coprecipitates were analysed by Western blot and detection with the HLA-G-specific antibody 4H84. In the HLA class I negative cell line K-562 the full length isoform HLAG1m and the soluble HLA-G1s were coprecipitated with b2-microglobulin, whereas HLA-G2m and HLA-G4 lacking the a2 and a3 domain respectively could not be detected. HLA-G1m and HLA-G2m but not HLA-G4 or HLA-G1s were associated with TAP. That is in contrast to the cell line 721.221 where all membrane bound isoforms coprecipitated with TAP. Differences in association with TAP could be due to HLA-E and HLA-F expression in the cell line 721.221. In HLA-G-transfected 721.221 the HLA-G isoforms could be coprecipitated with HLA-E using the HLA class I-specific antibody A1.4 that does not recognize HLA-G. In the cell line K-562 HLA-G1m is the only isoform that associates with both b2-microglobulin and TAP. The short HLA-G isoforms fail to interact with the necessary components for assembly and peptide loading. They cannot be detected at the cell surface by FACS analyses with several HLA- and HLA-

33rd Annual Meeting of the German Society of Immunology · 71 G-specific antibodies. Functional analyses with NK cells and the cell line NKL did not reveal cell surface expression of the truncated HLA-G isoforms. This work was supported by the DFG (SFB217). We thank M. McMaster and R. Tampé for kindly providing antibodies.

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Institute of Virology and Immunobiology, University of Würzburg, Würzburg, 2RWTHAachen, University Hospital, Aachen, Germany, 3Department of Microbiology and Immunology, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan, and 4 Institute of Infectious Diseases and Immunology, University of Utrecht, Utrecht, The Netherlands

E. 7 Changes in the CDR2 and the hypervariable region 4 (HV4/CDR4) of b 8.2) TCR-b b chain differentially affect recognition of MBP BV8S2 (Vb peptide 68-88 and the bacterial superantigens SEC1, MAS and YPM M. KREISS1, A. ASMUSS1, K. KREJCI1, L. LINK2, T. MYOSHI-AKIYAMA3, T. UCHIYAMA3, C.P.M. BROEREN4, D. LINDEMANN1, and T. HERRMANN1 BV8S2 (Vb8.2), which is preferentially used in the EAE inducing RT1Bl restricted gpMBP (68–88) response of the LEW rat, was compared with the highly homologous BV8S4 with respect to recognition of antigens and superantigens. To this end TCR-genes (AV23/J47, BV8S2/J1S3) of the encephalitogenic CD4 T cell line MBP13 were cloned, TCRb chains were mutagenized (swap mutants of CDR2 and/or HV4: BV8S2 vs BV8S4) and TCR were expressed in rat CD28 transduced BW58 TCR–/– mouse cells. The transduced cells were tested for reactivity to gpMBP (guinea pig myelin basic protein) and SEC1 (Staphylococcus enterotoxin C1) but also with two in terms of T cell recognition relatively poorly understood superantigens, MAS (Mycoplasma arthritidis superantigen) and YPM (Yersinia pseudotuberculosis mitogen). All ligands were presented by rat thymocytes as well as by RT1Bl transduced RAJI cells. BV8S2 wt TCR and the BV8S4 like double mutant reacted like their natural counterparts. BV8S2wt responded to MBP, SEC1, MAS and YPM, the double mutant responded only to YPM. The HV4 mutant responded to all ligands except SEC1, the CDR2 mutant to MAS and YPM but not MBP and SEC1. These results are consistent with current models of MHC class II restricted antigen recognition and SEC/TCR interaction but suggest also major differences in the mode of recognition of SEC1, MAS and YPM.

72 · 33rd Annual Meeting of the German Society of Immunology Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

E. 8 Small molecular compounds mediate ligand exchange reactions onto HLA-DR molecules by induction of a peptide receptive state V. MARIN ESTEBAN, K. FALK, and O. RÖTZSCHKE Small molecular hydrogen-bond (H-bond) donor compounds, with the capacity to disrupt hydrogen bonds, are able to accelerate ligand exchange reactions of HLA-DR molecules. Here we show that the catalytic effect of these H-bond donor compounds is not restrained to ligand exchange reactions but can be extended to the loading of empty HLA-DR molecules. Parachlorophenol (pCP) is one of the most effective H-bond donor compounds we have so far identified. Surface plasmon resonance experiments indicated that pCP accelerates the dissociation rate of peptide ligands, which can explain the effect of H-bond donor compounds on the peptide exchange reactions on HLA-DR molecules. However, the release of a previously bound peptide does not seem to be required to reach the rapid binding of a further peptide. Experiments with empty HLA-DR molecules indicated that small molecular H-bond donors rescue the peptide-receptive state of empty HLA-DR molecules thus allowing the rapid binding of a peptide ligand. In the absence of the small molecular compounds, the peptide-receptive state of empty HLA-DR molecules is rapidly lost but can be rescue at any time when the small molecular compound is added on. Thus, H-bond donor compounds accelerate both the dissociation rate of class II MHC–peptide complexes as well as the direct loading of peptides onto empty class II MHC molecules, displaying a catalytic effect analogous to HLA-DM, albeit at a physiological pH. These effects of small molecular H-bond compound have biological implications. MBP is an encephalitogenic and abundant protein in the myelin of the central nervous system. On the surface of antigen presenting cells, pCP catalyses with great efficiency exchange reactions of short MBP peptides but also of the full-length MBP protein either purified or present on spinal cord homogenates. This increase in antigen loading translates directly into an improved T cell specific response.

1 Section of Immunobiology, Institute of Zoophysiology and 3Institute of Medical Microbiology, University of Bonn, Bonn, and 2Institute of Virology, Charité, HumboldtUniversity, Berlin, Germany

E. 9 HSV envelope protein controls MHC class II assembly, transport and presentation J. NEUMANN1, E. SIEVERS1, M.J. RAFTERY2, G. SCHÖNRICH2, A.M. EIS-HÜBINGER3, and N. KOCH1 Many viruses evolved strategies to evade an immune response. During the long coevolution with their host herpesvirus acquired an astonishing variety of genes from the host genome. These viral homologues of cellular genes mimic or counteract key molecules of the host immune system. MHC molecules that present viral peptides to the T cell receptor are a main target of viral stra-

33rd Annual Meeting of the German Society of Immunology · 73 tegies to manipulate immune recognition. Little is known about viral strategies that interfere with the MHC class II antigen presentation pathway. We found a MHC class II binding site in the sequence of an HSV glycoprotein that was originally identified in human Ii. In addition, this viral sequence is adjacent to a highly conserved HLA-DR1 binding motif. Both viral sequences together resemble the class II binding site of human Ii, consisting of the MHC class II groove binding segment and a promiscuous binding site. We demonstrate binding and transport of the virus envelope protein with DR molecules in HSV infected B-lymphoma cells. With chimeric Ii/HSV fusion proteins we identified the viral sequences that mediates promiscuous or allotypespecific binding to the HLA-DR peptide-binding domain. The result indicates promiscuous binding of the virus sequence to HLA-DR molecules and suggests a potential of HSV to manipulate antigen processing and presentation.

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Department of Internal Medicine III, Johannes Gutenberg-University, Mainz, Germany and Department of Immunology, Roswell Park Cancer Institute, Buffalo NY, USA

E. 10 Regulation of the transporter associated with antigen-processing (TAP) subunits U. RITZ1, M. BOCK1, C. HUBER1, S. FERRONE2, and B. SELIGER1 The transporter associated with antigen processing (TAP) is composed of the TAP1 and TAP2 subunits. These two subunits demonstrate distinct biochemical and functional properties and play a key role in proper processing and presentation of MHC class I restricted antigens. Impaired TAP subunit expression has been frequently found in tumors of distinct origin and is accompanied by abnormalities of MHC class I surface expression and reduced anti-tumor response. However, the underlying molecular mechanisms of TAP subunit downregulation have only been investigated to a limited extent. A base pair deletion near the ATP-binding domain of TAP1 in a human melanoma cell line abrogated not only TAP1, but also TAP2 protein expression and function. TAP1 gene transfer into these TAP1-negative melanoma cells restored the TAPdeficient phenotype and caused an induction of TAP2 protein. The coordinated downregulation of TAP2 protein in the absence of TAP1 was also observed in B16 melanoma cells and TAP1–/– cells isolated from a patient with the HLA-deficiency syndrome, suggesting that TAP1 protein stabilizes TAP2. Treatment of TAP1 deficient cells with the proteasome inhibitor lactacystin, but not with other protease inhibitors could induce TAP2 protein expression suggesting that the TAP2 protein is degraded by the proteasome. However, this mechanism appears not to be general since a discordant TAP subunit expression has also been detected in some tumor cell lines. Thus, TAP1 and TAP2 protein expression is controlled at different levels.

74 · 33rd Annual Meeting of the German Society of Immunology Department of Molecular Cell Biology, Institute of Environmental Health Research, Düsseldorf, Germany

E. 11 Proteasome-dependent processing within the nucleus? T.D. ROCKEL and A. VON MIKECZ Cytoplasmic proteasomes have been shown to be the main proteolytic machinery for endogenous proteins in the cell. Proteasomal degradation plays an important role for regulation of proteins, recycling of amino acids and production of immunocompetent peptides for MHC-presentation. Although it is known, that proteasomes can also be found inside the nucleus, little is known about the function and activity of nucleoplasmic proteasomes. We monitored nuclear proteins and their relationship with the nucleoplasmic fraction of the proteasomes by confocal laser scanning microscopy in situ. Specific inhibition of proteasomal activity revealed, that proteins, colocalizing with proteasomes, also accumulate in the specific cell subcompartments. This suggests a physiological connection: Splicing factor SC35, splicing components U1–70k and Sm B/B’, histones or PML seem to be degraded by the proteasome under normal conditions, whereas centromeric and nucleolar proteins such as fibrillarin and UBF or topoisomerase I were not processed. These data suggest that nucleoplasmic proteasomes might contributed to protein degradation, as their cytoplasmic counterparts, and thus play a role in maintaining structure and function of the nucleus.

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Department of Immunology, Max-Planck-Institute of Infection Biology and 2Institute of Biochemistry, Charité, Humboldt-University, Berlin, Germany

E. 12 Organ-specific antigen processing by 20S proteasomes: Impact on autoimmunity U. STEINHOFF1, S.H.E. KAUFMANN1, P.M. KLOETZEL2, B. STREHL2, and U. KUCKELKORN2 Most antigenic peptides presented on MHC class I molecules are derived from degradation by proteasomes. Proteasomes are a complex of 700 kDA consisting of 28 different subunits which can be assigned to a or b subunits. While the a subunits form the external part of the barrel shaped proteasome mediating the contact to regulatory complexes the b subunits harbour the proteolytic active sites. We have recently shown that 20S proteasomes do not represent one entity but differ between various organs with respect to their structure and their function, including organ-specific processing of CD8 T cell epitopes. Further, the relevance of organ-specific antigen processing was demonstrated in a model of autoimmune pathology mediated by hsp60 specific CD8 T cells. Although the colon expressed much higher levels of hsp60, T cell mediated inflammation was restricted to the small intestine due to more efficient proteasomal processing in the latter organ. Further, bacterial infections have the potential to modulate the proteasomal activity with the result of enhanced processing of MHC class I restricted T cell epitopes of microbialand self origin. We therefore conclude that the activity of proteasomes controls organ-specifically protective and pathological CD8 T cell responses.

33rd Annual Meeting of the German Society of Immunology · 75 Molecular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany

E. 13 Loading of N-terminally extended class I-binding peptides requires the TAP-associated complex N. TIWARI, G.J. HÄMMERLING, and F. MOMBURG MHC class I-binding peptides are often transported into the endoplasmic reticulum by TAP as extended precursors that require trimming to their final size of 8–10 amino acids. It is controversial whether N-terminal (Nt) peptide trimming by ER-resident aminopeptidases acts on free TAP-transported peptides, or whether it is guided by peptide precursor binding to class I molecules inside or outside to the peptide loading complex (PLC) consisting of TAP1/2, tapasin, class I/b2-microglobulin, calreticulin, and the thioreductase ERp57. As a model system we used Kb molecules loaded with ovalbumin peptide 257–264 (OVAp) that can be recognized specifically by the monoclonal antibody 25.D1–16 and the T cell hybridoma B3Z. OVAp and various Nt extended versions were introduced into the ER in free form, or covalently linked to the Nterminus of Kbor b2m which can be more easily quantified. To study the role of the PLC for loading of extended peptides and Nt trimming, constructs were transfected into TAP/class I-deficient Ec7.1 cells, tapasin-deficient .220 cells, b2m-deficient EL4-S3 cells, as well as into P815 and RMA cells expressing the complete loading complex. In tapasin/TAP-positive P815, RMA, and EL4-S3 transfectants, efficient formation of the 25.D1–16 epitope from extended OVAp constructs was observed. By contrast, in Ec7.1 cells the 25.D1–16 epitope was produced from none of the constructs, and in .220 cells only from finally processed OVAp conjugates. These findings indicate that loading/trimming of extended class I ligands is dependent on the presence of tapasin and TAP and integration of class I into the PLC. The analysis by isoelectric focussing of Kband b2m conjugates with charge-tagged OVAp extensions will allow to quantify Nt trimming in the different transfectants.