242 MHC molecules: synthesis, assembly andpeptide binding 24 June 1997 - Poster presentations

P.1.05.28 Viral peptide specific inductlon of class II expression by murlne T cells

C.A. Smith, CM. Graham, D.B. Thomas. National institute for Medical Research. Division of Vimlogy, London, UK

Introduction: We have observed that CD4+ T cell clones, elicited after infection of C57BlllO mice with influenza virus, which are specific for the haemagglutinin pepttde HA1 186-205 are able to respond to peptide in the absence of APC as measured by IL-3 release, but are unable to proliferate. The purpose of this study was to determine whether or not MHC class II plays a role in this response.

Materials and Methods: Flow cytometry and cell sorting were used to study the induction and expression of surface MHC class II on T cell clones. R.T./PCR techniques were employed to study the expression of class II mRNA.

Reauftaz We have shown that murtne CD4+ T cell clones can express ctass II molecules on their surface. We have shown that viral peptide, in the absence of APC induces de novo synthesis and expression of class II IAb. This induction is peptidespectftcand can be inhibited by TCR-specific monoclonal antibodies. FAGS-sorted CD4+ class II- were shown to express dass II mRNA by R.T./PCR, and these cells became class II+ when cultured with peptkte.

Conduslon: We speculate that peptide specific induction of MHC class II expression may be a feature of Thl-type clones elicited by natural infection with virus.

1 P.l.05.30 1 A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in a lack of antigen presentation

F. Ossendorp ‘, M. Eggers 2, A. Neisig 3, T. Ruppert *, M. Groettrup 4, A. Sijts ’ , E. Mengede ‘, P.-M. Kloetzel 4, J. Neefies 3, U. Koszinowski *, C. Melief ‘. ’ Dept if Immunohematology; Unive&y Hospital Leiden, The Netherlands, *Netherlands Cancer Institute, Amsterdam, The Netherlands, 3 University of Heidelberg, Germany, 4 tiumboldi University Berlin, Germany

Introduction: The CTL epttope KSPWFtTL encoded by the AKWMCF type of Murine Leukemia Virus differs from the FMR (Frtend/Moloney/Rauscher) type in one residue, an arginine (R) instead of a lysine (K). Both peptides bind equally to the l@ MHC class I molecule and CTL can recognize RSPWFlTL synthetic pepttde but do not lyse tumor cells expressing FMR MuLV, suggesting a difference in peptide processing.

Material and Methods: Purified 20s proteasomes were used for digestion analysis of 26 mer synthetic pepttdes containing the epitope and natural flanking regions.

Results: Target cells loaded with digests of AKV-type peptides were recog- nized by CTL in contrast to digests of FMR-type peptides. Biochemical analysis of the digestion products revealed an important proteolytic cleavage site next to the R, leading to epitope destruction in the FMR sequence.

Analysis of the digested AVK-type peptides revealed IO- and 11 -mer frag- ments, whereas the naturally presented 8-mer peptide was not detectable. The IO- and II-mers peptides were efficiently translocated by TAP, in contrast to the 8-mer.

Conclusion: A single residue difference explains the lack of peptide pro- cessing in FMR MuLV by proteasomemediated epttope destruction, indicating a potential immune escape mechanism. The study also shows that longer pre- cursor peptides are trtmmed in the ER.

1 P.1.05.31 1 Flow cytometric detection of Interleukln-2 receptor and class II HLA-DR antigen on peritoneal fluid lymphocytes in women In endometriosis

M. Gooacz ‘, K. Kaminski *. J. Rolinski 3, J. Tomaszewskt ‘. J. Kotarski ‘. J.A. Jakowicki ’ ’ Dept. Gy&. Surg. Sch&/ of Medicine, L&in, Poland, iDepf. Perinatol. School of Medicine, L&/in, Poland. 3DeDt. C/in. Immunol. School of Medicine, Lublin, Poland

Introduction: Endometrtosis affects lO-15% women in reproductive age. In spite of the years of study, the etiology of the disease is still remains mys- tery. Many data confirm that endometrtosis can develop and progress only in woman with genetically or/and environmentally altered cell-mediated immune response. Dysfunction of cellular branch of immune system in peritoneal fluid was postulated to play an important role in the pathogenesis of the disease. The aim of this study was to evaluate the (I chain intedeukin-2 receptor (CD25) and class II HLA-DR antigen expression in peritoneal fluid (PF) thymus dependent lymphocytes (CD3+) in women with pelvic endometrtosis.

Matertals and Methods: Peritoneal fluid was obtained from 12 women with II and Ill AFS stage of pelvic endometrtoeis and from 7 women with cystade- noma of ovary (reference group of oatients). Interleukin-2 receotor and HLA-DR antigen were-determined on fresh cells at the time of sample submission using CD25 and HLA-DR monoclonal antibodies. Cell subpopulations were assessed by dual color flow cytometric method using combinations of phycoertthrin (PE) and fluorescein isothyocyanate (FITC) conjugated monoclonal antibodies and

IgGl antibody as a negative control. The CD3/19 antibody was used for pheno- typing T lymphocytes. All samples were measured on a Cytoron flow cytometer (Ortho Diagnostic System) and 1 x iti cells were analysed per test.

Reaultr: The mean PF volume from endometriotic patients was 9.1 ml vs 6.8 ml in reference group and mean cell count was respectively 1.72 x 10s vs 0.86 x lo3 cells/ul. Mean percentage of PF CD3t cells was significantly (p < 0.002) higher in reference group (38.7%) than in endometrtotic women (17.5%). The expression of CD25 in PF thymus dependent lymphocytes from women with endometrtosts was significantly higher than in non endometrtotic patients (23.4% vs 10.4%). The HLA-DR expression was also significantly higher in endometriotic patients (19.8% vs 11 .l%).

Conclusion: Such expression of CD25+ and HLA-DR+ on PF lymphocytes from endometriotic patients indicates that increased T lymphocytes activation can be associated with the presence of endometriotic lesions. Further study need to be done to determine if this findings can be useful in explaining the pathogenesis of the disease.

P.1.05.32 Clustering of MHC class II molecules on antigen presenting cells following binding of copolymer 1 and myelin basic protein

D. Teitelbaum, M. Fridkis-Hareli, I. Pecht, R. Amon, M. Sela. Department of Immunolog) The Weizmann institute of Science, Rehovot, Israel

Introduction: Copolymer 1 (Cop1 , Copaxonee), a synthetic copolymer of amino acids effective in suppression of experimental allergic encephalomyelitts and multiple sclerosis, and myelin basic protein (MBP) were shown to bind exten- sively and promiscuously to Class II MHC molecules on antigen presenting cells (APC) without prior processing. In the case of human APC, binding was demon- strated to DR but not DO or class I molecules. In the present study, we examined whether binding of Cop1 and MBP affects the MHC class II expression on the cell membrane.

Materials and Mathods: Biotinylated derivatives of Cop1 and MBP were used to directly monitor their binding to MHC molecules by flow cytometry using phycoerythrtne streptavidin. The levels of MHC surface expression were monitored by FACS analysis and fluorescence microscopy using fluorescein isothiocyanate (FITC) conjugated anti class I and Class II antibodies. The requirements for denovo protein synthesis and active transport to the membrane were studied using cycloheximide and Brefeldin A (BFA) respectively.

Results: When Cop1 or MBP were incubated with APC, the intensity of cell staining with anti DR, but not with anti DQ or anti class I antibodies was sig nificantly increased, compared to control APC staining which were not reacted with these antigens. In contrast, staining intensity was unaffected when p&4-102 of MBP or ovalbumin, a protein which undergoes proteolytic degradation prtor to binding, were incubated with the APC. Cycloheximide had no effect on the intensity of staining with anti-DR antibody, of cells treated with either Cop1 or MBP, whereas it inhibited the enhanced staining of both DR and DQ molecules caused by the respective antibodies, in the absence of these antigens. BFA decreased the level of staining with anti-DR and anti-W antibodies in both cases, with and without antigen added to the APC. Fluorescence microscopic analysis revealed that cells incubated wkh Cop1 or MBP, but not with ~84-102 or OVA, show clusters of aggregated DR molecules on the cell surface.

Conclusions: These observations indicate that Cop1 and MBP. being of polyvalent character, lead to increased fluorescence intensity of their com- plexes with HlA-DR, possibly due to recruitment and clustering of previously synthesized DR molecules.

P.1.05.33 In wirtv and in viva processing studies of peptide antigens in context of MHC class II presentation

H. Kalbacher, H. Beck, C. SchrOter, M. Deeg, T. Halder. Me&a/ and Natural Sciences Research Center Univemity of Ttibingen, Germany

Introduction: Generation of peptide antigens for MHC class II presentation requires a concerted action of a number of IysosomaVendosomal proteases (e.g. Cathepsins B, D, S) in specialized cellular compartments. To examine the processing events within an antigen presenting cell (APC) we developed an in vitro model to study the generation of peptide antigens.

Materials and Methods: For this purpose we digested model peptides, derived from the myelin basic protein (MBP) with defined endo- and exopep- tidases and identified the products by high sensitive liquidchromatography- mass spectrometry (LC-MS). Further we performed digestions by using en- dosomaulysosomal cellular fractions isolated from APC. Intracellular fractions were obtained by a combination of fractionated centrifugation/percoll density gradient centrifugation and by an additional free flow electrophoresis (FFE). The subcellular compartments were characterized by their enzymatic activity (Cathepsins B, L, S, D) and the amount of HLA class I, class II, invariant chain and HLA-DM. On the other hand, we performed pulse chase experiments of fluorescentty labeled peptide antigens with lymphoblastoid cell lines (LCL) as APC. After pulsing, we isolated HLA class II-bound peptides from the intracel-