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The relationship between peptide selectivity of HLA class I molecules and TAP transporters
240
MHC molecules: synthesis, assembly and peptide binding
P 1.05.19
The relationship between peptide aelectlvlty of HLA claaa I molecules and TAP transporters
S. Daniel ‘, V. Brusic2, S. Caillat-Zucman ‘, N. Petrovsky *, L. Harrlson *, D. Riganelli 3, F. Sinigaglia4, F. Gallaxzi 4, J. Hammer4, P.M. van Endert ‘. ’ lNSERh4 U25, 761 rue de SBvres, 75743 Paris Cedex 15, France. *The Walter and Eka Ha// Institute, The Royal Melbourne Hospital, Victoria 3650, Australia, 3lstituto d/ Medicina Interna, Universita di Perugia, 66166 Perugia, ItaM 4Roche Milan0 R/cerche, 56 Via Olgettina. 20132 M//an, /ta/y Intracellular antigen processing selects peptides for presentation by human leukocyte antigen (HLA) class I molecules according to unknown rules. To understand the role of the transporters associated with antigen processing (TAP) in peptide selection for presentation by HLA class I molecules, we undertook a comprehensive analysis of the selectiiity of human transporter in TAP/peptida binding assays. We found a pronounced and dominant effect of the 3 N-terminal and the C-terminal residues on binding of 8 to 1Cmer peptides. Binding data of 394 peptides were used to train artificial neural networks (ANNs) that successfully predicted TAP binding affinltles of 100 random sequence peptides. In ANN-simulated TAP binding experiments on almost 2000 ligands for HtA class I molecules, individual HLA class I alleles varied dramatically wlth respect to TAP affinities of their ligands. Some HLA class I alleles, especially HlA-827, should therefore be particularly efficient in presentation of antigens with low cytosolic abundance.
P1.05.20
Sllenclng of the HLA-B27 tranagane In spontaneous transgenlc mouse tumors
A. Koroknay, G. Brem ‘, G. Riethmiiller*, E.H. Weiss. institute ofAnthmpc/ogy and Human Genetics, University Munich, Gemrany. 2/n.st/tuteof Immunology, University Munich, Germany, ’ Institute of Molecular Animal Breeding, UniversiPyVienna, Germany
Introduction: Selective loss of locus-specific or allele-specific WA class I products is frequently observed in several metastatlc tumors. The mechanisms underlying these phenomena are difficult to investigate in autologous systems. CpG methylatlon in the HLA class I CpG islands is discussed to be involved in the transcriptional regulation or silencing, but experimental data are scarce. Here, we took advantage of a physiologically regulated HLA-B27 transgene to investigate site specific methylation in expressing and non expressing transgenie mouse tissues and tumor cell lines. Materials and Msthods: The establishment of cell lines from solid tumors was performed at normal cell culture conditions without additional growth factors. Cell surface expression of transgenic HlA-B27 and human 5&l as well as mouse MHC class I and 5sM gene products was investigated in freshly isolated tumor cells and early passages of the cell lines. Transcriptional silencing was confirmed by Norlhem blot analysis. The DNA-methylation pattern was examined bv classical Southern blot analysis, a new high resolution HTF mapping method and b/&f/t genom/c sequencing. Reeuk We could establish two cell lines, which are selectively deficient in HLA-B27 transgene expression, while endogenous MHC class I as well as human and murfne /JsM products can still be observed. In one cell line HLA-B27 is not induceable by IFNy and the transgene is heavily methyiated at CpGs in a heterogenous pattern. The transgene of the second cell line is intermediately methylated, and HLA-B27 can be induced by 5-azacytidine or IFNy. Interestingly, both cell lines are methylated to a high degree at the site (I located in the enhancer B, which is known to bind important tissue speclflc transcription factors in v/w. No CpG methylatlon of transgenic HLA-B27 promoter and 5’gene region was detected in brain, test& liver, kidney, lung or spleen of the mice. Conclusion: CpG-methylation of the HlA-B27 transgene is not involved in tissue specific transcriptional regulation. The heterogenous CpG methylation pattern focussed at cis-acting elements of the HLA-B27 transgene observed in our tumor cell lines points to an important role of this mechanism in transcriptional silencing of MHC class I genes in tumors. The methods established for the transgenic mouse tumor cell lines should be useful to investigate this phenomenon in human tumors and cell lines.
1P.1.05.21 ] Isolation of gluten-apaclflc, HLA-DO-reatrlcted T cells from the small Inteatlnal mucoaa of a coellac disease patient Y. van de Wal r, Y. Kooy I, M.L. Mearln *, A.S. Peha 3, F. Koning ‘. ‘Dept.of lmmunohaematobgy and Blcodbank. University Hcsp/ki/ Leiden, The Netherlands, *Dept. of Paediatrics, Univers/ty Hospita/ Leiden, The Netherlands, 3Dept. of Gastmentervlogy Free University Hospital Amsterdam, The Netherlands
Introduction: Coeliac Disease (CD) is a permanent intestinal intolerance to the dietary wheat gluten and related proteins. In genetically susceptible individuals ingestion of these proteins will result in intestinal villous atrophy, leading to
24 June 1997 - Poster presentations
malabsorption of dietary fats, proteins, carbohydrates etc.. The presence of activated, cytokine producing T cells in the active coeliac lesion and the strong HLA assoclatlon suggest that the cellular immune system plays a key role in the pathogenesis of CD. Currently available data indicate that CD is primarily associated with HLA-DQ2 (wCr1*0601, DQ51’02) and to a lesser extent with HlA-DQ6 (DQul’O301, DQ/Jl’O302). Materials andMethods:To Identify the T cell epitopes within gluten that may play a role in CD, we have established gluten-specific T cell clones (TCC) from small intestinal biopsy specimens of a DQ20Q9 hetemzygous CD patient. Primary stimulation was performed using autologous dendritlc cells (IL4/GMCSF method; Sailusto et al. J.Exp.Med. 179: 1109) that were loaded with a pep tic/tryptlc digest of gluten. Resuftm Outgrowing T cells were found to be gluten-speclfc. A large number of TCCs (all CD4+) were established by limiting dilution analysis; most of these TCCs are almost completely inhibited in their gluten-specific responses by anti-DQ, but not by anti-DR mAbs. In fact, no DR-restricted responses were found, which is in agreement with published data (Lundin et al. JExpMed. 179: 167). Conclusion and Future Strategy: Taken together, our results demonstrate that gluten-specific CD4+ T cells from the intestinal mucosa of a CD patient recognize glutenderived peptides predominantly when presented by HLA-DQ. Using different types of chromatography, we are currently purlfylng the gluten sample. Biologically active fractions are currently analyzed for sequence information by tandem mass spectrometry or edman degradation. Characterization of the gluten-specific TCCs and the identification of CD-specific T cell epitopes may lead to a better understanding of the pathogenesis of the disease.
P.1.05.22
Blochemlcal characterlzatlon of human Class I hlatocompatlblllty antigens
J.M. Escolano, P. Gonzalez-Poque, E. Rolddn, A. Moreno, J.C. Alvarez-Cermeho, A. Bootello, L.M. Vlllar. Serv/c/o de /nmunobg/a, Hospital Ram&y Cajal, Madrid, Spain
Introduction:sHLA is the major form of soluble Class I antigens present in serum and in human spleen membranes. It is secreted by T and B lymphocytes and the secretion increases upon activation. It can be released from spleen membranes by incubation at 37% in presence of protease inhibitors. MaterIsIssnd Methods:We have purlfled sHtA and classical HLA A. 8, C antigens from spleen membranes and from supematants and pellets of PBL cultured for three days in presence of PHA. Class I antigens have been purified by immunopreclpltatlon with W6/32 and protein G-sepharose or by affinity chromatography on a sepharose 4B-W6/32 column. After purification, soluble and classical Class I antigens were desyalized with neuraminidase type VIII, subjected to isoelectmfocusing on polyacrylamide gels in presence of 9 M urea and 2% NP40 and transferred to a PVDF membrane. lmmunodetection was carded out wlth HCI 0, a monoclonal antibody that recognizes a common determinant present in denatured heavy class I chains. Fteau~ Our results show differences between the pl of classical class I antigens and sHLA from the same individual. We have not detected variations in the pl of sHlA from different individuals. We have analyzed sHLA with three different manoclonal antibodies that recognize HLA G, one for the soluble form of the molecule and two for the complete membrane form. They did not recognize sHLA. Aminoterminal sequence analysis of sHlA from two different individuals did not show any difference between them. Our data show differences with HLA E and HiA F in this part of the molecule. Conoluslons: The data obtained from the aminoterminal sequence, the serological analysis and the IEF behaviour seems to indicate that sHLA are not HlA A, B, C, E, F or G molecules.
P.1.05.23
The MHC class lb molecule HLA-E binds signal sequence-derived peptldea wlth primary anchor realdues at poaltlona 2 and 9
V. Braud ‘, E.Y. Jones *, A. McMichael ’ . 1/n&//u/e of Mo/ecu/ar Medicine, John Radc//ge Hospital, Oxbrd, UK, *Laboratory of Molecular Med/c/ne, The Rex Richards BuiMing, Oxfcrd, UK Introduction: The human non classical HLA-E molecule is transcribed in most tissues and exhibits a limited polymorphism. Although no specific antlbodies are available, studies of HLA-A. -B, -C, -G null mutant cell line LCL721.221 and a murlne cell line X63 transfected with HLA-E and human 52 microglcbulin showed that HLA-E displays a very low cell surface expressions. HLA-E shares some structural similarities in the peptlde binding groove with the murine MHC class lb molecule Qa-l. Qa-1 has been shown to bind a peptlde from the leader sequence of H-2 D and H-2 L molecules. We were thus interested to test whether HLA-E was also able to bind signal sequence-derived peptides. Materlalsand Methods: We developed a peptlde binding assay in which the ability of peptides to stabilise HLA-E molecules in cell lysate was tested.
MHC molecules: synthesis, assembly and peptide binding
P 1.05.19
The relationship between peptide aelectlvlty of HLA claaa I molecules and TAP transporters
S. Daniel ‘, V. Brusic2, S. Caillat-Zucman ‘, N. Petrovsky *, L. Harrlson *, D. Riganelli 3, F. Sinigaglia4, F. Gallaxzi 4, J. Hammer4, P.M. van Endert ‘. ’ lNSERh4 U25, 761 rue de SBvres, 75743 Paris Cedex 15, France. *The Walter and Eka Ha// Institute, The Royal Melbourne Hospital, Victoria 3650, Australia, 3lstituto d/ Medicina Interna, Universita di Perugia, 66166 Perugia, ItaM 4Roche Milan0 R/cerche, 56 Via Olgettina. 20132 M//an, /ta/y Intracellular antigen processing selects peptides for presentation by human leukocyte antigen (HLA) class I molecules according to unknown rules. To understand the role of the transporters associated with antigen processing (TAP) in peptide selection for presentation by HLA class I molecules, we undertook a comprehensive analysis of the selectiiity of human transporter in TAP/peptida binding assays. We found a pronounced and dominant effect of the 3 N-terminal and the C-terminal residues on binding of 8 to 1Cmer peptides. Binding data of 394 peptides were used to train artificial neural networks (ANNs) that successfully predicted TAP binding affinltles of 100 random sequence peptides. In ANN-simulated TAP binding experiments on almost 2000 ligands for HtA class I molecules, individual HLA class I alleles varied dramatically wlth respect to TAP affinities of their ligands. Some HLA class I alleles, especially HlA-827, should therefore be particularly efficient in presentation of antigens with low cytosolic abundance.
P1.05.20
Sllenclng of the HLA-B27 tranagane In spontaneous transgenlc mouse tumors
A. Koroknay, G. Brem ‘, G. Riethmiiller*, E.H. Weiss. institute ofAnthmpc/ogy and Human Genetics, University Munich, Gemrany. 2/n.st/tuteof Immunology, University Munich, Germany, ’ Institute of Molecular Animal Breeding, UniversiPyVienna, Germany
Introduction: Selective loss of locus-specific or allele-specific WA class I products is frequently observed in several metastatlc tumors. The mechanisms underlying these phenomena are difficult to investigate in autologous systems. CpG methylatlon in the HLA class I CpG islands is discussed to be involved in the transcriptional regulation or silencing, but experimental data are scarce. Here, we took advantage of a physiologically regulated HLA-B27 transgene to investigate site specific methylation in expressing and non expressing transgenie mouse tissues and tumor cell lines. Materials and Msthods: The establishment of cell lines from solid tumors was performed at normal cell culture conditions without additional growth factors. Cell surface expression of transgenic HlA-B27 and human 5&l as well as mouse MHC class I and 5sM gene products was investigated in freshly isolated tumor cells and early passages of the cell lines. Transcriptional silencing was confirmed by Norlhem blot analysis. The DNA-methylation pattern was examined bv classical Southern blot analysis, a new high resolution HTF mapping method and b/&f/t genom/c sequencing. Reeuk We could establish two cell lines, which are selectively deficient in HLA-B27 transgene expression, while endogenous MHC class I as well as human and murfne /JsM products can still be observed. In one cell line HLA-B27 is not induceable by IFNy and the transgene is heavily methyiated at CpGs in a heterogenous pattern. The transgene of the second cell line is intermediately methylated, and HLA-B27 can be induced by 5-azacytidine or IFNy. Interestingly, both cell lines are methylated to a high degree at the site (I located in the enhancer B, which is known to bind important tissue speclflc transcription factors in v/w. No CpG methylatlon of transgenic HLA-B27 promoter and 5’gene region was detected in brain, test& liver, kidney, lung or spleen of the mice. Conclusion: CpG-methylation of the HlA-B27 transgene is not involved in tissue specific transcriptional regulation. The heterogenous CpG methylation pattern focussed at cis-acting elements of the HLA-B27 transgene observed in our tumor cell lines points to an important role of this mechanism in transcriptional silencing of MHC class I genes in tumors. The methods established for the transgenic mouse tumor cell lines should be useful to investigate this phenomenon in human tumors and cell lines.
1P.1.05.21 ] Isolation of gluten-apaclflc, HLA-DO-reatrlcted T cells from the small Inteatlnal mucoaa of a coellac disease patient Y. van de Wal r, Y. Kooy I, M.L. Mearln *, A.S. Peha 3, F. Koning ‘. ‘Dept.of lmmunohaematobgy and Blcodbank. University Hcsp/ki/ Leiden, The Netherlands, *Dept. of Paediatrics, Univers/ty Hospita/ Leiden, The Netherlands, 3Dept. of Gastmentervlogy Free University Hospital Amsterdam, The Netherlands
Introduction: Coeliac Disease (CD) is a permanent intestinal intolerance to the dietary wheat gluten and related proteins. In genetically susceptible individuals ingestion of these proteins will result in intestinal villous atrophy, leading to
24 June 1997 - Poster presentations
malabsorption of dietary fats, proteins, carbohydrates etc.. The presence of activated, cytokine producing T cells in the active coeliac lesion and the strong HLA assoclatlon suggest that the cellular immune system plays a key role in the pathogenesis of CD. Currently available data indicate that CD is primarily associated with HLA-DQ2 (wCr1*0601, DQ51’02) and to a lesser extent with HlA-DQ6 (DQul’O301, DQ/Jl’O302). Materials andMethods:To Identify the T cell epitopes within gluten that may play a role in CD, we have established gluten-specific T cell clones (TCC) from small intestinal biopsy specimens of a DQ20Q9 hetemzygous CD patient. Primary stimulation was performed using autologous dendritlc cells (IL4/GMCSF method; Sailusto et al. J.Exp.Med. 179: 1109) that were loaded with a pep tic/tryptlc digest of gluten. Resuftm Outgrowing T cells were found to be gluten-speclfc. A large number of TCCs (all CD4+) were established by limiting dilution analysis; most of these TCCs are almost completely inhibited in their gluten-specific responses by anti-DQ, but not by anti-DR mAbs. In fact, no DR-restricted responses were found, which is in agreement with published data (Lundin et al. JExpMed. 179: 167). Conclusion and Future Strategy: Taken together, our results demonstrate that gluten-specific CD4+ T cells from the intestinal mucosa of a CD patient recognize glutenderived peptides predominantly when presented by HLA-DQ. Using different types of chromatography, we are currently purlfylng the gluten sample. Biologically active fractions are currently analyzed for sequence information by tandem mass spectrometry or edman degradation. Characterization of the gluten-specific TCCs and the identification of CD-specific T cell epitopes may lead to a better understanding of the pathogenesis of the disease.
P.1.05.22
Blochemlcal characterlzatlon of human Class I hlatocompatlblllty antigens
J.M. Escolano, P. Gonzalez-Poque, E. Rolddn, A. Moreno, J.C. Alvarez-Cermeho, A. Bootello, L.M. Vlllar. Serv/c/o de /nmunobg/a, Hospital Ram&y Cajal, Madrid, Spain
Introduction:sHLA is the major form of soluble Class I antigens present in serum and in human spleen membranes. It is secreted by T and B lymphocytes and the secretion increases upon activation. It can be released from spleen membranes by incubation at 37% in presence of protease inhibitors. MaterIsIssnd Methods:We have purlfled sHtA and classical HLA A. 8, C antigens from spleen membranes and from supematants and pellets of PBL cultured for three days in presence of PHA. Class I antigens have been purified by immunopreclpltatlon with W6/32 and protein G-sepharose or by affinity chromatography on a sepharose 4B-W6/32 column. After purification, soluble and classical Class I antigens were desyalized with neuraminidase type VIII, subjected to isoelectmfocusing on polyacrylamide gels in presence of 9 M urea and 2% NP40 and transferred to a PVDF membrane. lmmunodetection was carded out wlth HCI 0, a monoclonal antibody that recognizes a common determinant present in denatured heavy class I chains. Fteau~ Our results show differences between the pl of classical class I antigens and sHLA from the same individual. We have not detected variations in the pl of sHlA from different individuals. We have analyzed sHLA with three different manoclonal antibodies that recognize HLA G, one for the soluble form of the molecule and two for the complete membrane form. They did not recognize sHLA. Aminoterminal sequence analysis of sHlA from two different individuals did not show any difference between them. Our data show differences with HLA E and HiA F in this part of the molecule. Conoluslons: The data obtained from the aminoterminal sequence, the serological analysis and the IEF behaviour seems to indicate that sHLA are not HlA A, B, C, E, F or G molecules.
P.1.05.23
The MHC class lb molecule HLA-E binds signal sequence-derived peptldea wlth primary anchor realdues at poaltlona 2 and 9
V. Braud ‘, E.Y. Jones *, A. McMichael ’ . 1/n&//u/e of Mo/ecu/ar Medicine, John Radc//ge Hospital, Oxbrd, UK, *Laboratory of Molecular Med/c/ne, The Rex Richards BuiMing, Oxfcrd, UK Introduction: The human non classical HLA-E molecule is transcribed in most tissues and exhibits a limited polymorphism. Although no specific antlbodies are available, studies of HLA-A. -B, -C, -G null mutant cell line LCL721.221 and a murlne cell line X63 transfected with HLA-E and human 52 microglcbulin showed that HLA-E displays a very low cell surface expressions. HLA-E shares some structural similarities in the peptlde binding groove with the murine MHC class lb molecule Qa-l. Qa-1 has been shown to bind a peptlde from the leader sequence of H-2 D and H-2 L molecules. We were thus interested to test whether HLA-E was also able to bind signal sequence-derived peptides. Materlalsand Methods: We developed a peptlde binding assay in which the ability of peptides to stabilise HLA-E molecules in cell lysate was tested.