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Molecular and cellular diversity of the DR2 haplotypes
136
Annual AACHT Meeting, 1985 method using DR, DQ, and D P specific c D N A probes. Adherent synovial lining cells were cultivated in vitro and characterized by classical morphological and immunocytochemical studies: While the cells are D R positives, on fresh tissue M H C class II expression was transitory in in vitro culture. The class II antigens were weakly expressed after 96 hr and undetectable at day 7 nor at the protein nor at the m R N A level. Although unlikely since the percentage of the three cell types (macrophages, fibroblasts, dentritic cells) remained stable, the lack of class II could be due to selection of an Ia-negative population. Alternatively it could be explained by an absence of signal for Ia expression delivered by the nonadherent cell population (T lymphocytes notably): We tested the ability of recombinant gamma interferon to reinduce Ia synthesis on adherent cells. Five units/ml of "ylFN were sufficient for reexpression of class II antigens and a maximal effect was obtained for doses over 50 units/ml. DR, DQ, and D P products were ex~ pressed under this stimulation in the whole population of cells, m R N A transcription was evidenced from the 24th hr of culture while protein expression was detectable only after 48 hr. Recent data argue for IFN synthesis in rheumatoid pannus. The HLA class II induction of normally Ia-negative cell populations by 5,IFN might thus take place in pathogenesis of rheumatoid synovitis.
NEW CLASS I IN MAN: SEROLOGICAL AND MOLECULAR CHARACTERIZATION. R. Fauchet, O. Bouhallier, M. Boscher, and D. Charron; C.R.T.S. Rennes: U A C.N.R.S 625 Pari~ France
We have previously demonstrated the existence of new class I (/3~-m associated markers expressed P H A T cells, EBV cell lines, lymphoblastic leukemia cells, and nonexpressed on T PBL and thymocytes. New antigens form an allelic polymorphic series in linkage disequilibrium with H L A - A antigens (AI, A2, AS, A9, A 10) that segregate as a genetic system. One selected serum 101 RE positive on A3 T P H A and A3 EBV cell lines was studied. The expression of the new class I molecules was correlated with D N A and R N A levels in P H A T cells and was different from the expression of the HLAA3 antigen in kinetic studies. A summation effect was observed with 101 RE and A3 allosera, suggesting that two different determinants were recognized on the same cells. They could be considered as markers of cellular activation and could correspond to the Qa-like system in man. 101 RE immunoprecipitated a 41-43k,-12k molecule from S ~5 labeled EBV lymphoblastoid cell lines as detected by one D-PAGE. After six cycles of s e quential immunoprecipitations, the lysate was completely depleted of A3 mob ecule and 101 RE was able to precipitate a 41-43k,-12k molecule. These results demonstrated that 101 RE recognized a molecular structure different from the A3 molecule.
MOLECULAR AND CELLULAR DIVERSITY OF THE DR2 HAPLOTYPES. V. David, S. De Roquefeuil, C. Freidel, L. Gebuhrer, V. Lepage, H. Betuel, P. Debr~, and D.J. Charron; Laboratoire d'Immunog~n~tique Humaine, Chu Pitie Salpetriere, Paris, France
We investigated the D R and D Q molecules in D R 2/2 cells belonging to five different cellular subtypes (Dw2, Dw12, FJO, DB9, and Dw-). N E P H G E 2D PAGE was used to analyze both the D R and D Q / 3 chains and IEF 2D PAGE was used for the D Q a chains. All haplotypes studied expressed two D R 13 chains when 35S labeled cells were immunoprecipitated with D1.12, a monomorphic anti-HLA-DR monoclonal antibody. One D R /3 chain (/31) is common to all subtypes and one (/32) varies according to the Dw type. Similarly, the D Q 3
Abstracts
137
chains (obtained with 625a or Leu 10 MoAbs) are polymorphic and one given DR/32 chain corresponds to unique D Q / 3 chain. These data suggest that both D R / 3 and D Q / 3 could contribute equally to the Dw reactivity. The D Q ce chain shows a structural variability although to a lesser extent. The D Q w l associated a chain appears identical in several Dw2 and FJO cells while a variant was detected for a Dw12 cell. In addition, the DR2, DQw3, DB9 cell possess a D Q chain different from all the other D Q ce chains. We have developed an antigen-specific human T cell clone from a DR2/5 individual. The pattern of restriction of this clone discriminates between two groups within a panel of DR2 cells. Group I (6 cells) were capable of presenting the antigen while group II (10 cells) were unable to stimulate the clone. D R matched individuals from the two groups are mutually stimulatory in MLR. Taken together, biochemical and functional studies emphasize the diversity of the.DR2 haplotypes.
A MONOCLONAL ANTIBODY WITH SPECIFICITY FOR AN EPITOPE SHARED BY FtLADRce, DR3, AND THE HEAVY CHA1N OF HLA CLASS I ANTIGENS. Paula M. Lutz and Peter Cresswell; Duke University, Durham NC The homology of class I and class II genes has suggested their divergence from a common ancestral gene. We report here a monoclonal antibody (MoAb), 2.B6, which has specificity for both class II c~ and/3 chains, as well as the heavy chain of class I. Western blot analysis of the binding of 2.B6 to Lens cu/inaris purified glycoproteins from TRal, a B-lymphoblastoid cell line (B-LCL), showed reactivity to proteins of MW 45kd, 33kd, and 29kd. Two-dimensional electrophoresis and Western blotting of TRal glycoproteins showed reactivity of this MoAb with proteins of the appropriate molecular weights and electrophoretic mobilities for class I heavy chain and class II c~ and/3 subunits, with X D 5 . A l l (a class 1I /3specific MoAb) and 3B10.7 (a class I heavy chain-specific MoAb donated by Dr. S. Radka) as controls. Two-dimensional Western blotting of DRa-/3-invariant chain complexes purified from TRal glycoproteins by affinity chromatography using the MoAbDA6.147 (specificity for D R a chains) showed that 2.B6 reacted with both DRc~ and DR/3 subunits. Parallel blots of X D 5 . A l l and DA6.147 served as controls. Heavy chains of class I HLA antigens purified from the BLCL JY on a W6/32 affinity column (a class I-specific MoAb) also reacted with 2.B6 in Western blot analysis (3B10.7 as a parallel control). 2.B6 does not react with cell surface class I and class II HLA antigens, indicating that the epitope is not exposed in the native molecules. (This work was supported by N I H grant AI 2O547.)
ISOLATION OF BIOSYNTHETIC INTERMEDIATES OF HLA CLASS II ANTIGENS. Drew N. Kelner and Peter Cre3swell; Duke University Medical Center, Durham, NC Previous work in this laboratory has demonstrated that treatment of B-lymphoblastoid cell lines (B-LCL) with the sodium ionophore monensin results in the intracellular accumulation of class II antigen complexes containing the associated invariant (I) chain. These complexes have been isolated from detergent extracts of monensin-treated B-LCL by immunoaffinity chromatography on class II-specific monoclonal antibody columns. Gel filtration of the purified complexes on Sephacryl S-300 revealed the presence of a 270,000 dalton complex that contained the a, /3, and I subunits as determined by two-dimensional gel electrophoresis. Metabolic labeling with [35S] sodium sulfate revealed the presence of a sulfated macromolecule associated with affinity-purified class II antigen complexes. The sulfated component was sensitive to digestion by testicular hyal-
Annual AACHT Meeting, 1985 method using DR, DQ, and D P specific c D N A probes. Adherent synovial lining cells were cultivated in vitro and characterized by classical morphological and immunocytochemical studies: While the cells are D R positives, on fresh tissue M H C class II expression was transitory in in vitro culture. The class II antigens were weakly expressed after 96 hr and undetectable at day 7 nor at the protein nor at the m R N A level. Although unlikely since the percentage of the three cell types (macrophages, fibroblasts, dentritic cells) remained stable, the lack of class II could be due to selection of an Ia-negative population. Alternatively it could be explained by an absence of signal for Ia expression delivered by the nonadherent cell population (T lymphocytes notably): We tested the ability of recombinant gamma interferon to reinduce Ia synthesis on adherent cells. Five units/ml of "ylFN were sufficient for reexpression of class II antigens and a maximal effect was obtained for doses over 50 units/ml. DR, DQ, and D P products were ex~ pressed under this stimulation in the whole population of cells, m R N A transcription was evidenced from the 24th hr of culture while protein expression was detectable only after 48 hr. Recent data argue for IFN synthesis in rheumatoid pannus. The HLA class II induction of normally Ia-negative cell populations by 5,IFN might thus take place in pathogenesis of rheumatoid synovitis.
NEW CLASS I IN MAN: SEROLOGICAL AND MOLECULAR CHARACTERIZATION. R. Fauchet, O. Bouhallier, M. Boscher, and D. Charron; C.R.T.S. Rennes: U A C.N.R.S 625 Pari~ France
We have previously demonstrated the existence of new class I (/3~-m associated markers expressed P H A T cells, EBV cell lines, lymphoblastic leukemia cells, and nonexpressed on T PBL and thymocytes. New antigens form an allelic polymorphic series in linkage disequilibrium with H L A - A antigens (AI, A2, AS, A9, A 10) that segregate as a genetic system. One selected serum 101 RE positive on A3 T P H A and A3 EBV cell lines was studied. The expression of the new class I molecules was correlated with D N A and R N A levels in P H A T cells and was different from the expression of the HLAA3 antigen in kinetic studies. A summation effect was observed with 101 RE and A3 allosera, suggesting that two different determinants were recognized on the same cells. They could be considered as markers of cellular activation and could correspond to the Qa-like system in man. 101 RE immunoprecipitated a 41-43k,-12k molecule from S ~5 labeled EBV lymphoblastoid cell lines as detected by one D-PAGE. After six cycles of s e quential immunoprecipitations, the lysate was completely depleted of A3 mob ecule and 101 RE was able to precipitate a 41-43k,-12k molecule. These results demonstrated that 101 RE recognized a molecular structure different from the A3 molecule.
MOLECULAR AND CELLULAR DIVERSITY OF THE DR2 HAPLOTYPES. V. David, S. De Roquefeuil, C. Freidel, L. Gebuhrer, V. Lepage, H. Betuel, P. Debr~, and D.J. Charron; Laboratoire d'Immunog~n~tique Humaine, Chu Pitie Salpetriere, Paris, France
We investigated the D R and D Q molecules in D R 2/2 cells belonging to five different cellular subtypes (Dw2, Dw12, FJO, DB9, and Dw-). N E P H G E 2D PAGE was used to analyze both the D R and D Q / 3 chains and IEF 2D PAGE was used for the D Q a chains. All haplotypes studied expressed two D R 13 chains when 35S labeled cells were immunoprecipitated with D1.12, a monomorphic anti-HLA-DR monoclonal antibody. One D R /3 chain (/31) is common to all subtypes and one (/32) varies according to the Dw type. Similarly, the D Q 3
Abstracts
137
chains (obtained with 625a or Leu 10 MoAbs) are polymorphic and one given DR/32 chain corresponds to unique D Q / 3 chain. These data suggest that both D R / 3 and D Q / 3 could contribute equally to the Dw reactivity. The D Q ce chain shows a structural variability although to a lesser extent. The D Q w l associated a chain appears identical in several Dw2 and FJO cells while a variant was detected for a Dw12 cell. In addition, the DR2, DQw3, DB9 cell possess a D Q chain different from all the other D Q ce chains. We have developed an antigen-specific human T cell clone from a DR2/5 individual. The pattern of restriction of this clone discriminates between two groups within a panel of DR2 cells. Group I (6 cells) were capable of presenting the antigen while group II (10 cells) were unable to stimulate the clone. D R matched individuals from the two groups are mutually stimulatory in MLR. Taken together, biochemical and functional studies emphasize the diversity of the.DR2 haplotypes.
A MONOCLONAL ANTIBODY WITH SPECIFICITY FOR AN EPITOPE SHARED BY FtLADRce, DR3, AND THE HEAVY CHA1N OF HLA CLASS I ANTIGENS. Paula M. Lutz and Peter Cresswell; Duke University, Durham NC The homology of class I and class II genes has suggested their divergence from a common ancestral gene. We report here a monoclonal antibody (MoAb), 2.B6, which has specificity for both class II c~ and/3 chains, as well as the heavy chain of class I. Western blot analysis of the binding of 2.B6 to Lens cu/inaris purified glycoproteins from TRal, a B-lymphoblastoid cell line (B-LCL), showed reactivity to proteins of MW 45kd, 33kd, and 29kd. Two-dimensional electrophoresis and Western blotting of TRal glycoproteins showed reactivity of this MoAb with proteins of the appropriate molecular weights and electrophoretic mobilities for class I heavy chain and class II c~ and/3 subunits, with X D 5 . A l l (a class 1I /3specific MoAb) and 3B10.7 (a class I heavy chain-specific MoAb donated by Dr. S. Radka) as controls. Two-dimensional Western blotting of DRa-/3-invariant chain complexes purified from TRal glycoproteins by affinity chromatography using the MoAbDA6.147 (specificity for D R a chains) showed that 2.B6 reacted with both DRc~ and DR/3 subunits. Parallel blots of X D 5 . A l l and DA6.147 served as controls. Heavy chains of class I HLA antigens purified from the BLCL JY on a W6/32 affinity column (a class I-specific MoAb) also reacted with 2.B6 in Western blot analysis (3B10.7 as a parallel control). 2.B6 does not react with cell surface class I and class II HLA antigens, indicating that the epitope is not exposed in the native molecules. (This work was supported by N I H grant AI 2O547.)
ISOLATION OF BIOSYNTHETIC INTERMEDIATES OF HLA CLASS II ANTIGENS. Drew N. Kelner and Peter Cre3swell; Duke University Medical Center, Durham, NC Previous work in this laboratory has demonstrated that treatment of B-lymphoblastoid cell lines (B-LCL) with the sodium ionophore monensin results in the intracellular accumulation of class II antigen complexes containing the associated invariant (I) chain. These complexes have been isolated from detergent extracts of monensin-treated B-LCL by immunoaffinity chromatography on class II-specific monoclonal antibody columns. Gel filtration of the purified complexes on Sephacryl S-300 revealed the presence of a 270,000 dalton complex that contained the a, /3, and I subunits as determined by two-dimensional gel electrophoresis. Metabolic labeling with [35S] sodium sulfate revealed the presence of a sulfated macromolecule associated with affinity-purified class II antigen complexes. The sulfated component was sensitive to digestion by testicular hyal-