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HLA-A2 epitopes recognized by alloantigens
168
Abstracts Genes coding for HLA molecules serologically related to HLA-A2 have been isolated using an A specific probe. Identity of the genes was determined by serological analysis of L cell transfectants. Protein encoding exons were selectively sequenced using oligonucleotide specific primers. Comparison of these sequences has allowed further definition of residues important for epitopes recognized by monoclonal antibodies and cytotoxic T lymphocytes. The relationship of the sequences indicates the multiple genetic mechanisms have been important in the diversification of human class I histocompatibility genes.
EXPRESSION OF CLASS I HISTOCOMPATIBILITY ANTIGENS ON RAT FIBROBLASTS. Bryan M. Gebhardt and Glenda Talley; LSU Eye Center, LSU Medical Center School of Medicine, New Orleans, LA The purpose of this investigation was to determine if fibroblasts derived from the cornea of the rat express the histocompatibility antigens of this species. Corneal fibroblasts derived from several strains of rats were grown in tissue culture in sufficient numbers so as to be sufficient to immunize an allogeneic strain. Groups of rats were injected with 0.1 x 106, 1 x 106, 10 x 106, and 20 x 106 tissue culture propagated allogeneic keratocytes. At 7-day intervals after immunization the rats were bled in order to obtain serum for testing for antibodies to cell membrane antigens of the appropriate corneal fibroblasts. It was found that 10 x 106 or more keratocytes were required to elicit a significant antibody response in an allogeneic animal. The presence of antibody was demonstrated by immunohistochemical staining and in both the hemagglutination assay using donor strain erythrocytes and in the lymphocytotoxicity assay using donor lymphocytes. Strong presumptive evidence that the antibody response to allogeneic keratocytes represents a response to class I major histocompatibility antigen was obtained by employing monoclonal antibodies against the monomorphic determinant of the rat class I antigen in hemagglutination and cytotoxicity inhibition assays. Immunohistochemical staining of cryostat sections of rat cornea using the sera from fibroblast immunized rats revealed that the corneal fibroblasts express the same antigens in vivo as in vitro indicating that they are not antigens expressed de novo as a result of tissue culture. Cornea fibroblasts from tissue culture and in situ fibroblasts did not exhibit class II histocompatibility antigens by immunohistochemical staining. These studies establish the antigenicity of rat corneal fibroblasts and indicate that they can be targets for cytotoxic cell attack during corneal allograft rejection. (Supported in part by PHS grant EY03150 from the National Eye Institute.) HLA-A2 EPITOPES RECOGNIZED BY ALLOANTIBODIES. Juan C. Scornik; Department of Pathology, University of Florida College of Medicine, Gainesville, FL Alloantibodies in broadly sensitized patients are difficult to characterize because they comprise mixtures with different specificities. By studying the ability of patients' sera to inhibit or enhance the biding of anti-HLA-A2 monoclonal antibodies, not only anti-A2 reactivity could be detected but several epitopes could be operationally defined. Two monoclonal antibodies were used: BB7.2 (antiA2, Aw69) and MA2.1 (anti-A2, B 17). They were labeled with fluorescein and their binding to A2 positive target lymphocytes was measured by flow cytometry (FACS II). Preincubation of target lymphocytes with sera from some patients produced a change in monoclonal antibody binding that allowed the recognition
Abstracts
169 of four different epitopes. Antibodies to epitope #1 (found in eight patients) inhibited both BB7.2 and MA2.1 and were absorbed out by A2 positive lymphocytes but not by A28 or B 17 positive lymphocytes. This epitope is probably identical or closely related to the one recognized by BB7.2. Antibodies to epitope #2 (three patients) also inhibited BB7.2 and MA2.1 but were absorbed out by A2 or B 17 positive cells. This epitope is probably identical or closely related to the one recognized by MA2.1. Antibodies to epitope #3 (three patients) inhibited MA2.1 but enhanced the binding of BB7.2; they were absorbed out also by either A2 or B17 positive cells. This epitope is related to epitope #2 but is clearly distinct from it. Antibodies to epitope #4 (two patients) inhibited MA2.1, did not inhibit BB7.2, and were absorbed out by A2 or A28 positive cells. This epitope is probably related to the A2-A28 public determinant. Thus, use of monoclonal antibodies allowed the detection of alloantibody specificities in complex sera that recognize different epitopes within the HLA-A2 molecule. (Supported by N I H Grant HL-33345.)
ELISA AND IMMUNOBLOTTING ANALYSES OF HUMAN PLATELETTROPHOBLAST-LYMPHOCYTE CROSS-REACTIVE (TLX) ANTIGENS. Toru Kazino, W. Page Faulk, and John A. McIntyre; Southern Illinois University School of Medicine, Spring/ield, IL; and Laboratories for Transplantation and Reproductive Biology, Methodist Hospital of lndianapolis, Indianapolis, IN Secondary, recurrent, spontaneous aborting women have high-titred, persistent antibodies which kill husbands' and third-party lymphocytes in complementdependent-cytotoxicity (CDC) assays. Cytotoxicity by these antibodies cannot be attributed to HLA specificities on a 55-member select-cell-panel and can be removed by absorption with HLA-negative trophoblast membranes or by HLAunrelated platelets. When secondary aborter sera were studied by CDC against paternal cells following absorption experiments, the results showed allotypic patterns of reactivity. We have designated these allotypic markers as trophoblast-lymphocyte cross-reactive (TLX) antigens. To further characterize TLX antigens and antibodies, we have developed an ELISA and studied 18 platelet membrane TLX preparations by using ten patients' and ten rabbits' anti-TLX sera. Factor analysis of the rabbit antisera showed 77 % of reactivity accountable to two factors, but the human sera accounted for 93% of variability within four factors. Some human IgG anti-TLX was found to react better in media containing high concentrations of albumin, and lgG:IgM anti-TLX ratios seemed to relate to pregnancy outcome. The antigen was resistant to chaotropic agents, heat, and 0.1 M EDTA but was destroyed by pronase and trypsin. IgM antibodies reacted better with neuraminidase-treated platelet membranes. The platelet TLX antigen can be harvested in high yield from the interface of chloroform-methanol extractions. Western blot analysis of the Folch extracted TLX antigens suggest molecular weights of 41, 135, and 180 kd. Their role in the biology and pathology of the human pregnancy allograft is under investigation in our laboratory. ANTI-TLX ANTIBODY IN HUMAN PREGNANCY: W. Page Faulk and John A. McIntyre; Laboratories for Transplantation and Reproductive Biology, Methodist Hospital of lndianapolis, IN Pregnant mammals produce antibodies which bind paternal lymphocytes in vitro. These immunoglobulins are not specific for class I or II major histocompatibility antigens, and it is not possible to describe their specificity by using traditional lymphocytotoxicity tests with MHC-typed donors. Lymphocytotoxicity of these
Abstracts Genes coding for HLA molecules serologically related to HLA-A2 have been isolated using an A specific probe. Identity of the genes was determined by serological analysis of L cell transfectants. Protein encoding exons were selectively sequenced using oligonucleotide specific primers. Comparison of these sequences has allowed further definition of residues important for epitopes recognized by monoclonal antibodies and cytotoxic T lymphocytes. The relationship of the sequences indicates the multiple genetic mechanisms have been important in the diversification of human class I histocompatibility genes.
EXPRESSION OF CLASS I HISTOCOMPATIBILITY ANTIGENS ON RAT FIBROBLASTS. Bryan M. Gebhardt and Glenda Talley; LSU Eye Center, LSU Medical Center School of Medicine, New Orleans, LA The purpose of this investigation was to determine if fibroblasts derived from the cornea of the rat express the histocompatibility antigens of this species. Corneal fibroblasts derived from several strains of rats were grown in tissue culture in sufficient numbers so as to be sufficient to immunize an allogeneic strain. Groups of rats were injected with 0.1 x 106, 1 x 106, 10 x 106, and 20 x 106 tissue culture propagated allogeneic keratocytes. At 7-day intervals after immunization the rats were bled in order to obtain serum for testing for antibodies to cell membrane antigens of the appropriate corneal fibroblasts. It was found that 10 x 106 or more keratocytes were required to elicit a significant antibody response in an allogeneic animal. The presence of antibody was demonstrated by immunohistochemical staining and in both the hemagglutination assay using donor strain erythrocytes and in the lymphocytotoxicity assay using donor lymphocytes. Strong presumptive evidence that the antibody response to allogeneic keratocytes represents a response to class I major histocompatibility antigen was obtained by employing monoclonal antibodies against the monomorphic determinant of the rat class I antigen in hemagglutination and cytotoxicity inhibition assays. Immunohistochemical staining of cryostat sections of rat cornea using the sera from fibroblast immunized rats revealed that the corneal fibroblasts express the same antigens in vivo as in vitro indicating that they are not antigens expressed de novo as a result of tissue culture. Cornea fibroblasts from tissue culture and in situ fibroblasts did not exhibit class II histocompatibility antigens by immunohistochemical staining. These studies establish the antigenicity of rat corneal fibroblasts and indicate that they can be targets for cytotoxic cell attack during corneal allograft rejection. (Supported in part by PHS grant EY03150 from the National Eye Institute.) HLA-A2 EPITOPES RECOGNIZED BY ALLOANTIBODIES. Juan C. Scornik; Department of Pathology, University of Florida College of Medicine, Gainesville, FL Alloantibodies in broadly sensitized patients are difficult to characterize because they comprise mixtures with different specificities. By studying the ability of patients' sera to inhibit or enhance the biding of anti-HLA-A2 monoclonal antibodies, not only anti-A2 reactivity could be detected but several epitopes could be operationally defined. Two monoclonal antibodies were used: BB7.2 (antiA2, Aw69) and MA2.1 (anti-A2, B 17). They were labeled with fluorescein and their binding to A2 positive target lymphocytes was measured by flow cytometry (FACS II). Preincubation of target lymphocytes with sera from some patients produced a change in monoclonal antibody binding that allowed the recognition
Abstracts
169 of four different epitopes. Antibodies to epitope #1 (found in eight patients) inhibited both BB7.2 and MA2.1 and were absorbed out by A2 positive lymphocytes but not by A28 or B 17 positive lymphocytes. This epitope is probably identical or closely related to the one recognized by BB7.2. Antibodies to epitope #2 (three patients) also inhibited BB7.2 and MA2.1 but were absorbed out by A2 or B 17 positive cells. This epitope is probably identical or closely related to the one recognized by MA2.1. Antibodies to epitope #3 (three patients) inhibited MA2.1 but enhanced the binding of BB7.2; they were absorbed out also by either A2 or B17 positive cells. This epitope is related to epitope #2 but is clearly distinct from it. Antibodies to epitope #4 (two patients) inhibited MA2.1, did not inhibit BB7.2, and were absorbed out by A2 or A28 positive cells. This epitope is probably related to the A2-A28 public determinant. Thus, use of monoclonal antibodies allowed the detection of alloantibody specificities in complex sera that recognize different epitopes within the HLA-A2 molecule. (Supported by N I H Grant HL-33345.)
ELISA AND IMMUNOBLOTTING ANALYSES OF HUMAN PLATELETTROPHOBLAST-LYMPHOCYTE CROSS-REACTIVE (TLX) ANTIGENS. Toru Kazino, W. Page Faulk, and John A. McIntyre; Southern Illinois University School of Medicine, Spring/ield, IL; and Laboratories for Transplantation and Reproductive Biology, Methodist Hospital of lndianapolis, Indianapolis, IN Secondary, recurrent, spontaneous aborting women have high-titred, persistent antibodies which kill husbands' and third-party lymphocytes in complementdependent-cytotoxicity (CDC) assays. Cytotoxicity by these antibodies cannot be attributed to HLA specificities on a 55-member select-cell-panel and can be removed by absorption with HLA-negative trophoblast membranes or by HLAunrelated platelets. When secondary aborter sera were studied by CDC against paternal cells following absorption experiments, the results showed allotypic patterns of reactivity. We have designated these allotypic markers as trophoblast-lymphocyte cross-reactive (TLX) antigens. To further characterize TLX antigens and antibodies, we have developed an ELISA and studied 18 platelet membrane TLX preparations by using ten patients' and ten rabbits' anti-TLX sera. Factor analysis of the rabbit antisera showed 77 % of reactivity accountable to two factors, but the human sera accounted for 93% of variability within four factors. Some human IgG anti-TLX was found to react better in media containing high concentrations of albumin, and lgG:IgM anti-TLX ratios seemed to relate to pregnancy outcome. The antigen was resistant to chaotropic agents, heat, and 0.1 M EDTA but was destroyed by pronase and trypsin. IgM antibodies reacted better with neuraminidase-treated platelet membranes. The platelet TLX antigen can be harvested in high yield from the interface of chloroform-methanol extractions. Western blot analysis of the Folch extracted TLX antigens suggest molecular weights of 41, 135, and 180 kd. Their role in the biology and pathology of the human pregnancy allograft is under investigation in our laboratory. ANTI-TLX ANTIBODY IN HUMAN PREGNANCY: W. Page Faulk and John A. McIntyre; Laboratories for Transplantation and Reproductive Biology, Methodist Hospital of lndianapolis, IN Pregnant mammals produce antibodies which bind paternal lymphocytes in vitro. These immunoglobulins are not specific for class I or II major histocompatibility antigens, and it is not possible to describe their specificity by using traditional lymphocytotoxicity tests with MHC-typed donors. Lymphocytotoxicity of these