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Long-term culture and characterization of alloreactive human T cell clones from renal needle biopsies
62
D. Bernard Amos Symposium
LONG-TERM CULTURE AND CHARACTERIZATION OF ALLOREACTIVE HUMAN T CELLCLONES FROM RENAL NEEDLE BIOPSIES. C. Miceli, R.S. Metzgar, and O.J. Finn: Dir. of Immunology. Duke Univ. Med. Ctr. Durham. NC Though little is known of cellular mechanisms involved in kidney rejection, successful diagnosis of severity of rejection based on infiltrates present in renal needle biopsies implies that the cellular component of the immune system plays a major role. Until recently, in vitro approaches toward analyzing cellular graft rejection were not feasible. The advent of T cell cloning technology provided methods for expansion and long-term culture of T cell infiltrates, which we have employed to study the specific T cell function in allograft rejection. Several T cell lines have been established by culturing renal needle biopsies from kidney recipients undergoing rejection episodes. Cultures are grown in the presence of IL-2 with weekly addition of irradiated donor lymphocytes as a source of antigen, obtained at the time of transplant and frozen or E.B.V. immortalized. All cells stain with OKT3, a Pan T marker. Expression of OKT8 and OKT4 antigens suggest cytotoxic as well as helper T cell functions. The IL-2 receptor and HLADR antigens are also expressed, consistent with the activated human T cell phenotype. Function and specificity of these lines are currently being investigated. One line, typed as 18% OKT8 positive, has demonstrated specific cytotoxicity for E.B.V. transformed donor cells. Efforts are underway to clone this and other T cell subpopulations and to study the specificity of each clone.
ANALYSIS OF HLA-DQ MOLECULESWITH MONOCLONAL ANTIBODIES SPECIFIC FOR DQ POLYMORPH1SMS. Susan F. Radka and Sandy J. Stewart; American Red Cross. Penn-Jersey Regional Blood Services, Philadelphia. PA We have produced several monoclonal antibodies detecting polymorphic determinants on HLA-DQ (MB,DC,DS) molecules. One antibody, SFR16-PI.2, detects a polymorphism absent from DQwl homozygous cells. Class II molecules isolated by this antibody from DR7 homozygous cell lines, when analyzed by two-dimensional gel electrophoresis, have beta chains with very basic electrophoretic mobility, similar to that originally described for DS (now renamed DQ). In addition, class II molecules having beta chains with acidic electrophoretic mobility are sometimes seen in metabolically labeled, but not surface-labeled DR7 cell line preparations. Depletion of DR molecules and subsequent immunoprecipitation with SFR16-PI.2 indicates that this antibody cross-reacts with an epitope on a biosynthetic intermediate of DR molecules. The broadly reactive polymorphic anti-DQ antibodies IVD12 (anti-DQw3), and Leu-10 when compared to SFR16-PI.2 on a DR5 homozygous BLCL isolate two DQ molecules, while SFR16-PI.2 isolates predominantly one. These data suggest that SFR16PI.2 detects allelic products of the DQ locus which encodes the molecules carrying the DQwl specificity. Results obtained with recently produced monoclonal antibodies having more restricted DQ specificity will also be presented.
SEGRATION OF POLYMORPHIC T CELL RECEPTOR GENES IN HUMAN FAMILIES. Mary Ann Robinson and Thomas J. Kindt; LIG, NIAID, NIH, Bethesda, MD Polymorphism in the genes encoding the constant region of the 3-chain of the T cell receptor (CT/3) have been detected by Southern blot analyses using a CT/3 probe and DNA extracted from B lymphoblastoid cell lines. The enzyme BglII
D. Bernard Amos Symposium
LONG-TERM CULTURE AND CHARACTERIZATION OF ALLOREACTIVE HUMAN T CELLCLONES FROM RENAL NEEDLE BIOPSIES. C. Miceli, R.S. Metzgar, and O.J. Finn: Dir. of Immunology. Duke Univ. Med. Ctr. Durham. NC Though little is known of cellular mechanisms involved in kidney rejection, successful diagnosis of severity of rejection based on infiltrates present in renal needle biopsies implies that the cellular component of the immune system plays a major role. Until recently, in vitro approaches toward analyzing cellular graft rejection were not feasible. The advent of T cell cloning technology provided methods for expansion and long-term culture of T cell infiltrates, which we have employed to study the specific T cell function in allograft rejection. Several T cell lines have been established by culturing renal needle biopsies from kidney recipients undergoing rejection episodes. Cultures are grown in the presence of IL-2 with weekly addition of irradiated donor lymphocytes as a source of antigen, obtained at the time of transplant and frozen or E.B.V. immortalized. All cells stain with OKT3, a Pan T marker. Expression of OKT8 and OKT4 antigens suggest cytotoxic as well as helper T cell functions. The IL-2 receptor and HLADR antigens are also expressed, consistent with the activated human T cell phenotype. Function and specificity of these lines are currently being investigated. One line, typed as 18% OKT8 positive, has demonstrated specific cytotoxicity for E.B.V. transformed donor cells. Efforts are underway to clone this and other T cell subpopulations and to study the specificity of each clone.
ANALYSIS OF HLA-DQ MOLECULESWITH MONOCLONAL ANTIBODIES SPECIFIC FOR DQ POLYMORPH1SMS. Susan F. Radka and Sandy J. Stewart; American Red Cross. Penn-Jersey Regional Blood Services, Philadelphia. PA We have produced several monoclonal antibodies detecting polymorphic determinants on HLA-DQ (MB,DC,DS) molecules. One antibody, SFR16-PI.2, detects a polymorphism absent from DQwl homozygous cells. Class II molecules isolated by this antibody from DR7 homozygous cell lines, when analyzed by two-dimensional gel electrophoresis, have beta chains with very basic electrophoretic mobility, similar to that originally described for DS (now renamed DQ). In addition, class II molecules having beta chains with acidic electrophoretic mobility are sometimes seen in metabolically labeled, but not surface-labeled DR7 cell line preparations. Depletion of DR molecules and subsequent immunoprecipitation with SFR16-PI.2 indicates that this antibody cross-reacts with an epitope on a biosynthetic intermediate of DR molecules. The broadly reactive polymorphic anti-DQ antibodies IVD12 (anti-DQw3), and Leu-10 when compared to SFR16-PI.2 on a DR5 homozygous BLCL isolate two DQ molecules, while SFR16-PI.2 isolates predominantly one. These data suggest that SFR16PI.2 detects allelic products of the DQ locus which encodes the molecules carrying the DQwl specificity. Results obtained with recently produced monoclonal antibodies having more restricted DQ specificity will also be presented.
SEGRATION OF POLYMORPHIC T CELL RECEPTOR GENES IN HUMAN FAMILIES. Mary Ann Robinson and Thomas J. Kindt; LIG, NIAID, NIH, Bethesda, MD Polymorphism in the genes encoding the constant region of the 3-chain of the T cell receptor (CT/3) have been detected by Southern blot analyses using a CT/3 probe and DNA extracted from B lymphoblastoid cell lines. The enzyme BglII