Biochemical studies of regulatory molecules in the immune response
1984
Macdonald, Peter Reg
Human T cell hybrids were produced following the polyethylene glycol-mediated fusion of concanavalin-A-activated peripheral blood lymphocytes with a 6-thioguanine-resistant subline of the human T cell acute lymphocytic leukemia line CCRF-CEM. Four T cell hybrid lines were established by hypoxanthine-aminopterin-thymidine selection and flow cytometric selection of the OKT3⁺ hybrids by sterile cell sorting on a FACS IV. Cell surface phenotype and the acquisition of functional hypoxanthine guanine phosphoribosyl transferase activity following hybridisation was used to confirm true human T cell hybrids were produced. Two assay systems, enabling the measurement of human in vitro antibody responses were developed and validated. Using one of these assays, a mitogen-dependent system, it was demonstrated that hybrid line TT-4 constitutively produced an activity which suppressed pokeweed mitogen-induced antibody production. In addition, it was demonstrated that the TT-4-derived activity was capable of suppressing the proliferative response of normal lectin-activated T cells to exogenous sources of interleukin-2. Preliminary biochemical studies of the hybrid-derived activity were undertaken. Sephacryl S-200 chromatography produced three major peaks of suppressor activity (>250, 120-140, 60-80 Kdal). The activity was partially acid and alkali labile, dithiothreitol sensitive, affected by the monosaccharides α-methyl mannoside, L-fucose and N-acetyl glucosamine but not by L-rhamnose. These studies confirm that somatic cell hybridisation techniques can be successfully applied for the production of human regulatory Lymphokines.
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