A hemolytic plaque assay for activated murine T cells.

Abstract

Murine spleen cells cultured with concanavalin (Con) A were shown previously to release into the culture supernatants helper and suppressor substances for antibody production. In this study, production of rabbit antisera against culture supernates from Con A-activated spleen cells and their use in a plaque assay for mitogen-activated T [thymus-derived] cells were described. The plaque assay, utilizing SRBC [sheep red blood cells] to which Staphylococcal protein A had been coupled, the developing anti-supernatant antiserum and the guinea pig complement readily detected secreting T cells. The T cell nature of the plaque-forming cells (PFC) was established principally by the following: the majority of lymphocytes in the centers of plaques were Thy-1-positive by fluorescence; spleen cells depleted of B [bone marrow-derived] cells by incubation in plastic dishes coated with rabbit anti-mouse Ig [immunoglobulin] antibody gave greatly enriched PFC responses; anti-Thy-1 and anti-Lyt-2.2 treatment of spleen cells almost completely depleted PFC; T cell mitogens (Con A and phytohemagglutinin) but not B cell mitogens (lipopolysaccharides) induced PFC responses and T cells maintained in culture for 10 days with Con A and T cell growth factor yield PFC. Kinetic and dose response studies showed that high doses of mitogen induced rapidly appearing T-PFC and the responses peaked at day 1-2 of culture. Lower doses of mitogen-induced PFC required longer periods of incubation for detection, indicating that cell activation and secretion may be different dose-dependent activities of mitogens. Another noteworthy finding was that antiserum reacted with surface antigens of T-PFC, indicating that secreted products are expressed on T cell membranes offering the possibility of isolating populations of cells with specific secretory potential. Although the precise nature of T cell products detected by the antiserum used in this assay are unresolved, 10% of the target-cell-adherent population from spleen cells of BALB/c mice sensitized to [neoplastic fibroblasts] L929 cells formed plaques. The antiserum apparently has significant activity against the products of cytotoxic T cells, a finding which accords with activity of anti-Lyt 2.2 serum against mitogen-induced T-PFC. The method offers new possibilities for analysis of T cells and their products and should provide an important approach to clonal analysis of lymphokine production.