Biologic Activity of Rabbit Antisera Directed against in Vitro Stimulated Guinea Pig T Lymphocytes

Abstract

In this communication we examined the functional activity of rabbit antisera produced against guinea pig T lymphocytes that had been stimulated in vitro by culture with macrophage-associated antigen. One antiserum (96-2) was raised against strain 13 T cells that were primed by culture for a week with trinitrophenyl-(TNP) modified syngeneic macrophages. Addition of 96-2 serum to TNP-primed strain 13 T cells or strain 13 T cells primed in a mixed leukocyte reaction (MLR) stimulated a dramatic increase in DNA synthesis in vitro. In contrast, strain 13 T cells primed in vitro with several protein antigens failed to be stimulated by the 96-2 serum. In addition, 96-2 serum produced little stimulation of TNP or MLR-primed strain 2 cells, or of strain 13 T cells taken directly from the animal. Another antiserum (144-2) was raised against glutamic acid-lysine copolymer (GL)-immune strain 2 T cells selected by culture with GL-pulsed strain 2 macrophages. Addition of 144-2 serum to GL-selected or MLR-primed strain 2 T cells also produced a dramatic proliferative response. However, 144-2 serum did not stimulate TNP-primed strain 2 or strain 13 T cells, or strain 2 T cells selected with unrelated protein antigens. By several techniques we showed that contaminating B cells in the primed T cell preparations were not responsible for the observed proliferation and that the antisera directly stimulated highly purified T cells. The proliferative activity of 96-2 and 144-2 could be removed by absorption with nonimmune spleen cells. Physicochemical analysis of the cellular antigens detected by the antisera showed molecular species of 98,000, 70,000, 33,000 and 25,000 daltons. By sequential immunoprecipitation analyses, the latter two peaks were determined to be Ia antigens, the 70,000 peak of in vitro primed cells contained some immunoglobulin-like molecules and another nonimmunoglobulin-like antigenic species, and the 98,000 species appeared unrelated to immunoglobulin or Ia antigens.