Antigen-induced proliferation of murine T-lymphocytes in vitro. I. Characterization of the lymphocyte culture system

Abstract

The present report describes further improvements in the methodology for antigen-specific T-lymphocyte activation in vitro. Following the experimental protocol, large numbers of enriched T-lymphocytes can be obtained from the draining lymph nodes of immunized mice after purification on a nylon column. These T-cells respond better to stimulation by the soluble immunizing antigen than do unfractionated lymph node cells. The magnitude of response, measured by the incorporation of [ 3H]thymidine, was dependent on culture conditions and percentage of macrophages added to the column-purified cells. The optimal specific proliferative response was achieved when lymphocytes were incubated with 50% spleen cells and then cultured in RPMI-1640 supplemented with 2-mercaptoethanol (5 × 10 −5 M), sodium pyruvate (1 mM), AB+ human serum (5%) and syngeneic mouse serum (0.5%). Under the optimal culture conditions the lymphocytes undergo two successive cycles of proliferation as a result of antigen or mitogen stimulation. Thus, our studies have defined the culture conditions which can support extensive antigen-induced proliferation of T-cells easily obtained in large numbers. This in vitro system of antigen-specific T-cells from lymph nodes of immunized mice is most suitable for studies on the mode of T-cell activation.