T-dependent B-cell activation is signalled by an early increase in potassium influx

When splenic lymphocytes from RNC nu/+ mice were enriched for T cells by lectin purification, mixed to constant cell density with splenocytes from syngeneic nude mice, and cultured with ConA, a proliferative response ensued which was greater than that expected from the T cells alone. This was shown both by incorporation of 3H-thymidine after 48 h of culture and by uptake of potassium (measured as 86Rb) after 15 h. Analysis of metaphase chromosomes stained with Hoechst dye 33258 from co-cultured T-enriched and nude lymphocytes (from female and male donors, respectively) mixed in the proportion 1:4 showed that 40% of the mitotic cells came from nude spleen. About half of the blast cells in such mixtures stained strongly with fluorescein-coupled goat anti-mouse immunoglobulin; T-cell blasts did not stain under these conditions. Treatment of the nu/nu cells with anti-Thy 1.2 and complement had no effect on their subsequent proliferation in coculture. B lymphocytes from nude mouse spleen were therefore activated to proliferate in this system. This B-cell activation can be detected by increased potassium uptake 15 h after the initiation of co-culture. Thus the increased monovalent cation flux (previously demonstrated when B and T lymphocytes were separately stimulated) also occurs when B cells are stimulated through cooperation with mitogen-activated T cells, and is also detectable early in culture. T-dependent activation of B cells is therefore detectable considerably earlier than by conventional assays (such as plaque formation).