In vitro activation of murine antigen-specific memory B cells by a T-dependent antigen

A procedure that generates an enriched population (60 to 85%) of memory B cells specific for TNP (TNP-MABC) was employed. The activation requirements of TNP-MABC for the T-dependent antigen TNP-KLH and carrier-primed helper T (T(h)) cells were compared to those for TNP-binding cells from non-immune mice (TNP-ABC). Proliferation and differentiation of TNP-MABC in response to cognate recognition of antigen requires less antigen and fewer carrier-primed T(h) cells than the activation of TNP-ABC. Furthermore, responses of the TNP-MABC were of a greater magnitude. Non-cognate activation induces a low level of proliferation of both TNP-ABC and TNP-MABC, but induces differentiation of TNP-MABC only. Percoll density fractionation of spleen cells prior to enrichment for TNP-MABC suggests that the small, dense cell population responds to cognate, but not to non-cognate activation. FACS separation of TNP-MABC by surface Ig isotype reveals that approximately 80% of the secondary IgG response is derived from cells expressing sIgG. Such cells constitute less than 10% of the total number of TNP-MABC. Limiting dilution studies with sorted TNP-MABC indicate that sIgG⁺ TNP-MABC are enriched for precursors that give rise to a large clone size. The in vitro results indicate the existence of three putative pathways for antigen-specific memory B cell activation by a T-dependent antigen: 1) sIgG⁺ cells differentiating into IgG-secreting cells; 2) sIgM⁺ cells differentiating into IgG-secreting cells; and 3) sIgM⁺ cells differentiating into IgM-secreting cells.