Hapten-specific murine colony-forming B cells: in vitro response of colonies to fluoresceinated thymus independent antigens.

The ability to clone hapten-specific B cells in agar and to subsequently trigger their clonal progeny to antibody synthesis was investigated. Fluorescein (FL) specific B cells were purified on FL-gelatin dishes and cultured in semisolid agar for 6 to 7 days; individual colonies were then picked for restimulation in microculture. FL-specific B cells could be cloned as efficiently as unpurified splenic B cells. The number of colonies formed depended on the presence of sheep erythrocytes (SRBC) or E. coli lipopolysaccharide (LPS) in the cultures. An additive number of colonies were observed with SRBC + LPS compared to that of SRBC or LPS alone. The colonies obtained from SRBC-containing cultures were stimulatable at high frequency by various FL-conjugated antigens to yield anti-FL PFC. However, colonies grown with LPS as the only additive were not stimulatable by any of the antigens tested. On the other hand, addition of M phi or SRBC as additional "mitogens" along with LPS in the agar resulted in progeny colonies that could respond in vitro. Although M phi did not increase the number of colonies, their presence enhanced the size and in some cases the frequency of stimulatable colonies. These data complement earlier observations in suggesting that different B cell subpopulations may grow under different cloning conditions. Moreover, the ability to stimulate the clonal progeny of single B cells to antibody synthesis should permit further definition of triggering and tolerance events at the single-cell level.