Development and maintenance of a B220(-) memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memo ry B cell subset rapidly dominates the secondary immune response to (4-hydr oxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP() memory B cells produce large numbers of B220(-)NP(+) B cells that can rap idly differentiate into plasma cells. Therefore, it is not clear whether th e novel B220(-) memory compartment is a consequence of secondary Ag challen ge or whether it develops as a stable memory subset after initial Ag challe nge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against ge rminal center localization and direct analysis in situ places a cohort of B 220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZ s. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellu lar dynamics and molecular attributes of these unique memory B cells sugges t they are distinct cellular products of the germinal center reaction in th e primary response and are maintained long-term in the spleen and bone marr ow.