The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.

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