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Spleen and bone marrow (BM) are the major sites of antibody production and anamnestic response in systemically immunized mice. We examined the VDJ segment repertoire of antibody plaque-forming cells (APFC) in those two sites in the course of antibody responses to the hapten nitrophenyl (NP). Individual IgG APFC expressed any one of 10 V(H) segments of the V186.2/V3 (J558) gene family: 186.2, 102, 23, C1H4, 165.l, CH10, 3, 593.3, 24.8 and 671.5. The majority of cells in both spleen and BM expressed the V186.2 gene joined to a D segment with Tyr95. During a 2-month period after a single immunization, the V186.2(+) APFC in BM accumulated 3 times as many somatic mutations than splenic APFC (average 8.5 versus 3 mutations/V(H)); this process was T(h) dependent as shown by in vivo depletion of CD4(+) lymphocytes. However, the V186.2(+) APFC in both spleen and BM shared a recurrent W33L replacement, indicating their common origin from germinal centers. The APFC expressing the other (analogue) V(H) segments were evenly represented in the spleen and BM, but they accumulated few, if any, mutations. The anamnestic V186.2(+) APFC were highly mutated both in the spleen and BM; they represented a new and unexpected clonotype. The V/D segments were joined by Gly95 instead of Tyr95, the W33L was absent and a new shared K58R replacement appeared. The APFC expressing the 'analogue' V(H) genes comprised approximately 20% of the anamnestic response and did not accumulate more mutations, but their affinities were in the range of the memory V186.2(+) cells. These data suggest that the late primary and secondary responses to a hapten may be born by different B cell lineages, and that some clonotypes may reach the memory pool without an extensive mutation and expansion.

Canonical germinal center B cells may not dominate the memory response to antigenic challenge