Bacterial cells were pelleted by centrifugation and incubated for 5 min with 0.5 mg/ml NP-OSu (Biosearchtech). polysaccharides that engage the B cell receptor and thus induce antigen-specific B cell responses. T-I type II antigens elicit robust and long-lasting primary antibody responses in mice (2), and polysaccharide vaccines such as Pneumovax and Menomune confer long-term humoral protection in adult humans. However, T-I type II antigens do not elicit a recall response; i.e., a boost in antibody production upon secondary immunization (3C6). Nevertheless, splenocytes from mice immunized with T-I type II antigens can respond to secondary challenge when adoptively transferred into naive irradiated recipients, and injection of immune serum into naive recipients before adoptive transfer suppresses this response (3, 4). T-D antigens elicit IGF1 memory B cells, which develop in T-D germinal centers and can be identified by somatic mutations in their Ig loci or by surface expression of secondary Ig isotypes (7, 8). T-I type II antigens stimulate extrafollicular foci of plasma cell production (2) and short-lived presumably abortive T-I YM-58483 germinal centers (9, 10). It is not known whether T-I type II immune responses generate memory B cells. Very low levels of somatic hypermutation (11) and low frequency of switching to secondary Ig isotypes during T-I type II responses hinder the identification of T-I memory B cells using these criteria, and it is widely accepted that memory B cells are derived only from T-D responses (1, 7, 8, 12, 13). Here we show that T-I type II immune responses generate memory B cells whose secondary activation by polysaccharides is stringently regulated by antigen-specific IgG antibodies. RESULTS AND DISCUSSION T-I type II immune responses generate memory B cells Memory B cells are quiescent B cells derived from proliferating antigen-experienced precursors (14). We used an in vivo BrdU pulse-chase strategy to test whether a model T-I type II antigen 4-hydroxy-3-nitrophenylacetyl (NP)-Ficoll elicits memory B cells. To ensure that the analysis was not confounded by BrdU incorporation into dividing bone marrow B cell precursors, we adoptively transferred allotype-marked (CD45.1+) splenic B cells from B1-8high IgH knock-in mice (15) into naive wild-type recipients before immunization and subsequently analyzed only the transferred population. Recipient mice were immunized with NP-Ficoll and fed BrdU for the duration of the proliferative phase of the T-I type II response (days YM-58483 1C5; reference 15), after which BrdU was withdrawn. Incorporation of BrdU into dividing B cells was assessed by flow cytometry immediately after BrdU withdrawal on day 5 after immunization. To detect quiescent long-term survivors derived from activated precursors, YM-58483 BrdU retention was assayed on days 15, 60, and 120 (Fig. 1 A). We detected allotype-marked BrdU-labeled B cells in the spleen of NP-Ficoll immunized recipients, but not in control recipients injected with PBS and fed BrdU (Fig. 1 B). BrdU-labeled YM-58483 B cells were Ig+ (Fig. 1 B) and thus NP-specific (15, 16). Because subsequent cell division in the absence of BrdU (days 5C120) would have resulted in loss of BrdU by dilution, the detected BrdU-labeled cells must be quiescent. Open in a separate window Figure 1. T-I type II immune response generates memory B cells. (A) BrdU pulse-chase strategy. (B) BrdU staining of adoptively transferred B1-8high B cells (top) and Ig staining of BrdU-gated B1-8high B cells (bottom) YM-58483 from NP-FicollCimmunized or naive wild-type recipients analyzed at the indicated time points. (C) B220 versus Syndecan-1 (top) and B220 versus IgG3 (bottom) staining of B1-8high B cells from naive recipients on day 15 after adoptive transfer (left) and of BrdU-gated B1-8high B cells from NP-FicollCimmunized recipients on day.