9×106 total cells/mouse; n = 9; and 54% B-1 cells). Thus, these data confirmed the presence of significant numbers of B-1 cells in the steady-state BM, where they are of a phenotype comparable with that of spleen but not PerC B-1 cells. To determine whether B-1 cells are the IgM-secreting cells in the BM we examined spontaneous IgM secretion in Ig-allotype-chimeric mice. Confirming our data from BALB/c mice (Fig. 1A), little spontaneous natural IgM secretion was detected by PerC B-1 cells (0.03 μg/mL from 2×105 total cells) in these animals (Fig. 4A). In contrast, higher concentrations of B-1 cell-derived IgM were found in cultures of spleen (0.72 μg/mL) and BM (0.73 μg/mL) (Fig.

4A). As BM cultures contained about 3-fold lower numbers learn more of IgM AFCs than spleen cultures (2.9±0.6×103 and 8.5±0.2×103 IgM AFCs per 106 cells respectively) antibody production per secreting cell was

highest in the BM. In the spleen, about 87% of natural IgM secreting cells were of B-1 (Igh-a) Alpelisib ic50 cell origin and in the BM that number was at least 95% (Fig. 4B). Given that 10–20% of host-derived Igh-b could be B-1 cells in the allotype-chimeras 26, we conclude that the IgM AFCs in spleen and BM are B-1 cells. In addition, comparing the frequencies of B-1 cells in spleen and BM and the number of AFCs, indicated that >80% of BM B-1 cells secreted IgM, whereas for spleen this number was closer to 40% (Fig. 4C). Consistent with results Fossariinae from BALB/c mice (Fig. 1E), a subset of spontaneous IgM-secreting B cells recognized influenza A/Mem/71, both in spleen and BM of Ig-allotype chimeras (Fig. 4D), further demonstrating that they are natural IgM-secreting

B-1 cells. To further demonstrate the role of BM B-1 cells in spontaneous IgM secretion, we conducted BM transfer experiments in which we transferred either entire BM or BM depleted of IgM-expressing cells into lethally irradiated RAG-1−/− mice. The results showed that IgM-expressing B cells contained all spontaneous IgM-secreting cells. None (n = 7) of the RAG-1−/− host mice that received surface IgM-depleted BM cells had any measurable serum IgM 6 weeks after transfer. In contrast, all mice (n = 8) that received complete BM had significant donor-derived IgM serum levels (Fig. 4E). Collectively, the data demonstrate the presence of a novel population of B-1 cells in the BM that spontaneously produces natural IgM and significantly contributes to steady-state serum IgM levels. We aimed to further characterize the BM B-1 cells and to compare these spontaneous natural IgM-secreting cells with resting B-2 cells, identified as CD19+ B220+ IgMlo IgDhi and classical B220lo CD43+ CD138+ plasma cells (Supporting Information Fig. 2). The latter were induced in mediastinal lymph nodes via infection of mice with influenza virus A/Mem71 for 10 days.