5c, top panel; see Supplementary material, Table S3). Although five Vκ segments were represented among 15 clones sequenced from B220lo CD19+ B cells, the 19–32 Vκ segment was highly over-represented among these clones, selleck being identified in 9/15 clones (60%) sequenced (Fig. 5c, top panel). Notably, 13/15 (87%) of these clones show a germ-line configuration, suggesting that the B220lo CD19+
B cells have not undergone somatic hypermutation in the germinal centre (Fig. 5c, lower right panel; see Supplementary material, Table S3). By contrast, the frequency of unmutated clones derived from B220hi CD19+ B cells is much lower, both in normal mice (5/13 clones; 38%) and dnRAG1 mice (19/38 clones; 50%). Accumulating B220lo CD19+ B cells resemble B1a B cells that are thought to be responsible for the production Dasatinib research buy of natural antibodies, so we wondered whether dnRAG1 mice might exhibit elevated levels of serum immunoglobulin. Surprisingly, however, measurements of serum IgM and IgG levels from unimmunized normal and dnRAG1 mice revealed that dnRAG1 mice have significantly lower levels (approximately threefold) of serum IgM and IgG than their WT counterparts (Fig. 6a).
To determine whether this outcome might be the result of defects in B-cell responsiveness toward antigenic stimulation, we measured the activation of WT or dnRAG1 splenocytes or sorted B220lo CD19+ B cells and B220hi CD19+ B cells using an MTT assay after mitogen treatment with lipopolysaccharide or BCR cross-linking using anti-IgM F(ab’)2 antibody. Metalloexopeptidase We found that both treatments stimulate splenocytes isolated from WT and dnRAG1 mice more than media alone, but dnRAG1 splenocytes showed a significantly diminished responsiveness toward stimulation by lipopolysaccharide or anti-IgM cross-linking than those isolated from WT mice (Fig. 6b, upper panel). Indeed, the level
of stimulation of dnRAG1 splenocytes by anti-IgM was not significantly different than a control F(ab’)2 antibody. Similar experiments conducted with sorted B220lo and B220hi B cells from WT and dnRAG1 mice revealed that while the B220hi and B220lo subsets are both stimulated by lipopolysaccharide, the level of stimulation is not significantly different between the subsets (Fig. 6b, lower panel). In contrast, B220hi B cells from WT mice responded significantly better to anti-IgM treatment than both B220hi and B220lo cells from dnRAG1 mice, with the difference being slightly greater for B220lo B cells (which showed no significant difference relative to treatment with a control F(ab’)2 antibody). The difference between WT and dnRAG1 B220hi B-cell responses is somewhat surprising, but it is likely that there is some heterogeneity in B220 expression levels among cells that are poorly responsive toward antigenic stimulation.