Figure 4.

Both CD5⁺ and CD5^- are negative for markers of activation, CD80 and CD86, directly ex vivo, which is increased following activation with CD40L and IL-21. CD5⁺ and CD5^- B cells were isolated and activated as previously described, taken at day 0 for purity stain and quantification of CD80 and CD86, or cultured in complete RPMI supplemented with IL-2 alone. At each indicated time point, cells were collected and surface stained for CD19, CD5, CD80, and CD86. For comparison of cell types, data is on gated CD19⁺ cells within the live lymphocyte gate. Representative flow plots for CD80 and CD86 expression at select times is shown in (A). Cell surface CD80⁺, CD86⁺, CD80⁺CD86⁺ or CD80^-CD86^- protein expression over time is shown for CD5⁺ B cells in panel (B) and CD5^- B cells in panel (C). Secreted IgM from CD5^+/- B cells +/- IL-21 and CD40L are shown in panel (D). Data shown are from 3 independent experiments assessing a total of 5 human donors. Significant differences compared to day 0 were determined within each cell type by a one-way ANOVA with a Tukey’s posttest where *p < 0.05, **p < 0.01, and ***p < 0.001. Significance between cell types at a given timepoint was determined with a two-way ANOVA with a Tukey’s posttest where ^ap < 0.05. Significance for IgM accumulation in culture supernatants between activated and non-activated cells was determined with a one-way ANOVA with a Tukey’s posttest where *p < 0.05 and ***p < 0.001.