Fig. 4. P2 and P4 B cells are more efficient than P1 B cells at suppressing allogeneic CD4⁺ T cell response elicited by allogeneic dendritic cells.

FACS-sorted P1, P2, and P4 B cells from the blood of healthy subjects were stimulated for 48 h with CpG-B and then cocultured for 6 days with CFSE-labeled autologous CD4⁺ T cells stimulated with allogenic dendritic cells (allo-DCs). CD4⁺ T cell proliferation was assessed by measuring CFSE dilution. Percent suppression was calculated using the formula as indicated method section. a, b Representative FACS data (left) and summarized data (right) showing CD4⁺ IFNγ⁺ (a) and CD4⁺ IL-17⁺ (b) T cell suppression by FACS-sorted P1, P2, and P4 B cell subsets. Cells were gated based on isotype antibody staining. c, d Summarized data showing suppression of CD4⁺ T cells proliferation, as well as IFNγ and IL-17 responses by P1, P2, and P4 B cells in the presence or absence of anti-IL-10/IL-10R (c) and anti-PD-L1 (d). FACS-sorted P1, P2, and P4 B cells were stimulated for 48 h with CpG-B, preincubated with anti-IL-10/IL-10R or anti-PD-L1 or isotypes, and then cocultured for 6 days with CFSE-labeled CD4⁺ T cells stimulated with allo-DCs as described in a and b. Data from five independent experiments performed with cells from different healthy subjects (n = 5). Error bars are mean ± SD. One-way ANOVA with Holm–Sidak’s multiple comparisons test (a, b) and two-way ANOVA with Sidak’s multiple comparison test (c, d) were used.