1. To study the differences in cytokine production between CD25+ and CD25− B cells, we used the TLR this website ligands, Pam3Cys, LPS and CpG stimulating TLR 2, 4 and 9, respectively. The results are summarized in Table 1. The levels of IL-6 in supernatants from CD25+ B cells were significantly higher when compared with

CD25− B cells following stimulation for 12 h with CpG-PS, LPS or Pam3Cys (P < 0.05, respectively). In addition, CD25+ B cells secreted significantly higher levels of INF-γ as well as IL-10 following 72 h stimulation with CpG-PS, LPS and Pam3Cys (P < 0.05, respectively). Finally, CD25+ B cells produced significantly higher levels of IL-4 following 72 h of stimulation with CpG-PS (P < 0.05) when compared with CD25− B cells. The levels of IL-2 and TNF were analysed at the different time points (24 and 72 h); however, no secretion was detected (data not shown). The increased cytokine production after TLR stimulation was not because of a higher proliferation rate within the CD25+ B-cell subset compared with CD25− B cell as we did not detect any difference in the proliferative ability of these cell populations (data not shown). To Trichostatin A investigate if there was any difference in the ability of CD25+ B cells to present antigens to CD4+ T cells, we used a mixed lymphocyte reaction (MLR) as

an alloantigenic stimulation. CD25+ B cells are significantly better at presenting alloantigen

to CD4+ T cells when compared with CD25− B cells (P < 0.05) (Fig. 2). To evaluate if there were any differences in spontaneous immunoglobulin secretion between naïve CD25+ and CD25− B cells, we performed ELISPOT assays detecting IgA, IgG and IgM secreting B cells and found that the frequency of CD25+ B cells secreting immunoglobulins of IgA, IgG and IgM class was significantly increased compared with CD25− B cells (P < 0.05, respectively) (Fig. 3A). To analyse the these ability of CD25+ B cells to produce antigen-specific antibody, mice were immunized with OVA. At day 14 after immunization, we found that the frequency of CD25+ B cells secreting OVA-specific IgM antibodies were significantly (P < 0.01) increased compared with CD25− B cells (Fig. 3B), whereas the difference regarding the IgG response was less pronounced (P < 0.05). The levels of IgA secretion were very low in both groups, and there was no significant difference in the number of IgA OVA-specific secreting cells between the populations. We found that CD25+ B cells migrated significantly better both spontaneously and towards the recombinant mouse chemokine CXCL13 (P < 0.05, respectively) than CD25− B cells (Fig. 4). The number of CD25+ B cells expressing homing receptors was significantly increased compared with CD25− B cells with respect to α4β7, CD62L, CXCR4 and CXCR5 (P < 0.01, and P < 0.05, respectively) (Fig. 5A–D).