No abstract

Singapore IL-10-producing regulatory B cells have been identified in mice and shown to downregulate inflammation, making them potentially important for maintenance of tolerance. In this study, we isolated B cells from human blood and spleen, and showed that after a short period of ex vivo stimulation a number of these cells produced IL-10. The IL-10-producing B cells did not fall within a single clearly defined subpopulation, but were enriched in both the memory (CD27 CD25À T-cell proliferation in vitro by a partially IL-10-dependent mechanism.These findings imply that manipulating IL-10 production by human B cells could be a useful therapeutic strategy for modulating immune responses in humans. 2686 Frontline Results and discussionIdentification of IL-10-producing B cells in human blood and spleen B cells in mice express IL-10 following stimulation with LPS or CpG, which bind TLR4 and TLR9, respectively [10,15]. Human B cells do not express TLR4 but do express TLR9. CpG-B 2006 is a ligand for human B-cell TLR-9 that induces IL-10 production [16][17][18]. We tested the ability of human B cells to produce IL-10 after a short stimulation, which decreased the likelihood of selective B-cell subset's expansion or surface molecule changes, which would not reflect the physiological condition. After stimulating purified B cells with CpG-B, PMA and ionomycin for 5 h, the frequency of IL-10-producing B cells from blood was 1.8% (n 5 10) and from spleen 1.1% (n 5 3) (po10 À6 and po0.05, compared with isotype control, respectively) (Fig. 1A). The combination of CpG-B with PMA and ionomycin was the most potent stimulator of IL-10 production, when compared with the other stimuli tested (anti-Ig, preplated CD40L transfected L cells or LPS, data not shown). The observed frequency of IL-10-producing B cells in blood differs from that in other studies [19][20][21][22], most likely because of the short-time stimulation system and the use of an intracellular cytokine assay instead of a cytokine secretion assay. In addition, our strategy employed a perfectly matched isotype control (same Fc fragment for the IL-10 and the control Ab from Miltenyi Biotec) to increase the specificity of IL-10 1 and IL-10 À B-cell discrimination, as opposed to the use of nonactivated B cells as a negative control which is prone to false positives. For the following phenotypic and functional experiments, only blood B cells were used due to the difficulty in obtaining sufficient spleen samples. CpG-B1anti-Ig stimulation triggers maximal IL-10 production by human blood B cells in vitroWe assessed the ability of different stimuli to induce maximal IL-10 production from purified B cells. Our cell selection method resulted in a 98% pure B-cell population (Fig. 1C, left Fig. 2B). This implies that other factors such as cell to cell contact may play a role in the effect observed, for example CD80 or CD86 interactions as suggested recently [23]. Using directly purified B cells activated with CpG-B 1anti-Ig, we also observed a high level of B-cell proliferati...