IL‐10 produced by activated human B cells regulates CD4⁺ T‐cell activation in vitro

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...

CD24hiCD27+ and plasmablast-like regulatory B cells in human chronic graft-versus-host disease

“…6,7 We evaluated IL-10-producing Bregs in healthy humans, AHSCT recipients without cGVHD, AHSCT patients with active cGVHD, and cGVHD in remission using 2 in vitro stimulation methods (CpG 6 CD40L).…”

“…[2][3][4][5] Our understanding of the function of Bregs in this disease is limited. IL-10-producing B cells have been shown to inhibit CD4 T-cell proliferation, 6 TNF-a and IFN-g production, 21 and monocyte activation 7 in in vitro coculture experiments.…”

“…7 In our previous work, we did not identify a particular IL-10-producing B-cell compartment after CpG sole stimulation but an enrichment of these cells in the CD27 hi and CD38 hi B-cell pools. 6 We evaluated the phenotype of IL-10-producing B cells in healthy individuals, and AHSCT recipients with and without cGVHD after CpG in vitro stimulation using a backgating strategy on IL-10 1 vs IL-10 2 B cells. Sixty-six hours of CpG stimulation induced a slight increase of the CD38 expression and decrease of the CD24 expression by B cells but no significant changes in the proportions of the different B-cell subsets (supplemental Figure 5).…”

“…The exact role of IL-10-producing regulatory B cells (Bregs) in cGVHD is yet to be understood. Bregs have been shown to downmodulate adaptive 6 or innate immune responses 7 in mice and humans. In cGVHD, B cells are essentially recognized as positive regulators of inflammation.…”

B cell immunosenescence: different features of naive and memory B cells in elderly

“…PBMCs were adjusted to 1 9 10 6 /ml in RPMI 1640 medium (Euroclone, Devon, UK) supplemented with 10% heatinactivated fetal calf serum (Euroclone), 1% penicillin/ streptomicin, 10 mM HEPES, and 1 mM L-Glutamin. B lymphocytes were separated from PBMCs by immunomagnetic sorting, as described by Miltenyi et al (1990) For intracellular IL-10 analysis, PBMCs (10 6 cells/ml) were suspended in complete medium with or without CpG-B 2006 (3 lg/ml, Tib Molbiol), PMA (50 ng/mL), ionomycin (1 mM) and monensin (2 mM; eBioscience) in 24-well flat-bottom plates for 5 h, at 37°C (CO 2 5%) (Bouaziz et al 2010). Cells were harvested, washed and stained with anti-IgD FITC , anti-CD19 PerCP and anti-CD27 APC (BD, Pharmingen).…”