Biotin
Upon priming by antigens, B cells undergo activation, proliferation, and differentiation into antibody-secreting cells. During thymus-dependent (TD) antibody responses, the proliferation and differentiation of antigen-primed B cells essentially rely on the helper function from CD4+ T cells. Follicular helper T (Tfh) cells constitute a specialized Th subset that localizes in close proximity to B cells and supports B cell proliferation and differentiation through co-stimulatory receptors and cytokines. Impaired Tfh-mediated B cell proliferation and differentiation were observed in patients with immunodeficiency, while overactivation of this process may lead to dysregulated immune responses seen in autoimmune disorders. Here, we describe an ex vivo coculture assay using circulating Tfh cells and B cells isolated from human blood. This method can be used to examine the function of patients' B cells for proliferation, differentiation, and antibody secretion, mediated by the physiological help from Tfh cells.
The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10- producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-? production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27- populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10-producing Bregs.