| Dendritic cells (DCs), the most potent professional antigen-presenting cells (APCs), have the unique capacity to integrate a wide array of incoming signals and convey them to lymphocytes, directing the appropriate immune responses. It is well known that immature DCs (imDCs) undergo rapid maturation following microbial infection or transplantation. Mature DCs lose endocytic activity, upregulate the surface expression of adhesion and costimulatory molecules, secrete proinflammatory cytokines and initiate primary T cell-mediated immune responses. In recent years, more and more studies have shown that DCs are heterogeneous cell population, with many kinds of DC subsets exhibiting different phenotype and functions. Among the DCs subsets, one population of DCs with negative immune regulation, so-called regulatory DCs, attracts much attention, and more evidences show that the regulatory DCs negatively regulate immune response by promoting naive CD4+ and CD8+ T cells to differentiate into IL-10-producing T regulatory/suppressor cells or inducing a preferential Th2 response. However, the machanisms by which regulatory DCs negatively regulate immune response remain to be fully understood to date.Our previous studies have shown that splenic stromal cells, mimicking the secondary lymph organ microenvironment, can drive mature DCs to proliferate and differentiate into a novel subtype of regulatory DCs (diffDCs), which express a higher level of IL-10 but minimal IL-12p70 and inhibit antigen-specific T-cell proliferation. Up-regulation of extracellular signal-regulated kinase (ERK1/2) activation was shown to be responsible for IL-10 preferential production, and suppression of p38 activation was for impaired IL-12p70 production in diffDCs. diffDCs can interact with other immune cells in physiology and pathology conditions. For example, diffDCs can active natural killer (NK) cells through diffDC-derived IL-10 and selectively recruit more Th1 cells through diffDC-derived IP-10 and inhibit Th1 proliferation. We have identified an in vivo counterpart of diffDCs in the spleen with similar phenotype and functions.B cells positively regulate immune responses through antibody production and optimal CD4+ T-cell activation. However, B cells possess additional immune functions, including the production of cytokines and the ability to function as APC. B cell development encompasses a continuum of stages that begin in primary lymphoid tissue; with subsequent functional maturation in secondary lymphoid tissue. Also, regulatory B cells have been recently identified and play important roles in the immune hematostasis. These findings suggest that secondary lymphoid tissue may be potential locations for diffDC-B cell interaction. However, whether diffDCs and B cells interact in these locations and what the consequence of these interactions may be have not been elucidated.Up to now, little is known about how regulatory diffDCs modulate the B cells function in the late period of the immune response. Therefore, in this study, we have two parts to investigate the effects of diffDCs on the function of B cells and then we further investigated the underlying mechanisms for the regulation of B cells by diffDCs.1. Regulatory dendritic cells (diffDCs) induce freshly isolated splenic B cells to differentiate into a novel subtype of regulatory B cell characterized by the ability to preferentially produce IL-10.The primary aim of this part of our study is to investigate whether diffDCs can interact with B cells and what the consequence of these interactions.Firstly, we analyzed the proliferation, apoptosis, antibody secretion and cytokine production by B cells after interaction with imDCs, maDCs or diffDCs. The proliferation and apoptosis of B cells were not significantly affected after interaction with imDCs, maDCs or diffDCs. Moreover, no difference in IgM and IgG2b production was detected between B cells after interaction with diffDCs and B cells after interaction with imDCs or maDCs. On the other hand, by screening cytokine production in B cells cocultured with diffDCs, we found there was no significant difference in production of IL-12, IFN-γ, IL-6, IP-10, PGE2 and TGF-βin B cells cocultured with diffDCs and cultured alone. Interestingly; B cells cocultured with diffDCs compared with B cells cocultured with imDCs or maDCs produced higher levels of IL-10 at varying ratios of DC: B. The ratio of 1:10 DC to B was closer to the ratio of DC to B in vivo. Therefore, cells were cocultured at the ratio of 1:10 DC to B in the following experiments. Furthermore, IL-10 production by B cells cocultured with diffDCs was increased gradually and reached a maximum after 48 hours of coculture and the increased IL-10 secretion was mainly from B cells as demonstrated by intracellular staining. Taken together, these results suggested that regulatory diffDCs can induce splenic B cells to differentiate into regulatory B cells with preferential IL-10 secretion.To investigate whether diffDCs could affect the expression of membrane molecules on B cells, experiments were performed in which the B-cell phenotype was analyzed by flow cytometry both in freshly isolated B-cell populations and in B cells that had been cultured for 48 hours with diffDCs. After culture with diffDCs, B cells displayed increased expression of CD19, CD62L and CD16/CD32. Available anti-CD16/CD32 monoclonal antibody recognizes activating receptors FcγRIII (CD16), FcγRIIa (CD32a) and inhibitory receptor FcγRIIb (CD32b). To investigate which receptor is overexpressed on CD19hiCD16/CD32hi B cells, FcγRIIb expression in splenic B cells and sorted CD19hiCD16/CD32hi B cells at mRNA level was analyzed. The FcγRIIb mRNA expression in CD19hi B cells was up-regulated more significantly than that in splenic B cells .These data suggested that it is FcγRIIb that is overexpressed on CD19hi B cells.We then wondered whether the CD19hiFcγRIIbhi B cells produce preferentially IL-10 than B cells. After cocultured with diffDCs, B cells could be divided into two subpopulations: CD19loFcγRIIblo and CD19hiFcγRIIbhi. We sorted CD19loFcγRIIblo and CD19hiFcγRIIbhi populations and stimulated them with polyI:C,LPS and CpG ODN for 24 hours respectively, then assayed IL-10 production in the supernatants. The results showed that sorted CD19hiFcγRIIbhi populations secreted significantly higher IL-10 production than CD19loFcγRIIblo populations or splenic B cells. Furthermore, LPS could stimulate CD19hiFcγRIIbhi populations to produce more IL-10 protein than for CD19loFcγRIIblo populations or splenic B cells. However, IL-10 production in CD19hiFcγRIIbhi populations could not be enhanced by stimulation with CpG ODN or polyI:C. Together, these results demonstrated that diffDCs can induce an IL-10-secreting B cell population with CD19hiFcγRIIbhi phenotype.In summary of this part of our study, we demonstrate that diffDCs induce splenic B cells to differentiate into a novel subtype of regulatory B cells (Bregs) with high CD19, FcγIIb and CD62L expression and high IL-10 secretion.2. The functions of CD19hiFcγIIbhi B cells and mechanisms underlying differentiation of CD19hiFcγIIbhi B cells driven by regulatory diffDCs. The primary aim of this part of our study is to investigate the functions of CD19 hiFcγIIbhi Bregs and the detailed mechanisms of CD19hiFcγRIIbhi Bregs differentiation and identification of an in vivo counterpart of these Bregs.As a very important immune regulatory molecule, FcγRIIb is overexpressed on the IL-10-producing Bregs identified in our system. It has been shown that Fc receptor mediates effective internalization of Ag-IgG complexes. To investigate whether FcγRIIb is involved in the phagocytosis of immune complex (IC), B cells and Bregs were incubated with FITC-OVA-IC, we found that Bregs had enhanced phagocytic capacity compared with that of B cells by FACS and immunofluorescence microscopy. To further confirm FcγRIIb mediates phagocytosis of FITC-OVA-IC by Bregs, FcγRIIb-/- Bregs were generated and then used to incubate with FITC-OVA-IC. The results showed that the ability of FcγRIIb-/- Bregs for the uptake of FITC-OVA-IC as determined by FACS significantly decreased as compared with that of CD19hiFcγRIIbhi Bregs. In addition, CD19hiFcγRIIbhi Bregs significantly inhibited maDC-induced CD4+ T cell proliferation compared with B cells, without depressing IL-2 or IFN-γsecretion. Our results indicated that CD19hiFcγRIIbhi Bregs can regulate immune response by inhibiting CD4+ T cell proliferation with the enhanced phagocytic capacity.To determine the differentiation process of CD19hiFcγRIIbhi Bregs depends on soluble mediators released by diffDCs or direct contact with diffDCs, we performed the experiments both in cell-cell contact and in transwell systems. Our data demonstrated that IL-10 production in CD19hiFcγRIIbhi Bregs depended on soluble mediators and also cell-cell contact. As reported previously by us, diffDCs secrete considerable amounts of IL-10, IL-6, IP-10, NO and IFN-β. We generated IL-10-/- diffDCs or used specific neutralizing mAbs and the selective NO synthase inhibitor dihydrobromide (1,4 PBIT) to test the role of these soluble mediators secreted by diffDCs in inducing IL-10-producing B cells, we found that antibody blockade of IFN-βand the NO synthase inhibitor PBIT partially inhibited IL-10 production by B cells induced by diffDCs. As reported that CD40L-CD40 pathway is an important pathway in DCs to B cells contact. CD40-/- B cells were generated to coculture with diffDCs. Surprisingly, absence of CD40 on B cells impaired their ability to produce IL-10, indicating that CD40 is also involved in the differentiation of CD19hiFcγRIIbhi Bregs. In contrast, B7H1 blockade had little effect on B-cell IL-10 production induced by diffDCs. Together, these data suggest that both diffDC-B direct contact in a CD40L-CD40 dependent way and diffDC-derived IFN-βand NO play critical roles in CD19hiFcγRIIbhi Bregs differentiation. Considering the above data are just based on the differentiated CD19hiFcγRIIbhi Bregs in vitro, we next investigated whether the natural counterpart of CD19hiFcγRIIbhi Bregs exist in vivo. We analyzed CD19+ B cells in spleen and lymph node of mice on the basis of the phenotype of CD19hiFcγRIIbhi, and only about 4.30% of CD19+ B cells were CD19hiFcγRIIbhi B cells, which were similar to CD19hiFcγRIIbhi B cells induced by diffDCs in vitro with respect to cytokine profile, phagocytic capacity and inhibition of CD4+ T cell proliferation. These results indicate that the natural counterpart of CD19hiFcγRIIbhi Bregs do exist in vivo.In conclusion, we show that diffDCs can induce freshly isolated splenic B cells to differentiate into a distinct regulatory B subset, CD19hiFcγIIbhi Bregs, which preferentially secret IL-10 and have regulatory functions in vitro and in vivo. These Bregs inhibit maDCs-initiated the proliferative response of antigen-specific CD4+ T cells. Also, CD19hiFcγIIbhi Bregs have enhanced phagocytic capacity as compared with that of B cells, and FcγRIIb mediates the uptake of IC by CD19hiFcγIIbhi Bregs. diffDC-derived IFN-β, NO and CD40L/CD40 interaction between diffDC/B cells play critical roles in the differentiation of CD19hiFcγIIbhi Bregs. Our results suggest that the regulatory DCs provide a new manner for negative feedback control of T cell immune response and maintenance of immune homeostasis by, at least partially, inducing differentiation of B cells into regulatory B cells. |
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