IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses.
ABSTRACT: A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-? production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.
Project description:CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-? production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.
Project description:BACKGROUND:An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B-cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients, the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling. METHODS:B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and sex-matched healthy volunteers were activated via CD40 and BCR, either alone or in combination. RESULTS:In tolerant patients, we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells compared with healthy controls. Furthermore, B cells from tolerant recipients had reduced extracellular signal-regulated kinase signalling after BCR-mediated activation compared with healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells. CONCLUSIONS:Altogether, our data suggest that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.
Project description:CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-?B activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase C?2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of I?B?, which we previously reported leads to NF-?B p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase C?2 and protein kinase C?/?(II), which we have previously reported leads to NF-?B (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.
Project description:Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naïve and memory allogeneic CD4+ and CD8+ T cell proliferation and IFN?, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8+ T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway.
Project description:IL-10 is a potent immunosuppressive cytokine that promotes the differentiation of tolerogenic dendritic cells (DC-10), and the subsequent induction of antigen-specific T regulatory type 1 (Tr1) cells, which suppress immune responses. However, IL-10 acts on multiple cell types and its effects are not solely inhibitory, therefore, limiting its use as immunomodulant. APVO210 is a bispecific fusion protein composed of an anti-CD86 antibody fused with monomeric IL-10 (ADAPTIR™ from Aptevo Therapeutics). APVO210 specifically induces IL-10R signaling in CD86+ antigen-presenting cells, but not in T and B cells. In this study, we tested whether APVO210 promotes the differentiation of tolerogenic DC-10 and the differentiation of antigen-specific CD4+ Tr1 cells in vitro. We compared the effect of APVO210 with that of recombinant human (rh) IL-10 on the in vitro differentiation of DC-10, induction of alloantigen-specific anergic CD4+ T cells, enrichment in CD49b+LAG3+ Tr1 cells mediating antigen-specific suppression, and stability upon exposure to inflammatory cytokines. APVO210 induced the differentiation of tolerogenic DC (DC-A210) that produced high levels of IL-10, expressed CD86, HLA-G, and intermediate levels of CD14 and CD16. These DC-A210 induced alloantigen-specific anergic T-cell cultures (T-alloA210) that were enriched in CD49b+ LAG3+ Tr1 cells, produced high levels of IL-10, and had suppressive properties. The phenotype and high IL-10 production by DC-A210, and the alloantigen-specific anergy of T-alloA210 were preserved upon exposure to the inflammatory cytokines IL-1?, IL-6, and TNF-?. The effects of APVO210 were comparable to that of dimeric rh IL-10. In conclusion, our data demonstrate that APVO210 drives the differentiation of tolerogenic DC and functional alloantigen-specific Tr1 cells in vitro. Since APVO210 specifically targets CD86+ cells, we hypothesize that it will specifically target CD86+ DC to induce Tr1 cells in vivo, and mediate antigen-specific immunological tolerance by induction of tolerogenic DC and Tr1 cells.
Project description:Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.
Project description:In addition to their use as colorants, anthraquinone derivatives have numerous medical applications, for example, as antibacterial and antiinflammatory agents. We confirmed that physcion (an anthraquinone derivative) induces TNF-alpha production by macrophages and increased the expressions of surface molecules (CD40, CD80, and CD86) and major histocompatibility complex (MHC) II. Based on these results, we hypothesized that physcion might induce the maturation of dendritic cells (DCs) to antigen-presenting cells (APCs), and decided to conduct in vitro experiments using bone-marrow-derived DCs (BMDCs). Physcion was not toxic to DCs and increased the expression of surface molecules (e.g., CD40, CD80, CD86, and MHC II) and the production of cytokines (e.g., IL-12p70, IL-1beta, IL-6, and TNF-alpha), but not of IL-10. To confirm that DCs matured by physcion induce T-cell-immune responses, naive CD4+ T cells were treated with physcion-treated DCs or their supernatants. Physcion induced the maturation of DCs, which promoted the polarization of Th1 cells. Our results show physcion-induced DC maturation via TLR4, and that mature DCs promote the differentiation of Th1 cells without affecting the differentiation of Th2 cells. These findings show that physcion has potential use as a treatment for inflammatory diseases associated with Th1/Th2 cell imbalance.
Project description:INTRODUCTION:Clonorchis sinensis is a major parasite affecting the Korea population. Despite the high infection rate and pathogenicity, very few studies have been conducted to investigate the immune responses against the proteins of C. sinensis. METHODS:In this study, in vitro immune response induced by a recombinant 21.6?kDa tegumental protein derived from C. sinensis (rCsTegu21.6) was confirmed in murine dendritic cells and T cells. For the in vivo analysis, each mouse was immunized three times. Total serum IgG and T cell cytokine production were determined by ELISA, while T cell proliferation was detected by a WST (Water-Soluble Tetrazolium salt)-1 assay. RESULTS:In vitro tests indicated that rCsTegu21.6 treatment increased the expression of surface molecules, such as CD40 (77%), CD80 (52%) and CD86 (46%), on murine dendritic cells and the secretion of cytokines (TNF-?, IL-6, IL-1?, IL-10, and IL-12p70). Moreover, co-culturing dendritic cells activated by rCsTegu21.6 with allogenic T cells induced T cell proliferation over time. rCsTegu21.6 also stimulated specific antibody production and cytokine secretion [IL-2, IL-4, and interferon (IFN)-?)] from T cells following immunization in vivo. Notably, rCsTegu21.6 predominantly induced IgG1 production and secretion of the Th2 cytokine IL-4, regardless of the type of adjuvant used. CONCLUSION:These results serve as a foundation for the development of tegumental protein-based vaccines against C. sinensis.
Project description:Natural rubber latex (NRL; Hevea brasiliensis) allergy is an IgE-mediated reaction to latex proteins. When latex glove exposure is the main sensitizing agent, Hev b 5 is one of the major allergens. Dendritic cells (DC), the main antigen presenting cells, modulated with pharmacological agents can restore tolerance in several experimental models, including allergy. In the current study, we aimed to generate DC with tolerogenic properties from NRL-allergic patients and evaluate their ability to modulate allergen-specific T and B cell responses. Here we show that dexamethasone-treated DC (dxDC) differentiated into a subset of DC, characterized by low expression of MHC class II, CD40, CD80, CD86 and CD83 molecules. Compared with LPS-matured DC, dxDC secreted lower IL-12 and higher IL-10 after CD40L activation, and induced lower alloantigenic T cell proliferation. We also show that dxDC pulsed with the dominant Hev b 5 T-cell epitope peptide, Hev b 5(46-65), inhibited both proliferation of Hev b 5-specific T-cell lines and the production of Hev b 5-specific IgE. Additionally, dxDC induced a subpopulation of IL-10-producing regulatory T cells that suppressed proliferation of Hev b 5-primed T cells. In conclusion, dxDC generated from NRL-allergic patients can modulate allergen-specific T-cell responses and IgE production, supporting their potential use in allergen-specific immunotherapy.
Project description:Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-? (IFN-?) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-?B p65 activation and IL-12 and IFN-? production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-? production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.