Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora

Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model

B cells have been reported to promote grafft rejectfion through alloantfibody productfion. However, there fis growfing evfidence that B cells can contrfibute to the mafintenance off tolerance. Here, we used a mouse model off MHC-class I mfismatched skfin transplantatfion to finvestfigate the contrfibutfion off B cells to grafft survfival. We demonstrate that adoptfive transffer off B cells prolongs skfin grafft survfival but only when the B cells were fisolated ffrom mfice housed fin low sterfilfity “conventfional” (CV) ffacfilfitfies and not ffrom mfice housed fin pathogen ffree ffacfilfitfies (SPF). However, prolongatfion off skfin grafft survfival was lost when B cells were fisolated ffrom IL-10 deficfient mfice housed fin CV ffacfilfitfies. The suppressfive ffunctfion off B cells fisolated ffrom mfice housed fin CV ffacfilfitfies correlated wfith an antfi-finlammatory envfironment and wfith the presence off a dfifferent gut mficrolora compared to mfice mafintafined fin SPF ffacfilfitfies. Treatment off mfice fin the CV ffacfilfity wfith antfibfiotfics abrogated the regulatory capacfity off B cells. Ffinally, we fidentfified transfitfional B cells fisolated ffrom CV ffacfilfitfies as possessfing the regulatory ffunctfion. These findfings demonstrate that B cells, and fin partficular transfitfional B cells, can promote prolongatfion off grafft survfival, a ffunctfion dependent on lficensfing by gut mficrolora

Dendritic cells license regulatory B cells to produce IL-10 and mediate suppression of antigen-specific CD8 T cells

Regulatory B cells (Bregs) suppress and reduce autoimmune pathology. However, given the variety of Breg subsets, the role of Bregs in the pathogenesis of type 1 diabetes is still unclear. Here, we dissect this fundamental mechanism. We show that natural protection from type 1 diabetes in nonobese diabetic (NOD) mice is associated with increased numbers of IL-10-producing B cells, while development of type 1 diabetes in NOD mice occurs in animals with compromised IL-10 production by B cells. However, B cells from diabetic mice regain IL-10 function if activated by the innate immune receptor TLR4 and can suppress insulin-specific CD8 T cells in a dendritic cell (DC)-dependent, IL-10-mediated fashion. Suppression of CD8 T cells is reliant on B-cell contact with DCs. This cell contact results in deactivation of DCs, inducing a tolerogenic state, which in turn can regulate pathogenic CD8 T cells. Our findings emphasize the importance of DC–Breg interactions during the development of type 1 diabetes

Galectin-1 is required for the regulatory function of B cells

Galectin-1 (Gal-1) is required for the development of B cells in the bone marrow (BM), however very little is known about the contribution of Gal-1 to the development of B cell regulatory function. Here, we report an important role for Gal-1 in the induction of B cells regulatory function. Mice deficient of Gal-1 (Gal-1−/−) showed significant loss of Transitional-2 (T2) B cells, previously reported to include IL-10+ regulatory B cells. Gal-1−/− B cells stimulated in vitro via CD40 molecules have impaired IL-10 and Tim-1 expression, the latter reported to be required for IL-10 production in regulatory B cells, and increased TNF-α expression compared to wild type (WT) B cells. Unlike their WT counterparts, T2 and T1 Gal-1−/− B cells did not suppress TNF-α expression by CD4+ T cells activated in vitro with allogenic DCs (allo-DCs), nor were they suppressive in vivo, being unable to delay MHC-class I mismatched skin allograft rejection following adoptive transfer. Moreover, T cells stimulated with allo-DCs show an increase in their survival when co-cultured with Gal-1−/− T2 and MZ B cells compared to WT T2 and MZ B cells. Collectively, these data suggest that Gal-1 contributes to the induction of B cells regulatory function