3B) These observations suggested that activation of the TLR2 sig

3B). These observations suggested that activation of the TLR2 signaling pathway conferred DCs the ability to diminish T1D in vivo. DCs play a crucial part in activating not only effector T cells, but also Tregs, and previous work has shown that CD4+CD25+ Tregs can be expanded with DCs in vitro and used to treat autoimmune diabetes in vivo 35, 36. Based on our results thus far, we assessed whether TLR2-mediated stimulation in vitro might expand Tregs capable of diminishing T1D see more in vivo. DCs and CD4+CD25+ T cells were purified from 9-wk-old NOD mice and cultured in the presence or absence of P3C. After 6 days, the DCs were depleted

from the culture, and the Tregs were counted and their phenotype assessed. CD4+CD25+ Tregs cultured with DCs and stimulated Selleck BGB324 through TLR2 in vitro had expanded five-fold, whereas cells cultured

in the absence of P3C showed no expansion in culture (Fig. 4A). Consistent with previous observations by others 29, 30, Foxp3 expression was reduced in TLR2-stimulated Tregs, although not completely lost, and surface expression of CD25 was increased (Fig. 4B), although modestly. Expression of CD127 and most notably PD-L1 was also increased on the surface of P3C-stimulated Tregs. Addition of an anti-CD3 antibody to the culture media further promoted the expansion of the Tregs but did not affect them in terms of expression of Foxp3 or CD127 (data not shown). Interestingly, P3C-mediated expansion of Tregs was associated

with IL-10 production and depended on the presence of DCs stimulated through TLR2 (either before or during culture with the Tregs) (Supporting Information Fig. 1). We then assessed the capacity of CD4+CD25+ T cells cultured with DCs and P3C to modulate T1D in vivo. While CD4+CD25+ Tregs cultured with DCs in the absence of P3C could diminish diabetes upon injection into 9-wk-old NOD mice, stimulation through TLR2 significantly ameliorated the tolerogenic function of these cells, which conferred efficient reduction of the disease (Fig. 4C). In sum, exposure of CD4+CD25+ Tregs to DCs stimulated through TLR2 promoted their selleck kinase inhibitor expansion and markedly increased their tolerogenic function in T1D in vivo. Based on our results thus far and our previous observations in virally mediated prevention of T1D 12, we addressed whether TLR2 neutralization in vivo concomitant to LCMV infection of NOD mice might affect the capacity of the virus to prevent autoimmunity. Anti-TLR2 blocking mAbs were administered to prediabetic, 9-wk-old NOD mice along with LCMV and again 5 days later, and development of diabetes was monitored. We observed that LCMV delayed the onset of diabetes but failed to significantly reduce disease incidence when administered to NOD mice in the context of TLR2 blockade (Fig. 5).

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