Supplementary MaterialsSupplementary File. hoc test. Sialic acid can be linked to the penultimate galactose of the complex, biantennary Fc glycan in either 2,3-, 2,6-, or 2,8-conformations. We have previously reported that only the 2 2,6-linked glycoform of sialic acid is biologically active (12). Our previous modeling data on the structural analysis of different Fc sialoforms (11) predicted that the Glu318/Lys340 pocket at the C2CC3 interface was required for the biological activity of 2,6-sial Fc and could uniquely accommodate this glycoform, whereas the 2 2,3-linked sialic acid would be sterically inhibited from fitting into this pocket. To test this prediction, we introduced an E318N point mutation into IgG1 Fc and compared its properties, when 2,6-sialylated, to Nepicastat HCl ic50 wild-type 2,6-sial Fc. Although comparable degrees of sialylation were achieved with the mutant compared with the wild type, only the wild-type sialylated Fc was capable of stimulating IL-33 expression in hDC-SIGN+ BMMs (Fig. S3were euthanized and Treg cells in spleens were analyzed by flow cytometry. Means SEM are plotted; * 0.05; ** 0.01; *** 0.001 determined by Tukeys post hoc test. IVIG- or F241A-Activated Treg Cells Suppress Pathogenic CD4+ T-Cell Responses in Vivo. To evaluate whether Treg-cell expansion in response Nepicastat HCl ic50 to IVIG, sFc, or F241A is able to suppress pathogenic CD4+ T-cell responses, we induced EAE in C57BL/6 wild-type mice by immunization with MOG35C55 peptide emulsified in complete Freund’s adjuvant (CFA). Starting 5 d postinduction, the mice were treated either with PBS, IVIG, or NA-IVIG every 5 d (at 1 g/kg). Clinical scores of EAE showed that mice that received IVIG had significantly reduced clinical scores compared with PBS-treated mice (Fig. 4and and 0.05; ** 0.01; *** 0.001 determined by Tukeys post hoc test. To assess whether the protection from EAE we observed in IVIG-treated animals was specifically mediated through activation and expansion of Treg cells, we tested the protective potential of F241A as a surrogate for IVIG in untreated and Treg cell-depleted mice. Treg-cell depletion was achieved by administration of an anti-CD25 antibody (PC61) 3 d before EAE induction and every 5 d thereafter. Mice treated with F241A (0.033 g/kg) displayed reduced disease severity compared with PBS-treated mice (Fig. 4and and 0.05; ** 0.01; *** 0.001 determined by Tukeys post hoc test. IL-33 Is a Critical Mediator of sFc-Triggered Treg-Cell Activation. We have Nepicastat HCl ic50 previously reported that the up-regulation of FcRIIB on effector macrophages by sialylated Fc critically depends on production and secretion of IL-33 (29). Recent findings have indicated that IL-33 has a positive effect on Nepicastat HCl ic50 Treg-cell stimulation and activation (31, 32) and thereby contributes to the suppression of inflammation in a mouse model of experimental colitis (33). To test the possibility that sFc-induced production of IL-33 may also contribute to Treg-cell stimulation, we confirmed that na?ve CD4+ T cells isolated from C57BL/6 wild-type mice, cultured for 3 d in the presence of anti-CD3 and anti-CD28 antibodies and TGF- to specifically drive Treg-cell differentiation and Rabbit Polyclonal to PARP4 then treated with IL-33, had a synergistic effect on Treg-cell differentiation as well as on Foxp3 expression (Fig. S6), as was previously reported by Schiering and coworkers (33). Moreover, IL-33 induced up-regulation of the IL-33 receptor ST2 on Treg cells. Addition of IL-23 to the Treg-cell culture counteracted the effect of IL-33 (Fig. S6), consistent with IL-23 being a negative regulator of ST2 (33, 34). We next determined whether administration of IL-33 also affects Treg cells in vivo. IL-33 (0.5 g) was given to C57BL/6 wild-type mice daily for 4 consecutive days. On day 5, spleens were analyzed for Treg-cell numbers. IL-33 administration resulted in a significant increase of Treg cells compared with PBS-treated control mice (Fig. 6 and and and and and 0.001 determined by Tukeys post hoc test. Because FcRIIB up-regulation on effector macrophages has been demonstrated to require the presence of basophils (29), we investigated whether basophils also.