Intracellular staining was carried out using a cytofix/cytoperm kit according to the manufacturer’s instructions (BD Biosciences). Cell suspensions were acquired with an LSR-II flow cytometer (BD Cytometry Systems). Analysis was carried out using FlowJo software (TreeStar, San Carlos, CA). Using Prism 4 software (GraphPad Software Inc., San Diego, CA), comparisons of selleckchem statistical significance between groups were assessed using the Mann–Whitney U-test. In inflammatory environments, recruited leucocytes may have emergent properties that are dependent on multiple local interactions with many different soluble signalling molecules. In EAU, accumulating Mϕ, derived from BM cells, infiltrate inflammatory sites in large numbers

and perform as professional APCs. They interact with T cells, both enhancing and regulating immunity. We have demonstrated that the Mϕ that accumulate in the target organ modify T cell responses, suppressing T cell proliferation but preserving cytokine secretion.10 These Mϕ express cell surface markers such as Gr1 and CD31 that are associated

with immune regulation, and to investigate AZD0530 in vivo the function of such cells, we generated Mϕin vitro from BM cells cultured in an inert environment (hydrophobic PTFE-coated tissue culture bags). We compared the ability of these cells to present antigen with other APCs. The OVA323–339-specific TCR transgenic OT-II CD4+ T cells were co-cultured with different populations of professional APCs in the presence or absence of cognate OVA peptide. Wild-type (WT) splenocytes, B cells and dendritic cells stimulated peptide-specific T-cell proliferation, but BM-Mϕ did not (Fig. 1a). To address whether this was the result of a failure of Mϕ to interact with T cells, we analysed other markers of T-cell activation. Despite

the lack of proliferation, we observed that, following co-culture with BM-Mϕ, OT-II T cells adopted an activated cell surface Fenbendazole phenotype and expressed high levels of CD69, CD44 and CD25 (Fig. 1b). The OT-II T cells activated by Mϕ also produced high levels of IFN-γ, the production of which was shown to be independent of TNFR1 signalling as BM-Mϕ derived from TNFR1 knockout (TNFR1−/−) mice stimulated T cells to produce similar amounts of IFN-γ. Interferon-γ activates Mϕ, which in turn leads to autocrine TNF-α signalling that further mediates Mϕ activation.11 Blocking Mϕ activation by neutralizing IFN-γ or TNF-α by the addition of anti IFN-γ mAb or sTNFR1-immunoglobulin fusion protein restored peptide-dependent T-cell proliferation (Fig. 1d), supporting our previous data that the regulation of T-cell proliferation by myeloid cells in the target organ during autoimmunity is dependent on the activation of myeloid cells by IFN-γ and TNF-α.10 Consistent with these in vitro blocking studies, TNFR1−/− Mϕ stimulated T-cell proliferation across a range of peptide concentrations, whereas WT Mϕ stimulated little proliferation (Fig. 1e).