10 This TNFR1-dependent correlation between myeloid regulatory function and NO production is consistent with previous findings in the mouse and rat that have associated NO with the inhibition of

T-cell proliferation.13–15 However, it was not established whether the abrogation of NO production, consequent to the absence of TNFR1, was sufficient to explain the relative increase in CD4+ T cells in EAU and the loss of regulatory function by target organ-infiltrating Mϕ. In addition, Mϕ-like cells, called MDSC,16 isolated under other chronic inflammatory situations, particularly from tumours, and that suppress anti-tumour immune responses, have been described previously.17–19 These cells exhibit a range of characteristics that are unlikely to Roscovitine price be controlled directly by TNFR1 signalling. Here we describe populations of Mϕ, generated in vitro, which can regulate T-cell responses. We show that the critical requirement for TNFR1 expression and signalling relates to the development of a regulatory myeloid cell phenotype, rather than being required for this

LEE011 in vitro regulatory function. Therefore, restoring signals downstream of TNF-α signalling leads to the generation of TNFR1-deficient cells that are competent to inhibit T-cell proliferation. We identify two independent processes that result from TNFR1 signalling that together play a critical role in the control of T-cell responses by Mϕ, which are mediated by IFN-γ and prostaglandin E2 (PGE2) respectively. In cells where the TNFR1−/− signalling

pathway is inhibited, the absence of these signals prevents Mϕ differentiation into myeloid cells that regulate T-cell proliferation. C57BL/6 mice were originally obtained from Harlan UK Limited (Oxford, UK), C57BL/6 TNFR1(p55)-deficient mice (TNFR1−/−) were obtained from The Jackson Laboratory (Bar Harbor, ME), and C57BL/6 OT-II transgenic mice20 expressing the T-cell receptor-specific for chicken ovalbumin323–339 (OVA) and I-Ab were a kind gift of Dr Steve Anderton (Department of Biological Sciences, University of Edinburgh, UK). All mice were housed under specific pathogen-free conditions Ponatinib datasheet with food and water continuously available. In all experiments, female mice aged between 6 and 8 weeks were used. Treatment of animals conformed to the regulations for animal research as set down by the Home Office, UK and also to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The OVA323–339 (ISQAVHAAHAEINEAGR) peptide (Sigma, Poole, UK) was at least 95% pure as determined by HPLC. Complete tissue culture medium was Dulbecco’s modified Eagle’s minimum essential medium (without phenol red) supplemented with 10% volume/volume (v/v) fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2mm l-glutamine, 1 mm sodium pyruvate and 5 μm 2-mercaptoethanol (all from Invitrogen, Paisley, UK).