O038 Role of human tolerogenic dendritic cell derived IL-35 in regulating T cell responses

Abstract

Introduction IL-35 is a novel cytokine of the IL-12 family, existing as a heterodimer of IL-12p35 and EBV-induced gene 3 (Ebi3). IL-35 is produced by natural and inducible regulatory T cells in humans and mice and seems required for optimal suppression. Dexamethasone (Dex) and 1, 25-dihydroxyvitamin D3 (D3) have previously been shown to inhibit IL-12 production in activated dendritic cells (DC) and suppress allogenic T cell responses. Tolerogenic DCs are potentially useful to specifically alleviate unwanted immune responses and induce immune tolerance in transplantation and autoimmunity. In this study, we investigated the regulation of production of various IL-12 family members in human tolerogenic DC (tDC), with a main focus on IL-35. Methods Monocytes were isolated from human PBMC and cultured for 6 days in the presence of IL-4 and GM-CSF to obtain immature DC. Tolerogenic dendritic cells were generated using the same culture conditions but with the addition of Dexamethasone for DexDC, or Dexamethasone with 1, 25-dihydroxyvitamin D3 for D3Dex. RT-PCR quantified IL-12A, IL-12B, IL-27A, IL-23A, EBI3 and IL-10. ELISA was used to measure IL-12p40, IL-12p70, IL-10 and IFN- γ in cellular supernatants. Intracellular expression of IL-12p35, Ebi3 and IL-12p40 was measured by flow cytometry. Expression of IL-12p35 and Ebi3 was evaluated using confocal microscopy and western blot. For T cell suppression assays naive CD4 + T cells were isolated form cord blood or PBMCs and stimulated with anti-CD3/28 in the presence or absence of DC derived supernatants. Proliferation was measured by 3 H- thymidine incorporation. Results We demonstrate by Q-PCR that, although tDC completely lack the expression of IL-12p40, they maintain mRNA expression of Ebi3 and IL-12p35. In line with this, tDC do not produce bioactive IL-12p70 or the homodimer IL-12p40. Using intracellular FACS and western blot we show that tDC maintain the protein expression of both Ebi3 and IL-12-p35. Expression of these proteins can be further enhanced upon stimulation with pro-inflammatory cytokines, TLR agonists and CD40 ligation. Functional inhibition studies show that tDC derived supernatants have the ability to suppress T cell proliferation. At present we are performing blocking experiments in tDC supernatants to fully evaluate the extent at which IL-35 contributes to the tolerogenic characteristic these cells possess in regulating T cell responses. Conclusion Taken together, our results suggest that aside from increased expression of previously described tolerance inducing markers (B7-H1, ILT3, etc.) and increased IL-10 production, tDC produce IL-35, providing an additional novel mechanism by which these cells elicit their tolerogenic potential.