Ectopic expression of IL-35Ig by dendritic cells induces tolerance via Arginase 1.

Introduction: Dendritic cells (DCs) are specialized antigen presenting cells playing a central role in determining the outcome of the immune response, forcing naïve T cells into either activation or differentiation into regulatory T cells (Tregs) depending on DC-extrinsic and DC-intrinsic factors, among which are the cytokine milieu and the immunoregulatory enzymes. Belonging in IL-12 family, IL-35 is a potent suppressive heterodimeric cytokine produced by T and B regulatory cells. Indoleamine 2,3-dioxigenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells, contribute to immunoregulation. Objectives: The aim of the study is to explore any possible link between IL-35 and the activity of IDO1 and Arg1 enzymes expressed by DCs. Materials & methods: We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. In vitro, modulation of Ido1 and Arg1 gene expression was evaluated by real-time PCR; in vivo, a negative vaccination strategy exploiting peptide-loaded DC35 was set up in order to induce antigen-specific tolerance in mice subsequently challenged with the same peptide in a delayed-type hypersensitivity (DTH) experiment. In all the experiments, DCs transfected with Ig tail (DCIg) were used as a control of DC35. Results: Unlike Ido1, Arg1 expression was induced in vitro in DC35, and it conferred an immunosuppressive phenotype on those cells, as revealed by a DTH assay. In particular, the loss of IDO1 function in DC35 (Ido–/– DC35) did not modify the unresponsiveness to skin test following wt DC35 administration. On the contrary, Arg1 inhibition in DC35 by the specific catalytic inhibitor nor-NOHA reverted the suppressive response to skin test observed with untreated DC35. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+CD39+, rather than Foxp3+, regulatory T cells. Conclusion: Arg1, but not Ido1, expression in DC35 appears to be an early event responsible for IL-35Ig–mediated immunosuppression observed in DC35-treated mice.