*Purpose: Induction of long term transplant survival by “costimulation blockade” (CoB) regimens is impaired by inflammatory responses. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, their target population and specific pathway used remain undefined. To better understand how TI-IFN could interfere with the induction of transplant tolerance, we studied their impact on the immunomodulatory properties of IL-10.

*Methods: Full mismatch (Balb/c into C57BL/6) skin transplant recipients received a peri-transplant regimen based on donor specific transfusion (DST) and anti-CD154 mAb (MR-1) +/- anti-IL-10R. Mouse T cells were isolated by negative-selection and Tmem and Treg subsets identified by flow cytometry. IL-10R expression and phospho-STAT3 induction after IL-10 or IL-6 stimulation in T cells were measured via flow cytometry. The gene expression profile of T cell subsets exposed to TI-IFN was assessed by microarray and quantitative PCR analysis. Protein levels were measured by Western Blot.

*Results: Our result showed that IL-10 has a fundamental role in the protective effect of CoB (transplant survival -MST 105d with CoB vs 47d with anti-IL-10R administration). We then studied the impact of T cell exposure to TI-IFN on IL-10 signaling. Following 48h of in vitro incubation with IFN-β, memory T cells (Tmem) and Tregs presented a dramatic defect in the production of phospho-STAT3 in response to IL-10. This effect was very selective, as IL-6 signaling (post IFN-β exposure) induced normal levels of phospho-STAT3. The reduced accumulation of phospho-STAT3 in conditioned cells resulted in the inhibition of the upregulation of mRNAs for LIGHT, Sphk1 and Tarm-1 – three genes we have discovered are induced by IL-10 in T cells. Encouragingly, this effect was slowly reversible with removal of TI-IFN, suggesting a “druggable” pathway. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10R expression or an increase in SOCS (Suppressor of Cytokine Signaling) 1 and 3, nor with reduced STAT3 cytoplasmic availability. Instead, this analysis suggested a novel role for the transcription factor STAT1 in dampening IL-10 signaling. Using T cells from STAT1-KO mice, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-β in Treg and Tmem, supporting our new model.

*Conclusions: Overall, these results highlight the importance of IL-10 in the therapeutic effect of CoB regimens and reveal a new molecular mechanism whereby TI-IFN interfere with IL-10 signaling, and ultimately with regulation of alloreactivity. Identifying a strategy to target this mechanism could provide a valuable aid in the clinical efficacy of CoB-based immunomodulatory strategies.

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