Purpose

A growing body of experimental evidence shows that engagement of toll like receptors (TLR) can abrogate or revert the tolerogenic effect of “costimulation blockade” in transplantation. Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, the target population and mechanism used to induce this effect remain unknown. To better understand this process we tested the hypothesis that TI-IFN could impact the immunomodulatory properties of IL-10.

Methods

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

Following 48h of bystander incubation with IFN-b, Tmem and Treg cells 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-b 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 inhibition of IL-10 signaling is slowly reversible with the removal of TI-IFN. Microarray and flow cytometry data indicated that this IL-10-specific unresponsiveness was not associated with any reduction of IL-10 receptor 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. IFN-b promotes a reversal of the STAT1/STAT3 protein ratio in T cells that favors a competitive role of STAT1. Using STAT1-KO cells, we show that the absence of STAT1 prevented IL-10 inhibition induced by IFN-b in Treg and Tmem.

Conclusion

Overall, these data reveal a new molecular mechanism whereby IFN-b interferes with IL-10 signaling and suppressive function in T cells. Thanks to its reversibility, targeting this mechanism could be a powerful tool to improve the efficacy of immunomodulatory strategies for transplant tolerance induction.

CITATION INFORMATION: Iglesias M, Arun A, Lam B, Lee W, Raimondi G, Brandacher G. Type-1 Interferon Impairs the Immunoregulatory Activity of IL-10: A Mechanism in the Abrogation of Transplant Tolerance. Am J Transplant. 2017;17 (suppl 3).

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