Background
A growing body of evidence shows that induction of long term transplant survival by costimulation blockade (CoB) regimens is impaired by inflammatory responses In particular, multiple studies reported that engagement of toll like receptors (TLR) abrogates the tolerogenic effect of CoB Despite the identification of type-1 interferons (TI-IFN) as mediators of this effect in multiple models, the target population and specific pathway used by TI-IFN to induce this effect remain unknown To better understand how an inflammatory environment, and more specifically IFN-b, could interfere with the induction of transplant tolerance we studied their impact on the immunomodulatory properties of IL-10.
Methods
Mouse bulk T cells were isolated by negative-selection and Tmem and Treg subpopulations identified by flow cytometry Phospho-STAT3 induction (a key signaling step) after IL-10 and IL-6 stimulation in indicated conditions in Tmem and Treg 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 48 h of bystander incubation with IFN-b, Tmem and Treg subpopulations present a dramatic defect in the production of phospho-STAT3 in response to IL-10, but not to IL-6 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 They suggested a role for STAT1 in this process After IFN-b exposure, there is a complete reversal of the STAT1/STAT3 ratio in T cells Using STAT1-KO cells, we show that the absence of STAT1 prevented/reduced IL-10 inhibition by IFN-b in Treg and Tmem, respectively.
Conclusions
Overall, these data reveal a promising new molecular mechanism where IFN-b interfere with IL-10 signaling in T cells via STAT1 Our data suggest that STAT1 exerts cross-competition with STAT3 for IL-10R binding, preventing its phosphorylation and activity A targeted regulation of this mechanisms that counteract IL-10 suppressive functions could be a powerful tool to improve the efficacy of immunomodulatory strategies for transplant tolerance induction.