P172 Combined computational and experimental analyses of IL-6 and IL-1 reveal distinct mechanisms of action in the modulation of regulatory T cell suppressive activity

Introduction The development of autoimmune diseases, cancer, and transplant outcomes are all influenced by the ability of regulatory T cells (Treg) to suppress the activity of conventional T cells (Tconv). Therefore, rational modulation of Tconv’ susceptibility to suppression by Tregs is highly desirable. However, our understanding of the factors and mechanisms that underlie this modulation is limited. We believe that inflammatory mediators, by contributing to the activation of Tconv while reducing Treg activity, limit the effectiveness of tolerogenic regimens. We aimed to identify principal inflammatory mediators, released by innate immune cells, that lessen T cell regulation and infer their mechanism of action. Methods Using a CFSE-based, in vitro mouse T cell activation system, we quantified Treg suppressive activity and assessed the influence of the supernatant of maturing dendritic cells sampled at multiple times after stimulation with LPS. In parallel, 24 cytokines were quantified using Luminex™ in each sample tested. Principal Component Analysis (PCA) was utilized to delineate likely modulators of Treg suppression; the results were subsequently validated experimentally. Results PCA suggested IL-6, a known modulator of Treg suppression, as one of the cytokines with the most relevant contribution to the variance of the system. Interestingly, the same analysis revealed a similar pattern of variance between IL-1 α and IL-1 β with IL-6 suggesting a modulatory role. The CFSE assay indicated that IL-6 did not directly interfere with the susceptibility of Tconv to suppression (as originally suggested), but counteracted regulation by promoting a higher frequency of proliferating cells (that remained susceptible to suppression). Conversely, IL-1 α and IL-1 β each interfered with Treg suppression by directly affecting their suppressive activity. Using combinations of IL-1R-knock-out Tconv or Treg, we established that this effect was exerted by acting on both cells. As the in vitro activity of Treg is counteracted by IL-2 availability, we next tested if the differential effect of IL-6 and IL-1 correlated with differences in IL-2 secretion by Tconv following activation (in the absence of Treg). While IL-6 did not significantly increase the frequency of IL-2-secreting cells, IL-1 doubled this number to values comparable to those seen upon exogenous costimulation, further underscoring the separate mechanisms of action of these cytokines. In a mouse model of inflammatory bowel disease, preliminary results indicate that blockade of IL-6 signaling delays disease development through inhibition of colitogenic T cells expansion allowing tighter control by Treg. Conclusion Our results suggest that combining computation with experimentation may help to dissect the complex inflammatory milieu that determines T cell susceptibility to suppression. Specifically, this approach identified complementary as well as synergistic activities of IL-6 and IL-1 ( α and β ). Elucidation of the differential intracellular mechanisms used will aid in identifying targets for therapeutic approaches to selectively promote (autoimmunity and transplantation) or prevent (cancer and vaccination) immuneregulation.