The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4+ conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only about 50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally-occurring Treg (nTreg) or induced Treg (iTreg) in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4β7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4β7 is dependent on activation of an mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, since specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3, did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [C] 2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes.

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PurPose: Tolerance induction to allografts in reconstructive transplantation remains an elusive goal, partly due to limitations in understanding the interplay between inflammatory mediators and activation/regulation of T lymphocytes. We believe specific inflammatory mediators contribute to the activation of alloreactive T cells via previously unappreciated mechanisms: i), direct costimulation, ii) simultaneous reduction of Treg suppressive activity, thereby limiting the effectiveness of tolerogenic regimens. We aimed to identify these inflammatory mediators that diminish T cell regulation and identify strategies to blunt their effect. Methods:In vitro assessment of inflammatory mediators and Treg activity on T cell activation and proliferation was determined via CFSE-suppression assay. The expression of 22 cytokines in the supernatant of maturing dendritic cells was quantified using Luminex. Bioinformatics analysis was used to determine which cytokines most significantly contributed to modulation of Treg suppression, and resultant cytokines were then tested in the CFSE-suppression assay. The immunomodulatory activity of specific cytokines in vivo was tested in a mouse model of inflammatory bowel disease (outcome measure=weight loss). Specifically, Rag-knockout mice were adoptively transferred with T cells (+/-Tregs), followed by injections of anti-IL-6R mAb or IL-1R antagonist (IL-1RA). To further dissect the role of IL-1 family members (e.g. IL-18), the experiment was repeated with T and Treg from MyD88 knockout mice. Production/accumulation of inflammatory cytokines in various tissues was also investigated after orthotopic hindlimb transplantation (OHTx) using Luminex at various timepoints. results: Bioinformatics identified IL-6 and IL-1α/β as potential modulators of Tcell/Treg activity. In vitro analysis confirmed that all three cytokines directly promoted T cell proliferation; however, IL-1α/β also directly inhibited Treg suppressive activity. In vivo analysis in Rag-/-mice confirmed that blockade of IL-6R after adoptive transfer of Tcells/Treg resulted in significantly delayed weight loss in this model (not observed without Treg injection), suggesting that blockade of IL-6 signaling sufficiently slowed activation of T cells permitting Treg to effectively control disease development. Unexpectedly, blockade of IL-1R did not delay disease progression, likely due to redundant function of IL-1 with IL-18. Therefore, the experiment was repeated using T and Treg from MyD88-/-mice (IL-1 and IL-18 use MyD88 for intracellular signaling) and only when MyD88 was absent from both injected populations (Tcells and Tregs) was there a significant delay in weight loss, corroborating our in vitro observations that IL-1α/β (and now IL-18) have a counter-regulatory effect on Tregs/Tcells. IL-6, IL-1α/β, and IL-18 were all confirmed to accumulate in various tissues after OHTx--further evidence of their role in the rejection response.ConClusion: Specific inflammatory cytokines promote activation of alloreactive T cells and reduce...

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Interleukin 10 (IL-10) is a fundamental immunoregulatory cytokine. Mice with memory T cells (Tmem)- or regulatory T cells (Treg)-specific depletion of the IL-10 receptor (IL-10R) develop fatal autoimmune diseases. However, no correlation is known between pathological conditions and altered IL-10 signaling in T cells. We investigated the influence of prolonged exposure to the supernatant of mouse maturing dendritic cells, exposed to LPS, on the integrity of the IL-10 signaling pathway in Tmem and Treg. Following 36-48h incubation, a block in phosphorylation of STAT3 in response to IL-10, but not in response to IL-6, was evident. This IL-10-specific unresponsiveness was not associated with lower IL-10R expression. Additionally, this inhibition was not induced by IL-6 or IL-1. Instead, incubation with IFN-β impaired IL-10 signaling in both Tmem and Treg. This effect was not blocked by AKT inhibition, suggesting a role for Jak/STAT or other IFN-β-initiated signaling pathways. In NOD mice, a model of human type-1 diabetes (T1D), diabetes development is linked to high levels of type-I interferons in pancreatic lymph nodes (PNC-LN). We analyzed the IL-10 response in T cells from spleen, PNC-LN, other LNs, and pancreata of NOD mice and discovered that starting at 4 weeks of age, Tmem and Treg in PNC-LN and pancreata display suppressed responses to IL-10. Overall, these data reveal a new molecular mechanism that controls the development of autoimmunity and the pathogenesis of T1D.