The transcription factor T-bet endows Treg with properties tosuppress Th1 inflammation. However, the mechanisms by which T-bet controls alloimmune responses are not fully understood. We hypothesized that T-bet regulates Treg distribution and suppressive functions, and uncovered a novel molecular mechanism for Treg migration and stability.

Methods: BALB/c (H-2^d) donor islets were isolated and transplanted to the renal capsule of streptozotocin-induced diabetic C57BL/6(H-2^b) wild type (WT) recipients. Blood glucose was monitored. CD4⁺CD25⁺Foxp3-GFPnatural Treg (nTreg) from WT or T-bet KO C57BL/6 were isolated and adoptively transferred to allograft recipients, or used for in vitro assays. Flow cytometry and qRT-PCR were performed to analyze expression of effector, adhesion and migration molecules.

Results: T-bet KO nTreg failed to prolong islet allograft survival as well as WT nTreg. T-bet KO nTreg suppressed effector T cell proliferation as well as WT in vitro, but failed to prevent antigen-specific CD4 T cell proliferation in vivo. Furthermore, T-bet KO nTreg did not prevent antigen-specific CD4 T cell infiltration into grafts and draining lymph nodes (dLN). Compared with naïve nTreg, T-bet expression was up-regulated by wild type nTreg recovered from the graft, suggesting that T-bet expressed by nTreg played an important role in suppression in the graft. T-bet KO nTreg remained within the graft compared to WT, and failed to traffic to the dLN from the graft afferent lymphatics. T-bet KO nTreg expressed more CCR4 and CD103 than WT nTreg, and in the graft the cognate ligands CCL22, CCL17 and E-cadherin were highly expressed. Imaging analysis showed that T-bet KO Treg remained associated with interstitial ligands and failed to access afferent lymph vessels. T-bet KO nTreg recovered from the graft expressed much less Foxp3 and the suppressor effector molecules IL-10, CTLA4, CD73 and CD39 than WT nTreg. More T-bet nTreg lost Foxp3 expression and became ex-Treg than WT nTreg.

Conclusion: T-bet regulates nTreg distribution through adhesion and migration molecules, resulting in graft retention that ultimately affects Treg stability, which is essential for in vivo suppression to protect islet allografts. These results demonstrate a novel and unique regulation of Treg migration, genetic stability and suppression. These functional and molecular interactions are foci for therapeutic interventions in immunity and tolerance.

CITATION INFORMATION: Xiong Y, Ahmad S, Iwami D, Brinkman C, Bromberg J. T-Bet Regulates nTreg Stability and Suppression in an Islet Allograft Model. Am J Transplant. 2016;16 (suppl 3).

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