Background: The transcription factor T-bet endows Treg with the functional and migratory properties to suppress Th1 inflammation. Previously, we showed that nTreg homing into draining lymph nodes (dLN) and prolongation of islet graft survival depended on T-bet. We hypothesized that nTreg utilized a novel molecular mechanism for homing into dLN to prolong graft survival.

Methods: BALB/c (H-2d) islets were transplanted to the renal capsule of streptozotocin-induced diabetic C57BL/6(H-2b) wild type (WT) recipients. CD4+CD25+ Treg from WT or T-bet knockout (KO) C57BL/6 mice were isolated and transferred to allograft recipients or mouse footpad, or used for in vitro assays. Flow cytometry and qRT-PCR were performed to analyze expression of effector molecules and adhesion and migration molecules. In vitro transwell migration assays toward various chemokines were performed.

Results: T-bet KO nTreg expressed more CCR4 and CD103 and less CXCR3 and S1P1 than WT nTreg. In vitro migration confirmed that T-bet KO nTreg migrated better toward CCL22, adhered better to E-cadherin, and migrated less toward S1P and CXCL10, compared to WT nTreg. T-bet KO nTreg migrated more toward, and adhered better to, allogeneic islets than WT Treg in vivo and in vitro. T-bet KO nTreg expressed more CD103 than WT in islet allografts in vivo. Isolated islets and islet grafts expressed significant amounts of CCR4 ligands CCL22 and CCL17 as well as CD103 ligand E-cadherin, suggesting that these interactions were important for Treg migration. This was confirmed by addition of soluble E-cadherin to Treg, which restored Treg migration toward CCL19 in vitro and homing into dLN after footpad injection. To determine the mechanism whereby graft retention led to poor graft protection by T-bet KO nTreg, Treg suppression of T cell proliferation was assessed. Although both WT and T-bet KO nTreg suppressed effector T cell proliferation in vitro and expressed identical levels of several effector molecules, T-bet KO nTreg failed to suppress antigen specific TCR transgenic CD4 T cell proliferation and infiltration into dLN and graft in vivo.

Conclusion: T-bet regulates migration and adhesion receptors required for Treg homing to dLN, which may be essential for in vivo suppression to protect islet allografts. These results demonstrate a novel and unique regulation of Treg migration and suppression, and the molecular interactions are potential foci for therapeutic intervention in immunity and tolerance.

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