Treg Require Transcription Factor T-bet to Traffic to Draining Lymph Nodes and Prolong Islet Allograft Survival.

Abstract

Introduction: Foxp3+ regulatory T cells (Treg) are required for tolerance. Treg must sequentially migrate from inflamed tissues to draining lymph nodes (dLN) to suppress the alloimmune response. The Th1 transcription factor T-bet is also essential for Treg to control Th1 inflammation. We hypothesized that T-bet regulates Treg function for prolonging allograft survival, and uncovered novel molecular mechanisms for Treg homing into dLN. Methods: BALB/c (H-2d) donor islets were isolated and islets transplanted to the kidney capsule of streptozotocin-induced diabetic C57BL/6 (H-2b) wild type (WT) recipients. Blood glucose was monitored. CD4+CD25+ Treg from WT or T-bet KO C57BL/6 were isolated and adoptively transferred to allograft recipients, or used for in vitro assays. Fluorescent flow cytometry for CCR2, CCR4, CCR5, CCR6, CCR7, CXCR3, ICAM-1, LFA-1, CD103, PSGL-1 and integrin alphav; and qRT-PCR for S1P1, CCR4, CCR7, TGF-b, IL-10 and CTLA-4, were performed. Purified CD4+CD25+ Treg were migrated across membranes in vitro to the indicated chemokines. Results: T-bet KO Treg failed to prolong islet allograft survival as well as WT Treg (15 vs. 30 days after iv transfer; 19 vs. 37 days after local transfer with islets). There was no difference in suppression of effector T cell proliferation in vitro between T-bet KO vs. WT Treg, nor in expression of suppressor effector molecules. T-bet KO nTreg migrated from blood to islets as effectively as WT nTreg, but failed to traffic to dLN from the graft, whereas WT nTreg homed to dLN. 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, compared to WT nTreg. T-bet KO nTreg migrated toward, and adhered better to, allogeneic islets than WT Treg, both in vivo and in vitro. Conclusions: T-bet regulates important migration and adhesion receptors that are required for Treg homing into dLN. Migration to dLN is required for full in vivo suppression to protect islet allografts. These results demonstrate novel and unique regulation of Treg migration and suppression, and the molecular interactions are potential foci for therapeutic intervention in immunity and tolerance.