Treg Require T-bet to Migrate Into Afferent Lymphatic Vessels.

Abstract

Introduction: Treg are the major T cell subset that migrates from skin to draining lymph nodes (dLN) during an immune response. However, the mechanisms that Treg use to traffic from tissues via afferent lymphatics to lymph nodes are unclear. The transcription factor T-bet is required for both CD4+ T cell and Treg trafficking to inflamed tissues. We hypothesized that T-bet is also required for Treg migration from inflamed grafts into afferent lymphatics and dLN. Methods: BALB/c (H-2d) donor islets were isolated and transplanted to the kidney capsule of streptozotocin-induced diabetic C57BL/6 (H-2b) wild type (WT) recipients. CD4+CD25+ Treg from WT or T-bet KO C57BL/6 were isolated and transferred to allograft recipients, or used for in vitro trans-well assays. Treg cell migration was quantified by 3D-confocal imaging and whole mount tissue staining. Results: T-bet KO nTreg migrated from blood to islet grafts with the same efficiency as WT nTreg, but failed to home from grafts into dLN. Immunohistochemical analysis of islet grafts showed more T-bet KO nTreg remained in grafts compared to WT nTreg, and T-bet KO nTreg migrated less efficiently into lymphatics than WT nTreg. Analysis of nTreg migration from tissues into afferent lymphatic vessels showed that a lower percentage of T-bet KO nTreg migrated into lymphatics compared to WT nTreg. In contrast to nTreg, both WT and T-bet KO CD4+ non-Treg T cells migrated into lymphatics with the same efficiency. Furthermore, the defect in lymphatic migration was characteristic of natural thymus derived Treg (nTreg) but not induced Treg (iTreg). T-bet KO nTreg expressed more CCR4 and CD103 and less CXCR3 and S1P1 than WT nTreg. However, T-bet KO iTreg and WT iTreg expressed similar amounts of chemokine receptors, integrins and adhesion molecules. In vitro migration confirmed that T-bet KO nTreg migrated better toward CCL22, and migrated less toward S1P, compared to WT nTreg. T-bet KO nTreg also migrated toward and adhered better to allogeneic islets and adhered better to E-cadherin than WT nTreg. In contrast CCR5-CCL5 and CCR7-CCL19 migration were similar between WT and T-bet KO nTreg. Conclusions: T-bet is required for efficient migration of nTreg into afferent lymphatic vessels and dLN, and for their full suppressive function. CD4+ non-Treg T cells and iTreg do not employ the same homing mechanisms for lymphatic migration. These unique distinctions between Treg subsets and between Treg and non-Treg provide novel loci for therapeutic manipulation of suppression in tolerance and immunity.