CCR7-amplified Regulatory T Cells Acquire Maximal Lymph Node Homing and Suppress Corneal Allograft Rejection

Abstract

Purpose: : Appropriate homing and migration are the pre-requisites for efficient functioning of regulatory T cells (Tregs). We herein investigated whether ex vivo amplification of Treg expression of CC chemokine receptor 7 (CCR7) would facilitate Treg homing to the reactive lymph nodes (LN) and promote corneal allograft survival.

Methods: : Fully disparate corneal grafts from C57Bl/6 (H-2^b) mice were orthotopically transplanted onto BALB/c (H-2^d) recipient mice. Naïve CD4+CD25+ Tregs (BALB/c) were cultured in a Treg-expansion cocktail (IL2, and CD3/28 Abs) with CCR7 ligand (CCL19 or CCL21). Cultured Tregs were then evaluated for the expression of CCR7, CD62L and CD103 homing receptors using flow cytometry. Tracking dye (PKH26)-labeled CCR7^high or CCR7^low Tregs were adoptively transferred (2x10⁵ cells/mice) to allograft recipients 24h post-transplantation. Peripheral blood and lymphoid tissues were harvested from allograft recipients at day 4 post-transplantation to identify the homing of labeled Tregs by flow cytometry. Graft survival was evaluated by slit-lamp biomicroscopy weekly up to 10 weeks.

Results: : Tregs cultured in the presence of CCL21, but not CCL19 upregulated Treg expression of CCR7 compared to those in media only. Furthermore, compared to Treg-expansion cocktail only, addition of CCL21 upregulated Treg expression of CCR7 (mean fluorescent intensity, MFI: 301 vs 351) and CD62L (MFI: 132 vs 196), and downregulated expression of CD103 (MFI: 768 vs 319). Adoptive transfer of PKH26-labeled Tregs to allograft recipients showed enhanced LN-homing of CCL21-stimulated CCR7^high expressing Tregs (>3-fold in ipsilateral reactive LN) compared to CCR7^low Tregs. Only the group that received CCL21-stimulated CCR7^high Tregs showed a significant increase in the allograft survival rate (87%, n=8/group, p<0.05). No improvement in graft survival was observed in the recipients which received CCR7^low expressing Tregs.

Conclusions: : Our data demonstrate that CCL21 conditioning of Tregs leads to a significant upregulation of CCR7 and CD62L expression, which promotes Treg homing to LN, and downregulation of CD103 which inhibits Treg retention in periphery. These CCL21-treated CCR7^high Tregs home more efficiently to the reactive LN of allograft recipients than CCR7^low Tregs, and prevent corneal allograft rejection.

Keywords: cornea: basic science • transplantation • immunomodulation/immunoregulation