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Sunil K. Chauhan, Daniel R. Saban, Reza Dana; CCR7-amplified Regulatory T Cells Acquire Maximal Lymph Node Homing and Suppress Corneal Allograft Rejection. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2368.
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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.
Fully disparate corneal grafts from C57Bl/6 (H-2b) mice were orthotopically transplanted onto BALB/c (H-2d) 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 CCR7high or CCR7low 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.
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 CCR7high expressing Tregs (>3-fold in ipsilateral reactive LN) compared to CCR7low Tregs. Only the group that received CCL21-stimulated CCR7high 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 CCR7low expressing Tregs.
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 CCR7high Tregs home more efficiently to the reactive LN of allograft recipients than CCR7low Tregs, and prevent corneal allograft rejection.
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