Regulatory T Cells Require CCR6 for Skin Migration and Local Suppression of Vitiligo

Abstract

Vitiligo is an autoimmune skin disease caused by melanocyte-targeting autoreactive CD8+ T cells. Regulatory T cells (Tregs) have been implicated in restraining vitiligo severity in both mouse models and human patients; however, whether they must be present in the skin for their suppressive function is still unclear. We observed uneven distribution of Tregs within different anatomical locations of mouse skin, which correlated with reduced depigmentation after vitiligo induction. We specifically depleted Tregs in our mouse model of vitiligo and observed increased disease. Next, we found that Tregs contact CD8+ T effector cells in vitiligo lesional skin and that Treg recruitment to the skin inversely correlated with disease severity, suggesting a critical role for Treg suppression within the skin. When we investigated the signals facilitating Treg migration to the skin, we found that although CXCR3 was dispensable for Treg migration and function in vitiligo, Tregs lacking CCR6 exhibited a reduced capacity to migrate to the skin and suppress depigmentation, despite normal systemic numbers in the skin-draining lymph nodes. Our observations highlight a key role for cutaneous Tregs in disease suppression during vitiligo and identify CCR6 as a chemokine receptor that contributes to Treg migration to the skin.

Figure 1:. Tregs suppress depigmentation in a mouse model of vitiligo.

(a) Total vitiligo scores from FoxP3-DTR mice treated with DTX or vehicle; (b) Representative images of ear and tail skin from FoxP3-DTR mice treated with PBS or DTX; (c) disease scores in the ear and tail (7 weeks post disease induction, N=12, two-tailed unpaired t test). (d) Sample flow plots and (e) quantification of Teffs in SDLN and skin of FoxP3-DTR mice treated with DTX (7 weeks post disease induction, N=8, two-tailed unpaired t test).

Figure 2:. Treg number in the skin correlates with disease severity.

(a) Vitiligo scores of the ears and tails of mice with a total score of at least 5 (N=177, two-tailed unpaired t test). (b) Sample flow plots and quantification of Teffs and Tregs in (c) tail skin, (d) ear skin, or (e) SDLNs in FoxP3-GFP mice with vitiligo at the indicated week after disease induction (N=10, bars represent S.E.M).

Figure 3:. Tregs directly contact Teffs in the skin during vitiligo.

(a) Representative en-face florescent microscopy image depicting GFP+ Tregs and RFP+ Teffs in lesional mouse ear skin; white arrows highlight clusters of Tregs and Teffs, 20x image, bar = 50μM. The area defined by the white box is magnified in (b), 20x image, bar = 50 μM. (c) Representative confocal microscopy image depicting GFP+ Tregs and RFP+ Teffs in lesional mouse ear skin, 40x image; bar = 50μM. The area defined by the black box is magnified in (d), 40x image; bar = 10μM. Imaging experiments were performed 5 weeks post disease induction.

Figure 4:. Trees require CCR6 for optimal suppression and migration to the skin in a mouse model of vitiligo.

(a) Ear vitiligo scores and (b) representative images of Rag⁻⁻ mice ears that received no Tregs, WT or CCR6⁻⁻ Tregs (5 weeks post disease induction, N=14, two-tailed unpaired t test). Quantification of (c) Teffs and (d) Tregs in the SDLNs and ear skin of Rag⁻⁻ mice (5 weeks post disease induction, N=8 No Treg, WT→Rag, N=9 CCR6⁻⁻→Rag two-tailed unpaired t test). (e) Quantification of WT and CCR6⁻⁻ Tregs in the SDLNs and ear skin of Rag⁻⁻ mice in a competitive Treg-adoptive transfer model of vitiligo (5 weeks post disease induction, N=8, two-tailed unpaired t test).