Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice
Michio Tomura, Tetsuya Honda, Hideaki Tanizaki, Atsushi Otsuka, Gyohei Egawa, Yoshiki Tokura, Herman Waldmann, Shohei Hori, Jason G. Cyster, Takeshi Watanabe, Yoshiki Miyachi, Osami Kanagawa, Kenji Kabashima
Michio Tomura, Tetsuya Honda, Hideaki Tanizaki, Atsushi Otsuka, Gyohei Egawa, Yoshiki Tokura, Herman Waldmann, Shohei Hori, Jason G. Cyster, Takeshi Watanabe, Yoshiki Miyachi, Osami Kanagawa, Kenji Kabashima
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Research Article

Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice

Abstract

Tregs play an important role in protecting the skin from autoimmune attack. However, the extent of Treg trafficking between the skin and draining lymph nodes (DLNs) is unknown. We set out to investigate this using mice engineered to express the photoconvertible fluorescence protein Kaede, which changes from green to red when exposed to violet light. By exposing the skin of Kaede-transgenic mice to violet light, we were able to label T cells in the periphery under physiological conditions with Kaede-red and demonstrated that both memory phenotype CD4+Foxp3– non-Tregs and CD4+Foxp3+ Tregs migrated from the skin to DLNs in the steady state. During cutaneous immune responses, Tregs constituted the major emigrants and inhibited immune responses more robustly than did LN-resident Tregs. We consistently observed that cutaneous immune responses were prolonged by depletion of endogenous Tregs in vivo. In addition, the circulating Tregs specifically included activated CD25hi Tregs that demonstrated a strong inhibitory function. Together, our results suggest that Tregs in circulation infiltrate the periphery, traffic to DLNs, and then recirculate back to the skin, contributing to the downregulation of cutaneous immune responses.

Authors

Michio Tomura, Tetsuya Honda, Hideaki Tanizaki, Atsushi Otsuka, Gyohei Egawa, Yoshiki Tokura, Herman Waldmann, Shohei Hori, Jason G. Cyster, Takeshi Watanabe, Yoshiki Miyachi, Osami Kanagawa, Kenji Kabashima

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Figure 4

Enhanced ear swelling response by Treg depletion and immunosuppressive activity of Treg subsets on T cell proliferation in vitro.

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Enhanced ear swelling response by Treg depletion and immunosuppressive a...
(A) The number of Tregs in the LNs after administration of Campath-1G Ab. (B) CHS: the Kaede/Foxp3hCD2/hCD52 mice were sensitized, and injected with vehicle or Campath-1G Ab before challenge (n = 8 for each group). (C–F) Immunosuppressive activity of Tregs. Kaede-red and Kaede-green Tregs were sorted from the Kaede/Foxp3hCD2/hCD52 mice, sensitized, challenged, and photoconverted. (C) Skin DLN cells of mice sensitized with DNFB were stimulated with DNBS in the presence or absence of Kaede-red Tregs or Kaede-green Tregs in vitro (n = 3). (D) Suppressive effect of Tregs in vitro. Kaede-red and Kaede-green Tregs were prepared as above and added to T cells stimulated with plate-bound anti-CD3 Ab. (E) Antigen specificity of Treg functions. LN cells from DNFB-sensitized or TNCB-sensitized mice were stimulated with DNBS or TNBS in vitro. Kaede-red and Kaede-green Tregs were added, and percentage inhibition of cell proliferation was evaluated as follows: (cell proliferation with DNBS or TNBS) – (cell proliferation with DNBS or TNBS in the presence of Tregs)/(cell proliferation with DNBS or TNBS) – (cell proliferation with vehicle) × 100. (F) Quantitative RT-PCR analysis on mRNA for Il10 (IL-10), Tgfb1 (TGF-β), and Ctla4 (CTLA-4) of Kaede-red Tregs and Kaede-green Tregs. The expression of each gene was normalized by the expression of Gapdh, and those in Kaede-green non-Tregs were normalized to 1 (n = 3). Data are representative of 3 independent experiments and presented as means ± SD (A–F). *P < 0.05 between the indicated groups (Student’s t test, A, B, E, and F; 1-way ANOVA followed by Dunnett multiple comparison test, C and D).