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. 2014 Sep;134(3):634-644.e10.
doi: 10.1016/j.jaci.2014.03.010. Epub 2014 Apr 24.

CCR10 regulates balanced maintenance and function of resident regulatory and effector T cells to promote immune homeostasis in the skin

Mingcan Xia  1 , Shaomin Hu  1 , Yaoyao Fu  1 , Wensen Jin  2 , Qiyi Yi  2 , Yurika Matsui  3 , Jie Yang  1 , Mary Ann McDowell  4 , Surojit Sarkar  1 , Vandana Kalia  1 , Na Xiong  5
Affiliations

CCR10 regulates balanced maintenance and function of resident regulatory and effector T cells to promote immune homeostasis in the skin

Mingcan Xia et al. J Allergy Clin Immunol. 2014 Sep.

Abstract

Background: CCR10 and CCL27 make up the most skin-specific chemokine receptor/ligand pair implicated in skin allergy and inflammatory diseases, including atopic dermatitis and psoriasis. This pair is thought to regulate the migration, maintenance, or both of skin T cells and is suggested to be therapeutic targets for treatment of skin diseases. However, the functional importance of CCR10/CCL27 in vivo remains elusive.

Objective: We sought to determine the expression and function of CCR10 in different subsets of skin T cells under both homeostatic and inflammatory conditions to gain a mechanistic insight into the potential roles of CCR10 during skin inflammation.

Methods: Using heterozygous and homozygous CCR10 knockout/enhanced green fluorescent protein knockin mice, we assessed the expression of CCR10 on regulatory and effector T cells of healthy and inflamed skin induced by chemicals, pathogens, and autoreactive T cells. In addition, we assessed the effect of CCR10 knockout on the maintenance and functions of different T cells and inflammatory status in the skin during different phases of the immune response.

Results: CCR10 expression is preferentially induced on memory-like skin-resident T cells and their progenitors for their maintenance in homeostatic skin but not expressed on most skin-infiltrating effector T cells during inflammation. In CCR10 knockout mice the imbalanced presence and dysregulated function of resident regulatory and effector T cells result in over-reactive and prolonged innate and memory responses in the skin, leading to increased clearance of Leishmania species infection in the skin.

Conclusion: CCR10 is a critical regulator of skin immune homeostasis.

Keywords: Chemokine receptor CCR10; Leishmania species; allergy; dermatitis; immune homeostasis; inflammation; maintenance; migration; regulatory T cells; skin infection; skin-resident T cells.

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Figures

Figure 1
Figure 1
Over-reactive innate response to stimulation in skin of CCR10−/− mice. (A) Ear thickness changes (ΔT) of CCR10−/− and CCR10+/− mice in a DNFB-induced CHS assay. N≥6. (B) Ear thickness changes of CCR10−/− and CCR10+/− mice after the one-time topical application of DNFB on the ear. N≥10. *P<0.05; **P<0.005; ***P<0.001 and NS: no significant difference (applied to all figures). (C) qRT-PCR analysis of RNA isolated from treated ears 3 days after the one-time DNFB application for TNF-α, IL-1β and IL-10. N=5 each. “No” indicates untreated skin samples (n=3). The values are relative levels normalized on β-actin. (D) Ear thickness changes of CCR10−/− and CCR10+/− mice after the one-time topical application of FITC (0.5%). N=21 for CCR10+/− mice and N=19 for CCR10−/− mice. (E) qRT-PCR analysis of RNA isolated from the treated skin 3 days after the one-time FITC-application for TNF–α, IL-1β and IL-10 as in (C). N=4 each. (F) Ear thickness changes of CCR10−/− and CCR10+/− mice after the one-time topical application of TPA. N≥10 for each genotype.
Figure 2
Figure 2
Imbalanced maintenance and dysregulated functions of Treg and Teff cells in the skin of CCR10−/− mice. (A) Flow cytometric (FACS) analysis of skin lymphocyte preparations of CCR10−/− and CCR10+/− mice for EGFP+ total CD3+ T cells (top) and γδT cells (bottom). The number next to each gate is the percentage (%) of the gated cells of total events in the histograph. The CD3highCCR10low cells are Vγ3+ γδT cells (21). (B) FACS analysis of gated skin EGFP+CD3+ T cells of CCR10−/− and CCR10+/− mice for CD4+ and CD8+ subsets. (C) Average percentages of EGFP+ CD8+ and CD4+ cells of skin lymphocyte preparations in CCR10+/− and CCR10−/− mice, calculated by multiplying % of total EGFP+ T cells (A) and % of CD4+ and CD8+ cells of the total EGFP+ T cells (B). (D) FACS analysis of gated skin EGFP+CD4+ T cells of CCR10−/− and CCR10+/− mice for Foxp3+ Treg cells. (E) Average percentages of Foxp3+CD25+ Treg cells of skin CD4+ T cells in CCR10+/− and CCR10−/− mice, calculated from the FACS analysis in (D). N≥10. (F) Average numbers of EGFP+ CD4+Foxp3+, CD4+ and CD4−(CD8+) T cells isolated from skin of CCR10+/− vs. CCR10−/− mice. N≥10. (G) FACS analysis of skin CD4+ T cells of CCR10−/− and CCR10+/− mice for the IL-10+ subset. Average percentages of IL-10+ cells of the EGFP+ or EGFP− CD4+ cells were shown on the left. N=4 each. (H) FACS analysis of skin CD4+ T cells of CCR10−/− and CCR10+/− mice for the IL-17A+ subset. The bar graphs show average percentages of IL-17A+ cells of EGFP+ or EGFP− CD4+ cells (middle) and their MFI for the IL-17A staining (right). N=6 each. (I) Levels of IL-17A production by purified CCR10+/− or CCR10−/− skin EGFP+CD4+CD25− T cells stimulated with IL-2 and coated anti-CD28/anti-TCRβ antibodies in culture. N=4–5. (J) Relative contribution of transferred CCR10−/− vs. CCR10+/− BM cells to the indicated skin T cell subsets in irradiated WT recipient mice. N=5 each. (K) FACS analysis of skin CD4+ T cells of CCR10−/− vs. CCR10+/− BM donor origins in recipient mice for the IL-17A+ cells, presented as in (H). N=5 each.
Figure 3
Figure 3
Treg cell-regulated immune homeostasis is critical for maintenance of CCR10+ memory-like resident T cells in the skin. (A) FACS analysis of T cells of untreated or inflamed skin of CCR10+/− mice for CCR10(EGFP) expression. The inflamed skin was of mice 1 day after DNFB-induced CHS response. The gray lines are of WT cells as negative controls for EGFP. N=2. (B–D) FACS analysis for EGFP on skin T cells in Rag1−/− mice transferred with CCR10+/− or CCR10−/− EGFP− splenic T cells 1 day (B) or 1 month (C, D) after the DNFB-induced CHS. Average percentages of EGFP+ skin T cells of CCR10+/− vs. CCR10−/− donors 1 month after the CHS induction are shown in (D). N=10 each. (E) Representative FACS analysis for molecules associated with memory on skin EGFP+ vs. EGFP− T cells of the CCR10+/− donor T cell origins in Rag1−/− mice 1 day after DNFB-induced CHS. N= 4. (F) FACS analysis of EGFP expression on Teff (top) and Treg (bottom) cells of the skin of Rag1−/− mice 7–8 weeks after they were transferred with EGFP− naïve splenic CD4+ Teff cells only (right) or a mixture of EGFP− naïve splenic CD4+ Teff and Treg cells (left). N=4. (G) FACS analysis for EGFP on skin T cells of one-month old CCR10+/− Scurfy and control mice. N=5.
Figure 4
Figure 4
CCR10 is critical in localization of CCR10+ T cells into the homeostatic skin. (A–B) Flow cytometry of gated CD3+CD8+ T cells isolated from OVA-treated inflamed ear skin (A) and untreated torso skin (B) for the donor-derived OT-I T cells (CD45.1−CD45.2+) (top) and expression of EGFP of the donor OT-I T cells (bottom). (C) Average numbers of EGFP+ CCR10+/− and CCR10−/− OT-1 T cells isolated from the inflamed ear skin and un-affected torso skin based on the analyses of panel B. N=5–6.
Figure 5
Figure 5
Defective resolution of immune memory responses in the skin of CCR10−/− mice. (A) Ear thickness changes of CCR10−/− and CCR10+/− mice in a DNFB-induced memory CHS assay. N≥6. (B) Representative H&E staining of ear sections 3 days after the DNFB-induced memory CHS response (X400). N=4. (C) qRT-PCR analysis of TNF-α, IL-1β and IL-10 transcripts in the treated skin 3 days after the DNFB-induced memory CHS. N=4. (D) Ear thickness changes of Rag1−/− mice transferred with EGFP− CCR10−/− and CCR10+/− splenic T cells in the DNFB-induced memory CHS. N≥6.
Figure 6
Figure 6
Accelerated clearance of L. major infection in skin of CCR10−/− mice. (A) Levels of Leishmania-specific 16S rDNA in the skin of CCR10−/− and CCR10+/− mice at different time-points after infection, determined by qPCR and normalized on mouse β-actin. N=11, 10 and 4 of each genotype for 3 days, 1 and 2 months post the infection respectively. (B) qRT-PCR analysis of TNF-α, IL-1β and IL-10 transcripts in the infected ears of CCR10−/− vs. CCR10+/− on Day 3 post the L. major infection. N=5 each. (C) Representative ear lesions at infection sites in CCR10−/− and CCR10+/− mice 1 month after the L. major injection. (D) Numbers of total, EGFP+ and EGFP− T cells in infected ears of CCR10−/− and CCR10+/− mice 1 month post the L. major infection. N=3 each.
Figure 7
Figure 7
Preferential expression of CCR10 by T cells of the healthy human skin. (A and B) Flow cytometric analysis of gated CD3+CD4+ T cells isolated from the healthy skin (A) and blood (B) of humans for the CCR10 expression. The number in each gate is the average percentage of cells in the gate of total events, expressed as means ± standard errors. N=3 individual samples for each analysis. The isotype control staining for the CCR10 staining was shown in the left panels.
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