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Developmentally programmed early-age skin localization of iNKT cells supports local tissue development and homeostasis

Nature Immunology volume 24, pages 225–238 (2023)Cite this article

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Abstract

Skin is exposed to various environmental assaults and undergoes morphological changes immediately after birth. Proper localization and function of immune cells in the skin is crucial for protection and establishment of skin tissue homeostasis. Here we report the discovery of a developmentally programmed process that directs preferential localization of invariant natural killer T (iNKT) cells to the skin for early local homeostatic regulation. We show that iNKT cells are programmed predominantly with a CCR10+ skin-homing phenotype during thymic development in infant and young mice. Early skin localization of iNKT cells is critical for proper commensal bacterial colonization and tissue development. Mechanistically, skin iNKT cells provide a local source of transferrin that regulates iron metabolism in hair follicle progenitor cells and helps hair follicle development. These findings provide molecular insights into the establishment and physiological functions of iNKT cells in the skin during early life.

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Fig. 1: iNKT cells are highly enriched in the skin of infant and young mice independent of the microbiota.
Fig. 2: A unique subset of CCR10+ NKT2/NKT1 cells dominates in the skin of infant and young mice.
Fig. 3: Preferential thymic generation of CCR10+ skin-homing NKT2 cells at young ages.
Fig. 4: iNKT cells regulate the skin microbiota and tissue homeostasis at young ages.
Fig. 5: Skin iNKT cells highly express transferrin and iron metabolism-related genes.
Fig. 6: Skin iNKT cells regulate local iron metabolism and help hair follicle development.

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Data availability

All data that support the findings of the present study are available from the corresponding authors upon request. Raw fastq files and processed reads of the transcriptional analyses generated during the present study have been deposited in the Gene Expression Omnibus under accession codes GSE205602 and GSE206066. The V4 16S rRNA gene-sequencing data have been deposited as fastq files in NCBI Sequencing Read Archive under BioProject number PRJNA859537. Source data are provided with this paper.

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Acknowledgements

Research reported in this publication was partly supported by the National Institute of Allergy and Infectious Diseases and the National Institute of Arthritis, Musculoskeletal and Skin Diseases of the National Institutes of Health under award numbers U01AI131393, R56AI071043, R01AR064831 and R01AR070887 (to N.X.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank the NIH Tetramer Core Facility for providing PBS57-loaded PE-conjugated CD1d tetramer and 5-OP-RU-loaded PE-conjugated MR1 tetramer. We also thank P. P. Pandolfi (Beth Israel Deaconess Medical Center) and D. Sant’Angelo (Rutgers RWJ Medical School) for providing PLZF-deficient mice and J. McCluskey and A. Corbett (The University of Melbourne) for 5-OP-RU-loaded PE-conjugated MR1 tetramer. We thank the Flow Cytometry Facility at Penn State University and the Flow Cytometry, Bioanalytics and Single-Cell, Genome Sequencing and Computational Biology and Bioinformatics Facilities of University of Texas Health Sciences Center, San Antonio, for technical support and service. We thank K. Restori for critical reading and editing of the manuscript.

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Author notes

  1. Luming Zhao

    Present address: School of Life Sciences, Westlake University, Hangzhou, China

  2. Chang Liao

    Present address: Department of Medicine, University of California San Francisco, San Francisco, CA, USA

Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center San Antonio, San Antonio, TX, USA

    Wei-Bei Wang, Yang-Ding Lin, Micha Davila & Na Xiong

  2. Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, USA

    Luming Zhao, Chang Liao, Micha Davila & Jasmine Sun

  3. Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA

    Yang Zhang

  4. Department of Population Health Sciences, and Greehey Children’s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Yidong Chen

  5. Department of Medicine–Division of Dermatology and Cutaneous Surgery, University of Texas Health Science Center San Antonio, San Antonio, TX, USA

    Na Xiong

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Contributions

W.-B.W., Y.-D.L., L.Z., C.L., Y.Z., M.D. and J.S. performed experiments. N.X. and W.-B.W. designed the study. W.-B.W., Y.-D.L., L.Z., C.L., Y.Z., Y.C. and N.X. analyzed the data. W.-B.W. and N.X. wrote the manuscript. All authors read and commented on the manuscript.

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Correspondence to Na Xiong.

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Nature Immunology thanks Thomas Kupper and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 iNKT cells are highly enriched in the skin of infant and young mice independently of microbiota exposure.

a, Representative FC plots of epidermal γδ T (CD3hiTCRβ-), dermal γδ T (CD3medTCRβ-), and αβ T (CD3medTCRβ+) cells from the skin of WT B6 mice at 3 weeks of age (top) and average percentages and numbers of epidermal and dermal γδ T cells in the skin of mice over time (bottom) (9d: n = 3, 12d: n = 16, 18d: n = 49, 25d: n = 34, 5wk: n = 32; 9–14wk: n = 13). b, Representative FC plots of iNKT (CD1d-tet+), Treg (FOXP3+) and CD8 αβ T cells (CD8+) of the gated skin αβ T cells (CD45+TCRβ+) from WT B6 mice at different ages. c, Representative FC plots of MAIT cells (MR1-5-OP-RU-tet+) of gated skin T cells (CD45+CD3med) at different ages (top). FC plots of MR1-Ac-6-FP tetramer staining are included as negative controls (bottom). d, FC analysis of expression of CD86 and CD8b in skin αβ T cells at 3 weeks of age. Boxplots indicate the value of median (horizontal line), 25th to 75th percentile (box), and min-max range (whisker) of the values. One dot represents one mouse.

Source data

Extended Data Fig. 2 Characterization of surface markers and transcription factors in skin iNKT cells.

a, Histograms of expression of PLZF, T-bet, and ROR𝞬t in skin iNKT cells of adult mice. b, FC analysis of expression of CCR6 and NK1.1 in CD1d-tetramer+ αβ T cells from the skin of adult mice. c, FC analysis of expression of CCR6 and NK1.1 in CD1d-tetramer+ αβ T cells from the skin of 26-day-old SPF versus GF mice. d, Histograms of expression of PLZF, T-bet, and ROR𝞬t in skin iNKT cells of 26-day-old SPF versus GF mice.

Extended Data Fig. 3 Characterization of CCR10+ immune cells in the skin, sLN, and spleen.

a, FC analysis of expression of CCR10(EGFP) in CD1d-tetramer+ αβ T cells from the skin, sLN, and spleen of Ccr10+/EGFP mice of different ages. b, FC analysis of expression of CCR10(EGFP) versus CD8 in MAIT cells from the sLN and skin of 3-week-old Ccr10+/EGFP mice. c, FC analysis of CCR10(EGFP) expression in CD8+ αβ T cells from the sLNs of Ccr10+/EGFP mice at different ages (left). Frequencies of CCR10+ cells in CD8+ αβ T are shown in bar graphs on the right. (9d: n = 2, 20d: n = 35, 4wk: n = 11, 5wk: n = 21; 9–14wk: n = 6). Boxplots indicate the value of median (horizontal line), 25th to 75th percentile (box), and min-max range (whisker) of the values. One dot represents one mouse.

Source data

Extended Data Fig. 4 iNKT cells are programmed with the CCR10+ skin homing property during their thymic developmental processes at early postnatal stages.

a, FC analysis of expression of CCR10 in thymic MAIT cells of different maturation stages in 4−week-old Ccr10+/EGFP mice. b, Representative FC analysis of CD8+CD4- thymocytes (left) for their expression of CCR10 and TCRβ in 2- to 3-week-old WT mice. c, FC analysis of iNKT cells in gated skin T (CD45+TCRβ+CD3+) cells from Cd1d+/− and Cd1d−/− mice at 2~3 weeks of age. d, e, Analysis of iNKT (CD1d-tet+TCRβ+) cells and their expression of CCR10 versus CD103 in the skin (d) and sLN (e) of Plzf+/+ (skin: n = 18, sLN: n = 34 or n = 4), Plzf+/− (skin: n = 25, sLN: n = 41 or n = 7), and Plzf−/− (skin: n = 14, sLN: n = 25 or n = 4) mice at 2~5 weeks of age. All mice used in this figure carry one CCR10-knockout/EGFP-knockin allele (Ccr10+/EGFP) for the purpose of reporting CCR10 expression with EGFP. Boxplots indicate the value of median (horizontal line), 25th to 75th percentile (box), and min-max range (whisker) of the values. Bars represent mean+s.e.m. One dot represents one mouse. Significance was determined by Kruskal-Wallis test with Dunn’s post test. NS, not significant.

Source data

Extended Data Fig. 5 Critical roles of iNKT cells in regulation of skin microbiota and tissue homeostasis at early postnatal stages.

a, Analysis of non-Treg Th (CD1d-tet−FOXP3−CD4+TCRβ+) cells in the skin of Cd1d+/− (n = 7) and Cd1d−/− (n = 9) littermate mice at 2–3 weeks of age. b, Bubble plots of the relative taxonomic abundance of bacteria in the skin of Cd1d+/− (n = 3) and Cd1d−/− (n = 5) littermate mice at 3 weeks of age. The size of each square is scaled logarithmically to represent the relative abundance of bacterial phylum for each indicated skin sample. c, Absolute read counts of indicated bacterial species in the skin of Cd1d+/− (n = 5) and Cd1d−/− (n = 10) littermate mice at 3 weeks of age. d, qRT-PCR analysis of Cramp, S100a7 (Psoriasin), S100a9, Defb3, Defb4, and Defb14 transcripts from the ear skin of 3-week-old Cd1d+/− (n = 3) and Cd1d−/− (n = 4) littermate mice. Relative expression was normalized to expression of Hprt. e,f, Heatmaps of relative expression of genes associated with developmental growth (e) and morphogenesis of an epithelium (f) in the skin of Cd1d +/− (n = 2) and Cd1d −/− (n = 3) littermate mice at 3 weeks of age. Bars represent mean+s.e.m. Short lines indicate means. One dot represents one mouse. Significance was determined by unpaired two-sided Student’s t-test. NS, not significant.

Source data

Extended Data Fig. 6 Comparison of gene expression profiles of skin and splenic iNKT cells of 3-week-old WT mice.

a, Hierarchical clustered heatmap of RNA-seq data comparing transcripts in splenic and skin iNKT cells of 3-week-old WT mice (n = 4). b,c, Antigen recognition capacity of iNKT cells from the skin. Donor iNKT cells were enriched from the thymus of wild type mice at 2–3 weeks of age and adoptively transferred into 1-week-old Cd1d+/+ (n = 4) or Cd1d−/− (n = 4) recipient mice. The skin cells from recipient mice were analyzed 1 week post transfer. Absolute numbers of donor iNKT cells (b) and histograms of expression and mean fluorescence intensity (MFI) quantification of Nur77 in donor iNKT cells (c) from the skin of Cd1d+/+ or Cd1d−/− recipient mice. d, RNA-seq data of expression of Il4 and Ifng between splenic and skin iNKT cells of 3-week-old WT mice (n = 4). e,f, and g, Heatmaps of expression of genes associated with positive regulation of immune response (e), natural killer cell-mediated cytotoxicity (f), and response to hypoxia (g) in splenic and skin iNKT cells of 3-week-old WT mice (n = 4). h, Histogram (left) and MFI quantification (right) of Hif1α expression in splenic and skin iNKT cells of 3-week-old WT mice (n = 4). i, RNA-seq data of expression of indicated genes encoding iron carriers in skin iNKT cells of 3-week-old WT mice (n = 4). Bars represent mean+s.e.m. One dot represents one mouse. Significance was determined by unpaired (b, c, and d) or paired (h) two-sided Student’s t-test. NS, not significant.

Source data

Extended Data Fig. 7 Expression of transferrin and other iron metabolism-associated genes in different subsets of skin T cells and splenic iNKT cells.

a, Expression of iron-associated genes from qRT-PCR analysis of immune cells sorted from the skin of WT mice (n = 4 per group). Relative expression was normalized to expression of Gapdh. b, Expression of iron-associated genes from qRT-PCR analysis of skin iNKT, splenic iNKT, and splenic NK1.1-CD4−CD8− (DN) iNKT cells from 3-week-old mice (n = 4 per group). Relative expression was normalized to expression of Gapdh. c,d, Histograms (left) and MFI quantification (right) of Trf (c) and CD71 (d) in whole and NK1.1- DN iNKT cells from the spleen of 3-week-old mice (n = 4). One dot represents one mouse. Significance was determined by paired two-sided Student’s t-test. NS, not significant.

Source data

Extended Data Fig. 8 Defects of iNKT cells in the skin but not spleens or thymi of Trf hpx/hpx mice.

a, b, Comparison of frequency of iNKT (CD3+CD1d-tet+) and Treg (CD3+FOXP3+) cells in spleens (a) and thymi (b) of Trf+/hpx (d6: n = 6, d12: n = 7) and Trf hpx/hpx (d6: n = 7, d12: n = 4) mice at 6 and 12 days of age. c, Comparison of absolute numbers of DETC (CD3hi TCRβ-), CD8T (CD3+CD8+), and MAIT cells (MR1-5−OP-RU-tet+TCRβ+) in the skin of Trf+/hpx (d6: n = 6, d12: n = 8 for DETC, d12: n = 6 for CD8T, d12: n = 3 for MAIT) and Trf hpx/hpx (d6: n = 3, d12: n = 7 for DETC, d12: n = 4 for CD8T, d12: n = 3 for MAIT) mice at 6 or 12 days of age. d, Histograms of expression of PLZF and T-bet in skin iNKT cells of BALB/c mice at 1–3 weeks of age. e, FC analysis of expression of CCR6 versus ROR𝞬t in skin iNKT cells of Trf+/hpx and Trf hpx/hpx mice at 12 days of age. f, Schematic of Trf administration into Trf hpx/hpx mice. g, Images of Trf +/hpx and Trf-treated Trf hpx/hpx mice at 12 days of age. h, Comparison of absolute numbers of iNKT (CD45+CD1d-tet+) and Treg (CD45+FOXP3+) cells in the skin of Trf+/hpx (n = 5) and Trf hpx/hpx (n = 5) mice after Trf treatment. i,j, Comparison of frequency of iNKT (CD3+CD1d-tet+) and Treg (CD3+FOXP3+) cells in the sLNs (i) and spleens (j) of Trf+/hpx (n = 5) and Trf hpx/hpx (n = 5) mice after Trf treatment. Short lines indicate means. Bars represent mean+s.e.m. One dot represents one mouse. Significance was determined by unpaired two-sided Student’s t-test (a, b, c, i, and j) or unpaired two-sided Mann-Whitney test (h). NS, not significant.

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Extended Data Fig. 9 iNKT cells regulate skin cell homeostasis through Trf-mediated regulation of iron homeostasis.

a, Histograms of expression of PLZF, T-bet, and ROR𝞬t in donor iNKT cells from the skin of 12-day-old Trf hpx/hpx mice that received transfer of WT iNKT cells. b, Images of Trf +/hpx and Trf hpx/hpx mice that received transfer of WT iNKT cells or PBS. c, Comparison of MFI of Ki67 expression in CD45−CD34+ hair follicle stem cells from the skin of Trf +/hpx mice (n = 6) and Trf hpx/hpx mice that received PBS (n = 4) or WT iNKT cells (n = 3). Data are representative of 3 independent experiments. d, Histogram (left) and MFI (right) of CD71 expression in CD45−CD49f+ hair follicle progenitor cells from the skin of Trf +/hpx (n = 13) and Trf hpx/hpx mice that received PBS (n = 8) or iNKT cells (n = 6). Data are representative of 6 independent experiments. e, Histogram (left) and MFI quantification (right) of intracellular ferrous (Fe2+) in skin CD45+ cells of Trf +/hpx (n = 7) and Trf hpx/hpx mice that received PBS (n = 4) or iNKT cells (n = 3). Data are representative of 3 independent experiments. Bars represent mean+s.e.m. One dot represents one mouse. Significance was determined by one-way ANOVA with Dunnett post test (c and d) or Brown-Forsythe and Welch ANOVA with Welch’s correction (e). NS, not significant.

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Extended Data Fig. 10 Adoptive transfer of iNKT cells from adult mice fail to rescue the impaired skin tissue development in Trf hpx/hpx mice of early ages.

a, Schematic of iNKT cell adoptive transfer strategy. Adult iNKT cells were isolated from the spleen of 14-week-old mice. b, FC analysis of CD45−CD49f+Ki67+ cells in the skin of Trf +/hpx mice (n = 3) and Trf hpx/hpx mice that received transfer of PBS (n = 3) or adult iNKT cells (n = 3). Bar graphs on the right compare average absolute numbers of skin CD45−CD49fmedKi67+ cells of 3 independent experiments. c, Histogram (left) and MFI quantification (right) of intracellular ferrous (Fe2+) in skin CD45-CD49f+ cells of Trf +/hpx (n = 3) and Trf hpx/hpx mice that received PBS (n = 3) or adult iNKT cells (n = 3). Data are representative of 3 independent experiments. Bars represent mean+s.e.m. One dot represents one mouse. Significance was determined by one-way ANOVA with Dunnett post test (b) or Brown-Forsythe and Welch ANOVA with Welch’s correction (c). NS, not significant.

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Wang, WB., Lin, YD., Zhao, L. et al. Developmentally programmed early-age skin localization of iNKT cells supports local tissue development and homeostasis. Nat Immunol 24, 225–238 (2023). https://doi.org/10.1038/s41590-022-01399-5

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