Research ArticleT CELLS

Human CD4+CD103+ cutaneous resident memory T cells are found in the circulation of healthy individuals

See allHide authors and affiliations

Science Immunology  05 Jul 2019:
Vol. 4, Issue 37, eaav8995
DOI: 10.1126/sciimmunol.aav8995
  • Fig. 1 CD4+CLA+CD103+T cells down-regulate CD69 and exit the skin.

    (A) Representative flow cytometric analysis of CD69 and CD103 expression by live gated CD8+ and CD4+ T cells from human skin. (B) Graphical summary of the proportions of CD69- and CD103-defined T cell populations among CD8+ and CD4+ skin T cells. (C) Representative flow cytometric analysis of CLA expression by live gated CD103+CD69+ TRM in human skin. (D) Human skin was adhered to tissue culture plates and cultured for 7 days submerged in medium. The ratio of CD4+ and CD8+ T cells and the expression of CLA and CD103 by T cells in the indicated samples were analyzed by flow cytometry. Representative data (n = 4). APC, allophycocyanin; PE, phycoerythrin. (E) Graphical summary of the proportion of CD69 cells among CD103+ or CD103 live gated CD45RACD4+CLA+ T cells from the indicated samples. Open symbols represent data from an individual with mammary carcinoma but no skin condition. Significance was determined by one-way repeated-measures ANOVA with Tukey’s posttest for pairwise comparisons. (F) Three 8-mm punch biopsies of healthy human skin per animal (n = 3) were placed on the back of NSG mice, and grafts and spleens were analyzed by flow cytometry 50 days later. Representative flow cytometric analysis of CLA and CD103 expression by live gated human CD45+CD3+CD4+CD25CD45RA T cells. (G) Graphical summary showing CD103 expression by live gated human CD45+CD3+CD4+CD25CD45RACLA+ T cells from skin grafts and spleens of skin-grafted NSG mice.

  • Fig. 2 CD4+CLA+CD103+ T cells constitute a unique cell population in human blood.

    (A) Left: Mass cytometry analysis of CD45RA and CLA expression by live gated CD3+CD45+ PBMCs showing the gate used to define CD3+CLA+ T cells for subsequent clustering analysis. Right: t-SNE analysis and clustering of CD3+CLA+ T cells from blood of five healthy donors based on the expression of CD4, CD8, CCR7, CD103, β7 integrin, CXCR3, CCR6, CCR4, CCR10, Foxp3, CD27, CD25, CD161, and CD56. (B) Heat map showing relative expression of the indicated markers in each of the CD3+CLA+ cell clusters. (C) t-SNE analysis of CD3+CLA+ T cells overlaid with relative expression of the indicated markers.

  • Fig. 3 Shared phenotype of CD4+CLA+CD103+ T cells from human blood and skin.

    (A) Representative flow cytometric analysis of CD45RA and CLA expression by live gated CD4+ T cells from blood and skin of healthy donors. (B) Representative flow cytometric analysis of CCR7 and CD103 expression by live gated CD4+CD45RACLA+ memory T cells from blood and skin of healthy donors. (C) Graphical summary of the proportions of CCR7- and CD103-defined T cell populations among CD4+CD45RACLA+ T cells from blood and skin. (D and E) Representative flow cytometric analysis and graphical summary of expression of the indicated markers by CD4+ T cell populations in the blood and skin as indicated. Significance was determined by one-way repeated-measures ANOVA with Tukey’s posttest for pairwise comparisons.

  • Fig. 4 CD4+CLA+CD103+ T cells from human blood and skin share a transcriptional profile.

    Whole-transcriptome profiling by RNA-seq was performed on sorted CLA+ T cell subsets from blood or skin. (A) Venn diagram showing the number of significantly differentially expressed genes [FDR < 0.05 and log2 fold change (FC) > 1] between CLA+CD103+ T cells and either CLA+CD103CCR7+ or CLA+CD103CCR7 T cells as indicated. The overlapping 83 genes were designated as the CD103+ gene signature. (B) Barcode plot showing the distribution of the CD103+ signature genes (red, up-regulated in CD103+; blue, down-regulated in CD103+) relative to gene expression changes comparing CD103+ and CD103CCR7 T cells from the blood or skin as indicated. Significance was determined by rotation gene set testing for linear models. (C) Heat map and hierarchical clustering of RNA-seq samples from the indicated blood and skin cell populations based on the CD103+ gene signature. (D) Venn diagram showing functional annotation of key genes up- or down-regulated by CLA+CD103+ T cells in blood or skin identified in our phenotypic, functional, and transcriptional analyses. Category names were assigned on the basis of described functions of the indicated genes in the published literature. Underlined gene names indicate proteins whose expression pattern was validated by flow cytometry in Figs. 3 and 5.

  • Fig. 5 CD4+CLA+CD103+T cells from human blood and skin share a functional profile.

    (A) Representative flow cytometric analysis of CD103 and CLA expression by live gated CD4+CD45RA T cells from blood and skin of healthy donors. (B to D) Representative flow cytometric analysis of indicated CLA/CD103 subpopulations of blood and skin CD4+CD45RA T cells producing IL-13, IL-4, IL-22, IL-17A, IFN-γ, and GM-CSF as indicated upon ex vivo stimulation with PMA/ionomycin and intracellular cytokine staining. (E) Graphical summary of the proportions of CLACD103, CLA+CD103, and CLA+CD103+ live gated CD4+CD45RA T cells producing cytokines as indicated. Open symbols represent data from an individual with mammary carcinoma. Significance was determined by one-way repeated-measures ANOVA with Tukey’s posttest for pairwise comparisons.

  • Fig. 6 CD4+CLA+CD103+ T cells in skin and blood are clonally related.

    TCRβ sequencing was performed on sorted CLA+ memory CD4+ T cell subsets (as in Fig. 4) from blood and skin sorted on the basis of the expression of CD103 and CCR7 as indicated. (A) Left: Circle plot of unique productive TCRβ sequences from each of the indicated populations of CLA+ T cells from one representative donor (donor 1). Connections highlight sequences from skin CLA+CD103+ T cells found in each of the other populations. For visualization, sequences were downsampled (weighted for relative abundance) for populations containing >1000 unique sequences. Right: Graphical summary of the Morisita index as a measure of TCR repertoire similarity over all productive rearrangements between CLA+CD103+ T cells in the blood and each of the indicated populations across all four donors examined. (B) Circle plot (left) and graphical summary of the Morisita index (right) as in (A) using CLA+CD103+ T cells from blood as the reference population. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test.

  • Fig. 7 CD4+CLA+CD103+T cells are present in human lymph.

    (A) Representative flow cytometric analysis of CD45RA and CLA expression by live gated CD4+ T cells from blood and TDL. (B) Representative flow cytometric analysis of CCR7 and CD103 expression by live gated CD4+CD45RACLA+ memory T cells from blood and TDL. (C) Graphical summary of the proportions of CCR7- and CD103-defined T cell populations among CD4+CD45RACLA+ T cells from blood and TDL. (D) Representative flow cytometric analysis and graphical summary of expression of the indicated markers by CD4+ T cell populations in the blood and TDL as indicated. Significance was determined by one-way repeated-measures ANOVA with Tukey’s posttest for pairwise comparisons.

  • Fig. 8 CD4+CLA+CD103+TRMcan exit the skin and reseed distant skin sites in a xenograft model.

    (A) In vitro expanded human keratinocytes and fibroblasts were grafted onto the backs of NSG mice using a grafting chamber. After 99 days of healing and differentiation, the ES or adjacent murine skin was excised, frozen in O.C.T., and stained either with hematoxylin and eosin (H&E) (left) or with anti-human type VII collagen before immunofluorescence analysis (right). Human skin from a healthy donor was used as control. (B) Experimental schematic for the generation of ES followed by xenografting human skin onto NSG mice. (C) Representative photograph of ES and skin grafts on day 144. (D) Representative flow cytometric analysis and (E) graphical summary of CLA+CD103+ T cells by live gated human CD45+CD3+CD4+CD45RA T cells from skin grafts, spleen, and ES (3 to 5 weeks after skin grafting). Open and filled symbols denote samples derived from two different skin donors. Each symbol represents data from one recipient animal. (F) Representative flow cytometric analysis and graphical summary of expression of CD27, CD9, and CD69 by live gated CD45+CD4+CD45RACD103+CLA+ T cells in the skin grafts, spleen, and ES 5 weeks after skin grafting (day 145 relative to ES generation). Significance was determined by one-way ANOVA with Tukey’s posttest for pairwise comparisons. (G) Experimental schematic for the generation of ES followed by adoptive transfer of 2.5 × 106 PBMCs (autologous to the ES)/mouse into NSG mice. i.v., intravenous. (H) Representative flow cytometric analysis and (I) graphical summary of CLA+CD103+ T cells by live gated human CD45+CD3+CD4+CD45RA T cells from spleen and ES 25 days after PBMC transfer. Each symbol represents data from one recipient animal. Significance was determined by paired t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/37/eaav8995/DC1

    Fig. S1. Representative flow cytometry gating strategies used to identify T cell subsets in human blood and skin.

    Fig. S2. CD103 expression is not induced on human CD4+ T cells in NSG mice.

    Fig. S3. CyTOF analysis of CLA+ T cells in PBMCs.

    Fig. S4. Frequencies of CLA+ T cell subsets in blood and skin.

    Fig. S5. Experimental schematic of cell isolation and sort gates for RNA-seq.

    Fig. S6. Gene expression in the different populations of CD4+CLA+ T cells from blood and skin as determined by RNA-seq.

    Fig. S7. CLA+CD103+ T cells from human blood and skin coproduce IL-22 and IL-13.

    Fig. S8. Analysis of TCRβ repertoire overlap and Vβ gene usage in CLA+ T cells from blood and skin.

    Fig. S9. Human skin–derived immune cells do not infiltrate murine skin.

    Table S1. Detailed list of antibodies and reagents.

    Table S2. RNA-seq pairwise comparisons.

    Table S3. Raw data file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Representative flow cytometry gating strategies used to identify T cell subsets in human blood and skin.
    • Fig. S2. CD103 expression is not induced on human CD4+ T cells in NSG mice.
    • Fig. S3. CyTOF analysis of CLA+ T cells in PBMCs.
    • Fig. S4. Frequencies of CLA+ T cell subsets in blood and skin.
    • Fig. S5. Experimental schematic of cell isolation and sort gates for RNA-seq.
    • Fig. S6. Gene expression in the different populations of CD4+CLA+ T cells from blood and skin as determined by RNA-seq.
    • Fig. S7. CLA+CD103+ T cells from human blood and skin coproduce IL-22 and IL-13.
    • Fig. S8. Analysis of TCRβ repertoire overlap and Vβ gene usage in CLA+ T cells from blood and skin.
    • Fig. S9. Human skin–derived immune cells do not infiltrate murine skin.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Detailed list of antibodies and reagents.
    • Table S2 (Microsoft Excel format). RNA-seq pairwise comparisons.
    • Table S3 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article