Research ArticleBARRIER IMMUNITY

The human tissue-resident CCR5+ T cell compartment maintains protective and functional properties during inflammation

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Science Translational Medicine  04 Dec 2019:
Vol. 11, Issue 521, eaaw8718
DOI: 10.1126/scitranslmed.aaw8718
  • Fig. 1 CD4 and CD8 TRM are abundant in healthy oral mucosal tissue.

    Human gingival tissue was obtained from routine oral surgeries. A piece of each tissue was saved for histology, including evaluation by a pathologist (scoring criteria are listed in Material and Methods). The remaining tissue was processed for flow cytometry. (A) Gating strategy for CD8 and CD4 T cells (middle) in the blood (top) and mucosa (bottom). Memory (CCR7 and CD45RA) and tissue-resident (CD69 and CD103) phenotype of CD8 T cells (left) and CD4 Tconv cells (right). Numbers indicate percentages of the parent population. Percentage of CD69+CD103 (orange), CD69+CD103+ (blue), CD69CD103+ (pink), and CD69CD103 (green) on (B) CD8 T cells and (C) CD4 T cells across n = healthy tissues from 14 donors. Error bars show SD from the mean. ****P ≤ 0.0001. (D) Mucosal tissue with 4′,6-diamidino-2-phenylindole (DAPI; white) staining depicting how epithelium (Epi), subepithelium (SE), and stroma compartments were drawn in HALO. (E) Representative healthy tissue stained with CD4 (red), CD8 (green), and DAPI (blue). Dotted line indicates the epithelial boundary. (F) IFDP analysis ratio of CD4 to CD8 T cells across the tissue compartments, n = healthy tissues from 10 donors. **P ≤ 0.01 generated by repeated measures one-way ANOVA with Tukey’s posttest.

  • Fig. 2 CCR5 is highly expressed by CD4 Tconv and Treg in healthy oral mucosa.

    (A) Representative flow plots of CCR5 and CD69 expression on CD4 Tconv cells from the blood and healthy mucosa. (B) Quantification of CCR5 expression on CD69+ and CD69 populations, numbers indicate the mean percentage for each population (n = 17 donors). (C) CCR5+CD69+ percentage of CD4 Tconv cells in healthy mucosa (n = 17). (D) Representative image from a healthy tissue showing CD4 (red), CD69 (blue), and CCR5 (green) staining. Detailed set of four images shows combined and individual staining of cells from white box with DAPI (gray). Arrows indicate all triple-positive cells. (E) Gating strategy for Treg in the blood (top) and mucosa (bottom). Numbers indicate percentages of the parent population. (F) Representative histograms depicting Foxp3 (top) and CTLA4 (bottom) expression on CD25neg/lo (blue) and CD25hi Treg (red) in the blood (filled) and mucosa (open). (G) Treg percentage of CD4 T cells in the blood and mucosa (n = 9 donors). (H) Proportion of CD69+CD103 (orange), CD69+CD103+ (blue), CD69CD103+ (pink), and CD69CD103 (green) in Treg cells from the blood and mucosa (n = 9 donors). Error bars show SD from the mean. (I) Expression of CCR5 on Treg from the blood and healthy mucosa. Numbers indicate the mean percentage for each population (n = 9 donors). *P < 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, generated with paired t test.

  • Fig. 3 Inflammation is associated with an increase in CD69+ and CD69 CD4 T cells in distinct tissue compartments.

    (A) CD4 (green) T cells in healthy (left) and inflamed (right) representative oral mucosa tissues with DAPI (blue). Dotted lines indicate the base of the epithelium, and solid lines mark the divide between subepithelium and stroma compartments. (B) IFDP quantification of CD4 T cells in the epithelium (left), subepithelium (middle), and stroma (right) stratified by inflammation score. n = 5 to 7 donors per score (total n = 23 donors analyzed). (C) Flow data of CD69+ percentage of CD4 T cells stratified by inflammation. Numbers indicate how many tissues were analyzed for each score. (D) IFDP quantification of CD69+ percentage of CD4 T cells stratified by inflammation for each compartment, n = 4 donors per score. Slope, R2, and P values for slope indicated on each graph were generated with linear regression. Black symbols indicate flow cytometry, and blue symbols indicate IFDP analysis. NS, not significant.

  • Fig. 4 CCR5 is expressed on resident and transient CD4 Tconv and Treg populations in inflamed oral mucosa.

    (A) Expression of CCR5 on CD4 Tconv cells from the blood and inflamed mucosa determined by flow cytometry. Numbers indicate the mean percentage for each population (n = 20). (B) Flow cytometry data depicting CCR5+ percentage of CD4 T cells stratified by inflammation score. Numbers indicate how many tissues were analyzed for each score. (C) Representative image from a healthy tissue showing CD4 (red), CD69 (blue), and CCR5 (green) staining. Detailed set of four images shows combined and individual staining of cells from white box with DAPI (gray). Arrows indicate all triple-positive cells. Dashed line indicates the epithelial boundary. (D) IFDP quantification of CD4 T cells expressing CCR5 in the epithelium (top), subepithelium (middle), and stroma (bottom) stratified by inflammation with n = 4 per score. (E) Treg percentage of CD4 T cells stratified by inflammation. Numbers indicate how many tissues were analyzed for each score. (F) Expression of CCR5 on CD4 Treg cells from the blood and inflamed mucosa determined by flow cytometry. Numbers indicate the mean percentage for each population (n = 17). (A and F) ***P ≤ 0.001, ****P ≤ 0.0001, generated with paired t test. (B, D, and E) Slope, R2, and P values for slope indicated on each graph were generated with linear regression. Black symbols indicate flow cytometry, and blue symbols indicate IFDP analysis.

  • Fig. 5 Tissue localization imparts a distinct gene expression profile on CCR5-expressing CD4 T cells.

    (A) Sorting scheme for CD4 T cells from the blood (CCR5+CD69, red) and oral mucosa (CCR5+CD69+, orange; CCR5+CD69, teal) for RNA-seq experiments. (B) Heatmap of genes differentially expressed with inflammation score (FDR, <0.25). Grayscale and numbers indicate inflammation score. (C) Heatmap of genes differentially expressed between the blood and oral mucosa (FDR, <0.05; expression fold change, ≥2). Letters designate individual donors; colors for sample origin correspond to those in (A). (D) Gene set enrichment analysis (GSEA) for the “TRM Core Signature” gene set in our oral mucosa CD69+ samples versus CD69 samples. The y axis is the enrichment score, and the x axis is genes ranked according to the absolute value of log fold change between the mucosa CD69+ and CD69 samples. P < 0.001 for TRM Core Signature enrichment. (E) Volcano plot showing genes up-regulated in CD69 (teal) and CD69+ (orange) CD4 T cells from the oral mucosa. Highly significant genes associated with a TH17 signature are denoted. Dotted lines show an FDR of 0.05 (horizontal) and a fold change of ±2 (vertical).

  • Fig. 6 TH17 CD4 T cells with a TRM signature are a distinct subset within the tissue CCR5+CD69+ tissue population.

    (A) tSNE plot of the single-cell RNA-seq data from six blood and oral mucosa subsets, each assigned a unique color: blood total CD4 (purple), blood CD4 CCR5+CD69 (red), mucosa CD4 CCR5 (green), mucosa CD4 CCR5+ (blue), mucosa CD4 CCR5+CD69 (teal), and mucosa CD4 CCR5+CD69+ (orange). Asterisks indicate samples that correspond to bulk sorted populations from previous experiments. (B) Heatmap of the differentially expressed genes defining each population (FDR, <0.01). (C) Mean expression of TH17 and TRM Core Signature (only using genes up in CD69+ cells in the TRM Core Signature) gene sets in individual CD4 T cells from blood CCR5+CD69 (left), mucosa CCR5+CD69 (middle), and mucosa CCR5+CD69+ (right). Cells were sorted from a single donor.

  • Fig. 7 CD4 TRM in human oral mucosa rapidly produce inflammatory cytokines.

    (A) Blood and mucosa CD4 T cell production of IL-17A/F and IFNγ after 6 hours without stimulation or TCR stimulation (anti-CD3/CD28 beads) from a single donor. Mucosa stimulation data are shown for CD69 (left) and CD69+ (right) populations. (B) IL-17A/F and (C) IFNγ production from n = 12 donors. *P < 0.05, **P ≤ 0.01, generated with paired t test. NS, not significant.

  • Fig. 8 CCR5+ CD4 TRM are stably maintained in human rectal mucosa during treatment with the CCR5 antagonist Maraviroc.

    (A) Diagram of sample collection for rectal biopsies used from the CHARM-03 study. (B) Representative flow plots showing CD69 and CCR5 expression on live, CD3+CD4+ T cells. Numbers indicate a percentage of parent population. (C) Proportions of CCR5+CD69+ (dark orange), CCR5CD69+ (light orange), CCR5+CD69 (teal), and CCR5CD69 (gray) as a percentage of CD4 T cells. Pre-Tx, pretreatment; MVC, maraviroc. (D) Representative immunofluorescence images of CD4 (red) and CCR5 (green) from a male donor before treatment (left), after oral Maraviroc (middle), and after topical Maraviroc (right). n = 5 male and n = 5 female participants.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/521/eaaw8718/DC1

    Material and Methods

    Fig. S1. Oral mucosa pathology assessment.

    Fig. S2. CD4 TRM phenotype and localization in human oral mucosa.

    Fig. S3. T cells as a percentage of total nucleated cells in healthy oral mucosal tissue.

    Fig. S4. CD8 T cell and CD4 T cell changes with inflammation in human oral mucosa.

    Fig. S5. Network analysis and GSEA for bulk RNA-seq of CD4 T cells from blood and oral mucosa samples.

    Fig. S6. scRNA-seq.

    Fig. S7. CD4 T cell functional potential.

    Fig. S8. Impact of the CCR5 antagonist Maraviroc on CD4 T cell subsets in human rectal mucosa.

    Table S1. List of donors used in this study.

    Table S2. DE gene list comparing CCR5+ CD4 T cells from human blood and oral mucosa from bulk RNA-seq.

    Table S3. List of TH17-associated genes used for scRNA-seq analysis.

    Table S4. List of commercially available antibodies used for IF experiments.

    Table S5. List of DE genes between CCR5+ (fold change up) and CCR5 (fold change down) cells from scRNA-seq in human oral mucosa.

    Data file S1. Primary data.

    References (6165)

  • The PDF file includes:

    • Material and Methods
    • Fig. S1. Oral mucosa pathology assessment.
    • Fig. S2. CD4 TRM phenotype and localization in human oral mucosa.
    • Fig. S3. T cells as a percentage of total nucleated cells in healthy oral mucosal tissue.
    • Fig. S4. CD8 T cell and CD4 T cell changes with inflammation in human oral mucosa.
    • Fig. S5. Network analysis and GSEA for bulk RNA-seq of CD4 T cells from blood and oral mucosa samples.
    • Fig. S6. scRNA-seq.
    • Fig. S7. CD4 T cell functional potential.
    • Fig. S8. Impact of the CCR5 antagonist Maraviroc on CD4 T cell subsets in human rectal mucosa.
    • References (6165)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of donors used in this study.
    • Table S2 (Microsoft Excel format). DE gene list comparing CCR5+ CD4 T cells from human blood and oral mucosa from bulk RNA-seq.
    • Table S3 (Microsoft Excel format). List of TH17-associated genes used for scRNA-seq analysis.
    • Table S4 (Microsoft Excel format). List of commercially available antibodies used for IF experiments.
    • Table S5 (Microsoft Excel format). List of DE genes between CCR5+ (fold change up) and CCR5 (fold change down) cells from scRNA-seq in human oral mucosa.
    • Data file S1 (Microsoft Excel format). Primary data.

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