Figure Lengend Snippet: Long-lived, conventional CD4 + and CD8 + T cell, but not unconventional T cell, populations can persist for at least 5 years in the human intestine (A) Representative flow cytometry plot of HLA-A2 expression on T cells from the blood, the recipient native intestinal mucosa, and the intestinal transplant graft demonstrating identification of donor- and recipient-derived populations by HLA mismatch. (B) Representative chip cytometry image of intestinal graft mucosa demonstrating the presence of donor-derived (HLA-A3 + , yellow arrows) and recipient-derived (HLA-A3 − , white arrows) CD3 + T cells in the lamina propria. Cytokeratin (gray); CD3 (purple); HLA-A3 (green). (C) Percentage of recipient-origin CD3 + T cells in intestinal grafts, categorized by time after transplant (n = 37; 16 subjects; means ± SEM). (D) Percentage of recipient-origin CD3 + T cells in intestinal grafts, categorized by history of graft rejection (n = 37; 16 subjects; means ± SEM). (E) Flow cytometry plot of HLA-A3 expression on graft-derived T cells in one subject who demonstrated persistent donor chimerism in the intestinal graft 1,865 days (5 years and 1 month) after transplant. (F) Percentage of donor-origin CD3 + T cells in the blood of intestinal transplant recipients, categorized by time after transplant (n = 13; means ± SEM). Dashed line at 4% represents the cutoff for significant macrochimerism from prior studies ( Fu et al., 2019 ; Zuber et al., 2015 ). (G) Conventional CD8 + and CD4 + T cell subsets in the small intestinal graft as a proportion of total T cells, categorised by time post-transplant (n = 39; 18 subjects; means ± SEM). (H) The percentage of recipient-derived T cells within conventional CD8 + and CD4 + T cell subsets in the intestinal graft, categorized by time after transplant (n = 37; 16 subjects; means ± SEM). (I) Unconventional non-Vδ2 + γδ T cell, Vδ2 + γδ T cell, and Vα7.2 + CD161 + CD8 + T cell (mucosal-associated invariant T cell) subsets in the small intestinal graft as a proportion of total T cells, categorized by time after transplant (n = 39; 18 subjects; means ± SEM). (J) The percentage of recipient-derived T cells infiltrating the intestinal graft within unconventional non-Vδ2 + γδ T cell (n = 27; 12 subjects), Vδ2 + γδ T cell (n = 25; 12 subjects), and Vα7.2 + CD161 + CD8 + T cell (n = 20; 12 subjects) subsets, categorized by time after transplant (means ± SEM). (K) Representative flow cytometry plot of CD69 and CD103 expression on donor- and recipient-derived CD8 + and CD4 + T cells in the intestinal graft. (L) Percentage of donor- and recipient-derived CD8 + and CD4 + T cells in the intestinal graft co-expressing CD69 and CD103 categorized by time after transplant (n = 35; 16 subjects; median marked with black line). For further analysis of surface marker expression, rare populations with fewer than 10 cells were excluded from the analysis (J and L). Statistical analysis performed with one-way ANOVA with Dunnett’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01.

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi Biotec , Cat# 130-113-591.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Chip Cytometry, Marker, Comparison

CD103 + and CD103 − CD8 + T cells display distinct phenotypes in healthy and transplanted intestine (A) Representative expression of CD161, CD7, CD127 (IL7R), β2-integrin (ITGB2), and granzyme K on CD103 − (red) and CD103 + (black) donor-derived T cells from the intestinal transplant graft. (B and C) Phenotypic analysis of CD8 + T cell populations in healthy small intestine. (B) Representative flow cytometry plots and histograms of CD8 + T cell phenotype from spectral flow cytometry, with expression of the following markers plotted against CD103: CD69, CD161, CD7, CD127, β2-integrin, granzyme K, KLRG1, and Ki-67. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (C) Proportion of positive cells (CD161, KLRG1, and Ki-67) or MFI (CD7, CD127, β2-integrin, and granzyme K) of CD8 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). Mean percentage or MFI represented by bars. Connecting lines represent populations from the same subject. (D–I) Phenotypic analysis of recipient-derived CD8 + T cell populations infiltrating the intestinal transplant graft. (D) The proportion of recipient-derived CD8 + T cells co-expressing CD69 and CD103 in intestinal transplant grafts, categorized by time after transplant (n = 35; 16 subjects). (E–I) Proportion of CD161 + cells (E), MFI of CD127 (F), β2-integrin (G), and granzyme K (H), or proportion of Ki-67 + cells (I) of recipient-derived CD8 + T cells, categorized by CD69 and CD103 expression and time after transplant in intestinal transplant grafts (n = 23; 12 subjects). Mean percentage or MFI represented by bars. Black lines connect populations from the same subject. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Journal: Cell Reports

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

doi: 10.1016/j.celrep.2020.108661

Figure Lengend Snippet: CD103 + and CD103 − CD8 + T cells display distinct phenotypes in healthy and transplanted intestine (A) Representative expression of CD161, CD7, CD127 (IL7R), β2-integrin (ITGB2), and granzyme K on CD103 − (red) and CD103 + (black) donor-derived T cells from the intestinal transplant graft. (B and C) Phenotypic analysis of CD8 + T cell populations in healthy small intestine. (B) Representative flow cytometry plots and histograms of CD8 + T cell phenotype from spectral flow cytometry, with expression of the following markers plotted against CD103: CD69, CD161, CD7, CD127, β2-integrin, granzyme K, KLRG1, and Ki-67. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (C) Proportion of positive cells (CD161, KLRG1, and Ki-67) or MFI (CD7, CD127, β2-integrin, and granzyme K) of CD8 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). Mean percentage or MFI represented by bars. Connecting lines represent populations from the same subject. (D–I) Phenotypic analysis of recipient-derived CD8 + T cell populations infiltrating the intestinal transplant graft. (D) The proportion of recipient-derived CD8 + T cells co-expressing CD69 and CD103 in intestinal transplant grafts, categorized by time after transplant (n = 35; 16 subjects). (E–I) Proportion of CD161 + cells (E), MFI of CD127 (F), β2-integrin (G), and granzyme K (H), or proportion of Ki-67 + cells (I) of recipient-derived CD8 + T cells, categorized by CD69 and CD103 expression and time after transplant in intestinal transplant grafts (n = 23; 12 subjects). Mean percentage or MFI represented by bars. Black lines connect populations from the same subject. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi Biotec , Cat# 130-113-591.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Control, Comparison

Graft-infiltrating, recipient-derived T cells take on a T RM cell phenotype over time, with CD103 + and CD103 − CD8 + T cells displaying distinct phenotypes (A) Fluorescence microscopy chip cytometry image of small intestinal transplant mucosa from a single subject 3 months after transplant. The donor was HLA-A3 + , and the recipient was HLA-A3 − . False-color fluorescence imaging for cytokeratin (gray), CD3 (purple), CD8 (red), CD103 (blue), and HLA-A3 (green). Donor-derived CD8 + CD103 + and CD103 − T cells are indicated by white and yellow arrows, respectively. (B) Phenotypic analysis of CD8 + T cell populations in healthy small intestinal epithelium and lamina propria. Proportion of positive cells (CD161) or MFI (CD7, CD127, β2-integrin, and KLRG1) of intraepithelial lymphocyte (IEL) or lamina propria lymphocyte (LPL) CD8 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 4). Mean percentage or MFI represented by bars. Connecting lines represent populations from the same subject. (C) Fluorescence microscopy chip cytometry of representative CD103 − (cells 1 and 2) and CD103 + (cells 3 and 4) donor-derived CD8 + T cells, showing the expression of CD18 (β2-integrin), KLRG1, and granzyme K. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Journal: Cell Reports

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

doi: 10.1016/j.celrep.2020.108661

Figure Lengend Snippet: Graft-infiltrating, recipient-derived T cells take on a T RM cell phenotype over time, with CD103 + and CD103 − CD8 + T cells displaying distinct phenotypes (A) Fluorescence microscopy chip cytometry image of small intestinal transplant mucosa from a single subject 3 months after transplant. The donor was HLA-A3 + , and the recipient was HLA-A3 − . False-color fluorescence imaging for cytokeratin (gray), CD3 (purple), CD8 (red), CD103 (blue), and HLA-A3 (green). Donor-derived CD8 + CD103 + and CD103 − T cells are indicated by white and yellow arrows, respectively. (B) Phenotypic analysis of CD8 + T cell populations in healthy small intestinal epithelium and lamina propria. Proportion of positive cells (CD161) or MFI (CD7, CD127, β2-integrin, and KLRG1) of intraepithelial lymphocyte (IEL) or lamina propria lymphocyte (LPL) CD8 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 4). Mean percentage or MFI represented by bars. Connecting lines represent populations from the same subject. (C) Fluorescence microscopy chip cytometry of representative CD103 − (cells 1 and 2) and CD103 + (cells 3 and 4) donor-derived CD8 + T cells, showing the expression of CD18 (β2-integrin), KLRG1, and granzyme K. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi Biotec , Cat# 130-113-591.

Techniques: Derivative Assay, Fluorescence, Microscopy, Chip Cytometry, Imaging, Expressing, Control, Comparison

CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Journal: Cell Reports

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

doi: 10.1016/j.celrep.2020.108661

Figure Lengend Snippet: CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi Biotec , Cat# 130-113-591.

Techniques: Functional Assay, Expressing, Control, Derivative Assay, Comparison

Journal: Cell Reports

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

doi: 10.1016/j.celrep.2020.108661

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Article Snippet: Anti-human CD161 (clone 191B8) APC , Miltenyi Biotec , Cat# 130-113-591.

Techniques: Control, Recombinant, Staining, Activation Assay, Infection, Software

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