Fig 2: Graft-infiltrating, recipient-derived T cells take on a TRM 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.

Fig 3: 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.