Fig 2: CD103+ CD8+ intestinal T cells demonstrate greater capacity for cytokine production(A–D) Cytokine production by small intestinal CD8+ T cells. Representative histograms of expression, and group summaries of proportion of CD8+ T cells expressing TNF-α (A), IFN-γ (B), CCL4 (C), and IL-2 (D) 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).(E) Mean proportion of CD8+ T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-α, IFN-γ, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10).(F) Mean percentage (± SEM) of CD8+ T cells co-expressing TNF-α, IFN-γ, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression.Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

Fig 3: 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 4: Alterations of dNK subsets in the decidua of RM pregnancyA. Representative flow cytometry plots showing the strategy to sort dNK cells in human decidual tissues at gestational weeks 6–8, including dNK1 (CD3−CD56+CD39+), dNK3 (CD3−CD56+CD39−CD103+), and dNK4 (CD3−CD56+CD16+). B. Quantification of cell population of dNK1 and dNK3 (left panel), as well as dNK4 (right panel) subsets in decidual tissues from Ctrl (n = 5) and RM patients (n = 5 in left panel, and n = 11 in right panel). Significant difference between the two groups is analyzed using unpaired t-test. *, P < 0.05; **, P < 0.01. C. Violin plots illustrating the expression of typical differential genes (absolute log2 FC > 0.5 and Bonferroni-adjusted P < 0.05) in dNK1, dNK3, and dNK4 cells from Ctrl and RM patients. The differential expression analysis is performed using the method of non-parametric two-sided Wilcoxon rank sum test in Seurat.

Fig 5: 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.