Fig 2: Subset defining genes of lamina propria CD8+CD69+CD103–and CD8+CD69+CD103+T cells. (A) Volcano plot of the expressed genes, with a nominal P value <0.99, comparing lamina propria CD8+CD69+CD103+ to CD8+CD69+CD103– T cells; selected genes are highlighted. On the x-axis, the log2 fold change (log2FC) is shown, and on the y-axis, the -log10 P value (-log10P) is shown. Gray indicates not significantly differentially expressed genes, yellow indicates genes with a log2FC >0.25 and -log10P > 10 × 10–2.5, green indicates genes with a log2FC >0.25 and -log10P < 10 × 10–2.5. (B) Representative flowcytometric dotplots, including Fluorescence Minus One (FMO) control, of Itgb2, GzmK, KLRG1, and EOMES (upper row) and quantification of the respective marker (lower row) comparing lamina propria CD8+CD69+CD103+ and CD103– T cells in healthy control subjects (n = 6–7; circles), CD patients from inflamed (diamonds) and noninflamed (triangles) ileum (paired, n = 4–6). Bars represent median and interquartile range. Comparison was performed with a paired 1-tailed t test. NS, not significant.

Fig 3: Visualization of CD103–and CD103+CD8+T cells in the human ileum. (A) Representative composite images of imaging mass cytometry performed on human ileum sections showing an overlay of E-cadherin (blue), CD8 (red in lamina propria, pink in epithelium), and CD103 (cyan, colors white in CD8 T cells) for a healthy control (HC) subject (left), noninflamed ileum of a CD patient (middle), and inflamed ileum of a CD patient (right). The magnified section of the HC subject shows an example of CD103+ (white arrows) and CD103– (orange arrows) CD8+ T cells within the lamina propria and epithelium. HC subjects: n = 2; CD patients: n = 3 (paired). (B) Quantification of CD103+ within CD8+ T cells in both the epithelium and lamina propria of the HC, CD noninflamed, and CD inflamed ileum. Every value is an average of 2 (HC subjects and noninflamed CD patients) or 3 (inflamed CD patients) samples measured using imaging mass cytometry. For each of the samples, 2 independent counts were performed.

Fig 4: During inflammation, most upregulated genes are found in CD8+Trm cells from the epithelium. (A) Diagram showing the differentially expressed genes that are specific (ie, not differentially expressed in the other disease state) for healthy control (HC) subjects (left) or CD patients (right). The upper part depicts the number of genes that are upregulated and the lower depicts part those that are downregulated in intraepithelial compared with lamina propria CD8+CD69+CD103+ T cells from the ileum. (B) Selection of top genes up- and downregulated as per panel A. (C) Pathway terms related to the genes specific for CD8+ T cells from HC subjects or CD patients for both lamina propria and epithelium derived from panel A (colors reflect the colors of panel A).

Fig 5: Location shapes the profile of intestinal CD103+CD69+CD8+T cells. (A) Volcano plot of the expressed genes, with a nominal P value <0.99, comparing intraepithelial to lamina propria (LP) CD8+CD69+CD103+ T cells; selected genes are highlighted. On the x-axis, the log2 fold change (log2FC) is shown, and on the y-axis, the -log10 P value (-log10P) is shown. Gray indicates not significantly differentially expressed genes, yellow indicates genes with a log2FC >0.25 and -log10P >10 × 10–2.5, green indicates genes with a log2FC > 0.25 and -log10P < 10 × 10–2.5. (B) Heatmap of the top 200 differentially expressed genes comparing CD103+ intraepithelial and lamina propria T cells with hierarchical clustering on the columns concerning compartment, status, and patient. Rows are z score normalized. (C) Pathway terms related to the 321 genes upregulated in intraepithelial (top) and 413 genes upregulated in LP (bottom) CD8+CD69+CD103+ T cells. (D) Messenger RNA (mRNA) expression (log2 counts; right) and percentage (left) of ileal CD8+CD69+CD103+ T cells expressing IL7R (CD127) in healthy control subjects (n = 3–5; diamonds) and CD patients from inflamed (circles) and noninflamed (triangles) ileum (paired, n = 4–5). Comparison was performed with Wald’s statistic and a paired 2-tailed t test, respectively. (E) Gene set enrichment analysis of Trm genes in humans (identified by Hombrink et al)25 in pairwise comparisons involving intraepithelial and LP CD8+CD69+CD103+ T cells derived from the ileum of healthy adult control subjects and CD patients pooled, represented by the normalized enrichment score and FDR statistical value (FDRq). (F) Representative gating strategy including Fluorescence Minus One (FMO) control (upper panel) and quantification (lower panel) of CD63 in CD8+CD69+CD103+ T cells comparing epithelium (IE) and LP. Bars represent median and interquartile range. Comparison was performed with a paired 1-tailed test. Symbols as per panel D. Healthy control subjects: n = 7; CD patients: n = 3–6. (G) As per panel F but for CXCR3. (H) As per panel F but for TIM-3 (left) and TIGIT (right). Comparison was performed with a paired 2-tailed t test. NS, not significant.