Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn's disease

Abstract

T cells differentiate into highly diverse subsets and display plasticity depending on the environment. Although lymphocytes are key mediators of inflammation, functional specialization of T cells in inflammatory bowel disease (IBD) has not been effectively described. Here, we performed deep profiling of T cells in the intestinal mucosa of IBD and identified a CD4⁺ tissue-resident memory T cell (Trm) subset that is increased in Crohn's disease (CD) showing unique inflammatory properties. Functionally and transcriptionally distinct CD4⁺ Trm subsets are observed in the inflamed gut mucosa, among which a CD-specific CD4⁺ Trm subset, expressing CD161 and CCR5 along with CD103, displays previously unrecognized pleiotropic signatures of innate and effector activities. These inflammatory features are further enhanced by their spatial proximity to gut epithelial cells. Furthermore, the CD-specific CD4⁺ Trm subset is the most predominant producer of type 1 inflammatory cytokines upon various stimulations among all CD4⁺ T cells, suggesting that the accumulation of this T cell subset is a pathological hallmark of CD. Our results provide comprehensive insights into the pathogenesis of IBD, paving the way for decoding of the molecular mechanisms underlying this disease.

Keywords: T-cell immunity; inflammatory bowel disease; mass cytometry; single-cell RNA-seq; tissue-resident T cell.

Fig. 1.

Proteomic profiles of intestinal lymphocytes in IBD revealed by mass cytometry. (A) Phenograph-based visualization on the tSNE plot of CD3⁺ T cells from frozen LPMC of the first batch of the first cohort of patients. Annotation of each subcluster on merged tSNE. CD4⁺ and CD8⁺ T cells are circled by red and blue dashed lines, respectively, and Trm by solid lines in their respective colors. Each sample (n = 4 per group) was concatenated together. (B) Heat map of mean expression values of T cell markers. (C) Cell type proportions of CD3⁺ T cells in the first cohort of patients (n = 8 per group, batches 1 and 2). *P < 0.05, **P < 0.01, ****P < 0.0001, mean ± SEM, one-way ANOVA (Tukey’s multiple comparison test). (D) Expression level of each cell surface marker associated with CD- and UC-predominant CD4⁺ T cell subsets was overlaid on the tSNE plot. Representative markers high in CD- and UC-predominant T cell subsets are shown in Upper and Lower rows, respectively. (E) Proportions of CD4⁺ Trm, CDpop, and the rest of CD4⁺ Trm (non-CDpop Trm) among CD4⁺ T cells in each sample. *P < 0.05, **P < 0.01, ***P < 0.001, n = 18 per group, one-way ANOVA (Tukey’s multiple comparison test). (F) UMAP of the second cohort of patients (n = 18 per group). tSNE overlay of selected markers is shown. (G) Proportion of IFN-γ⁺ and GZMA⁺ cells in CD4⁺ Trm and CDpop derived from CD patients after stimulation with PMA/ionomycin for 2 h. **P < 0.01, n = 6 per group, mean ± SEM, two-tailed paired Student’s t test. (H) Serum CRP levels of CDpop^high (n = 11) and CDpop^low (n = 15) patients before surgery. *P < 0.05, mean ± SEM, two-tailed paired Student’s t test.

Fig. 2.

Single-cell transcriptional landscape of colonic lymphocytes in IBD. (A) UMAP plot of FACS-sorted CD3⁺ T cells in fresh colonic samples (n = 4 per group). Cluster number is in parentheses. Data from CD, UC, and controls were merged. (B) UMAP plot of FACS-sorted CD3⁺ T cells in each group. (C) Cell type proportions of each CD4⁺ T cell subset in CD3⁺ T cells. *P < 0.05, **P < 0.01, ****P < 0.0001, mean ± SEM, one-way ANOVA (Tukey’s multiple comparison test). (D) Volcano plot of genes differentially expressed between CD and UC CD4⁺ T cell subclusters. ADT: antibody-derived tag. (E) Gene expression plot of representative transcription factor transcripts that were significantly up-regulated in CD (PRDM1) or in UC (LEF1, TCF7, and BCL6) was overlaid on the UMAP plot. (F) MRtree that reveals the transcriptional distinctions and similarities between cell types. The pie charts on tree nodes represent the cell type composition in each cluster.

Fig. 3.

Transcriptional signatures of CD-predominant CD4⁺ Trm. (A) Protein expression of cell surface markers defining CDpop. (B) CDpop gated by the positive expression of CD4, CD103, CCR5, and CD161, and negative expression of CD27 and CCR7 was overlaid on cluster 12 of the UMAP plot. (C) Gene expression plot of secretory proteins overlaid on CD3⁺ T cells (Upper row) and cluster 12 (Lower row) of the UMAP. (D) Volcano plot of genes differentially expressed between CDpop and the rest of the CD4⁺ T cells. (E) CDpop was reclustered (upper row), and CDpop in each condition was overlaid on its UMAP (Lower row). (F) Gene expression plot of GZMA and IFNG overlaid on the UMAP. (G) Violin plot of GZMA and IFNG in each cluster. (H) Proportion of CDtrm in CD4⁺ T cells. (I) Diagram showing the relationships between CD4⁺ Trm, CDpop, and CDtrm. (J) Volcano plot of genes differentially expressed between CDtrm and the rest of CDpop. (K) Bar graph of the top 20 enriched terms across genes that were significantly up-regulated in CDtrm compared with the levels in the rest of CDpop.

Fig. 4.

Responsiveness of CD-predominant CD4⁺ Trm to cytokines. (A) Left: Proportion of IFN-γ⁺ cells in CDpop under cytokine stimulation in CD samples. *P < 0.05, n = 8 per group, mean ± SEM, two-way RM ANOVA (Tukey’s multiple comparison test). Right: Representative images of flow cytometry analysis. (B) Proportions of IFN-γ⁺ cells with or without stimulation in CDpop and CDpop with negative selection of MAIT (TCRVα7.2⁺) and γδT cells. *P < 0.05 and **P < 0.01, n = 5 per group, mean ± SEM, RM one-way ANOVA (Tukey’s multiple comparison test). (C) Left: Proportions of IFN-γ⁺ cells in CDpop and CD4⁺ T cells after cytokine stimulation. Right: The corresponding samples are connected with a line. *P < 0.05, n = 7 per group, mean ± SEM, two-tailed paired Student’s t test. (D) Left: Proportions of IFN-γ⁺ cells in CDpop and CD4⁺ T cells after TCR or cytokine cocktail. Right: The corresponding samples are connected with a line. *P < 0.05, n = 6 per group, mean ± SEM, two-way RM ANOVA (Sidak’s multiple comparison test). (E) Left: Cytokine/TCR ratio for IFN-γ secretion of each sample. Right: The corresponding samples are connected with a line. *P < 0.05, n = 6 per group, mean ± SEM, two-tailed paired Student’s t test. (F) Left: Proportions of TNF-γ⁺ cells in CDpop and CD4⁺ T cells after TCR or cytokine stimulation. Right: The corresponding samples are connected with a line. *P < 0.05, n = 6 per group, mean ± SEM, two-way RM ANOVA (Sidak’s multiple comparison test). (G) Phenograph-based visualization on the tSNE plot of frozen colonic CD4⁺ T cells from LPMC from four CD patients and two controls (n = 6 per condition and n = 12 merged) stimulated with or without cytokine cocktail. Heat map of mean expression values of T cell markers is shown on the right. (H) tSNE overlay of selected surface marker expression on merged plot. (I) tSNE overlay of cytokine markers in stimulated and unstimulated samples. (J) Proportion of each cytokine-positive cell in total CD4⁺ T cells, CD103⁻ counterpart of CDpop, and CDpop after stimulation by cytokine cocktail. ***P < 0.05, **P < 0.01, ***P < 0.001, n = 6 per group, mean ± SEM, two-way RM ANOVA (Sidak’s multiple comparison test).

Fig. 5.

Cytotoxic effect of CD-predominant CD4⁺ Trm on epithelia. (A) Representative images of immunohistochemistry of LP from CD patients. (Scale bar, 20 μm.) Red, CD4; green, CD103; and blue, DAPI. White arrows show CD4⁺ CD103⁺ Trm in LP. (B) Percentage of LDH release in the culture supernatant. *P < 0.05, n = 4 per group, mean ± SEM. shown, two-way ANOVA (Sidak’s multiple comparison test). (C) Average spheroid damage scores of four independent experiments. *P < 0.05, n = 4 per group, mean ± SEM. shown, two-way ANOVA (Sidak’s multiple comparison test). (D) Representative images of spheroids cocultured with or without CDpop under the cytokine stimuli. (Scale bar, 200 μm.) (E) The effect of IFN-γ neutralizing antibody (nAb) on cytokine-stimulated CDpop evaluated by relative LDH release in the culture supernatant. *P < 0.05, n = 4 per group, mean ± SEM. shown, Dunn’s multiple comparison test. (F) Representative images of spheroids cocultured with or without CDpop and nAb. (Scale bar, 150 μm.)