Beta Integrin Expression Defines Functionally Distinct CD4⁺ T-Cell Subsets That Are Altered in Ulcerative Colitis

In ulcerative colitis (UC), a chronic inflammatory condition affecting over 5 million people worldwide, colonic damage is driven by T cells.¹ T cells enter the gut through integrin expression, either α₄β₇ that binds to mucosal addressin cell adhesion molecule (MadCAM) on epithelial cells or α₄β₁ that binds to vascular cell adhesion molecule (VCAM) on endothelial cells.² Vedolizumab inhibits α₄β₇ and MadCAM interactions and is commonly used to treat UC, but treatment failures are common (>50%).³ Increased proportions of α₄β₁⁺ T cells have been associated with poor response to vedolizumab and worse UC severity.^2,4 Interestingly, in mouse models of colitis, blocking α₄β₁ was superior to blocking α₄β₇ for reducing gut inflammation.⁵ We therefore hypothesized that α₄β₁ and α₄β₇ T cells may have unique functional properties in UC.

We used flow cytometry to analyze associations between CD4⁺ T-cell integrin expression and secretion of interferon-gamma (IFN-γ) and interleukin-17A/F (IL-17A/F), cytokines important in intestinal immunity. We isolated lamina propria mononuclear cells (LPMCs) from biopsies of UC patients (n = 8; Supplementary Table 1) and controls (n = 12; Supplementary Table 1), stimulated them with phorbol myristic acid and ionomycin for 4 hours, and stained with a 24-color antibody panel (Supplementary Table 2). All UC patients were vedolizumab-naive.

Unsupervised clustering analysis identified 14 unique CD4⁺ T-cell clusters (Figure 1A), with clusters 1 and 12 significantly higher and cluster 11 significantly lower in UC compared to controls (Figure 1B). Protein expression within each cluster was analyzed as a mean fluorescence intensity (MFI) percentile. Cluster 1 was enriched for regulatory T cell (Treg) markers CD25, FOXP3, and HELIOS; cluster 12 expressed β₁ and IL-17A/F (Th17 cell-like); and cluster 11 was defined by high expression of β₁, β₇, and IFN-γ (Th1 cell-like) (Figure 1C). Using supervised hierarchical gating, we confirmed that, compared to controls, at the single-cell level, UC LPMCs had significantly increased proportions of CD25^highFOXP3⁺HELIOS⁺ Treg cells (Figure 1D) and MFI of IL-17A/F within IL-17A/F⁺ cells. In contrast, UC LPMCs had significantly lower frequencies of IFN-γ⁺ T cells and IFN-γ MFI (Figure 1E). These data support reports of enriched Th17 and Treg cells in UC inflamed gut.⁶

Figure 1.

Increased α₄β₁⁺CD4⁺ T cells in UC LPMCs. LPMCs from UC patients (n = 6–8) or controls (n = 10–12) were stimulated with PMA and ionomycin for 4 hours and stained with panels in Supplementary Table 3. (A) FlowSOM-generated CD4⁺ T-cell clusters on opt-SNE-1 plots. (B) EdgeR determined significantly altered cluster frequencies between UC and controls. Significant clusters were circled in A. (C) Heatmap showing the MFI of cluster-defining markers. Manual gating was used to analyze (D) CD25^highFOXP3⁺HELIOS⁺ Tregs and (E) the proportion and mean fluorescence intensity (MFI) of IFN-γ⁺ and IL-17A/F⁺ cells. (F) Representative gating and ratios of β₁⁺ and β₇⁺ cells within CD4⁺α₄⁺CD45RA^neg cells. (G) Correlations between the ratio of β₁:β₇ integrin expression and cytokine MFI (within cytokine⁺ cells). Analysis used Wilcoxon signed rank tests or Spearman’s correlation. ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001. Graphs show median values. PMA, phorbol myristic acid.

Next, we studied β integrin expression patterns in CD4⁺ T-cell subsets. Compared to controls, CD4⁺α₄⁺CD45RA^neg T cells from UC LPMCs had a significantly higher ratio of β₁ to β₇ expression (Figure 1F). Similar results were found when CD4⁺ T cells were split into conventional T cells and Tregs (Supplementary Figure 1A gating in Supplementary Figure 2B). Notably, these differences between UC and controls were only present in LPMC and not peripheral blood mononuclear cells (PBMCs) (Supplementary Figure 1B). Interestingly, the ratio of β₁:β₇ integrins correlated negatively with IFN-γ MFI and positively with IL-17A/F MFI (Figure 1G, P = .001 and .029, respectively).

Interestingly, irrespective of disease state, α₄β₇⁺ T cells had significantly higher proportions of IFN-γ⁺ cells and lower IL-17A/F⁺ cells than α₄β₁⁺ T cells (Supplementary Figure 2A). To ask if the link between integrin expression and cytokine secretion was restricted to LPMCs, we analyzed PBMCs from healthy controls (n = 8). Consistent with LPMC data, α₄β₁⁺ CD4⁺ conventional T cells from healthy and UC PBMCs had lower amounts of IFN-γ and higher frequencies of IL-17A/F⁺ cells compared to α₄β₇⁺ cells (Figure 2B and C, gating in Supplementary Figure 2B). Thus, the relationship between β integrin expression, IFN-γ, and IL-17A/F is a common feature of CD4⁺ T cells in PBMC and LPMC in both healthy and UC individuals.

Figure 2.

Integrin expression defines T cells with unique cytokine profiles. (A) Representative gating and compiled data of IFN-γ⁺ or IL-17A/F⁺ cells in α₄β₁⁺ and α₄β₇⁺ T cells (CD4⁺CD45RA^-) in LPMC from UC and controls. (B) Control and (C) UC PBMCs were stimulated with PMA and ionomycin for 4 hours and IFN-γ and IL-17A/F production from α₄β₁ and α₄β₇ Tcon measured (gating in Supplementary Figure 2, n = 8). (D) α₄β₁ or α₄β₇ cells were sorted from PBMC (gating in Supplementary Figure 2), expanded with αCD3/CD28 for 7 days and integrin expression measured. (E and F) Expanded cells were stimulated with PMA and ionomycin for 6 hours and IFN-γ and IL-17A/F production measured by flow cytometry (n = 7). Analysis used Wilcoxon signed rank tests. ∗P < .05, ∗∗P < 1. Graphs show median values. PMA, phorbol myristic acid.

To test stability of differential cytokine production in α₄β₁⁺ and α₄β₇⁺ CD4⁺ T cells, these subsets were sorted from PBMCs and expanded with αCD3/CD28 for 7 days. Expanded cells retained their characteristic integrin expression (>92% purity, Figure 2D), and, similar to ex vivo cells, expanded α₄β₁⁺ cells had a reduced IFN-γ ΜFI and higher proportion of IL-17A/F⁺ cells compared to α₄β₇⁺ cells (Figure 2E and F). These data confirm α₄β₇⁺ T cells produce higher levels of IFN-γ, whereas α₄β₁⁺ cells are enriched for IL-17⁺ cells. This echoes reports of increased β₁ expression on Th17 cells in multiple sclerosis patients⁷ and downregulated β₇ integrin expression on mucosal-associated invariant T cells linked to reduced IFN-γ production in patients with type 1 diabetes.⁸

We found LPMCs of UC patients have significantly lower proportions of α₄β₇⁺ IFNγ⁺ CD4⁺ T cells compared to controls, with corresponding enrichment in α₄β₁⁺IL-17A/F⁺ cells. Reduced α₄β₇ expression in UC LPMCs has been previously reported,⁹ and increased α₄β₁⁺ T cells have been associated with disease severity, with intestinal VCAM-1 (the ligand for α₄β₁⁺) also increased in active UC.² Here, we extend these findings by linking integrin expression to cytokine polarization. Expression of α₄β₁⁺ associated with IL-17 production, while α₄β₇ cells produced more IFN-γ. This parallels a study, suggesting α₄β₇-mediated costimulation is inferior to CD28 for inducing Th17 polarization.¹⁰

In summary, this study to define the characteristics of α₄β₁⁺ vs α₄β₇⁺ CD4⁺ T cells in UC led to the unexpected discovery that β integrin expression is linked to unique cytokine production profiles, a finding that held true in PBMC, LPMCs, and following in vitro expansion. Thus, alterations in the balance of β₁- vs β₇-expressing cells may be a phenomenon common to many inflammatory diseases.