Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity

Abstract

Background: Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. CD4⁺ T_H2 cells play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting their cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4⁺ T cell subsets and their effector molecules may drive airway inflammation and remodeling.

Methods: We performed single-cell transcriptome analysis of >50,000 airway CD4⁺ T cells isolated from bronchoalveolar lavage samples from 30 patients with mild and severe asthma.

Findings: We observed striking heterogeneity in the nature of CD4⁺ T cells present in asthmatics' airways, with tissue-resident memory T (T_RM) cells making a dominant contribution. Notably, in severe asthmatics, a subset of CD4⁺ T_RM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4⁺ T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor activation (HLA-DRB1, HLA-DPA1) and cytotoxicity (GZMB, GZMA) and, following stimulation, expressed high levels of transcripts encoding for pro-inflammatory non-T_H2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling.

Conclusions: Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease.

Funding: This research was funded by the NIH.

Keywords: CD4+ tissue-resident memory T cells; Translation to patients; airway T cells; asthma; cytotoxic CD4+ T cells; severe asthma; single-cell RNA sequencing.

Figure 1.. Single-cell transcriptomic analysis reveals heterogeneity among airway CD4⁺ T cells.

(A) Study overview. (B) Uniform manifold approximation and projection (UMAP) visualization of Seurat-based clustering analysis of 27,771 single-cell transcriptomes of ex vivo sorted CD4⁺ T cells obtained from 9 mild and 16 severe asthmatic patients. Each dot represents a cell and is colored based on cluster type. Proportion of cells in each cluster is shown (parenthesis). (C) Heatmap of row-wise z-score-normalized mean expression of significantly enriched transcripts in each cluster. Adjusted P-value < 0.05 and log₂ (fold change) > 0.25. (D) Row-wise z-score-normalized mean expression (color scale) and percent of expressing cells (size scale) plot for a selection of marker genes in each cluster. (E) UMAP shows T_RM signature score (color scale) for each cell. Clusters are delineated by colored lines. (F) GSEA between CD103⁺ T_RM cluster (top) and CD103^– T_RM cluster (bottom) versus all non-T_RM clusters using published T_RM signature gene lists (Table S3A). NES, normalized enrichment score; q, false discovery rate. (G) Violin plot displays normalized expression of ITGAE (CD103) in T_RM clusters (CD103⁺ T_RM and CD103^– T_RM) compared to T_CM cluster. (H) Violin plots show normalized protein expression of CD103 and CD69 in T_RM clusters compared to T_CM cluster (analysis done for 6 severe asthmatic patients).

Figure 2.. CD103⁺ T_RM cells are significantly increased in the airways of males with severe asthma.

(A) Pie charts represent average proportions of CD4⁺ T cell subsets in the 4 clinical groups: mild asthmatic (MA) and severe asthmatic (SA) patients separated by sex. Colors correspond to cluster type. (B) Normalized stacked bar charts represent the proportions of CD4⁺ T cell clusters per donor for the 4 clinical groups. Colors correspond to cluster type. (C) Dot plots show proportions of CD103⁺ T_RM, CD103^– T_RM, T_CM, T_REG, and T_FH clusters for the 4 clinical groups (*, P < 0.05; **, P < 0.01; Mann-Whitney U test). (D) Dot plots show proportions of CD103⁺ T_RM, CD103^– T_RM, non-T_RM, T_REG, and T_FH cells for the 4 clinical groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001; Mann-Whitney U test). (E) Representative contour plot showing the expression of CD69 versus CD103 from CD4⁺ T cells by flow cytometry from two donors, one mild and one severe asthmatic. (F) Dot plots show proportions of CD103⁺ T_RM cluster in severe asthmatics off (–) or on (+) oral corticosteroids (OCS; left) or biologics (right) treatment separated by sex (Mann-Whitney U test). (G) Scatter correlation plots between proportions of cells in CD103⁺ T_RM (top) or CD103^– T_RM (bottom) cluster with clinical features (composite asthma severity score and 100% - post-bronchodilator FEV1/FVC %). Each dot represents data from a single patient and are colored and shaped based on the 4 clinical groups. Correlation coefficient r and P value were computed using Spearman correlation analysis (trendline black). (H) Dot plots show proportions of CD4⁺CD69⁺CD103⁺ T_RM cells (top) and CD4⁺CD69⁺CD103^– T_RM cells (bottom) in BAL samples measured by flow cytometry from healthy (green) donors and asthmatic (blue = mild to moderate asthma; red = severe asthma) donors (unspecified sex) (**, P < 0.01; Kruskal-Wallis one-way test followed by Dunn’s post-hoc test). Data obtained from published datasets #1, #2, and #3. (C, D, F, H) Each dot represents data from a single subject, bars represent the mean, and t-lines represent SEM.

Figure 3.. CD103⁺ T_RM subset displays qualitative features linked to TCR activation and cytotoxicity.

(A) Volcano plot shows false discovery rate (FDR) (-log₁₀ adjusted P-value) and log₂ (fold change) in expression for genes differentially expressed in CD103⁺ T_RM versus CD103^– T_RM clusters using sex as covariate. Dots are colored according to the mean of expression (log₂) and sized based on the difference in the percentage of cells expressing the given gene, both derived from the group in which the gene is upregulated. Gray dotted lines represent the statistical threshold values: log₂(fold change) > 0.25 and -log₁₀(FDR > 1.3 (adjusted P-value < 0.05). (B) Heatmap of row-wise z-score-normalized mean expression of 1001 differentially expressed genes between CD103⁺ T_RM and CD103^– T_RM clusters in male and female patients separately. Adjusted P-value < 0.05 and log₂ (fold change) > 0.25. (C) IPA shows top 10 pathways enriched for genes with increased expression in CD103⁺ T_RM cluster compared to CD103^– T_RM cluster. Numbers show matching genes from dataset and IPA gene lists (Table S3B). (D, G) Violin plots show normalized expression for example genes up-regulated in CD103⁺ T_RM cluster linked to TCR signaling (D) or cytotoxicity (G). Color code represents the fraction of cells expressing the indicated gene in each cluster. (E, H) GSEA shows enrichment of genes linked to TCR signaling (E) or cytotoxicity (H) in CD103⁺ T_RM cluster compared to CD103^– T_RM cluster. NES, normalized enrichment score; q, false discovery rate. (F, I) GSVA shows TCR signaling (F) or cytotoxicity (I) enrichment scores per donor in CD103⁺ T_RM and CD103^– T_RM clusters (***, P < 0.001 and ****, P < 0.0001, respectively; Mann-Whitney U test). (E, F, H, I) Gene lists in Table S3A. (J) Single-cell pseudotime trajectory analysis of airway CD4⁺ T cell subsets. Trajectory constructed using the Monocle3 algorithm. (K) Dot plots show Shannon-Wiener (top) and Inverse Simpson (bottom) TCR diversity indexes in bulk samples from CD103⁺ T_RM, CD103^– T_RM, and non-T_RM cells (**, P < 0.01; ***, P < 0.001; Kruskal-Wallis one-way test followed by Dunn’s post-hoc test). (F, I, K) Each dot represents data from a single patient, bars represent the mean, and t-lines represent SEM. (L) Bar chart shows number of TCR clonotypes specific (left) or shared (right) between bulk samples from CD103⁺ T_RM, CD103^– T_RM, and non-T_RM cells. Total number of clones in each sample group (left bottom corner) is shown.

Figure 4.. Molecules that restrain T cell activation and effector functions are reduced in severe asthma.

(A) Crater plot shows the log₂ (fold change) expression of genes between severe and mild asthma in males (x-axis) and females (y-axis). Dotted lines indicate the statistical threshold value of fold change for gene filtering (adjusted P-value < 0.05 and log₂ (fold change) > 0.25). (B) Plot shows row-wise z-score-normalized mean expression (color scale) and percent of expressing cells (size scale) for indicated genes in each cluster per disease. (C) GSEA plot shows enrichment of genes linked to cAMP immunoregulation pathway in cells from severe compared to mild asthmatics, in males (left) and females (right). NES, normalized enrichment score; q, false discovery rate. (D, E) GSVA shows cAMP signaling enrichment scores per donor grouped by disease per cluster (D) or per donor in CD103^– T_RM (left) and CD103⁺ T_RM (right) clusters for the 4 clinical groups (E) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Mann-Whitney U test). (C, D, E) Gene lists in Table S3A. (F) Violin plots show normalized expression for genes down-regulated in severe asthma in the T_FH (top) and T_REG (bottom) clusters. Color code represents the fraction of cells expressing the indicated gene in each cluster.

Figure 5.. Pro-inflammatory cytokines are expressed by airway CD4⁺ T cells from severe asthmatics.

(A) Crater plot shows the log₂ (fold change) expression of genes between severe and mild asthma in males (x-axis) and females (y-axis). Dotted lines indicate the statistical threshold value of fold change for gene filtering (adjusted P-value < 0.05 and log₂ (fold change) > 0.25). (B) Plot shows row-wise z-score-normalized mean expression (color scale) and percent of expressing cells (size scale) for indicated genes in resting and stimulation conditions per disease. (C) Volcano plot shows false discovery rate (FDR) (-log₁₀ adjusted P-value) and log₂ (fold change) in expression for genes differentially expressed in GZMB⁺ cells versus GZMB^– cells. Dots are colored according to the mean of expression (log₂) and sized based on the difference in the percentage of cells expressing the given gene, both derived from the group in which the gene is up-regulated. Gray dotted lines represent the statistical threshold values: log₂(fold change) > 0.25 and -log₁₀(FDR > 1.3 (adjusted P-value < 0.05). (D) Plot shows row-wise z-score-normalized mean expression (color scale) and percent of expressing cells (size scale) for indicated genes in all GZMB^–, mild asthma (MA) GZMB⁺, and severe asthma (SA) GZMB⁺ cells. (E) Scatter plots show co-expression of CCL3 with other cytokine genes transcripts in GZMB-expressing CD4⁺ T cells in stimulation condition. Percentage of co-expressing cells is indicated (top right). Each dot represents one cell. Cells are colored based on density value. Dotted lines indicate threshold of Seurat normalized gene expression (log2FC) (> 0.5).