Single-Cell RNA sequencing reveals clonally-expanded CD4+ tissue-resident memory T cells in Histidyl-tRNA Synthetase-induced myositis

Figures

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  Figure 1. Bulk RNA sequencing analysis of human myositis biopsy specimens. (A) Left: Volcano plot displaying differentially expressed genes (DEGs) in anti–Jo-1 myositis patients (n = 37) compared with healthy controls (n = 37), identified by Wilcoxon’s rank-sum test, with adjusted P value < 0.05 and log2 fold change (FC) > 1 or < –1. Genes with log2 FC > 1 and adjusted P < 0.05 were designated as upregulated (Up); those with a log2 FC < –1 and adjusted P < 0.05 were classified as downregulated (Down). Right: Bar plot demonstrating Gene Ontology (GO) enrichment analysis comparing anti–Jo-1 myositis with healthy muscle, ordered by normalized enrichment scores. FDR < 0.05; minimum gene IDs in category = 20. (B) Left: Volcano plot displaying DEGs in anti–Jo-1 myositis patients (n = 37) compared with all other myositis subtypes (n = 625). Right: Bar plot demonstrating GO enrichment analysis ordered by normalized enrichment scores. FDR < 0.05; minimum gene IDs in category = 20. (C) The depicted heatmap demonstrates relative expression of key genes representative of resident memory T cells (TRMs) in different subsets of inflammatory myopathy, genetic myopathies, and healthy controls. Genes…

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  Figure 2. Single-cell profile in murine muscle with histidyl-tRNA synthetase–induced myositis. (A) Histidyl-tRNA synthetase (HRS) immunization protocol for induction of experimental murine myositis. (B) UMAP visualization of 8 clusters identified in muscle-infiltrating cells of mice with HRS-induced myositis, 2 weeks after immunization (n = 28,903 cells). (C) UMAP visualization of 8 clusters split into 2 groups corresponding to HRS-immunized versus PBS control mice. While HRS group A and PBS control group A each consisted of pooled muscle cell–infiltrating cells from 2 mice, HRS groups B–D and PBS control groups B–D each consisted of pooled cells from 3–5 mice. (D) Percentage of each cell cluster in total cell counts across different sample groups (HRS A–D, PBS A–D). (E) The normalized expression of Cd3e and Cd28 visualized in feature plots.

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  Figure 3. T/NK cell subclusters in muscle tissue from HRS-induced myositis. (A) UMAP visualization of subsets of T/NK cells isolated from muscle tissue in HRS-induced myositis. (B) Percentage of each subcluster in total T/NK cell counts displayed across different samples. (C) Dot plot displaying the expression level of highly expressed genes for each subcluster. (D) Normalized expression of Cd4, Cd8a, Cd69, Cd103, Cxcr6, and Ifng visualized in violin plots. (E) Cells isolated from muscle were analyzed by spectral flow cytometry. Data show CD3+CD4+, CD3+FoxP3+, and CD3+CD161+ cells; more detailed assessment of CD3+CD4+CD69+ cells demonstrates a CD44+CD62LloCCR7loCXCR6hi phenotype. Also shown is IFN-γ expression with or without in vitro stimulation with αTCR activation beads and PMA/ionomycin. (F) Pseudotime trajectory combined with identified cell clusters in T/NK subsets, visualized in the depicted UMAP. Color corresponds to the clusters shown in A. (G) Pseudotime values across different T/NK subsets, visualized via violin plots and aligned from low to high.

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  Figure 4. Functional characterization of TRMs and interactions between TRMs and other cells. (A and B) Pathway analysis based on DEGs of CD4+ TRMs in T/NK subsets. Pathways generated from GO were selected from the top 40 pathways identified (A), and pathways generated from KEGG were selected from the top 70 (B). (C and D) Interaction strength between CD4+ TRMs and all clusters, visualized by network and scatter plots. Interaction strength represents ligand-receptor–mediated intercellular communication probability, measured by CellChat. Vertex size is proportional to cell counts in each cluster (C). In scatter plots displaying sources and targets across all clusters (D), dot size is proportional to the number of inferred outgoing and incoming links associated with each cell cluster. (E) Heatmap visualizing TNF and IFN-γ signaling networks across different clusters. Horizontal and longitudinal axes indicate incoming and outgoing signals, respectively. (F and G) Interaction strength between CD4+ TRMs and other subsets of T/NK cells, again depicted as network and scatter plots with parameters as described for plots shown in C and D. (H) Heatmap visualizing TNF signaling network across…

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  Figure 5. Expanded clonotypes are identified in muscle tissue from HRS-induced myositis. (A) While the bar graphs demonstrate counts of T cell clonotypes (cell counts from a specific clonotype) across 5 independent experimental groups of mice (n = 4 HRS-immunized, n = 1 PBS control group), the checkerboard matrix depicts the number of overlapping clonotypes as a heatmap. Clonotypes were divided into 4 groups based on the number of identical TCR clones (5 < large ≤ 15; 3 < medium ≤ 5; 1 < small ≤ 3; single = 1). (B) Complementary analyses show clonotype counts across 7 subsets of T/NK cells (bar graphs), with the number of overlapping clonotypes illustrated by the accompanying heatmap. (C) Clustering network of different clonotypes from CD4+ TRMs with high sequence similarity, where each dot represents a specific clonotype and cluster size indicates the number of connections with different clonotypes. The accompanying sequence logo plot shows CDR3 (TCRα and TCRβ) sequence alignments of 3 clusters of clonotypes selected from this plot (circled and numbered). (D) Clusters of closely related clonotypes with a high degree of similarity in TCR sequence were divided into 4 groups based on cell…

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  Figure 6. CD4+ TRM profile is maintained from 2 to 6 weeks after HRS immunization. (A) HRS immunization protocol for assessment of experimental murine myositis at different time points. (B) UMAP visualization of 8 clusters identified in muscle-infiltrating cells from mice with HRS-induced myositis, with the percentage of each cell cluster relative to total cell number shown in the bar graphs at right. Data were pooled from mice immunized with recombinant HRS at 2 weeks (n = 2) and 6 weeks (n = 3) after immunization. (C) UMAP visualization of T/NK cell subclusters, along with the percentage of each subcluster of T/NK cells (bar graphs, right). (D) Average expression level of signature genes in CD4+ TRMs at 2 versus 6 weeks after immunization.

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  Figure 7. Expanded T cell clonotypes 2 weeks and 6 weeks after immunization. (A) Counts of T cell clonotypes (segregated by clonotype size) at 2 versus 6 weeks after immunization. (B and C) Counts of T cell clonotypes residing in 7 subsets of T/NK cells 2 weeks (B) and 6 weeks (C) after immunization. (D and E) Shared clonotypes between designated subclusters of T/NK cells 2 weeks (D) and 6 weeks (E) after immunization.