Single-cell characterisation of tissue homing CD4 + and CD8 + T cell clones in immune-mediated refractory arthritis

Abstract

Background: Immune-mediated arthritis is a group of autoinflammatory diseases, where the patient's own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients.

Methods: In this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues.

Results: Our results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue.

Conclusions: Our findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis.

Keywords: Arthritis; Autoimmunity; T cell; TCR-seq; scRNA-seq.

Fig. 1

Phenotypic characterization of CD45 + cells in the cohort. a Overview of the study—Patient cohort and study samples. b Uniform Manifold Approximation and Projection (UMAP) representation and unsupervised clustering of all cells from the IMA samples (n = 3) profiled with scRNA + TCRαβ-seq. c Left: UMAP representation of the cells from ST and PB in each cluster as identified in our study. Middle: UMAP representation of the cells as per sorting strategy in each cluster as identified in our study. Right: UMAP representation of the cells representing different disease types. d Dot plot with scaled expression of top 8 DE genes between IMA phenotype clusters (Padj < 0.05, calculated as Bonferroni corrected Wilcoxon test). e Number of DE genes both upregulated and downregulated between cells from ST and PB for each cluster (Padj < 0.05, |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test). f Pathway enrichment analysis with GO biological processes (Hypergeometric testing). g Left: Focused UMAP of all cells with TCR from panel 1b. Right: Proportion of the cells identified as Hyperexpanded (> 9), Expanded (> 1) and Singlet as identified in the UMAP. h Cells detected as expanded versus cells detected as singlets in ST and PB in our data shows that expanded cells are enriched in ST (Padj < 0.05, Benjamini–Hochberg corrected Fisher’s one-sided exact test)

Fig. 2

CD4 + and CD8 + T cells and their phenotypes in IMA. a Feature plot of top DE genes between ST and PB CD4 + T cells (Padj < 0.05, |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test). b Left: UMAP representation of the re-clustered CD4 + T cells. Right: UMAP representation of the tissue origin. c UMAP representation of TCR clones identified as Hyperexpanded (> 9), Expanded (> 1) and Singlet. d Violin plot of top DE genes between ST and PB in re-clustered Tregs (Padj < 0.05,  |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test). e Proportion of cells in different phases of cell cycle in all CD4 + cells in PB and ST. f Left: UMAP representation of the re-clustered CD8 + T cells. Right: UMAP representation of the tissue origin. g UMAP representation of TCR clones identified as Hyperexpanded (> 9), Expanded (> 1) and Singlet. h Feature plot of top DE genes that are different between ST and PB in CD8 + T cells (Padj < 0.05, |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test)

Fig. 3

Clonal trafficking of CD4 + and CD8 + T cells between synovial tissue and peripheral blood. a All common clones (defined by the exact same CDR3b sequence) in CD4 + cells between ST and PB presented in the UMAP representation from panel 2b. b Cells with clones CASSPGETQYF (left panel) and CASSLSGGAGELFF (right panel) as projected in UMAP representation from 2b. c Left: DE genes between ST and PB cells sharing the same CDR3b sequence CASSPGETQYF in Pt1-JIA (Padj < 0.05, |Log2FC| > 0.5 calculated with Bonferroni corrected Wilcoxon test). Right: DE genes between ST and PB cells sharing the same CDR3b sequence. CASSLSGGAGELFF in Pt1-JIA (Padj < 0.05, |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test). d Number of cells with CDR3b clones CASSPGETQYF and CASSLSGGAGELFF in different clusters as identified in UMAP representation from 2b. e DE genes of all CD4 + T cells that are enriched in ST as compared to PB (Padj < 0.05,  |Log2FC| > 0.5 calculated with Bonferroni corrected two-sided t-test). f Top upregulated GO-BP pathways in CD4 + T cells that are enriched in ST as compared to PB (Padj < 0.05, Benjamini–Hochberg corrected Fisher’s one-sided exact test on differentially expressed genes). g UMAP representation of all CD4 + T cells predicted to have a motif in GLIPH2 analysis as seen in expanded versus non expanded cells. h DE genes of CD4 + T cells that are predicted to have a pattern in GLIPH2 and are enriched to ST as compared to PB. i All common clones (defined by the exact same CDR3b sequence) in CD8 + T cells between ST and PB presented in the UMAP representation from panel 2f. j Cells with clones CASRGGTSITDTQYF as projected in UMAP representation from 2f. k DE genes between ST and PB cells sharing the same CDR3b sequence CASRGGTSITDTQYF in Pt2-SP (Padj < 0.05, calculated with Bonferroni corrected two-sided t-test)