Single cell immune profiling in ankylosing spondylitis reveals resistance of CD8+ T cells to immune exhaustion

Abstract

Persistent chronic inflammation is a hallmark of ankylosing spondylitis (AS), with cytotoxic T cells (CTLs) increasingly implicated in its pathogenesis. Ordinarily, T cell exhaustion follows sustained, persistent T cell activation to limit collateral tissue damage. Using mass cytometry and single-cell RNA sequencing (scRNA-seq), we identified a clonally expanded CTL subset in AS synovial fluid that expresses inhibitory receptors (PD-1, TIGIT, LAG-3) yet retains its effector capacity to express granzymes, perforin, TNF-α, and IFN-γ. Gene expression profile of this CTL subset shows the downregulation of canonical exhaustion markers. At the protein level, TOX, a critical transcription factor regulating CTL exhaustion, is downregulated in PD-1⁺TIGIT⁺LAG-3⁺CTLs. In-silico trajectory analyses suggest that these cells may differentiate into other effector CTL subsets. Our findings reveal a checkpoint-expressing CTL population in AS that resists exhaustion and retains an activated, effector phenotype. We propose that failure to undergo exhaustion may be a fundamental mechanism sustaining AS chronic inflammation.

Keywords: Immune response; Immunology; Transcriptomics.

Graphical abstract

Figure 1

Dysregulation of immune checkpoint expression on peripheral cytotoxic cells (CTLs) in patients with ankylosing spondylitis (A) PD-1 and TIGIT expression on peripheral CTLs from age and sex-matched healthy control subjects (n = 10) and patients with active axial spondyloarthritis (axSpA/ankylosing spondylitis/AS) (n = 18). ∗p < 0.05, calculated by Mann-Whitney U test. (B) Uniform manifold approximation and projection (UMAP) plot shows CTL PhenoGraph clusters in AS and healthy control (HC) PBMCs. Clusters are annotated by cluster number (Right). (C) CTL clusters with the proportions significantly increased in AS PBMC versus HC PBMC (red, Log2 FC > 0) or significantly decreased (blue, Log2 FC < 0). Clusters in gray: no significant proportion difference. (D) Bar Graph shows the log2 fold proportional change (Log2 FC) for each cluster between the two groups (AS PBMC vs. HC PBMC). ∗∗p < 0.005, ∗p < 0.05. The p values are calculated by the diffcyt R package. (E) For each PhenoGraph cluster, the colors represent the row Z score values of the median expression values of the indicated markers. Heatmap is generated by the ComplexHeatmap R package. (F) Stacked bar plot shows the frequencies of each PhenoGraph cluster as a percentage of total CD8⁺ T cells for 10 HC PBMCs and 18 AS PBMCs. (G) Frequency of biaxially gated- CD38 expressing effector CTLs defined as CD3⁺ CD8⁺ CD45RA- CCR7- CD27⁺ CD28⁺ for 10 HC PBMCs and 18 AS PBMCs. ∗p < 0.05, calculated by Mann-Whitney U test.

Figure 2

Inhibitory receptor expressing CTLs are highly enriched in the synovial fluid of patients with ankylosing spondylitis (A) Uniform manifold approximation and projection (UMAP) plot shows CTL PhenoGraph clusters in AS SFMCs and matched AS PBMCs. Clusters are annotated by cluster numbers (Right). (B) CTL clusters with the proportions significantly increased in AS PBMC versus HC PBMC (red, Log2 FC > 0) or significantly decreased (blue, Log2 FC < 0). Clusters in gray: no significant proportion difference. (C) Bar Graph shows the log2 fold proportional change (Log2 FC) for each cluster between the two groups (AS SFMC vs. matched PBMC). ∗∗∗p < 0.0005, ∗∗p < 0.005, ∗p < 0.05. The p values are calculated by the diffcyt R package. (D) For each PhenoGraph cluster, the colors represent the row Z score values of the median expression values of the indicated markers. Heatmap is generated by the ComplexHeatmap R package. (E) Stacked bar plot shows the frequencies of each PhenoGraph cluster as a percentage of total CD8⁺ T cells for 8 pairs of SFMCs-PBMCs. (F) Mass cytometric analyses to quantify proportions of CD127- PD-1+ TIGIT+ CTLs from synovial fluid of patients with AS. ∗p < 0.05, calculated by Friedman repeated measures test with Bonferroni correction.

Figure 3

Single cell RNA sequencing (scRNA-seq) of AS synovial fluid CTLs (A) Uniform manifold approximation and projection (UMAP) plots display the transcriptomics profiles of sorted CD45RO + CTLs from the indicated groups. HC- Healthy control CD45RO + CTL from PBMC; AS CD45RO + CTL from PBMC; AS CD45RO + CTL from SFMC; PD-1+ TIGIT+ CD127- CTL from SFMC. (B) CTL clusters with the proportions significantly increased (red, Log2 FC > 0) or significantly decreased (blue, Log2 FC < 0) in PD-1+ TIGIT+ CD127- CD45RO + CD8⁺ T cells versus matched peripheral blood (from the same patient) CD45RO + CD8⁺ T cells. Clusters in gray: no significant proportion difference. (C) Bar graph shows the log2 fold proportional change (Log2 FC) for each scRNA-seq cluster between PD-1+ TIGIT+ CD127- SF CTL and matched peripheral blood CD45RO + CTL. ∗∗∗p < 0.001, ∗p < 0.05. p-values are calculated by the diffcyt R package. (D) Dot plot shows the relative, scaled expression of the indicated exhaustion-related genes (ENTPD1, CD244, CD101, CDKN2A, TIGIT, PDCD1, CTLA4, LAG3, and HAVCR2; x axis) for each Seurat cluster (y axis). (E) UMAP clustering of CTLs captured by sequencing mature CD45RO + CTLs from PBMCs of healthy control (n = 4) and patients with AS (n = 4); SFMCs of patients with AS (n = 4) and PD-1+ TIGIT+ CD127- CTL sorted from SFMCs of patients with AS (n = 3).

Figure 4

Trajectory analyses depicting the developmental relationship between CTL clusters identified by scRNA-seq (A) UMAP plot displays Slingshot trajectory inferences generated by “GEX_lineage_trajectories” function from the Platypus R package. (B) UMAP plot displays Monocle3 trajectory inferences. The principal roots of the trajectories were defined from early differentiated memory CTL clusters- 1, 3, and 8. Rectangular box depicts possible developmental trajectories within cluster 2. (C) UMAP plot (left) displays scaled expression of the TOX transcript. Dot plot (right) representation of scaled expression of TOX within each indicated Seurat cluster (y axis). (D) Pseudo-time analysis of the development trajectories. UMAP plot shows Monocle3 trajectory inferences overlaying with pseudo-time values generated by Monocle3. (E) Boxplot representation of average pseudo-time values for each indicated cell type, defined in the cluster identification analysis.

Figure 5

Synovial fluid CD127- PD-1+ TIGIT+ CTLs retain some capacity to produce cytolytic molecules and retain the capacity to produce inflammatory cytokines upon stimulation (A and B) Mass cytometric analyses to quantify median expression levels of intracellular granzyme B (A) and perforin (B) peripheral blood (PB) CTLs and synovial fluid (SF) CTLs from 8 pairs of active patients with AS. ∗p < 0.05 calculated with Friedman repeated measures tests with Bonferroni correction. (C) Mass cytometric analyses to quantify median expression levels of cell surface CD38 in peripheral blood (PB) CTLs and synovial fluid (SF) CTLs from 8 pairs of patients with active AS. ∗p < 0.05 calculated with Friedman repeated measures tests with Bonferroni correction. (D) Cytokine release assay; Gating strategy to measure intracellular IFNγ and TNFα. CD127+ and CD127- PD-1+ TIGIT+ CTLs were FACS-sorted from paired SFMC and PBMC samples, then stimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A. Intracellular IFNγ and TNFα were measured by flow cytometry. (E and F) Graphs display proportions of CD8⁺ T cells expressing intracellular IFNγ (E) and TNFα (F) ∗p < 0.05; calculated by Friedman repeated measures test with Bonferroni correction.

Figure 6

Downregulation of TOX expression in CD127- PD-1+ CTLs in SFMCs from patients with ankylosing spondylitis (A) Representative flow cytometric analyses of blood or synovial fluid (SF) PD-1+ CD127+ and CD127- CTL for TOX transcription factor expression. CD127+ and CD127- CTLs were gated from live CD3⁺ CD8⁺ PD-1+ cells. Fluorescence minus one (FMO) control were used to define CD127+, CD127-and PD-1+ CD8⁺ T cells. (B) TOX expression, measured by median fluorescence intensities (MFI), in CD127- (left) and CD127+ (right) PD-1+ CTLs in paired blood and SF from 6 patients. ∗p < 0.05, calculated by two-sided Wilcoxon-signed ranked test; ns-not significant. (C) UMAP clustering (top) of CTLs captured by sequencing mature CD45RO + CTLs from PBMCs of healthy control (n = 4) and patients with AS (n = 4); SFMCs of patients with AS (n = 4) and PD-1+ TIGIT+ CD127- CTL sorted from SFMCs of patients with AS (n = 3). Dot plot (bottom) shows the relative, scaled expression of the indicated genes (TIGIT, PDCD1, CTLA4, LAG3, HAVCR2, TOX, and TOX2; x axis) for each Seurat cluster (y axis).