Single Cell Immune Profiling in Ankylosing Spondylitis Reveals Resistance of CD8+ T cells to Immune Exhaustion

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-a, and IFN-g. Gene expression profile of this CTL subset show 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 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. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) isolated from AS patients annd healthy control subjuects were thawed, and then FACS (Fluorescence activated cell sorting)-sorted for CD45RO+ CD3+ CD8+ cells for single cell RNA sequencing. For three patients, CD45RO+ CD3+ CD8+ PD-1+ TIGIT+ CD127- cells were also FACS-sorted. We generated libraries for single cell immune profiling (10x Genomics; 5' Gene expression integrated with VDJ profiling).