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.
© 2025 The Authors.

CD8+ T cell exhaustion (Tex) limits immune control of cancer, but the underlying molecular drivers are unclear. In the present study, we identified the prostaglandin I2 (prostacyclin) receptor PTGIR as a cell-intrinsic regulator of T cell exhaustion. Transcriptomic profiling of terminally exhausted (Ttex) CD8+ T cells revealed increased activation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response pathway. Enhancing NRF2 activity (by conditional deletion of Kelch-like ECH-associated protein 1 (KEAP1)) boosts glutathione production in CD8+ T cells but accelerates terminal exhaustion. NRF2 upregulates PTGIR expression in CD8+ T cells. Silencing PTGIR expression enhances T cell effector function (that is, interferon-γ and granzyme production) and limits Ttex cell development in chronic infection and cancer models. Mechanistically, PTGIR signaling impairs T cell metabolism and cytokine production while inducing transcriptional features of Tex cells. These findings identify PTGIR as a NRF2-dependent immune checkpoint that regulates balance between effector and exhausted CD8+ T cell states.
© 2025. The Author(s).

Long-lived memory CD8+ T cells are essential for the control of persistent viral infections. The mechanisms that preserve memory cells are poorly understood. Fate mapping of the transcriptional repressor GFI1 identified that GFI1 was differentially regulated in virus-specific CD8+ T cells and was selectively expressed in stem cell memory and central memory cells. Deletion of GFI1 led to reduced proliferation and progressive loss of memory T cells, which in turn resulted in failure to maintain antigen-specific CD8+ T cell populations following infection with chronic lymphocytic choriomeningitis virus or murine cytomegalovirus. Ablation of GFI1 resulted in downregulation of the transcription factors EOMES and BCL-2 in memory CD8+ T cells. Ectopic expression of EOMES rescued the expression of BCL-2, but the persistence of memory CD8+ T cells was only partially rescued. These findings highlight the critical role of GFI1 in the long-term maintenance of memory CD8+ T cells in persistent infections by sustaining their proliferative potential.
© 2025. Crown.

NR1B1/RARα expression is dynamically regulated in cytotoxic lymphocytes (CTLs) in tumors, but the importance of its expression in anti-tumor CTLs remains unknown. RARα gene expression is upregulated in CTLs in tumor microenvironments (TME), but its protein expression is downregulated by retinoic acid. The role of RARα expression in regulating anti-tumor effector CTL (Teff) differentiation is reported. Mice that over-express RARα in T cells are defective in early Teff differentiation and fail to populate tumors. In contrast, RARα-deficient CTLs are hyper-active in making tumor-populating Teff cells, suggesting that RARα represses Teff differentiation. Moreover, RARα negatively controls the trafficking receptor switch from the lymphoid to an effector type. Generation of chimeric antigen receptor (CAR) T cells with reduced RARα expression produces highly effective CAR T cells with enhanced anti-tumor cytotoxicity. Mechanistically, upregulated RARα expression decreases the nuclear histone acetylase (HAT) activity, required for TCF1 to BATF transcription factor and trafficking switches during Teff differentiation. Additionally, RARα and BATF closely associate with each other on Teff-associated genes on the chromatin for possible cross-regulation. In sum, T cell-expressed RARα is identified as a novel negative regulator and potential target of intervention in promoting anti-cancer T cell immunity.
© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.

Chimeric antigen receptor T cells (CAR T cells) with T stem (TSCM) cell-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM cell-like CAR T cells. We identified three KIs, UNC10225387B, UNC10225263A and UNC10112761A, that combined in vitro increased the frequency of CD45RA+CCR7+TCF1hi TSCM cell-like CAR T cells from both healthy donors and patients with cancer. KI-treated CAR T cells showed enhanced antitumor effects both in vitro and in vivo in mouse tumor models. The KI cocktail maintains TSCM cell-like phenotype preferentially in CAR T cells originating from naive T cells and causes transcriptomic changes without arresting T cell activation or modulating the chromatin organization. Specific kinases, ITK, ADCK3, MAP3K4 and CDK13, targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM cell-like CAR T cells. Knockdown of these kinases individually or in combination enriches for TSCM cell-like CAR T cells, but only CAR T cells generated in the presence of the KI cocktail show robust expansion and differentiation on stimulation with tumor cells. Overall, transient pharmacological inhibition of strategically targeted kinases maintains stem-like features in CAR T cells and improves their antitumor activity.
© 2025. The Author(s).