GPR25 as a novel target to boost anti-tumor TRM responses 2300820

Abstract

Abstract Introduction Tissue-resident memory CD8 T (TRM) cells are central to durable antiviral and antitumor immunity, yet the molecular cues that program their formation remain incompletely defined. We identified the orphan G protein—coupled receptor GPR25 as enriched in TRM cells and induced by TGF-β, suggesting a role in TRM differentiation and maintenance. Methods Using adoptive-transfer models of viral infection and tumor challenge, we compared trafficking, differentiation and recall responses of wild-type and Gpr25-deficient CD8 T cells. Single-cell RNA sequencing dissected transcriptional programs altered by Gpr25 deficiency. We validated TGF-β downstream signaling by modulating GPR25 expression and tested reported ligands (CXCL17 and cleaved 4CysCXCL17) in orthogonal β-arrestin and calcium-mobilization assays. Finally, we implemented a high-throughput screening (HTS) pipeline–based on inducible GPR25 reporter cell lines and calcium/β-arrestin readouts–to discover small-molecule GPR25 agonists. Results Gpr25-deficient CD8 T cells trafficked normally but failed to develop and sustain TRM populations in lung and liver, with reduced stem-like features (including TCF1), and exhibited impaired secondary TRM expansion after rechallenge. Transcriptomes of Gpr25-deficient cells exhibited reduced TGF-β—responsive signatures, and SMAD2/3 phosphorylation in vitro. Besides, we were unable to replicate reported CXCL17—GPR25 agonism across multiple assay platforms. Leveraging inducible GPR25 reporter lines, our HTS workflow reliably detects ligand-dependent and ligand-enriched activities and is poised to identify small-molecule agonists. Conclusion GPR25 is a key regulator of TRM differentiation and maintenance through enhancement of TGF-β signaling. Failure to validate CXCL17 as the endogenous ligand underscores the need for systematic deorphanization. Our HTS platform positions us to discover GPR25 agonists with the potential to augment TRM-mediated antitumor immunity and translate into novel immunotherapeutics. Funding Source William K. Bowes Jr Foundation (to P.V.), Whittaker Foundation (to c.h.o.), curebound Discovery grant 24Dg05 (to P.V. and m.R.J.) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)