Postdoctoral Researcher La Jolla Inst. for Immunol., United States
Disclosure(s):
Han Feng, PhD: No financial relationships to disclose
Introduction/Rationale: 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.
