Work

241 Engineering CAR-T cell resistance to CAF-derived paracrine cAMP-mediated immunosuppression

Year: 2026

Type: conference-abstract

Abstract: Background CAR-T cell therapy has shown limited activity in solid tumors, where suppressive features of the tumor microenvironment restrict T cell activation, function, and durability. We recently demonstrated that the protein kinase A (PKA)–cyclic AMP responsive element modulator (CREM) pathway is activated downstream of CAR and cytokine signaling and promotes activation-associated dysfunction, limiting anti-tumor efficacy. 1 Given the role of cyclic AMP (cAMP) as a key second messenger for immunosuppressive metabolic cues and the metabolic activity of cancer-associated fibroblasts (CAFs), we hypothesized that CREM integrates CAF-driven metabolic stress and that CREM disruption in CAR-T cells would overcome this suppression.Methods Primary human CAR-T cells were exposed to immunosuppressive metabolites known to elevate cAMP. T cell function was assessed by flow cytometry and Incucyte-based CAR-directed killing in 3D spheroids and 2D rechallenge assays. CREM expression was measured by RT-qPCR, and its functional role was tested using CRISPR/Cas9-mediated disruption via electroporation. To evaluate whether stromal cAMP-associated programs were linked to clinical resistance, we analyzed single-cell RNA-seq data from longitudinal on-treatment RCC biopsies stratified by clinical outcome. We then modeled stromal-mediated immunosuppression using mixed spheroids of 786-O RCC tumor cells and immortalized stellate CAFs.Results Lactic acid, PGE 2, and adenosine receptor agonists suppressed T cell effector function across complementary assays, including cytokine production, proliferation, and CAR-mediated tumor killing, with PGE2 producing the strongest and most consistent impairment. PGE2 rapidly induced CREM mRNA with isoform-specific patterns and kinetics distinct from other cAMP-inducing stimuli, including T cell receptor ligation. In longitudinal RCC scRNA-seq data, CAFs enriched at disease progression displayed an LRRC15+, TGFβ-associated myofibroblast phenotype with glycolytic, hypoxic, and cAMP-associated metabolic programs. These stromal programs correlated with exhausted, cAMP-high T cells bearing an active CREM regulon. In mixed spheroids, CAFs inhibited CAR-T cell-mediated tumor killing, recapitulating clinical observations, and this effect was further enhanced by hypoxic preconditioning. CAF-conditioned media, but not tumor-conditioned media, impaired T cell cytokine production and induced CREM in a pattern resembling PGE2 stimulation, supporting a role for CAF-derived soluble factors. CREM isoform-specific knockout rescued CAR-T cell killing in the presence of cAMP-inducing metabolites or CAFs and improved CAR-T cell effector function in vivo.Conclusions These findings identify CAF-derived paracrine cAMP-inducing signals as mediators of CAR-T cell dysfunction in solid tumors and nominate CREM as a targetable node for engineering resistance to stromal immunosuppression. Our CAF-rich modeling platform provides a framework to dissect stromal-metabolic suppression and guide targeting of cAMP-dependent pathways.Reference Rafei H, Basar R, Acharya S, et al. CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells. Nature 2025;643:1076–1086. doi:10.1038/s41586-025-09087-8.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0241