Abstract LB293: Cystine restriction enhances CD8+ CAR-T potency by promoting OXPHOS via GCN2-eIF2α-SLC1A5 axis

Abstract Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors, primarily due to functional exhaustion and impaired metabolic fitness of CD8⁺ T cells. Targeting immunometabolic checkpoints may provide a strategy to overcome these limitations. However, the metabolic pathways governing CD8⁺ T cell exhaustion in solid tumors remain incompletely defined.To identify metabolic regulators of CD8⁺ T cell exhaustion, we established an in vitro exhaustion model and performed transcriptomic profiling, which revealed significant upregulation of the cystine/glutamate antiporter SLC7A11 in exhausted CD8⁺ T cells. Elevated SLC7A11 expression was further confirmed in PD-1⁺TIM-3⁺ exhausted CD8⁺ T cells from colorectal cancer patient samples. We generated and humanized a monoclonal antibody targeting SLC7A11, and its specificity and binding affinity were validated by flow cytometry and surface plasmon resonance analysis.Functional studies demonstrated that pharmacologic blockade or genetic inhibition of SLC7A11 alleviated CD8⁺ T cell exhaustion, promoted the expansion of stem-like memory (TSCM) cells, and reduced the expression of exhaustion markers. In CEA-specific CD8⁺ CAR-T cells, SLC7A11 inhibition enhanced tumor cytotoxicity, decreased exhaustion-associated transcriptional programs, and upregulated memory-associated gene signatures. Metabolic analyses revealed that limiting cystine uptake increased mitochondrial oxidative phosphorylation, ATP production, mitochondrial mass, and cristae density, indicating improved bioenergetic fitness.Mechanistically, cystine restriction activated the integrated stress response, characterized by activation of the GCN2-eIF2α-ATF4 pathway and transcriptional upregulation of the glutamine transporter SLC1A5. Increased glutamine uptake and mTORC1 signaling further supported mitochondrial metabolism. Chromatin immunoprecipitation confirmed direct binding of ATF4 to the SLC1A5 promoter, establishing a mechanistic link between cystine restriction and metabolic reprogramming in CAR-T cells. In vivo, treatment with the anti-SLC7A11 antibody significantly enhanced CAR-T cell-mediated tumor control in colorectal and breast cancer cell-derived xenograft models, outperforming either monotherapy. Consistently, genetic ablation of Slc7a11 in mice reduced tumor burden and synergized with immune checkpoint blockade. Single-cell RNA sequencing of tumor-infiltrating lymphocytes revealed a shift toward naïve, stem-like, and memory CD8⁺ T cell states, reduced exhausted populations, and altered clonal dynamics following Slc7a11 deletion. Collectively, our findings identify cystine metabolism as a previously underappreciated immunometabolic checkpoint regulating CD8⁺ T cell exhaustion and demonstrate that targeting SLC7A11 represents a promising strategy to enhance CAR-T cell efficacy in solid tumors. Citation Format: Xiaoxue Pan, Yuhang Yin, Pengyuan Wang, Shanwen Chen. Cystine restriction enhances CD8+ CAR-T potency by promoting OXPHOS via GCN2-eIF2α-SLC1A5 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB293.