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Background The ideal product features required to achieve durable response following CAR T-cell therapy are unknown. Here, we performed a comprehensive analysis encompassing functional experiments in vitro paired with pre-clinical studies using infused product, and multi-omics analysis of the anti-CD19 CAR T-cell therapy Axicabtagene Ciloleucel (axi-cel) to identify mechanisms leading to treatment resistance.Methods We conducted genomic and functional profiling of axi-cel products from 36 patients with relapse/refractory LBCL from Zuma-1 and correlated findings with in vivo efficacy, clinical outcomes, and post-treatment patient CAR T-cell expansion and cytokine levels. Cytokine data were validated in an expanded multi-trial meta-cohort of 586 patients treated with axi-cel. Product characterization was performed with scRNAseq and scATACseq, while cytokine secretion was measured following stimulation with CD19+ tumor cells or CD19-coated red blood cells. In vivo efficacy was evaluated in tumor-bearing NSG-MHC I/II DKO mice, through survival, as well as peripheral blood CAR T-cell kinetics and cytokine levels ( figure 1).Results In a B-cell lymphoma model, mice treated with CAR T products from patients with durable responses demonstrated superior tumor clearance and survival. Mouse survival significantly correlated with clinical response in ZUMA-1 patients (R=0.60; P = 0.007, figure 2) and with CAR T-cell expansion in both mice and patients.scRNAseq of axi-cel products revealed 13 transcriptional clusters, with disease progression significantly associated (P = 0.029) with a CD8+ T-cell cluster, overexpressing Th2 master regulator, GATA3, and with a hypoproliferative, exhausted cluster (P < 0.05, figure 2). Both clusters showed enrichment of the exhaustion marker TIGIT. Matched scATAC-seq presented an enrichment in chromatin accessibility at the GATA3 promoter in CD8+ T cells from non-responders. Immunophenotyping and intracellular cytokine staining post stimulation corroborated Th2 polarized, IL-4-producing GATA3+CD8+ T cells as a distinguishing feature of non-responders. In mice, axi-cel products enriched with this subset were associated with reduced survival.CD19 antigen stimulation of axi-cel products induced production of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-5, IL-13) cytokines, highlighting differences in Th1/Th2 polarization: a Th1-Th2 index strongly associated with clinical efficacy (P < 0.05), confirming Th2 cytokine production was enriched in axi-cel products from non-responders. In a pooled axi-cel cohort (n=586, ZUMA-1, -5, -7, -12), post-infusion peak serum cytokine-calculated Th1-Th2 index associated strongly with PFS (P = 0.0062) and ongoing response (P < 0.0005).Conclusions GATA3+CD8+ T cells and Th1-Th2 index were found to be determinants of axi-cel efficacy, offering insights into mechanisms of resistance, risk stratification and guiding next-generation CAR T therapies.Abstract 266 Figure 1Schematic of the ex vivo functional profiling workflow applied axi-cel products from ZUMA-1Abstract 266 Figure 2Results from in vivo lymphoma model and genomics profiling of axi-cel products from ZUMA1