Immunometabolic Adaptation of CD19-Targeted CAR T Cells in the Central Nervous System Microenvironment of Patients Promotes Memory Development.

Metabolic reprogramming is a hallmark of T-cell activation, and metabolic fitness is fundamental for T-cell-mediated antitumor immunity. Insights into the metabolic plasticity of chimeric antigen receptor (CAR) T cells in patients could help identify approaches to improve their efficacy in treating cancer. Here, we investigated the spatiotemporal immunometabolic adaptation of CD19-targeted CAR T cells using clinical samples from CAR T-cell-treated patients. Context-dependent immunometabolic adaptation of CAR T cells demonstrated the link between their metabolism, activation, differentiation, function, and local microenvironment. Specifically, compared with the peripheral blood, low lipid availability, high IL15, and low TGFß in the central nervous system microenvironment promoted immunometabolic adaptation of CAR T cells, including upregulation of a lipolytic signature and memory properties. Pharmacologic inhibition of lipolysis in cerebrospinal fluid led to decreased CAR T-cell survival. Furthermore, manufacturing CAR T cells in cerebrospinal fluid enhanced their metabolic fitness and antileukemic activity. Overall, this study elucidates spatiotemporal immunometabolic rewiring of CAR T cells in patients and demonstrates that these adaptations can be exploited to maximize the therapeutic efficacy of CAR T cells. SIGNIFICANCE: The spatiotemporal immunometabolic landscape of CD19-targeted CAR T cells from patients reveals metabolic adaptations in specific microenvironments that can be exploited to maximize the therapeutic efficacy of CAR T cells.

Single-cell analysis by mass cytometry reveals CD19 CAR T cell spatiotemporal plasticity in patients.

The adaptive T cell immune response requires cellular plasticity to generate distinct subsets with diverse functional and migratory capacities. Studies of CAR T cells have primarily focused on a limited number of phenotypic markers in blood, representing an incomplete view of CAR T cell complexity. Here, we adapted mass cytometry to simultaneously analyze trafficking and functional proteins expression in CD19 CAR T cells across patients' tissues, including leukapheresis T cells, CAR product, CAR T cells in peripheral blood, bone marrow, and cerebrospinal fluid post infusion and correlate them with phenotypes. This approach revealed spatiotemporal plasticity of CAR T cells. Patients' CAR product revealed upregulation in many trafficking and activation molecules compared to leukapheresis T cells as baseline. Including statistically significant upregulation in CD4 and CD8 integrin-ß7, CD4 granzyme B, and CD11a as well as CD8 CD25 and CD95. Moreover, patients' tissues showed spatiotemporal alteration in trafficking, activation, maturation, and exhaustion features, with a distinct signature in the central nervous system niche. Compared to peripheral blood samples, cerebrospinal fluid samples were statistically significant enriched in CD4 and CD8 trafficking and memory phenotype proteins integrin ß7, CCR7, CXCR4, and CD8 CD69. Our data provide a potential framework to remodel CAR T cells and enhance immunotherapy efficacy.