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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-β7, 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 β7, CCR7, CXCR4, and CD8 CD69. Our data provide a potential framework to remodel CAR T cells and enhance immunotherapy efficacy. [ABSTRACT FROM AUTHOR]

High-grade (WHO grades III-IV) glioma remains one of the most lethal human cancers. Adoptive transfer of tumor-targeting chimeric antigen receptor (CAR)-redirected T cells for high-grade glioma has revealed promising indications of anti-tumor activity, but objective clinical responses remain elusive for most patients. A significant challenge to effective immunotherapy is the highly heterogeneous structure of these tumors, including large variations in the magnitudes and distributions of target antigen expression, observed both within individual tumors and between patients. To obtain a more detailed understanding of immunotherapy target antigens within patient tumors, we immunochemically mapped at single cell resolution three clinically-relevant targets, IL13Rα2, HER2 and EGFR, on tumor samples drawn from a 43-patient cohort. We observed that within individual tumor samples, expression of these antigens was neither random nor uniform, but rather that they mapped into local neighborhoods – phenotypically similar cells within regions of cellular tumor – reflecting not well understood properties of tumor cells and their milieu. Notably, tumor cell neighborhoods of high antigen expression were not arranged independently within regions. For example, in cellular tumor regions, neighborhoods of high IL13Rα2 and HER2 expression appeared to be reciprocal to those of EGFR, while in areas of pseudopalisading necrosis, expression of IL13Rα2 and HER2, but not EGFR, appeared to reflect the radial organization of tumor cells around hypoxic cores. Other structural features affecting expression of immunotherapy target antigens remain to be elucidated. This structured but heterogeneous organization of antigen expression in high grade glioma is highly permissive for antigen escape, and combinatorial antigen targeting is a commonly suggested potential mitigating strategy. Deeper understanding of antigen expression within and between patient tumors will enhance optimization of combination immunotherapies, the most immediate clinical application of the observations presented here being the importance of including (wild-type) EGFR as a target antigen. [ABSTRACT FROM AUTHOR]