Single-Cell Analysis By Mass Cytometry Reveals CD19 CAR T Cell Spatiotemporal Plasticity in Patients
Authors: Goldberg Lior , Haas Eric R , Vyas Vibhuti , Urak Ryan , Forman Stephen J , Wang Xiuli 
Published: November 2021

Abstract
T cell adaptive immune response requires cellular plasticity to generate distinct subsets with diverse functional and migratory capacities. By expressing unique patterns of trafficking molecules, T cells interact with tissue-specific vascular endothelia for preferential recruitment to distinct tissues. Chimeric antigen receptors (CARs) are synthetically engineered receptors that redirect the specificity, effector function, and metabolism of transduced T cells. 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. To address this need, we used cytometry by time of flight (CyTOF) which enables high dimensional, in-depth, proteomic analysis of immune cells at the single-cell level. We designed an integrative panel with antibodies specific for CAR, cell lineage, activation, maturation, trafficking, and exhaustion markers (Figure 1A), and investigated the spatiotemporal landscape of CD19 CAR T cells across patients' tissues.
To interrogate the spatiotemporal dynamics of CAR T cells in vivo we analyzed clinical samples from three patients treated with CD19 CAR T cells for B cell hematological malignancies. For each patient we sampled the leukapheresis T cells, CAR product, PMBCs on days 7 and 28, BM on day 28 and cerebrospinal fluid on day 7 or 28 post CAR T cell infusion (Figure 1B). Patients CAR product revealed upregulation in most of the trafficking and activation molecules compared to leukapheresis T cells as baseline. Remarkably, the human tissue samples showed diverse spatiotemporal landscapes. For example, CSF samples were enriched in activation markers such as CD25, CD27, CD95, granzyme B; trafficking proteins such as integrin β7, CD11a, CD49d, CD62L, CD69, CCR7, CXCR3, CXCR4 and had diminished exhaustion marker PD-1 (Figure 1C).
To further explore the spatiotemporal dynamics, we employed FlowSOM which identified 21 distinct clusters (Figure 1D). Quantitation of clusters frequency and protein expression revealed significant heterogeneity, individual clusters showed different patterns of expansion and contraction by time and place (Figure 1E). For example, leukapheresis T cells were solely enriched in clusters 1-2 marked by CD45RA CD27, and CCR7 expression, while CAR product was enriched in clusters 9,11, and 13 marked by CXCR3, integrin β7 trafficking molecules and granzyme B expression. Furthermore, day 7 peripheral blood samples were solely enriched in clusters 19-20 marked by the expression of the activation markers HLA-DR, CD25, and granzyme B, in addition to the trafficking proteins CD49d, CD62L and CXCR3. Notably, cluster 12 which had low abundance in the leukapheresis T cells and was depleted in the CAR product, dominated day 7 peripheral blood samples, and persisted to a lesser degree in all tissue samples. Moreover, clusters 3 and 4 that were enriched in the CSF expressed high CXCR3, CXCR4, CCR7, integrin β7, CD62L, and CD49b which may attribute to their migration between blood and CSF.
Since CAR T cells are genetically modified T cells with distinct properties, we next asked to evaluate the phenotypic similarities between patients' tissue CAR T cells and T cell subsets in healthy donor PBMCs. Spearman correlation analysis demonstrated strong positive correlation between leukapheresed T cells and Naïve/stem cell memory phenotypes. Whereas, peripheral blood samples correlated the most with effector and effector memory phenotype, notably on day 28 post infusion. Interestingly, CSF samples possessed features of central/stem cell memory phenotype, mirroring our preclinical findings where CAR T cells exposed into CSF are memory-like T cells. (Wang, et al, Cancer Immunol Res, 2021), and BM CAR T cells, correlated with both effector and memory phenotypes, highlighting the role of the BM as a T cell memory niche.
In summary, mass cytometry enabled us for the first time, to our knowledge, to provide insights into the spatiotemporal plasticity of CAR T cell therapy. We revealed remarkable diversification within patients CAR T cells and their spatiotemporal relationship to T cells specialization within the human immune system and identified tissue specific CAR T cell expression profiles. Our work provides a potential framework to remodel CAR T cells for enhanced immunotherapy efficacy.
Figure 1 Figure 1.

Disclosures
Forman: Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy. Wang: Pepromene Bio, Inc.: Consultancy.