Browsing by Author "Singh, Harjeet"
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Item FUNCTIONAL METRICS OF EFFICACIOUS CAR T CELLS REVEALED BY MULTI-DIMENSIONAL SINGLE-CELL PROFILING(2022-12-13) Rezvan, Ali; Varadarajan, Navin; Willson, Richard C.; Conrad, Jacinta C.; Mohan, Chandra; Singh, Harjeet; Zhang, Xiaoliu ShaunChimeric antigen receptors (CAR) T cells for B-cell malignancies serve as a model for identifying T-cell subsets with superior clinical activity. We profiled the infusion products (IP) of patients with large B-cell lymphoma (LBCL) using multi-omic single-cell assays to reveal the therapeutic potential of CD19-CAR+ T cells. Functional profiling using timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed high migration and serial killing. Cellular profiling using confocal microscopy revealed that migration is correlated with both mitochondrial and lysosomal volume; and molecular profiling scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and in vivo persistence. T cells enriched for migratory capacity showed sustained serial killing and optimal in vivo efficacy. Our results demonstrate that migration is a cell-intrinsic biomarker desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.Item Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells(Cancer Immunology Research, 2016-05) Liadi, Ivan; Singh, Harjeet; Romain, Gabrielle; Rey-Villamizar, Nicolas; Merouane, Amine; Adolacion, Jay R.T.; Kebriaei, Partow; Huls, Helen; Qiu, Peng; Roysam, Badrinath; Cooper, Laurence J.N.; Varadarajan, NavinT cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR.Item Single-cell Functional Profiling of Lymphocytes for Cancer Immunotherapy(2019-08) An, Xingyue; Varadarajan, Navin; Cirino, Patrick C.; Rimer, Jeffrey D.; Peng, Weiyi; Singh, HarjeetImmunotherapy by harnessing patients’ the immune system has changed the landscape of cancer therapeutics and shown promising and remarkable clinical responses. However, not all the patients would be beneficial from the treatment. Lymphocytes are a significant target in anti-tumor immunotherapy, and the functional assessment of lymphocytes will provide insights on their functional biology and will provide a direct path to the improvement of the treatment efficacy. In the first part of this dissertation, we developed and implemented a methodology based on Timelapse Imaging Microscopy in Nanowell Grids (TIMING) platform that integrates phenotypic profiling and dynamic cytokine secretion with single-cell resolution. Analysis of hundreds of human peripheral nature killer cells (NK cells) suggested that CD56dimCD16+ NK cells are immediate interferon gamma (IFN-γ) secretor upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and no evidence of cooperation between NK cells to synergistic activation or faster IFN-γ secretion. These results establish our technology as an investigational tool for cellular phenotyping and real-time protein secretion of individual cells in a high-throughput manner and demonstrate that the conventional phenotypic based functional annotation of NK cells might be overly simplistic. In the second part of this dissertation, we performed whole transcriptomic profiling on T cells from acute myeloid leukemia patients (responders and non-responders) who were treated with combination therapy of a hypomethylating agent (5-azacytidine) and an immune checkpoint inhibitor (nivolumab, programmed cell death protein 1/PD-1 inhibitor). Sixty-four patient-derived T cells from peripheral blood or bone marrow (site of disease), which were collected before the initiation of the therapy (baseline, T0) and after the first round of treatment (end of cycle one, EC1), were evaluated. Our results demonstrate (1) treatment-induced gene expression changes on circulating CD8 T cells, and (2) the ratios of effector and exhausted CD8 T cells has the potential to serve as a biomarker for patient stratification.