Browsing by Author "Cooper, Laurence J.N."
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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 Quantitative High-Throughput Single-Cell Cytotoxicity Assay for T cells(Journal of Visualized Experiments, 2013) Liadi, Ivan; Roszik, Jason; Romain, Gabrielle; Cooper, Laurence J.N.; Varadarajan, NavinCancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5. Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.