Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Department of Chemical and Biomolecular Engineering, University of Houston.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2013; DOI: 10.3791/50058
Source: PubMed


Cancer 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 trials(1-4). 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 toxicity(5). 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 vivo(6,7). 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 targets(8). Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes(9). 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.

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    • "Unlike most cytotoxicity assays, which indirectly relate cell death to the release of cytoplasmic contents into a bulk solution, this technique allows the researcher to obtain more accurate cytotoxicity data by directly reporting the number of living and dead cells. Such single-cell analysis capabilities [28], [29] were critically important to achieving the high precision observed in these assays, enabled the use of much smaller cell samples, and were required for the analysis of cytotoxicity in whole blood. This is particularly powerful when combined with the automated imaging and data analysis platforms that are available from a number of different vendors. "
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