High-Throughput Single-Cell Functional and Molecular Profiling of Immune Cells in Cancer Immunotherapy



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Immunotherapy has revolutionized the treatment of cancer and newer approaches including the adoptive transfer of genetically modified T cells reprogrammed to target tumor antigens have shown remarkable responses. Despite their promise, the efficacy of adoptive immunotherapy remains unpredictable due to the heterogeneous nature of the infusion products, patients’ characteristics, treatment regimens, and tumor burdens. Specifically with regards to the T-cell infusion product, there is a need to develop methodologies that allow for definition of potencies to understand the phenotypic, molecular, and functional contribution of infusion products at single-cell level. In the first part of this dissertation, we implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to demonstrate that while CD4+CAR+ (CAR4) cells killed at slower rate, most likely due to lower granzyme B content, they benefited from apoptosis resistance compared to CD8+CAR+ (CAR8) cells. These findings suggest that overall potency of multi-killing should be evaluated together in their context to resist apoptosis. In the second part of this dissertation, we developed single-cell multiplexed platforms comprising beads biosensors for detecting protein secretion, TIMING to monitor motility and cell-cell interactions, and microfluidic qPCR for transcriptional profiling. Analysis of thousands of single-cell interactions for over 5 hours revealed that the integrated behavior of polyfunctional T cells that kill and secrete IFN-γ was similar to those without IFN-γ secretion, suggesting cytolysis to be the dominant determinant of the interaction behavior and that killing enables faster synapse termination. In addition, tracking the speed of these cells by TIMING indicated that serial killer T cells may be identified based on their high out-of-contact basal motility. Transcriptional profiling of these single-cells confirmed that the motile cells expressed increased amounts of perforin and displayed an activated phenotype. In summary, these results highlight the heterogeneity of immune cells and thus, the need for definition of potency prior to infusion. We propose that single-cell platforms as demonstrated here are suitable to uncover the diversity and to help identify optimal functional and molecular biomarkers for applications in the clinic.



Single cell, TIMING, High-throughput, CAR T cells, Cytokine, Single-cell gene expression, Beads


Portions of this document appear in: Liadi, Ivan, Harjeet Singh, Gabrielle Romain, Nicolas Rey-Villamizar, Amine Merouane, Jay R. T. Adolacion, Partow Kebriaei et al. "Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells." Cancer immunology research 3, no. 5 (2015): 473-482. DOI: 10.1158/2326-6066