Single-cell Functional Profiling of Lymphocytes for Cancer Immunotherapy



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Immunotherapy 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.



Cancer Immunotherapy, Single cell, Immune checkpoint, Immunology, T cells, PD-1, Lymphocyte


Portions of this document appear in: An, Xingyue, and Navin Varadarajan. "Single-cell technologies for profiling T cells to enable monitoring of immunotherapies." Current opinion in chemical engineering 19 (2018): 142-152. And in: An, Xingyue, Victor G. Sendra, Ivan Liadi, Balakrishnan Ramesh, Gabrielle Romain, Cara Haymaker, Melisa Martinez-Paniagua et al. "Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells." PloS one 12, no. 8 (2017): e0181904.