Date of Award
10-14-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Natural killer (NK) cells are innate lymphoid cells that are a first line of defense against virally infected or malignant cells. Their ability to recognize and kill target cells is controlled by engagement of inhibitory, activating, and cytokine receptors. This leads to release of granules that contain perforin and granzymes, expression of death receptor ligands, and production of cytokines and chemokines. An effective NK cell response results in target cell death, along with changes in the nearby cellular microenvironment to promote immunity. As such NK cells play an essential role in tumor immunosurveillance, and there is an inverse correlation between circulating NK cells and the presence of metastases in various cancers. Numerous studies have also demonstrated a high frequency of dysfunctional NK cells in cancer patients. In addition, immunotherapy utilizing adoptively transferred conventional (c) NK cells do not have durable responses due to lack of persistence and poor function in patients. Therefore, strategies to restore and augment NK cell function are a nascent immunotherapy approach. This premise of NK cell therapy is well supported by pre-clinical studies in mouse and human models of hematological malignancies that demonstrate effective anti-tumor responses. One approach to overcome the current limitations of cNK cells is the initiation and differentiation of memory-like programs, which was pioneered by our group at Washington University. This approach utilizes brief initiating cytokine receptor signals via (IL)-12, -15, and -18 (IL-12/15/18) which results in the differentiation of memory-like (ML) NK cells after 5-7 days. ML NK cells are not limited by antigen specificity and exhibit enhanced functionality after different types of restimulation (e.g. with IL-12/15 or tumor target cells). Also, ML NK cells continue to demonstrate their enhanced functional responses after multiple cell divisions suggesting that there are molecular mechanisms that imprint a heritable epigenetic state. Moreover, ML NK cells have improved cytotoxicity and IFN-γ production, reduced sensitivity to KIR inhibition, enhanced in vivo persistence, as well as a unique multidimensional phenotype including increased expression of activating receptors and granzyme B. This in-depth functional characterization of ML NK cells has allowed for their advancement into the clinic. ML NK cells are being investigated to augment hematopoietic cell transplantation (HCT) and treat post-HCT relapse in leukemia patients. In these trials, ML NK cells were able to expand and persist for months in vivo, quantified using a unique multidimensional mass cytometry and CITE-seq. While much is known about their functional characteristics, the underlying mechanisms involved in programming conventional (c)NK cells into ML NK cells are poorly understood. Further, whether all cNK cells that are stimulated with IL-12/15/18 become memory-like NK cells is also unclear. In this research study, we discovered that IL-12/15/18 activation/initiation and subsequent ML differentiation resulted in multiple cell fates, including an enriched ML population (eML), as well as effector (eff)cNK cells that are similar to cytokine-primed cNK cells. We identified marked transcriptional and chromatin accessibility changes that occur during early IL-12/15/18 initiation of the ML program, some of which persist in the fully differentiated ML NK cell state while others arise during differentiation. eML NK cells have unique transcriptional, epigenetic, and functional programs compared to effcNK and cNK cells, and unexpectedly could be identified by expression of ENTPD1 (CD39). eML NK cells identified by flow cytometry had the highest functional (IFN-γ) response to cytokine or tumor target stimulation, as well as robust cytotoxicity. Importantly, eML NK cells had a unique global ATAC-seq signature after differentiation that aligned with their enhanced functionality, while the epigenetic profile of effcNK cells was similar to cNK cells. The interrogation of mechanisms driving the unique eML NK cell epigenetic profiles was further defined by distinct DNA methylation patterns and histone modifications. Within eML NK cells, two distinct subsets were identified by CITE-seq, which have distinct transcription factor signatures. Moreover, these two subsets also have unique cell origins arising from the CD56bright subset (eML-1) or CD56dim subset (eML-2) of cNK cells. In patients treated with ML NK cell therapy on clinical trials, both eML-1 and eML-2 subsets were readily identified by scRNA-seq and flow cytometry several months after transfer. Using a functional CITE-seq assay to interrogate single-cell NK cell responses following cytokine and tumor cell stimulation, eML-1 NK cells have a unique transcriptional response including increased IFNG transcript, while eML-2 NK cells had molecular features of exhaustion. Although, eML-2 NK cells have features associated with exhaustion, this was uncoupled with their potent functionality. Collectively, this study establishes a new cellular and molecular model of cytokine-induced ML NK cell initiation and differentiation and defines epigenetic and transcriptional mechanisms that drive ML NK cell programs. These findings reveal novel features of ML NK cell subset biology, as well as inform new strategies to further improve IL-12/15/18-induced ML NK cells as a cancer therapy.
Language
English (en)
Chair and Committee
Todd Fehniger
Committee Members
David Spencer; Jacqueline Payton; Marco Colonna; Megan Cooper; Ting Wang
Recommended Citation
Tran, Jennifer, "Elucidating the Heterogeneity and Epigenetic Determinants of Memory-like Natural Killer Cell Differentiation" (2024). Arts & Sciences Electronic Theses and Dissertations. 3265.
https://openscholarship.wustl.edu/art_sci_etds/3265