Traditionally, natural killer (NK) cells are defined as effector lymphocytes of innate immunity. However, during the last decade, adaptive features of NK cells have been demonstrated in animal models and humans. In humans, a specific subset of NK cells exhibiting the adaptive features of clonal expansion, altered effector functions, and long-term persistence have been found in approximately one third of healthy subjects. Despite this prevalence, the immune biology of adaptive NK cells, including their origin and the basis of their altered functions and phenotypes, are not well understood. In this dissertation work, I have conducted studies to address the mechanisms of altered functional activities and the development of human adaptive NK cells.

Human cytomegalovirus (HCMV) infection arguably plays a role in the induction of adaptive NK cells. Investigations of HCMV-induced adaptive NK cells have led to the recognition of significant changes in their transcriptional profiles, including a great number of genes regarded as potential regulators that might determine phenotypes and functions of adaptive NK cells. NK cells are notoriously challenging targets for genetic modification. Due to the lack of methods to study individual genes, the identities of key factors that regulate adaptive features of NK cells remained unclear. By integrating NK cell in vitro expansion, electroporation and CRISPR-Cas9 technologies, I successfully established a gene-editing platform for NK cells, and demonstrated that the deficiency of FcRγ (a signal transducing adaptor protein) directly contributes to many altered characteristics of adaptive NK cells. Studies in this area are presented in Chapters 2 - 4.

As adaptive NK cells are highly heterogeneous, it has been difficult to study their development/differentiation. To study this aspect of adaptive NK cells, a population of individuals that are at high risk of HCMV infection would be invaluable. For this work, I therefore analyzed NK cells in immunocompromised bone marrow transplantation (BMT) recipients, in whom NK cell populations could be monitored over time. By longitudinally analyzing NK cells in BMT patients, I provided new insights into the phenotypic configuration of adaptive NK cells and proposed mechanisms that regulate their expansion and function. Findings pertinent to this topic are presented in Chapter 5.

Finally, knowing that NK cells may execute cytotoxicity through exocytosis of cytoplasmic granules containing cytolytic toxins, I chose to study this functional activity of adaptive NK cells; an activity that was largely unexplored due to the lack of appropriate experimental reagents. I validated the applicability of reagents and profiled the expression of cytolytic granule contents of adaptive NK cells. I revealed that protein expression of granzyme H, a protein of unknown biological function, is significantly higher in adaptive NK cells compared to conventional NK cells. This discovery laid the foundation for new exploration of the cytotoxicity of adaptive NK cells, and is presented in Chapter 6.

Observations of the altered effector functions of human adaptive NK cells has inspired the potential therapeutic use of these cells in relevant clinical settings, including infectious disease, cancer, and autoimmunity. The research presented in this dissertation has now expanded our understanding of the biology of adaptive NK cells, which may in turn advance the development of NK cell-based immunotherapies to improve human health.