Differential regulation of cell cycle entry and apoptosis in cytolytic lymphocytes informed by primary immunodeficiency - PROJECT SUMMARY: Human natural killer (NK) cells are critical for control of viral infection and fighting cancer. Their importance is underscored by human diseases that arise from impaired or absent NK cells. The underlying genetic lesions that cause these human diseases can be used to uncover unique aspects of NK cell biology. Here, we focus on human disease-causing genetic variants that lead to impaired function of the replisome, a protein complex that carries out DNA replication. Defects in replisome function makes cells vulnerable to under-replication of DNA, which can lead to impaired cell cycle progression, DNA damage, and/or apoptosis. While regulation of cell cycle progression and response to DNA damage are critical functions for all effector lymphocytes, including T and NK cells, the lineage-specific sensitivity of NK cells to replisome activity is not understood and most studies of cell cycle regulation have been performed with non-immune cell types. In preliminary results, we found that isolated NK cell deficiency arises from disease-causing variants in replisome proteins including MCM4, MCM10, GINS1, and GINS4. We also demonstrated, using patient-derived and control induced pluripotent stem cell differentiation, that disease-causing replisome variants specifically affected the NK cell lineage and that mature NK cells were highly susceptible to mild replication stress that was tolerated by T cells. Our central hypothesis is that NK cells and T cells differ in both their lineage-dependent regulation cell cycle entry and their response to replication stress, rendering NK cells more sensitive than T cells to changes in replisome activity. This proposal will study how differential regulation of replisome protein expression and responses to replication stress are controlled in NK and T cells versus in hematopoietic stem and progenitor cells (HSPCs). In Aim 1, we will use biochemical, single molecule imaging, and genetic/epigenetic approaches to define how replisome protein function and cell cycle progression is regulated in lymphoid progenitors and mature cells. In Aim 2, we will identify lymphocyte lineage-specific sensors that control survival or apoptosis decisions and manipulate the apoptotic pathway and test for rescue of the NK cell deficiency that occurs in response to mild replication stress in vitro. Better understanding of the mechanisms by which sensitivity to apoptosis is conferred in human NK cells has important implications for physiological contexts that rely on NK cell activation, such as during viral infection or cancer. We are also developing innovative single-cell microscopy-based tools and techniques that will potentially be applicable to a broad diversity of fields and cell types. Importantly, the results of these experiments and basic insights gained could have innovative therapeutic applications. NK cell-based therapeutics are becoming significant tools in our modern arsenal for cancer treatment; understanding the weak points in their proliferation and differentiation could allow researchers to circumvent barriers to their in vitro generation.
