Highlights d Deletion of CISH in human NK cells leads to improved antitumor activity d CISH À/À NK cells demonstrate more efficient glycolytic and OxPhos activity d The improved metabolic profile is mediated by mTOR signaling d CISH À/À NK cells more effectively treat AML in vivo with longer NK cell persistence

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a “two cell type” whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.

Mouse and human pluripotent stem cells have been widely used to study the development of the hematopoietic and immune systems. While not all cells can be derived with the same efficiency, immune cells such as natural killer (NK) cells and macrophages can be easily produced from pluripotent stem cells to enable development of new cell-based therapies. NK cells and macrophages are part of the innate immune system, the first line of defense against malignancies and infectious disease. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)-derived NK cells can be produced at a clinical scale suitable for translation into clinical trials. Additionally, pluripotent stem cells can be genetically modified to produce hESC and iPSCderived human NK cells with enhanced anti-tumor activity. These engineered NK cells can express a stabilized version of the high-affinity Fc receptor CD16, chimeric antigen receptors (CARs), or other strategies to enable more potent and targeted cellular immunotherapies. Moreover, macrophages can also be routinely and efficiently produced from hESCs and iPSCs as a tool to expand our knowledge of the basic biology of these cells. hESC/iPSC-derived macrophages can also be employed as a novel approach for cancer immunotherapy, as well as a strategy to repair or regenerate diseased/damaged tissues and organs. Background:Mouse and human pluripotent stem cells have been now used for decades in key studies to better understand mammalian hematopoiesis. Studies with mouse ESCs to create genetic knock-out mice have been instrumental to define key genetic mechanisms that mediate development of different hematopoietic lineages (1, 2). In an effort to move this work into a human system, our group was the first to derive blood cells (mainly myeloid and erythroid cells) from hESCs (3) and later the first to produce lymphocytes (4). These initial studies on hESC-derived lymphocytes produced NK cells with phenotype and function similar to NK cells isolated from peripheral blood, including the ability to kill diverse tumor cells in vitro

Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56 + NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34 + hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.