Here, we noticed the paradoxical activity of the IL-2 receptor chains in mice, but in light of published studies, our findings suggest the potential for translational crossover to humans during treatment of cancers with anti-CD25 antibodies. most cancers, the role of radiation therapy on NK cells is not well characterized. Experimental Design: This study combines radiation, immunotherapies, genetic mouse models, and antibody depletion experiments to identify the role of NK cells in overcoming resistance to RT in orthotopic models of head and neck squamous cell RWJ 50271 carcinoma. Results: We have found that NK cells are a crucial component in the development of an anti-tumor response, as depleting them removes efficacy of the previously successful combination treatment of RT, anti-CD25 and anti-CD137. However, in the absence of NK cells, the effect can be rescued through treatment with FLT3L. But neither RT with FLT3L therapy alone nor RT with anti-NKG2A yields any meaningful tumor growth delay. We also identify a role for IL-2 in activating NK cells to secrete FLT3L. This activity, we show, is usually mediated through CD122, the intermediate affinity IL-2 receptor and can be targeted with anti-CD25 therapy. Conclusions: These findings highlight the complexity of using radio-immunotherapies to activate NK cells within the tumor microenvironment, and the importance of NK cells in activating dendritic cells for increased tumor surveillance. Introduction Head and Neck Squamous Cell Carcinoma (HNSCC) is usually a spontaneously arising malignancy that affects over 600,000 patients annually (1). For locally advanced disease, chemotherapy and radiation therapy (RT) or surgery and risk-adapted adjuvant chemo-radiotherapy remain the standard of care, with a five-year survival rate of around 50% (1). Clinical trials are currently underway for immunotherapies, but HPV-negative HNSCCs are characterized by low immune cell infiltration, and have exhibited poor response to immunotherapy (2). Preclinical evidence shows a benefit of combination therapies, in which RT is added to immunotherapy to sensitize the tumor microenvironment (TME) to checkpoint inhibition immunotherapies, such as programmed death receptor ligand 1 (PD-L1) (3). Regrettably, even in this context, the development of adaptive resistance to radioimmunotherapy remains a confounding factor (4,5), and the mechanisms that mediate resistance to combination radioimmunotherapy remain unknown. Many new therapies focus on switching the largely inhibitory immune response found in most HNSCC to a pro-inflammatory one by removing inhibitory signals, such as checkpoint inhibitors (4). However, preclinical evidence suggests that these immunotherapies are ineffective at eliminating resistance to radiation (6,7), and alternate options require further inquiry. In some mouse models of HPV-negative HNSCC, regulatory T Rabbit polyclonal to ANKRD40 cells (Tregs) have been identified as key regulators of immunosuppression within the TME, and depletion of Tregs through anti-CD25 antibody can lead to tumor eradication (5). However, a variety of cells express CD25, the alpha subunit of the IL-2 receptor, leaving the potential for off target effects. Natural killer (NK) cells are one such population of CD25-expressing cells. In addition to their ability to contribute to killing virally infected or cancerous cells through cytotoxic activity, NK cells are known to regulate the immune response in melanoma through production of FMS-like tyrosine 3 ligand (FLT3L) (8), allowing for growth of dendritic cells (DCs) in the TME, which increases tumor surveillance and antigen presentation (9,10). Activating NK RWJ 50271 cells, as a form of immunotherapy, represents an understudied area with high potential. Monalizumab, a monoclonal antibody targeting the inhibitory receptor NKG2A on NK RWJ 50271 cells, has previously been proposed as an immunotherapy to activate NK cells (11). However, as we show here, anti-NKG2A antibody fails to activate NK cells in the HNSCC TME, even when combined with RT. For activation by cytokines, NK RWJ 50271 cells rely on IL-15 (12-14) and to some extent IL-2, even though role RWJ 50271 or the latter is not completely understood (14,15). The IL-2 receptor is composed of CD25 (IL-2r alpha), CD122 (IL-2r beta), and CD132 (IL-2r gamma). Both the IL-2 and IL-15 receptors are heterotrimeric, and share the same beta and gamma subunits, which could result in overlapping functions (16). There is some evidence that in humans, IL-2 can stimulate CD25+ NK cells from your liver to proliferate (17), and studies in multiple sclerosis have identified a.