Multiple breakthroughs in immunology such as chimeric antigen receptor (CAR) T/NK cells and immune checkpoint inhibitors (ICIs) have pushed immunotherapy to the first-line treatment in multiple malignancies. However, since AIDS compromises the capacity of the immune system to fight infections and other diseases, people living with HIV (PLWH) are traditionally excluded from these clinical trials. HIV, head and neck squamous cell carcinoma (HNSCC), and immunotherapy: Antiretroviral therapy (ART) have pushed the overall survival of PLWH beyond 40 years. Thus, as PLWH’s life expectancy increases, the rate of HIV-associated co-morbidities such as HNSCC rises. In this context, HIV destroys the CD4+ T cells, severely compromising the capacity of the immune system to respond against infections such as human papillomavirus, which commonly leads to HNSCC. Therefore, multiple studies have highlighted the potential advantages of CAR NK cells in PLWH given their capacity to destroy tumor cells independently of CD4+ T cells. The need for better in vitro models to evaluate CAR NK cell immunotherapy in HIV+ HNSCC: Solid tumors, including HNSCC, generate a immunosuppressive microenvironment characterized by hypoxia, nutrient starvation, acidic pH, and waste product accumulation. These environmental factors impose an overwhelming burden on NK cells, severely limiting their capacity to destroy tumor cells and leading to NK cell exhaustion. However, mimicking the structural and environmental complexity of HNSCC using traditional cell culture platforms based on Petri dishes is extremely challenging. Thus, here we propose to evaluate the potential of CAR NK cells and ICIs for HIV+ HNSCC through our unique combination of microphysiological systems, genetically-modified HIV latency models, CAR NK cells, and FDAapproved ICIs. Aim 1. Evaluation of CAR NK cells for HNSCC using microphysiological models. Our microphysiological models include the culture of a patient-derived HNSCC organoid in a 3D collagen matrix with two flanking blood vessel surrogates to perfuse media and nourish the system. Autologous human CD4+ T cells, infected with HIV-1 will be embedded in the collagen matrix to mimic HIV infection in PLWH. Finally, CAR NK cells will be perfused through the blood vessels to evaluate their capacity to destroy the HNSCC cells in the presence of HIV+ scenarios. Aim 2 Study potential synergy between CAR NK cells and ICIs using microphysiological models. We will evaluate potential synergy between CAR NK cells and pembrolizumab (i.e., PD-1 antibody approved by the FDA for HNSCC) using our HNSCC microphysiological model. The different cell populations (e.g., tumor, NK cells) will be retrieved from models harboring different levels of CD4+ T cells to evaluate potential immune exhaustion mechanisms caused by HIVassociated CD4+ T cell depletion combined with the suppressive tumor environment. The long-term of this proposal is the development of guidelines to design the first H...