Project Summary Recently we showed in HNSCC models that effective checkpoint blockade is dependent upon intact lymphatic drainage for dendritic cell trafficking and that elective nodal radiation abrogated the effectiveness of immunotherapy, possibly explaining the lack of efficacy seen in prior studies. Neoadjuvant immunotherapy preserves draining lymphatics, however, emerging trials testing PD-1i prior to surgery have documented major pathological response rates of only 5%. To enhance the effectiveness of single agent PD-1i, improve clinical outcomes and explore the immunomodulatory capacity of hypofractionated radiation, we recently completed a first in human phase I trial testing neoadjvuant immunoradiotherapy (NIRT) in locally advanced HPV+ and HPV- HNSCC patients. Patients received nivolumab x 3 doses in combination with stereotactic body radiation therapy (SBRT) to gross tumor volume of 8Gy x5 or 8Gy x3, followed by definitive surgical resection. Astonishingly, the pathologic complete response rate in HPV+ patients was 90% and no patient required adjuvant radiation or chemoradiation. A cohort of 5 patients with stage III-IVA HPV-negative HNSCC treated with 8GyX3 plus nivolumab demonstrated a 60% major pathologic (>25%) response and clinical to pathologic downstaging of 100%. Transcriptional signatures from these surgical specimens compared to baseline are characterized by increased phagocytic and macrophage activation pathways, Th1 and Th2 cell differentiation, antigen presentation and peptide loading onto MHC class I, as well as a loss of cell proliferation pathways that are distinctly different from those treated with radiation alone, strongly suggesting a synergistic effect. Given our previous work showing that prognosis of patients with HPV- HNSCC is significantly affected by spatial interactions between the immune effector and suppressor cells in the tumor microenvironment (TME), particularly tumor-reactive, antigen-specific CD8 T-cells, we hypothesize th