Project Summary/Abstract Head and neck squamous cell carcinoma (HNSCC) are often associated with human papillomavirus (HPV) infection. The incidence of HPV+ HNSCC has been rapidly rising during the past 2-3 decades. Thus, it is critical to gain better understanding of the pathogenic mechanisms of HPV+ HNSCCs in the context of immune recognition and elucidate the mechanistic determinants underlying therapy responsiveness vs. resistance in HPV+ HNSCC. The effects of immune checkpoint inhibitors (ICI) are highly heterogeneous in HNSCC patients; however, it remains incompletely understood whether anti-HPV responses contribute to ICI efficacy. Recent studies showed that HPV+ HNSCC responded better than HPV− HNSCC in a neoadjuvant anti-PD1 trial, suggesting that effective HPV-related responses may benefit ICI treatment. The goal of this proposal is to determine whether and how effective anti-HPV immune responses may contribute to ICI efficacy by testing if the breadth and depth of T cell receptor (TCR) responses against HPV antigens affect the outcomes and therapy responses of HPV+ HNSCC using human samples from clinical trials and murine HNSCC models. Prior studies focused on defined E7-derived peptides for HPV-related cellular therapy. However, HNSCC tumor-infiltrating lymphocytes (TILs) contain much more HPV E2-reactive CD8 T cells than E7-reactive ones. Hence, we will test TCRs against different HPV antigens (e.g., E6/E7 vs. E2) and elucidate the mechanistic connection between HPV-specific TCRs and therapeutic responses. Our proposed studies may overcome the barrier of the HNSCC field and develop a new toolbox of targeting HPV+ HNSCCs with maximal breadth and depth of TCRs. We will employ a novel “window of opportunity” trial open at our institution (HCC 18-139) that tests the effects of anti-PD1 (nivolumab) vs. anti-PD1 + anti-CTLA4 or anti-LAG-3 in a neoadjuvant setting before surgery resection of locally advanced resectable HPV+ HNSCC. Additionally, we will employ a HNSCC mouse model (mEER) expressing HPV16 E6/E7 antigen in which a small fraction of tumor-bearing recipients responded to anti-PD-L1 treatment, allowing unambiguous identification of responders vs. non-responders. Using these neoadjuvant trial samples, we will test whether anti-HPV immune responses underlie ICI responses and examine whether HPV-reactive TCR clonal dynamics and T cell transcriptomics in TILs correlate with ICI responses. Our mouse model allows functional validation of expanded HPV-reactive TCR clonotypes correlating with ICI responses and permits antigen manipulation to enhance clinical responsiveness as well as lay the groundwork for future clinical and targetable trials.