Abstract: More than one million cases of cutaneous squamous cell carcinoma (cSCC) are diagnosed annuallyin the US and approximately 4% of patients develop metastases and 2% die of cSCC; thus a similar numberof people die each year from cSCC as melanoma. Immune checkpoint inhibitors (ICI) are a new class of drugsthat have transformed the therapy of multiple cancer types but only half of cSCC patients respond to ICItreatment. ICI target receptors on T cells such as PD-1 that are expressed after activation and function to turnoff T cell responses. The response of cSCC patients to ICI demonstrates the ability of T cells to constraincSCC growth. However it remains unclear the extent to which CD8 and in particular CD4 T cells contribute toimmune-mediated control of cSCC. While the focus of anti-tumor T cell responses has been on MHC class Ineoantigens that elicit cytotoxic CD8 T cell responses there is growing evidence that MHC class IIneoantigens eliciting CD4 T cell responses are critical in constraining tumor growth and enhancing response toICI. Thus there is a critical need to understand the role of CD8 and CD4 T cells especially the role ofneoantigen-specific T cells in controlling cSCC growth. We generated a novel physiologic cSCCtransplantable model on the BALB/c background from a solar simulated light-induced invasive cSCC tumor.Preliminary data supports that T cells constrain the in vivo tumor growth in the cSCC model and that this modelis sensitive to anti-PD-1 treatment. Using bioinformatic approaches with whole exome and RNA sequencingdata we have identified immunogenic MHC class I and II neoantigens predicted to elicit a T cell responsebased on the binding affinity and presentation of the neoantigen:MHC complex and neoantigen expression.Using melanoma patient data our lab has previously demonstrated that these characteristics accuratelypredict the ability of a neoantigen to elicit a T cell response. The central hypothesis is that both neoantigen-specific CD8 and CD4 T cells contribute to immune-mediated control of cSCC growth and response totreatment with vaccination with immunogenic neoantigens alone or in combination with anti-PD-1. To addressthis hypothesis we will determine the role of CD8 and CD4 T cells in controlling tumor growth identify MHCclass I and II neoantigens that elicit in vivo T cell responses and evaluate the expression of functional andinhibitory neoantigen-specific CD8 and CD4 T cells throughout cSCC tumor growth. Then we will vaccinatemice with dendritic cells loaded with irradiated tumor cells or immunogenic MHC class I and/or II neoantigensand compare the efficacy of these vaccination strategies in inducing CD8 and/or CD4 T cells to prevent cSCCgrowth and treat cSCC alone or in combination with anti-PD-1. We will demonstrate the requirement for CD8and/or CD4 T cells through antibody depletion and adoptive transfer. The impact of this project is to 1) identifythe contributions of neoantigen-specific CD8 and CD4 T cells in control of cSCC growth and 2) advance theapplication of personalized neoantigen vaccines to treat cSCC alone or in combination with anti-PD-1.