Project Abstract Pediatric high-grade glioma (pHGG), diffuse midline glioma (DMG), recurrent medulloblastoma (MB), and recurrent ependymoma are devastating diseases. The median overall survival (OS) of these diagnoses are 8 to 12 months. Brain cancer is the most common cause of cancer death in patients aged 0-19 and improvements in survival remain elusive. Current treatments for these malignant tumors typically entail surgical resection or biopsy, radiation, and sometimes chemotherapy. These treatments are rarely curative. There is a profound knowledge gap in understanding how to improve the survival and quality of life in children with these malignant brain tumors. In contrast to conventional chemotherapy/radiation, immunotherapy offers a more precise treatment approach. The human cytomegalovirus (CMV) antigen, pp65, is found in the majority of HGG and MB but not in adjacent brain, making it a promising immunotherapeutic target. Limited data from multiple labs suggests CMV antigens may also be present in ependymoma. However, this has not been confirmed in large samples nor is it known if CMV antigens are preferentially expressed in certain ependymoma subtypes. Preliminary results from a Phase 1 clinical trial of a peptide vaccine directed at pp65 (PEP-CMV) to treat recurrent pHGG/DMG and MB has demonstrated significant antigen-specific T cell immune responses and prolonged clinical/imaging responses in many patients. Additionally, in an ongoing study of PEP-CMV in adults with newly-diagnosed glioblastoma, there was a significant increase in the cytokine/chemokines G-CSF, GM-CSF, IFN-γ, IL-10, IL-2, IL-8, MIP1-α, and TNF-α levels after PEP-CMV delivery. The addition of checkpoint blockade has not been explored in immunotherapies targeting CMV, however, it has enhanced the immunogenicity of cancer vaccines in preclinical brain tumor models other non-brain solid tumors. We hypothesize that CMV antigens are present in a subset of ependymomas and adding checkpoint blockade to PEP-CMV will increase T cell and cytokine responses. Our objectives of this pilot study are to 1) determine if ependymoma is a putative target of PEP-CMV and 2) improve the immunogenicity of PEP-CMV. To attain the overall objectives, the following specific aims will be pursued. Aim 1: Establish if CMV antigens are expressed in ependymoma. Utilizing validated and replicated methods, we will analyze 60 human ependymoma specimens for expression of CMV antigens. Aim 2: Establish if checkpoint blockade will increase the immunogenicity of PEP-CMV. To accomplish this, we will use a validated transgenic HLA-A2 mouse model to determine if IFN-γ generation via ELISpot and cytokine generation responses are increased by adding checkpoint blockade. Our long-term objective is to use data from this pilot study to conduct a Phase 1 clinical trial of PEP-CMV plus checkpoint blockade for the treatment of recurrent ependymoma, MB, and pHGG/DMG.