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Background Neuroblastoma is the second most common solid pediatric tumor, with ~50% of patients having high-risk disease (HR-NBL), which has a poor response-rate. HR-NBL patients are currently treated with an immunotherapy regimen that includes anti-GD2 (aGD2). Considered a ‘cold’ tumor, most HR-NBLs have infiltration of suppressive immune cells, poor anti-tumor immune infiltrate, and low-to-no expression of MHCI. Suppressive myeloid cells express CD40, thus anti-CD40 agonist (aCD40) may reprogram them to have antitumor activity. Since T cells are engaged via interactions with MHC, low expression of MHCI may contribute to reduced T cell involvement during immunotherapy. Diminished MHCI expression on HR-NBLs can occur through multiple mechanisms, including epigenetic modifications (EMs). In NBL, EMs contribute to disease progression and therapy resistance, and regulate tumor immunophenotypes, including MHCI expression. Key EMs include DNA methylation (DNMT), histone deacetylation (HDAC), and histone methylation (HMT), all of which are reversible with epigenetic inhibitors (EMis). We identified inhibitors of DNMTs (guadecitabine, ‘Guad’) and HDACs (entinostat, ‘Ent’) that, when combined with aGD2 and aCD40, cause tumor regression.Methods HR-NBL cells were treated with EMis and assessed for MHCI expression changes via qPCR and flow cytometry. Using two separate murine models of ‘cold’ HR-NBL tumors (TAM6 in SVJae; 9464D in C57Bl/6), we investigated whether inhibitors of EMs (EMis) + aCD40 could improve the anti-tumor efficacy of anti-GD2. Increased anti-tumor immune infiltration may lead to upregulation of immune checkpoints. We also tested whether inclusion of the immune checkpoint inhibitor (ICI) may further improve response. Mice were treated with combinations of aGD2+/-EMi+/-aCD40+/-ICIs and monitored for response and immune infiltration.Results In vitro treatment of HR-NBL with EMis showed that MHCI-related genes are expressed. In both tumor models, mice bearing HR-NBL tumors the best response (tumor regression, tumor-free mice, and overall survival) to therapy was achieved using therapies that include aGD2/EMis/aCD40/ICIs. T cells are involved in the antitumor response following treatment with aGD2/EMis/aCD40/ICIs.Conclusions Using a combination therapy that includes aGD2/EMis/aCD40/ICIs, the tumor microenvironment is reinvigorated, enabling increased activity of immune cells, including T cells. We hypothesize that the T cell engagement may be via the addition of EMis, coupled with aCD40 reprograming of suppressive cells while simultaneously activating macrophages and DCs to enhance the efficacy of aGD2. Therapies geared towards restoring MHCI expression, combined with effective immunotherapy regimens that allow for persistent immune responses, might augment immune responses and improve efficacy of combination immunotherapy for HR-NBL.Ethics Approval Our research is conducted under strict oversight by the IACUC and in compliance with all applicable federal and institutional policies.Acknowledgements This research was supported by Midwest Athletes Against Childhood Cancer; and by public health service grants from the National Cancer Institute: Alex’s Lemonade Stand Foundation, the Hartwell Foundation, Midwest Athletes Against Childhood Cancer, R35 CA197078, U54 CA232568 as well as the Data Science Initiative grant from the University of Wisconsin-Madison Office of the Chancellor and the Vice Chancellor for Research and Graduate Education (with funding from the Wisconsin Alumni Research Foundation). We are also grateful to the UW Flow Cytometry Core Facility for the resources and expertise that they provide, and the UW-Madison Hilldale Awards for undergraduate student research support.