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Background Malignant gliomas (MG) are the most common primary malignancies of the central nervous system and virtually incurable. Thus, novel treatments are urgently needed. Oncolytic HSVs (oHSV) are promising immunotherapies capable of direct oncolysis and stimulating immune activity. Our gene expression studies from a Phase Ib clinical trial of oHSV-treated then resected tumors found IL-27 expression directly correlated with survival. We therefore developed an IL-27 expressing oHSV (C027) and showed that it improves survival in syngeneic murine glioma models.Methods We hypothesize that oHSV IL-27 expression enhances anti-glioma activity by promoting CD4 T cell activation, Th1 polarization, and memory formation. To test this, CT-2A MG brain tumors were orthotopically established in (C57BL/6) mice and treated 7d later with equivalent virus dose of C027 (C134+IL27) or parent virus (C134) or vehicle control. To assess immunophenotypic changes, tumors and blood were collected 7d post-treatment and analyzed by flow cytometry. Replicate experiments were conducted with antibody depletion (αCD4 or isotype) to determine CD4 T cells’ requirement for the C027 anti-glioma effects. To assess functional memory response in survivors, tumor rechallenge experiments were performed on long-term survivors ([LTS] >60d) alongside age-matched naïve controls.Results Relative to controls, C027-treated tumors had increased proportions of total CD4 T cells, central memory (CD62L +CD44+), and effector/effector memory (CD62L-CD44+) CD4 T cells. Systemically, C027 treated mice had an increase in Th1-like (T-bet+IFNγ+) and central memory (CD62L+CD44+) CD4 T cells relative to parent and vehicle controls. Relative to isotype controls, C027 treated mice lacking CD4 T cells had significantly reduced survival (p=0.028). Interestingly, CD4 depletion reduces overall C027 survival yet there were some long-term survivors. However, while CD4-intact C027 survivors rejected tumor growth on flank rechallenge relative to age-matched naïve controls, C027 survivors that lacked CD4 T cells during initial tumor challenge (CD4-intact on rechallenge) failed to reject their tumors on rechallenge.Conclusions IL-27 expression by oHSV increases memory and effector CD4 T cell populations systemically and intratumorally. C027-treated mice also had increased circulating Th1-like CD4 T cells. Mechanistically, IL-27-oHSV therapeutic activity is abrogated in the absence of CD4 T cells. Within this CD4 depleted cohort, ~10% survive in repeated studies but these LTS mice fail to reject their tumors on tumor rechallenge, unlike CD4-intact C027-LTS mice, suggesting CD4 T cells are essential for the development of durable immune memory. IL-27 expression by oHSV is a promising approach to enhance anti-glioma activity and immune memory formation.Ethics Approval All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Nationwide Children’s Hospital (AR16-00057, AR21-00145).