Human cytomegalovirus (HCMV) antigens in glioblastoma (GBM) present opportunities for personalised immunotherapy. However, their presence in GBM tissue is still under debate, and evidence of their impact on functional immune responses and prognosis is sparse. Here, we investigated the presence of pp65 (UL83) and immediate early 1 (IE-1) HCMV antigens in a cohort of Norwegian GBM patients (n = 177), using qPCR, immunohistochemistry, and serology. HCMV status was then used to investigate whether viral antigens influenced immune cell phenotype, infiltration, activation and patient survival. Pp65 and IE-1 were detected by qPCR in 23% and 43% of GBM patients, respectively. Furthermore, there was increased seropositivity in GBM patients relative to donors (79% vs. 48%, respectively; Logistic regression, OR = 4.05, 95%CI [1.807-9.114], P = 0.001, also when adjusted for age (OR = 2.84, 95%CI [1.110-7.275], P = 0.029). Tissue IE-1-positivity correlated with increased CD3⁺CD8⁺ T-cell infiltration (P < 0.0001), where CD8⁺ effector memory T (T_EM) cells accounted for the majority of CD8⁺T cells compared with peripheral blood of HCMV⁺ patients (P < 0.0001), and HCMV⁺ (P < 0.001) and HCMV^- (P < 0.001) donors. HLA-A2/B8-restricted HCMV-specific CD8⁺ T cells were more frequent in blood and tumor of HCMV⁺ GBM patients compared with seronegative patients, and donors irrespective of their serostatus. In biopsies, the HCMV-specific CD8⁺ T_EM cells highly expressed CTLA-4 and PD-1 immune checkpoint protein markers compared with populations in peripheral blood (P < 0.001 and P < 0.0001), which expressed 3-fold greater levels of CD28 (P < 0.001 and P < 0.0001). These peripheral blood T cells correspondingly secreted higher levels of IFNγ in response to pp65 and IE-1 peptide stimulation (P < 0.001). Thus, despite apparent increased immunogenicity of HCMV compared with tumor antigens, the T cells were tolerised, and HCMV status did not impact patient survival (Log Rank_3.53 HR = 0.85 95%CI [0.564-1.290], P = 0.45). Enhancing immune functionality in the tumor microenvironment thus may improve patient outcome.

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