Adenoviral delivery of the <i>CIITA</i> transgene induces T‐cell‐mediated killing in glioblastoma organoids

The immunosuppressive nature of the tumor microenvironment poses a significant challenge to effective immunotherapies against glioblastoma (GB). Boosting the immune response is critical for successful therapy. Here, we adopted a cancer gene therapy approach to induce T-cell-mediated killing of the tumor through increased activation of the immune system. Patient-based three-dimensional (3D) GB models were infected with a replication-deficient adenovirus (AdV) armed with the class II major histocompatibility complex (MHC-II) transactivator (CIITA) gene (Ad-CIITA). Successful induction of surface MHC-II was achieved in infected GB cell lines and primary human GB organoids. Infection with an AdV carrying a mutant form of CIITA with a single amino acid substitution resulted in cytoplasmic accumulation of CIITA without subsequent MHC-II expression. Co-culture of infected tumor cells with either peripheral blood mononuclear cells (PBMCs) or isolated T-cells led to dramatic breakdown of GB organoids. Intriguingly, both wild-type and mutant Ad-CIITA, but not unarmed AdV, triggered immune-mediated tumor cell death in the co-culture system, suggesting an at least partially MHC-II-independent process. We further show that the observed cancer cell killing requires the presence of either CD8⁺ or CD4⁺ T-cells and direct contact between GB and immune cells. We did not, however, detect evidence of activation of canonical T-cell-mediated cell death pathways. Although the precise mechanism remains to be determined, these findings highlight the potential of AdV-mediated CIITA delivery to enhance T-cell-mediated immunity against GB.