Background Previous trials testing oncolytic adenovirus Delta-24-RGD in malignant gliomas has shown encouraging results. Since oncolytic viruses can stimulate immune reactions against both virus and tumor, immunodominance of viral antigens may prevent the preferred generation of tumor-specific clones. In this work, we tested the extent to which nanoparticle-induced immune tolerance to viral antigens shift the focus of the immune system to tumor antigens and improves the therapeutic outcome.

Methods Genomic DNA from surgical samples of 9 glioma patients treated with Delta-24-RGD and flash-frozen brains of virus-treated GL261 glioma-bearing mice were examined with T cell receptor sequencing. Nanoparticles (NP) encapsulating specific antigens were administered to mice intravenously, and isolated T cells were used for bulk RNA-sequencing. Survival and interferon-gamma (IFNγ) production from tumor-infiltrating lymphocytes were evaluated in GL261-bearing mice treated with virotherapy and NP. Brain-infiltrating lymphocytes were interrogated with flow cytometry.

Results In patients treated with Delta-24-RGD, the 10 most common T cell clones represented 60% of the total T cell frequencies, confirming the presence of immunodominance in these patients. In mice, intravenously administered NP accumulated in the liver (P<0.0001), specifically in CD11b+ myeloid cells. Antigen presenting cells in the liver have been reported to delete self-reactive immune cells through peripheral immune tolerance. T cells isolated after NP injection displayed transcriptomic signatures associated with immune tolerance including increased expression of PD-1 and reduced expression of granzymes (P<0.0001). In glioma-bearing mice treated with virotherapy, NP administration reduced immune responses of tumor-infiltrating lymphocytes against viral epitopes (P<0.0001) and increased the response against tumor antigens (P<0.0001), which suggested a subversion of immunodominance by viral antigens. Flow cytometry analyses revealed comparable frequencies of CD4+ and CD8+ T cells in the tumors of mice with or without NP, suggesting that this shift does not require a global change in the tumor microenvironment. Upon NP treatment, the frequencies of virus-specific T cells decreased (P<0.0001), whereas the frequencies of tumor-specific clones increased compared to virus treatment alone (P<0.0001). Importantly, the combination treatment enhanced the survival of glioma-bearing mice compared to virus-treatment alone (P<0.001).

Conclusions Our data suggest that it is possible to re-focus the immune response of the patient using nanoparticle administration to induce viral antigens tolerogenicity. Our data from patients and clinically relevant animal models should propel the development of a future clinical trial aimed to utilize tolerogenic strategies to prioritize anti-tumor immune response during oncolytic virotherapy for brain tumors.

Ethics Approval This study involves human material and was approved by the MD Anderson Cancer Institutional Review Board for the clinical trial under number NCT00805376. The evaluation of de-identified patient samples was approved under ID01-310. All experimental procedures involving the use of mice were done in accordance with protocol 00001420-RN approved by the Animal Care and Use Committee of MD Anderson Cancer Center, according to National Institutes of Health and USA Department of Agriculture guidelines.