Combatting Against Cancer Using Virus: Oncolytic Virus M1 Reinvigorates T-cell immunity against glioblastoma

Glioblastoma multiforme (GBM), usually arising from glial precursor cells or neural stem cells, is the most common and lethal primary brain cancer in adults. It has very limited treatment options and a median life expectancy less than 2 years after diagnosis. Systemic and local immunosuppression induced by GBMs largely contributes to malignancy aggressiveness and resistance to multiple immunotherapies, such as immune checkpoint blockade (ICB) therapy, which is powerful for primary care of other types of solid tumors. 

Oncolytic virotherapy has attracted growing awareness as a potential transformative cancer therapy in recent years. It involves delivery of a genetically modified version of virus into the host and it would selectively destroy tumor cells while leaving neighboring healthy cells undamaged, overcoming a common limitation of chemotherapy. This targeted destruction is either achieved through direct oncolysis ("onco-" meaning cancer-related, "-lysis" meaning burst) or immune-mediated attack. Oncolytic therapy using alphacirus M1(OVM), which was originally isolated from Culex mosquitoes, recently emerged as a potential glioma therapeutic approach. The virus is delivered through intravenous administration and the main advantages are OVM's ability to selectively replicate in tumor cells overexpressing matrix remodeling-associated 8 (the marker allowing them to recognize and attach) and efficiency in penetrating through the blood brain barrier, which is a dense layer of blood vessels and cells that protect our brain from harmful substances but makes drug delivery difficult at the same time. Even though we might be worried that using virus as medium can be dangerous by itself at the first glance, the toxic genes are being taken out in advance and it's previously shown to be very safe: GBM patients showed high tolerance towards OVM in previous clinical trials and repeated intravenous administration of OVM is non-pathogenic for nonhuman primates (Zhang et al., 2016).

Despite of its huge therapeutic potential, the knowledge regarding how OVM initiates adaptive immunity against gliomas and how the interactions between OVM and our immune system would influence the therapeutic effects of ICB is still lacking. Hence the researchers are seeking to address these two knowledge gaps.

First, they confirmed the efficacy of OVM in reducing glioma progression. The immunocompromised mice were injected intracranially with GBM cell lines (GL261-Luciferase, GL261 and CT2A) to induce a glioma in the brain on day 0 and followed by daily tail intravenous injection of OVM or vehicle from day 5 to day 9. Vehicle includes the buffer solutions that researchers use to deliver OVM but containing no OVM, acting as the control condition. GL261-Luciferase is a cell line where it's infected with lentivirus whose genome was engineered to contain the luciferase gene so that wherever the tumor develops, it would glow. A stronger luminescence intensity (red) indicates the progression of glioma while bluish color indicates little tumor growth. It was found that OVM significantly inhibited the growth of glioma and prolonged the survival of glioma-bearing mice. 

Moving on to investigate the immune response, the researchers found that OVM successfully induced immunogenic cell death and reverse the GBM-induced immunosuppression both locally and systemically. Immunogenic cell death is characterized by the release of damage-associated molecular patterns (DAMPs) by tumor cells, which are molecules that normally stay inside the cell and becomes 'danger signals' recognized by immune cells when being released outside, driving adaptive-immunity elimination in tumor microenvironment. They examined the release of DAMPs, revealing significant CRT (externalized calreticulin, a multifunctional protein) exposure and elevated extracellular ATP (adenosine triphosphate, energy source for our cells) in the supernatant collected from glioma cell lines with OVM treatment. This indicates the occurrence of immunogenic cell death induced by OVM. 

Additionally, the number of CD4+ and CD8+ T cells increased significantly in peripheral blood of glioma-bearing mice following intravenous OVM treatment. Spleen atrophy and loss of splenic T-cell population were also recovered. Similarly, in the tumor local environment, they also found rapid increase in tumor-specific CD8+ T cells in the spleen. The measurement is achieved by orthotopical implantation of GL261 glioma cells expressing OVA (full-length ovalbumin, processed into SIINFEKL) and H-2Kb-SIINFEKL tetramer that binds to T cell receptors where H-2Kb is the mouse version of MHC class I protein. The recognition of glioma-derived-antigen-MHC complex by T cells allows the accurate tracking of tumor-specific T cells (Hsu et al., 2025). Hence, with OVM infection, both local and systemic immunity are boosted and recovered.

  

Before diving deeper into the mechanism, the researchers digressed a bit and addressed a main concern about the efficacy of OVM: Spleen is the major body 'filter' consisting of large number of immune cells that's found previously able to eliminate delivered oncolytic viruses and OVM is often 'trapped' inside the spleen of various host animals. Therefore, the researchers removed the spleen (splenectomy) from mice to find out the role of spleen in OVM treatment. To their surprise, opposite to the greater reduction in glioma progression that they have expected, the antitumor activity of OVM treatment was completely abrogated after removal. Splenectomy-OVM mice almost have the same low survival probability as vehicle control and no expansion of T-cell population in the peripheral blood and tumor microenvironment.

Consequently, the researchers tried to find out the specific cell population in the spleen that's mediating the reversal of immunosuppression and antitumor activity. They first employed a broad RNA sequencing and computational screening and found that B cells have the highest abundance and showed strongest predicted interactions with T cells after OVM treatment. To move beyond correlation, a series of validation experiment were conducted: In vitro co-culture experiments demonstrated that B cells from OVM-treated mice significantly enhanced activation of T cells but not other splenic immune populations and this activation is dose-dependent. Further, after blocking the B cells with anti-CD19 antibody, OVM administration failed to prolong the survival of glioma-bearing mice and increase the infiltration of T cell populations in the blood and tumor microenvironment. 

To elucidate the mechanism by which splenic B cells reverse immunosuppression, the researchers compared the interactions of B cells with CD8+ T cells in the OVM-treated group and the vehicle control group. They found that OVM treatment leads to much more frequent physical contacts between B and T cells, visualized through live cell imaging.

To determine the functional significance of this cell-to-cell contact, they created two conditions where the cells can directly interact with each other and where they are forced to be separated from each other by a membrane (transwell chamber). CD8+ T cell proliferation is measured by CFSE dilution (carboxyfluorescein succinimidyl ester,  a fluorescence dye that's split between new-born cells for every division) and CD8+ T cell activation is measured by GZMB+ (granzyme B, the 'weapon' T cells produce to kill target cancer cells). Using these two tracking tools, they discovered that T cell proliferation and activation is induced only when B cells and T cells that can freely contact with each other but not in transwell-separated groups. These results together suggests that the direct contact between B and T cells is essential for T-cell expansion and activation required for OVM-mediated immune enhancement.

In order to validate this finding in vivo, researchers targeted the Major Histocompatibility Complex class I (MHC-I), a critical component of the structural interface between B and T cells. B cells communicate and 'educate' T cells by presenting a fragment of tumor on MHC-I to activate them, which process is known as antigen presentation. Blocking MHC-I with a specific antibody completely abolished B-cell-induced CD8+ T cell proliferation and activation. Collectively, these results confirm that the direct physical interface is crucial for immunity restoration by OVM treatment. Later the researchers identified the specific subset of B cells that's responsible for this interaction to be Bst2+ B cells by computational screening and in-vivo evidence: Bst+B cells significantly prolonged the survival of B-cell-deficient, GL261-OVA-bearing mice after OVM stimulation while Bst2-B cells and B cells from vehicle control group did not.

The interface between B and T cells involves B cells 'presenting' a peptide derived from cancer cells or other sick cells on MHC-I protein, which is then recognized by T cell receptors (TCR). 

Finally, to bridge the above mechanistic findings with clinical application, the researchers tested if OVM treatment can help overcome immunosuppression that previously has prevent immune checkpoint blockade (ICB) therapy from functioning in glioblastoma patients. ICB therapy works by removing the "brake" on the T cells. Our bodies have these natural immune checkpoint proteins that's naturally used to prevent excessive inflammation from happening that would otherwise harm other healthy cells. But at the same time, this also grant tumor cells a tool to escape from immune responses. ICB drugs block these inhibitory signals and boost the working capacity of our immune system. 

PD-L1 and PD-1 are the immune checkpoint proteins that 'put a brake' on the T cell when bound. If either of them are blocked, which is what the researchers and ICB drugs do, T cells are free to attack the tumor cells. 

To test the synergy between ICB and OVM therapy, immunocompromised mice was injected intracranially on day 0 and from day 5 to day 9, the mice is divided into different groups and treated daily intravenously with different combinations of immune checkpoint protein antibodies, including isotype antibody as control, PD-1 and PD-L1 antibodies, with or without OVM. The researchers revealed that, comparing with other 5 combinations, OVM treatment and PD-1 inhibitor together yielded the greatest recruitment of CD8+ T cells into tumor microenvironment, suppression of intracranial glioma growth as well as restoration of splenic size and weight. Systematically, massive expansions of effector (activated) CD8+ and CD4+ T cells in peripheral bloodstream are also observed. Therefore, OVM synergizes with PD-1 inhibitors for glioma treatment by simultaneously reshaping systemic immunity and the local immune environment in the brain.

isotype= control isotype antibody; PD-1 and PD-L1 Ab= PD-1 and PD-L1 antibodies (ICB therapy); Vehicle= control for OVM treatment; OVM= oncolytic viral therapy. 

This study provides profound mechanistic details for how emerging OVM treatment reverses the immunosuppressive environment of GBM, which is incurable and has grim prognosis. Most significantly, it establishes OVM as a transformative catalyst for ICB therapy, which is historically ineffective for GBM. The findings also illuminate a remarkable capability of OVM bypassing the 'toxic', immunosuppressed tumor microenvironment in the brain, and re-directing the site of immune cell communication to the 'clean sanctuary', the spleen. Within this protected environment, the recovery of B- and T-cell physical interface structures allows for robust antigen presentation, subsequent T cell activation, and eventually elimination of the tumor cells. The study has a strong advantage in its comprehensive validation of each step. For instance, when assessing the efficacy of the therapies, they would use several measures like the size of the spleen, tumor growth and immune cells activation level. Also, when confirming the determining role of splenic B cells, they combined both computational tools and biological checks, including knockout and correlation experiments. Moving forward, these discoveries open a new frontier for translational medicine. While mouse models provide an essential foundation, future research should evaluate OVM in non-human primates and eventually human clinical trials to characterize its safety profile in more complex in vivo environment. Long-term studies are also preferable because so far only the decrease in size of tumor is. reported but future relapse is still possible, especially given the ability of tumors to develop resistance mutations. Furthermore, the mechanism behind OVM-directed B-cell migration to the spleen and T-cell migration to the brain can be elucidated to identify potential signaling molecule targets.

Citations:

For images:

https://www.researchgate.net/figure/The-origin-of-glioblastoma-During-normal-embryonic-development-and-in-the-adult-brain_fig2_371833392

https://vyriad.com/science/oncolytic-virus-platforms/
https://www.cancerresearch.org/immunotherapy-by-treatment-types/oncolytic-virus-therapy
https://www.nature.com/articles/s41592-020-01031-0
https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/checkpoint-inhibitors

https://rcastoragev2.blob.core.windows.net/13974d8439d3384abb2302e11f2da069/PMC9563749.pdf

For academic literature:
the primary article:
‌Han, Yu, et al. “Oncolytic Virus M1 Reinvigorates CD8+ T-Cell Immunity against Glioblastoma through B-Cell-Dependent Antigen Cross-Presentation in the Spleen.” Cellular & Molecular Immunology, 4 Mar. 2026, www.nature.com/articles/s41423-026-01396-w/figures/8, https://doi.org/10.1038/s41423-026-01396-w. Accessed 26 Mar. 2026.

Others:

‌National Cancer Institute. “Https://Www.cancer.gov/Publications/Dictionaries/Cancer-Terms/Def/Blood-Brain-Barrier.” Www.cancer.gov, 2 Feb. 2011, www.cancer.gov/publications/dictionaries/cancer-terms/def/blood-brain-barrier.

‌Hsu, Chung-Yao, et al. “Polymerised Superparamagnetic Antigen Presenting Cell Lymphocyte Capture for Enriching Tumour Reactive T-Cells and Neoantigen Identification.” Nature Communications, vol. 16, no. 1, 2 June 2025, www.nature.com/articles/s41467-025-60321-3, https://doi.org/10.1038/s41467-025-60321-3. Accessed 26 Mar. 2026.

“Up Close: How Immune Checkpoint Inhibitors Revolutionize Cancer Treatment | What’s up at Upstate | SUNY Upstate.” Www.upstate.edu, www.upstate.edu/whatsup/2023/100523-up-close-how-immune.php.

Zhang, Haipeng, et al. Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates after Multiple Rounds of Repeated Intravenous Injections. Vol. 27, no. 9, 1 Sept. 2016, pp. 700–711, https://doi.org/10.1089/hum.2016.038. Accessed 1 Aug. 2023.

‌“Defined Immune Response Tracking in Mice: OVA MHC Tetramers – Caltag Medsystems.” Caltagmedsystems.co.uk, Mar. 2024, www.caltagmedsystems.co.uk/information/defined-immune-response-tracking-in-mice-ova-mhc-tetramers/. Accessed 29 Apr. 2026.

‌“Oncolytic Virus Therapy and Its Side Effects.” Cancer.org, 2023, www.cancer.org/cancer/managing-cancer/treatment-types/immunotherapy/oncolytic-virus-therapy.html.

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