BTK inhibition enhances immunovirotherapy in glioblastoma via tertiary lymphoid structure modulation

Authors

Kyritsi, K., Zhu, D., Ding, H., Friedman, G. K., Lv, D., Wang, M., Mivechi, N. F., Kollhe, R., Johnson, T. S., Kaur, B., Munn, D. H., Hong, B.

Abstract

Glioblastoma (GBM) is a highly aggressive type of glioma that is resistant to immunotherapy and is associated with poor prognosis, largely due to its immunosuppressive tumor microenvironment. Brutons tyrosine kinase (BTK) is a non-receptor kinase that not only plays an important role in oncogenic signaling, particularly in tumor growth, but also regulates the activity of tumor-infiltrating myeloid cells, including dendritic cells, macrophages, and microglia in brain tumors. High BTK expression is associated with poor survival in patients with glioma. Oncolytic herpes simplex virus type 1 (oHSV) derived virotherapy, a novel treatment strategy, has demonstrated effectiveness against GBM; however, its efficacy is limited by the tumor microenvironment. In this study, we found that BTK is predominantly expressed in GBM-infiltrating myeloid cells. Intratumoral injection of oHSV not only promotes infiltration of myeloid cells and T cells but also activates BTK in these myeloid cells, thereby limiting oHSV infection and replication in tumor cells. Combination treatment with BTK inhibitor ibrutinib improves anti-tumor efficacy of oHSV in both human GBM12 xenograft and syngeneic murine GSC005 models. Mechanistically, BTK inhibition increases oHSV-mediated tumor cell death (cleaved caspase-3) and cytotoxic CD8 T cell infiltration, while decreasing tumor cell proliferation (Ki-67). BTK inhibition not only suppresses oHSV clearance by tumor-infiltrating microglia and macrophages but also reduces their pro-invasive effects on tumor cells. Addition of IDO inhibitor, an immune modulator, further prolongs survival in tumor-bearing mice in a syngeneic GBM model. Single-cell mRNA sequencing (scRNA seq) analysis indicates that combination treatment modifies key signaling pathways in both tumor-infiltrating myeloid cells (macrophages and microglia) and CD8 T cells. Further analysis shows that BTK inhibition, with or without IDO inhibition, promotes the formation of tumor-infiltrating tertiary lymphoid structures (TLS) during intratumoral oHSV treatment, subsequently remodeling T cell, NKT cell, and monocyte, macrophage populations. These results indicate that BTK inhibition exerts multifaceted effects in enhancing the anti-tumor efficacy of oHSV therapy.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 05 Jun 2026.

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