IL-23-engineered oncolytic vaccinia virus enhances pancreatic cancer control through inflammasome-driven Th17-skewed CD4+ T-cell responses.

Abstract

Background: Oncolytic viruses represent a promising immunotherapeutic strategy for converting pancreatic and other immunologically cold tumors. While their ability to stimulate innate immune signaling and promote antitumor immunity is established, mechanisms enabling durable adaptive responses remain poorly defined.

Methods: We used CRISPR-Cas9-based homologous recombination to generate a thymidine kinase (TK) and B2R double-deleted backbone virus, VV∆TK∆B2R (VV-DD). On this backbone, we engineered VV∆TKmp40∆B2Rmp19 (VV-DDIL23), which expresses the p40 and p19 subunits of interleukin-23 (IL-23) separately to mimic natural heterodimer assembly. Its antitumor efficacy was evaluated in immunosuppressive pancreatic ductal adenocarcinoma models and compared with a single-chain IL-23 fusion construct (VV-DDIL23F). In parallel, unbiased single-cell RNA sequencing was employed to systematically dissect the immune mechanisms underlying the potent antitumor activity of VV-DDIL23.

Results: VV-DDIL23 exhibited potent antitumor efficacy and significantly outperformed VV-DDIL23F. Single-cell RNA sequencing revealed that VV-DDIL23 enhanced antigen processing and presentation in macrophages, characterized by elevated major histocompatibility complex-II expression, upregulation of co-stimulatory molecules, and production of cytokines including interferon-I and IL-1β. This was accompanied by the emergence of a distinct population of tumor-infiltrating CD4⁺ T cells that mediated long-term memory immune surveillance against tumors. Mechanistically, VV-DDIL23 infection triggered tumor cell-mediated activation of the macrophage inflammasome, leading to IL-1β release. Notably, IL-1β synergized with virally encoded IL-23 to promote the differentiation of Th17-skewed Cxcr6⁺Rora⁺ CD4⁺ T cells with sustained antitumor functions.

Conclusion: This work reveals a previously unrecognized pathway linking innate viral recognition by macrophages to CD4⁺ T cell-directed differentiation and tumor clearance, providing a novel therapeutic strategy to overcome immunosuppression in pancreatic cancer.