Type I interferon reprograms neutrophil-T cell interactions to enhance antitumor immunity and informs a combinatorial strategy for esophageal cancer

Background: Type I interferons (IFN-I) integrate antiviral and antitumor responses by coordinating innate and adaptive immunity. Although their canonical function involves dendritic-cell (DC)-mediated antigen presentation, we identified a DC-independent mechanism whereby IFN-I directly reprograms neutrophils and reshapes the tumor immune microenvironment (TME) in esophageal squamous cell carcinoma (ESCC). This study pursued three aims: (1) to define the cellular compartments required for IFN-I signaling; (2) to dissect how IFN-I-reprogrammed neutrophils potentiate T-cell antitumor function; and (3) to evaluate translational strategies combining IFN-I agonism, TGF-beta and PD-1 blockade. Methods: Differentiated neutrophil-like cells were stimulated with IFN-alpha or IFN-I inducers (RIG-I-2CARD, STING-CTD ± c-di-GMP). N1/N2 polarization markers (CD54/ICAM-1, CD86, SELL, ARG1) were analyzed by flow cytometry and Western blot. Co-cultures of neutrophils, T cells, and KYSE esophageal carcinoma cells quantified T-cell cytotoxicity. Mechanistic assays used ICAM-1/LFA-1 blockade, metabolic inhibitors (nor-NOHA, DPI, NAC), and Transwell tests. For in-vivo validation, Ifnar1 fl/fl with CD4-Cre, Itgax-Cre, and S100a8-CreERT2 mice restricted IFN-I signaling to T cells, DCs, or neutrophils. MC38 tumor (C57BL/6) and KYSE450 xenograft (HSC-NSG) were established. IFN-I activation used Ad5-IFNA4 (10⁷-10⁸ PFU i.v., weekly) or c-di-GMP (20 µg i.p., q3d). Combination arms received TGF-beta-trap and/or anti-PD-1 (10 mg/kg i.p.). Tumor growth, N1/N2 ratio, and CD8⁺ Teff infiltration were assessed by flow cytometry. Results: IFN-I signaling acted as the dominant upstream regulator of neutrophil-dependent tumor control. IFN-alpha and IFN-I inducers consistently polarized neutrophils toward an N1-like phenotype. Only neutrophils pretreated with IFN-alpha enhanced T-cell-mediated tumor killing; untreated cells failed. Direct addition of IFN-alpha to the culture had no effect, confirming that neutrophil priming by IFN-I is required. Blocking ICAM-1/LFA-1 or separating cells by Transwell reduced T-cell killing, indicating contact dependence. In vivo, Ad5-IFNA4 and c-di-GMP were well tolerated and increased intratumoral CD54⁺ N1-like neutrophils. Ongoing studies in Ifnar1 conditional and HSC-NSG models are testing whether sustained IFN-I activation synergizes with PD-1 and TGF-beta blockade to enhance CD8⁺ Teff infiltration and antitumor efficacy. Conclusions: Type I interferon signaling links neutrophil reprogramming to T-cell activation. IFN-I drives N1 polarization, promotes ICAM-1/LFA-1-dependent contact, and may relieve ARG1/NOX2-mediated suppression to restore T-cell cytotoxicity.These findings support a therapeutic framework combining IFN-I agonists with TGF-beta and PD-1 blockade for durable antitumor immunity in ESCC.