Abstract 1569: Role of NK and CD8+T cells in the antitumor effect elicited by combined treatment with radiation, tumor-specific antibody, and IL2 in a syngeneic murine model of head and neck squamous cell carcinoma
Authors: Jin Wonjong 
Published: July 2021

Abstract
Background: Radiation therapy (RT) has been shown to activate an in situ vaccine (ISV) effects. Recently, we reported that combining tumor-antigen-specific monoclonal antibody (mAb), RT, and intratumoral (IT) injection of IL2 activates a much greater ISV response compared to RT alone. In this combination, RT can enhance the mAb-dependent cell-mediated cytotoxicity response by NK cells when delivered with a tumor-specific mAb. Here we evaluated the role of NK cells in activating the CD8+ T cell-dependent ISV effect following RT, mAb, and IT-IL2.
Methods: MOC2 murine head and neck squamous cell carcinoma (HNSCC) cells expressing human epidermal growth factor receptor (MOC2-huEGFR+) were engrafted on the right flank of C57BL/6 mice. Tumors were treated with RT, the anti-huEGFR mAb cetuximab, and IT-IL2. In mice rendered tumor-free, immune memory was tested by re-engraftment with MOC2 wild-type tumor cells on the left flank. Splenic cells were isolated from a cohort of treated mice and cultured with MOC2 cells. These were analyzed by flow cytometry to quantify IFNγ expression. To test ISV efficacy in metastatic settings, mice were engrafted with MOC2-huEGFR+ right flank tumors and MOC2 wild-type left flank tumors. After treatment, immune infiltration was analyzed in both tumors, blood, and spleen. To assess the necessity of immune cells, CD4+, CD8+, or NK1.1+ cells were depleted.
Results: Combining RT and IT cetuximab and IL2 resulted in greater tumor regression and overall survival compared to single or dual treatments in mice with MOC2-huEGFR+ tumors. Six of 7 disease-free mice exhibited immune memory. Coculture of splenocytes from ISV treated mice with MOC2-huEGFR cells in vitro showed increased IFNγ expression in CD4+, CD8+, and NK cells. However, when splenocytes were first sorted and then co-cultured, only CD8+ T cells showed increased IFNγ expression after coculture with MOC2-huEGFR+. In a two-tumor model, delivering this ISV regimen to a right flank MOC2-huEGFR+ tumor increased CD8+ T cell infiltration in a distant left flank MOC2 wild-type tumor without affecting the number of, or IFNγ expression in, CD4+ or NK cells. Depletion of CD8+ cells eliminated antitumor response at both the right and left flank tumors in this model. Interestingly, NK cells, while not necessary for tumor response at the ISV-targeted right flank, were essential to the activation of a systemic CD8+ T cell response at the distant left flank tumor.
Conclusions: In a syngeneic murine model of HNSCC, the local combination of RT, cetuximab, and IL2 destroys the targeted tumor and enables this site to serve as a potent immune stimulus and personalized source of antigenicity for activation of adaptive T cell immunity. NK cells are necessary for the activation of this ISV effect and the resulting CD8+ T cell-dependent systemic antitumor response.
Citation Format: Wonjong Jin. Role of NK and CD8+T cells in the antitumor effect elicited by combined treatment with radiation, tumor-specific antibody, and IL2 in a syngeneic murine model of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1569.