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Supplementary Data 6 from Inhibition of EphB4–Ephrin-B2 Signaling Reprograms the Tumor Immune Microenvironment in Head and Neck Cancers

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journal contribution
posted on 2023-03-31, 01:42 authored by Shilpa Bhatia, Ayman Oweida, Shelby Lennon, Laurel B. Darragh, Dallin Milner, Andy V. Phan, Adam C. Mueller, Benjamin Van Court, David Raben, Natalie J. Serkova, Xiao-Jing Wang, Antonio Jimeno, Eric T. Clambey, Elena B. Pasquale, Sana D. Karam

Total number of CD4+T cells remain unchanged following Control-Fc and ephrin-B2-Fc (20 μg/ml) treatment of T cells (A). Correlation analysis was also performed between TGF-β and EFNB2, EphB4 on R2 analysis platform (B-C). Alterations of cell growth and survival markers in western blots performed using lysates of T cells treated with ephrin-B2-Fc and control-Fc (D). These samples were run on the same gel.


National Institute of Dental and Craniofacial Research


Golfers Against Cancer

Cancer League of Colorado

University of Colorado

Paul Sandoval Grant,Wings of Hope grant



Identifying targets present in the tumor microenvironment that contribute to immune evasion has become an important area of research. In this study, we identified EphB4–ephrin-B2 signaling as a regulator of both innate and adaptive components of the immune system. EphB4 belongs to receptor tyrosine kinase family that interacts with ephrin-B2 ligand at sites of cell–cell contact, resulting in bidirectional signaling. We found that EphB4–ephrin-B2 inhibition alone or in combination with radiation (RT) reduced intratumoral regulatory T cells (Tregs) and increased activation of both CD8+ and CD4+Foxp3− T cells compared with the control group in an orthotopic head and neck squamous cell carcinoma (HNSCC) model. We also compared the effect of EphB4–ephrin-B2 inhibition combined with RT with combined anti-PDL1 and RT and observed similar tumor growth suppression, particularly at early time-points. A patient-derived xenograft model showed reduction of tumor-associated M2 macrophages and favored polarization towards an antitumoral M1 phenotype following EphB4–ephrin-B2 inhibition with RT. In vitro, EphB4 signaling inhibition decreased Ki67-expressing Tregs and Treg activation compared with the control group. Overall, our study is the first to implicate the role of EphB4–ephrin-B2 in tumor immune response. Moreover, our findings suggest that EphB4–ephrin-B2 inhibition combined with RT represents a potential alternative for patients with HNSCC and could be particularly beneficial for patients who are ineligible to receive or cannot tolerate anti-PDL1 therapy. These findings present EphB4–ephrin-B2 inhibition as an alternative to anti-PDL1 therapeutics that can be used in combination with radiation to induce an effective antitumor immune response in patients with HNSCC.

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