Abstract C058: Myeloid-T cell interactions and B cell activation mediate response to treatment with entinostat and checkpoint inhibitors in metastatic breast cancer

Abstract Complications due to metastasis to vital organs accounts for most deaths due to breast cancer. Immune checkpoint inhibitors (ICIs) activate T cells to mount anti-tumor responses, but have proved ineffective against breast cancer. Macrophages and myeloid-derived suppressor cells (MDSCs) are abundant in breast tumors and suppress anti-tumoral responses by T cells, contributing to treatment resistance. The combination of ICIs anti-PD-1 and anti-CTLA-4 with the histone deacetylase inhibitor, entinostat, has been shown to inhibit breast-to-lung metastasis in the 4T1 model of triple-negative breast cancer (TNBC) and increased survival in 4T1 and the NT2.5 model of HER2 + breast cancer. This treatment combination also resulted in a 25% objective response rate in a phase Ib clinical trial composed of patients with metastatic breast cancer. Using our recently published NT2.5-LM model of HER2+ breast-to-lung metastasis, we demonstrated increased survival and decreased lung metastasis using entinostat, anti-PD-1, and anti-CTLA-4 (EPC). EPC increased B cell activation in lung metastases, B cell and CD8+ T cell activation in lymph nodes, and cancer cell-targeting antibodies, as observed via flow cytometry. A cell chat analysis of a single cell RNA sequencing data from treated NT2.5-LM lung metastases revealed a decrease in interactions between CD8+ T cells and macrophages involving ICAM-1 and Mac-1 (CD11b/CD18) with EPC treatment. Decreased ICAM-1 expression was confirmed in effector CD8+ T cells via flow cytometry. In the 4T1 model, EPC increased the proportions of effector CD8+ T cells and activated Th2 cells in lung metastases. To assess functional changes in immune cells with EPC treatment, we co-cultured healthy, splenic CD8+ T cells with macrophages or granulocytic MDSCs (G-MDSCs) from treated NT2.5-LM or 4T1 lung metastases. Although EPC did not result in any changes to macrophage suppression of T cell proliferation in NT2.5-LM or G-MDSC suppression in either model, simulating the effects of EPC treatment ex-vivo via ICAM-1 blockade increased the proliferation of CD8+ T cells in co-cultures with macrophages. Blockade of ICAM-1 in-vivo was not sufficient to increase survival either as a single agent or in combination with anti-PD-1 and anti-CTLA-4. An imaging mass cytometry analysis of metastatic tumors from patients treated with EPC showed increased CD8+ T cell proportions, expression of activation marker CD137 in B cells and CD8+ T cells, and distances between CD8+ T cells and macrophages with EPC in treatment in responders. In addition, only responders had detectable germinal centers or mature tertiary lymphoid structures. A flow cytometry analysis of patient blood revealed an increase in plasma cell proportions and upregulation of HLA-DR in B cells in responders with EPC treatment. Overall, our results suggest that B cell activation and reduction of myeloid immunosuppression increase the efficacy of ICIs in metastatic breast cancer; these mechanisms should be carefully considered when using ICIs in clinical settings. Citation Format: Edgar Gonzalez, Evanthia T. Roussos Torres, Adam L. MacLean, Yingtong Liu, Xiaojun Wu, Jesse Kreger, Arianna Barbetta, Juliet Emamaullee, Vered Stearns, Roisin Connolly, Won Jin. Ho, Sarah M. Shin, Julie Jang. Myeloid-T cell interactions and B cell activation mediate response to treatment with entinostat and checkpoint inhibitors in metastatic breast cancer abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C058.