Abstract PS4-04-22: Bacterial-tumor antigen cross reactive T-cells are enriched in tumors of newly diagnosed breast cancer patients

Abstract Background: The breast tumor immune microenvironment is characterized by a Type II signature which is an early event in breast cancer and portends more aggressive disease. We have previously demonstrated that bacteria-specific T-cells, derived from the gut, also recognize tumor antigens with which they share a high degree of homology at the level of Class II epitopes presented in MHC molecules. These peripheralized bacterial-tumor antigen cross reactive T-cells are tolerogenic, secrete high levels of IL-10 and, in mouse models, when adoptively transferred, traffic specifically to breast tumors and accelerate tumor growth. We questioned if the Type II signature present in human breast cancer is due to these bacterial-tumor antigen (BAC-TA) cross reactive T-cells. Methods: PBMC and stool were collected from newly diagnosed, treatment naive breast cancer patients (n = 56) or volunteer aged matched donors (n = 14). PBMC were screened with a panel of nine previously identified BAC-TA epitopes by IL-10 ELISPOT. Tumor biopsies were collected from three patients at the time of surgery. BAC-TA-specific T-cell lines were generated from PBMC from these three patients that responded with high magnitude IL-10 in the initial screen to BIRC5-p13-30 for Donor 1 (mean, 178±59 SPW / 106; p = 0.0002) and Donor 2 (mean, 1612±234 SPW / 106; p = 0.0011) and PRL3-p104-118 for Donor 3 (mean, 365±100 SPW / 106 T-cells; p = 0.03). Extracted DNA from the PBMC, T-cell lines and tumor biopsy was subjected to TCRb sequencing. Metagenomic sequencing was performed on stool samples. Results: A higher precursor frequency of BAC-TA cross reactive IL-10-secreting T-cells were observed in 60% of breast cancer patients (median, 1:160,000 PBMC) as compared to 28% of the volunteer donors (median <1:1, 000,000 PBMC; p = 0.039). To determine if these BAC-TA cross reactive T-cells were found in the tumors of newly diagnosed breast cancer patients, we matched T-cell clones in the BAC-TA-specific T-cell line against T-cells in the corresponding donor’s tumor. Three BIRC5-p13-30-specific T-cell clones in Donor 1 were found in the tumor biopsy. Furthermore, the levels of three bacteria with >50% homology to BIRC5-p13-30 were enriched in the stool of Donor 1 as compared to the levels in the stool of the 56 breast cancer patients evaluated in this study (L. lactis; enriched by 186-fold and S. oralis and P. fluorescens; both enriched by 3-fold). Similar trends were observed in the other two donors. Eleven BIRC5-p13-specific T-cell clones from Donor 2 were found in the matching tumor biopsy, with enrichment of two bacterial species with high BIRC5-p13-30 sequence homology (K. pneumoniae; 256-fold increase and P. distasonis; 4-fold increase). 25 PRL3-p104-specific T-cell clones in Donor 3 were found to be identical between the tumor biopsy and T-cell line. The stool of Donor 3 demonstrated a 15-fold and 3.5-fold increase in P. copri and P. fluorescens, respectively, two bacteria with at least 50% homology to PRL3-p104-118. Conclusion: IL-10-secreting BAC-TA T-cells can be found in high numbers in the peripheral blood of of newly diagnosed breast cancer patients and can be identified infiltrating the tumor. IL-10 is a key immunosuppressive cytokine that plays a central role in dampening immune responses and maintaining immune homeostasis. IL-10 inhibits the production of pro-inflammatory cytokines such as TNF-α and IFN-γ and can limit antigen-presenting cell function, leading to the reduction of Th1, Th17 and cytotoxic CD8+ T-cell activation. These data lay the foundation for a study that will follow newly diagnosed breast cancer patients correlating levels of BAC-TA cross reactive T-cells in blood with prognosis or response to therapy. Citation Format: D. Cecil, J. Childs, M. Disis. Bacterial-tumor antigen cross reactive T-cells are enriched in tumors of newly diagnosed breast cancer patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-22.