Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

Significance The essence of the adaptive immune response depends on the specificity of antigen receptors. This report identifies shared alpha–beta T-cell receptor (TCR) pairs from the tissues of HLA-A2+ patients with breast cancer and control donors. Using an emulsion-based RT-PCR assay, we analyzed TCR sequences from tissues ex vivo. We identified multiple TCR pairs shared between tumors, but not control samples. Although recent reports have concluded that anticancer T-cell responses depend on patient-specific mutation-associated neoantigens, this study provides evidence that T cells also recognize shared antigens. This approach has broad application to a variety of research questions where the end goal is to examine T-cell repertoires and/or identify T-cell antigens. Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.