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

Abstract

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.T-cell receptors | breast cancer | emulsion RT-PCR | high-throughput sequencing | T-cell repertoire profiling I nfiltration of numerous tumors by CD8 + alpha-beta T cells is associated with better outcomes and longer survival times for patients with breast cancer (1-5). Targeted immune-based therapies hold great promise toward improving breast cancer treatments (6, 7). Studies have examined the immune phenotype of breast cancer tumor-infiltrating lymphocytes (TILs), suggesting that activated nonsuppressive T cells are of most benefit (8). Further assessment of the TIL repertoire has broad implications for breast cancer therapies in antigen discovery, cancer vaccines, and adoptive cell therapies (7).Unique genetic recombination events are required to produce the T-cell receptor (TCR) (reviewed in 9). The alpha-and beta-chains of the TCR undergo V(D)J recombination and heterodimerize in the thymus, resulting in a diverse T-cell repertoire that specifically recognizes peptide-MHC complexes. Many studies have analyzed the alpha-and beta-chains of TIL TCRs separately using highthroughput sequencing to describe the diversity of TILs (10-13). Pairs are readily identified after expansion of T-cell clones, although culture of T cells can lead to substantial skewing of the repertoire (14), which may select for T cells of varied affinity or avidity (15). Single-cell sequencing identifies alpha-beta pairs, but is often laborious and has rela...

Harnessing shared antigens and T-cell receptors in cancer: Opportunities and challenges

“…The best-studied examples of the former are melanocyte differentiation antigens (e.g., MelanA) studied in the context of melanoma. Increased levels of MelanA-specific T cells could be detected in tumor draining lymph nodes in melanoma (12), similar to the findings described by Munson et al (6). Epitopes derived from viral oncoproteins in virus-induced malignancies or recurrent oncogenic mutations could, in principle, also be shared across several patients and provide an attractive target for immunotherapy.…”

“…Restricted TCR use is also a feature of emerging families of unconventional T cells that include autoreactive T cells and can recognize nonpeptide ligands (10). The findings of Munson et al (6), although provocative, should be viewed as preliminary. In addition to the current limitation of small numbers of patients studied thus far, evaluation of TCRs from different regions of the same tumor, different lesions within the same patient, and lesions with different T-cell subsets will further refine the conclusions.…”

“…There is an unmet need to understand and harness the properties of T cells infiltrating such tumors better. In PNAS, Munson et al (6) identify several shared T-cell receptors in T cells infiltrating HLA A2…”

“…Most of the current studies are based on sequencing isolated single (e.g., TCR-β) chains, although singlecell or computational methods to analyze αβ pairs are emerging (7). Munson et al (6) adapt an emulsionbased RT-PCR-based approach (8), wherein initial amplification of TCRs from limited numbers of TCRs within a lipid droplet allows identification of dominant TCR α/β partners. Analysis of TCRs from TILs or blood of patients with breast cancer revealed several shared TCR sequences with an enrichment of TCRs from the TRBV7 family.…”

Shared αβ TCR Usage in Lungs of Sarcoidosis Patients with Löfgren’s Syndrome

“…The open-source Galaxy Platform (usegalaxy.org) was utilized to process the raw sequencing files, as outlined below and as previously described (19). For each of the iRepertoire or scPCR samples, the raw read 1 and read 2 sequences were paired in the Galaxy using the Pear function, and the samples were then split into individual files via the Barcode Splitter function and utilized for MiTCR identification (see below).…”