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The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires., Author summary More than 1,100 cancer treatments are currently in clinical testing. Of these, 295 are immune-oncology medicines and vaccines. For Checkpoint Immunotherapy alone, 52,539 patients were enrolled in 2017. Of these treatments, 108 are for breast cancer, the leading cancer diagnosed in women. Although checkpoint immunotherapy has yet to be proven beneficial in most types of breast cancer, current observations of which specific T cell go into the tumor, and how these cells are connected to the response to treatment, have lead to a new understating of the role of the immune system. Yet, and in spite of this intense research into the role of T cells in tumors, very little is known about how clones of T cells behave during tumor formation. The paper we present here studies this clonal behavior over time. We study it during mammary tumor development in mice. We see that a set of T cell clones, which comes from a diversity of different nucleotide sequences, stands out in its behavior compared with other T cell clones. Importantly, we show that these mouse T cell clones have a strong connection to human breast cancer. The specific identification of a subset of T cell clones, together with the behavior of these clones over time, is critical to our understanding of the effect of modifications to the immune system, such as the modifications made by immunotherapy treatments, and to our understanding of how to better control the behavior of the immune system.

Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data. Although several hierarchical clustering-based methods have been proposed, they vary in distance and linkage methods and have not yet been rigorously compared. In this study, we use a combination of human experimental and simulated data to characterize the performance of hierarchical clustering-based methods for partitioning sequences into clones. We find that single linkage clustering has high performance, with specificity, sensitivity, and positive predictive value all >99%, whereas other linkages result in a significant loss of sensitivity. Surprisingly, distance metrics that incorporate the biases of somatic hypermutation do not outperform simple Hamming distance. Although errors were more likely in sequences with short junctions, using the entire dataset to choose a single distance threshold for clustering is near optimal. Our results suggest that hierarchical clustering using single linkage with Hamming distance identifies clones with high confidence and provides a fully automated method for clonal grouping. The performance estimates we develop provide important context to interpret clonal analysis of repertoire sequencing data and allow for rigorous testing of other clonal grouping algorithms.

Cancer immunotherapy by checkpoint blockade proves that an effective immune response to a tumor can be induced clinically. However, little is known about the evolution of tumor-associated T-cell receptor (TCR) repertoires without intervention. Here we studied TCR repertoire evolution in mice spontaneously developing mammary tumors; we sequenced peripheral blood alpha and beta TCRs of CD4+CD62L+CD44− T cells monthly for 8 months in 10 FVB/NJ mice transgenic at the Erbb2 locus, all developing tumors; 5 FVB/NJ mice without the transgene were age-matched controls. Sequences were either private (restricted to one mouse) or public (shared among mice); public sequences were either exclusive to the tumor group or inclusive among different groups. We now report that 1), public AA sequences were each encoded by many different nucleotide sequences (NT) recombinations (convergent recombination; CR); 2) mice developing tumors evolved tumor-exclusive public sequences, derived initially from private or from inclusive public sequences; and 3) tumor-exclusive public sequences in mice were also present among published public TCR sequences from human breast cancer patients. These cross-species tumor-exclusive TCR sequences manifested high CR; but the AA sequences shared by mice and humans did not share NT sequences. Thus, tumor-exclusive TCR AA sequences across species are selected from different NT recombination events. The roles of tumor-exclusive TCR repertoires in advancing or inhibiting tumor development and the effects of tumor immunotherapy on these T cells remain to be seen.