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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.

Summary Biology of the response to anti-CTLA-4 involves the dynamics of specific T cell clones. Reasons for clinical success and failure of this treatment are still largely unknown. Here, we quantified the dynamics of the T cell receptor (TCR) repertoire, throughout 4 weeks involving treatment with anti-CTLA-4, in a syngeneic mouse model for colorectal cancer. These dynamics show an initial increase in clonality in tandem with a decrease in diversity, effects which gradually subside. Furthermore, response to treatment is tightly connected to the shared and public parts of the T cell repertoire. We were able to recognize time-dependent behaviors of specific TCR sequences and cell types and to show the response is dominated by specific motifs. We see that a single, specific time point might be useful to inform a physician of the true response to treatmentThe research further highlights the importance of temporal analyses of the immune response., Graphical Abstract, Highlights • Response to ICI is associated with pre-treatment TCR repertoire in mice • TCR repertoire goes through distinct, ICI-dependent changes with time • Tumor size and its response to ICI can be tracked by TCR repertoire metrics • A single time point is found to be a focal point of the immune response, Immunology; Cell Biology: Bioinformatics

We describe and provide an array of diverse methods to predict cellular positions in tissue from RNA-seq data selecting mapping genes according to their spatial/statistical properties and their effect on improving the cell positioning., Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pair-rule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes than in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues.

The introduction of novel cancer drugs and innovative treatments brings great hope for cancer patients, but also an urgent need to match drugs to suitable patients, since certain drugs that benefit one patient may actually harm others. The newly developed poly-ADP ribose polymerase (PARP) inhibitors (PARPis) are a group of pharmacological enzyme inhibitors used clinically for multiple indications. Several forms of cancer tend to be PARP dependent, making PARP an attractive target for cancer therapy. Specifically, PARPis are commonly used in BRCA-associated breast cancers patients, since unrepaired single-strand breaks are converted into double-strand breaks and BRCA-associated tumors cannot repair them by homologous recombination so that PARPi leads to tumor cell death, by a mechanism called “Synthetic Lethality”. Unfortunately, not all patients respond to PARPi, and it is not currently possible to predict who will or will not respond. Here, we present a specific genomic marker, which reflects a single-nucleotide polymorphism of human PARP1 and correlates in vitro with response to PARPi, throughout all indications. In addition, we report that this SNP is associated with re-shaping mRNA, and mRNA levels, and influences the final protein structure to expose new binding sites while hiding others. The status of the SNP is therefore critical to patients’ care, as it relates responses to PARPi to the PARP1-SNP carried.

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.

SummaryThe T cell receptor (TCR) controls the cellular adaptive immune response to antigens, but our understanding of TCR repertoire diversity and response to challenge is still incomplete. For example, TCR clones shared by different individuals with minimal alteration to germline gene sequences (public clones) are detectable in all vertebrates, but their significance is unknown. Although small in size, the zebrafish TCR repertoire is controlled by processes similar to those operating in mammals. Thus, we studied the zebrafish TCR repertoire and its response to stimulation with self and foreign antigens. We found that cross-reactive public TCRs dominate the T cell response, endowing a limited TCR repertoire with the ability to cope with diverse antigenic challenges. These features of vertebrate public TCRs might provide a mechanism for the rapid generation of protective T cell immunity, allowing a short temporal window for the development of more specific private T cell responses.