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Supplementary Tables 3, 6, 7, 9, 11, 12

Supplementary Tables 1, 2, 4, 5, 8, 10, 13, 14, 15

In vitro killing assay movies. Co-culture of 12T melanoma cells with irrelevant 108TILs

2P microscopy movies of in vivo experiments

Supplementary Figures 1-17

Supplementary methods

A T‐cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune‐privileged, and in the tumor. However, CTAs are self‐antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA‐A2‐MAGE‐A1278‐286. We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN‐γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy., The human hT27 TCR recognizes the cancer testis antigen HLA‐A2‐MAGE‐A1278‐286 with low affinity and avidity. We enhanced the avidity of this TCR using somatic hypermutation (SHM) in BWZ.36‐derived cells, a T‐cell line. The resulting mutant TCRs had vastly improved anti‐tumor activity and are better suited for cancer immunotherapy.

One of the feats of adaptive immunity is its ability to recognize foreign pathogens while sparing the self. During maturation in the thymus, T cells are selected through the binding properties of their antigen-specific T-cell receptor (TCR), through the elimination of both weakly (positive selection) and strongly (negative selection) self-reactive receptors. However, the impact of thymic selection on the TCR repertoire is poorly understood. Here, we use transgenic Nur77-mice expressing a T-cell activation reporter to study the repertoires of thymic T cells at various stages of their development, including cells that do not pass selection. We combine high-throughput repertoire sequencing with statistical inference techniques to charactarize the selection of the TCR in these distinct subsets. We find small but significant differences in the TCR repertoire parameters between the maturation stages, which recapitulate known differentiation pathways leading to the CD4+ and CD8+ subtypes. These differences can be simulated by simple models of selection acting linearly on the sequence features. We find no evidence of specific sequences or sequence motifs or features that are suppressed by negative selection. These results are consistent with a collective or statistical model for T-cell specificity, where negative selection biases the repertoire away from self recognition, rather than ensuring lack of self-reactivity at the single-cell level.

The creation and evolution of the T cell receptor repertoire within an individual combines stochastic and deterministic processes. We systematically examine the structure of the repertoire in different T cell subsets in young, adult and LCMV infected mice, from the perspective of variable gene usage, nucleotide sequences and amino acid motifs. Young individuals share a high level of organization, especially in the frequency distribution of variable genes and amino acid motifs. In adult mice, this structure relaxes and is replaced by idiotypic evolution of the effector and regulatory repertoire. The repertoire of CD4+ regulatory T cells was more similar to naïve cells in young mice, but became more similar to effectors with age. Finally, we observed a dramatic restructuring of the repertoire following infection with LCMV. We hypothesize that the stochastic process of recombination and thymic selection initially impose a strong structure to the repertoire, which gradually relaxes following asynchronous responses to different antigens during life.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies. Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366–75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity. DOI: http://dx.doi.org/10.7554/eLife.22057.001

Introduction The heterogeneous fates of metastatic breast cancer (MBC) preclude our understanding of both resistance to therapy and escape from cancer immunoediting. Here, we performed a comprehensive molecular analysis of lethal MBC patients (pts), interrogating both the malignant and immune tumour microenvironment (TME) compartments, and T-cell receptor (TCR) repertoires, across multiple metastases (mets). Material and methods Multi-platform profiling of mets (n=182 mets to 22 organs, 5–36 mets/pt), primary tumours (n=6) and ctDNA from body fluids (4.7/pt) in 10 warm autopsies of MBC pts (5 ER+/HER2-, 3 ER+/HER2+, 1 ER-/HER2+, 1 ER-/HER2-), included exome seq (n=86), shallow whole genome seq (n=168), RNA seq (n=61), ultra-deep targeted seq (TS) (n=243), TCRseq (n=70) and IHC (n=102). State-of-the-art bioinformatics was applied to integrate the data. Results and discussions Mutation (mut) burden landscapes varied between pts (11 579 mut, median 255.41 mut/pt) and across mets within each pt (median 122 mut/met; were greater than TCGA mut burden (median 63.5 mut/primary, p=4.927e-14). Landscape of mut and predicted neo-antigen were dominated by stem (present in all mets/pt) or clade (some mets/pt), but not private (one met/pt). TS data confirmed that all primary tumours contained the clonal ancestors of 10 pts, and characterised ctDNA bathing organs. Copy number alteration profiles were remarkably similar across mets in 9 of the 10 pts, except in a ER+/HER2- pt, whose mets shared a common ancestor (1q gain/16q loss), then early sub-clonal evolution occurred. Mets were grouped into phylogenic clades that shared common genomic ancestry and accumulated previously unknown mutation signatures. Mets evolved as communities of clones as a fraction of the metastatic stem and clade mutations were sub-clonal. Immune TME was either homogeneous in a particular metastatic clade, or different across mets to a particular organ. Stem and clade clonotypes prevailed across TCR landscape within each pt. TCR repertoires revealed adaptive immune responses to co-evolve with the metastatic genomes. Conclusion The genomic and immune landscapes demonstrate an unprecedented integrated view of the heterogeneous landscape of genomic aberrations, TME features and T-cell adaptive immune responses in lethal MBC.

1009Background: The heterogeneous fates of MBC preclude our understanding of both resistance to therapy and escape from cancer immunoediting. Here, we performed a comprehensive molecular analysis o...

Summary The detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer., Graphical Abstract, Highlights • Genomic and transcriptomic landscapes for 10 lethal breast cancers • Within a patient, metastases group in limited clades with shared genomic ancestry • Tumor immune microenvironments across metastases are not uniform • Phylogenetic trees are correlated with TIL-TCR trees across metastases, De Mattos-Arruda et al. profiled multiple metastases from autopsies of patients with therapy-resistant breast cancer, showing that multi-clonal spreading occurs in a small number of founder events. The analysis characterizes predicted neo-antigen landscapes, tumor microenvironments, and accumulation of HLA LOH. T cell immune responses appear to co-evolve with metastatic cancer genomes.