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In tumors, a subset of CD8 + T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1 + CD8 + T cells revealed that while intratumoral TCF-1 + CD8 + T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1 + CD8 + T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1 + CD8 + T cell subsets developed over time-a proliferative SlamF6 + subset and a non-cycling SlamF6 - subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6 + TCF-1 + CD8 + T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6 + T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1 + CD8 + T cells and their decrease contributes to failed anti-tumor immunity., Competing Interests: Declaration of interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. He is the President of Break Through Cancer. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J. laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and The Lustgarten Foundation for Pancreatic Cancer Research, but this funding did not support the research described in this manuscript. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov until July 31, 2020. From August 1, 2020, A.R. is an employee of Genentech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1 + progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1 + cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6 + TCF1 + cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6 + TCF1 + cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors., Competing Interests: Declaration of interests T.J. is on the board of directors of Amgen and ThermoFisher Scientific, a co-founder of Dragonfly Therapeutics and T2 Biosystems, an SAB member of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics and is president of Break Through Cancer. The T.J. lab receives funding from the Johnson & Johnson Lung Cancer Initiative and The Lustgarten Foundation for Pancreatic Cancer Research, but this funding did not support this research. A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and until August 31, 2020, was an SAB member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov, and ThermoFisher Scientific. A.R. is an employee of Genentech (Roche Group) from August 1, 2020. S.S. is a consultant for RareCyte, Inc. A.M.C. interned at Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS., Competing Interests: Declaration of Interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific, and a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. T.J. also received funding from Calico and currently receives funding from Johnson & Johnson, but this funding did not support the research described in this manuscript. A.R. is a co-founder and equity holder in Celsius Therapeutics and a SAB member for Thermo Fisher, Asimov, Neogene Therapeutics, and Syros Pharmaceuticals, and an equity holder of Immunitas Therapeutics. C.M.R. serves on the SAB of Bridge Medicines and Harpoon Therapeutics, and has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Bicycle, Celgene, Daiichi Sankyo, Genentech, Ipsen, Loxo, Pharmamar, and Vavotek. None of the affiliations listed above represent a conflict of interest with the design or execution of this study or interpretation of data presented in this manuscript. Other authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Regulatory T cells (T regs ) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. T regs in human tumors span diverse transcriptional states distinct from those of peripheral T regs , but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of T regs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive T regs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. T reg -specific deletion of ST2 alters the evolution of effector T reg diversity, increases infiltration of CD8 + T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in T reg -mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6 + cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6 + cells impairs airway injury repair in vivo. Distinct Lgr5 + cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural killer (NK) cells function in the recognition and destruction of host cells infected with pathogens. Many regulatory mechanisms govern the potent responses of NK cells, both at the cellular and molecular level. Ablation of microRNA (miRNA) processing enzymes demonstrated that miRNAs play critical roles in NK cell differentiation and function; however, the role of individual miRNAs requires further investigation. Using mice containing a targeted deletion of microRNA-155 (miR-155), we observed defects in NK cell maintenance and maturation at steady state, as well as in homeostatic proliferation in lymphopenic mice. In addition, we discovered that miR-155 is up-regulated in activated NK cells during mouse cytomegalovirus (MCMV) infection in response to signals from the proinflammatory cytokines IL-12 and IL-18 and through signal transducer and activator of transcription 4 (STAT4) signaling. Although miR-155 was found to be dispensable for cytotoxicity and cytokine production when triggered through activating receptors, NK cells lacking miR-155 exhibited severely impaired effector and memory cell numbers in both lymphoid and nonlymphoid tissues after MCMV infection. We demonstrate that miR-155 differentially targets Noxa and suppressor of cytokine signaling 1 (SOCS1) in NK cells at distinct stages of homeostasis and activation. NK cells constitutively expressing Noxa and SOCS1 exhibit profound defects in expansion during the response to MCMV infection, suggesting that their regulation by miR-155 promotes antiviral immunity.

MicroRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity of the miRNA seed to the 3' untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNA-miR-155-in a genetically controlled manner. We found that approximately 40% of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these noncanonical sites feature extensive complementarity to the miRNA seed with one mismatch. These noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. Thus, noncanonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedia's use for 3D visualization in structure-related pedagogy., (Copyright © 2011 Elsevier Inc. All rights reserved.)