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.
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•Lgr5 and Lgr6 mark mesenchymal cells in adult lungs•Single-cell transcriptome analysis defines mesenchymal heterogeneity•Distinct mesenchymal niches drive airway and alveolar differentiation•Wnt activity affects epithelial differentiation specified by mesenchymal cells
Heterogeneous mesenchymal cell populations in the lung play a central role in epithelial maintenance and alveolar differentiation.
DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not ...durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.
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.
► Differential CLIP-Seq reveals transcriptome-wide sites of miR-155 binding ► Many miR-155 binding sites are noncanonical and lack a perfect seed match ► Most noncanonical binding sites contain a mismatch to the canonical seed motif ► Noncanonical sites mediate gene regulation, albeit weaker than canonical sites
Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct ...from those of peripheral Tregs, 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 Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs 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. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention.
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•Single-cell profiling of CD4 T cells along tumor development in a mouse model•Treg diversity shifts to a Klrg1+Areg+ (KA) effector phenotype in advanced tumors•Il1rl1 (encoding ST2)+ Tregs have higher expression of KA effector Treg genes•Treg-specific ST2 loss enhances CD8+ T cell infiltration and decreases tumor burden
Li et al. show in a genetic mouse model of lung adenocarcinoma that during tumor development regulatory T cell (Treg) diversity shifts from an interferon-responsive to a ST2-positive, Klrg1+Areg+ effector-like phenotype. Treg-specific deletion of ST2 alters Treg heterogeneity, increases tumor infiltration by CD8+ T cells, and decreases tumor burden.
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.
BackgroundDNA mismatch repair deficiency (MMRd) drives the accumulation of somatic mutation-derived antigens (neoantigens), which underlies the remarkable response rates of this class of cancer to ...immune checkpoint blockade (ICB) therapy. Nevertheless, most advanced MMRd tumors do not respond durably to ICB, despite elevated tumor mutation and neoantigen burden. Critical questions remain regarding tumor mutation burden and immunosurveillance in the evolution and therapy resistance of these cancers.MethodsWe developed autochthonous mouse models of lung and colon cancer with perturbations of MMR genes (e.g., Msh2) via in vivo CRISPR-Cas9 and conditional knockout (Msh2flox ) and characterized their genomic profiles via whole exome and RNA sequencing. We performed randomly-enrolled and blinded preclinical trials of ICB (anti-PD-1/CTLA-4) with and without immunogenic chemotherapy (oxaliplatin/cyclophosphamide). We also established cell lines from these tumors with re-expression of Msh2 to arrest mutagenesis, performed sub-cloning, identified bona fide MHC-I neoepitopes, and tracked tumor growth, response to ICB, and neoantigen-specific T cell responses upon re-transplantation of these lines at different clonal fractions. Finally, we performed a meta-analysis of the predictive power of neoantigen burden and clonality in ICB response of human MMRd gastric and colorectal cancer.ResultsAs expected, MMRd resulted in elevated mutation and neoantigen burden with signatures consistent with the human disease. Surprisingly, this was not accompanied by an increase in T cell infiltration and the lung and colon cancer models were uniformly non-responsive to ICB, even when combined with immunogenic chemotherapy. Mechanistically, we showed that tumors harbored extensive intratumoral heterogeneity of mutations, which facilitated immune evasion of otherwise immunogenic subclones. Furthermore, neoantigen-specific T cells responses decreased in magnitude and effector differentiation with decreasing neoantigen clonal fraction. Interestingly, we found that immunosurveillance did not alter overall tumor mutation or neoantigen burden but shaped the clonal architecture of neoantigens arising spontaneously during tumorigenesis. Finally, we showed that clonal, but not subclonal, neoantigen burden was predictive of ICB response in clinical trials of MMRd gastric and colorectal cancer.ConclusionsThese results provide valuable context for understanding how immunosurveillance shapes the evolution of cancers undergoing sustained mutagenesis, as occurs in human cancer. Our findings point to a critical role of intratumoral heterogeneity in predicting response to immunotherapy and justify deeper clinical exploration of this biomarker in MMRd cancers. Finally, these results have major implications for therapies aimed at deliberately increasing tumor mutation burden. We predict that such therapies would increase intratumoral heterogeneity and undermine anticancer immunity.Ethics ApprovalAll animal use was approved by the Department of Comparative Medicine (DCM) at MIT and the Institutional Animal Care and Use Committee (IACUC) under protocol number 0714–076-17.
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.
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•Longitudinal scRNA-seq of tumor-specific TCF-1+ CD8+ T cells in KP lung adenocarcinoma•Identified a proliferative Slamf6+ TCF-1+ T cell subset and a non-cycling SlamF6− subset•The lymph node contains a recruitable reservoir of functional TCF-1+ CD8+ T cells•Flt3L+CD40 boosts cDC1, increases TCF-1+CD8+ T cell frequencies, decreases tumor burden
In tumors, TCF-1+ CD8+ T cells drive the response to immune checkpoint blockade. Schenkel, Herbst et al. reveal that, in lung adenocarcinoma, a reservoir of TCF-1+ CD8+ T cells is maintained in tumor draining lymph nodes by conventional type I dendritic cell (cDC1). Decrease of these cDC1 as the tumor progresses contributes to failed anti-tumor immunity.
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.
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•Lung cancer progression is accompanied by a stereotypic expansion of heterogeneity•Cell state heterogeneity arises largely independently of genetic variation•State transitions occur via an HPCS harboring high differentiation and growth capacity•The HPCS is drug resistant and portends poor patient survival across all cancers
Cellular states capable of promoting tumor progression and resisting therapies exist in heterogeneous tumors. Marjanovic et al. discover that a high-plasticity cell state common to mouse and human lung tumors drives cellular heterogeneity, is highly tumorigenic and drug resistant, and associates with poor patient prognosis.
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.
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•Peptide-MHC binding properties underlie antigen dominance hierarchies in a lung cancer model•CD8 T cells responding to subdominant antigens are enriched in TCF1+ progenitors•CCR6+ TCF1+ progenitor cells contribute poorly to checkpoint blockade response•Vaccination can eliminate CCR6+ TCF1+ cells and improve the subdominant response
Analysis of antigen dominance hierarchy and dynamics in a lung cancer model highlights the opportunity of optimizing T cell responses against subdominant neoantigens for better checkpoint blockade response through vaccination that adjusts T cell progenitor subpopulations
Abstract
CD8+ T cells drive protective responses against infection and cancer. In the context of chronic antigen stimulation, CD8+ T cells become progressively dysfunctional, losing the ability to ...proliferate, secrete cytokines, and kill target cells. While dysfunctional CD8 T cells have been characterized in infection, studies examining longitudinal responses in tumors have been limited. Here, in an autochthonous model of lung adenocarcinoma using high dimensional flow cytometry and single cell RNA sequencing, we demonstrate that tumor specific CD8 T cell heterogeneity is dynamic, revealing multiple dysfunctional-like CD8 T cell populations. Among these states, we identify TCF-1+ CD8 T cells, a population previously associated with superior functionality. Temporal analysis revealed heterogeneity within the TCF-1+ CD8+ T cell compartment - SlamF6+ CD8 T cells were predominant in early stage disease, while SlamF6- CD8 T cells appeared later and were the majority during progression. Functionally, the SlamF6+ population was proliferative and expressed more inhibitory receptors than SlamF6- cells. However, SlamF6+ CD8 T cells gradually lost the ability to divide and secrete cytokines over the course of tumorigenesis, demonstrating that they become dysfunctional over time. Collectively, our results provide new insights into longitudinal response of TCF-1+ T cells over the course of tumorigenesis and have therapeutic implications for modulating the anti-tumor T cell response.
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