Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice ...deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.
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•Anti-PD-1 efficacy depends on intratumoral activity of the CXCR3 chemokine system•CD103+ dendritic-cell-derived CXCL9 and CXCR3 on CD8+ T cells are required•CXCR3 ligands are positive indicators of responsiveness to anti-PD-1 therapy•Inducing CXCR3 ligands in non-responsive tumors restores sensitivity to anti-PD-1
Chow et al. find the CXCR3 chemokine system is not required for CD8+ T cell migration into the tumor, but rather for the enhancement of the intratumoral CD8+ T cell response in the context of PD-1 blockade. The CXCR3 chemokine system might serve as a biomarker for sensitivity to PD-1 blockade and a target for improving clinical outcomes.
Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms ...responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.
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•Mechanisms of inflammatory adverse events induced by checkpoint blockade•Colitis-associated differentiation of CD8 Trm cells to cytotoxic effector cells•Persistence and expansion of CTLA-4+ Treg cells•Inflammatory pathways provide opportunities for therapeutic intervention
Single-cell analyses of immune checkpoint blockade-associated colitis patient samples reveal enrichment of regulatory T cells in colitic lesions and nominate inflammatory pathways for potential therapeutic intervention.
The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop ...two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.
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•RAISIN RNA-seq of 1.6 million cells and 6,513 enteric neurons from adult mice and humans•Dozens of neuron subsets that vary by location, circadian phase, age, and species•Putative interactions between neurons and epithelial, stromal, and immune cells•Enteric neurons express risk genes for extraintestinal diseases with gut dysmotility
Comprehensive mapping and comparison of the adult mouse and human enteric nervous system is achieved with development of methods to isolate intact nuclei with ribosome-bound mRNA and label-free profiling of rare cell types. The atlases reveal the neuronal diversity of the ENS; its potential to link the gut, immune system, and brain; and contributions to disease.
Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches ...require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
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•Somatic mtDNA mutations can track cellular relationships and hierarchies in vitro•Single-cell genomic assays faithfully detect mtDNA mutations•Lineage inference can be combined with gene expression or chromatin state profiles•mtDNA mutations enable studies of clonal architecture in human health and disease
Using single-cell sequencing technologies, somatic mutations in mtDNA can be used as natural genetic barcodes to study cellular states and clonal dynamics.
Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses
; however, the relationship between phenotypic characteristics and TCR ...properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8
T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.
Natural mitochondrial DNA (mtDNA) mutations enable the inference of clonal relationships among cells. mtDNA can be profiled along with measures of cell state, but has not yet been combined with the ...massively parallel approaches needed to tackle the complexity of human tissue. Here, we introduce a high-throughput, droplet-based mitochondrial single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), a method that combines high-confidence mtDNA mutation calling in thousands of single cells with their concomitant high-quality accessible chromatin profile. This enables the inference of mtDNA heteroplasmy, clonal relationships, cell state and accessible chromatin variation in individual cells. We reveal single-cell variation in heteroplasmy of a pathologic mtDNA variant, which we associate with intra-individual chromatin variability and clonal evolution. We clonally trace thousands of cells from cancers, linking epigenomic variability to subclonal evolution, and infer cellular dynamics of differentiating hematopoietic cells in vitro and in vivo. Taken together, our approach enables the study of cellular population dynamics and clonal properties in vivo.
Treatment of cancer has been revolutionized by immune checkpoint blockade therapies. Despite the high rate of response in advanced melanoma, the majority of patients succumb to disease. To identify ...factors associated with success or failure of checkpoint therapy, we profiled transcriptomes of 16,291 individual immune cells from 48 tumor samples of melanoma patients treated with checkpoint inhibitors. Two distinct states of CD8+ T cells were defined by clustering and associated with patient tumor regression or progression. A single transcription factor, TCF7, was visualized within CD8+ T cells in fixed tumor samples and predicted positive clinical outcome in an independent cohort of checkpoint-treated patients. We delineated the epigenetic landscape and clonality of these T cell states and demonstrated enhanced antitumor immunity by targeting novel combinations of factors in exhausted cells. Our study of immune cell transcriptomes from tumors demonstrates a strategy for identifying predictors, mechanisms, and targets for enhancing checkpoint immunotherapy.
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•Single-cell RNA-seq reveals distinct CD45+ cells associated with clinical outcome•The balance between two CD8+ T cell states is linked with tumor regression•TCF7+CD8+ T cell frequency in tumor tissue predicts response and better survival•Dual blockade of CD39 with different checkpoint proteins enhances immunity
Single-cell analysis of immune cells from melanoma patients treated with immune checkpoint therapy uncovers a TCF7+ memory-like state in the cytotoxic T cell population and demonstrates its association with a positive outcome.
Treatment of anti-PD-1 refractory melanoma remains a challenge. Intratumoral injection of ONCOS-102, a chimeric oncolytic adenovirus expressing GMCSF, into anti-PD-1-resistant melanoma with ...administration of pembrolizumab was safe and effective. Response to therapy was associated with increased lymphocyte infiltration and expression of cytotoxicity and costimulatory genes. See related article by Shoushtari et al., p. 100.
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