Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors.

It is currently accepted that cancer-associated fibroblasts (CAF) participate in T-cell exclusion from tumor nests. To unbiasedly test this, we used single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors. We identified four main CAF populations, two of which are associated with T-cell exclusion: (i) MYH11+αSMA+ CAF, which are present in early-stage tumors and form a single cell layer lining cancer aggregates, and (ii) FAP+αSMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both populations orchestrate a particular structural tissue organization through dense and aligned fiber deposition compared with T cell-permissive CAF. Yet they produce distinct matrix molecules, including collagen IV (MYH11+αSMA+ CAF) and collagen XI/XII (FAP+αSMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T-cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors. SIGNIFICANCE: The cellular and molecular programs driving T-cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T-cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to developing new strategies targeting T cell-excluding CAF. See related commentary by Sherman, p. 2501. This article is highlighted in the In This Issue feature, p. 2483.

Single-cell analysis of human non-small cell lung cancer lesions refines tumor classification and patient stratification.

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and pathology of multiple organs in individuals under 21 years of age in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although an autoimmune pathogenesis has been proposed, the genes, pathways and cell types causal to this new disease remain unknown. Here we perform RNA sequencing of blood from patients with MIS-C and controls to find disease-associated genes clustered in a co-expression module annotated to CD56dimCD57+ natural killer (NK) cells and exhausted CD8+ T cells. A similar transcriptome signature is replicated in an independent cohort of Kawasaki disease (KD), the related condition after which MIS-C was initially named. Probing a probabilistic causal network previously constructed from over 1,000 blood transcriptomes both validates the structure of this module and reveals nine key regulators, including TBX21, a central coordinator of exhausted CD8+ T cell differentiation. Together, this unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

Author Correction: Sampling the host response to SARS-CoV-2 in hospitals under siege.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8 + T-cells and CD56 dim CD57 + NK cells. Bayesian network analyses revealed nine key regulators of this module, including TBX21 , a central coordinator of exhausted CD8 + T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.