The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.

Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis, and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA sequencing (scRNA-seq) to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes spanning new fibroblastic and osteoblastic subpopulations including distinct osteoblast differentiation trajectories. Emerging acute myeloid leukemia impaired mesenchymal osteogenic differentiation and reduced regulatory molecules necessary for normal hematopoiesis. These data suggest that tissue stroma responds to malignant cells by disadvantaging normal parenchymal cells. Our taxonomy of the stromal compartment provides a comprehensive bone marrow cell census and experimental support for cancer cell crosstalk with specific stromal elements to impair normal tissue function and thereby enable emergent cancer.

Signaling abnormalities in immune responses in the small intestine can trigger chronic type 2 inflammation involving interaction of multiple immune cell types. To systematically characterize this response, we analyzed 58,067 immune cells from the mouse small intestine by single-cell RNA sequencing (scRNA-seq) at steady state and after induction of a type 2 inflammatory reaction to ovalbumin (OVA). Computational analysis revealed broad shifts in both cell-type composition and cell programs in response to the inflammation, especially in group 2 innate lymphoid cells (ILC2s). Inflammation induced the expression of exon 5 of Calca, which encodes the alpha-calcitonin gene-related peptide (α-CGRP), in intestinal KLRG1+ ILC2s. α-CGRP antagonized KLRG1+ ILC2s proliferation but promoted IL-5 expression. Genetic perturbation of α-CGRP increased the proportion of intestinal KLRG1+ ILC2s. Our work highlights a model where α-CGRP-mediated neuronal signaling is critical for suppressing ILC2 expansion and maintaining homeostasis of the type 2 immune machinery.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

Tumor-only genomic testing can uncover somatic and germline pathogenic variants [pathogenic/likely pathogenic (P/LP)] in cancer predisposition genes. We describe the prevalence of P/LPs in BRCA1/2 and PALB2 (B1B2P2) across malignancies and the frequency of clinical germline testing (CGT) in patients with P/LPs in B1B2P2 identified on tumor-only testing.

The molecular underpinnings of HER2-low and HER2-0 (IHC 0) breast tumors remain poorly defined. Using genomic findings from 1039 patients with HER2-negative metastatic breast cancer undergoing next-generation sequencing from 7/2013-12/2020, we compare results between HER2-low (n = 487, 47%) and HER2-0 tumors (n = 552, 53%). A significantly higher number of ERBB2 alleles (median copy count: 2.05) are observed among HER2-low tumors compared to HER2-0 (median copy count: 1.79; P = 2.36e-6), with HER2-0 tumors harboring a higher rate of ERBB2 hemideletions (31.1% vs. 14.5%). No other genomic alteration reaches significance after accounting for multiple hypothesis testing, and no significant differences in tumor mutational burden are observed between HER2-low and HER2-0 tumors (median: 7.26 mutations/megabase vs. 7.60 mutations/megabase, p = 0.24). Here, we show that the genomic landscape of HER2-low and HER2-0 tumors does not differ significantly, apart from a higher ERBB2 copy count among HER2-low tumors, and a higher rate of ERBB2 hemideletions in HER2-0 tumors.

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.

Evolution drives changes in a protein's sequence over time. The extent to which these changes in sequence lead to shifts in the underlying preference for each amino acid at each site is an important question with implications for comparative sequence-analysis methods, such as molecular phylogenetics. To quantify the extent that site-specific amino acid preferences shift during evolution, we performed deep mutational scanning on two homologs of human influenza nucleoprotein with 94% amino acid identity. We found that only a modest fraction of sites exhibited shifts in amino acid preferences that exceeded the noise in our experiments. Furthermore, even among sites that did exhibit detectable shifts, the magnitude tended to be small relative to differences between nonhomologous proteins. Given the limited change in amino acid preferences between these close homologs, we tested whether our measurements could inform site-specific substitution models that describe the evolution of nucleoproteins from more diverse influenza viruses. We found that site-specific evolutionary models informed by our experiments greatly outperformed nonsite-specific alternatives in fitting phylogenies of nucleoproteins from human, swine, equine, and avian influenza. Combining the experimental data from both homologs improved phylogenetic fit, partly because measurements in multiple genetic contexts better captured the evolutionary average of the amino acid preferences for sites with shifting preferences. Our results show that site-specific amino acid preferences are sufficiently conserved that measuring mutational effects in one protein provides information that can improve quantitative evolutionary modeling of nearby homologs.

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells.

Massively parallel single-cell and single-nucleus RNA sequencing has opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so is the need for computational pipelines for scaled analysis. Here we developed Cumulus-a cloud-based framework for analyzing large-scale single-cell and single-nucleus RNA sequencing datasets. Cumulus combines the power of cloud computing with improvements in algorithm and implementation to achieve high scalability, low cost, user-friendliness and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies.

Fetal growth restriction (FGR) affects 5-10% of pregnancies, and can have serious consequences for both mother and child. Prevention and treatment are limited because FGR pathogenesis is poorly understood. Genetic studies implicate KIR and HLA genes in FGR, however, linkage disequilibrium, genetic influence from both parents, and challenges with investigating human pregnancies make the risk alleles and their functional effects difficult to map. Here, we demonstrate that the interaction between the maternal KIR2DL1, expressed on uterine natural killer (NK) cells, and the paternally inherited HLA-C*0501, expressed on fetal trophoblast cells, leads to FGR in a humanized mouse model. We show that the KIR2DL1 and C*0501 interaction leads to pathogenic uterine arterial remodeling and modulation of uterine NK cell function. This initial effect cascades to altered transcriptional expression and intercellular communication at the maternal-fetal interface. These findings provide mechanistic insight into specific FGR risk alleles, and provide avenues of prevention and treatment.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) are effective for many patients with lung cancer with EGFR mutations. However, not all patients are responsive to EGFR TKIs, including even those harboring EGFR-sensitizing mutations. In this study, we quantified the cells and cellular interaction features of the tumor microenvironment (TME) using routine H&E-stained biopsy sections. These TME features were used to develop a prediction model for survival benefit from EGFR TKI therapy in patients with lung adenocarcinoma and EGFR-sensitizing mutations in the Lung Cancer Mutation Consortium 1 (LCMC1) and validated in an independent LCMC2 cohort. In the validation data set, EGFR TKI treatment prolonged survival in the predicted-to-benefit group but not in the predicted-not-to-benefit group. Among patients treated with EGFR TKIs, the predicted-to-benefit group had prolonged survival outcomes compared with the predicted not-to-benefit group. The EGFR TKI survival benefit positively correlated with tumor-tumor interaction image features and negatively correlated with tumor-stroma interaction. Moreover, the tumor-stroma interaction was associated with higher activation of the hepatocyte growth factor/MET-mediated PI3K/AKT signaling pathway and epithelial-mesenchymal transition process, supporting the hypothesis of fibroblast-involved resistance to EGFR TKI treatment.

In an ongoing, open-label, single-arm phase II study ( NCT02927301 ), 181 patients with untreated, resectable, stage IB-IIIB non-small cell lung cancer received two doses of neoadjuvant atezolizumab monotherapy. The primary end point was major pathological response (MPR; ≤10% viable malignant cells) in resected tumors without EGFR or ALK alterations. Of the 143 patients in the primary end point analysis, the MPR was 20% (95% confidence interval, 14-28%). With a minimum duration of follow-up of 3 years, the 3-year survival rate of 80% was encouraging. The most common adverse events during the neoadjuvant phase were fatigue (39%, 71 of 181) and procedural pain (29%, 53 of 181), along with expected immune-related toxicities; there were no unexpected safety signals. In exploratory analyses, MPR was predicted using the pre-treatment peripheral blood immunophenotype based on 14 immune cell subsets. Immune cell subsets predictive of MPR in the peripheral blood were also identified in the tumor microenvironment and were associated with MPR. This study of neoadjuvant atezolizumab in a large cohort of patients with resectable non-small cell lung cancer was safe and met its primary end point of MPR ≥ 15%. Data from this single-arm, non-randomized trial suggest that profiles of innate immune cells in pre-treatment peripheral blood may predict pathological response after neoadjuvant atezolizumab, but additional studies are needed to determine whether these profiles can inform patient selection and new therapeutic approaches.

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.