Summary The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce “weighted-nearest neighbor” analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide 1 . Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care 2 . Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines 3,4 that may be produced by a subset of inflammatory monocytes 5,6 , lymphopenia 7,8 and T cell exhaustion 9,10 . To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.To profile the peripheral immune response to severe COVID-19, we performed Seq-Well-based 11,12 massively parallel single-cell RNA sequencing (scRNA-seq) on eight peripheral blood samples from seven hospitalized patients with polymerase chain reaction with reverse transcription (RT-PCR)-confirmed SARS-CoV-2 infection and six healthy controls. The demographics and clinical features of these patients are listed in Fig. 1a. The seven patients profiled were male, aged 20 to >80 years. We collected samples between 2 and 16 days following symptom onset; healthy controls were asymptomatic, four male and two female, and aged 30-50 years (Fig. 1a and Extended Data Fig. 1). Four of eight COVID-19 samples were collected from ventilated patients who were diagnosed with acute respiratory distress syndrome (ARDS; Fig. 1a). One patient (C1) was sampled twice: at nine days post-symptom onset while only requiring supplemental oxygen and at 11 days post-symptom onset following intubation. Three patients received azithromycin, which

Background There is no effective pharmacotherapy for the acute respiratory distress syndrome (ARDS), and mortality remains high. Preclinical studies support the efficacy of mesenchymal stem (stromal) cells (MSCs) in the treatment of lung injury. The aim of this phase one clinical trial was to test the safety of a single dose of allogeneic bone marrow-derived MSCs in patients with moderate-to-severe ARDS. Methods The STem cells for ARDS Treatment (START) trial was a multi-center, open-label, dose-escalation phase one clinical trial of a single dose of intravenous MSCs in patients with moderate-to-severe ARDS. The trial is registered with clinicaltrials.gov number [NCT01775774]. The first three patients were treated with low dose MSCs (1million cells/kg predicted body weight (PBW)); the next three patients received intermediate dose MSCs (5 million cells/kg PBW); and the final three patients received high dose MSCs (10 million cells/kg PBW). Primary outcomes included the incidence of pre-specified infusion associated events and serious adverse events. Secondary outcomes included standard respiratory and systemic endpoints, 28- and 60-day mortality, and measurement of biologic markers of inflammation and endothelial and epithelial injury. The trial completed enrollment in January 2014. Findings There were no pre-specified infusion associated events or treatment-related adverse events in any of the nine patients in this trial. Serious adverse events (SAEs) were subsequently observed in three patients during in the weeks following the infusion: two patients expired >seven days after the MSC infusion, and one patient was discovered to have multiple embolic infarcts of the spleen, kidneys, and brain that were age-indeterminate but thought to have occurred prior the MSC infusion based on MRI results. None of these SAEs were thought to be MSC-related. Interpretation A single intravenous infusion of allogeneic, bone marrow-derived human MSCs was well tolerated in 9 patients with moderate to severe ARDS. Based on this phase one experience, we have proceeded to phase two testing of MSCs for moderate to severe ARDS. Funding The trial was funded by the National Heart, Lung and Blood Institute (NHLBI U01HL10871301).

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

Sepsis is a common cause of death, but outcomes in individual patients are difficult to predict. Elucidating the molecular processes that differ between sepsis patients who survive and those who die may permit more appropriate treatments to be deployed. We examined the clinical features, and the plasma metabolome and proteome of patients with and without community-acquired sepsis, upon their arrival at hospital emergency departments and 24 hours later. The metabolomes and proteomes of patients at hospital admittance who would die differed markedly from those who would survive. The different profiles of proteins and metabolites clustered into fatty acid transport and β-oxidation, gluconeogenesis and the citric acid cycle. They differed consistently among several sets of patients, and diverged more as death approached. In contrast, the metabolomes and proteomes of surviving patients with mild sepsis did not differ from survivors with severe sepsis or septic shock. An algorithm derived from clinical features together with measurements of seven metabolites predicted patient survival. This algorithm may help to guide the treatment of individual patients with sepsis.

Rationale: Despite advances in clinical management, there are currently no reliable diagnostic and therapeutic targets for acute respiratory distress syndrome (ARDS). The inflammasome/caspase-1 pathway regulates the maturation and secretion of proinflammatory cytokines (e.g., IL-18). IL-18 is associated with injury in animal models of systemic inflammation. Objectives: We sought to determine the contribution of the inflammasome pathway in experimental acute lung injury and human ARDS. Methods: We performed comprehensive gene expression profiling on peripheral blood from patients with critical illness. Gene expression changes were assessed using real-time polymerase chain reaction, and IL-18 levels were measured in the plasma of the critically ill patients. Wild-type mice or mice genetically deficient in IL-18 or caspase-1 were mechanically ventilated using moderate tidal volume (12 ml/kg). Lung injury parameters were assessed in lung tissue, serum, and bronchoalveolar lavage fluid. Measurements and Main Results: In mice, mechanical ventilation enhanced IL-18 levels in the lung, serum, and bronchoalveolar lavage fluid. IL-18-neutralizing antibody treatment, or genetic deletion of IL-18 or caspase-1, reduced lung injury in response to mechanical ventilation. In human patients with ARDS, inflammasome-related mRNA transcripts (CASP1, IL1B, and IL18) were increased in peripheral blood. In samples from four clinical centers, IL-18 was elevated in the plasma of patients with ARDS (sepsis or trauma-induced ARDS) and served as a novel biomarker of intensive care unit morbidity and mortality. Conclusions: The inflammasome pathway and its downstream cytokines play critical roles in ARDS development.

In this paper, Choi and colleagues analyzed levels of mitochondrial DNA in two prospective observational cohort studies and found that increased mtDNA levels are associated with ICU mortality, and improve risk prediction in medical ICU patients. The data suggests that mtDNA could serve as a viable plasma biomarker in MICU patients.

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.