Integrated analysis of multimodal single-cell data.

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.

The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.

Structure and Degradation of Circular RNAs Regulate PKR Activation in Innate Immunity.

Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.

Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

BCG vaccination alters the epigenetic landscape of progenitor cells in human bone marrow to influence innate immune responses.

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r > 0.8) with the interleukin (IL)-1ß secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

Cellular spermine targets JAK signaling to restrain cytokine-mediated autoimmunity.

Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-ß-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.

Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans.

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP.

Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections.

Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

Host and Environmental Factors Influencing Individual Human Cytokine Responses.

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.

Linking the Human Gut Microbiome to Inflammatory Cytokine Production Capacity.

Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.

The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease.

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1-/- mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective.

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

Delivering three punches to knockout intracellular bacteria.

Cytotoxic lymphocytes kill bacteria-infected cells, but the mechanisms at work remain unclear. Walch et al. show that these lymphocytes deliver a toxic molecular trio in a two-step process, penetrating first the infected cells and then delivering bactericidal granzymes into the intracytoplasmic bacteria.

Global analyses of human immune variation reveal baseline predictors of postvaccination responses.

A major goal of systems biology is the development of models that accurately predict responses to perturbation. Constructing such models requires the collection of dense measurements of system states, yet transformation of data into predictive constructs remains a challenge. To begin to model human immunity, we analyzed immune parameters in depth both at baseline and in response to influenza vaccination. Peripheral blood mononuclear cell transcriptomes, serum titers, cell subpopulation frequencies, and B cell responses were assessed in 63 individuals before and after vaccination and were used to develop a systematic framework to dissect inter- and intra-individual variation and build predictive models of postvaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation cell populations alone, which were validated using independent baseline time points. Most of the parameters contributing to prediction delineated temporally stable baseline differences across individuals, raising the prospect of immune monitoring before intervention.

Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.

Dissecting human population variation in single-cell responses to SARS-CoV-2.

Humans display substantial interindividual clinical variability after SARS-CoV-2 infection1-3, the genetic and immunological basis of which has begun to be deciphered4. However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells-from 222 healthy donors of diverse ancestries-that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk.