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Infection with West Nile Virus (WNV) can drive a wide range of responses, from asymptomatic to flu-like symptoms/fever or severe cases of encephalitis and death. To identify cellular and molecular signatures distinguishing WNV severity, we employed systems profiling of peripheral blood from asymptomatic and severely ill individuals infected with WNV. We interrogated immune responses longitudinally from acute infection through convalescence at 3 months and 1 year employing multiplexed single cell protein and transcriptional profiling (CyTOF and Seq-Well) complemented with matched serum proteomics and metabolomics. At the acute time point, we detected both an elevated proportion of pro-inflammatory markers in innate immune cell types and reduced frequency of regulatory T cell activity in participants with severe infection compared to those with asymptomatic infection. Single-cell transcriptomics of paired samples revealed that asymptomatic donors had higher expression of genes associated with innate immune pathways, in particular anti-inflammatory CD16+monocytes at the acute time point. A multi-omics analysis identified factors--beyond those from individual analyses--that distinguished immune state trajectory between severity groups. Here we highlighted the potential of systems immunology using multiple cell-type and cell-state-specific analyses to identify correlates of infection severity and host cellular activity contributing to an effective anti-viral response.

Alzheimer’s disease, the most common age-related neurodegenerative disease, is closely associated with both amyloid-ß plaque and neuroinflammation. Two thirds of Alzheimer’s disease patients are females and they have a higher disease risk. Moreover, women with Alzheimer’s disease have more extensive brain histological changes than men along with more severe cognitive symptoms and neurodegeneration. To identify how sex difference induces structural brain changes, we performed unbiased massively parallel single nucleus RNA sequencing on Alzheimer’s disease and control brains focusing on the middle temporal gyrus, a brain region strongly affected by the disease but not previously studied with these methods. We identified a subpopulation of selectively vulnerable layer 2/3 excitatory neurons that that were RORB-negative and CDH9-expressing. This vulnerability differs from that reported for other brain regions, but there was no detectable difference between male and female patterns in middle temporal gyrus samples. Disease-associated, but sex-independent, reactive astrocyte signatures were also present. In clear contrast, the microglia signatures of diseased brains differed between males and females. Combining single cell transcriptomic data with results from genome-wide association studies (GWAS), we identifiedMERTKgenetic variation as a risk factor for Alzheimer’s disease selectively in females. Taken together, our single cell dataset revealed a unique cellular-level view of sex-specific transcriptional changes in Alzheimer’s disease, illuminating GWAS identification of sex-specific Alzheimer’s risk genes. These data serve as a rich resource for interrogation of the molecular and cellular basis of Alzheimer’s disease.

Autoimmune diseases, among the most common disorders of young adults, are mediated by genetic and environmental factors. While CD4+Foxp3+regulatory T cells (Tregs) play a central role in preventing autoimmunity, the molecular mechanism underlying their dysfunction is unknown. Here, we performed comprehensive transcriptomic and epigenomic profiling of Tregs in the autoimmune disease multiple sclerosis (MS) to identify central transcriptional programs regulating human autoimmunity. We discovered that upregulation of a primate-specific shortPRDM1isoform (PRDM1-S) inducesSGK1independent from evolutionally conserved longPRDM1, leading to destabilization of Foxp3 and Treg dysfunction. This aberrantPRDM1-S/SGK1axis is shared among other autoimmune diseases. Furthermore, by chromatin landscape profiling in MS Tregs we identified aPRDM1-Sspecificcis-regulatory element associated with enriched binding of AP-1/IRF transcription factors. Our study identifies evolutionally emergedPRDM1-Sand epigenetic priming of AP-1/IRF as key drivers of pathogenic Treg programs leading to human autoimmune disease.

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder where recent evidence suggests pathogenesis may be mediated by inflammatory processes. The molecular architecture of the disease remains to be fully elucidated. We performed single-nucleus transcriptomics and unbiased proteomics using postmortem tissue obtained from the prefrontal cortex of 12 individuals with late-stage PD and age-matched controls. We analyzed ∼80,000 nuclei and identified eight major cell types, including brain-resident T cells, each with distinct transcriptional changes in line with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem tissue, we found that α-synuclein pathology is inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in prefrontal cortex that were preferentially downregulated in PD. Strikingly, comparing this dataset to a regionally similar published analysis for Alzheimer’s disease (AD), we found no common differentially expressed genes in neurons, but identified many shared differentially expressed genes in glial cells, suggesting that disease etiology in PD and AD are likely distinct. These data are presented as a resource for interrogating the molecular and cellular basis of PD and other neurodegenerative diseases.One Sentence SummaryWe provide an extensive single cell analysis profiling nearly 80,000 brain nuclei from prefrontal cortex of late-stage Parkinson’s disease brains, demonstrate that α-synuclein pathology is inversely correlated with chaperone expression in excitatory neurons, found a selective abatement of neuron-astrocyte interactions with enhanced neuroinflammation, and augmented the study with proteomic analysis and cross-comparisons with Alzheimer’s disease datasets, providing valuable insights into the pathways of neurodegeneration and a deep definition of the underlying molecular pathology for Parkinson’s disease.

Compared with sequencing-based global genomic profiling, cytometry labels targeted surface markers on millions of cells in parallel either by conjugated rare earth metal particles or Unique Molecular Identifier (UMI) barcodes. Correct annotation of these cells to specific cell types is a key step in the analysis of these data. However, there is no computational tool that automatically annotates single cell proteomics data for cell type inference. In this manuscript, we propose an automated single cellproteomics dataannotation approach calledProtAnnoto facilitate cell type assignments without laborious manual gating. ProtAnno is designed to incorporate information from annotated single cell RNA-seq (scRNA-seq), CITE-seq, and prior data knowledge (which can be imprecise) on biomarkers for different cell types. We have performed extensive simulations to demonstrate the accuracy and robustness of ProtAnno. For several single cell proteomics datasets that have been manually labeled, ProtAnno was able to correctly label most single cells. In summary, ProtAnno offers an accurate and robust tool to automate cell type annotations for large single cell proteomics datasets, and the analysis of such annotated cell types can offer valuable biological insights.

Autoimmune and inflammatory diseases are polygenic disorders of the immune system. Many genomic loci harbor risk alleles for several diseases, but the limited resolution of genetic mapping prevents determining if the same allele is responsible, indicating a shared underlying mechanism. Using a collection of 129,058 cases and controls across six diseases, we show that ∼40% of overlapping associations are due to the same allele. We improve fine-mapping resolution for shared alleles two-fold by combining cases and controls across diseases, allowing us to identify more eQTLs driven by the shared alleles. The patterns of sharing indicate widespread shared mechanisms, but not a single global autoimmune mechanism. Our approach can be applied to any set of traits, and is particularly valuable as sample collections become depleted. One sentence summary Genetic mapping in autoimmune diseases increases genetic mapping resolution and reveals shared mechanisms

SummaryA dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5−CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited “B cell help” signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFNγ, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

SUMMARYMultisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV2 infection in otherwise healthy children. Here, we define immune abnormalities in MIS-C compared to adult COVID-19 and pediatric/adult healthy controls using single-cell RNA sequencing, antigen receptor repertoire analysis, unbiased serum proteomics, andin vitroassays. Despite no evidence of active infection, we uncover elevated S100A-family alarmins in myeloid cells and marked enrichment of serum proteins that map to myeloid cells and pathways including cytokines, complement/coagulation, and fluid shear stress in MIS-C patients. Moreover, NK and CD8 T cell cytotoxicity genes are elevated, and plasmablasts harboring IgG1 and IgG3 are expanded. Consistently, we detect elevated binding of serum IgG from severe MIS-C patients to activated human cardiac microvascular endothelial cells in culture. Thus, we define immunopathology features of MIS-C with implications for predicting and managing this SARS-CoV2-induced critical illness in children.

Understanding the relationship between tumor and peripheral immune environments could allow longitudinal immune monitoring in cancer. Here, we examined whether T cells that share the same TCRαβ and are found in both tumor and blood can be interrogated to gain insight into the ongoing tumor T cell response. Paired transcriptome and TCRαβ repertoire of circulating and tumor-infiltrating T cells were analyzed from matched tumor and blood from patients with metastatic melanoma at the single cell level. We found that in circulating T cells matching clonally expanded tumor-infiltrating T cells (circulating TILs), gene signatures of effector functions, but not terminal exhaustion, reflect those observed in the tumor. In contrast, features of exhaustion are displayed predominantly by T cells present only in tumor. Finally, genes associated with a high degree of blood-tumor TCR sharing were overexpressed in tumor tissue after immunotherapy. These data demonstrate that circulating TILs, identified by TCRs shared with T cells in tumors, have unique transcriptional expression patterns that may have utility for the interrogation of T cell function in cancer immunotherapy.SummaryCombining transcriptomic and TCRαβ repertoire analysis of circulating and tumor-infiltrating CD8 T cells from patients with metastatic melanoma, we identify a blood-based population with effector properties that reflect those of clonally related tumor-resident T cells.

A dysregulated immune response against the SARS-CoV-2 virus plays a critical role in severe COVID-19. However, the molecular and cellular mechanisms by which the virus causes lethal immunopathology are poorly understood. Here, we utilize multiomics single-cell analysis to probe dynamic immune responses in patients with stable or progressive manifestations of COVID-19, and assess the effects of tocilizumab, an anti-IL-6 receptor monoclonal antibody. Coordinated profiling of gene expression and cell lineage protein markers reveals a prominent type-1 interferon response across all immune cells, especially in progressive patients. An anti-inflammatory innate immune response and a pre-exhaustion phenotype in activated T cells are hallmarks of progressive disease. Skewed T cell receptor repertoires in CD8+ T cells and uniquely enriched V(D)J sequences are also identified in COVID-19 patients. B cell repertoire and somatic hypermutation analysis are consistent with a primary immune response, with possible contribution from memory B cells. Our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19, which may contribute to delayed virus clearance and has implications for therapeutic intervention.

FoxP3 positive regulatory T cells (Tregs) rely on fatty acid β-oxidation (FAO)-driven OXPHOS for differentiation and function. Recent data have demonstrated a role for Tregs in the maintenance of tissue homeostasis with tissue-resident Tregs possessing tissue-specific transcriptomes. However, specific signals that establish these tissue-resident Tregs programs are largely unknown. As Tregs metabolically rely on FAO, and considering the lipid-rich environments of tissues, we hypothesized that environmental lipids drive Treg homeostasis. Using human adipose tissue as a model for tissue residency, we identify oleic acid as the most prevalent free fatty acid in human adipose tissue. Mechanistically, oleic acid amplifies Treg FAO-driven OXPHOS metabolism, creating a positive feedback mechanism that induces the expression of Foxp3 and enhances phosphorylation of STAT5, which acts to stabilize the Treg lineage and increase suppressive function. Comparing the transcriptomic program induced by oleic acid to that of the pro-inflammatory arachidonic acid, we find that Tregs sorted from peripheral blood and adipose of healthy donors transcriptomically resemble the oleic acid in vitro treated Tregs, whereas Tregs obtained from the adipose tissue of relapsing-remitting MS patients more closely resemble an arachidonic acid profile. Finally, we find that oleic acid concentrations are reduced in the fat tissue of MS patients, and exposure of dysfunctional MS Tregs to oleic acid restores defects in their suppressive function. These data demonstrate the importance of fatty acids in regulating tissue inflammatory signals.

To identify co-inhibitory immune pathways important in the brain, we hypothesized that comparison of T cells in lesions from patients with MS with tumor infiltrating T cells (TILs) from patients with GBM may reveal novel targets for immunotherapy. Focusing on PD-1 and TIGIT, we found that TIGIT and its ligand CD155 were highly expressed on GBM TILs but were near-absent in MS lesions, while lymphocytic expression of PD-1/PDL-1 was comparable. TIGIT was also upregulated in peripheral lymphocytes in GBM, suggesting recirculation of TILs. These data raise the possibility that anti-TIGIT therapy may be beneficial for patients with glioblastoma.

Foxp3+regulatory T cells (Tregs) are the central component of peripheral immune tolerance. While dysregulation of the Treg cytokine signature has been observed in autoimmune diseases such as multiple sclerosis (MS) and type 1 diabetes, the regulatory mechanisms balancing pro- and anti-inflammatory cytokine production are not known. Here, we identify imbalance between IFNγ and IL-10 as a shared Treg signature, present in patients with MS and under high salt conditions. By performing RNA-seq analysis on human Treg subpopulations, we identify β-catenin as a key regulator that controls the expression of IFNγ and IL-10. The activated β-catenin signature is enriched specifically in IFNγ+Tregs in humans, and this was confirmedin vivowith Treg-specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional, IFNγ-producing, Treg phenotype. Moreover, we identify PTGER2 as a major factor balancing IFNγ and IL-10 production in the context of a high salt environment, with skewed activation of the β-catenin/SGK1/Foxo axis in IFNγ+Tregs. These findings identify a novel molecular mechanism underlying inflammatory Tregs in human autoimmune disease and reveal a new role for a PTGER2-β-catenin loop in Tregs linking environmental high salt conditions to autoimmunity.

Central nervous system (CNS) immune activation in an important driver of neuronal injury during several neurodegenerative and neuroinflammatory diseases. During HIV infection, CNS immune activation is associated with high rates of neurocognitive impairment, even with sustained long-term suppressive antiretroviral therapy (ART). However, the cellular subsets that drive immune activation and neuronal damage in the CNS during HIV infection and neurological conditions remain unknown, in part because CNS cells are difficult to access in living humans. Using single cell RNA sequencing (scRNA-seq) on cerebrospinal fluid (CSF) and blood from adults with HIV, we identified a rare (

Multiple sclerosis is a common, complex neurological disease, where almost 20% of risk heritability can be attributed to common genetic variants, including >230 identified by genome-wide association studies (Patsopoulos et al., 2017). Multiple strands of evidence suggest that the majority of the remaining heritability is also due to the additive effects of individual variants, rather than epistatic interactions between these variants, or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that as much as 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common variant signals, which highlight a key role for regulatory T cell homeostasis and regulation, IFNγ biology and NFκB signaling in MS pathogenesis. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

Epigenetic annotation studies of genetic risk variants for multiple sclerosis (MS) implicate dysfunctional lymphocytes in MS susceptibility; however, the role of central nervous system (CNS) cells remains unclear. We investigated the effect of the risk variant, rs7665090G, located near NFKB1, on astrocytes. We demonstrated that chromatin is accessible at the risk locus, a prerequisite for its impact on astroglial function. The risk variant was associated with increased NF-κB signaling and target gene expression driving lymphocyte recruitment in cultured human astrocytes and astrocytes within MS lesions, and with increased lesional lymphocytic infiltrates. In MS patients, the risk genotype was associated with increased lesion volumes on MRI. Thus, we established that the rs7665090G variant perturbs astrocyte function resulting in increased CNS access for peripheral immune cells. MS may thus result from variant-driven dysregulation of the peripheral immune system and the CNS, where perturbed CNS cell function aids in establishing local autoimmune inflammation.One Sentence SummaryThe NF-κB relevant multiple sclerosis risk variant, rs7665090G, drives astrocyte responses that promote lesion formation.