Coordinated immune dysregulation in juvenile dermatomyositis revealed by single-cell genomics.

Juvenile dermatomyositis (JDM) is one of several childhood-onset autoimmune disorders characterized by a type I IFN response and autoantibodies. Treatment options are limited due to an incomplete understanding of how the disease emerges from dysregulated cell states across the immune system. We therefore investigated the blood of patients with JDM at different stages of disease activity using single-cell transcriptomics paired with surface protein expression. By immunophenotyping peripheral blood mononuclear cells, we observed skewing of the B cell compartment toward an immature naive state as a hallmark of JDM at diagnosis. Furthermore, we find that these changes in B cells are paralleled by T cell signatures suggestive of Th2-mediated inflammation that persist despite disease quiescence. We applied network analysis to reveal that hyperactivation of the type I IFN response in all immune populations is coordinated with previously masked cell states including dysfunctional protein processing in CD4+ T cells and regulation of cell death programming in NK cells, CD8+ T cells, and γδ T cells. Together, these findings unveil the coordinated immune dysregulation underpinning JDM and provide insight into strategies for restoring balance in immune function.

Coordinated immune dysregulation in Juvenile Dermatomyositis revealed by single-cell genomics.

Juvenile Dermatomyositis (JDM) is one of several childhood-onset autoimmune disorders characterized by a type I interferon response and autoantibodies. Treatment options are limited due to incomplete understanding of how the disease emerges from dysregulated cell states across the immune system. We therefore investigated the blood of JDM patients at different stages of disease activity using single-cell transcriptomics paired with surface protein expression. By immunophenotyping peripheral blood mononuclear cells, we observed skewing of the B cell compartment towards an immature naive state as a hallmark of JDM. Furthermore, we find that these changes in B cells are paralleled by signatures of Th2-mediated inflammation. Additionally, our work identified SIGLEC-1 expression in monocytes as a composite measure of heterogeneous type I interferon activity in disease. We applied network analysis to reveal that hyperactivation of the type I interferon response in all immune populations is coordinated with dysfunctional protein processing and regulation of cell death programming. This analysis separated the ubiquitously expressed type I interferon response into a central hub and revealed previously masked cell states. Together, these findings reveal the coordinated immune dysregulation underpinning JDM and provide novel insight into strategies for restoring balance in immune function.

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling.

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.

Single-cell RNA-seq reveals cell type-specific molecular and genetic associations to lupus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon-stimulated genes (ISGs) in monocytes, reduction of naïve CD4+ T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH+ CD8+ T cells. Cell type-specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type-specific cis-expression quantitative trait loci and to link SLE-associated variants to cell type-specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.

Monoclonal antibody-mediated neutralization of SARS-CoV-2 in an IRF9-deficient child.

We describe an unvaccinated child at risk for life-threatening COVID-19 due to an inherited deficiency of IRF9, which governs ISGF-3-dependent responses to type I and III interferons (IFN). She was admitted, with a high nasal SARS-CoV-2 load on day 1 of upper respiratory tract infection. She was viremic on day 2 and received casirivimab and imdevimab. Her clinical manifestations and viremia disappeared on days 3 and 4, respectively. Circulating SARS-CoV-2 virus induced the expression of IFN-stimulated genes in leukocytes on day 1, whereas the secretion of blood type I IFNs, which peaked on day 4, did not. Antibody-mediated SARS-CoV-2 neutralization is, therefore, sufficient to overcome a deficiency of antiviral IFNs.

SCITO-seq: single-cell combinatorial indexed cytometry sequencing.

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 105 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling.

Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19.

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN­specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non­COVID-19 controls revealed a lack of type I IFN­stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN­specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN­specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms.

XYZeq: Spatially resolved single-cell RNA sequencing reveals expression heterogeneity in the tumor microenvironment.

Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue.

Longitudinal single-cell epitope and RNA-sequencing reveals the immunological impact of type 1 interferon autoantibodies in critical COVID-19: Anti-IFN antibodies in critical COVID-19 correlate with poor ISG response and upregulation of LAIR1 surface protein in PBMCs.

Type I interferon (IFN-I) neutralizing autoantibodies have been found in some critical COVID-19 patients; however, their prevalence and longitudinal dynamics across the disease severity scale, and functional effects on circulating leukocytes remain unknown. Here, in 284 COVID-19 patients, we found IFN-I autoantibodies in 19% of critical, 6% of severe and none of the moderate cases. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 COVID-19 patients, 15 non-COVID-19 patients and 11 non-hospitalized healthy controls, revealed a lack of IFN-I stimulated gene (ISG-I) response in myeloid cells from critical cases, including those producing anti-IFN-I autoantibodies. Moreover, surface protein analysis showed an inverse correlation of the inhibitory receptor LAIR-1 with ISG-I expression response early in the disease course. This aberrant ISG-I response in critical patients with and without IFN-I autoantibodies, supports a unifying model for disease pathogenesis involving ISG-I suppression via convergent mechanisms.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Ex vivo cytosolic delivery of functional macromolecules to immune cells.

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach's potential to engineer cell function is demonstrated in HIV infection studies.

Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform.

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ∼ 5 min without calcium vs. ∼ 30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods.

A vector-free microfluidic platform for intracellular delivery.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.