Coordinated, multicellular patterns of transcriptional variation that stratify patient cohorts are revealed by tensor decomposition.

Tissue-level and organism-level biological processes often involve the coordinated action of multiple distinct cell types. The recent application of single-cell assays to many individuals should enable the study of how donor-level variation in one cell type is linked to that in other cell types. Here we introduce a computational approach called single-cell interpretable tensor decomposition (scITD) to identify common axes of interindividual variation by considering joint expression variation across multiple cell types. scITD combines expression matrices from each cell type into a higher-order matrix and factorizes the result using the Tucker tensor decomposition. Applying scITD to single-cell RNA-sequencing data on 115 persons with lupus and 83 persons with coronavirus disease 2019, we identify patterns of coordinated cellular activity linked to disease severity and specific phenotypes, such as lupus nephritis. scITD results also implicate specific signaling pathways likely mediating coordination between cell types. Overall, scITD offers a tool for understanding the covariation of cell states across individuals, which can yield insights into the complex processes that define and stratify disease.

Protracted neuronal recruitment in the temporal lobes of young children.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1-4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

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.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells, 1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

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.

CRISPR activation and interference screens decode stimulation responses in primary human T cells.

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.

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.

Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins.

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

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.

Changes in gene expression variability reveal a stable synthetic lethal interaction network in BRCA2-ovarian cancers.

Synthetic lethal interactions (SLIs) are robust mechanisms that provide cells with the ability to remain viable despite having mutations in genes critical to the DNA damage response, a core cellular process. Studies in model organisms such as S. cerevisiae showed that thousands of genes important in maintaining DNA integrity cooperated in a SLI network. Two genes participate in a SLI when a mutation in one gene has no effect on the cell, but mutations in both interacting genes are lethal. Furthermore in C. elegans, a mutation in a critical gene that is important for development induced a change in expression variability in the synthetic lethal interactor. In cancer, targeting SLIs shows promise in selectively killing cancer cells. For example, targeting PARP1 is an effective treatment for BRCA1/2- breast and ovarian cancers. Although PARP1 is already identified as having a SLI with BRCA1/2-, computationally searching for other genes that cooperate in the SLI network could highlight genes that may have promise for being a cancer-specific drug target. Using RNA sequencing data for ovarian cancer patients with BRCA2 mutations and the R Bioconductor package pathVar, we showed that genes whose expression changes to an invariant, stable expression state are likely candidates for SLIs with BRCA2. Our results highlight the interactions between the genes with predicted SLIs and protein-coding genes that are functionally important in the DNA damage response. The method of analyzing expression variability to computationally identify genes with SLIs can be applied to query SLIs in other tumor types.

Recombinant rubistatin (r-Rub), an MVD disintegrin, inhibits cell migration and proliferation, and is a strong apoptotic inducer of the human melanoma cell line SK-Mel-28.

Disintegrins are low molecular weight peptides isolated from viper venom. These peptides bind to integrin receptors using a conserved binding motif sequence containing an RGD or similar motif. As a consequence, disintegrins can inhibit platelet aggregation and inhibit cell migration, proliferation, and initiate apoptosis in cancer cell lines. Rubistatin is a MVD disintegrin cloned from a Crotalus ruber ruber venom gland. The biological activity of MVD disintegrins is poorly understood. Recombinant rubistatin (r-Rub) was cloned into a pET32b plasmid and expressed in reductase-deficient Escherichia coli. Expression was induced with IPTG and the resulting fusion peptide was affinity purified, followed by thrombin cleavage, and removal of vector coded sequences. r-Rub peptide inhibited ADP-induced platelet aggregation by 54% ± 6.38 in whole blood. We assessed the ability of r-Rub to initiate apoptosis in three human cancer cell lines. Cultures of SK-Mel-28, HeLA, and T24 cells were grown for 24 h with 2.5 µM r-Rub followed by Hoechst staining. Chromatin fragmentation was observed in treated SK-Mel-28, but not in T24 or HeLA cells. A TUNEL assay revealed that 51.55% ± 5.28 of SK-Mel-28 cells were apoptotic after 18 h of treatment with 3.5 µM of r-Rub. Cell migration and proliferation assays were performed in order to further characterize the biological effects of r-Rub on SK-Mel-28 cells. At 3 µM, r-Rub inhibited cell migration by 44.4% ± 0.5, while at 3.5 µM it was able to inhibit cell proliferation by 83% ± 6.0.