Mycobacterium tuberculosis Rv0928 protein facilitates macrophage control of mycobacterium infection by promoting mitochondrial intrinsic apoptosis and ROS-mediated inflammation.

Macrophages are the main target cells for Mycobacterium tuberculosis (Mtb) infection. Previous studies have shown that Mtb actively upregulates phosphorus transport proteins, such as Rv0928 protein (also known as PstS3), to increase inorganic phosphate uptake and promote their survival under low phosphorus culture conditions in vitro. However, it is unclear whether this upregulation of PstS3 affects the intracellular survival of Mtb, as the latter is also largely dependent on the immune response of infected macrophages. By using Rv0928-overexpressing Mycobacterium smegmatis (Ms::Rv0928), we unexpectedly found that Rv0928 not only increased apoptosis, but also augmented the inflammatory response of infected macrophages. These enhanced cellular defense mechanisms ultimately led to a dramatic reduction in intracellular bacterial load. By investigating the underlying mechanisms, we found that Rv0928 interacted with the macrophage mitochondrial phosphate carrier protein SLC25A3, reduced mitochondrial membrane potential and caused mitochondrial cytochrome c release, which ultimately activated caspase-9-mediated intrinsic apoptosis. In addition, Rv0928 amplified macrophage mitochondrial ROS production, further enhancing pro-inflammatory cytokine production by promoting activation of NF-κB and MAPK pathways. Our study suggested that Mtb Rv0928 up-regulation enhanced the immune defense response of macrophages. These findings may help us to better understand the complex process of mutual adaptation and mutual regulation between Mtb and macrophages during infection.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Mucosal Vaccination with Cyclic Dinucleotide Adjuvants Induces Effective T Cell Homing and IL-17-Dependent Protection against Mycobacterium tuberculosis Infection.

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines.

Probabilistic harmonization and annotation of single-cell transcriptomics data with deep generative models.

As the number of single-cell transcriptomics datasets grows, the natural next step is to integrate the accumulating data to achieve a common ontology of cell types and states. However, it is not straightforward to compare gene expression levels across datasets and to automatically assign cell type labels in a new dataset based on existing annotations. In this manuscript, we demonstrate that our previously developed method, scVI, provides an effective and fully probabilistic approach for joint representation and analysis of scRNA-seq data, while accounting for uncertainty caused by biological and measurement noise. We also introduce single-cell ANnotation using Variational Inference (scANVI), a semi-supervised variant of scVI designed to leverage existing cell state annotations. We demonstrate that scVI and scANVI compare favorably to state-of-the-art methods for data integration and cell state annotation in terms of accuracy, scalability, and adaptability to challenging settings. In contrast to existing methods, scVI and scANVI integrate multiple datasets with a single generative model that can be directly used for downstream tasks, such as differential expression. Both methods are easily accessible through scvi-tools.

Inference of single-cell phylogenies from lineage tracing data using Cassiopeia.

The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis.

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

Simulating multiple faceted variability in single cell RNA sequencing.

The abundance of new computational methods for processing and interpreting transcriptomes at a single cell level raises the need for in silico platforms for evaluation and validation. Here, we present SymSim, a simulator that explicitly models the processes that give rise to data observed in single cell RNA-Seq experiments. The components of the SymSim pipeline pertain to the three primary sources of variation in single cell RNA-Seq data: noise intrinsic to the process of transcription, extrinsic variation indicative of different cell states (both discrete and continuous), and technical variation due to low sensitivity and measurement noise and bias. We demonstrate how SymSim can be used for benchmarking methods for clustering, differential expression and trajectory inference, and for examining the effects of various parameters on their performance. We also show how SymSim can be used to evaluate the number of cells required to detect a rare population under various scenarios.

Natural selection interacts with recombination to shape the evolution of hybrid genomes.

To investigate the consequences of hybridization between species, we studied three replicate hybrid populations that formed naturally between two swordtail fish species, estimating their fine-scale genetic map and inferring ancestry along the genomes of 690 individuals. In all three populations, ancestry from the "minor" parental species is more common in regions of high recombination and where there is linkage to fewer putative targets of selection. The same patterns are apparent in a reanalysis of human and archaic admixture. These results support models in which ancestry from the minor parental species is more likely to persist when rapidly uncoupled from alleles that are deleterious in hybrids. Our analyses further indicate that selection on swordtail hybrids stems predominantly from deleterious combinations of epistatically interacting alleles.