Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, ...differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.
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•Determined differentiation trajectory and location of Tex cells and their precursors•cDC1s are essential for viral control but not for Tpex cell expansion•cDC1s maintain Tpex cell niches in an MHC-I-dependent manner•cDC1s limit activation-driven T cell exhaustion and immunopathology
Dähling et al. establish the role of cDCs for immunotherapy and find that cDC1s are required to mediate viral control. cDC1s provide a niche to maintain the precursors of exhausted T (Tpex) cell population and prevent their overactivation and subsequent immunopathology. These findings reveal the importance of cDC1s in maintaining Tpex cells to balance viral control, exhaustion, and immunopathology.
JAK2V617F mutation is associated with an increased risk for athero-thrombotic cardiovascular disease, but its role in aortic disease development and complications remains unknown. In a cohort of ...patients with myeloproliferative neoplasm, JAK2V617F mutation was identified as an independent risk factor for dilation of both the ascending and descending thoracic aorta. Using single-cell RNA-seq, complementary genetically-modified mouse models, as well as pharmacological approaches, we found that JAK2V617F mutation was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages, and dissecting aneurysm through the recruitment of circulating monocytes at later stages. Finally, genetic manipulation of tissue-resident macrophages, or treatment with a Jak2 inhibitor, ruxolitinib, mitigated aortic wall inflammation and reduced aortic dilation and rupture. Overall, JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm.
Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA ...sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.
Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson's disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic ...protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c
cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c
cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c
cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.
Abstract
Aims
Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field ...is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice.
Methods and results
We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor sequencing. Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- to 18-month-old hearts and integrated our data with publicly available cardiomyocyte single-cell sequencing datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity.
Conclusions
By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.
Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge ...that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting ...organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host
. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility
. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding
. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition ...and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34
SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9
FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19
and Cxcl9
FRCs in LNs of Irf3
mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.
The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. ...Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.
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