Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunization in the pig lung.

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1β.

A metabolic pacer ensures smooth running of the lymphocyte activation race.

Upon lymphocyte stimulation, accumulation of intracellular NAD(H) reflects the strength of antigen receptor signals and controls the rate of cell cycle entry and proliferation (see related Research Article by Turner .).

Cytotoxic T lymphocytes require transcription for infiltration but not target cell lysis.

Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. CTL T cell receptor (TCR) ligation not only drives the delivery and release of cytolytic granules but also initiates a new wave of transcription. In order to address whether TCR-induced transcriptomic changes impact the ability of CTLs to kill, we asked which genes are expressed immediately after CTLs encounter targets and how CTL responses change when inhibiting transcription.

ChAdOx1 nCoV-19 vaccination generates spike-specific CD8 T cells in aged mice.

Effective vaccines have reduced the morbidity and mortality caused by severe acute respiratory syndrome coronavirus-2 infection; however, the elderly remain the most at risk. Understanding how vaccines generate protective immunity and how these mechanisms change with age is key for informing future vaccine design. Cytotoxic CD8 T cells are important for killing virally infected cells, and vaccines that induce antigen-specific CD8 T cells in addition to humoral immunity provide an extra layer of immune protection.

Super-killer CTLs are generated by single gene deletion of Bach2.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8 T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8 T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type.

Divide and Conquer: Phenotypic and Temporal Heterogeneity Within CD8 T Cell Responses.

The advent of technologies that can characterize the phenotypes, functions and fates of individual cells has revealed extensive and often unexpected levels of diversity between cells that are nominally of the same subset. CD8 T cells, also known as cytotoxic T lymphocytes (CTLs), are no exception. Investigations of individual CD8 T cells both and have highlighted the heterogeneity of cellular responses at the levels of activation, differentiation and function.

Staggered starts in the race to T cell activation.

How T lymphocytes tune their responses to different strengths of stimulation is a fundamental question in immunology. Recent work using new optogenetic, single-cell genomic, and live-imaging approaches has revealed that stimulation strength controls the rate of individual cell responses within a population. Moreover, these responses have been found to use shared molecular programs, regardless of stimulation strength.

Longitudinal proteomic profiling of dialysis patients with COVID-19 reveals markers of severity and predictors of death.

End-stage kidney disease (ESKD) patients are at high risk of severe COVID-19. We measured 436 circulating proteins in serial blood samples from hospitalised and non-hospitalised ESKD patients with COVID-19 (n = 256 samples from 55 patients). Comparison to 51 non-infected patients revealed 221 differentially expressed proteins, with consistent results in a separate subcohort of 46 COVID-19 patients.

ILC2-driven innate immune checkpoint mechanism antagonizes NK cell antimetastatic function in the lung.

Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden.

Stimulation strength controls the rate of initiation but not the molecular organisation of TCR-induced signalling.

Millions of naïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leading to T cell activation.

Circulating TNF-like protein 1A (TL1A) is elevated early in rheumatoid arthritis and depends on TNF.

Background: The tumor necrosis factor (TNF) superfamily cytokine TNF-like protein 1A (TL1A) and its receptor DR3 are essential for diverse animal models of autoimmune disease and may be pathogenic in rheumatoid arthritis (RA). However, the relationship of TL1A to disease duration, activity, and response to anti-TNF and other therapies in RA is not clear.

Methods: We measured soluble TL1A in synovial fluid (SF), serum, or plasma from RA first-degree relatives (FDRs) and in early RA and established disease.

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes.

How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2.

Reduced monocyte and macrophage TNFSF15/TL1A expression is associated with susceptibility to inflammatory bowel disease.

Chronic inflammation in inflammatory bowel disease (IBD) results from a breakdown of intestinal immune homeostasis and compromise of the intestinal barrier. Genome-wide association studies have identified over 200 genetic loci associated with risk for IBD, but the functional mechanisms of most of these genetic variants remain unknown. Polymorphisms at the TNFSF15 locus, which encodes the TNF superfamily cytokine commonly known as TL1A, are associated with susceptibility to IBD in multiple ethnic groups.

Correcting the Mean-Variance Dependency for Differential Variability Testing Using Single-Cell RNA Sequencing Data.

Cell-to-cell transcriptional variability in otherwise homogeneous cell populations plays an important role in tissue function and development. Single-cell RNA sequencing can characterize this variability in a transcriptome-wide manner. However, technical variation and the confounding between variability and mean expression estimates hinder meaningful comparison of expression variability between cell populations.

T cell cytolytic capacity is independent of initial stimulation strength.

How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8 T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells.

Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells.

TNF superfamily cytokines play major roles in the regulation of adaptive and innate immunity. The TNF superfamily cytokine TL1A (TNFSF15), through its cognate receptor DR3 (TNFRSF25), promotes T cell immunity to pathogens and directly costimulates group 2 and 3 innate lymphoid cells. Polymorphisms in the gene are associated with the risk for various human diseases, including inflammatory bowel disease.

Testing for differential abundance in mass cytometry data.

When comparing biological conditions using mass cytometry data, a key challenge is to identify cellular populations that change in abundance. Here, we present a computational strategy for detecting 'differentially abundant' populations by assigning cells to hyperspheres, testing for significant differences between conditions and controlling the spatial false discovery rate. Our method (http://bioconductor.

Targeted genomic analysis reveals widespread autoimmune disease association with regulatory variants in the TNF superfamily cytokine signalling network.

Background: Tumour necrosis factor (TNF) superfamily cytokines and their receptors regulate diverse immune system functions through a common set of signalling pathways. Genetic variants in and expression of individual TNF superfamily cytokines, receptors and signalling proteins have been associated with autoimmune and inflammatory diseases, but their interconnected biology has been largely unexplored.

Methods: We took a hypothesis-driven approach using available genome-wide datasets to identify genetic variants regulating gene expression in the TNF superfamily cytokine signalling network and the association of these variants with autoimmune and autoinflammatory disease.

Insight into Genotype-Phenotype Associations through eQTL Mapping in Multiple Cell Types in Health and Immune-Mediated Disease.

Genome-wide association studies (GWAS) have transformed our understanding of the genetics of complex traits such as autoimmune diseases, but how risk variants contribute to pathogenesis remains largely unknown. Identifying genetic variants that affect gene expression (expression quantitative trait loci, or eQTLs) is crucial to addressing this. eQTLs vary between tissues and following in vitro cellular activation, but have not been examined in the context of human inflammatory diseases.

The TNF-family cytokine TL1A: from lymphocyte costimulator to disease co-conspirator.

Originally described in 2002 as a T cell-costimulatory cytokine, the tumor necrosis factor family member TNF-like factor 1A (TL1A), encoded by the TNFSF15 gene, has since been found to affect multiple cell lineages through its receptor, death receptor 3 (DR3, encoded by TNFRSF25) with distinct cell-type effects. Genetic deficiency or blockade of TL1A-DR3 has defined a number of disease states that depend on this cytokine-receptor pair, whereas excess TL1A leads to allergic gastrointestinal inflammation through stimulation of group 2 innate lymphoid cells. Noncoding variants in the TL1A locus are associated with susceptibility to inflammatory bowel disease and leprosy, predicting that the level of TL1A expression may influence host defense and the development of autoimmune and inflammatory diseases.

The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease.

Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.

Background: Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings.