a-semiautomated-chip-seq-procedure-for-large-scale-epigenetic-studies

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

La Jolla Institute for Immunology

Allergic asthma and rhinitis are two common chronic allergic diseases that affect the lungs and nose, respectively. Both diseases share clinical and pathological features characteristic of excessive allergen-induced type 2 inflammation, orchestrated by memory CD4(+) T cells that produce type 2 cytokines (Th2 cells). However, a large majority of subjects with allergic rhinitis do not develop asthma, suggesting divergence in disease mechanisms. Because Th2 cells play a pathogenic role in both these diseases and are also present in healthy nonallergic subjects, we performed global transcriptional profiling to determine whether there are qualitative differences in Th2 cells from subjects with allergic asthma, rhinitis, and healthy controls. Th2 cells from asthmatic subjects expressed higher levels of several genes that promote their survival as well as alter their metabolic pathways to favor persistence at sites of allergic inflammation. In addition, genes that enhanced Th2 polarization and Th2 cytokine production were also upregulated in asthma. Several genes that oppose T cell activation were downregulated in asthma, suggesting enhanced activation potential of Th2 cells from asthmatic subjects. Many novel genes with poorly defined functions were also differentially expressed in asthma. Thus, our transcriptomic analysis of circulating Th2 cells has identified several molecules that are likely to confer pathogenic features to Th2 cells that are either unique or common to both asthma and rhinitis.

Transcriptional Profiling of Th2 Cells Identifies Pathogenic Features Associated with Asthma.

CD4 cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4 memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the T (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4 T cells in the central memory (T) and effector memory (T) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-T cells show marked clonal expansion compared with T and T cells and that most of CD4-T were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-T was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the T subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.

Precursors of human CD4 cytotoxic T lymphocytes identified by single-cell transcriptome analysis.

While many genetic variants have been associated with risk for human diseases, how these variants affect gene expression in various cell types remains largely unknown. To address this gap, the DICE (database of immune cell expression, expression quantitative trait loci [eQTLs], and epigenomics) project was established. Considering all human immune cell types and conditions studied, we identified cis-eQTLs for a total of 12,254 unique genes, which represent 61% of all protein-coding genes expressed in these cell types. Strikingly, a large fraction (41%) of these genes showed a strong cis-association with genotype only in a single cell type. We also found that biological sex is associated with major differences in immune cell gene expression in a highly cell-specific manner. These datasets will help reveal the effects of disease risk-associated genetic polymorphisms on specific immune cell types, providing mechanistic insights into how they might influence pathogenesis (https://dice-database.org).

Impact of Genetic Polymorphisms on Human Immune Cell Gene Expression.

High numbers of tissue-resident memory T (T) cells are associated with better clinical outcomes in cancer patients. However, the molecular characteristics that drive their efficient immune response to tumors are poorly understood. Here, single-cell and bulk transcriptomic analysis of T and non-T cells present in tumor and normal lung tissue from patients with lung cancer revealed that PD-1-expressing T cells in tumors were clonally expanded and enriched for transcripts linked to cell proliferation and cytotoxicity when compared with PD-1-expressing non-T cells. This feature was more prominent in the T cell subset coexpressing PD-1 and TIM-3, and it was validated by functional assays ex vivo and also reflected in their chromatin accessibility profile. This PD-1TIM-3 T cell subset was enriched in responders to PD-1 inhibitors and in tumors with a greater magnitude of CTL responses. These data highlight that not all CTLs expressing PD-1 are dysfunctional; on the contrary, T cells with PD-1 expression were enriched for features suggestive of superior functionality.

Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

CD4 T helper (T) cells and regulatory T (T) cells that respond to common allergens play an important role in driving and dampening airway inflammation in patients with asthma. Until recently, direct, unbiased molecular analysis of allergen-reactive T and T cells has not been possible. To better understand the diversity of these T cell subsets in allergy and asthma, we analyzed the single-cell transcriptome of ~50,000 house dust mite (HDM) allergen-reactive T cells and T cells from asthmatics with HDM allergy and from three control groups: asthmatics without HDM allergy and nonasthmatics with and without HDM allergy. Our analyses show that HDM allergen-reactive T and T cells are highly heterogeneous and certain subsets are quantitatively and qualitatively different in individuals with HDM-reactive asthma. The number of interleukin-9 (IL-9)-expressing HDM-reactive T cells is greater in asthmatics with HDM allergy compared with nonasthmatics with HDM allergy, and this IL-9-expressing T subset displays enhanced pathogenic properties. More HDM-reactive T and T cells expressing the interferon response signature (TIFNR and TIFNR) are present in asthmatics without HDM allergy compared with those with HDM allergy. In cells from these subsets (TIFNR and TIFNR), expression of  was enriched; its product, tumor necrosis factor-related apoptosis-inducing ligand, dampens activation of T cells. These findings suggest that the TIFNR and TIFNR subsets may dampen allergic responses, which may help explain why only some people develop T2 responses to nearly ubiquitous allergens.

Article	Year
Transcriptional Profiling of Th2 Cells Identifies Pathogenic Features Associated with Asthma. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27271570	2016
Precursors of human CD4 cytotoxic T lymphocytes identified by single-cell transcriptome analysis. Science immunology\| PubMed ID: 29352091	2018
Impact of Genetic Polymorphisms on Human Immune Cell Gene Expression. Cell\| PubMed ID: 30449622	2018
Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer. The Journal of experimental medicine\| PubMed ID: 31227543	2019
Publisher Correction: Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nature immunology\| PubMed ID: 31624378	2019
Single-cell transcriptomic analysis of allergen-specific T cells in allergy and asthma. Science immunology\| PubMed ID: 32532832	2020

Pandurangan Vijayanad

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