Gene expression profiles of CD4+CD25+ regulatory T cells during acute and chronic viral infection
ABSTRACT: Regulatory T (Treg) cells act as terminators in the case of T cell immunity during the acute phase of viral infection. However, their roles in chronic viral infection are not completely understood. We compared the phenotype and function of Treg cells during acute and chronic viral infection using lymphocytic choriomeningitis virus-infected mouse models. Chronic infection, unlike acute infection, led to induction of Treg cells and upregulation of various inhibitory receptors. Treg cells isolated from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting T cell proliferation and subsequent cytokine production than those from naïve (naive Treg cells) or acutely infected mice (acute Treg cells). These gene expression profiles provided evidence that chronic Treg cells display characteristics distinct from either naive or acute Treg cells. Mouse splenic CD4+CD25+ regulatory T cells were analyzed at 0 day and 16 day after acute or chronic viral infection with LCMV Arm or CL13, respectively.
Project description:Although several markers have been associated with the characterization of regulatory T cells (Treg) and their function, no studies have investigated the dynamics of their phenotype during infection. Since the necessity of Treg to control immunopathology has been demonstrated, we used the chronic helminth infection model S. mansoni to address the impact on the Treg gene repertoire. Before gene expression profiling we first chose to study the localization and antigen-specific suppressive nature of classically defined Treg during infection. Presence of Foxp3+ cells were found especially in the periphery of granulomas and isolated CD4+CD25hiFoxp3+ Treg from infected mice blocked IFN-gamma and IL-10 cytokine secretion from infected CD4+CD25- effector T cells (Teff). Furthermore the gene expression patterns of Treg and Teff showed that in total 474 genes were significantly regulated during chronic schistosomiasis. Upon k-means clustering we identified genes exclusively regulated in all four populations including Foxp3, CD103, GITR, OX40 and CTLA-4: classical Treg markers. During infection however, several non-classical genes were up-regulated solely within the Treg population such as Slpi, Gzmb, Mt1, Fabp5, Nfil3, Socs2, Gpr177 and Klrg1. Using RT-PCR we confirmed aspects of the microarray data and in addition showed that the expression profile of Treg from S. mansoni-infected mice is simultaneously unique and comparative with Treg derived from other infections Regulatory T cells (Treg) or effector T cells (Teff) were FACS-sorted as CD4+CD25+ or CD4+CD25- from mesenteric lymph nodes (MLN) of naive mice or from mice infected with Schistosoma mansoni. Affymetrix MOE430A 2.0 genechips were used to identify genes differentially expressed in Treg or Teff under resting or infected conditions.
Project description:During acute viral infections, naïve CD4+ T cells differentiate into effector CD4+ T cells and, after viral control, into memory CD4+ T cells. Memory CD4+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD4+ T cells become less functional. To compare the development of functional memory T cells with poorly functional T cells from chronic viral infection, we generated longitudinal transcriptional profiles for each. Naive CD44Lo CD4+ T cells were isolated and sorted from uninfected C57BL/6 mice and H2-IAb GP66-specific CD4+ T cells were sorted using MHC-II tetramers at d6, 8, 15, and 30 p.i. with either LCMV Arm or LCMV clone 13. RNA from these CD4+ T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays.
Project description:T follicular helper (TFH) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (TFR) cells limit GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Here we show that SOCE is required for the differentiation and function of both TFH and TFR cells. Conditional deletion of Stim1 and Stim2 genes in T cells or Treg cells results in spontaneous autoantibody production and humoral autoimmunity. Conversely, antibody-mediated immune responses following viral infection critically depend on SOCE in TFH cells. Mechanistically, STIM1 and STIM2 control early TFR and TFH cell differentiation through NFAT-mediated IRF4, BATF and Bcl-6 expression. SOCE plays a dual role in GC response by controlling TFH and TFR cell function, thus enabling protective B cell responses and preventing humoral autoimmunity. RNAseq analyses of WT and Stim1Stim2 DKO follicular T cells and non-follicular T cells; 4-6 mice per cohort in duplicates. Mice were infected for 10 days with LCMV.
Project description:Rationale: COPD is characterized by an abnormal regulatory T cell (Treg) response and increases in Th1 and Th17 cell responses. It is unclear if dysregulation of miRNAs within Treg alters the adaptive immunophenotype in COPD. Objectives: To compare the miRNA profile of COPD Treg cells with that of healthy controls and to explore the function of differentially expressed miRNAs. Methods: Treg cells (CD4+CD25+CD49-CD127-) and T effector (CD4+CD25-) cells were obtained from the peripheral blood of 4 nonsmokers, 4 healthy current smokers, and 4 COPD current smokers for analysis of miRNA expression, matching them for age and sex. We assessed expression initially by microarray analysis on the Illumina miRNA platform then conducted real time RT-PCR validation of the microarray results. 24 samples were analyzed. 8 from each patient group of healthy Normal, Healthy Smoker, and COPD Smoker.
Project description:Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute and chronic infectious diseases remains unclear. We recently demonstrated that IFN- receptor (IFNAR) signaling promotes Treg function in autoimmunity. To dissect the functional role of IFNAR-signaling in Tregs during acute and chronic viral infection, we infected Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice with LCMV Armstrong and Clone-13. In both models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced expression of Treg associated activation antigens. The enhanced activated phenotype was also seen when we compared the transcriptomes of IFNARfl/flxFoxp3YFP-Cre and wild type (WT) Tregs by RNA-Seq on day 25-post Clone-13 infection. LCMV-specific CD8+ T cells from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral IFN and TNF in both acute and chronic LCMV. In the chronic model, the numbers of anti-viral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs than WT mice during chronic infection. Thus, type I IFN signaling in Tregs is context-dependent, resulting in enhanced suppressor function in some models of autoimmunity, but decreased suppressor function in acute and chronic viral infection. Overall design: mRNA from Treg cells from 5 WT and 5 IFNAR deficient mice were analyzied by RNA-seq using Illumina HiSeq
Project description:Cytolytic activity by CD8+ cytotoxic T lymphocytes (CTL) is a powerful tactic in the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTL is progressively dampened during chronic infection - yet the environmental cues and molecular pathways controlling immune “exhaustion” remain unclear. We find CTL immunity is regulated by the central transcriptional response to hypoxia, mediated by the von-Hippel-Lindau/Hypoxia-Inducible-Factor (VHL/HIF) pathway. Deletion of VHL, the primary negative regulator of HIF, leads to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTL display enhanced control of persistent viral infection and neoplastic growth. We find HIF and oxygen influence expression of pivotal CTL transcription, effector and costimulatory-inhibitory molecules, which is relevant to strategies to promote viral and tumor clearance. To understand the role of the VHL/HIF pathway in regulating T cell responses to acute and persistant antigen in vivo, a mixture of ~10^4 WT and Vhl KO virus-specific CD8+ T cells (P14s) was transferred iv into uninfected WT host mice. After infection with either LCMV-Armstrong (acute viral infection) or LCMV-clone13 (persistent viral infection) we double-FACS isolated the responding P14 donor cells from pooled spleens from two sets of host mice to obtain duplicates for microarray for the four conditions, resulting in eight samples (2 WT Arm, 2 VHL KO Arm; 2 WT cl13, 2 VHL KO cl13) at 6 to 7 days post-infection. All conditions were sorted on KRLG1lo P14 cells. Note this was a mixed P14 transfer, so WT and KO cells were responding to infection in the same WT host mice to aid in normalizing effects such as antigen load and cytokine environment.
Project description:CD4 and CD8 T cells display functional defects during chronic infection such as loss of certain cytokines. Recent studies have suggested that CD4 T cells may actually gain other functions, however. Here, we analyzed gene expression profiles from LCMV-specific CD4 and CD8 T cells throughout the response to either acute LCMV or chronic LCMV infection. This alllowed us to identify CD4-specific changes during chronic infection compared to acute infection but also revealed shared core regulators between CD4 and CD8 T cells. LCMV-specific CD4 and CD8 T cells were isolated 6, 8, 15 and 30 days post infection with LCMV Armstrong or LCMV clone 13. Naïve CD4 and CD8 T cells were also isolated from naïve mice as comparisons. Four replicates of each sample were hybridized. The only exception is LCMV-specific CD4 T cells isolated 6 days post infection with LCMV-Arm where only three replicates were hybridized.
Project description:Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute/chronic infectious diseases and in the tumor microenvironment remains unclear. We recently demonstrated that IFN-a/b receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here, we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic LCMV Clone-13 infection, and in a transplantable colon adenocarcinoma model. In both viral and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. The enhanced activated phenotype was also seen when we compared the transcriptomes of IFNARfl/flxFoxp3YFP-Cre mice Tregs with the wild type (WT) Tregs by RNA-Seq on day 5 Armstrong and day 25-post Clone-13 infection. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-g and TNF-a. In the chronic infection model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and a higher tumor burden than the WT controls. Thus, type I IFN signaling in Tregs is context-dependent, resulting in enhanced suppressor function in some models of autoimmunity, but decreased suppressor function in acute/chronic viral infection and in the tumor microenvironment. Overall design: mRNA from Treg cells from 4 WT and 4 IFNAR deficient mice were analyzed by RNA-seq using Illumina HiSeq 5 days post LCM Armstrong infection.
Project description:CD4+CD25+FOXP3+ human regulatory T cells (Treg) are essential for self-tolerance and immune homeostasis. Here, we generated genome-wide maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation for CD4+CD25highCD45RA+ naive and CD4+CD25highCD45RA- memory Treg and their CD25- conventional T cell (Tconv) counterparts after in vitro expansion . In addition we generated genome-wide maps of the transcription factors STAT5, FOXP3, RUNX1 and ETS1 in expanded CD4+CD25highCD45RA+ Treg- and CD4+CD25- Tconv to elucidate their role in cell type-specific gene regulation. ChIP-seq of 2 histone marks and transcription factors ETS1, STAT5, FOXP3 and RUNX1 in expanded T cell subpopulations
Project description:The aim of this work was to identify functional features that are specific of human Treg cells, through the identification of genes that are differentially expressed: 1/ in activated Treg clones versus activated Thelper clones; 2/ in Th clones activated in the presence versus the absence of TGFb; 3/ in suppressed Th clones, i.e. Th clones activated in the presence of Treg clones, versus controls. Experiment Overall Design: Two to four million cells of human T cell clones were collected at rest (time point = 0) or after activation with coated anti-CD3 and soluble anti-CD28 antibodies (time points = 6, 24 or 41hrs). RNA was extracted for hybridization on Affymetrix microarrays.