RNA expression of reactivated Antigen-specific memory CD4 T cells previously exposed to tolerogenic or immunogenic stimulation
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ABSTRACT: Activated and memory CD4 T cells play important roles in many autoimmune diseases often acting as upstream co-ordinators of inflammation and tissue destruction. A major therapeutic strategy is to turn off or tolerize these CD4 T cells thereby providing a cure. While much is understood about inducing tolerance in naïve CD4 T cells, little is known about whether or how to induce tolerance in previously activated or memory CD4 T cells. Here, we use RNA-sequencing to investigate the consequences of activating mouse CD4 memory T cells with antigen delivered in the absence of adjuvant, a classic tolerance-inducing strategy for naïve T cells. To examine the long term consequences of exposing memory CD4 T cells to tolerizing signals, we first reactivated them with antigen delivered with (control) or without (experimental) adjuvant, rested the cells and then reactivated them with antigen and adjuvant 5 days prior to isolating RNA for gene expression analysis.
Project description:CD4+ T-cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. We here reveal how “help” signals delivered during priming impact memory differentiation of CTLs, as informed by genome-wide analyses. “Help” signals promoted the IL-15-dependent maintenance of central memory (TCM) cells. However, they had a much larger impact on the generation of effector memory (TEM) cells and their gene expression program . CD4+ T-cell help created TEM cells that produced Granzyme B and IFNg after antigen-independent recall with IL-12 and IL-18. Furthermore, “helped” memory CTLs expressed the effector program characteristic of “helped” primary CTLs upon recall with MHC class I-restricted antigen only, due to both epigenetic imprinting and sustained mRNA expression of relevant genes . Thus, CD4+ T-cell help delivered during priming creates CD8+ TEM cells with innate and help-independent recall capacities.
Project description:The intestinal immune system must concomitantly tolerate food and commensals and protect against pathogens. Antigen-presenting cells (APCs) orchestrate these immune responses by presenting luminal antigens to CD4+ T cells and inducing their differentiation into regulatory (pTreg) or inflammatory (Th) subsets. Here, we used LIPSTIC to identify APCs that presented dietary antigens under tolerizing and inflammatory conditions. We show that helminth infections disrupted tolerance proportionally to the reduction in ratio between tolerogenic, including migratory cDC1s and Rorγt+ APCs, and inflammatory APCs, represented primarily by cDC2 subsets. However, the inflammatory subset of cDC2s expanded by helminth infection did not present dietary antigens, thus avoiding diet-specific TH2 cell differentiation. Our data uncover cellular mechanisms by which tolerance to food is induced and can be disrupted by infection.
Project description:Foxp3+ regulatory T cells (Treg) play a central role for tolerance against self and innocuous environmental antigens. However, the role of antigen-specificity for Treg-mediated tolerance is only incompletely understood. Here we show by direct ex vivo characterization of human CD4+ T cells, that the response against innocuous airborne antigens, such as plant pollen or fungal spores, is dominated by memory-like antigen-specific Treg. Surprisingly, breakdown of tolerance in atopic donors was not accompanied by a quantitatively or qualitatively altered Treg response, but instead correlated with a striking dichotomy of Treg versus Th2 target specificity. Allergenic proteins, are selectively targeted by Th2 cells, but not Treg. Thus human Treg specific for airborne antigens maintain tolerance at mucosal sites and the failure to generate specific Treg against a subgroup of antigens provides a window of opportunity for allergy development. PBMCs from sex and age matched birch pollen allergic patients and healthy controls, were stimulated (7h) with airborne fungal (A. fumigatus) or birch pollen antigen (birch) and sorted into antigen specific conventional and regulatory T cells according to their expression of CD154+ and CD137+ on CD4+ T cells, respectively. Number of samples per group in parentheses: Healthy controls stimulated with A. fumigatus (n=5), allergic patients stimulated with A. fumigatus (n=6), healthy controls stimulated with birch (n=6), allergic patients stimulated with birch (n=4).
Project description:CD4 T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for longlived antibody responses. However, it remains unclear whether there are CD4+ memory T cells committed to the Tfh lineage after antigen clearance. Using adoptive transfer of antigen-specific memory CD4+ subpopulations (based on CXCR5 and Ly6c expression)in the LCMV infection model, we found that there are distinct memory CD4+ T cell populations with commitment to the Tfh and Th1 lineages. Our conclusions are based on gene expression profiles, epigenetic studies and phenotypic and functional analysis. The gene expression profiles of virus-specific CD4 T cell subets at effector and memory stages is presented here. The SMARTA TCR transgenic / adptive transfer system was used to identify and sort subsets of antigen-specific CD4 T cells (based on their expression of Ly6c and CXCR5) elicited after acute infection with LCMV (Arm).
Project description:C57/BL6 to C57/BL6; Balb/c to C57/BL6; Balb/c to C57/BL6 with anti CD80/86 mAb; Identificaton of gene expression profile in tolerizing murine cardiac allograft by co-stimulatory blockade. The induction of specific tolerance would be the ultimate achievement in transplant immunology, but the precise mechanisms of immunological tolerance remain largely unknown. Here, we investigated global gene expression analysis in tolerizing murine cardiac allografts by means of oligonucleotide microarrays. Tolerance induction was achieved in cardiac allografts from BALB/c to C57BL/6 mice by daily intraperitoneal injection of anti-CD80 and CD86 mAbs. Comparative analysis revealed 64 genes to be induced more extensively in the tolerizing than in the syngeneic isografts, and 16 genes than in the rejecting allografts. Two genes were specifically upregulated in the tolerizing allografts. In the tolerizing allografts there were induced marked expressions of a number of genes for proinflammatory factors, including interferon-gamma inducible cytokines and chemokines, as well as apoptosis-related genes which were also upregulated in the rejecting allografts. Moreover, these gene expression patterns continued to be upregulated more than 70 days post transplant. These results provide evidence that immunological tolerance can be induced and maintained in the presence of prominent proinflammatory gene expression in vivo.
Project description:C57/BL6 to C57/BL6 Balb/c to C57/BL6 Balb/c to C57/BL6 with anti CD80/86 mAb Identificaton of gene expression profile in tolerizing murine cardiac allograft by co-stimulatory blockade. The induction of specific tolerance would be the ultimate achievement in transplant immunology, but the precise mechanisms of immunological tolerance remain largely unknown. Here, we investigated global gene expression analysis in tolerizing murine cardiac allografts by means of oligonucleotide microarrays. Tolerance induction was achieved in cardiac allografts from BALB/c to C57BL/6 mice by daily intraperitoneal injection of anti-CD80 and CD86 mAbs. Comparative analysis revealed 64 genes to be induced more extensively in the tolerizing than in the syngeneic isografts, and 16 genes than in the rejecting allografts. Two genes were specifically upregulated in the tolerizing allografts. In the tolerizing allografts there were induced marked expressions of a number of genes for proinflammatory factors, including interferon-gamma inducible cytokines and chemokines, as well as apoptosis-related genes which were also upregulated in the rejecting allografts. Moreover, these gene expression patterns continued to be upregulated more than 70 days post transplant. These results provide evidence that immunological tolerance can be induced and maintained in the presence of prominent proinflammatory gene expression in vivo. Keywords: other
Project description:Our previous work showed that vaccine elicited Klebsiella pneumoniae specific CD4+ tissue resident memory (TRM) cells in the lung provide serotype independent immunity against K. pneumoniae for up to 6 months. However, vaccine efficacy wanes in proportion to lung CD4+ TRM cell number. To study what controls TRM maintenance, apart from antigen, we developed an LTA/1Ovalbumin (OVA) model and tetramer pulldown method to enrich OVA+ lung CD4+ TRM cells over time. We applied single-cell RNA-sequencing (scRNA-seq) of these cells, collected on days 30, 90 and 184 after prime immunization with OVA and our prior Th17 adjuvant, E. coli heat labile toxin A1 (LTA1). As control, we used splenic CD4+ T cells from naive mice
Project description:Memory antigen-specific CD4+ T cells against Chlamydia trachomatis are necessary for protection against secondary genital tract infection. While it is known that naïve antigen-specific CD4+ T cells can traffic to the genital tract in an antigen-specific manner, these T cells are not protective during primary infection. Here, we sought to compare the differences between memory and naïve antigen-specific CD4+ T cells in the same mouse following secondary infection using transgenic CD4+ T cells (NR1 T cells). Using RNA sequencing, we found that there were subtle but distinct differences between these two T cell populations. Naïve NR1 T cells significantly upregulated cell cycle genes and were more proliferative than memory NR1 T cells in the draining lymph node. In contrast, memory NR1 T cells were more activated than naïve NR1 T cells and were enriched in the genital tract. Together, our data provide insight into the differences between memory and naïve antigen-specific CD4+ T cells during C. trachomatis infection.
Project description:While memory T-cells (Tmem) represent a hallmark of adaptive immunity, and despite extensive characterization of CD8+ Tmem, little is known about regulation of CD4+ Tmem cell survival. In this study, we analyzed antigen-specific CD4+T cell memory populations in mice and human to characterize their unique genetic and surface phenotypes. First, using microarray technology, we studied dynamic gene expression of antigen specific CD4+ T cells during infection, memory differentiation, and survival up to nearly a year. In murine CD4+T cells, we observed reduced expression of genes that induce cell proliferation and increased expression of anti-apoptotic and pro-survival genes. Importantly, these genetic programs revealed multiple transmembrane markers enriched in the murine CD4+ Tmem population. We verified the ability of these markers to denote CD4+ Tmem populations using influenza vaccination, and observed enrichment of these surface markers in antigen-specific cells. Finally, we tested the ability of these markers to denote human memory CD4+ T cells. We found that their expression exclusively co-localized with existing human memory marker CD45R0, and that sorting of cells positive for these markers recovered more responding antigen-specific CD4+T cells than the general CD4+T cell population. In sum, this study presents unique gene signatures of long-lived murine CD4+ Tmem cells along with new surface markers some of which were validated in human. The new information can improve our assessment of CD4 memory T cells can be incorporated for novel therapeutics and vaccine design.