IFNγ signaling endows DCs with the capacity to control type I inflammation during parasitic infection through promoting T-bet+ regulatory T cells
ABSTRACT: Transcriptome analysis of IFNγ-insensitive DCs IFNγ signaling drives dendritic cells (DCs) to promote type I T cell (Th1) immunity. Here, we show that activation of DCs by IFNγ is equally crucial for the differentiation of a population of T-bet+ regulatory T (Treg) cells specialized to inhibit Th1 immune responses. Conditional deletion of IFNγ receptor in DCs but not in Treg cells resulted in a severe defect in this specific Treg cell subset, leading to exacerbated immune pathology during parasitic infections. Mechanistically, IFNγ-unresponsive DCs failed to produce sufficient amount of IL-27, a cytokine required for optimal T-bet induction in Treg cells. Thus, IFNγ signalling endows DCs with the ability to efficiently control a specific type of T cell immunity through promoting a corresponding Treg cell population. We analyzed saliva from 3 WT DC samples and 3 IFNγR2 KO DC samples isolated from unmanipulated mice. In addition, we analyzed saliva from 3 WT DC samples and 3 IFNγR2 KO DC samples isolated from mixed BM chimeras (WT + IFNgR2KO) day 8 T. gondii infected.
Project description:Role of bromodomain and extra-terminal motif (BET) proteins in GATA1-null erythrolbasts (G1E) and in differentiation induced by activation of conditional GATA1 tested by addition of BET inhibitor JQ1 (250nM) Array protocols were conducted as described in the Ambion WT Expression Manual and the Affymetrix GeneChip Expression Analysis Technical Manual by the University of Pennsylvania Molecular Profiling Core. Two-factor design (+/- JQ1, +/- GATA1). External RNA spike-in controls (ERCC controls, Ambion) added to each sample in proportion to cell number at the time of RNA harvest.
Project description:CD4+Foxp3+ Treg cells are essential for maintaining self-tolerance and preventing excessive immune responses. In the context of Th1 immune responses, co-expression of the Th1 transcription factor T-bet with Foxp3 is essential for Treg cells to control Th1 responses. T-bet-dependent expression of CXCR3 directs Tregs to the site of inflammation, however, the suppressive mediators enabling effective control of Th1 responses at this site are unknown. In this study, we determined the signature of CXCR3+ Treg cells arising in Th1 settings and defined universal features of Treg cells in this context using multiple Th1-dominated models. Our analysis defined a set of Th1-specific co-inhibitory receptors that are specifically expressed in Treg cells during Th1 immune responses. Among these, we identified the novel co-inhibitory receptor CD85k as a functional mediator of the enhanced suppression of Th1 effector cells by CXCR3+ Treg cells.
Project description:Xbp1 is a major transcription factor in the unfolded protein response. To uncover its function in DCs we generated a conditional KO for Xbp1 in dendritic cells. We here compare the expression of mRNAs in two different splenic DC subpopulations, CD8a and CD11b DCs in both WT and KO mice. Reference: Inositol-requiring enzyme 1-alpha regulates CD8a dendritic cell function via regulated mRNA decay. Osorio et al, Nature Immunology (2014) Primary DC subsets were isolated and sorted from spleens from 3 different WT or CD11c-cre Xbp-1fl/fl mice. RNA was isolated, converted to cDNA and then hybridised on Affymetrix GeneChip Mouse Gene 1.0 ST Arrays (GPL6246).
Project description:Gene expression profiling of Treg-conditioned dendritic cells (DCs) compared to LPS-matured DCs and immature DCs. The hypothesis tested in the present study was that Treg conditioning alters the gene expression profile of DCs to result in that of a semi-mature phenotype. The results provide important information on how Tregs modulate DC function. Overall design: Total RNA was obtained from mature DC and Treg-conditioned DC following 24 hr 100 ng/ml LPS treatment. RNA was isolated from immature DC at the same time-point to serve as a control.
Project description:Abstract Background: Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GMCSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-g (IFN-g) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs. Methods: Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-g and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis. Results: After 24 hours of LPS and IFN-g stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs. Conclusion: DCs, matured with LPS and IFN-g, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy. Overall design: Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-g and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.
Project description:This a model from the article:
Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12.
Schulz EG, Mariani L, Radbruch A, Höfer T.
Differentiation of naive T lymphocytes into type I T helper (Th1) cells requires interferon-gamma and interleukin-12. It is puzzling that interferon-gamma induces the Th1 transcription factor T-bet, whereas interleukin-12 mediates Th1 cell lineage differentiation. We use mathematical modeling to analyze the expression kinetics of T-bet, interferon-gamma, and the IL-12 receptor beta2 chain (IL-12Rbeta2) during Th1 cell differentiation, in the presence or absence of interleukin-12 or interferon-gamma signaling. We show that interferon-gamma induced initial T-bet expression, whereas IL-12Rbeta2 was repressed by T cell receptor (TCR) signaling. The termination of TCR signaling permitted upregulation of IL-12Rbeta2 by T-bet and interleukin-12 signaling that maintained T-bet expression. This late expression of T-bet, accompanied by the upregulation of the transcription factors Runx3 and Hlx, was required to imprint the Th cell for interferon-gamma re-expression. Thus initial polarization and subsequent imprinting of Th1 cells are mediated by interlinked, sequentially acting positive feedback loops of TCR-interferon-gamma-Stat1-T-bet and interleukin-12-Stat4-T-bet signaling.
The original model was created by:
Edda G. Schulz
Theoretical Biophysics, Institute of Biology, Humboldt Universität, Invalidenstrasse 42, 10115 Berlin, Germany.
Project description:IL-27 is a potent antagonist of TH1-mediated inflammation, but the basis for this effect is not fully understood. Recent studies identified a population of T-bet+ CXCR3+ Treg that limit TH1-mediated immune pathology. The studies presented here demonstrate that IL-27-mediated STAT1 activation promotes Treg expression of T-bet and CXCR3. Infection with Toxoplasma gondii induced a similar Treg population that limits T cell responses and this population at mucosal sites is IL-27-dependent. Furthermore, transfer of Treg ameliorated the infection-induced CD4+ T cell-mediated pathology observed in IL-27p28-/- mice. Although IFN-γ promoted a similar population of cells in the periphery, it did not compensate for the absence of IL-27 at mucosal sites and microarray analysis revealed that Treg exposed to either cytokine have distinct transcriptional profiles. These findings suggest that IFN-γ and IL-27 have different roles in Treg biology but define IL-27 as a key cytokine that promotes the development of Treg specialized to control TH1 immunity. Three conditions were analyzed across two timepoints. Inducible regulatory T cells (iTreg) were generated in vitro in the presence of IL-27, IFNg or under 'Neutral' conditions as a control. Samples were collected at 10 hours and 2 days during the culture period. Three biological replicates were used for each condition.
Project description:The AhR is a ligand activated transcription factor that may be important in normal skin physiology. We compared gene expression profiles between AhR Wt and AhR KO primary mouse keratinocyte cultures. We identified 391 genes that were differentially expressed with a 1.5 fold cutoff and p<.05, and identified the AhR as an important regulator of genes involved in normal epidermal differentiation. AhR Wt primary keratinocyte cultures (n=4) were compared with AhR KO primary keratinocyte cultures (n=3)
Project description:Thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs) have been described to play a critical role in thymic Treg generation. Our findings could show that both these thymic APCs can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs. In order to elucidate the unique properties of thymic APCs, gene expression profiling was performed in comparison to splenic DCs. Transcriptome analysis of thymic APCs revealed differential expression of costimulatory molecules that could be involved in stable Treg generation. Importantly, both mTEC- and t-DC- induced alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to alloantigen-specific Tregs generated by splenic DCs. Overall design: Thymic APCs, including mTECs and t-DCs and splenic DCs were isolated ex vivo from thymus as CD45-EpCAM+Ly51- (mTECs) and CD45+EpCAM-CD11chiLin- (t-DCs) and from spleen as CD11chiLin- (splenic DCs) (Lin is defined as CD90, CD49b, F4/80 and CD19), respectively.
Project description:Our group showed that DC-instrinsic C3ar1/C5ar1 signals are required for TLR-initiated DC maturation in vivo. To more broadly analyze how local complement signaling affects DC maturation process in response to TLR9 stimulation, WT or C3ar1-/-C5ar1-/- mice were stimulated with CpG (i.v. 100 micrograms) or vehicle control. 4hrs later, splenic CD11c+DCs were isolated and RNAs from the cells were purified for microarray analyses. Overall design: The experiment include samples from WT unstimulated DCs (n=2), WT CpG-stimulated DCs (n=3), C3ar1-/-C5ar1-/- unstimulated DCs (n=2), and C3ar1-/-C5ar1-/- CpG-stimulated DCs (n=3).