Identification of IL-23 target genes in intestinal CD4+ T cells
ABSTRACT: IL-23 negatively regulates St2 and Gata3 expression in intestinal CD4+ T cells Colitis was induced by co-transfer of WT and Il23r-/- naïve CD4 T cells into Rag1-/- recipients. Upon development of clinical signs of inflammation WT and Il23r-/- CD4+ T cells were sort-purified from the colon lamina propria on the basis of congenic markers. Total RNA was extracted immediately after the sort without further manipulation.
Project description:The IL-33 receptor ST2 is differentially expressed by colonic lamina propria Treg cells Microarray of sort-purified Foxp3+ Treg cells from colonic lamina propria over mesenteric lymph node
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines. PBMCs were isolated from five healthy donors. The cells were stimulated with PMA/Ionomycin for 6 h, and subsequently sorted into the CD4+CD45RA-CD40L+, CD8+CD45RA-CD40L-, CD8+CD45RA-CD40L+ populations by FACS-sort.
Project description:CCR6+ innate lymphoid cells were sorted from the mesenteric lymph node of naïve C57BL/6 mice Cells were sort purified as lineage negative, CD127+ CD90.2+ CCR6+ ST2- 4 replicates were performed
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines PBMCs were isolated from five healthy donors. The cells were stimulated with PMA/Ionomycin for 6 h, and subsequently sorted into the CD4+CD45RA-CD40L+, CD8+CD45RA-CD40L-, CD8+CD45RA-CD40L+ populations by FACS-sort
Project description:Analyses of ILC3s in Rorc floxed control and Id2iΔRORγt mice following daily treatments of tamoxifen for two weeks. Cells were sort purified as lineage negative, CD127+ CD90.2+ CCR6+ ST2- CCR6+ ILCs were sorted from the mesenteric LN of Rorc floxed control and Id2iΔRORγt mice following daily treatments of tamoxifen for two weeks.
Project description:GPR15 is an orphan G-protein coupled receptor and its expression is abundant among T cells in the large intestine lamina propria. We used microarrays to examine charateristics of GPR15- vs GPR15+ CD4+ T cells in LILP and identified distinct classes of up-regulated genes in GPR15+ CD4+ T cells. We have generated GFP knock-in mice in GPR15 locus and used GFP as a maker for GPR15 expression. In heterozygous mice, GPR15+CD4+ T cells and GPR15-CD4-T cells were sorted and RNA was prepared from them.
Project description:CD4+ T cells that selectively produce interleukin (IL)-17, are critical for host defense and autoimmunity1-4. Crucial for T helper17 (Th17) cells in vivo5,6, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been argued to be the factors responsible for initiating specification7-10. Herein, we show that Th17 differentiation occurs in the absence of TGF-β signaling. Neither IL-6 nor IL-23 alone efficiently generated Th17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naïve precursors, independently of TGF-β. Epigenetic modification of the Il17a/Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed Rorγt and T-bet. T-bet+Rorγt+ Th17 cells are generated in vivo during experimental allergic encephalomyelitis (EAE), and adoptively transferred Th17 cells generated with IL-23 in the absence of TGF-β1 were more pathogenic in this experimental disease. These data suggest a new model for Th17 differentiation. Consistent with genetic data linking the IL23R with autoimmunity, our findings re-emphasize the role of IL-23 and therefore have important implications for the development of new therapies. Mouse T helper 17 cell differentiation with or without TGFB
Project description:Gene expression profiling of human CD8+ CD161hi and CD161lo central and effector memory and naïve T cell subsets. The mechanisms by which IL-17 secreting cells are regulated have not been completely elucidated. We previously identified a population of rhodamine-effluxing memory CD8+ T cells with high expression of CD161 that contributes to immune reconstitution after lymphopenia-inducing chemotherapy. Here we find that CD161hi CD8+ T cells share transcriptional programming with Th17 cells, but most do not secrete IL-17 or proliferate to stimulation through the T cell receptor (TCR). Transcriptional analysis of subsets identified by expression of CD161 and CD62L revealed a novel mechanism of TCR signaling pathway regulation in CD161hi CD8+ T cells that is distinct from that described in anergic or tolerant cells and renders them functionally dependent on costimulation through innate cytokine receptors or CD28. CD161hi CD8+ T cells, induced to proliferate by a TCR signal delivered with costimulation, demonstrated plasticity that was dependent on the nature of costimulation and resulted in expansion of IL-17 secreting cells that could not proliferate to a TCR signal alone or differentiation to Tc1-like cells that proliferated to TCR stimulation in the absence of costimulation. The data show an association between TCR signaling pathway downregulation and type 17 programming in CD161hi CD8+ T cells, whose dysregulation could mediate IL-17 dependent inflammatory diseases. T cell subsets were sort-purified from healthy adults and analyzed by gene expression profiling. Isolation: Effluxing CD161hi and non-effluxing CD161lo CD8+ TCM and TEM subsets were purified using magnetic bead separation and cell sorting to achieve >98% purity, as previously described (35). CD8+ T cells were positively selected using CD8 Microbeads (Miltenyi Biotec, Germany), loaded with Rh123 (Sigma, St. Louis, MO) and cultured for 30 min to allow Rh123 efflux, then labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCRγδ, Vα24, CD8, CD95, CD62L and CD161. CD161hi and CD161lo TCM and TEM subsets were sort-purified on a FACS ARIA equipped with 405 nm, 488 nm and 633 nm lasers (BD Biosciences). CD161hi TCM and TEM subsets were identified as CD62L+/Rh123lo/CD161hi and CD62L-/Rh123lo/CD161hi events, respectively, in the CD4-/CD16-/TcRγδ-/Vα24-/CD8+/CD95+ population. CD161lo TCM and TEM subsets were identified as CD62L+/Rh123hi/CD161int/neg and CD62L-/Rh123hi/CD161int/neg events, respectively, in the CD4-/CD16-/TcRγδ-/Vα24-/CD8+/CD95+ population. Gene expression profiling: RNA was extracted from sort-purified subsets from 6 healthy individuals using the RNeasy Plus Minikit (Qiagen, Valencia, CA) and biotinylated, followed by generation of amplified cRNA for array hybridization using the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems, Foster City, CA). Amplified biotinylated cRNA was then purified before quality control to ensure high quality cRNA was available for hybridization. Labeled cRNA was hybridized to Illumina WG-6 Expression BeadChips v3.0 (Illumina, San Diego, CA), which quantitate expression of 48,802 transcripts derived from the NCBI RefSeq (Build 36.2, Release 22) and UniGene databases (Build 199). BeadChips were washed before reading and image extraction, and then scanned on an Illumina BeadArray Reader. The resulting TIFF images were processed using GenomeStudio Gene Expression Module (GEM) software. Data quality was assessed using the Control Summary feature in GenomeStudio GEM. For a given analysis set, a GenomeStudio Probe-level Final Report was generated by combining the Sample Probe Profile and Control Probe Profile tables. The Final Report comprising the full dataset was initially processed using the Bioconductor package lumi by employing a background correction estimate and quantile normalization. A small adjustment (i.e. 20 counts) was made to the entire dataset to make all intensity signals non-negative and these values were log2-transformed. The dataset was initially filtered using the ‘shorth’ function of the Bioconductor package genefilter, resulting in retention of 31,084 of 48,802 original probes. Each pairwise comparison was further filtered by discarding probes whose signal intensity was less than a defined signal “noise floor” across all arrays in the data subset. This was achieved by calculating the median of the ‘negative control’ probe signals for each array, averaging these values, and setting the noise floor as 3 times this average. Differential gene expression was then determined using the Bioconductor package limma, and a false discovery rate (FDR) method applied to correct for multiple testing. Significant differential gene expression was defined by a log2 ratio > 0.585 (± 1.5-fold) and FDR (adjusted p value) < 0.05.
Project description:We report gene expression changes in Cul3 deficient thymic CD4+ T cells We used microarrays to detail the global programme of gene expression changes upon removal of Cul3 during thymic development CD25-, CD1dPBS57-, gdTCR - CD4+ Single positive cells from thymocytes of CD4Cre-Cul3fl/fl (LM) and CD4 Cre+Cul3fl/fl (KO) were sort purified and total RNA was extracted using Trizol and analysed by microarray.