Comparison of intestinal surface CD3- refractory celiac disease type II (RCDII) cell lines with intestinal surface CD3+ cell lines of non-RCDII patients
ABSTRACT: Analysis of transcriptional differences between aberrant (surface CD3-) RCDII cell lines and regular (surface CD3+) intraepithelial cell lines in order to characterize aberrant RCDII cells. All cell lines were cultured from duodenal biopsies of RCDII or non-RCDII (either celiac or non-celiac) patients. We compared 3 RCDII cell lines from RCDII patients P1-P3 with the following control cell lines: CD3+CD4+ T cell line from P1 and P3, CD3+CD8+ T cell line from P3 and mixed CD4/CD8+ T cell lines from non-RCDII patients. All control cells matched the phenotype of mature T cells (CD3+CD4+ and/or CD3+CD8+). Samples were randomly distributed among two BeadChips and measured in duplicates.
Project description:We have found that knockdown of the human telomerase RNA template, hTR, induces Bim-mediated apoptosis independent of telomere length in primary human CD4 T cells, whereas knockdown of the telomerase enzymatic protein, hTERT does not induce apoptosis in the timeframe of our studies. We used microarray analysis to determine any gene changes in human primary CD4 T cells that could provide mechanistic insight into hTR knockdown induced apoptosis. Cells with control shScramble, shTERT, and shTR were used in this experiment Primary human CD4 T cells were trandsuced in biological triplicates with lentivirus containing shRNAs against a scrambled sequence, hTERT, and hTR 24 hours after CD3 CD28 stimiulation. Cells were cultured in the presence of puromycin to select for cells with shRNAs. shTERT and shTR were compared to shScramble results.
Project description:It is known that ubiquitination is important for T cell receptor (TCR) signaling during T cell activation but the breadth of ubiquitination events triggered during TCR signaling is not completely understood. This dataset utilizes di-glycine remnant profiling combined with mass spectrometry to identify a global landscape of ubiquitination events downstream of the TCR and to quantify changes ubiquitin abundance in response to TCR stimulation. Additionally, whole cell proteomics data were generated to measure protein abundances during TCR stimulation. Mouse primary T cells were isolated, proliferated and either remained resting or stimulated with CD3/CD28 to activate downstream signaling through the TCR and co-stimulatory pathways. Di-glycine remnant profiling and whole cell proteomics was performed on rested cells and cells that had undergone CD3/CD28 TCR stimulation for 4 hours. These data were analyzed to identify the ubiquitination events during TCR activation and to quantify the change in peptide-based ubiquitin abundance and total protein abundance over the course of the 4 hour TCR stimulation. Integration of di-glycine and whole cell proteomics was used to generate protein-specific predictions of whether ubiquitination events downstream of TCR signaling lead to a decrease in associated protein abundance. The analysis of these data suggests that T cell activation leads to an increase in ubiquitination that is not associated with proteasomal or lysosomal degradation.
Project description:During persistent antigen stimulation, CD8+ cytolytic T cells (CTL) show a gradual decrease in effector function, or “exhaustion”, which impairs the immune response to tumors and infections. Here we show that NFAT, a transcription factor with an established role in T cell activation, in parallel controls a second transcriptional program conferring the characteristic features of CD8+ T cell exhaustion, including upregulation of genes encoding inhibitory cell surface receptors and diminished TCR signaling. Expression of an engineered NFAT1, which induces this negative regulatory program in the absence of the effector program, interferes with the ability of CD8+ T cells to protect against Listeria infection or attenuate tumor growth in vivo. NFAT elicits this second program of gene expression in large part by binding to a subset of the sites occupied by NFAT during a typical effector response, suggesting that a balance between the two pathways determines the outcome of TCR signaling. Determination of NFAT1 binding sites in CD8 T cells in vitro
Project description:Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10. Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions. For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads in 96-well flat bottom plates. MACSi Beads were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to separate IL-10-secreting and non-secreting cells. Using a BD Aria or DIVA cell sorter (Becton Dickinson), living CD4+ IL-10-secreting and non-secreting cells, with (co-culture with Dll4; 'TH1Notch') or without ('TH1Control') activation of the Notch signaling pathway, were isolated. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The preparation for the hybridization to the chip was done using the GeneChip 3' IVT Express Kit. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 4 mouse genome 430 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.1.1., both Affymetrix. The data was analyzed using the original GCOS CHP-file Signals, Excel and AmiGO website.
Project description:CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and na ve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80°C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray. CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and naïve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80°C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-039A.html, SABiosciences Frederick, MD). Briefly, total RNA was harvested from each individual T cell subset from each patient or healthy control and contaminating DNA was digested with DNase. Messenger RNA was converted to cDNA and loaded onto PCR array plates for quantitative real-time PCR. Quantification of transcript levels was determined by normalizing to 5 housekeeping genes from each individual sample. Relative gene expression levels for each subset were averaged and compared between cell populations from the patient group and healthy controls. Because of the multiple comparisons only p values ≤ 0.01 were considered significant.
Project description:The experiment was designed to assess how intestinal epithelial cells respond to different soluble factors produced by intraepithelial lymphocytes (IEL) and how this response is altered when the epithelial cells express Btnl1. Total RNA of MODE-K cells transduced with Btnl1-GFP or GFP alone and stimulated with TNF+IL-1_ in comparison to medium and with supernatant from anti-CD3-activated IEL in comparison to supernatant from unstimulated IEL
Project description:We used the NanoString mouse nCounter miRNA expression platform to compare the miRNA expression in double-positive (DP) thymocytes that developed in the presence (GFP+) or absence (GFP-ve) of ectopic mLin28 expression. Transduced (GFP+) and untransduced (GFP-ve) CD4+ CD8+ CD3- thymocytes were FACS sorted and pooled from three recipients of hematopoietic stem and progenitor cells transduced with Lin28-RV six weeks post-reconstitution (bone marrow chimeric mice). Total RNA was extracted and used for sample preparation and hybridization per manufacturer's recommendations.
Project description:The goal of the study was to identify the genes which are regulated by Interleukin-2 in the CD4+ T cells of the scurfy mice during regulatory T-cell deficiency. Scurfy (Sf) mice bear a mutation in the forkhead box P3 (Foxp3) transcription factor, lack regulatory T-cells (Treg), develop multi-organ inflammation, and die prematurely. The major target organs affected are skin, lungs, and liver. Sf mice lacking the Il2 gene (Sf.Il2-/-), despite devoid of Treg, did not develop skin and lung inflammation, but the inflammation in liver, pancreas, submandibular gland and colon remained. Genome-wide microarray analysis revealed hundreds of genes were differentially regulated among Sf, Sf.Il2-/-, and B6 CD4+ T-cells but the most changes were those encoding receptors for trafficking/chemotaxis/retention and lymphokines. Our study suggests that IL-2 controls the skin and lung inflammation in Sf mice in an apparent "organ-specific" manner through two novel mechanisms: by regulating the expression of genes encoding receptors for T-cell trafficking/chemotaxis/retention and by regulating Th2 cell expansion and lymphokine production. Thus, IL-2 is a master regulator for multi-organ inflammation and an underlying etiological factor for various diseases associated with skin and lung inflammation. Methods: CD4+ T cells were purified by Fluorescence Assisted Cell Sorting from the peripheral lymph nodes of (A) three individual Scurfy (Sf; B6.Cg-Foxp3sf/J) male mice, (B) three individual Sf.Il2-/- male mice (Scurfy mice carrying a null Interleukin (IL)-2 gene (B6.129P2-Il2tm1Hor/J)) and (C) a pooled sample of lymph nodes from two B6 (C57BL/6J) mice. All the mice were 3 weeks old. Total RNA was prepared using RNeasy mini kit (Qiagen). RNA samples were converted to cRNA, labeled and hybridized to Affymetrix Mouse 430_2 chips (Mouse Genome 430 2.0 Array, Affymetrix, Santa Clara, CA) at the University of Virginia DNA Sciences Core Facility. 1. RNA from CD4+ T cells purified from pooled peripheral lymph nodes of two 3-week old B6 mice) - 1 biological replicate 2. RNA from CD4+ T-cells purified from peripheral lymph nodes of 3-week old scurfy (Sf) mice - 3 biological replicates. 3. RNA from CD4+ T cells purified from peripheral lymph nodes of Sf.Il2-/- mice - 3 biological replicates.
Project description:The discovery of TET proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T cell development in the thymus and T cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages, and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers, and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T cell development and differentiation. Study the distribution of 5hmC in 7 T cell subsets at sequential steps of development, differentiation and specification