Transcription profiling by array of mouse CD8+ naive T cells from mice expressing normal IL-7R or conditional IL-7R alpha switched on or off for different amounts of time
ABSTRACT: The transcriptional profile of CD8+ naive T cells was compared from F5 transgenic mice expressing normal IL-7R or a conditional IL-7R alpha that was either switched on, or switched off for different amounts of time.
Project description:Effects of IL-4 on CD8 T cells functions are largely unknown. IL-4 induces survival and proliferation of CD8 T cells, but several studies suggest that IL-4 could also affect several functions of CD8 T cells such as cytotoxicity. Our team has shown that IL-4 repress the expression of Ccl5 in vitro. To define more precisely the impact of IL-4 on CD8 T cells, we performed a whole genome expression microarray analysis of naive and memory CD8 T cells cultured in presence or absence of IL-4. This approach allowed us to define the IL4-gene-expression signature on CD8 T cells. 18 samples were processed. Two populations of F5 naive CD8 T cells were FACS-sorted: samples from each population were incubated 20 hours with IL-7 in presence or absence of IL-4. Thus, a total of 6 “Naive” samples were processed. In addition, 4 populations of F5 TIM memory CD8 T cells were FACS-sorted: samples from 2 of these populations were incubated 20 hours in presence of IL-7 and/or IL-4, or in medium alone. Thus, 12 “Memory” samples were processed.
Project description:We report that a large percentage of thymic B cells undergo class switching intrathymically. Thymic B cell class switching requires cognate T-B interaction. To determine whether B cell specificity was also important for thymic B cell class-switching, we sorted class-switched thymic B cells (CD19+B220+IgM-IgD-), unswitched B cells (CD19+B220+IgM+IgD+) and bulk splenic B cells in 3H9 heavy chain-fixed mice and performed high throughput sequencing analysis of the light chain of these populations. Results of this analysis indicated that class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells and splenic B cells. Further reactivity tests indicated that a large part of BCRs enriched in class-switched thymic B cells are autoreactive. These data suggest that autoreactive B cells are selected into class-switched population and expanded in the thymus. Light chain repertoire profiles of class-switched thymic B cells, unswithced thymic B cells and splenic B cells from 3H9 mice were generated by deep sequencing.
Project description:We report that a large percentage of thymic B cells undergo class switching intrathymically. Thymic B cell class switching requires cognate T-B interaction. To determine whether B cell specificity was also important for thymic B cell class-switching, we sorted class-switched thymic B cells (CD19+B220+IgM-IgD-), unswitched B cells (CD19+B220+IgM+IgD+) and bulk splenic B cells in 3H9 heavy chain-fixed mice and performed high throughput sequencing analysis of the light chain of these populations. Results of this analysis indicated that class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells and splenic B cells. Further reactivity tests indicated that a large part of BCRs enriched in class-switched thymic B cells are autoreactive. These data suggest that autoreactive B cells are selected into class-switched population and expanded in the thymus. Overall design: Light chain repertoire profiles of class-switched thymic B cells, unswithced thymic B cells and splenic B cells from 3H9 mice were generated by deep sequencing.
Project description:The expansion, trafficking and functional effectiveness of adoptively transferred CD8+ T-cells play a critical role in mediating effective anti-tumor immunity. However, the mechanisms which program the highly proliferative and functional state of CD8+ T-cells are not completely understood. We hypothesized that IL-12, a cytokine commonly induced by TLR activation, could enhance T-cell priming by altering responsiveness to antigen and cytokines. Priming of tumor specific CD8+ T-cells in the presence of IL-12 induced the acquisition of a 'polyfunctional' effector response and increased the generation of memory cells. Moreover, IL-12 priming also promoted high levels of the IL-2 receptor alpha-chain (CD25) and robust IL-2 mediated activation of STAT5. This sensitivity to IL-2 translated into enhanced in vivo proliferation of adoptively transferred CD8+ T-cells. Furthermore, real-time, in vivo imaging of T-cell trafficking confirmed the ability of IL-12 priming to drive in vivo proliferation. IL-12 priming enhanced the anti-tumor function of adoptively transferred cells by reducing established subcutaneous tumor burden, and significantly increasing survival in an established intracranial tumor model. Finally, IL-12 priming of human PBMCs generates tumor specific T-cells phenotypically and functionally similar to IL-12 primed Pmel-1 T-cells. These results highlight IL-12 as an important mediator of CD8+ T-cell effector function and anti-tumor immunity. We primed Pmel-1 TCR transgenic CD8+ T-cells with cognate antigen and either IL-2 or IL-12 and compared their gene expression profiles. This was used to identify pathways or genes necessary for anti-tumor activity in vivo. RNA was isolated from Pmel-1 T-cells primed with antigen and cytokine for 6 days and hybridized to Affymetrix arrays.
Project description:We used microarray analysis to identify specific molecular mechanisms controlling IL-5 transcription in memory Th2 cells. IL-5+ and IL-5- memory Th2 cells were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Transcriptional profiling and gene expression profiling analysis of sorted CD8+IL-10+ T cells compared to CD8+IL-10- T cells using IL-10-GFP(tiger) reporter mice Two sample, CD8+IL-10+ T cells vs CD8+IL-10- T cells. Three replicate per array.
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:The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4+ T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with Pneumonia Virus of Mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo. Genome-wide transcription factors mapping and binding of STAT3, H3K4me3, H3K27me, H3K4me1, H3K27ac in mouse CD4+ T cells and dendritic cells in WT and Stat3-/- mice. RNA-Seq is performed in mouse CD4+ T cells and dendritic cells in WT mice, with or without indicated cytokines.
Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong