CD3/CD28 and IL-2 stimulated mouse CD8 T cells with or without anti-CD132 blocking antibody
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ABSTRACT: Mouse CD8 T cells were isolated from total splenocytes from C57BL/6 mice by MACS separation. Cells were incubated for 48 h with CD3/CD28 DynaBeads and 50 ng/ml IL-2 for 48 h with or without 100 nm anti-CD132 blocking antibody. Cells were collected and RNA was isolated.
Project description:CD8+T cells are immune cells that recognize foreign antigens on infected and tumor cells, leading to cytokine-dependent expansion and activation of cytotoxicity towards the targets. To identify Runx3 regulated genes, CD8+ T cells were isolated from spleen of WT and Runx3-/- mice . Six samples (3 WT and 3 Runx3-/-) of CD8+ T cells were separately obtained from individual mice, TCR activated and cultured for 4 days with IL-2.
Project description:Transcriptional profiling of Double Negative (CD4-/CD8-) T-cells isolated from SIV infected Sooty Mangebys. DN T-cells were stimulated through the T-Cell receptor using anti CD3/CD28 antibodies for 4 hours and compared to unstimulated DN T-cells. Two condition experiment, Stimulated vs Unstimulated. 4 animals tested, each with Stim vs Unstim, a dye flip of Stim vs Unstim, Stim vs Stim and Unstim vs Unstim.
Project description:Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical grade tumor-redirected TSCM cells starting from naïve precursors. CD8+CD62L+CD45RA+ naïve T cells enriched by streptamer-based serial positive selection were activated by CD3/CD28 engagement in the presence of IL-7, IL-21 and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions allowed for the generation of CD19-CAR modified TSCM cells that were phenotypically, functionally and transcriptomically equivalent to their naturally occurring counterpart. Compared with T cell products currently under clinical investigation, CD19-CAR modified TSCM cells exhibit enhanced metabolic fitness, persistence and anti-tumor activity against systemic acute lymphoblastic leukemia xenografts. Based on these findings, we have initiated a phase 1 clinical study to evaluate the activity of CD19-CAR modified TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation. Three healthy human blood donors provided lymphocyte-enriched apheresis blood for this study after informed consent. From all samples, total RNA was isolated using an miRNeasy Mini Kit (Qiagen), processed by Ambionâ??s WT expression kit, fragmented and labeled with a WT Terminal Labeling Kit (Affymetrix), hybridized to WT Human Gene 1.0 ST arrays (Affymetrix) and stained on a Genechip Fluidics Station 450 (Affymetrix), all according to the respective manufacturer's instructions. Samples represent exon-level and gene-level analyses.
Project description:Investigation of proteostatic effects of HSP90 inhibition by 17-AAG and differentiation of maturation state dependent HSP90 requirements of proteins in activated T-cells.
Project description:To determine functional overlap between cMyc and AP4 in CD8+ T cell priming, we retrovirally expressed cMyc or AP4 in cMyc-deficient CD8+ T cells and examined gene expression after activation. Naive CD8+ T cells from Myc conditional knockout mice with a tamoxifen inducible Cre transgene were retrovirally transduced with Myc or AP4 followed by a treatment with 4-hydroxytamoxifen in the presence of IL-7 for 2 days. RNA was harvested 48 hours after restimulation of transduced cells with anti-CD3 antibody and gene expression was compared by microarray. CD8+ T cells from littermate wildtype mice that were transduced with an empty retrovirus were used as control.
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
Project description:Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.