Expression data from wild-type and microRNA-155 (miR-155) deficient CD8 T cells
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ABSTRACT: MicroRNA-155 (miR-155) is upregulated in primary effector CD8 T cells but is expressed at low amounts in naïve cells. Anti-viral CD8 T cell responses and viral clearance were impaired in miR-155 deficient (bic-/-) mice, and this defect was intrinsic to CD8 T cells, as adoptively transferred bic-/- CD8 T cells generated greatly reduced primary and memory responses during infection. To understand the mechanism by which miR-155 regulates CD8 T cell activation, we analyzed the gene expression profiles of naive and in vitro activated wild-type and bic-/- CD8 T cells. CD8 T cells were purified from uninfected C57BL/6 mice and stimulated in vitro with plate-bound anti-CD3 and anti-CD28 antibodies for 48 h or left unstimulated. RNA from these CD8 T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays.
Project description:MicroRNA-155 (miR-155) is upregulated in primary effector CD8 T cells but is expressed at low amounts in naïve cells. Anti-viral CD8 T cell responses and viral clearance were impaired in miR-155 deficient (bic-/-) mice, and this defect was intrinsic to CD8 T cells, as adoptively transferred bic-/- CD8 T cells generated greatly reduced primary and memory responses during infection. To understand the mechanism by which miR-155 regulates CD8 T cell activation, we analyzed the gene expression profiles of naive and in vitro activated wild-type and bic-/- CD8 T cells.
Project description:Transcriptome analysis of bic-deficient T cells identified 152 upregulated genes 99 of which contain miR-155 binding sites in their 3'UTRs.
Project description:Determing the influence of lipid metabolism on murine T cell blastogenesis. Gene expression studies from purified spleen and lymph node T cells with conditional deletion of the SREBP Cleavage Activating Protein (SCAP) ex vivo or activated with plate-bound anti-CD3 and CD28 antibodies for 6 h. CD8 T cells were purified from T cell specific Scap deficient mouse spleens (KO, n=3) and control littermate (WT, n=3). The cells from each mouse were used for RNA extraction at either quiescent (T0) or 6 h activation (T6).
Project description:CD8+ T cells isolated from HCC tissue were divided into three groups: PD1-TIM3- CD8+ TILs, exhibiting full effector function; PD1-intTIM3+ CD8+ TILs, exhibiting partial exhaustion; and PD1-hiTIM3+ CD8+ TILs, exhibiting severe exhaustion, as reflected by the differences in their ability to produce effector cytokines respectively. Transcriptome sequence analysis was performed to investigate the gene expression profile was performed.
Project description:The activation of TLR-MyD88 (Toll like receptor- Myeloid differentiation factor 88) signaling within T cells functions as a potent costimulatory signal that boosts antitumor and antiviral responses. However, the molecular mechanisms underlying the costimulatory processes are poorly understood. We compared microarray gene analysis data between TLR1-TLR2 stimulated and unstimulated T cell receptor transgenic âpmelâ and MyD88-/-pmel CD8+ T cells and identified changes in the expression levels of several TNF family members. In particular, TLR-stimulation increased 4-1BB levels in pmel but not in MyD88-/-pmel T cells. A link between 4-1BB and TLR1-TLR2 signaling in CD8+ T cells was highlighted by in fact that 4-1BB-/- T cells exhibited suboptimal responses to TLR1-TLR2 agonist, but responded normally to CD28 or OX40 costimulation. Moreover, blocking 4-1BB signaling with antibodies also hindered the costimulatory effects of the TLR1-TLR2 agonist. The elevated levels of 4-1BB transcripts in TLR1-TLR2âstimulated cells were not due to increased mRNA stability nor increased histone activation but instead were associated with increased binding of p65 and c-Jun to two distinct 4-1BB promoter sites. Combining TLR1-TLR2 ligand with an agonistic anti-4-1BB antibody enhanced the antitumor activity in mice with established melanoma tumors. These studies reveal that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells are in part mediated by 4-1BB and are important for mounting an effective antitumor immune response. CD8+ T cells from the B6.Cg-Thy1/Cy Tg(TcraTcrb)8Rest/J mice, referred to as âpmelâ T cells or from MyD88 knockout pmel mice (MyD88â/âpmel) were sorted. pmel and MyD88â/âpmel T cells were activated using MyD88â/â CD8 T cell-depleted splenocytes pulsed with 10ng/ml of mgp100. This was with or without 10µg/ml of Pam3CSK4. pmel or MyD88â/âpmel CD8 T cells were enriched and used for the extraction of RNA used for genomic analysis.
Project description:The negative feedback mechanism is essential to maintain effective immunity and tissue homeostasis. 1,25-dihydroxyvitamin D (1,25(OH)2D3) modulates innate immune response, but the mechanism remains poorly understood. Here we report that vitamin D receptor (VDR) signaling attenuates Toll-like receptor-mediated inflammation by enhancing the negative feedback inhibition. VDR inactivation leads to a hyperinflammatory response in mice and macrophage cultures when challenged with lipopolysaccharide (LPS) due to miR-155 overproduction that excessively suppresses SOCS1, a key regulator that enhances the negative feedback loop. Deletion of miR-155 attenuates vitamin D suppression of LPS-induced inflammation, confirming that 1,25(OH)2D3 stimulates SOCS1 by down-regulating miR-155. 1,25(OH)2D3 down-regulates bic transcription by inhibiting NF-kappaB activation, which is mediated by a kappaB cis-DNA element located within the first intron of the bic gene. Together these data identify a novel regulatory mechanism for vitamin D to control innate immunity.
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.
Project description:To investigate the mechanism by which Cul5 regulates CD8+ T cell cytokine-dependent differentiation and TCR-dependent activation, we performed quantitative mass spectrometry (MS) by data-independent acquisition (DIA)-MS of total proteins in the Cul5 KO and NC primary CD8+ T cells in the following conditions: 1) Cytokine dependent expansion and differentiation (T0); 2) 8 hours cytokine withdraw prior to TCR stimulation (T8); and 3) 16 hours TCR stimulation post 8-hour cytokine withdraw (T16). Principal component analysis (PCA) and correlation analysis revealed that replicates in each condition clustered together while different conditions separated from each other, suggesting significant proteomic changes among different conditions of the same T cells as well as between Cul5 KO and NC T cells in each condition. Together with similar total protein quantities among all detected samples, DIA-MS analyses were of high quality. Additionally, the strong reductions of Cul5 abundances in the Cul5 KO cells from all three conditions compared to the NC cells confirms high KO efficiency. Of note, the proteomic analysis showed that the Cul5 protein level was significantly increased upon TCR stimulation post cytokine starvation in the NC cells, suggesting a potential negative feedback regulatory role of Cul5 in CD8+ T cell activation. Consistent with this idea, we observed more markedly upregulation of Cul5 expression upon of TCR stimulation of na•ve primary CD8+ cells. To identify Cul5 interacting proteins in CD8+ T cells, we overexpressed Cul5 with a C terminal HA-tag (Cul5-HA) in mouse primary CD8+ T cells by retroviral transduction. The cells were subjected to TCR stimulation for 12 hr, and Cul5-HA was immunoprecipitated by anti-HA, followed by DIA-MS analysis (co-IP-MS). Compared to the negative control samples (cells transduced with the empty vector), 65 proteins were enriched (p value <0.05 and fold change >1.5) in the anti-HA IP samples. Altogether, we report that Cul5 KO alters CD8+ T cell proteome and the Cul5 interactome.
Project description:Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a and Stat5b double knock-in (DKI) N-domain mutant mice that form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined consensus motifs for dimers versus tetramers. Whereas Stat5- deficient mice exhibited perinatal lethality, DKI mice were viable, indicating that STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4+CD25+ T cells, NK cells, and CD8+ T cells, with impaired cytokine-induced proliferation and homeostatic proliferation of CD8+ T cells. DKI CD8+ T cell proliferation following viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is dispensable for survival but is critical for cytokine responses and normal immune function. Genome-wide mapping of STAT5A,STAT5B binding in mouse WT and DKI T cells (cultured with or without IL-2 for 1 hr) was conducted. RNA-Seq is conducted in mouse CD8+ T cells (WT and DKI, non-treated or treated with IL-2/IL-15 for 4 hr, 24 hr, 48 hr and 72 hr)
Project description:The transcription factor BATF is required for Th17 and TFH differentiation. Here, we show that BATF also has a fundamental role in regulating effector CD8+ T cell differentiation. BATF-deficient CD8+ T cells show profound defects in effector expansion and undergo proliferative and metabolic catastrophe early after antigen encounter. BATF, together with IRF4 and Jun proteins, binds to and promotes early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors, while paradoxically repressing genes encoding effector molecules (IFNg and granzyme B). Thus, BATF amplifies TCR-dependent transcription factor expression and augments inflammatory signal propagation but restrains effector gene expression. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved. P14 TCR transgenic CD8+ T cells from wild-type or BATF-/- mice were examined either as naïve cells or after 3 days of in vitro stimulation with antibodies to CD3 and CD28 in the presence of IL-2