Project description:To understand the gene expression programs regulated by IRF8 in B cells responding to LPS stimulation we generated IRF8 deficient B cells by CRISPR/Cas9. IRF8 knockout and wild-type CD43- splenic B cells were isolated and directly used for RNA-seq (naive B cells) or simulated to differentiate with LPS, IL2, and IL5 for 3 days. At this time activated Bcells and plasmablasts were sorted for RNA-seq.
Project description:To understand the differentiation program in monocyte/macrophage differentiation, we performed ChIP-seq for IRF8 and H3K4me1 together with gene expression profiling during IRF8-induced monocyte differentiation. Both promoter-proximal and -distal binding of IRF8 associated with induction of the genes especially those related to monocytes/macrophages and immunity. DNA motif analysis for cis-regulatory elements of indirect IRF8 target genes predicted KLF4, essential for Ly6C+ monocyte development, to be a downstream transcription factor regulating the indirect target gene expression. Introduction of KLF4 into an Irf8-/- myeloid progenitor cell line induced a subset of IRF8 target genes and partially induced monocyte/macrophage differentiation. Together, this study revealed the genome-wide behavior of IRF8 and the IRF8-KLF4 axis during monocyte differentiation. Gene expressions in monocyte-like cells differentiated by IRF8 or KLF4 were measured at day 4 after retroviral transductions to myeloid progenitor cell line, Tot2. Two independent experiments were performed.
Project description:Transcriptional profiling of IRF8-regulated genes in mouse CD11b-positive primary cells. CD11b-positive cells were isolated from spleens of age-matched wt and IRF8 KO mice, the compared for their gene expression profiles. The objective is to identify genes that are regulated by IRF8 in CD11b-positive primary cells. Two-condition experiments. Wt vs IRF8 KO cells. Biological replicates: 2 replicates for the comparison.
Project description:To understand the differentiation program in monocyte/macrophage differentiation, we performed ChIP-seq for IRF8 and H3K4me1 together with gene expression profiling during IRF8-induced monocyte differentiation. Both promoter-proximal and -distal binding of IRF8 associated with induction of the genes especially those related to monocytes/macrophages and immunity. DNA motif analysis for cis-regulatory elements of indirect IRF8 target genes predicted KLF4, essential for Ly6C+ monocyte development, to be a downstream transcription factor regulating the indirect target gene expression. Introduction of KLF4 into an Irf8-/- myeloid progenitor cell line induced a subset of IRF8 target genes and partially induced monocyte/macrophage differentiation. Together, this study revealed the genome-wide behavior of IRF8 and the IRF8-KLF4 axis during monocyte differentiation.
Project description:Conditional IRF8 KO mice (mice with a conditional allele of Irf8 crossed with CD19-Cre mice) showed increased numbers of both Gene expression data spleen marginal zone (MZ) and Gene expression data spleen follicular (FO) B cells compared to control mice. To evaluate gene expression patterns that distinguished FO or MZ B cells derived from conditional KO and control mice, we used Affymetrix GeneChip® Mouse gene 1.0 ST Array. FACS-sorted MZ and FO B cells from individual mouse were used for RNA extraction and Affyarray hybridization. There were six independent biological replications in each group - six cases of MZ B cells and FO cells in IRF8 conditional KO mice and six cases of MZ B cells and FO cells in control WT mice.
Project description:Conditional IRF8 KO mice (mice with a conditional allele of Irf8 crossed with CD19-Cre mice) showed increased numbers of both Gene expression data spleen marginal zone (MZ) and Gene expression data spleen follicular (FO) B cells compared to control mice. To evaluate gene expression patterns that distinguished FO or MZ B cells derived from conditional KO and control mice, we used Affymetrix GeneChip® Mouse gene 1.0 ST Array.
Project description:Transcriptional profiling of IRF8-regulated genes in mouse CD11b-positive primary cells. CD11b-positive cells were isolated from spleens of age-matched wt and IRF8 KO mice, the compared for their gene expression profiles. The objective is to identify genes that are regulated by IRF8 in CD11b-positive primary cells.
Project description:Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is still incompletely understood. We report here that the interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increases p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significant higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis
Project description:IRF8, a transcriptional factor, has the heightened expression in germinal center(GC) B cell and GC-origin B cell lymphoma. To identify IRF8 direct targets in GC B cells, ChIP-chip ananlysis was done in three different GC-origin diffuse large B cell lymphoma cell lines. IRF8-negative cell lines, MMS1, was also used as a negatvie control. IRF8 ChIP in three different cell lines (ODH1, VAL and LY1:high level of IRF8) and in one negative control cell lines (MMS1:negatvie for IRF8). Total four different samples. One sample per a set of two arrays (promoter1 and promoter2).
Project description:IRF8, a transcriptional factor, has the heightened expression in germinal center(GC) B cell and GC-origin B cell lymphoma. To identify IRF8 direct targets in GC B cells, ChIP-chip ananlysis was done in three different GC-origin diffuse large B cell lymphoma cell lines. IRF8-negative cell lines, MPC11, was also used as a negatvie control. IRF8 ChIP in three different cell lines (NFS201, NFS202 and NFS205:high level of IRF8) and in one negative control cell lines(MPC11:negatvie for IRF8). Total four different samples. One sample per a set of two arrays (promoter1 and promoter2).