Project description:Peritoneal macrophages were obtained by intraperitoneal injection of cold PBS from Ldlr−/− mice reconstituted with Lyz2Cre+/− Nrp1flox/flox or Lyz2Cre+/− Nrp1+/+ bone marrow.
Project description:T follicular helper (TFH) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (TFR) cells limit GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Here we show that SOCE is required for the differentiation and function of both TFH and TFR cells. Conditional deletion of Stim1 and Stim2 genes in T cells or Treg cells results in spontaneous autoantibody production and humoral autoimmunity. Conversely, antibody-mediated immune responses following viral infection critically depend on SOCE in TFH cells. Mechanistically, STIM1 and STIM2 control early TFR and TFH cell differentiation through NFAT-mediated IRF4, BATF and Bcl-6 expression. SOCE plays a dual role in GC response by controlling TFH and TFR cell function, thus enabling protective B cell responses and preventing humoral autoimmunity. RNAseq analyses of WT and Stim1Stim2 DKO follicular T cells and non-follicular T cells; 4-6 mice per cohort in duplicates. Mice were infected for 10 days with LCMV.
Project description:Single-cell RNA-seq analysis of mouse CD4 T cells to understand the function of HDAC1 for pathogenic Th2 cells. Wildtype and HDAC1 KO cell from several sorted populations are covered. Hashtag oligos were used to enable multiplexing before analysis on a 10x chromium.
Project description:The deletion of the POZ domain of the transcription factor Miz1 leads to a late onset neuropathy with subsequent spontaneous regeneration in mice. In this study, we aim to identify genes that are involved in the development of this neuropathy. We traced first gene regulatory changes in sciatic nerve tissue from Miz1∆POZ mice to 30 days after birth. At this time point, we harvested samples of sciatic nerve tissue from several mice and performed RNAseq analysis to identify differentially expressed genes between Miz1∆POZ and wild type mice.
Project description:The role of FoxP3+ regulatory T (Treg) cells in the maintenance of immunological tolerance is well established. Recently, genome-wide association studies (GWAS) in humans have associated polymorphisms within the BACH2 locus encoding the transcription factor BTB and CNC homology 1, basic leucine zipper transcription factor 2 (Bach2) with diverse allergic and autoimmune diseases including asthma, multiple sclerosis, Crohn's disease, celiac disease, generalized vitiligo and type 1 diabetes. Common to these diseases is a failure to adequately maintain immunological tolerance. However, a role for Bach2 in this process has not been established. Here, by assessing the phenotype of mice in which the Bach2 gene is disrupted, we demonstrate a non-redundant role for Bach2 in the prevention of a spontaneous lethal inflammatory disorder predominantly affecting the lung and gut with excessive T helper 2 (Th2) responses and formation of circulating autoantibodies. Bach2 was necessary for efficient induction of FoxP3 expression both during thymopoesis and upon stimulation of naïve peripheral CD4+ T cells under Treg polarizing conditions in vitro. Consequently, in bone marrow reconstitution experiments, Bach2 expression within the haematopoetic system was necessary for suppression of lethal autoimmunity in a manner that was FoxP3 dependent. These findings demonstrate a requirement for Bach2 in early lineage commitment of both thymic and induced Treg cells and point to shared mechanisms that underlie diverse allergic and autoimmune disorders that may serve as targets in the development of novel therapeutic strategies. Six samples were collected from separate mice: three Ly5.1+ wildtype thymocyte samples (biological replicates) and three Ly5.1- Bach2 knockout thymocyte samples (biological replicates).
Project description:CTLA-4 is thought to inhibit effector T cells both intrinsically, by competing with CD28 for B7 ligands, and extrinsically, through the action of regulatory T cells. We studied in vivo responses of normal and CTLA-4-deficient antigen-specific murine effector CD4+ T cells. In order to do these studies in a physiological model of immunity to foreign antigen, we transferred small numbers of congenically marked RAG2-deficient 5C.C7 T cells with either a normal or knockout allele of CTLA-4 into normal syngeneic B10.A recipient mice. The T cells were then activated by immunization with MCC peptide and LPS. To look for transcriptional signatures of negative regulation of T cell responses by CTLA-4, we used microarray analysis to compare transcripts in wild type and CTLA-4 KO 5C.C7 T cells four days after immunization. This is the first instance in which differences are observed in extent of accumulation of wild type and CTLA-4 KO 5C.C7 T cells. To compare the gene expression profile between wild type and CTLA-4 KO adoptively transferred T cells 4 days after immunization.
Project description:Murine WBP1L-deficient (Wbp1l-/-) hematopoietic stem and progenitor cells engraft significantly better than wild-type (WT) cells in competitive transplantation assays. To analyze the mechanism, Wbp1l-/- or WT bone marrow cells (both Ly5.2+Ly5.1-) were each mixed 1:1 with WT competitor cells (Ly5.2+Ly5.1+) and the mixture was transplanted into the lethally irradiated recipient mice. 22 weeks after the transplantation the donor Ly5.2+Ly5.1- LSK cells (Wbp1l-/- or WT) were purified from the bone marrow of recipient mice, RNA was extracted and subjected to RNA sequencing.
Project description:Eight weeks old male Adrb1tm1BkkAdrb2tm1Bkk/J , stock number 003810, were purchased from The Jackson Laboratory. These mice are homozygous null for the Adrb1 and Adrb2 genes, and are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Mice were euthanized, and the whole bone marrow was extracted using established methods.1 Whole bone marrow cells from Adrb1tm1BkkAdrb2tm1Bkk/J mice were reconstituted into lethally- irradiated (950 Rad) C57BL/6J mice using a single retro-orbital injection at a ratio of 1:4 (Adrb1tm1BkkAdrb2tm1Bkk/J to C57BL/6J). All reconstituted mice were recovered for 2-3 months prior to tissue collection for mRNA arrays (1) The FASEB Journal 2015:29(1),652.13. Seven control animals and seven animals that received new bone marrow from double adrenergic receptor knockout (treated KO chimera). Following recovery, tissues that were harvested included bone marrow, brain, and distal colon.
Project description:Eight weeks old male Adrb1tm1BkkAdrb2tm1Bkk/J , stock number 003810, were purchased from The Jackson Laboratory. These mice are homozygous null for the Adrb1 and Adrb2 genes, and are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Mice were euthanized, and the whole bone marrow was extracted using established methods.1 Whole bone marrow cells from Adrb1tm1BkkAdrb2tm1Bkk/J mice were reconstituted into lethally- irradiated (950 Rad) C57BL/6J mice using a single retro-orbital injection at a ratio of 1:4 (Adrb1tm1BkkAdrb2tm1Bkk/J to C57BL/6J). All reconstituted mice were recovered for 2-3 months prior to tissue collection for mRNA arrays (1) The FASEB Journal 2015:29(1),652.13. Eight control animals and eight animals that received new bone marrow from double adrenergic receptor knockout (treated KO chimera). Following recovery, tissues that were harvested included bone marrow, brain, and distal colon.
Project description:Eight weeks old male Adrb1tm1BkkAdrb2tm1Bkk/J , stock number 003810, were purchased from The Jackson Laboratory. These mice are homozygous null for the Adrb1 and Adrb2 genes, and are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Mice were euthanized, and the whole bone marrow was extracted using established methods.1 Whole bone marrow cells from Adrb1tm1BkkAdrb2tm1Bkk/J mice were reconstituted into lethally- irradiated (950 Rad) C57BL/6J mice using a single retro-orbital injection at a ratio of 1:4 (Adrb1tm1BkkAdrb2tm1Bkk/J to C57BL/6J). All reconstituted mice were recovered for 2-3 months prior to tissue collection for mRNA arrays (1) The FASEB Journal 2015:29(1),652.13. Eight control animals and eight animals that received new bone marrow from double adrenergic receptor knockout (treated KO chimera). Following recovery, tissues that were harvested included bone marrow, brain, and distal colon.