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Next generation sequencing compares CNS-infiltrating CD4 cells versus draining lymph node effector CD4 cells in experimental autoimmune encephalomyelitis

ABSTRACT: Next generation sequencing compares CNS-infiltrating CD4 cells versus draining lymph node effector CD4 cells in experimental autoimmune encephalomyelitis

PROVIDER: PRJNA793585 | ENA |

REPOSITORIES: ENA

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Next generation sequencing compares CNS-infiltrating CD4 cells versus draining lymph node effector CD4 cells in experimental autoimmune encephalomyelitis

Project description:The immunology has been advanced mostly by studying peripheral blood in human and peripheral immune organ in mouse. However, a long standing problem in develop therapeutics treating autoimmune diseases is that we are short of knowledge about inflammatory-tissue infiltraing CD4 cells, which are terminally differentiated effector that exactly mediate the tissue damage. Thus, to address this issue, we induced experimental autoimmune encephalomyelitis (EAE) in mouse and sorted out both CNS infiltrating CD4 cells and draining lymph node effector CD4 cells at disease onset, and compared their transcriptome

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Transcriptome of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells and control CD4 T cells from draining lymp nodes and spleens of mice during experimental autoimmune encephalomyelitis (EAE)

Project description:MOG-reactive CD4 T cells were isolated from draining lymph nodes (dLN) and spleen of C57BL6/N female mice on day 10 after subcutaneous immunization with MOG(35-55) emulsified in Complete Freund’s adjuvant (CFA). Some mice received the CpG-B-1826 (50 microg; TCCATgACgTTCCTgACgTT; TIB MOLBIOL Syntheselabor GmbH), which was added to the MOG(35-55)/CFA mixture before emulsification. To isolate MOG-reactive CD4 T cells, dLN and spleens were harvested from mice on day 10 post-immunization, and single cell suspensions were re-stimulated for 5 hours using a mixture of MOG(35-55) (20 microg/ml), anti-CD40, anti-CD28, and anti-CD40L-PE (all from a commercial kit – Miltenyi Biotec – cat number 130-093-129). After 5 hours, CD4-positive cells were enriched magnetically by MACS using anti-CD4 microbeads (Miltenyi Biotec GmbH, cat. 130-049-201, clone L3T4), and subsequently subjected to FACS sorting to isolate CD4+CD40L+ and CD4+CD40L- cells after staining with anti-CD4 (clone RM4-5, conjugated to Pacific Blue), including a bin channel for CD8-positive cells (clone 53-6.7) and a marker for staining dead cells (NHPO). Sorted CD4+CD40L+ and CD4+CD40L- cells were then quickly checked for purity and directly lysed in 350 microlitre RLT buffer containing 1% beta-mercaptoethanol (Invitrogen). Purity was >95%. Mice were 6-10 weeks of age at the time of immunization. The goal was to identify genes differentially expressed between MOG-reactive CD4 T cells in mice treated or not with CpG-B, and to characterize the transcriptome of autoreactive CD4 T cells in draining lymph nodes and spleen on day 10 after immunization.

2023-04-26 | GSE205600 | GEO

Quantitative proteome profiling of CNS-infiltrating autoreactive CD4+ cells reveals selective changes during experimental autoimmune encephalomyelitis

Project description:Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4+ lymphocytes into the CNS where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labelling approach to investigate the proteome of CD4+ cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models; PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease and remission phases of disease and of these, 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (Annexin A1), which represent key events during EAE progression were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4+ cell-driven processes that occur during the initiation of relapse and remission stages of disease.

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Project description:Experimental autoimmune encephalomyelitis (EAE)-susceptible DA and EAE-resistant congenic R23 rats were immunized with myelin oligodendrocyte glycoprotein (MOG) to induce an autoimmune response. Seven days later draining inguinal lymph nodes were removed. 2 conditions were examined: 'ex vivo' and 'MOG restimulated', which involved 24hrs of incubation with an encephalogenic MOG 91-108 peptide.

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Comparative genomic hybridization of experimental autoimmune encephalomyelitis (EAE)-susceptible DA and EAE-resistant PVG rat strains stimulated with myelin oligodendrocyte glycoprotein (MOG) to identify the alternative splicing.

Project description:Experimental autoimmune encephalomyelitis (EAE)-susceptible DA and EAE-resistant PVG rats were immunized with myelin oligodendrocyte glycoprotein (MOG) to induce an autoimmune response. Seven days later draining inguinal lymph nodes were removed. 2 conditions were examined: 'ex vivo' and 'MOG restimulated', which involved 24hrs of incubation with an encephalogenic MOG 91-108 peptide.

2009-11-01 | E-MEXP-2237 | biostudies-arrayexpress

Transcriptome of cells from draining lymph nodes of mice during experimental autoimmune encephalomyelitis (EAE)

Project description:Cells were prepared from draining lymph nodes (dLN) from naïve C57BL6/N female mice, as well as from C57BL6/N female mice at day 1 and day 3 after subcutaneous immunization with MOG(35-55) emulsified in Complete Freund’s adjuvant (CFA). Some mice received the CpG-B-1826 (50 microg; TCCATgACgTTCCTgACgTT; TIB MOLBIOL Syntheselabor GmbH), which was added to the MOG(35-55)/CFA mixture before emulsification. Cells from dLN were directly lysed in 350 microlitres RLT buffer containing 1% beta-mercaptoethanol (Invitrogen). Mice were 6-10 weeks of age at the time of immunization. The goal was to identify genes differentially expressed between dLN cells from mice immunized with the classical EAE protocol versus mice immunized with the addition of CpG-B-1826 to the immunization cocktail in order to gain some insights into the immune-modulatory effect of this treatment using an unbiased approach.

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Murine CD4+ T cells: naive vs. experimental autoimmune uveitis

Project description:miRNA expression profiling of CD4+ T cells comparing naïve mice and experimental autoimmune uveitis (EAU) mice. EAU was induced by immunization of retinal antigen (IRBP1-20) in complete Freund’s adjuvant (CFA). CD4+ T cells were isolated and purified from the spleen and draining lymph nodes 13 days after immunization.

2022-09-10 | GSE213034 | GEO

Transcriptomic profiling of choroid plexus in experimental autoimmune encephalomyelitis

Project description:Multiple sclerosis (MS) is a demyelinating disease causing major neurological disability in young adults. We characterized the transcriptome of the choroid plexus (CP), which is part of the blood-brain barriers and the major site of cerebrospinal fluid (CSF) production, in the experimental autoimmune encephalomyelitis mouse model of MS. Genes encoding for adhesion molecules, chemokines and cytokines displayed the most altered expression, supporting the CP as a site of immune-brain interaction in MS. The gene encoding for lipocalin 2 (LCN2) was the most up-regulated, and CSF LCN2 levels coincided with the phases of active disease. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After quality assessment using the Agilent Bioanalyzer (Agilent Technologies, CA, USA), 100ng of total RNA from 3 pooled controls and 3 or 2 pooled samples of each experimental autoimmune encephalomyelitis (EAE) phase were amplified and labelled with the Illumina TotalPrep RNA Amplification Kit (Illumina Inc., San Diego, CA, USA). The labeled cRNA was then hybridized using the recommended protocol in a total of two Illumina Whole-Genome MouseRef-8 expression Beadchips (Illumina Inc., San Diego, CA, USA).

2012-06-29 | E-GEOD-35363 | biostudies-arrayexpress

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