TCR-αβ+CD3+CD4?CD8? “double negative” T cells are expanded in the peripheral blood

TCR-αβ+CD3+CD4?CD8? “double negative” T cells are expanded in the peripheral blood of patients with systemic lupus erythematosus (SLE) and lupus-prone mice. of both and genes remain elusive. Rabbit Polyclonal to CAMK5. Here we demonstrate that CREMα orchestrates epigenetic remodeling of the cluster through the recruitment of DNA methyltransferase (DNMT) 3a and histone methyltransferase G9a. Thus we propose that CREMα is essential for the expansion of double negative T cells in SLE. CREMα blockade may have therapeutic value in autoimmune disorders with DN T cell PD153035 expansion. mice. We recently demonstrated that DN T cells in humans and MRL/mice derive from CD8+ T cells by down-regulating CD8 surface-receptor expression (1). The regulation of CD8 has been studied in humans and mice. In both species mature CD4+ and CD8+ T cells derive from CD4?CD8? double negative thymocytes that convert into CD4+CD8+ double positive progenitor cells which later on during their differentiation into mature T cells down-regulate either CD4 or CD8 (2 3 Thymus-derived CD8+ T cells express heterodimers of CD8α and CD8β on their surface whereas gut-derived CD8+ T cells or CD8+ dendritic cells express CD8α homodimers (2 3 We have reported that during the TCR activation-induced transformation of CD8+ T cells into PD153035 peripheral DN T cells the transcription factor cAMP responsive element modulator (CREM) α promoter in human CD8+ T cells. locus which are syntenic with six in the human cluster (2 3 Transgenic reporter systems allowed PD153035 the identification of several enhancer elements within the cluster (E8I-E8IV) (2 3 5 This enhancer network is required for lineage-specific regulation of CD8α and CD8β PD153035 during T cell development and its elements undergo epigenetic remodeling during T cell development either allowing or prohibiting the expression of CD8A and/or CD8B (3). Epigenetic mechanisms regulate gene expression by influencing the accessibility of chromatin to transcription factors and RNA polymerases (16). The addition of methyl groups to the 5′-carbon end of cytidine residues in cytidine-phosphate-guanosine sequences of the genomic DNA and post-translational modifications to the amino terminus of histone proteins represent the two main mechanisms during chromatin remodeling (16). It has been demonstrated that the cluster in mice undergoes epigenetic remodeling during T cell development in the thymus (4). Low degrees of DNA methylation in CD4+CD8+ double positive and PD153035 CD8+ T cells allow the expression of murine and and genes in CD4+ and DN T cells prohibit gene expression (4). In this study we asked whether the cluster undergoes epigenetic remodeling in CD8+ T cells in response to TCR stimulation. We investigated whether the transcription factor CREMα which is induced in response to TCR stimulation and expressed at increased levels in T cells from SLE patients induces chromatin remodeling of the CD8 cluster in response to TCR activation. We demonstrate that CREMα is recruited to several conserved non-coding regions within the human cluster mediating epigenetic silencing of and mice were purchased from The Jackson Laboratory (Bar Harbor ME) and housed in specific pathogen-free conditions. Experimental procedures were approved by the BIDMC Animal Care and Use Committee. Flow Cytometry and Cell Sorting Anti-CD4-PB anti-CD8-PE and anti-CD3-APC/Cy7 were purchased from BioLegend. Samples were acquired on a LSR II flow cytometer (BD Biosciences) and data were analyzed FlowJo version 7.2.2 (Tree Star). For the analysis of T lymphocyte populations a first gate that included live cells was used. CD3+ T lymphocytes were then plotted in a CD4+ CD8+ graphic that allowed the identification of discrete CD4+ CD8+ and double negative T lymphocyte populations. For some experiments stained cells were sorted in a FACSAria flow cytometer (BD Biosciences) post-sorting purity was >98%. Semi-quantitative Real-time Polymerase Chain Reaction Total RNA from control and SLE T lymphocytes was isolated using the Qiagen RNeasy Mini Kit (Qiagen). cDNA was generated using a first strand cDNA synthesis kit (Invitrogen). For gene expression analyses real-time PCR was performed using SYBR.