cAMP-responsive element modulator α (CREMα) trans-represses the transmembrane glycoprotein CD8 and contributes to the generation of CD3+CD4-CD8- T cells in health and disease - PubMed (original) (raw)

Clinical Trial

. 2013 Nov 1;288(44):31880-7.

doi: 10.1074/jbc.M113.508655. Epub 2013 Sep 18.

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Clinical Trial

cAMP-responsive element modulator α (CREMα) trans-represses the transmembrane glycoprotein CD8 and contributes to the generation of CD3+CD4-CD8- T cells in health and disease

Christian M Hedrich et al. J Biol Chem. 2013.

Abstract

T cell receptor-αβ(+) CD3(+)CD4(-)CD8(-) "double-negative" T cells are expanded in the peripheral blood of patients with systemic lupus erythematosus and autoimmune lymphoproliferative syndrome. In both disorders, double-negative T cells infiltrate tissues, induce immunoglobulin production, and secrete proinflammatory cytokines. Double-negative T cells derive from CD8(+) T cells through down-regulation of CD8 surface co-receptors. However, the molecular mechanisms orchestrating this process remain unclear. Here, we demonstrate that the transcription factor cAMP-responsive element modulator α (CREMα), which is expressed at increased levels in T cells from systemic lupus erythematosus patients, contributes to transcriptional silencing of CD8A and CD8B. We provide the first evidence that CREMα trans-represses a regulatory element 5' of the CD8B gene. Therefore, CREMα represents a promising candidate in the search for biomarkers and treatment options in diseases in which double-negative T cells contribute to the pathogenesis.

Keywords: Autoimmune Diseases; CD8; CREMα; Cell Differentiation; Cellular Immune Response; Double-negative T Cells; Gene Regulation; SLE; T Cell.

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Figures

FIGURE 1.

FIGURE 1.

Human CD8 protein and mRNA expression in response to TCR stimulation. A, primary human CD8+ T lymphocytes from healthy individuals were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 120 h. Most stimulated CD8 T cells were alive as shown by their forward (FSC) and side (SSC) scatter distribution (left panel) and expressed CD3 (middle panel). A large proportion of CD8+ T lymphocytes down-regulated CD8 and transformed into DN T cells (right panel). B, TCR stimulation with anti-CD3 and anti-CD28 antibodies for 120 h significantly decreased CD8A and CD8B mRNA expression in primary human CD8+ T lymphocytes from healthy individuals. Values are means ± S.D. NS, non-stimulated T cells; ST, TCR-stimulated T cells. C, kinetics of CD8A and CD8B mRNA expression in response to TCR stimulation with anti-CD3 and anti-CD28 antibodies over 120 h in primary human CD8+ T lymphocytes from healthy individuals. Values indicate -fold changes over the relative gene expression in unstimulated T cells, which were assigned a relative expression of 1. NS indicates time points at which TCR stimulation did not result in significant changes. p values indicate statistically significant changes in gene expression after 48 and 72 h (CD8A) or 96 and 120 h (CD8A and CD8B).

FIGURE 2.

FIGURE 2.

CD8 expression in MRL/lpr mice is regulated at the transcriptional level. A, CD8a and CD8b mRNAs were differentially expressed in CD4+, CD8+, and DN T lymphocytes sorted from 14-week-old MRL/lpr mice. B and C, CD8+ T cells from MRL/lpr mice down-regulated CD8 surface receptors in response to TCR stimulation with anti-CD3 and anti-CD28 antibodies. This was more pronounced in 14-week-old symptomatic animals compared with younger animals (6 week old) before the onset of symptoms. Also, a subset of CD4+ T cells down-regulated CD4 in response to stimulation. However, the CD4-to-DN conversion rate was significantly lower than the CD8-to-DN conversion rate. Values are means ± S.D. D, CD4+ and CD8+ T cells from 6-week-old “healthy” MRL/lpr mice expressed reduced levels of CREMα compared with 14-week-old diseased animals. CD8+ T cells expressed more CREMα than CD4+ T cells from the same mouse, reflecting their capacity to transform into DN T lymphocytes in response to TCR stimulation.

FIGURE 3.

FIGURE 3.

CREMα governs CD8 expression. A, CREMα enhanced the generation of DN T cells from primary human CD8+ T cells from healthy individuals (120 h). CD8− T cells were increased in response to CREMα compared with controls (empty vector (EV)). CREMα increased the relative numbers of DN T cells (left panel) by down-regulating CD8 surface expression on individual T cells (right panel). MFI, mean fluorescence intensity. B, CREMα negatively regulated CD8A and CD8B mRNA expression in primary human CD8+ T cells compared controls (24 h). C, CREM knockdown resulted in increased CD8A and CD8B expression in primary human CD8+ T cells compared with cells transfected with scrambled siRNA (24 h).

FIGURE 4.

FIGURE 4.

_trans_-Regulation of the CD8 cluster. A, to investigate _trans_- and _cis_-regulatory elements across the human and murine CD8 locus, we defined regions of interest based on bioinformatic approaches. We aligned the mouse and human CD8 genes (VISTA Genome Browser) and searched for CNS sites. CNS sites were defined as regions of >200 bp with >70% homology between human and mouse. Eight regions of interest (CNS1–CNS8) were defined based on the degree of sequence conservation and the presence of reported regulatory regions. C II–C IV are previously reported DNase hypersensitivity clusters with regulatory capacities; E8I–E8IV are previously defined enhancer elements (6). B, reporter constructs of CNS regions within the human CD8 cluster were generated and transfected into Jurkat T cells. CNS2 is syntenic to the murine CD8b promoter, and CNS7 and CNS8 are within previously reported regulatory elements. All constructs exhibited enhanced activity over an empty pGL3 plasmid (empty vector (EV)). C, CREMα reduced the activity of CNS2, whereas CREB enhanced promoter activity. Both CREMα and CREB did not show significant effects on either CNS7 or CNS8.

FIGURE 5.

FIGURE 5.

CREMα represses the activity of CNS2 through binding to CRE motifs. A, CNS2, which is _trans_-regulated by CREB and CREMα, harbors two CREs that bind CREB or CREMα. Displayed are the consensus sequence of the palindromic CRE site (upper) and two putative CRE sites within CNS2 (lower). B, deletion of either of the two CRE sites within CNS2 resulted in reduced promoter activity and abrogated CREMα effects after transfection into Jurkat T cells. C, CREMα and phospho-CREB (p-CREB) competed for the recruitment to CNS2 as assessed by ChIP. CD8+ T cells from healthy donors exhibited mostly phospho-CREB and almost no CREMα recruitment to CNS2 (bars 1 and 3). In response to TCR stimulation with anti-CD3 and anti-CD28 antibodies, phospho-CREB was partially replaced with CREMα (bars 2 and 4). D, in stimulated CD8+ T cells from SLE patients, CREMα recruitment to CNS2 was even more pronounced (bars 1 and 2). Phospho-CREB was almost completely replaced with CREMα in SLE T cells. NS, non-stimulated cells; ST, T cells after TCR stimulation with anti-CD3 and anti-CD28 antibodies for 120 h; rel., relative; EV, empty vector.

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