Helle Jensen

Molecular Regulation of MHC Class I Chain-Related Protein A Expression after HDAC-Inhibitor Treatment of Jurkat T Cells

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-κB activity can be induced by HDAC treatment. However, nuclear translocation of NF-κB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of ∼3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position −113 to −93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.

2-Deoxy d-Glucose Prevents Cell Surface Expression of NKG2D Ligands through Inhibition of N-Linked Glycosylation

NKG2D ligand surface expression is important for immune recognition of stressed and neotransformed cells. In this study, we show that surface expression of MICA/B and other NKG2D ligands is dependent on N-linked glycosylation. The inhibitor of glycolysis and N-linked glycosylation, 2-deoxy-d-glucose (2DG), potently inhibited surface expression of MICA/B after histone deacetylase inhibitor treatment; the inhibition occurred posttranscriptionally without affecting MICA promoter activity. Transient overexpression of MICA surface expression was also inhibited by 2DG. 2DG blocks N-linked glycosylation of MICA/B by a reversible mechanism that can be alleviated by addition of d-mannose; this does not, however, affect the inhibition of glycolysis. Addition of d-mannose restored MICA/B surface expression after 2DG treatment. In addition, specific pharmacological or small interfering RNA-mediated targeting of glycolytic enzymes did not affect MICA/B surface expression, strongly suggesting that N-linked glycosylation, and not glycolysis, is essential for MICA/B surface expression. Corroborating this, tunicamycin, a selective inhibitor of N-linked glycosylation, abolished MICA/B surface expression without compromising activation of MICA promoter activity. NK cell-mediated killing assay and staining with a recombinant NKG2D–Fc fusion protein showed that all functional NKG2D ligands induced by histone deacetylase inhibitor treatment were abolished by 2DG treatment and fully reconstituted by further addition of d-mannose. Our data suggest that posttranslational N-linked glycosylation is strictly required for NKG2D ligand surface expression. Cancer and infection often result in aberrant glycosylation, which could likely be involved in modulation of NKG2D ligand expression. Our data further imply that chemotherapeutic use of 2DG may restrict NKG2D ligand surface expression and inhibit secretion of immunoinhibitory soluble NKG2D ligands.

Propionic Acid Secreted from Propionibacteria Induces NKG2D Ligand Expression on Human-Activated T Lymphocytes and Cancer Cells

We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could directly stimulate functional MICA/B surface expression and MICA promoter activity by a mechanism dependent on intracellular calcium. Deletion and point mutations further demonstrated that a GC-box motif around −110 from the MICA transcription start site is essential for propionate-mediated MICA promoter activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate and CMV. In contrast, compounds with histone deacetylase-inhibitory activity such as butyrate and FR901228 stimulated MICA/B expression through a pathway that was not affected by inhibition of glycolysis, clearly suggesting that MICA/B is regulated through different molecular mechanisms. We propose that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic.

Vesicular Stomatitis Virus Infection Promotes Immune Evasion by Preventing NKG2D-Ligand Surface Expression

Vesicular stomatitis virus (VSV) has recently gained attention for its oncolytic ability in cancer treatment. Initially, we hypothesized that VSV infection could increase immune recognition of cancer cells through induction of the immune stimulatory NKG2D-ligands. Here we show that VSV infection leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV) and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused an active suppression of NKG2D-ligand surface expression, affecting both endogenous and histone deacetylase (HDAC)-inhibitor induced MICA, MICB and ULBP-2 expression. The classical immune escape mechanism of VSV (i.e., the M protein blockade of nucleocytoplasmic mRNA transport) was not involved, as the VSV mutant strain, VSVΔM51, which possess a defective M protein, prevented MICA surface expression similarly to wild-type VSV. The VSV mediated down modulation of NKG2D-ligand expression did not involve apoptosis. Constitutive expression of MICA bypassed the escape mechanism, suggesting that VSV affect NKG2D-ligand expression at an early post-transcriptional level. Our results show that VSV possess an escape mechanism, which could affect the immune recognition of VSV infected cancer cells. This may also have implications for immune recognition of cancer cells after combined treatment with VSV and chemotherapeutic drugs.

N-glycosylation of asparagine 8 regulates surface expression of major histocompatibility complex class I chain-related protein A (MICA) alleles dependent on threonine 24.

Background: Immune activation through surface expression of the human NKG2D ligand MICA is important in clearance of virus-infected or cancerous cells. Results: Molecular characterization of N-glycosylation in regulation of MICA cell surface expression. Conclusion: Surface expression of MICA alleles vary in dependence for N-glycosylation. Significance: We identify N-glycosylation as an allele-specific regulatory mechanism of MICA and pinpoint the essential residues. NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.

BEYOND "MEDIA PANICS": Reconceptualising public debates about children and media

This paper presents a critical review of the use of “moral panics” theory as a means of understanding public debates about children and media, and specifically of the notion of “media panics.” Following a brief presentation of the origins and key aspects of media panics theory, it focuses on six key issues that are at stake. It argues that there are some problems with the epistemology of media panics theory, with its account of intentionality, and with its claim to rationality. It further suggests that media panics theory tends to provide a “presentist” view of history, and a rather oversimplified account of cultural and generational politics; and that it neglects the positive positions that are also often rehearsed in public debate. The final section of the article provides brief discussions of two approaches that might provide productive alternatives to media panics theory: social constructionism and new cultural history.

Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

We show that inhibition of HDAC activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC inhibitor‐mediated Hsp70 surface expression was confined to the apoptotic Annexin V‐positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis such as etoposide and camptothecin also led to a robust induction of Hsp70 surface expression. Hsp70 expression was, however, not caused by induction of apoptosis per se, as activated CD4 T cells remained Hsp70 surface‐negative despite effective induction of apoptosis. Interestingly, inhibition of endolysosomes or normal ER/Golgi transport did not affect Hsp70 surface expression. Intracellular calcium and the transcription factor Sp1, which has been shown previously to be important for the intracellular stress mediated by HDAC inhibitors, were not involved in Hsp70 surface expression. We also found that HDAC inhibitors decreased cellular PMET activity and that a selective inhibition of PMET activity with extracellular NADH induced a robust Hsp70 surface expression. Our data suggest that inhibition of HDAC activity selectively induces surface expression of Hsp70 on hematopoietic cancer cells and that this may increase immunorecognition of these cells.

Regulation of NKG2D-ligand cell surface expression by intracellular calcium after HDAC-inhibitor treatment.

In this study we demonstrate that histone deacetylase (HDAC)-inhibitor mediated cell surface expression of the structural different NKG2D-ligands MICA/B and ULBP2 is calcium-dependent. Treatment with the calcium chelator EGTA inhibited constitutive as well as HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression on melanoma cells and Jurkat T-cells. A NKG2D-dependent cytolytic assay and staining with a recombinant NKG2D-Fc fusion protein showed that calcium chelation impaired the functional ability of NKG2D-ligands induced by HDAC-inhibitor treatment. The HDAC-inhibitor induced cell surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T-cells. We further show that secretion and cell surface binding of the calcium-regulating protein galectin-1 is enhanced upon HDAC-inhibitor treatment of melanoma cells. However, binding of galectin-1 to cell surface glycoproteins was not critical for constitutive or HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression. We provide evidence that MICA/B and ULBP2 cell surface expression is controlled differently by calcium, which adds to the increasing perception that cell surface expression of MICA/B and ULBP2 is controlled by distinct signal transduction pathways.

TV as Children's Spokesman: Conflicting notions of Children and Childhood in Danish Children's Television around 1968

“We are not the teachers’ spokesmen, but the children’s. This is also established in the guidelines which the Board of Directors has made for our department: ‘the producers should try to step into the children’s shoes, take their problems seriously, and suggest solutions if such are available . . . The Board finds it reasonable that children make themselves heard—even though their criticism of the milieu they live in might sometimes be hasty, [and only] sometimes right.’”2

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression.