Søren Skov

Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B.

We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3 by small interfering RNA or by different inhibitors showed that GSK-3 activity is essential for the induced MICA/B expression. We thus present evidence that cancer cells which survive the direct induction of cell death by HDAC inhibitors become targets for NKG2D-expressing cells like NK cells, gammadelta T cells, and CD8 T cells.

IL-2 and IL-15 Regulate CD154 Expression on Activated CD4 T Cells

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.

A maternal gluten-free diet reduces inflammation and diabetes incidence in the offspring of NOD mice

Early-life interventions in the intestinal environment have previously been shown to influence diabetes incidence. We therefore hypothesized that a gluten-free (GF) diet, known to decrease the incidence of type 1 diabetes, would protect against the development of diabetes when fed only during the pregnancy and lactation period. Pregnant nonobese diabetic (NOD) mice were fed a GF or standard diet until all pups were weaned to a standard diet. The early-life GF environment dramatically decreased the incidence of diabetes and insulitis. Gut microbiota analysis by 16S rRNA gene sequencing revealed a pronounced difference between both mothers and their offspring on different diets, characterized by increased numbers of Akkermansia, Proteobacteria, and TM7 in the GF diet group. In addition, pancreatic forkhead box P3 regulatory T cells were increased in GF-fed offspring, as were M2 macrophage gene markers and tight junction-related genes in the gut, while intestinal gene expression of proinflammatory cytokines was reduced. An increased proportion of T cells in the pancreas expressing the mucosal integrin α4β7 suggests that the mechanism involves increased trafficking of gut-primed immune cells to the pancreas. In conclusion, a GF diet during fetal and early postnatal life reduces the incidence of diabetes. The mechanism may involve changes in gut microbiota and shifts to a less proinflammatory immunological milieu in the gut and pancreas.

Mode of Delivery Shapes Gut Colonization Pattern and Modulates Regulatory Immunity in Mice

Delivery mode has been associated with long-term changes in gut microbiota composition and more recently also with changes in the immune system. This has further been suggested to link Cesarean section (C-section) with an increased risk for development of immune-mediated diseases such as type 1 diabetes. In this study, we demonstrate that both C-section and cross-fostering with a genetically distinct strain influence the gut microbiota composition and immune key markers in mice. Gut microbiota profiling by denaturing gradient gel electrophoresis and 454/FLX-based 16S rRNA gene amplicon sequencing revealed that mice born by C-section had a distinct bacterial profile at weaning characterized by higher abundance of Bacteroides and Lachnospiraceae, and less Rikenellaceae and Ruminococcus. No clustering according to delivery method as determined by principal component analysis of denaturing gradient gel electrophoresis profiles was evident in adult mice. However, the adult C-section–born mice had lower proportions of Foxp3+ regulatory T cells, tolerogenic CD103+ dendritic cells, and less Il10 gene expression in mesenteric lymph nodes and spleens. This demonstrates long-term systemic effect on the regulatory immune system that was also evident in NOD mice, a model of type 1 diabetes, born by C-section. However, no effect of delivery mode was seen on diabetes incidence or insulitis development. In conclusion, the first exposure to microorganisms seems to be crucial for the early life gut microbiota and priming of regulatory immune system in mice, and mode of delivery strongly influences this.

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.

Gut microbiota regulates NKG2D ligand expression on intestinal epithelial cells

Intestinal epithelial cells (IECs) are one of a few cell types in the body with constitutive surface expression of natural killer group 2 member D (NKG2D) ligands, although the magnitude of ligand expression by IECs varies. Here, we investigated whether the gut microbiota regulates the NKG2D ligand expression on small IECs. Germ‐free and ampicillin‐treated mice were shown to have a significant increase in NKG2D ligand expression. Interestingly, vancomycin treatment, which propagated the bacterium Akkermansia muciniphila and reduced the level of IFN‐γ and IL‐15 in the intestine, decreased the NKG2D ligand expression on IECs. In addition, a similar increase in A. muciniphila and a decreased NKG2D ligand expression was seen after feeding with dietary xylooligosaccharides. A pronounced increase in NKG2D ligand expression was furthermore observed in IL‐10‐deficient mice. In summary, our results suggest that the constitutive levels of NKG2D ligand expression on IECs are regulated by microbial signaling in the gut and further disfavor the intuitive notion that IEC NKG2D ligand expression is caused by low‐grade immune reaction against commensal bacteria. It is more likely that constitutively high IEC NKG2D ligand expression is kept in check by an intestinal regulatory immune milieu induced by members of the gut microbiota, for example A. muciniphila.

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.

Signal transduction by the major histocompatibility complex class I molecule

Ligation of cell surface major histocompatibility class I (MHC‐I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC‐I‐mediated signaling can lead to both increased and decreased activity of the MHC‐I‐expressing cell depending on the fine specificity of the anti‐MHC‐I antibodies, the context of CD8 ligation, the nature and cell cycle state of the MHC‐I‐expressing cell and the presence or absence of additional cellular or humoral stimulation. This paper reviews the biochemical, physiological and cellular events immediately after and at later intervals following MHC‐I ligation. It is hypothesized that MHC‐I expression, both ontogenically and in evolution, is driven by a cell‐mediated selection pressure advantageous to the MHC‐I‐expressing cell. Accordingly, in addition to their role in T‐cell selection and functioning, MHC‐I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems.

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.

Bi-phasic Effect of Interferon (IFN)-α IFN-α UP- AND DOWN-REGULATES INTERLEUKIN-4 SIGNALING IN HUMAN T CELLS

Interferon (IFN)-α/β is produced by virally infected cells and is believed to play an important role in early phases of the innate immune response. In addition, IFN-α/β inhibits interleukin (IL)-4 signaling in B cells and monocytes, suggesting that IFN-α/β (like IFN-γ) is a Th1 cytokine. Here, we study cross-talk between IFN-α and IL-4 in human T cells. As expected, stimulation with IFN-α for 12–24 h inhibits IL-4 signaling. Surprisingly, however, IFN-α has the opposite effect on IL-4 signaling at earlier time points (up to 6 h). Thus, IFN-α enhances IL-4-mediated STAT6 activation in both CD4+ and CD8+ human T cells. The effect is specific because (i) another interferon, IFN-γ, does not enhance IL-4-mediated STAT6 activation, (ii) IFN-α-mediated STAT1 and STAT2 activation is not modulated by IL-4, and (iii) activation of Janus kinases is not enhanced or prolonged by simultaneous stimulation with IFN-α and IL-4. Moreover, co-stimulation results in a selective increased STAT6/STAT2 association and an association between IFNAR/IL-4R components, suggesting that the IFNAR provides an additional STAT6 docking site via STAT2, leading to a more efficient dimerization/activation of STAT6 only. The co-stimulatory effect on STAT6 activation correlates with a cooperative increase in nuclear translocation, DNA binding, transcriptional activity, and mRNA expression of STAT6 target genes (IL-4Rα and IL-15Rα). In conclusion, we provide evidence that IFN-α both up- and down-regulates IL-4-mediated STAT6 signaling and thereby regulates the sensitivity to IL-4 in human T lymphocytes. Thus, our findings suggest that IFN-α has a complex regulatory role in adaptive immunity, which is different from the “classical” Th1 profile of IFN-γ.