Alcohol Facilitates CD1d Loading, Subsequent Activation of NKT Cells, and Reduces the Incidence of Diabetes in NOD Mice

Background: Ethanol ('alcohol') is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. Methods: The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. Results: Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. Conclusion: Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases

Cord-Blood-Stem-Cell-Derived Conventional Dendritic Cells Specifically Originate from CD115-Expressing Precursors

Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime na&#239;ve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34+ stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34+ stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115+-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity

Generation of a cord blood-derived Wilms Tumor 1 dendritic cell vaccine for AML patients treated with allogeneic cord blood transplantation

The poor survival rates of refractory/relapsed acute myeloid leukemia (AML) patients after haematopoietic cell transplantation (HCT) requires the development of additional immune therapeutic strategies. As the elicitation of tumor-antigen specific cytotoxic T lymphocytes (CTLs) is associated with reduced relapses and enhanced survival, enhanced priming of these CTLs using an anti-AML vaccine may result in long-term immunity against AML. Cord blood (CB), as allogeneic HCT source, may provide a unique setting for such post-HCT vaccination, considering its enhanced graft-versus-leukemia (GvL) effects and population of highly responsive naïve T cells. It is our goal to develop a powerful and safe immune therapeutic strategy composed of CB-HCT followed by vaccination with CB CD34(+)-derived dendritic cells (DCs) presenting the oncoprotein Wilms Tumor-1 (WT1), which is expressed in AML-blasts in the majority of patients. Here, we describe the optimization of a clinically applicable DC culture protocol. This two-step protocol consisting of an expansion phase followed by the differentiation toward DCs, enables us to generate sufficient cord blood-derived DCs (CBDCs) in the clinical setting. At the end of the culture, the CBDCs exhibit a mature surface phenotype, are able to migrate, express tumor antigen (WT1) after electroporation with mRNA encoding the full-length WT1 protein, and stimulate WT1-specific T cells

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation

Background: Natural killer T (NKT)cells in adipose tissue (AT)contribute to whole body energy homeostasis. Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes. Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC)as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i)natural killer T (NKT)cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt)5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes

Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p

Patient characteristics (patients included in the SHIP activity assay).

<p>Patient characteristics (patients included in the SHIP activity assay).</p

Aberrant SHIP1 activity and expression in adult CD patients.

<p>(A) PBMCs from CD patients and healthy controls (HC) were lysed, phosphatase assay performed. Examples of 3 of 5 CD patients with abrogated SHIP1 expression are shown. Lower panels show SHIP1, PTEN and actin protein in the same PBMC lysates of CD patients and healthy controls. Phosphatase activity in these patients was tested in duplicate, with identical results. (B) Correction of SHIP1 phosphatase activity for the amount of SHIP1 protein in the lysates shows that intrinsic SHIP1 enzymatic activity is significantly lower in CD patients compared to HC. (C) Increased mRNA and protein (examples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2A Fig</a>) expression compensates for the reduced intrinsic enzymatic activity (CD patients [n = 47] vs healthy controls [n = 42], 1.68 fold increase, p = 0.0042). RNA was available of three of the five SHIP1-deficient patients (open circles). (C) PTEN protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown. (D,E) PTEN (D) and SHIP2 (E) protein expression in PBMC lysates from CD patients and healthy controls (HC) were determined by Western blot analysis, and corrected for Actin levels in the same samples. Quantification of individual experiments, including mean ±SD are shown (examples in Fig 2A lower panel and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182308#pone.0182308.s003" target="_blank">S2B Fig</a>).</p

Specific SHIP1 activity measurement.

<p>(A) Specificity of the assay was determined by immunoprecipitation (IP) of SHIP1 from multiple myeloma cell lines (U266 and OPM2) that express SHIP1 (see Western blot insert of whole cell lysates) and breast cancer cell line (MCF7) that does not. Phosphatase activity of immunoprecipitates was determined by adding the SHIP1 substrate PtdIns(3,4,5)P₃, followed by Malachite Green reaction. Human recombinant SHIP2 was used as positive control for the phosphatase assay. Only immunoprecipitates from SHIP1-competent cells show phosphatase activity. (B) OCRL1, SHIP2 or SHIP1 were immunoprecipitated from SHIP1-competent U266 cells and SHIP1-negative MDA-MB-231 (MDA) breast cancer cells. IPs were run on Western blot and probed for SHIP1, SHIP2 and OCRL1 (upper panel). Whole cell lysates (WCL) were also subjected to Western blot analysis of these phosphatases (lower panels). OCRL1 and SHIP2 were precipitated from both MDA-MB-231 and OPM2 cells, whereas SHIP1 was only observed in OPM2, emphasizing specificity of the IP. (C) Malachite Green phosphatase assay (wells shown in lower panels) shows no activity in SHIP1 precipitates from MDA-MB-231 cells, whereas SHIP1 activity was observed in U266 cells. In contrast, OCRL1 phosphatase activity was detected in both cell lines.</p