UL49.5 peptide inhibits TAP-dependent presentation of antigen to CD8⁺ T cell clones.

<p>A-B) Murine DCs were incubated with 0.5 mg/ml OVA and the indicated concentrations of the UL49.5-NP. DCs were then co-cultured with OT-I T (A) or OT-II (B) cells and supernatants were analyzed for IL-2 by ELISA. Shown is the mean and standard error of 2 independent experiments. * p = 0.07; ** p = 0.01; *** p = 0.007 (Student's two-tailed t test). C) Human monocyte derived DC were incubated for 1 hr with UL49.5-NP (5 uM) or UL49.5-SCR (5 uM) peptides and then infected with H37Rv-eGFP (MOI = 10) overnight. IFNγ production by the TAP-dependent HLA-E-restricted T cell clone D160 1-23 or the TAP-independent Class II clone D454 E12 was assessed by ELISPOT. IFNγ response by each T cell clone following UL49.5-NP treatment was normalized to the response in the presence of UL49.5-SCR. Shown is the mean response and standard error from at least 4 independent experiments. *** denotes significantly reduced IFNγ production by D160 1–23 in the presence of UL49.5-NP compared to D454 E12 (Student's two-tailed t test, p<0.001).</p

Adenoviral Vectors Stimulate Glucagon Transcription in Human Mesenchymal Stem Cells Expressing Pancreatic Transcription Factors

<div><p>Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising <em>in vitro</em> and <em>in vivo</em>. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.</p> </div

E1-deleted adenoviral vector transduction triggers epigenetic changes in BM-MSC.

<p>(A–B) LV-CMV-GFP or LV-CMV-Pdx-1-modified BM-MSC were treated for 3 days with TSA (1 µM). Pdx-1 and glucagon gene expression was assessed by RT-PCR (A) or qPCR (B). GAPDH was used as reference. (C) ChIP analysis of acetylated histone H4 in the human glucagon gene in BM-MSC modified by adenoviral vector (Ad) or in control non-transduced cells (NT). Position of the primer sets over the glucagon gene are indicated: GCG-207 = glucagon promoter; GCG+297 = Intron 1; GCG+3142 Exon 2. (D) Micrococcal accessibility assay of the glucagon promoter upon adenoviral transduction. DNA from BM-MSC (GFP) or adenovirus-modified BM-MSC (Pdx-1/Ngn-3/Maf-A) was extracted and digested with 1000 mU of micrococcal nuclease. Digested DNA was compared to undigested DNA by qPCR (set to 100%).</p

Genes retained in E1- deleted adenoviral vector affect glucagon gene expression.

<p>(A) LV-CMV-Pdx-1 modified cells were transduced with HAdV-5/fib50-EF1α-DsRed (MOI = 30) or HD-HAdV-5/Fib50. Four days post transduction glucagon gene expression was evaluated by qPCR. GAPDH was used as reference. (B) BM-MSC(GFP) or BM-MSC (Pdx-1) were transduced with HAdV-5/fib50-EF1α-DsRed or LV-CMV-E4-ORF3 (MOI = 2). Four days post transduction glucagon gene expression was evaluated by PCR (C) or qPCR (D). GAPDH was used as reference.</p

BILF1 associates with MHC class I molecules at the cell surface and increases their rate of internalization.

<p>(A) BILF1 is predominantly localized at the cell surface. 293 cells stably transduced with BILF1 retrovirus were grown on glass slides, fixed and permeabilized, then stained with rat anti-HA (3F10) primary antibodies and Alexa Fluor® 594 goat anti-rat IgG. The nuclei were counterstained with DAPI. The stained slides were analyzed with a laser scanning confocal microscope, and the three photographs show different 1 micron-thick sections through representative cells. BILF1 stained red, and the nuclei stained blue. (B) BILF1 and MHC class I molecules co-precipitate at the cell surface. 293 cells (2×10⁶) stably transduced with control (c) or BILF1 (b) retrovirus were incubated with saturating concentrations of antibodies specific for MHC class I (W6/32), TfR (H68.4) or HA tagged BILF1 (3F10) on ice. After washing away excess antibody, the cells were lysed with NP40 detergent buffer, then precipitated with protein A/G beads and subjected to western-blotting as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000255#ppat-1000255-g006" target="_blank">Fig. 6B</a>, using antibodies specific for MHC class I (HC10) and HA tagged BILF1 (3F10). (C) BILF1 increases the rate of internalization of MHC class I, but not class II, from the cell surface. MJS cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32; top graph) or MHC class II (L234; bottom graph), then washed and incubated at 37°C for different periods of time. The cells were subsequently stained with PE-conjugated goat anti-mouse IgG antibody, and analyzed by flow cytometry. The mean fluorescence intensities of staining were averaged for triplicate samples, and normalized to the initial time 0 min samples. (D) BILF1 increases the rate of internalization, but not the rate of appearance, of MHC class I at the cell surface. Top graph: 293 cells stably transduced with control or BILF1 retrovirus were incubated at 0°C with saturating concentrations of mAb to MHC class I (W6/32), then treated exactly as for the internalization assay performed with MJS cells in panel C. Bottom graph: replicate aliquots of the saturated W6/32-bound cells were harvested at the indicated time points, and the appearance of new MHC class I molecules was assayed by staining with PE-conjugated W6/32 antibody. The mean fluorescence intensities of staining were averaged for triplicate samples.</p

Effect of BILF1 on maturation and degradation of MHC class I molecules.

<p>(A) Acquisition of endoglycosidase H (Endo H) -resistance of MHC class I heavy chain. 293 cells (2×10⁶) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min with ³⁵S-methionine/cysteine and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 and treated with Endo H enzyme. Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. (B) Kinetics of MHC class I molecule degradation. 293 cells (2×10⁶) stably transduced with control or BILF1 retrovirus were metabolically labeled for 15 min and chased for the indicated time periods. After lysis in NP-40 detergent buffer, samples were immunoprecipitated with mAb W6/32 (HLA class I heavy chain and β₂-microglobulin) or H68.4 (TfR). Protein samples were separated by 10% SDS/PAGE gel, dried and exposed to autoradiography.</p

Ectopic Pdx-1 expression in combination with HAdV-5/fib50-EF1α-DsRed infection induces glucagon gene expression in bone marrow-derived MSC.

<p>(A) BM-MSC were transduced with LV-CMV-Pdx-1 (MOI = 2), and 4 days post-transduction the modified cells were exposed to HAdV-5/fib50-EF1α-DsRed at an MOI = 30. Glucagon gene expression was evaluated by qPCR and GAPDH was used as reference. (B–C) Adenovirus transduction does not stimulate CMV-promoter driven transgene. BM-MSC were transduced with LV-CMV-GFP and increasing amounts of HAdV-5/fib50-EF1α-DsRed virus (+: MOI = 10; ++: MOI = 30). Percentage of GFP and DsRed positive cells (B) and mean fluorescent intensity (C) were evaluated by FACS (Values are indicated +/− SD). (D) Western blot analysis of LV-CMV-Pdx-1 modified cells transduced with HAdV-5/fib50-EF1α-DsRed (MOI = 30). Pdx-1 expression level was evaluated by western blot using anti-Pdx-1 antibody; anti-actin was used as loading control.</p