DNA replication and virus release of hexon-modified Ad vector in pancreatic cells.

<p>1 x 10⁶ hPSCs or 2.5x10⁶ A549, Panc1 or UlaPaCa cells were seeded. On the next day, replicating Ad vectors (AdhWt or AdhCKS17) were added at an infectious MOI of 20. Six hours post infection cells were washed with PBS to remove unbound virus, and fresh medium was added to the cells. The supernatants and the cell lysates were collected during the course of infection (at 48 and 72 hours post infection). (<b>A</b>) To determine the number of physical particles found in the supernatant at the indicated time points, viral DNA was isolated and subjected to quantitative PCR to analyse the Ad genome number. (<b>B</b>) The numbers of infectious particles per cell (viral release) within the supernatants or the cell lysates (indicated by “-”or “+”) were assessed by plaque assay. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, n = 2.</p

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220⁺ Double-Negative T Lymphocytes of Autoimmune MRL/<em>lpr</em> Mice

<div><p>Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including <em>Fas Ligand</em> (FasL) and <em>P2X7 receptor</em> (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/<em>lpr</em> mice (<em>lpr</em> mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/<em>lpr</em> mice carry a <em>P2X7R</em> allele, which confers a high sensitivity to ATP. However, during aging, the MRL/<em>lpr</em> T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.</p> </div

ATP-induced shedding of CD62L in CD4⁺, CD8⁺ and DN T lymphocytes expressing the transmembrane tyrosine phosphatase B220.

<p>Spleen cells from 3 to 5-mo-old MRL<i>^+/+</i>, MRL<i>/lpr</i>, B6 and B6/<i>lpr</i> mice (n = 5 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) for 45 min at 37°C. Spleen cells were then stained with anti-CD90, anti-B220, anti-CD62L and anti-CD4 or anti-CD8 mAb to assess CD62L expression on B220<i>^–</i> and B220⁺ CD90⁺ T cells (either CD4⁺ or CD8⁺) from MRL<i>^+/+</i>, MRL<i>/lpr</i>, B6 and B6/<i>lpr</i> mice as well as on B220⁺ DN CD90⁺ T cells from MRL<i>/lpr</i> and B6/<i>lpr</i> mice. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Cellular uptake inhibition experiments with soluble fiber knob protein and TGFBRII-specific antibody.

<p>(<b>A</b>) Lysates from pancreatic hPSCs and tumor cells were analyzed by SDS-PAGE and immunoblotting. TGFBRII was detected by an anti-TGFBRII-specific polyclonal antibody from rabbit (sc-1700). A549 cells were pre-incubated with soluble fiber knob protein, TGFBRII-specific antibody or rabbit serum (serving as isotype control), respectively, and transduced with the replication-deficient vectors AdGFPhCKS17 and AdGFPhWt (control) at a particle MOI of 100. After two hours the medium was replaced with fresh medium and incubated for additional 2 and 24 hours to (<b>B</b>) analyze GFP expression by flow cytometry or to (<b>C</b>) determine relative Ad genome levels by qPCR using total DNA isolated from cells. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, n = 3.</p

Dose–response experiments for ATP-induced CD62L shedding on T lymphocytes.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> (<i>solid line</i>), MRL/<i>lpr</i> (<i>dashed line</i>) and P2X7-deficient B6<i>^P2X7R−/−</i> (<i>dotted line</i>) mice (n = 3 mice/group) were treated for 45 min at 37°C with doses of ATP ranging from 100 to 5000 µM. Spleen cells were then triple-stained with anti-CD90, anti-CD19 and anti-CD62L mAb to assess by flow cytometry: (A) the percentage of CD62L-expressing CD19^–CD90⁺ T cells and (B) MFI of CD62L on CD19^–CD90⁺ T cells. Results are expressed as the mean percentage of initial expression ± SE.</p

P2X7R antagonist KN-62 inhibits ATP-induced CD62L shedding and pore formation in MRL^+/+ T lymphocytes.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> mice (n = 4 mice/group) were preincubated for 15 min at 37°C with or without 0.5, 1 and 5 µM KN-62 and then stimulated with 500 µM ATP for 30 min. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L or YO-PRO-1 to assess by flow cytometry: (A) CD62L shedding or (B) pore formation in the gated CD90⁺ T-cell population stimulated (▪) or not (□) with ATP. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Influence of hexon modification on biodistribution.

<p>For Kupffer cell depletion, 200 μl of clodronate liposomes were injected into the tail vein of BALB/c mice. After 24 hours, 3x10¹⁰ viral particles of AdGFPhWt or AdGFPhCKS17 were injected intravenously into the tail vein of mice (n = 4 or 5), and organs were collected 45 minutes or 72 hours later. (<b>A</b>) Relative Ad genome levels were determined from total DNA isolated from liver obtained 45 minutes or 72 hours after infection. (<b>B</b>) GFP expression was measured in liver lysates obtained 72 hours after infection by fluorimetry. (<b>C</b>) Analysis of relative Ad genome levels from total DNA isolated from spleen obtained 45 minutes or 72 hours after infection. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.005, n = 4–5.</p

Metalloprotease inhibitors GM6001 and Ro 31–9790 inhibit PMA/Iono- and ATP-induced shedding of CD62L on MRL^+/+ T lymphocytes.

<p>Spleen cells from MRL<i>^+/+</i> and MRL/<i>lpr</i> mice (n = 5 mice/group) were preincubated with or without 10, 25, 50, 100 µM GM6001 or 3, 30, 100 µM Ro 31–9790 for 15 min at 37°C, and then either left unstimulated (□) or stimulated with 500 µM ATP (▴) or 5 ng/ml PMA plus 0.5 µg/ml Ionomycin (○) for 45 min at 37°C. Cells were subsequently stained with anti-CD90 and anti-CD62L mAb. Cell surface expression of CD62L was assessed in the gated CD90⁺ T cells by flow cytometry.</p

Limitations of Ad vector-mediated transduction of early passage pancreatic cancer cell lines and primary hPSCs.

<p>(<b>A</b>) 2x10⁴ hPSCs or 2x10⁵ tumor cells were transduced with ΔE1 Ad1stGFP at a MOI of 5. To determine the Ad transduction rate the cells were subjected both to flow cytometry for detection of GFP expression (24 h.p.i.) expressed as the percentage of GFP-positive cells and to slot blot analysis to determine their relative Ad genome content (2 h.p.i.). (<b>B</b>) CAR levels on different cell lines and hPSCs were determined by flow cytometry as detailed in the Materials and Methods section. To analyze Ad replication rates and production of progeny virions in different cells, respectively, wildtype Ad5 (Ad5Wt) was used for infection of different cell lines at a slot blot adjusted actual MOI of 1. (<b>C</b>) Cells were harvested 2 and 48 hours p.i., genomic DNA was isolated and subjected to slot blot analysis using an Ad5 fiber-specific probe. Ad replication is expressed as the ratio of Ad genomes 48 h.p.i./ 2 h.p.i. in comparison to A549 cells (set to 100). (<b>D</b>) Forty-eight hours after infection cells harvested, lysed by repeated freezing and thawing, and treated with Benzonase. Two and 10 microliters of the lysate were used to reinfect A549 cells. The number of infectious particles was determined by DNA slot blot analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117254#pone.0117254.ref040" target="_blank">40</a>] using a Ad5 fiber-specific probe and the number of infectious particles per tumor cell was calculated. * P < 0.05, ** P < 0.01 *** P < 0.005, n = 2–4.</p

Real-time kinetic analysis of calcium influx in B220^– and B220⁺ T lymphocytes stimulated with ATP.

<p>Spleen cells from 4-mo-old MRL<i>^+/+</i> (<i>red</i>) MRL/<i>lpr</i> (<i>blue</i>) and B6<i>^P2X7−/−</i> (<i>black</i>) mice (n = 3 mice/group) were loaded with calcium-reactive fluorescence probe, Oregon Green 488 BAPTA-1. After loading, cells were stained with anti-CD90, anti-CD19 and anti-B220 mAbs. Baseline MFI of the calcium probe was recorded in the gated B220^–CD90⁺ (○) and B220⁺CD90⁺ (▵) T cells during 50 s by flow cytometry, and was followed by the addition of 500 µM ATP (at time t0, arrow). Changes over time in MFI values of the calcium probe were monitored in the T-cell subsets by flow cytometry. MFI values were normalized by subtracting the baseline MFI.</p