Dose-dependent Toxicity of Panenza on Memory T cells specific for influenza vaccine.

<p>A- PBMC from patients vaccinated with the seasonal flu vaccine (Mutagrip) and the pandemic 2009 H1N1 vaccine (Pandemrix) were collected 21 days after vaccine administration. Cells (5×10⁵ per well) were stimulated for 5 days with Mutagrip or the nonadjuvanted pandemic 2009 H1N1 Panenza vaccine at the indicated concentrations (vaccine concentration is expressed as the final concentration of HA). Cells were also stimulated with CMV or EBV peptides at 0.25 μg/ml, tetanus toxoid (TT) or tuberculin PPD at 5 μg/ml. Cultures were pulsed with 1 μCi per well of [3H] thymidine over the final 16 h of culture and cell proliferation expressed in cpm. All tests were done in triplicate and results show the mean values for three patients. The bars indicate the mean and standard deviation. Asterisks indicate significant <i>P-</i>values (p = 0.03) for comparison of cultures stimulated with Panenza at 0.02 to 0.5 μg/ml to cultures stimulated with 0.01 μg/ml of Panenza (ANOVA test). B- PBMC from Pandemrix vaccinated patients were incubated with soluble Panenza at indicated concentrations, or added to plates coated overnight at 4°C with indicated concentrations of Panenza. H3TdR (1μCi/well) was added at day 4 and T-cell proliferation was assessed at day 5. The bars indicate the mean and standard deviation. Asterisks indicate significant <i>P-</i>values (* <0.05, ** <0.01, ***<0.001) for comparison of cultures stimulated with coated Panenza to cultures stimulated with soluble Panenza. C- PBMC from a patient vaccinated with Mutagrip and then with Pandemrix were incubated overnight with culture medium, Mutagrip or Panenza at 0.25 μg/ml and the percentage of apoptotic cells was determined by flow cytometry combining 7-AAD staining with CD3 and CD4 membrane detection. Dot plots represent the co-expression of CD4 and 7-AAD on gated CD3⁺ T cells. D- PBMC from a patient vaccinated with Mutagrip and Panenza were incubated overnight with either vaccine at various concentrations and the percentage of apoptotic (7-AAD⁺) cells within indicated subsets was determined combining 7-AAD staining with CD19, CD3, CD8, CD4, CD56, CD16 or CD14. B cells were CD3⁻CD19⁺, T cells were CD3⁺CD8⁺ or CD3⁺ CD4⁺, NK cells were CD3⁻CD56⁺CD16⁺, monocytes were CD3⁻CD19⁻CD14⁺.</p

ROS induction by Thimerosal in TCR-activated T cells and anti-apoptotic effect of NAC.

<p>A- PBMC from a HD were stimulated overnight with anti-CD3 mAbs in the presence of thimerosal at 3 μg/ml, and NAC (2.5 μM) was added in half of the cultures. ROS production was detected with HE, ΔY_m was assessed using the DiOC₆(3) probe, and apoptosis was measured with 7-AAD. The dot plots show combined analysis of ΔY_m and apoptosis or ΔY_m and ROS production on gated CD4⁺ T cells, in a representative experiment out of three experiments performed with three different HD. B- PBMC from a HD were either unstimulated (left panel) or stimulated overnight with anti-CD3 mAbs in the presence or absence of thimerosal at 3 μg/ml (right panel). The anti-oxidant NAC (2.5 μM) was added in some cultures. The percentages of CD4⁺ and CD4⁻ T cells with a concomitant drop in ΔY_m and increased oxidizing ability (DIOC₆^high 7-AAD^neg) under the indicated culture conditions are shown. The bars indicate the mean and standard deviation from 3 experiments with three different donors. Asterisks indicate significant <i>P-</i>values (** <0.001, ***0.0001) for comparison of cultures stimulated with thimerosal and NAC to cultures stimulated with thimerosal only.</p

Thimerosal induces cytochrome c release and caspase-3 activation.

<p>A- Confocal analysis of PBMC incubated 16 hours either in the presence of thimerosal (0.9 or 3 μg/ml), or with staurosporine (1 μg/ml) as a positive control. Cells were processed for cytochrome c staining (green) and co-stained with DAPI (blue) to detect nuclei modifications. B-C Western blot analysis of active caspase-3 expression in PBMC incubated for 16 h in medium (unstimulated) (B) or stimulated with anti-CD3 mAbs (C). The impact of indicated concentrations of thimerosal or staurosporine on active caspase-3 expression is shown, together with the influence of the broad caspase inhibitor QVD and the anti-oxidant NAC.</p

Toxicity of thimerosal on TCR-activated T cells through alterations in mitochondrial membrane potential.

<p>A- Left panel: PBMC from a HD were stimulated overnight with anti-CD3/CD28 mAbs in the presence or absence of thimerosal at indicated concentrations. Cell viability was assessed using the MTT assay. Right panel: Kinetics of thimerosal effect on the viability of PBMC from a HD activated overnight with anti-CD3/CD28 mAbs, assessed using the MTT assay. Cultures were done in triplicate and these data are representative of at least three experiments performed with three different donors. The bars indicate the mean and standard deviation. Asterisks indicate significant <i>p-</i>values (** <0.05, ***0.01, ANOVA test) for comparison of cultures stimulated in the presence of thimerosal to cultures stimulated without thimerosal. B- Induction by thimerosal of apoptotic cell death and drop in the mitochondrial transmembrane potential ΔY_m in unstimulated PBMC incubated overnight with the indicated concentrations of the agent. Staurosporine was used as a positive control. Apoptosis was measured with 7-AAD and variations in ΔY_m were assessed using the DiOC₆(3) probe. Dot plots in the upper panel show the dose-dependent apoptotic cell death induced by thimerosal in CD3⁺CD4⁺ and CD3⁺CD4⁻ T cells, and dot plots in the lower panel show the evolution of ΔY_m in association with cell death on gated CD3⁺CD4⁺ T cells. C- Evolution of ΔY_m in association with cell death on gated CD4⁺ T cells following overnight stimulation of PBMC with anti-CD3/CD28 mAbs in the presence of thimerosal or staurosporine. The four transition states (A to D) from living cells with intact ΔY_m (DIOC₆^high 7-AAD^neg) to apoptotic cells (DIOC₆^low 7-AAD^high) are calculated from the dot plots and shown in pie charts. Data of one representative experiment among six are shown.</p

Thimerosal inhibits T-cell proliferation and induces a G0/G1 cell cycle arrest.

<p>A- PBMC from a HD were labeled with CFSE and stimulated during 4 days with anti-CD3/CD28 mAbs in the presence or not of thimerosal. At the end of the culture, cells were co-stained with anti-CD4 mAbs and 7-AAD, and the rate of proliferation in CD4 T cells was determined (upper panel) combined with their priming for apoptosis (lower panel). B- The experiment of panel A was performed with 7 different HD and the bars indicate the mean and standard deviation. The left panel shows the percentage of total proliferating cells, the middle panel shows the proliferation of living cells and the right panel shows the proliferation of dying cells. Asterisks indicate significant <i>P-</i>values (* <0.05, ANOVA test) for comparison of cultures stimulated in the presence of thimerosal at 180 ng/ml to cultures stimulated in the absence of thimerosal. C: Cell cycle analysis of PBMC stimulated with anti-CD3/CD28 mAbs in the presence of thimerosal at three different concentrations. The pie charts show the repartition of cells in the various phases of the cell cycle. D- Curves show the mean values from 7 HD, demonstrating the accumulation of cells in G0/G1 phase and the disappearance of cells in the S phase after exposure to increasing concentrations of thimerosal. Asterisks indicate significant <i>P-</i>values (** <0.01, ***0.001) for comparison of the proportions of cells in G0/G1 or in S phase with or without thimerosal.</p

Impact of thimerosal on cytokine and chemokine release upon TCR ligation.

<p>Pattern of cytokines and chemokines simultaneously quantified by multiplex bead assay arrays in supernatants from freshly isolated PBMC following 16-hour stimulation with anti-CD3/CD28 mAbs in the absence or presence of three different concentrations of thimerosal. The bars indicate the mean and standard deviation from experiments with three different donors. Asterisks indicate significant <i>P-</i>values (* <0.05, ANOVA test) for comparison with cultures without thimerosal.</p