Anti-tumor effect of immunizations in C57BL/6 mice bearing subcutaneous tumors.

<p>(a) Mice were injected with E.G7-OVA cells on day 0 and then immunized on days 4 and 18 (arrows) with [pOVA30-melanin + CpG], [melanin + CpG], [pOVA30 + CpG], or [pOVA30-melanin] (n = 10 mice/group with pooled data from two different experiments). (b) Effect of the in vivo CD8 depletion in C57BL/6 mice bearing subcutaneous E.G7-OVA tumors and immunized on days 4 and 18 (arrows) with [melanin + CpG] (n = 4 mice) or [pOVA30-melanin + CpG] (n = 8 mice). The latter group received either anti-CD8 or an isotype-matched control mAb. Tumor growth was assessed twice a week by measuring the size of tumors with calipers. Results are expressed as the mean ± SEM of tumor volumes. * p < 0.05; ** p < 0.001, when compared with control groups.</p

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8⁺ T-cell responses

<div><p>Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8–35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8⁺ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.</p></div

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

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

Phenotype of SIINFELKL-specific T-cells in mice immunized on days 0 and 14 with [pOVA30 + CpG] or [pOVA30-melanin + CpG].

<p>(a) gating strategy and representative results (in one mouse) for T-Bet, CD62L and granzyme expression. (b) mean ± SD expression of T-bet, CD62L and granzyme within the CD8⁺dextramer⁺ population (n = 8 mice/group, with pooled data from two different experiments of 4 mice each). ** p<0,01; ***p<0,001.</p

Preparation and characterization of synthetic melanin and peptide vaccine formulations.

<p>(a) Schematic of the chemical process for obtaining water-soluble peptide-melanin complexes. Evolution over time of b) the UV-visible spectrum during gp100-melanin synthesis and c) the absorbance ratio at 350 and 280 nm (A350/A280) for melanin (black squares) and for gp100-melanin (red circles). (d): TEM image of gp100-melanin after 18 h of incubation time (inset: high magnification). (e) SDS-PAGE showing the migration of the unbound gp100 peptide within the resolving gel after different incubation times in the presence of oxidizing L-Dopa leading to gp100-melanin formation (following electrophoresis, the gels were stained with Coomassie Blue). (f) FTIR spectra in deuterated solution for melanin (black line) and gp100-melanin (red line); the amide II' band at 1455 cm-1 (blue hatching) is characteristic of peptides.</p

Distribution of melanin in draining lymph nodes.

<p>Macroscopic aspect of the draining inguinal lymph nodes (arrows) of BALB/c mice 2 days after injections with gp100-melanin (a) or saline (b). Fontana-Masson staining of a draining lymph node 2 days after injection with gp100-melanin, showing melanin-laden macrophages in the sinuses (c) and in the paracortical area (d).</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

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

T-cell response after subcutaneous immunizations in C57BL/6 mice.

<p>Mice were immunized with pOVA35 or pOVA35-melanin (pOVA35-Mel), on days 0 and 14 and sacrificed on day 21. Splenocytes were re-stimulated in vitro either with a) the MHC class II epitope (CD4) or b) the MHC class I-epitope (CD8) (non conjugated to melanin). The numbers of IFNg-SFCs (Spot forming cells) were measured. Each point represents an individual mouse (n = 8 mice/group with pooled data from 2 different experiments of 4 mice each). Bars = median. ** p < 0.01 (Mann–Whitney test).</p