Comparison of the gene expression patterns between donors and treatments.
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The image depicts the log of the fold change of NK genes regulated due to co-culture with 3D7-infected erythrocytes (iRBCs) or with the cytokines IL-12 andIL-18.
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NK92/iRBCs co-culture and flow cytometry
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NK92 cells were kept in two different environments for 24 hours prior to the co-culture: in normal cell medium'>medium medium'>(+rIL2; NK92 nm) and in cell medium'>medium without rIL-2 (starvation medium'>medium; NK92s).
- Cells from both environments were co-cultured with 3D7 schizont-infected erythrocytes and uRBCs (NK92-RBCs ratio: 1:3) in their respective growth medium.
- As a positive control, cells were also incubated with a mixture of IL-12 and IL-18 (and MBL, respectively; 100 ng/106 cells each).
- After the indicated time of incubation at 37°C and 5% CO2, NK cells from the co-culture as well as cells incubated without RBCs were stained for 30 min at 4°C with fluorochrome-conjugated antibodies for surface CD56 (APC), CD3 (PE), CD16 (FITC), CD69 (FITC), CD25 (PE) in parallel with the appropriate isotype controls.
- Cells were also internally stained with the IFN-γ (PE) antibody (all…
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NK92 cells were kept in two different environments for 24 hours prior to the co-culture: in normal cell medium'>medium medium'>(+rIL2; NK92 nm) and in cell medium'>medium without rIL-2 (starvation medium'>medium; NK92s).
- Cells from both environments were co-cultured with 3D7 schizont-infected erythrocytes and uRBCs (NK92-RBCs ratio: 1:3) in their respective growth medium.
- As a positive control, cells were also incubated with a mixture of IL-12 and IL-18 (and MBL, respectively; 100 ng/106 cells each).
- After the indicated time of incubation at 37°C and 5% CO2, NK cells from the co-culture as well as cells incubated without RBCs were stained for 30 min at 4°C with fluorochrome-conjugated antibodies for surface CD56 (APC), CD3 (PE), CD16 (FITC), CD69 (FITC), CD25 (PE) in parallel with the appropriate isotype controls.
- Cells were also internally stained with the IFN-γ (PE) antibody (all ).
- Dead cells were excluded from the analysis based on scatter signals and 7AAD fluorescence.
- Acquisition of samples was carried out in a FACS canto flow cytometer .
- Data were analysed with BD FACS Diva 6.0 software.
- Gates were set on the events compatible to lymphocytes regarding "size of the cells" × "internal complexity" .
- A total of 10.000 events were collected for each sample.
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In vitro Induction of Allogeneic Th1 and Th2 cell Lines
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Purified human naïve CD4 T cells were stimulated with irradiated (100Gy) allogeneic Epstein-Barr virus (EBV) – transformed B cells (1∶1 ratio) in complete medium'>RPMI-8 medium at 105 cells/mL in round-bottom 96-well plate.
- Th1-biased cultures contained recombinant human IL-2 (5 ng/mL), recombinant human IL-12 (20 ng/mL) and anti-IL-4 (5 µg/ml& ).
- Th2-biased cultures contained recombinant human IL-2 (5 ng/mL), recombinant human IL-4 (20 ng/mL& ), anti-IL-12 (5 µg/ml, ebioscience) and anti-IFNγ (5 µg/ml& ).
- Fresh medium containing 5 ng/mL IL-2 was added if necessary to cultures showing strong proliferation.
- The cultures were restimulated and expanded every seven days.
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IFN-λ treatment-induced inhibitory DCs phenotype is dependent on IL-10 and PD-1/PD-L1 ligand.
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Co-culture supernatants from indicated groups were analyzed for IL-2 , IL-12 by ELISA; mean±SD pg/ml shown.
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CTL Induction
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Naïve CD8+ T cells were negatively isolated from PBMCs of A24-positive donors using the CD8+ T cell isolation kit II and anti-CD45RO microbeads .
- The isolated cells were more than 95% pure CD45RO− CD8+ populations.
- These CD8+ T cells (1×106 cells/well) were cocultured with irradiated (100 Gy) aAPCs (1×105 cells/well) in 2 mL X-VIVO20 supplemented with 5% human AB serum (MP, , ) in wells of a 12-well plate in the presence of 10 ng/mL IL-12 .
- On day 3, 10 ng/mL IL-7 and IL-15 were added.
- Every 3 days, half the medium was exchanged for fresh medium containing 10 ng/mL IL-15.
- On day 12, the T cells were restimulated with γ-irradiated aAPCs.
- One day thereafter, IL-2 was added, to a final concentration of 20 U/mL.
- celltype'>To establish celltype'>T cell clones, a limiting dilution of the polyclonal Ccelltype'>TL was performed as…
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Naïve CD8+ T cells were negatively isolated from PBMCs of A24-positive donors using the CD8+ T cell isolation kit II and anti-CD45RO microbeads .
- The isolated cells were more than 95% pure CD45RO− CD8+ populations.
- These CD8+ T cells (1×106 cells/well) were cocultured with irradiated (100 Gy) aAPCs (1×105 cells/well) in 2 mL X-VIVO20 supplemented with 5% human AB serum (MP, , ) in wells of a 12-well plate in the presence of 10 ng/mL IL-12 .
- On day 3, 10 ng/mL IL-7 and IL-15 were added.
- Every 3 days, half the medium was exchanged for fresh medium containing 10 ng/mL IL-15.
- On day 12, the T cells were restimulated with γ-irradiated aAPCs.
- One day thereafter, IL-2 was added, to a final concentration of 20 U/mL.
- celltype'>To establish celltype'>T cell clones, a limiting dilution of the polyclonal Ccelltype'>TL was performed as previously described
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IL-21 secretion is increased from CD4+ T cells from aged subjects A, B, C Bar graph depicts the levels of IL-21 (A), IFN-γ (B), IL-17 (C) in the supernatant from aged and young total CD4+ T cells after stimulation with IL-12 for 5 days.
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D, F. Bar graph depicts the levels of IL-21 secreted from aged and young, Naïve CD4+ T cells and memory CD4+ T cells after stimulation with IL-12 for 5 days.
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Vaccination and tumour challenge
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At 6 weeks HIS mice were distributed into two groups with matched levels of CD45+ human circulating cells.
- A group was subjected to two cycles of anti-HER-2 cell vaccination according to a schedule previously reported highly effective in HER-2/neu transgenic models (6 mitomycin-treated cells (to block proliferation) in 0.4 ml PBS.
- In the third week mice received five times daily administration of recombinant human IL-12 in 0.2 ml PBS supplemented with 0.01% mouse serum albumin .
- The dose of IL-12 was 50 ng per mouse per day in the first 4-week cycle and 100 ng per mouse per day in all subsequent cycles.
- The fourth-week mice received no treatment.
- The 4-week vaccination cycle was repeated twice.
- Non-vaccinated group received the injection of PBS or PBS supplemented with mouse serum albumin.
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Blood samples, cells and cell cultures
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Peripheral blood mononuclear cells (PBMCs) were prepared on a Ficoll-Paque density gradient by centrifugation (800 g, 30 min at room temperature), washed twice and frozen in RPMI 1640-FCS (5%)-DMSO (8.7%)-methyl-cellulose (0.1%).
- NK cells were selected from PBMCs by magnetic cell sorting using indirect NK isolation kit according to manufacturer's recommendations.
- Average celltype'>NK cell purity checked by flow cytometry (CD3−/CD16+/−/CD56+/−) was 97.51% ± 2.47 (standard deviation) across all donors.
- NK cells were cultured in complete RPMI 1640 medium, including 1 mM sodium pyruvate, and 1% heat-inactivated fetal calf serum.
- Recombinant human IL-2 and IL-12 were obtained from .
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Establishment of antigen specific T-cell cultures and clones
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Peripheral blood lymphocytes (PBLs) isolated from a melanoma patient were stimulated with irradiated (25 Gy) autologous IκB10-loaded DCs (PBL:DC ratio = 3 × 106:3 × 105).
- The following day IL-7 (5 ng/mL) and IL-12 (10 ng/mL) were added.
- Stimulation of the cultures were performed every 8 d with IκB10-loaded irradiated autologous DCs (2×) followed by IκB10-loaded irradiated autologous PBLs (3×).
- After each stimulation, 120 U/mL IL-2 was added.
- The established cultures were tested for specificity for IκB10 after 4th stimulation.
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Functional characterization of FC preparations.
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The mean concentrations of active TGF-β1 , IL-12p70 , IL-10 , and HSP90α derived from four types of FC preparations (Mock/FCs, TGF-β/FCs, E-Mock/FCs, and E-TGF-β/FCs) (n = 4) were analyzed by ELISA.
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