Supplementary MaterialsFigure S1: TEM analysis of CNF samples. Co.) and 50 ng/mL Neratinib inhibition of interferon (IFN)- (R&D Systems, Minneapolis, MN, USA), or remaining untreated for the next 16 hours. In some experiments, anti-IL-6 receptor (R) (tocilizumab [Actemra?; Roche Diagnostics], 20 g/mL) and/or IL-6 (40 ng/mL; R&D systems) were added during differentiation of DC, as explained in the CNF in a different way impair differentiation and subsequent maturation of DC section. Mixed cell ethnicities Before cocultivation experiments with T cells, DC were filtered through sterile 30 m pore-size filters (Miltenyi Biotec) and washed twice in total RPMI medium to prevent transfer of free CNF and stimuli. DC (0.25104C0.5104/well in 96-well plate) were cocultivated with MACS-purified allogeneic T cells (1105/well) for 5 days. For proliferation assays, CD3+ T cells were pre-labeled with carboxyfluorescein succinimidyl ester (CFSE, 2 M; Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers protocol. For cytokines analysis, the supernatants of DC/CD3+ T-cell cocultures were collected after addition of phorbolmyristate acetate (PMA) (20 ng/mL) and ionomycin (500 ng/mL) (both from Sigma-Aldrich Co.) for the last 4 hours of incubation. For the circulation cytometric detection of intracellular cytokines, the cocultures were treated with PMA/ionomycin and monensin (3 M; Sigma-Aldrich Co.) for the last 3 hours of incubation. In some experiments, CD3+ or CD8+ T cells (5105/well inside a 24-well plate) were primed for 3 days with DC (1104/well), either in the presence or absence of 1-methyl-tryptophan (1-MT, 0.3 mM; Sigma-Aldrich Co.), anti-ILT-3, and anti-ILT-4 antibody (Ab) (both at 2 g/mL; R&D Systems) or isotype control Ab (anti-rat IgG2b; Thermo Fisher Scientific), and then treated with IL-2 (3 ng/mL; R&D Systems) for an additional 3 days. Additional control included the T cells cultivated similarly, but in the absence Sele of Neratinib inhibition DC. The primed T cells were analyzed phenotypically or used in the suppression assay in which different numbers of primed T cells (0.5105C1105/well inside a 96-well plate) were cocultivated with responder allogeneic CFSE-labeled CD3+ T cells (2105/well) in the presence of plate-bonded anti-CD3 (5 g/mL) Abdominal and soluble anti-CD28 Abdominal (1 g/mL) (both from eBioscience, San Diego, CA, USA) for 5 days. The cytotoxic activity of CD8+ T cells (0.5105 cells/sample) primed with HEp-2 lysate-pulsed syngeneic DC was evaluated by their co-incubation with CFSE-labeled HEp-2 target cells (1105 cells/sample) for 4 hours, as described previously.34 PBMC (10106/mL) were cryopreserved in 10% dimethyl-sulfoxide/FCS at ?80C for 5 days, and utilized for the isolation of syngeneic CD8+ T cells about day time of cocultivation with HEp-2 lysate-pulsed DC. The viability of CD8+ T cells after the thawing of PBMC and MACS sorting was more than 95%, relating to Trypan blue exclusion test. Cell viability, proliferation, and cytokine production The analysis of DC viability after 4 days of cultivation with or without CNF and APA samples was carried Neratinib inhibition out after staining the cells with Trypan blue (1% in physiological answer), or propidium iodide (PI, 10 g/mL; Sigma-Aldrich Co.), as explained earlier.34 HEp-2 cell death in coculture with DC-primed CD8+ T cells was analyzed by circulation cytometry (Sysmex Partec Cube 6) based on PI staining of CFSE-labeled HEp-2 cells. The proliferation of CFSE-labeled CD3+ T cells in response to DC, or CD3/CD28 activation, was analyzed within PI? populace by circulation cytometric measurement of CFSE dilution Neratinib inhibition during cell division.34 The Proliferation Index, ie, the average quantity of cells derived from an initial cell, was calculated using proliferation fit statistics in FCS Express 4 (De Novo Software, Glendale, CA, USA). The cytokine concentrations in cell tradition supernatants were determined by appropriate enzyme-linked immunosorbent assay (ELISA) packages (R&D Systems). Circulation cytometry Phenotype analysis of Neratinib inhibition DC and T cells after the ethnicities was carried out using circulation cytometer (Sysmex Partec Cube 6) after staining the.