Medium and Reagents
The cell culture medium used throughout these experiments was RPMI 1640 Glutamax (Invitrogen, Paisley, UK) supplemented with 1 mmol/L sodium pyruvate (Sigma-Aldrich, l’Isle d’Abeau, France), non-essential amino acids (Invitrogen), 20ng/ml gentamicin (Invitrogen) and 10% heat inactivated human AB serum (referred as complete medium).
Immunophenotyping was performed by using Fluorescein Isothiocyanate (FITC)-, Phycoerythrin (PE)-, PE-Cyanin5 or 7 (PC5 or PC7)- and Allophycocyanin (APC)-conjugated and purified mouse monoclonal antibodies from Beckman Coulter (Marseille, France). IgG2a isotypic control, IFNγ and IL-4 antibodies were purchased from BD Biosciences (Pont de Claix, France).
Cells were stained with IgG1, IgG2a, CD11a, CD14, CD40, CD54, CD58, CD80, CD83, CD86, CD209, HLA-I, HLA-II and HLA-DR markers. DioC6 (3,3-dihexyloxacarbocyanine iodide) and OVA-FITC were purchased from Invitrogen. Lucifer Yellow was purchased from Sigma-Aldrich. The 7-Aminoactinomycin D (7AAD) viability dye was purchased from Beckman Coulter. The 8-methoxypsoralen (8-MOP) was provided by our cell therapy unit. Flow cytometric analyses were performed using a 4 colors FACScalibur flow cytometer and the CellQuest-Pro software (BD Biosciences).
Blood samples were collected from three healthy volunteers who gave their written informed consent. The peripheral blood mononuclear cells (PBMC) were separated by density gradient on lymphocyte separation medium (Eurobio, Les Ulis, France). Monocytes and T cells were purified according to the manufacturer instructions by using the human monocyte enrichment cocktail RosetteSep and from PBMC using the human T cells enrichment cocktail EasySep kit (Stem Cell Technologies Inc, Vancouver, Canada) respectively. Cells were cryopreserved in liquid nitrogen until use.
Monocyte-derived dendritic cells (moDC) generation
Purified monocytes were seeded at 0.5×106
cells/ml in a complete medium in the presence of 50U/ml of GM-CSF (Leucomax, Shering Plough, France) and 10 ng/ml of IL-4 (Tebu, Le Perray-en-Yvelines, France) as previously described(18
). Both cytokines were added at day 0 and additional IL-4 at day 3. After 6 days immature moDCs were harvested and verified as CD14-
by flow cytometry.
Experimental Psoralen/UV-A treatment (PUVA)
Purified monocytes were seeded at 1×106cells/ml in a complete medium. Cells were incubated for 15 min at 37°C with 200ng/ml of 8-MOP in the dark and were exposed to 2 J/cm2 365nm wavelength UV-A radiation (Bio Sun, Vilbert-Lourmat). After PUVA treatment, cells were washed in a complete medium and then cultured in the same medium at 37°C in 5% atmosphere CO2.
Apoptotic cells were quantified by DioC6/7AAD staining as described previously(19
). DioC6 was used to monitor mitochondrial transmembrane potential disruption. Monocytes (1×106
cells in 1ml) were incubated with DioC6 at a final concentration of 1 nM for 15 min at 37°C. Cells were washed in PBS and then analyzed by flow cytometry.
Percentage of specific apoptosis was calculated by taking into account of the number and the viability of cells as follows:
Endocytosis of monocytes was assayed by Lucifer Yellow and Ovalbumine-FITC after PUVA treatment. 2×105 monocytes in 200μl of RPMI with 10% Fetal Calf Serum (FCS) medium were incubated with 1 mg/ml of Lucifer yellow or 0.1mg/ml of FITC-conjugated ovalbumine in polypropylene tubes (BD Bioscience) for 3 hours at 37°C in 5% CO2 to allow endocytosis. Negative control was performed at 4°C. For ovalbumin-macropinocytosis -negative control, cells were incubated at 37°C in the presence of 10μg/ml of Cytochalasin D (Sigma-Aldrich). After 3 hours, cells were centrifuged twice in HBSS with 2% FCS and then the pellet was incubated with CD11b antibody. Phagocytic cells were determined as CD11b-FITC/Lucifer Yellow (FL-2) or Ova-FITC/CD11b-PE double positive cells by flow cytometry.
Cells activation and Cytokine production
Monocytes (1×106) were cultured in 1 ml of complete medium and activated with 1μg/ml LPS (Sigma-Aldrich) for 24hours. Immature moDCs were produced as described above. At day 6, they were activated with 1μg/ml LPS and 100ng/ml IFNγ (Peprotech Neuilly sur Seine, France) for 48 hours.
Culture supernatants from activated monocytes and moDCs were collected after 24 and 48 hours respectively. The contents were analyzed for IL-1, IL-6, IL-8, IL-10, IL-12 p70 and TNFα by Cytokine Bead Array (CBA) inflammation kit (BD Biosciences) according to the manufacturer’s recommendations with FACScan (BD Bioscience). Data were analyzed with CBA software (BD Biosciences).
Transwells chemotaxis assay
Migration assays were carried out by using transwells (6.5mm diameter, COSTAR, Cambridge, MA, USA) with 5.105 cells/well. Control or treated PBMCs were washed, preincubated for 1 h at 37°C in RPMI with FCS 2 % and then placed for 2 h in 5 μm pore size inserts at 37°C. The migrating monocytes were harvested and immunostained with CD14-FITC. The results were shown as the percentage of monocytes which have migrated relative to the input number of monocytes. The chemokines used were MCP-1/CCL2 (CCR2), MIP-1β/CCL4 (CCR5), RANTES/CCL5 (CCR1) and SDF-1α/CXCL12 (CXCR4), at 100, 50, 50 and 100 ng/mL respectively (R&D systems, MN, USA).
Antigen specific T cell amplication
Autologous monocytes (1×106/ml) were pulsed with 10μM of the influenza M1 peptide (GILGFVFTL; HLA-A2 restricted) for 3h at 37°C in RPMI. Then 5×104 of control or treated Flu-monocytes were co- cultured with 1×105 T cells for 7 days. PE - Flu-M1/HLA-A2 tetramers (Beckman Coulter, France) were used for the staining of the M1 specific CD3+CD8+ T cells according to the manufacturer’s recommendations.
Mixed lymphocyte reaction
Responding T lymphocytes and stimulating monocytes were purified from PBMC of two healthy volunteers. Unidirectional MLR were conducted in quadruplicate of 96-well flat-bottom plates (BD Biosciences) by mixing 5×104
responding purified CD3+ T cells and 5×103
monocytes or moDC +/− PUVA (ratio responding/stimulator cells from 1/1 to 1/0.1) in a final volume of 200μl (20
). After five days, [3
H]thymidine was added to each well and cell were harvested after 18 h later (Cell harvester: Inotech IH 280 Berthold) [3
H]thymidine incorporation was measured by a liquid scintillatrion counter, TopCount NXT (Perkin Elmer).
T cell polarization
A mixed lymphocyte reaction was performed as described above. After 6-day culture, cells were harvested and T cells were restimulated with 0.5ng/ml of PMA and 5ng/ml of Ionomycine (Sigma-Aldrich) for 6 hours. The secretion pathway was inhibited by using Monensine after 1 hour. At the end of the culture, cells were permeabilized by cytofix/cytoperm Kit (BD Bioscience) allowing the intracellular staining of IFNγ and IL-4 (BD Bioscience). Analysis was performed by using a FACSCanto II cytometer (BD Bioscience).
The unpaired student’s T-test or two-way ANOVA were used to determine if the differences observed between control and treated conditions were significant or non-significant. A value of p < 0.05 was considered significant.