Generation and treatment of DC
PBMC were isolated from human peripheral blood of healthy donors by standard density gradient centrifugation on Ficoll-Hypaque. Mononuclear cells were separated from PBL by centrifugation on a 50% Percoll solution (Amersham Biosciences, Uppsala, Sweden). Monocytes were purified by immunomagnetic depletion (Dynal, Oslo, Norway) using a cocktail of monoclonal Ab anti-CD19 (4G7 hybridoma), anti-CD3 (OKT3, ATCC, Rockville, MD, USA) and anti-CD56 (NKH1, Beckman Coulter, Fullerton, CA, USA). Monocytes (purity > 90%) were differentiated to immature DC (iDC) during 7 days with 40 ng/ml human rGM-CSF and 250 U/ml human rIL-4 in RPMI 1640 (Abcys, Paris, France) supplemented with 2 mM glutamine, 10 mM Hepes, 40 ng/ml gentamycin (Life Technologies, Rockville, MD, USA) and 10% FCS. Differentiating monocytes were treated at day 5 with 1 mM 1-methyl-DL-tryptophan or 2,5 mM of Trp or 60 μM of kynurenine (Sigma-Aldrich, St Quentin-Fallavier, France) and at day 6 with 1 μg/ml LPS (Escherichia coli, serotype 0127:B8, Sigma-Aldrich), 10 μg/ml polyI:C (pIC - Amersham Biosciences), 10 μg/ml peptidoglycan (PGN) of Staphylococcus aureus (Sigma-Aldrich) or 10 μg/ml of Pam3CSK4 (Pam - Axxora, San Diego, CA). All cells and supernatants were collected at day 7. Control mature DC (mDC) were obtained by adding TLR ligands at day 6 for 24 h. When indicated, 40 μM PD98059, an inhibitor of MEK1/2 (Biomol, Plymouth Meeting, PA, USA), or 25 μM SB203580, an inhibitor of p38-MAPK (Biomol), were added 30 min before 1-MT treatment. All DC were more than 95% pure as assessed by CD14 and CD1a labeling.
Phenotype was analyzed on a FACScalibur (BD Biosciences, Le Pont de Claix, France) using FITC-conjugated anti-CD14, -HLA-DR, -CD80, -CD54, and PE-conjugated anti-CD1a, - CD86, -CD83 and -CD40 (Beckman Coulter).
Culture supernatants were stored at −80°C. IL-6, IL-10, IL-1β, TNFα and IL-13 levels were determined using cytokine-specific ELISA kits (Endogen, Woburn, MA, USA). IL-12 p40 and p70 were assayed using ELISA kits from Biosource (Camarillo, CA, USA). IL-2, IL-4, IL-5, IL-10 and IFNγ were determined using the human Th1-Th2 cytokine CBA kit I (BD Biosciences).
Mixed Lymphocyte Reaction
T lymphocytes were purified after Ficoll-Hypaque and Percoll gradient centrifugation by immunomagnetic depletion using a cocktail of monoclonal Ab anti-CD19 (4G7), anti-CD56 (NKH1), anti-CD16 (3G8), anti-CD14 (RMO52) and anti-glycophorin A (11E4B7.6) (Beckman Coulter). T lymphocytes were more than 95% pure as assessed by CD3 labeling. Primary MLR were conducted in 96-well flat-bottom culture plates. DC recovered at day 7 were extensively washed and resuspended in complete RPMI 1640/10% FCS. Cells were cocultured in triplicates with 2.105 allogeneic T cells in 200 μl at DC/T cell ratios ranging from 1/10 to 1/40. Supernatants were recovered at indicated time points for IL-2, IL-4, IL-5, IL-10, IL-13 and IFNγ measurement.
T cell response against tetanus toxin
DC were treated as for MLR and autologous CD3 T cells were purified as described above from frozen PBL. DC recovered at day 7 were extensively washed and resuspended in complete RPMI 1640/10% FCS. Cells were cocultured in triplicates with 2.105 allogeneic T cells in 200 μl at 1/20 DC/T cell ratio. 25 μg/ml purified tetanus neurotoxin (kindly provided by Dr Villiers, INSERM U548, CEA Grenoble, France) was then added to cocultures. Supernatants were recovered after 5 days of coculture for IL-2, IL-4, IL-5, IL-10, IL-13 and IFNγ measurement. Tetanus neurotoxin has no effect on DC or T cells alone (data not shown).
Intracellular staining of cytokines
MLR were conducted for 5 days and T cells were expanded for 7 days with 25 U/ml rhIL-2 (Biosource), washed and restimulated with 10 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (VWR International, Fontenay-sous-Bois, France) for 5 h. 10 ng/ml Brefeldin A (Sigma-Aldrich) was added during the last 2 h. Cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences). Intracellular staining was performed using FITC-labeled anti-IFNγ monoclonal Ab and PE-labeled anti-IL-5 and IL-13 monoclonal Ab (BD Biosciences).
IDO expression and activity
Total RNA was extracted from cells collected at day 7 using RNeasy Mini kit (Qiagen, Courtaboeuf, France). 100 ng of total RNA was reverse transcribed using the thermoscript RT-PCR system (Life Technologies). Primers used for PCR amplification are: 5′-GCTTTCACACAGGCGTCATA-3′ and 5′-GGTCATGGAGATGTCCGTAA-3′ for IDO, and 5′-GGAGGTGTAATGGACGTTA-3′ and 5′-CTGAGACTCCTTGCCATAG-3′ for S12. The amplified products were analyzed by gel electrophoresis (691 bp for IDO and 311 bp for S12).
Trp is converted by IDO to N-formylkynurenine which is further catabolized to kynurenine. Quantification of kynurenine in supernatants thus reflects IDO activity. Kynurenine was measured in fresh supernatants of DC collected at day 7 as described previously (44
). Briefly, 100 μl of 30% TCA was added to 200 μl of supernatant and vortexed. After centrifugation, 125 μl of supernatant was incubated with 125 μl of Ehrlich reagent (p-dimethylaminobenzaldehyde; Sigma-Aldrich) in a microtiter plate for 10 min at room temperature. Optical density was measured at 490 nm. Values were referred to a standard curve with defined kynurenine concentrations (0–120 μM, Sigma-Aldrich) and normalized to 106
Phosphorylation of p38-MAPK, ERK and c-Fos
For studies on ERK and p38 phosphorylation, 2.106 differentiating monocytes were treated at day 5 with 1 mM 1-MT and collected at day 6. Cells were extensively washed and starved for 2 h in complete RPMI 1640 medium without serum. Cells were treated with the different TLR ligands for 5, 10, 15, 30 or 45 min. Cells were washed twice with cold PBS and pellets were lyzed in RIPA buffer containing 1 mM PMSF and 1% protease inhibitors. Phosphorylated and total ERK and p38 MAPK were quantified by specific ELISA (Assay Designs Inc, Ann Arbor, MI, USA).
For studies on c-Fos phosphorylation, 2.106 differentiating monocytes were treated at day 5 with 1 mM 1-MT and collected at day 6. Cells were extensively washed and resuspended in RPMI/0,3% delipidated BSA. Cells were treated with the different TLR ligands for 1, 2, 4 or 6 hours. Cells were then washed twice with cold PBS and pellets were lyzed in RIPA buffer containing 1 mM PMSF and 1% protease inhibitors. Phosphorylated c-Fos was quantified by a chemoluminescent ELISA (Endogen) and normalized to the amount of protein determined with the microBCA assay kit (Pierce, Rockford, IL, USA).