Baboons (Papio anubis, from the CNRS Primatology Center, Rousset, France) were negative for all quarantine tests, including a tuberculin skin test. Animals were housed at the large animal facility of our laboratory following the recommendations of the Institutional Ethical Guidelines of the Institut National de la Santé Et de la Recherche Médicale, France. All experiments were performed under general anaesthesia with Zoletil (Virbac, Carron, France). Pharmacokinetic and pharmacodynamic

studies were performed during DTH experiments on five baboons receiving an i.v. bolus of either 1 mg/kg or 0·1 mg/kg of chimeric A9H12. Chimeric A9H12 was quantified in baboon sera using a specific sandwich ELISA. LAG-3-Ig (Immutep, Orsay, France) was immobilized on plastic at pH 9·5 overnight at a concentration of 5 µg/ml. After saturation with

5% gelatin at 37°C for 2 h, serum diluted PD98059 in PBS-0·05% Tween 20 were incubated for 4 h at room temperature, washed and revealed with a mouse anti-human IgG kappa chain Compound Library price antibody (EFS, Nantes, France) at a 1:2000 dilution, followed by peroxidase-labelled goat anti-mouse antibody (Jackson Immunoresearch, Westgrove, PA, USA) at a 1:5000 dilution. Optical density was recorded at 450 nm after a tetramethylbenzidine (TMB) revelation period of 10 min at room temperature in the dark and addition of 25 µl 1 N sulphuric acid/well. Baboons were immunized intradermally (i.d.) twice with a bacillus Calmette–Guérin (BCG) vaccine (0·1 ml; 2–8 × 105 UFS; Sanofi Pasteur MSD, Lyon, France) in the upper region of the leg, 4 and 2 weeks before the DTH skin test. To investigate antigen-specific T cell immunity before

DTH skin testing, successful immunization was confirmed by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay (non-human primate IFN-γ ELISPOT kit; R&D Systems, Minneapolis, MN, USA) on freshly isolated Adenosine triphosphate PBMC, according to the manufacturer’s instructions. Intradermal reactions (IDR) were performed with duplicate intradermal injections of two doses (2000 UI or 40 UI) of tuberculin-purified protein derivative (PPD; Symbiotics Corporation, San Diego, CA, USA) in 0·1 ml in the skin on the right back of the animals. Saline (0·1 ml) was used as a negative control. Dermal responses at the injection sites were measured using a caliper square. The diameter of each indurated erythema was measured by two observers from days 3–8, and were considered positive when > 4 mm in diameter. The mean of the reading was recorded. Skin biopsies from the DTH or control (saline) site were performed at day 4 on one duplicate and placed in Tissue Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Villeneuve d’Ascq, France) for immunohistochemical analysis. A second IDR was performed after a 3-week washout period and animals received one i.v. injection of either 1 mg/kg or 0·1 mg/kg of chimeric A9H12 1 day before this second challenge with PPD.