Cpg1668

Bone marrow cells were cultured as described previously [47 (link),48 (link)]. In brief, bone marrow cells from Cnr1^−/−, Cnr2^−/− and Cnr1^−/−/Cnr2^−/− mice were seeded at 5 × 10⁵ cells/mL in DMEM supplemented with 10% FCS, 1% MEM/NEAA, 1% Penicillin/Streptavidin 0.1% β-Mercaptoethanol (all from Thermo Fisher Scientific, Inchinnan, UK) and 10% conditioned medium of B16 cells. Medium was exchanged on day 3 and cells stimulated on day 5 with either 100 ng/mL Escherichia coli Lipopolysaccharide (LPS) Serotype 0127:B8 (Sigma-Aldrich, Saint Louis, MO, USA) or 1 nmol/mL CpG1668 (TIB MOLBIOL, Berlin, Germany). After 16 h of stimulation, cells were harvested and analyzed by FACS or transferred in the migration assay.

Dlg2^ΔE9/ΔE9 [39 (link)] mice were a kind gift of Seth G. N. Grant (Edinburgh University, Edinburgh, UK). Bicistronic Interferon β/YFP reporter knock-in mice (mob: messenger of IFN beta; IFNβ^mob/mob) have been described previously [40 ]. IFNβ^mob/mob, Dlg2^ΔE9/ΔE9 mice, and their wild type (WT) littermates are on C57BL/6 N background and were kept under pathogen-free conditions. The mice were euthanized by cervical dislocation. The experiments were approved by the government of North-Rhine Westphalia. Where indicated, mice were injected i.v. with 10 μg CpG 1668 (TIB MOLBIOL) complexed to DOTAP (Roche) for 6 h, or as indicated.

Microglia were plated at 30x10³/96-well in 200 μl DMEM supplemented with 10% heat inactivated FCS, 1% penicillin/streptomycin and left to adhere overnight. After removal of 100 μl of media cells were stimulated with the TLR ligands Pam3CysSK4 (100ng/ml), imiquimod (10μg/ml) (all from InvivoGen, Toulouse, France), LPS (100ng/ml, Enzo Life Sciences GmbH, Lörrach, Germany), CpG 1668 (1μM, TIB MolBiol, Berlin, Germany) for 24 h. Subsequently, conditioned microglial supernatants were transferred to naïve γδ T cells (30x10³/96-well in 100 μl complete RPMI), or naïve γδ T cells were co-cultured with stimulated microglia at a 1:1 ratio. After indicated time points cells were collected for flow cytometry and supernatants were recovered for ELISA or multiplex analysis of cytokines, as indicated. TLR stimulation of bone marrow-derived macrophages was carried out likewise. For neutralization of IL-1β and IL-23, conditioned microglial supernatants were pre-incubated for 1 h at 4°C with 10 μg/ml anti-IL-1β (clone B122), anti-IL-23 (p19, clone MMp19B2) or respective isotype controls (all obtained from BioLegend, San Diego, USA) before supernatants were used for incubation of naïve γδ T cells.