Cpg b

Fresh human or NHP PBMCs were labeled with 0.25 µM CellTrace Violet (Invitrogen, C34557) at a cell concentration of 1 million/mL for 20 min at 37 °C. Labeled PBMCs were then incubated with testing antibodies, 1 µg/ml CpG-B (Invivogen, tlrl-2006) or left untreated in complete media (RPMI1640, 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin) and were cultured for 5 days. Some samples were cultured for 5 days under 10 ng/mL IL-4 and 50 ng/mL IL-21 supplemented condition as described earlier [32 (link)]. After culture, cells were washed with PBS and stained with LIVE/DEAD prior to FcR Blocking as described above. Samples were then surface stained with a panel of fluorescently labeled antibodies (Table S1). After staining and washing, PBMCs were resuspended in 1% PFA and acquired on an LSRFortessa flow cytometer.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of healthy adult donors using standard ficoll density gradient separation. Cells were frozen and stored in liquid nitrogen until use. PBMCs (2x10⁵ cells/well) were cultured in RPMI with 10% heat-inactivated fetal bovine serum and 10 μg/ml gentamicin in 96 well flat-bottomed plates with CpG class B (Integrated DNA Technologies) (referred to here as CpG-B (PS)) with or without TFAM at the indicated concentrations. For blocking experiments, we used colchicine (150 ng/ml; Sigma, St. Louis, MO), an inhibitor of microtubule-mediated uptake, and ODN TTAGGG (“iODN”; 10:1 ratio of iODN to CpG-B (PS), Invivogen, San Diego, CA), an inhibitory oligonucleotide and a TLR9 antagonist. After 7 days, culture supernatants were tested for IgG antibody production by enzyme-linked immunosorbent assay (ELISA) as previously described [43 (link)]. The Colorado Multiple Institutional Review Board (COMIRB) specifically approved this study and the use of all samples in these studies (#050993). Participants provided their written informed consent to participate in this study.

PBMCs were cultured at a concentration of 2×10⁶ cells/ml in RPMI-1640 containing L-glutamine, 10% FCS and antibiotics in medium only or stimulated with 10 μg/ml CpG-B (InvivoGen, California, USA) and 1 μg/ml CD40L (InvivoGen) for 48 h. For the last 5 hours of the culture, an additional stimulation with 50 ng/ml PMA and 1 μg/ml Inomycin (Sigma-Aldrich, MO, USA) in the presence of Golgistop used at 1:1000 dilution (BD) was performed. Cells were first stained for cell surface markers PerCp anti-CD19, FITC anti-CD10 and V450 anti-CD27 in addition to LIVE/DEAD staining. In next step cells were fixed and permeabilized using BD Cytofix/Cytoperm kit and stained with PE anti-IL-6 (MQ2-6A3) and matching isotype control, both from (BD). Cells were washed and fixed in 1% formaldehyde before the analysis were conducted using a Cyan flow cytometer. Collected data were analysed using FlowJo, version 9.4.11 (Tree star).

pDC-Gen2.2 cells were cultured at 1–2 10^6 cells/ml in complete RPMI 1640 on the MS-5 feeder cells. Before the experiment, the non-adherent fraction of the culture was harvested, pelleted at 1500 rpm and re-suspended in a fresh medium. 50,000 cells were plated in 96-well round bottom plates and stimulated with 0.25μM CpGA (Invivogen, tlrl-2216), 0.25μM CpGB (Invivogen, tlrl-2006), 3.75μg/ml R848 (Invivogen, tlrl-r848), 0.5μg/ml LPS (Invivogen, tlrl-b5lps) or were left unstimulated (Mock) in total volume of 150μl. The stimulation assay was performed with and without 5μM RBV (1221 ng/ml) (Sigma, R9644). At time points (indicated in figures), the culture supernatants were analyzed for IFNα by ELISA and cells were harvested for RNA isolation and qRT-PCR analysis of gene expression. Where total PBMCs were used, 50,000 cells were stimulated with 0.25μM CpGA +/- RBV and analyzed as above.

To generate apoptotic cells, thymocytes were cultured with 1 uM Dexamethasone (Sigma) for 4 h at 37°C. Staining with 7AAD and Annexin V (BD Biosciences) determined that this treatment typically resulted in 45% of Annexin V⁺ apoptotic thymocytes and in 1% of 7AAD⁺ Annexin V^- necrotic thymocytes. Marginal zone and follicular B cells were sorted from purified splenic CD43⁻ B cells as IgM⁺CD21⁺CD23⁻ for MZ B cells and IgM⁺CD21⁻CD23⁺ for FO B cells, using a FACS Aria-II cell sorter (BD Biosciences). Post-sorting purity of either subset was greater than 90%. The sorted MZ or FO B cells were co-cultured at a 1∶7 ratio with apoptotic thymocytes and 1 ug/ml CpG-B (Invivogen) for 3 d at 37°C. Cytokines were quantified from extracted RNA and culture supernatant by qPCR and ELISA, respectively.

CpG-A (ODN 2,216), CpG-B (ODN 2006), CL413 (Adilipolin), CL267 and R848 were obtained from InvivoGen (San Diego, USA) for use in in vitro PBMC stimulation assays, and GS-9620 was a gift from Gilead Sciences. All of them were used at concentration recommended by manufacturer for optimal in vitro stimulation. Recombinant IFN-α-2a and IFN-λ3 were obtained from PBL. Anti-Human Interferon Lambda Receptor 1 (IFNLR), clone MMHLR-1, neutralizing (MAb) was from PBL; Anti-IFN-α/β Receptor Chain 2 Antibody, clone MMHAR-2, MAB1155 was from EMD Millipore.

Bone marrow (BM) cells removed from tibiae, femurs and hips of 8- to 14-week-old C57BL/6J mice were incubated in low endotoxin RPMI-1640 medium (Fisher Scientific, Illkirch, France) supplemented with 10% (vol/vol) FCS and 1% antibiotics (penicillin and streptomycin) for 18 h with 1 μg/ml of the oligodeoxynucleotide CpG 1668 (CpG-B) (Eurogentec, Angers, France) or with the respective agonists of TLR1-9, supplied in a commercial kit (InvivoGen, Toulouse, France), including: Pam3CSK4 (0.5 μg/ml), FSLI (1 μg/ml), HKLM (2 × 10⁶ cells/ml), Poly I:C (HMW) (5 μg/ml), Poly I:C (LMW) (5 μg/ml), LPS-EK standard (1 μg/ml), FLA-ST standard (1 μg/ml), ssRNA40/LyoVec (2 μg/ml) as well as CpG 1826 (CpG-B) (1 μg/ml). c-kit⁺ BM cells were sorted by immuno-magnetic separation using a RoboSep automaton (StemCell Technologies, Grenoble, France). Sorted cells were further stained with appropriate fluorochrome-conjugated mAbs against Sca-1, B220 (BD Biosciences, Le Pont de Claix, France), PDCA-1 (eBioscience, Paris, France or BioLegend, London, UK) and electronically sorted as a c-kit⁺Sca-1⁺B220⁺PDCA-1⁺ cell subset using a FACS-Aria IIIu (BD Biosciences).