Soluble cd40l

Anti-CD40L antibodies used were: hu5c8, a humanized anti-human CD40L intact IgG₁ monoclonal antibody (Biogen Idec, Cambridge, MA, USA); aglycosyl hu5c8, a mutant form of hu5c8 with reduced FcR binding (Biogen Idec, MA, USA); CDP7657, a monovalent Fab' PEGylated anti-CD40L antibody (UCB Pharma, Slough, UK); M90, a mouse anti-human CD40L intact IgG₁ monoclonal antibody (hybridoma from ATCC, Manassas, VA, USA); MR1 Fab' PEG, a monovalent, murinized Fab' PEGylated anti-mouse CD40L (UCB Pharma, Slough, UK). Soluble CD40L was from Peprotech (Rocky Hill, NJ, USA), phycoerythrin (PE)-labeled CD40-Fc fusion protein was from Ancell (Bayport, MN, USA) and thrombin receptor agonist peptide (TRAP) was from Sigma (St Louis, MO, USA).

Peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). B cells were purified using Human B Cell Isolation Kit II (Miltenyi Biotec), and naïve B cells were isolated using Human Naïve B Cell Isolation Kit (Miltenyi Biotec). The ethics committee of the University of Tokyo Hospital approved this study (No. 10154 and G3582). All subjects provided written informed consent, and the study was conducted in accordance with relevant guidelines.
Unless otherwise indicated, cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS (Equitech Bio), 100 μg/ml L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 50 μM 2-ME (Sigma). In some experiments, cells were cultured in X-VIVO15 (Lonza) to exclude the effect of TGF-β in FCS.
TGF-β1 and β3 (R&D) were used at 1 ng/ml unless otherwise indicated. IL-21 (PeproTech), IL-4 (R&D), soluble CD40L (PeproTech), and CpG-ODN2006 (Enzo Life Sciences) were used at 50 ng/ml, 100U/ml, 2 μg/ml, and 6 μg/ml respectively, and BCR stimulation was induced using goat anti-human IgA + IgG + IgM (H+L) (Jackson ImmunoResearch) at 2.5 μg/ml.

CFPAC-1 cells transduced with either 50 MOI RAdMock or RAdnCD40L or un-transduced for 24 hours were co-cultured with iDC at a ratio of 1:2 cells to iDC. The uninfected CFPAC-1 and iDC co-culture were either treated with soluble CD40L (1 µg/ml) (PeproTech) or maturation cocktail or left untreated as a negative control for 48 hours. For IL-10 and IL-12p70 assessment in the culture media, samples were collected at 24 and 48 hours following iDC-CFPAC-1 co-cultures, pre-cleared by centrifugation and assayed by Enzyme-Linked Immune Sorbent Assay (ELISA) kit (eBioscience, Hatfield, UK) according to the manufacturer’s instructions utilizing the automated Modulus Microplate reader (Turner BioSystems, USA).