Cd40 clone 5c3

Monocyte derived DC were generated by standard methods [38 (link)] from PBMC obtained from anonymous donors (Continental Blood Services Inc, Miami) and plated on 6 well plates. DC were then transduced with Ad5-ΔLMP1-MAVS or Ad5-Gag control (MOI = 50). DC were incubated with virus at 4°C for 1 h, followed by 3 h at 37°C. Complete media was then added to 2ml. As a positive control, DC were matured with cytokine mix Mimic (5 ng/ml TNF-α (Peprotech), 5ng/ml IL-1b (Peprotech), 750ng/ml IL-6 (Peprotech), and 1ug/ml PGE2 (Sigma)). Cells were incubated for 36 hours at 37°C, harvested, and stained with the following antibodies: CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 (CCR7) clone 3D12, and CD11c clone 3.9) (BD Bioscience). After flow cytometry analysis, the mean fluorescence intensity (MFI) for each antibody was calculated for CD11c+ dendritic cells under each experimental condition. FlowJo 7.6.4 flow cytometry analysis software (FlowJo, Ashland, OR) was used for analysis. Three independent wells were analyzed for each condition.

Titrated Alexa Fluor 647 labelled-VLP stocks were used to stain cDCs, LNCaP and HEK 293 TT cells at the indicated concentration. VLP attachment was detected by flow cytometry gated on DAPI negative cells. To assess DC activation, cells were incubated for 24 hours with 10³ VLPS/cell, 10³ BKPyV particles/cell or with 100ng/mL LPS and 1μg/mL R848 (Invivogen, San Diego, CA). Antibodies to CD40 (clone 5C3; BD Biosciences), CD80 (clone L307, BD Biosciences), CD83 (clone HB15e, BD Biosciences), CD86 (clone IT2.2, BD Biosciences), CCR6 (clone 11A9, BD Biosciences), CCR7 (clone 3D12, BD Biosciences) and HLA-DR (clone G46-6, BD Biosciences) were used to monitor DC maturation. Whole blood staining was performed on 500μl blood samples from healthy donors with or without Fc fragment receptor blockers (Miltenyi Biotec). Whole blood staining was done with Alexa Fluor 647 labelled-VLPS (2.5μg/ml) and cell subsets were discriminated using the following antibody panel: CD45 (Clone J33; Beckman Coulter, Brea, CA), CD11c (Clone BU15; Beckman Coulter), HLA-DR (Clone L243; BD Biosciences), CD123 (Clone 9F5; BD Biosciences) and Lineage (Lin 1; BD Biosciences). FACS analyses were mainly performed on a LSR II flow cytometer (BD Biosciences).

The pHAECs or iHAECs grown in 1% gelatin-coated tissue culture plates up to about 90% confluence were collected with TE/TN. Cells were washed, fixed, permeabilized using the Fix/Perm kit (Invitrogen Corporation, Frederick, MD), and incubated with FcBlock (BD Biosciences, San Jose, CA) and 10% goat serum to block nonspecific staining. Cells were stained with the primary antibodies for 2 h on ice, washed, and incubated with the secondary fluorescently labeled antibodies for 2 h on ice, followed by flow cytometry analysis (BD LSR II; Becton Dickinson, Franklin Lakes, NJ). Data were analyzed with FlowJo (FlowJo, LLC, Ashland, OR). The following antibodies were used: mouse anti-human CD31 (clone JC/70A; Pierce/Thermo Fisher Scientific, Rockford, IL); CD40 (clone 5C3; BD Biosciences); MHC-II (clone TÜ39; BD Biosciences); VE-cadherin (clone 123413; R&D Systems, Minneapolis, MN); rabbit anti-human ICAM-1 (polyclonal antibody; NeoScientific, Woburn, MA); CD14, eNOS, TLR4, vWF, and ZO-1 (all polyclonal antibodies; Bioss Antibodies, Inc., Woburn, MA); and secondary antibodies goat anti-mouse Alexa Fluor 488 (AF488) and goat anti-rabbit Alexa Fluor 647 (AF647) (both from Molecular Probes, Eugene, OR).