Anti tnf apc

For analysis of LMP1&2-specific and EBNA1-specific T-cell frequencies, AdE1-LMPpoly-expanded products were stimulated for 4 hours in the presence of GolgiPlug (BD Biosciences) with a pool of defined epitopes from LMP1&2 or EBNA1 or with an overlapping set of peptides encompassing the whole EBNA1 protein (all from Mimotopes, GenScript or JPT Technologies), and then assessed for the intracellular production of IFN-γ. For multiparametric analysis, cells were stimulated for 4 hours in the presence of GolgiPlug and GolgiStop (BD Biosciences) with the peptides listed above and anti-CD107a-FITC (BD Biosciences). Cells were then washed and stained with anti-CD8-PerCPCy5.5 (eBioscience) and anti-CD4-PECy7 (BD Biosciences), fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences), washed again and stained with anti-IFN-γ-AF700, anti-IL-2-PE and anti-TNF-APC (all from BD Biosciences). After a further wash, cells were resuspended in PBS and acquired using a BD LSR Fortessa with FACSDiva software (BD Biosciences). Post-acquisition and Boolean analysis was performed using FlowJo software (TreeStar).

To study CD8⁺ T cell proliferation and functionality, 5000 DCs were stimulated as indicated and co‐cultured with 20 000 allogeneic naïve CD8⁺ T cells (CD8⁺, CD27⁺, CD45RO⁻, and CD45RA⁺) in the presence of 1 pg/mL Staphylococcus aureus enterotoxin B (SEB; Sigma–Aldrich). To determine proliferation, CD8⁺ T cells were incubated with 0.5 μM CFSE (Invitrogen) and washed extensively prior to co‐culture. At day 3 or 4, cells were incubated overnight with the modified thymidine analogue EdU (Click‐iT kit; Invitrogen) and further processed according to the manufacturer's instructions. The percentage of divided cells (EdU⁺ or CFSE⁻) was determined by flow cytometry (Canto II, BD Biosciences). To determine intracellular granzyme B expression, cells were harvested at day 4 or 5, washed with PBS, fixated with 4% formaldehyde (Sigma–Aldrich) for 15 min, washed again, permeabilized with 0.5% saponin (Calbiochem) in PBS containing 0.5% BSA (PAA) and 0.1% sodium azide (Merck), and stained with anti‐granzyme B‐PE (Sanquin Blood Supply) and analyzed by flow cytometry. For intracellular IFN‐γ or TNF staining, CD8⁺ T cells were restimulated at day 4 or 5 with 100 ng/mL PMA, 1 μg/mL ionomycin, and 10 μg/mL brefeldin A (all Sigma–Aldrich) for 6 h, washed, fixated, and permeabilized as described above, stained with anti‐IFN‐y‐ FITC and anti‐TNF‐APC (both BD Biosciences) and analyzed by flow cytometry.

Sorted CD8⁺CD44^lo T cells stimulated at 2 x 10⁴ cells per well in mAb-coated 24-well plates for up to 72 hours (h) were incubated in the presence of 10 U/mL recombinant human IL-2 (Roche Diagnostics, Mannheim, Germany). The mAb were coated onto Nunc plates overnight in PBS at the following concentrations: anti-CD3ε (clone 145-2C11) at 10 μg/mL; anti-CD8α (clone 53–6.7) at 10 μg/mL; anti-CD11a (clone I21/7.7) at 5 μg/mL; anti-CD28 (clone 37.51) at 10 μg/mL [16 (link)]. To permit intracellular cytokine staining, GolgiPlug at a 1:1000 dilution (BD Biosciences, San Diego, CA, USA) was added for the last 5 h of the incubation. After incubation, cells were surface-stained using anti-CD8α-PE (BD Pharmingen; clone 53–6.7), fixed and permeabilised using Cytofix/Cytoperm buffer and 1 × Perm/Wash buffer (BD Biosciences) according to manufacturer’s instructions and stained with anti-IFNγ-FITC (BD Pharmingen; clone XMG1.2) and anti-TNF-APC (BD Pharmingen; clone MP6- XT22) [17 (link)]. Cells were then acquired using FACSCalibur flow cytometer (BD Biosciences) and data were analyzed by using FlowJo software (Versions 9&10) (FlowJo LLC, Ashland, Oregon).