Anti tnfα clone mab11

Freshly thawed PBMCs were cultured overnight at 37°C with 5% CO₂ in supplemented RPMI with 10% Human-Ab serum (GeminiBio). The rested cells were washed, and2million PBMCs were stimulated using aAPC described previously (23 (link)) in supplemented RPMI with 10% Human-Ab serum plus mixed cytokines (4 ng/mL IL-2, 0.3 ng/mL IL-4, 0.4 ng/mL IL-6, 0.2 ng/mL IL-1b, and 1 ng/mL IFN-γ). On day 7, 2 million thawed PBMCs were replated with aAPCs. The cells were fed on days 3, 5, 10, and 12. On day 14, cells were stimulated with aAPCs and Golgi plug for 6 hours and then stained with tetramers (LLL and YLQ, NIH Tetramer Core Facility; M1 and MART1 from MBL; and LLY from Tetramer Shop) at room temperature for 30 minutes before staining with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen) and anti-CD3 (clone SK7) and anti-CD8 (clone SKI) antibodies at 4°C for 30 minutes. Cells were subsequently fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences — Pharmingen) and stained with anti–IFN-γ (clone 4S.B3) and anti–TNF-α (clone MAb11) antibodies at 4°C for 30 minutes. Antibodies were purchased from Biolegend unless otherwise stated. Samples were acquired using the Attune flow cytometer (Invitrogen), and analyses were performed using FlowJo 10.7.1 software.

To determine by flow cytometry the phenotype and cytokine profiles of vaccine-specific CD4+ and CD8+ T-cells 12 days after in vitro stimulation (IVS), patient PBMCs were re-challenged with individual peptides overnight at 37 °C, 5%CO2 in presence of Brefeldin A (51-2301KZ, BD Bioscience). The next day, the cells were washed with PBS and stained for 20 min on ice with anti-CD3 (clone SK7; # 344840, Biolegend), anti-CD8 (clone RPA-T8; #301042, Biolegend), anti-CD4 (clone RPA-T4; #558116, BD Biosciences), and a viability dye Zombie UV (#77474, Biolegend). After fixation and permeabilization for 20 min at 4 °C (BD Bioscience Cytofix/Cytoperm Kit), anti-CD154 (CD40-L, clone 24–31; #310826, Biolegend), anti-Granzyme B (clone GB11; #GRB17, Invitrogen), anti-Perforin (clone B-D48; #353310, Biolegend), anti-IL-2 (clone MQ1-17H12; #559334, BD Biosciences), anti-TNF-α (clone MAb11; #557647, BD Biosciences), and anti-IFN-γ (clone B27; #554702, BD Biosciences). All samples were acquired on a 5-laser BD FORTESSA instrument equipped with the FACS DiVa software. Analyses were performed with FlowJo v10.5 (FLOWJ,.LLC, Ashland, OR, USA) and SPICE 6 software.

Mononuclear cells were incubated with PMA (50 ng/mL) and ionomycin (500ng/mL) or cultured in RPMI containing 10% FBS (R10) alone. Samples were incubated at a concentration of 10 µg/mL, and Golgi Plug (brefeldin A) and Golgi Stop (monensin) were included at 10 µg/mL. Samples were incubated for overnight at 37°C in 5% CO2 and then permeabilized using Fix & Perm reagents (BD Bioscience) and stained intracellularly with anti–IFN-γ (clone B27) and anti–TNF-α (clone Mab11). At the end of stimulation, cells were washed once with FACS wash (PBS containing 2% [vol/vol] FBS and 0.25% of sodium azide) and surface stained with anti-CD3, anti-TCR7.2 (3C10), anti-Ki67 (B56) anti-CD8 (SK1), anti-CD4 (OKT4) Cell stain at room temperature for 30 minutes. Cells were then fixed with Cytofix/Cytoperm (BD Pharmingen) for 20 minutes at 4°C and washed with Perm wash (BD Pharmingen). Cells were then incubated for 30 minutes at 4°C with antibodies specific to IFN-γ and TNF-α, washed once with Perm wash and once with FACS wash, and resuspended in PBS containing 1% formalin. Cells were acquired on a BD FACS Canto II Immunocytometric system. FlowJo for Windows, Ver.10.0.8 (FlowJo LLC, Ashland, OR, USA) was used to perform the analysis. At least 100,000 events were acquired for each samples.