Anti ctla4

To generate DCs, CD14⁺ monocytes were cultured (3-5 x 10⁶/well; 6-well plate; total volume, 6 ml; 37°C/5% CO₂) for 8 days in R9 medium (RPMI 1640, 100 µg/ml penicillin, 100 U/ml streptomycin, 25 µg/ml transferrin, 10% human AB serum [Innovative Research], 25 mM HEPES buffer, 2 mM L-glutamine) supplemented with GM-CSF and IL-4 (800 U/ml) [PeproTech Ltd]. To induce DC maturation, 25 ng/ml TNF-α [PeproTech Ltd] and 1 µg/ml lipopolysaccharide [Sigma-Aldrich Ltd, E.coli 0111:B4] were added on the penultimate day of culture. Naïve or memory CD3⁺ T-cells, with Tregs removed, were cultured (2.5 x 10⁶/well; 24 well plate; total volume, 2 ml) with autologous mature DCs (0.8 x 10⁵/well) and SMX-NO (50 µM) for 8 days. To specific wells, human targeted anti-PD-L1, anti-CTLA4, or anti-TIM-3 antibodies (Biolegend, London, UK; anti-PD-L1, 5 µg/ml; anti-CTLA4, 10 µg/ml; anti-TIM-3, 7.5 µg/ml) were added prior to the addition of SMX-NO and incubated for ≥ 30 min (37°C/5% CO₂). Dose ranging studies were performed around those directed by the supplier to ascertain optimal antibody concentrations. Alternatively, specific quantities of autologous CD25⁺ Tregs were added to individual wells. Experiments were repeated a minimum of three times.

Surface staining of the single cell suspensions was performed for 30 min at 4°C, and viability was assessed using LIVE/DEAD Fixable viability dye as per the manufacturer’s instructions (Thermo Fisher Scientific).
The following surface markers antibodies from Biolegend were used: anti-CD3 (clone: 145–2C11), anti-CD4 (RM4–5), anti-CD8a (53–5.8), anti-CD45 (30-F11), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD19 (6D5), anti-TCRβ (H57–597), anti-NK1.1 (PK136), anti-KLRG1 (2F1), anti-PD-1 (29F.1A12), anti-CD25 (PC-61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-Thy1.1 (OX-7), anti-CCR2 (SA203G11), anti-CXCR3 (CXCR3–173), anti-CXCR5 (L138D7), anti-CXCR6 (SA051D1), anti-CD103 (2E7). Samples were then fixed overnight at 4°C using the using 100 μL of Foxp3 Fix/Perm buffer (eBioscience). After membrane permeabilization using 1X permeabilization buffer (eBioscience) for 5 min, intracellular staining was performed for 120 min at room temperature using the following antibodies: anti-Ctla-4 (UC10–4B9, Biolegend), anti-Helios (22F6, Biolegend), anti-Foxp3 (FJK16, Thermofisher), anti-Gata3 (TWAJ, Thermofisher), anti-RORγ (AFKJS-9, Thermofisher), anti-Ki-67 (16A8, Biolegend). Cells were acquired with an Aurora flow cytometer (Cytek Biosciences) or a FACSymphony flow cytometer (BD Biosciences). Data were analyzed using FlowJo software version 10 (TreeStar, BD LifeSciences).

Freshly thawed PBMC from CMV seropositive and seronegative donors were stained with CellTrace™ Violet and stimulated with CMV antigen in growth medium in the presence of neutralizing mouse monoclonal antibodies (mAb) anti-CTLA-4 (Biolegend; clone L3D10) and/or anti-PD-1 (Biolegend; clone A17788B). After 6 days of incubation, CellEvent™ Caspase-3/7 Green Detection Reagent was added to PBMC cultures 30 min, followed by Zombie Yellow™ viability dye, followed by anti-CD3 Alexa-Fluor 700 (eBiosciences) and anti-CD4 PC 5.5 (Beckman Coulter) for 30 minutes at room temperature. Lastly, cells were washed with Annexin V buffer diluted to 1X (eBiosciences) followed by Annexin V APC (eBiosciences). Cells were analyzed using a Gallios Flow Cytometer.