Nivolumab

The stimulatory function of the electroporated DCs was assessed by their ability to induce in vitro proliferation of autologous nonadherent PBMCs used as a source of enriched T-cells. These T-cells (2 × 10⁵ cells/well) were incubated in X-VIVO 10 medium with the DCs (6 × 10⁴ cells/well) previously electroporated with TMEP-B or TMEP-Bmod mRNA, and then they were seeded in 96 round-well plates at 37 °C in a humid 5% CO₂ atmosphere at a ratio of 1 DC to 3 T-cells. Mock electroporated DCs were used as negative controls. To determine the roles of Nivolumab (Anti-PD1) and Vesatolimod (TLR7 agonist) on T-cell proliferation, we added 20 µg/mL of Nivolumab (MedChemExpress) and 1000 nM of Vesatolimod into the DC/T-cells co-culture either separately or together. Both agents were added when setting up the co-cultures that also contained the cytokine cocktails. To assess T-cell proliferation, these were stained with carboxyfluorescein succinimidyl ester (CFSE) using the Cell Trace CFSE proliferation kit (Invitrogen) according to the manufacturer’s instructions. T-cell proliferation was measured by flow cytometry of CFSE dilution after 6 days of co-culture and expressed as the percentage of CFSE^low cells after 6 days of co-culture under the different conditions.

PD1 expression on T-cells was blocked by incubating with 20 µg/mL Nivolumab (anti-PD1, MedChemExpress, NJ, USA) for 30 min at 37 °C. After incubation, cells were washed twice with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide, then washed once with PBSx1 before being centrifuged at 1500 rpm for 5 min. Next, cells were incubated with Fc Block (Milteny Biotech, Bergisch Gladbach, Alemania) for 30 min at 4 °C and then washed with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide before being centrifuged at 1500 rpm for 5 min. The cells were then subjected to surface staining using anti-Nivolumab PE (Anti-Human IgG4 pFc, Abcam, Cambridge, United Kingdom), anti-PD1 PE-Cy7 (Clone: EH12.1, BD Pharmingen, San Jose, CA, USA), and CD3 PERCP (Clone: HIT3a, BD Pharmingen) [27 (link)]. The corresponding isotypes and tube with cells without previous Nivolumab incubation were used as controls. PD1 expression and Nivolumab binding through human IgG4 were evaluated by flow cytometry using FACSCanto II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).