Clone a15153g

We selected cryopreserved PBMCs from S1 (n=10) and S2 (n=10). Samples were previously characterized by the expression of TIGIT and TIM-3 on total CD8+ T-cells. PBMCs were thawed and rested for four hours at 37 °C in a 5% CO₂ incubator. Next, cells were incubated under RPMI complemented medium 10% FBS with 1 μl/ml of anti-CD28/CD49d and 1 μl/ml of Monensin A overnight at 37 °C in a 5% CO₂. PBMCs are divided in the following conditions; (1) unstimulated, (2) SEB (1 μg/ml, Sigma-Aldrich) and (3) HIV-1-Gag peptide pool (2 μg/peptide/ml) in the absence or presence of αTIGIT and/or αTIM-3, and its respective isotype antibodies. For the single blockade of TIGIT (αTIGIT), we included Ultra-LEAF purified anti-human TIGIT antibody (10 μg/ml, clone A15153G, Biolegend) or its control isotype Ultra-LEAF purified mouse IgG2a antibody (10 μg/ml, MOPC-173, Biolegend). For single TIM-3 blockade (αTIM-3), we used Ultra-LEAF purified anti-human TIM-3 antibody (10 μg/ml, clone F38-2E2, Biolegend) or its respective isotype Ultra-LEAF purified mouse IgG1 antibody (10 μg/ml, MOPC-21, Biolegend). Finally, we included αTIGIT+αTIM-3 or their respective IgG2 + IgG1 isotypes for a combinational blockade. The next day, PBMCs were surface and intracellularly stained with the panel of antibodies and the methodology described in the section above.

Peripheral blood mononuclear cells (PBMCs) were collected from two OS samples (BC3 and BC16) using density centrifugation with Lymphocyte Separation Medium (MP Biomedicals). Then CD3⁺ T cells were isolated with magnetic-activated cell sorting system (MACS; Miltenyi Biotec) according to the manufacturer’s protocol. For the T-cell activation assays, CD3⁺ cells were seeded in 24-well plates and stimulated for three days with interferon-γ (IFN-γ, 1000 U/mL; Peprotech), IL-2 (600 U/mL, Peprotech), and anti-CD3 antibody (5 ng/mL, clone OKT3; Biolegend). Subsequently, TIGIT was blocked for 24 h with anti-TIGIT antibodies (50 μg/mL, clone #A15153G, Biolegend). 143B and U2OS cells were seeded in 96-well plates, incubated overnight, and then added to CD3⁺ T cells at effector-to-target (E:T) ratios of 4:1 and 8:1. Co-culture systems were incubated for 8 h. The supernatant was harvested and analyzed using the CytoTox 96® non-radioactive cytotoxicity assay (Promega, CTB163, USA). Each group had three parallel wells, and all experiments were performed at least three times^{96 (link)}. According to the manual, the killing effect of T cells against target cells was assessed with the following equation: Cytotoxicity = (Experimental–Effector Spontaneous–Target Spontaneous)/(Target Maximum–Target Spontaneous) × 100%.