Clone f38 2e2

Freshly isolated Tros were seeded at a density of 2 × 10⁵ cells/ml per well in Matrigel (Coring, U.S.A)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, U.S.A). Equal numbers of dCD14⁺ cells or pCD14⁺ cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87 G; Biolegend, U.S.A) were added. dCD14⁺ cells were also cultured with HTR8/Svneo cells (ATCC, www.atcc.org) or DSCs for 48 h. In some wells, dCD14⁺ cells (2 × 10⁵ cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while Tros were plated in the lower chamber to establish indirect cell contact.
Freshly isolated dCD4⁺ T cells co-cultured with Tim-3⁺dMφs or Tim-3⁻dMφs at a 1: 1 ratio. In some experiments, Tim-3⁺dMφs were pretreated with anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), or anti-CD132 (10 μg/ml, clone TUGh4, BioLegend, U.S.A.), or anti-IL-4 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.). Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.), were added 4 h before the end of the 48 h co-culture. The supernatants were then collected for intracellular cytokine analysis of T cells.

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.

dCD8⁺ T cells were cultured (5 × 10⁵ per well) in the presence of anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, USA), anti-CTLA-4 (10 μg/ml, clone L3D10, BioLegend, USA), both of the two antibodies, or isotype control for 48 h. Brefeldin A, PMA, and ionomycin was added 4 h before the end of the culture. The cells were then collected for further analysis by flow cytometry.