Anti il 10

Cell supernatants were collected on day 3 and frozen until further use. Immunol 2HB microtiter plates (Thermo Scientific) were coated with anti-cytokine antibodies anti-IL-6 (Clone #6708), anti-IL-10 (Clone #127107), anti-TNFα (Clone #28401), and anti-M-CSF (Clone #21113) (R&D Systems, Minneapolis, MN, USA) and then blocked with PBS/2% BSA. Serially diluted standards and culture supernatants were added to these plates overnight. Plates were incubated with biotinylated anti-cytokine Ab (R&D Systems), followed by phosphatase-streptavidin (BD Biosciences) and K-Gold PNPP Substrate (Neogen Corporation, Lexington, KY, USA). Human IL-12p70 Quantikine, IL-4 Quantikine, and TGFβ1 Quantikine ELISAs were performed based on manufacturer’s instructions (R&D Systems). ELISAs were read using a SpectraMax M5 Microplate Reader and SoftMax Pro Acquisition and Analysis Software (both Molecular Devices, Sunnyvale, CA, USA).

Cells were thawed, washed and reconstituted in AIM V serum-free medium (Life Technologies, Oslo, Norway) containing 0.1% human serum albumin. After an overnight rest, cells were pulse-labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies) at a concentration of 2 μM for 5 minutes. The following blocking antibodies were added to parallel culture wells at a final concentration of 10 μg/mL: anti-IL-10 (R&D Systems, MN, USA; clone 23738), anti-TGF-β (R&D; clone 1D11), anti-PD-L1 (eBioscience, CA, USA; clone MIH1), and anti-HVEM (R&D; clone 94801).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO₂ for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).

Proliferation Assays. CD4⁺CD25^high (all Foxp3⁺) cells isolated from the secondary lymphoid organs were magnetically sorted from the spleen of Foxp3-GFP-KI C57BL/6J mice. They were loaded with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) and cultured (5× 10⁴ cells per well) in RPMI medium 1640 supplemented with 5% (vol/vol) FCS (Life Technologies), 1% antibiotics, and 5 × 10⁻⁵ M β-mercaptoethanol. Cells were plated in 96-well round-bottomed culture plates, either alone or with sorted MPP at 1:1 T:MPP cell ratios, and stimulated with 2.5 μg/ml of anti-CD3 mAb (clone 145–2C11) and 5 μg/ml of anti-CD28 mAb (clone 37.51, eBioscience) for 4 days. Inhibitors were added at 5 to 20 µg/ml: anti-CD137L (clone TKS-1, Biolegend), anti-CD80 (clone 16-10A1, Biolegend), anti-GITRL (clone 5F1, Biolegend), anti-CD86 (clone GL-1, BD Biosciences), anti-OX40 (polyclonal goat IgG, R&D Systems), anti-TGFβ (clone 2G7, grown in our laboratory), anti-IL10 (clone JES052A5, R&D).