Anti human il 4

PBMCs were incubated with CD4 microbeads (Miltenyi, German) according to manufacturer’s instructions. CD4 T cells positive separations were performed by MiniMACS ™ Separator. CD4 T cells were resuspended in RPMI1640 supplemented with 10% FBS, 1 mM l-glutamine, 55 uM β-Mercaptoethanol, 200U/ml penicillin, and 100 ug/ml streptomycin. The cells were seeded at 5 × 10⁵/mL in 24-well plates which were precoated with 2 ug/ml anti-CD3 (BioGems, USA), and stimulated with 4 ug/ml anti-CD28 (BioGems, USA) and 10 ng/ml IL-2 (PeproTech, USA). IL-12 (50 ng/ml, PeproTech, USA) and anti-human IL-4 (10 ug/ml, PeproTech, USA) were used for T helper1 (Th1) culture. IL-4 (50 ng/ml, PeproTech, USA) and anti-human IFN-γ (10 ug/ml, PeproTech, USA) were used for Th2 culture. IL-6 (50 ng/ml, PeproTech, USA), IL-1β (10 ng/ml, PeproTech, USA), IL-23 (10 ng/ml, PeproTech, USA), TGF-β (10 ng/ml, PeproTech, USA), anti-human IFN-γ (10 ug/ml), and anti-human IL-4 (10 ug/ml) were used for Th17 culture. TGF-β (10 ng/ml), anti-human IFN-γ (10 ug/ml), and anti-human IL-4 (10 ug/ml) were used for regulatory T cells (Tregs) culture.

Buffy coats of leukocytes were obtained from anonymous HIV- donors (New York Blood Center). Peripheral blood mononuclear cells (PBMCs) were harvested by density centrifugation with Ficoll-Paque (GE Healthcare). Naïve CD4⁺ T cells were isolated by MACS negative selection using the human naïve T-cell isolation kit (Miltenyi Biotec) to yield a population with the phenotype CD4⁺ CD45RA⁺ CD45RO⁻ CCR7⁺ CD62L⁺ CD27⁺ with purity levels equal or higher than 95%. Cells were then activated for 72 hours with beads coated with αCD3 and αCD28 antibodies, Dynabeads Human T-Activator CD3/CD28 for T cell Expansion and Activation, (ThermoFisher Scientific) under non-polarizing (NP) conditions, in the presence of 10 ng/ml of TGF-β1, 2 μg/ml of anti-Human IL-12 and 1 μg/ml of anti-Human IL-4 (all from Peprotech). Dynabeads were removed on day 3 and consequently cultured at a density of 1 × 10⁶ cells/ml in complete medium with 30 IU/ml of rhIL-2 (R&D Systems). Cells were infected by spinoculation on Day 5: 10⁶ cells were infected with 300 ng/mL p24 for 2 hours at 2900 rpm and 37 °C in 1 mL. After infection, cells kept in culture at 1 × 10⁶ cells/ml in complete medium with 30 IU/ml of rhIL-2 at 37 °C for 7 days prior to further experimentation, during which actively infected cells die, leaving a mixed population of latently infected or uninfected cells.