Il 32γ

CD4⁺ T-cells were isolated from total PBMCs by negative selection using EasySep Human CD4 T Enrichment columns (StemCell, Cat #19052). Purity of CD4 was typically >98% as determined by surface staining of CD4⁺ T-cells with CD3-Pacific Blue (UCHT1) and CD4-Alexa Fluor 700 (RPA-T4), (both from BD) and FACS analysis (BD LSRII analyser). Purified CD4⁺ T-cells were stimulated with plate-coated anti-CD3 antibody (0.5μg/ml) and soluble anti-CD28 antibody (0.5μg/ml) (both from BD Biosciences, Cat ##555329, 5555726 respectively) for 48 h in the presence or absence of 500 ng IL-32α (BioLegend, Cat # 551004) or IL-32γ (R&D Cat # 4690-IL-025/CF). Secreted cytokines were measured from the supernatant of activated cells using the LEGENDplex™ Human Th Cytokine Panel (13-plex) (BioLegend, Cat # 740001) according to manufacturer’s directions. Infections of PBMCs stimulated with PHA and IL-2 (0.25μg/ml, and 100 units/ml, respectively) or resting cells were carried out using 50 ng of the laboratory strain HIV-BaL per 10⁶ cells by Spinoculation61 (link) (2 hours/Room temperature). Following infection, PBMCs were washed twice by PBS and centrifuged at 1500 rpm for 5 min then were re-suspended in RPMI-1640 medium supplemented with 10% FBS. Cells were kept in culture for 48 h before collection of supernatants and cells for cytokine measures.

PBMCs were resuspended at 2 million cells/ml in RPMI 1640 medium (Gibco by Life Technologies, Waltham, MA) complemented with 10% FBS and stimulated with 500 ng/ml of IL-32α, IL-32β, or IL-32γ (R&D Systems, Minneapolis MN) in final volume of 1 ml. Cells were incubated for 5 days at 37°C and 5% CO₂. For cell proliferation, PBMC were labeled with 2.5 μM of 5-(and-6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) according to manufacturer’s instructions (Sigma-Aldrich, St Louis, MO) and stimulated with 1 µg/ml of phytohemagglutinin-L (PHA-L) (Sigma) and 10 ng/ml IL-2 (R&D systems) for 4 days.