Human il 7

Fresh lung ILC2s (CD45⁺Lin⁻CD127⁺CD90.2⁺ST2⁺) sorted from IL33-treated mice were cultured in 96-well round-bottom plates at a density of 5 × 10³ cells per well in RPMI-1640 medium containing 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in the presence of 10 ng/ml human IL-2 (PeproTech), 20 ng/ml mouse IL-7 (PeproTech, 217-17-100), 1 ng/ml mouse IL-33 (BioLegend, 580506), 20 ng/ml human IL-7 (R&D Systems, 207-IL-010) and 50 ng/ml human IL-33 (R&D Systems, 3625-IL-010) with or without α-MSH (10 ng/ml) or β-endorphin (MCE, HY-P1502, 10 ng/ml). Then, the levels of cytokines in the culture supernatants were measured by ELISA (mouse IL-5 ELISA kit, Invitrogen, 88-7054-76; mouse IL-13 ELISA kit, Invitrogen, 88-7137-76; human IL-5 ELISA kit, Invitrogen, 88-7056-88; and human IL-13 ELISA kit, Invitrogen, 88-7439-88). For intracellular cytokine staining, 50 ng/ml PMA, 1 µg/ml ionomycin, and 1 µg/ml BFA were added to the cultures 2–4 h before staining.

HLACs, PBMCs, or SupT1-R5 (10⁶ cells/mL) were mock-treated or infected with F4.HSA at a final concentration of 1500–3000 ng/mL p24^Gag, and cultured for 3 days at 37˚C. Where appropriate, primary cells were pre-activated with 10 μg/ml PHA (Sigma) or with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher), using the appropriate media supplemented with 100 U/mL human interleukin-2 (IL-2) (Life Technologies). To assess infection rates, cells were subjected to flow cytometric analysis as detailed below. For experiments where samples were sorted after infection, cells were first enriched by negative selection for CD4+ T cells using the EasySep Human CD4+ T Cell Enrichment Kit (STEMCELL Technologies) to minimize sort time. Where indicated, sorted cells were mock-treated or stimulated for 2–3 days with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) at a ratio of 1 bead/cell in the presence of 60U/ml IL-2 (Life Technologies), 12 nM PEP005 (Ingenol 3-angelate, Sigma-Aldrich), 40 nM Romidepsin (Sigma-Aldrich), or 10 ng/ml human IL-7 (R&D System), and then harvested for flow cytometric analysis as detailed below.