Il 17a bv510

Exogenous full-length, N-terminal, and C-terminal CRDs at a concentration of 350 nM were incubated with 1 × 10⁶ PBMCs of HIV-infected patients in a humidified 5% CO₂ incubator at 37°C for 24 h. Cells were stained with antibodies against surface markers like CD3 APC H7, CD8 BUV737, Tim-3 PECF594, CD45RA PECy5, and CCR7 BUV395 (BioLegend and BD Biosciences, USA) antibodies for 30 min at room temperature. The cells were permeabilized (Permeabilizing Solution II, BD Biosciences, USA) and then stained with an intracellular antibody cocktail for 30 min at room temperature against cytokines and proliferation markers interleukin (IL)-2 BV605, IL-17a BV510, IL-10 PE, interferon (IFN)-γ PECy7, Ki-67 BV786, and FITC P24 (KC-57) (BioLegend, BD Biosciences, Beckman Coulter, USA). The cells were acquired on FACS Fusion I (BD Biosciences, USA) within 24 h to get 50,000 of the gated events of lymphocytes. The data analysis was done by using FACSDiva software version 9.0.1 and FlowJo version 8.0.3.

Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 10⁶ single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO₂. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).