Il 2 bv605

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.

All cell culture supplies were obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA), unless otherwise specified. Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). All TLR7/8 compounds were synthesized and characterized as previously reported^{27 (link)}. Frozen human PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH). Fluorophore labelled anti-human monoclonal antibodies (CD3-FITC, CD56-PE/Cy7, CD11c-PE/Cy7, CD19-PE, GranzymeB-PE, IL-6-APC, TNF-α-APC/Cy7, IFNγ-BV421 and IL-2-BV605) and Brefeldin A were purchased from Biolegend (San Diego, CA).