Cd3 apc h7

Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7).

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.

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD4 Alexa Fluor® 700, CD8 APC, CD39 PE-Cy7, CD73 FITC, and CD73 eFluor450, CCR7 (CD197) PE-Cy7, PD1 (CD279) PE (eBioscience); CCR7 PE-CF594, CD45 AmCyan and CD45 FITC (Becton Dickinson), CD3 APC-H7, CD25 FITC (BioLegend) and CD25 PE (MACS Miltenyi). All mAbs were titrated using normal PBL to establish optimal dilution. T_reg were defined by their ability to produce extracellular adenosine as previously described by Deaglio and Borselino [28 (link), 29 (link)]. We have previously demonstrated that these CD4⁺ CD39⁺ CD25⁺ T_reg are FOXP3⁺ CD127^neg [24 (link)].