Cd8 apc

Whole blood was labeled with CD3 PC7, CD4 PE and CD8 APC (Becton Dickinson, San Jose, CA, USA). Erythrocytes were lysed twice with EasyLyse (Dako) before washing in PBS. Cells were resuspended in AnnexinV buffer and stained with AnnexinV FITC (Tau technologies, Kattendijke, The Netherlands) and Dapi (Sigma-Aldrich) before assessment of apoptosis by flow cytometry on Fortessa X-20 (Becton Dickinson). Data were analyzed using Kaluza 2.0 software.

Intracellular cytokine staining (ICS) assays were performed as previously described [19 (link)]. In brief, splenocytes and lung lymphocytes were isolated and seeded into 96-well plates, incubated with peptide pools of Ag85A or Mtb32 (2 μg/ml per peptide) or with 40 ng/ml Phorbol 12-myristate-13-acetate (PMA) and 1000 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO). One hour later, brefeldin A (10 μg/ml, BD Biosciences, San Diego, CA) was added and the PMA+ionomycin-stimulated cells were incubated for additional 5 h, whereas the peptide pool-stimulated cells were incubated for additional 10 h. The cells were harvested and stained with surface antibodies (CD3-PerCP, CD4-FITC, CD8-APC; BD Biosciences, San Diego, CA) for 1 h and were then washed, permeabilized, and stained with intracellular antibodies (IFN-γ-PE; BD Biosciences, San Diego, CA). Finally, the cells were detected with a BD Accuri™ C6 instrument.
To assess Tregs in the spleen and mediastinal lymph nodes (MLN), lymphocytes were isolated and stained with surface antibodies (CD3-Pacific Blue, CD4-FITC, CD25-APC; BD Biosciences, San Diego, CA) for 1 h and then washed, permeabilized, and stained with intracellular antibody (FoxP3-PE; BD Biosciences, San Diego, CA). The cells were detected with a BD Accuri™ C6 instrument.

PHA-blast target cells from 10 HIV-uninfected donors, either BAGN-treated or -untreated, and stained for surface activation markers and HIV co-receptors expression, were further used for the VRAs. At day 3, both BAGN-treated and -untreated target cells were infected (MOI = 0.001) with the viral stocks produced with or without BAGN in the culture medium. Cells were cultured for 7 days in R20/50, with or without 0.2 mM BAGN, at 37°C in a 5% CO₂ containing atmosphere. Secretion of p24 into the culture supernatant was measured by ELISA (Fujirebio) and intracellular p24 production was measured by flow cytometry. Briefly, cells were stained with Live/Dead violet fluorescence fixable dead cell stain reactive kit (Invitrogen) followed by extracellular staining with antibodies: CD3-APC-H7, CD4-PreCp, and CD8-APC (BD Biosciences). The cells were fixed and permeabilized (Fix and Perm, Invitrogen) while staining for HIV Gag p24 using the KC57-FITC antibody (Coulter). As before, cells were collected on an LSR II instrument (Becton Dickinson) and analysis performed using the FlowJo software.

All mice were on the C57BL6/J background. PD-1^−/− mice were kindly provided by Professor Arlene Sharpe, Harvard Medical School (30 (link)). Wild-type (WT) mice were purchased from Janvier, France. Twelve-week-old mice were sacrificed. The spleens were homogenized through a 70 µm filter and washed prior to red blood cell lysis (Sigma-Aldrich). The cells were subsequently washed twice and seeded in a 6-well plate with 30 × 10⁶ cells/well. Cells were stimulated with plate-bound anti-mouse CD3, 1 µg/ml (clone: 145-2C11, BD) and anti-mouse CD28, 2 µg/ml (clone: 37.51, BD) in EV free media. After 72 h of stimulation EVs were collected from WT cells according to the previously described protocol for human cells. The collected EVs were co-cultured with PD-1^−/− cells and anti-CD3/anti-CD28 for 48 h. Cells were analyzed by flow cytometry, using anti-mouse PD-1 BV421 (clone: 29F.1A12, Biolegend), -CD3 PerCP-Cy5.5 (clone: 145-2C11), -CD4 PE (clone: GK1.5), -CD8 APC (clone: 53-6.7), -CD19 FITC (clone: ID3) (all from BD), and Live/Dead Aqua fix (Thermo-Fischer). Antibodies were used in the concentrations recommended by the manufacturer. Doublets and dead cells were excluded and gating was done on CD3⁺CD4⁺ T cells.