Ki 67 pe

The cells were treated with an FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated with each fluorochrome-conjugated antibody or appropriate isotype control at 4 °C for 30 minutes in the dark. For hnRNPA27B1 staining, cells were fixed for 15 minutes at 4 °C with 2% paraformaldehyde (PFA) and washed with staining buffer. The T-cell phenotype was determined using CD25 PerCP-Cy™5.5, PD-1 (CD279) PE, CD4 FITC, CD8 PE all purchased from BD Biosciences. Intracellular staining for hnRNPA2/B1 (Abcam, cat. no. ab6102; dilution 1:200, Milan, Italy) and Ki67 PE (BD, Bioscience) was performed using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences, Milan, Italy) according to the manufacturer’s instructions. Secondary antibodies were incubated for 1 h at 4 °C (Alexa Flour 488- or PE-conjugated anti mouse IgG was used at dilution 1:300, Thermo Fisher Scientific, Milan Italy) and samples washed twice before reading. All data were acquired on a FACSCalibur and analysed using CELLQuest Pro software (BD Pharmingen, San Jose, CA). Compensation controls for each fluorochrome with positive and negative populations, unstained and IgG controls were acquired using the BD Calibrite 3-Color Kit Beads (Catalog n° 340486, BD, Milan, Italy) and single/double positive antibodies for the FL1, FL2 channels using fresh activated PBMCs.

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside-treated and -untreated PHA-blasts were used to monitor expression of surface markers of cell activation and HIV co-receptor CCR5 and CXCR4. Briefly, PHA-blasts were prepared from 20 HIV-negative donors by stimulation with PHA for 2 days. One-half of the cells from each sample was then treated with 2 mM of BAGN overnight. The next day, cells were stained for viability markers (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) and markers of T cell activation (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, CD8-V500, CD25-APC, HLA-DR-FITC, and CD38-PerCP-Cy5.5; BD Biosciences). Cells were fixed and permeabilized (Fix and Perm, Invitrogen) to stain with Ki67-PE (BD Biosciences). Additional aliquots of BAGN-treated and -untreated PHA-blasts were used to stain for viability and T cell subsets as before and for HIV co-receptors (antibodies: anti-human CD184-APC (CXCR4), CD195-PE (CCR5); BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and analysis was performed using FlowJo software.

Collected cells (from cultures or disintegrated tumor tissue) were fixed for 15 min in 1.6% paraformaldehyde (PFA) at room temperature (RT) and permeabilized with 100% ice cold methanol. Up to four samples were given a fluorescent “barcode” by adding pacific orange (PO) dye (Life Technologies, Carlsbad, CA) at different concentrations ranging from 0 to 2 ng/μl (see Supplementary Figure S8B). For the analysis of the in vivo samples, we also included a “spike” control (Melmet 5 from in vitro), which received no PO (see Supplementary Figure S8C). After incubation at RT for 30 min, followed by washing, the barcoded samples were combined for simultaneous staining with antibodies against pS6-Alexa 647 (Cell Signaling Technology, #4851 dilutions 1:80 for in vitro and 1:50 for in vivo samples), pERK-PE (Cell Signaling Technology, #5315; dilution 1:5) or Ki-67-PE (BD Biosciences, #556027; dilution 1:5) for 30 min at RT. For cell cycle analysis, pS6-Alexa 647 stained cells were additionally stained with 1.5 μg/ml Hoechst 33258 (Life Technologies, #H3569) for 30 min at 37°C. The samples were analyzed on an LSR II flow cytometer (BD Bioscience, San Jose, CA). BD FACS Diva^™ software was used to control the flow cytometer and Flow Jo software (FlowJo, Ashland, OR) was used to analyze the data.

The following conjugated Anti-human monoclonal antibodies (mAbs) were purchased from BD Pharmingen, USA: CD3 PE-Cy7, CD4 FITC, CD4 PerCP-Cy5.5, CD4 APC, CD8 PerCP-Cy5.5, CD14 PErCP-Cy5.5, CD14 PE, CD80 FITC, CD86 PE, CD152 PE, HLA-DR APC, Ki67 PE, Annexin V FITC, and Propidium Iodide. CD28 FITC and CD45 Pacific blue were purchased from eBioscience, USA.

The following antibodies were used in this study: CD3ε-eFlour 450 (145-2C11, eBioscience), CD3ε-APC-eFluor 780 (17A2, eBioscience), CD8α-APC-eFluor 780 (53-6.7, eBioscience), CD8α-BV605 (53-6.7, Biolegend), CD8α-PerCP-Cy5.5 (53-6.7, eBioscience), CD44-PE-Cy7 (IM7, Biolegend), CD62L-APC (MEL-14, eBioscience), CD62L-BV510 (MEL-14, Biolegend), CD69eFluor 450 (H1.2F3, eBioscience) CD69-biotin (H1.2F3, eBioscience), CD122-FITC (TM-B1, BD Biosciences), CD127-BV605 (A7R34, Biolegend), and Streptavidin PerCP-Cy5.5 (BD Biosciences). The MHC class I tetramers H2-D^bGP_33–41 APC and H2-D^bNP_396–404 PE were kind gifts from Dr. Ramon Arens (LUMC, Leiden, The Netherlands). Cells were fixed with Foxp3/Transcription Factor Staining buffer set (eBioscience) and stained with Ki-67 PE or Ki-67 FITC (B56, BD Biosciences). Samples were acquired with the LSR Fortessa (BD) and analyzed with FlowJo software (Tree Star, Inc.).

Total and resting CD4+ T cells were isolated as described previously from buffy backs from three HIV-uninfected individuals from the Red Cross blood transfusion service. CD4+ T cells were plated at 500,000 cells per well in a 96-well plate and stimulated with LRAs for 1, 3 and 6 days as described above. Following stimulation, cells were stained with live/dead cell viability dye (Life Technologies, cat # L34955) for 15 min at room temperature followed by surface staining for CD3-BV711 (BD Biosciences, cat # 563725, RRID:AB_2744392) and CD4-APC (BD Biosciences, cat # 555349, RRID:AB_398593) on ice for 30 min. Cells were then intracellularly stained for Ki-67-PE (BD Biosciences, cat # 556027, RRID:AB_2266296) using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences, cat # 554714) according to the manufacturer's protocol. Flow cytometry was assessed on an LSRFortessa (BD Biosciences) and data were analyzed using FlowJo v.10.