Anti cd3 apc h7

As the source of effector cells, we used both PBMCs and BM samples derived from MM patients at different state disease and PBMCs from healthy donors. K562 were used as target cells that were co-incubated with effector cells in complete medium at 2.5:1 effector/target (E/T) ratio for 2 h at 37 °C and 5% CO₂ [11 (link)]. In some experiments, different human cell lines such as SKO-007(J3), ARK and ARP and primary malignant plasma cells were used as targets. Thereafter, cells were washed with PBS/2% FCS and stained with the lysosomal marker CD107a/APC (BD Biosciences, San Jose, CA) and anti-CD3/APC-H7, anti-CD56/PE, anti-CD45/PE-Cy7, anti-CD16/PerCP for 45 min at 4 °C. All the samples were acquired using a FACSCanto II (BD Biosciences, San Jose, CA) and data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).

To quantify the expression of CD38 and HLA-DR on CD8⁺ T cells, cryopreserved PBMC were thawed and stained with the following antibodies: anti-CD3 APC-H7, anti-CD4 PECF594, anti-CD8 APC, anti-CD38 BB515, and anti-HLA-DR PE (BD Biosciences, United States), and acquired using a BD FACSAria IIu Flow Cytometer (BD Biosciences, United States). The Fixable Viability Stain 450 (FVS 450-BD Biosciences, United States) was used to exclude non-viable cells. Flow cytometric analyses were performed with FlowJo v.10.0.7 (Tree Star Inc., Ashland, OR, United States). Plasmatic levels (pg/mL) of IP-10, IL-18, RANTES, CTACK, and PDGF-AA were measured using the human Magnetic Luminex Performance Assay (R&D systems, United States), following manufacturer’s instruction and the analyses were performed on a Luminex 200 System (Luminex, United States).

Single cell suspensions of leukocytes from blood, ascites, tumor or omental metastases were stained with the following monoclonal antibodies: anti-CD3 APC-H7 (BD Pharmingen, 560176), anti-CD4 eFlour 450 (eBioscience, 48-0048-42), anti-CD8 Pacific Orange (Invitrogen, MHCD0830), anti-CD25 APC (BD Pharmingen, 555434), or anti-CTLA-4 APC (BD Pharmingen, 555855), anti-CD28 PerCpCy5.5 (eBioscience, 45-0289-42), anti-CD38 PE-TR (Invitrogen, MHCD3817), anti-HLA-DR PE-Cy7 (BD Biosciences, 335795), anti-FoxP3 PE (eBioscience, 12-4777-42), anti-ki67 FITC (BD Pharmingen, 556026) or CD39 FITC (BD Pharmingen, 561444) or anti-Helios (Biolegend, 137214). Cells were stained in FACs buffer (1%FBS in PBS with 0.01%NaN₃) and fixed according to the ebioscience FoxP3 Fix-Perm kit protocol (eBioscience, 00-5521-00). All samples were run on a BD LSRII Flow cytometer and analysed by FACSDiva BD. Briefly, for every single flow cytometric antibody, we have used Fluorescent Minus One (FMO), to discriminate between positive and negative cells [35 (link)].

For staining of inhibitory receptors, stimulated PBMCs were surface stained with anti-CD4 (V500; BD Biosciences), anti-CD3 (APC-H7; BD Biosciences), anti-CD8 (Alexa Fluor 405; Invitrogen), anti-PD-1 (FITC; BD Bioscience) and anti-human TIM-3 antibody (PE; R&D Systems) for 30 min at 4°C. Cells were washed, fixed, permeabilized (Invitrogen), and intracellularly stained with anti-IFN-γ (PE-Cy7; BD Biosciences), anti-IL-2 (BV605; BD Biosciences), and anti-CTLA-4 (APC; BD Biosciences) for 30 min at 4°C, washed and fixed with 1% formaldehyde. Cells were analyzed using a LSR-II flow cytometer (BD Immunocytometry Systems). One million events were collected. Electronic compensation was performed with Ab capture beads (BD Biosciences) stained separately with individual monoclonal Abs used in the test samples. The data files were analyzed using Diva (BD) and FlowJo Software (Treestar, Co). The expression of TIM-3, PD-1 and CTLA-4 was examined on cytokine-producing cells with frequencies ≥0.03% above the background (media control tube) to ensure an adequate number of events for analysis as previously validated by our laboratory [15 (link)–18 (link)]. Flourescence minus one (FMO) controls were used to set the gates for determining the mean fluorescent intensity (MFI) of TIM-3, PD-1 and CTLA-4 on Gag-responsive T cells expressing IFN-γ or IL-2.