Ficoll histopaque 1077 gradient

EBV B95-8 virus was produced from the B95-8 Z-HT cell line, as previously described [36 (link)]. Buffy coats were obtained from normal donors through the Gulf Coast Regional Blood Center and peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll Histopaque-1077 gradient (Sigma, St. Louis, MO, USA #H8889). Primary human B cells were cultured in Roswell Park Memorial Institute medium (RPMI)1640 supplemented with 15% fetal bovine serum (FBS), 2 mM l-glutamine, penicillin, and streptomycin (1X, Sigma, #G6784) (R15) as well as 0.5 µg/mL Cyclosporin A (CsA) (Sigma, #30024). Bulk infections were performed by incubating cells with B95-8 Z-HT supernatant at 500 μL per 10⁶ B cells calculated from within the PBMC population for 1 h at 37 °C in a CO₂ incubator. Incubation was followed by washing in PBS and resuspending in R15 media supplemented with CsA. Bulk infections were conducted on 5 × 10⁸ PBMCs. LCLs were generated from normal donors by continuous outgrowth of EBV-infected primary B cells for greater than 35 days. LCLs were cultured in RPMI media supplemented with 10% FBS (R10). Bloom syndrome (BLM) deficient LCLs (GM16377, GM16375, and GM09960) were obtained from Coriell Institute (Coriell Institute, Camden, NJ, USA).

Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from the Gulf Coast Regional Blood Center (Houston, TX) via centrifugation over a Ficoll Histopaque-1077 gradient (Sigma, catalog no. H8889). The B95-8 strain of Epstein-Barr virus was generated from the B95-8 Z-HT cell line as previously described (39 (link)). Virus infections were performed by adding either 100 μl of filtered B95-8 Z-HT supernatant to 10⁶ PBMCs or 500 μl of B95-8 Z-HT per 10⁶ B cells, as determined by FACS analysis.
Cell lines were cultured in RPMI 1640 media supplemented with 10% (LCLs) or 15% (primary B cells) fetal bovine serum (FBS) (Corning), 2 mM l-glutamine (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). All cells were maintained at 37°C in a humidified incubator with 5% CO₂.

The cell lines MCF7 (human breast cancer, ATCC), Me30966 and Me501 (human metastatic melanoma), and SW480 (human colon carcinoma) supplied by Fondazione IRCCS Istituto Nazionale dei Tumouri, Milan, Italy [23] (link), [31] (link), were cultured in RPMI 1640 (Gibco Laboratories, Grand Island, NY, USA) supplemented with antibiotics (Sigma-Aldrich, St. Louis, MO) and 10% fetal bovine serum (FBS, Gibco) in humidified 5% CO₂ and 95% air atmosphere. Human PBMC (Peripheral Blood Mononuclear Cells) were isolated from buffy coats by Ficoll-Histopaque 1077 gradient (Sigma-Aldrich). Buffy coats were provided by Centro Trasfusionale Universitario Azienda Policlinico Umberto I in Rome, Italy (the study was approved by the ethical committee of Istituto Superiore di Sanità, Rome, Italy, and donors gave written-informed consent to participate). Unbuffered culture medium (UNB) was prepared without sodium bicarbonate. Different pH mediums were controlled by a pH meter (Metrohm AG, mod. 691, Herisau, Switzerland). Experiments were performed in buffered medium (pH 7.4), unbuffered medium (UNB w/o sodium bicarbonate, initial pH 7.2) or buffered acidic medium (pH 5.0 or 6.0). The cell lines were negative for mycoplasma contamination, as routinely tested by modified nested polymerase chain reaction (VenorGeM Kit, Minerva biolabs, Germany).

Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Histopaque^® 1077 gradient (Sigma-Aldrich, Lyon, France). From PMBCs derived from fresh blood, innate lymphoid cells were enriched by negative immunomagnetic selection (EasySep Human Pan-ILC Enrichment Kit, cat# 17975, StemCell). The kit contained a cocktail of magnetic antibodies targeting the major cell lines of the immune system except ILCs. Enrichment was performed according to the manufacturer’s instructions (36 ). Freshly enriched ILCs were cultured in a complete medium consisting of RPMI supplemented with 10% human serum AB (sHAB), 10 ng/mL IL-7 (PeproTech^®, Neuilly sur Seine, France), 1 mM sodium pyruvate (Gibco) and antibiotics (penicillin 100 I.U./mL and streptomycin 100 μg/mL). Different combinations of media were tested in the presence of IL-2, IL-12, and/or IL-7 at different concentrations (at 5, 10, 20, 30, or 90 ng/mL) or a commercial medium specific for NK cells (NK MACS Medium, cat# 130-114-429, MiltenyiBiotec) with lower efficacy. From the ~60 donors, we obtained, after enrichment, between 2.7 x 10⁵ and 9 x 10⁵ cells/donor, with an average of 6.3 x 10⁵ cells/donor. The percent yield of this enrichment protocol was from 0.02% to 0.7% with a mean of 0.18%. The predicted yield is 0.05 to 0.07% of mononuclear cells.

Blood of 20 healthy donors and 20 melanoma patients was provided by Centro Trasfusionale Universitario and Clinica Dermatologica of Azienda Policlinico Umberto I, University Sapienza, Rome, Italy. The study was approved by the ethical committee of Azienda Policlinico Umberto I, and subjects gave written informed consent to participate. Human PBMC were isolated with Ficoll-Histopaque 1077 gradient (Sigma-Aldrich, St. Louis, MO, United States). Ex vivo expanded human NK cells were obtained as previously described (22 (link)). Briefly, PBMCs from buffy coats were cocultured with cobalt-irradiated B lymphoblastoid Roswell Park Memorial Institute (RPMI) 8866 cells. On day 7, cells were incubated with human rIL-2 (100 U/ml; Hoffman-La Roche, Nutley, NJ, United States) for 3 days. The resulting NK cell population was >80% CD56⁺, CD3⁻, and CD14⁻ as assessed by flow cytometry analyses (cell viability, >90%). Using this culture method, an average of 30–40-fold increase in activated NK cell number was obtained. The supernatant of NK cell culture was properly frozen at −80°C for NKEVs isolation.

Collected tissues (lung and spleen) were homogenized with a stomacher lab blender (Seward, England) into single‐cell suspensions in cold SB. Red blood cells (RBCs) were removed from samples by suspension in appropriate amount of RBC lysis buffer (Sigma‐Aldrich, USA) for 3‐5 minutes and washed with SB. Samples were then filtered with 100‐μm cell strainers (Fisher Scientific, China). To isolate mononuclear cells from the lungs, we used Ficoll gradient Histopaque‐1077 (Sigma‐Aldrich, USA) as previously described.16 Isolated cells from the lungs and spleens were counted with trypan blue stain (Sigma‐Aldrich, USA) using a Neubauer haemato‐cytometer chamber (VWR Scientific, USA). For T cell subset determination, isolated cells were stained with the following antibodies: CD3‐FITC or APC, CD4‐PE/CY7 or APC, and CD8‐PE (Biolegend, USA). Stained samples were analysed with a flow‐cytometer FC500 (Beckman Coulter Inc, USA).