Rpmi 1640

Bone marrow-derived cells from C57BL/6 mice were cultured in RPMI 1640 media supplemented with 10% FBS and 1% penicillin/streptomycin and differentiated to macrophages (bone marrow-derived macrophages (BMDMs)) by recombinant murine GM-CSF (25 ng/ml; Miltenyi Biotech) for 7 days. Peritoneal macrophages (PMs) were isolated from the peritoneal cavity 3 days after the intraperitoneal injection of a 3% thioglycollate solution (Fluka, Sigma-Aldrich, St. Louis, MO, USA). PMs, the murine-derived macrophage cell line RAW 264.7 cells, were cultured using RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 1% (v/v) penicillin/streptomycin (Sigma-Aldrich). All types of cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

Gag‐specific CD4⁺ T cell clones (F12) are specific for HIV Gag‐p24 (gag2: aa 271–290) and restricted by HLA‐DRβ1*01 as previously described (Moris et al, 2006 (link); Coulon et al, 2016 (link)). HCMV‐specific CD4⁺ T cell clones are specific for HCMV pp65 antigen (pp65: aa 108–127) and restricted by HLA‐DRβ1*01. Pp65‐specific clones were isolated from PBMCs of healthy donors after several rounds of in vitro stimulation with synthetic peptide corresponding to immunodominant epitopes from the pp65 protein. Pp65‐specific cells were isolated using the IFNγ secreting assay from Miltenyi Biotec and cloned by limiting dilution. F12 and pp65 clones were restimulated and expanded, as previously described (Moris et al, 2006 (link)), using irradiated feeders and autologous or HLA‐matched lymphoblastoid cell lines loaded with cognate peptides in T cell cloning medium: RPMI 1640 containing 5% human AB serum (Institut Jacques Boy), recombinant human IL‐2 (100 IU/ml, Miltenyi Biotec), PHA (0,25 μg/ml, Remel), nonessential amino acids, and sodium pyruvate (both from Life Technologies). At least 1 h before co‐culture with HeLa‐CIITA cells, T cell clones were thawed and allowed to rest at 37°C in RPMI containing DNAse (5 μg/mL, New England Biolabs).

Single liver cell suspensions were prepared by first homogenizing the whole tissue in RPMI 1640 using a gentle MACS dissociator (Miltenyi Biotech, Auburn, CA, USA). The resulting homogenate was then centrifuged at 931 g, and the cell pellet was mixed with 33% Percoll (Sigma-Aldrich) in RPMI 1640 solution (Invitrogen). The cell suspension was then centrifuged at 931 g for 20 min at room temperature. The cell pellet was removed and washed, and red blood cells were lysed with ACK lysis buffer (prepared in house). Single cell suspensions from all tissues analyzed were then washed with RPMI 1640 (Invitrogen) containing 10% FBS, and viable cells were counted via trypan blue (MP Biomedicals, Solon, OH, USA) exclusion.

Nine human NK/T‐cell lines, including NK‐S1, MEC04, YT, KAI‐3, KHYG1, NKYS, SNK6, SNK1, and NK92 cells, were used in our study. NK‐S1 cells were derived from an NK lymphoma xenograft.⁵¹ MEC04 was a gift from Dr. Paul Coppo and Dr. Philippe Gaulard, and YT was a gift from Dr. C. Clayberger. KAI‐3 and KHYG1 cells were purchased from JCRB Cell Bank, and NK92 cells were purchased from ATCC. SNK1, SNK6, and NKYS were kindly provided by Dr. Norio Shimizu. YT cells were cultured in IMDM (Life Technologies) with 20% fetal bovine serum (FBS, HyClone) and 1% sodium pyruvate (Life Technologies). NK‐S1 cells were grown in DMEM (Life Technologies) with 10% FBS and 10% horse serum (HS, Life Technologies). MEC04 cells were maintained in RPMI 1640 medium with 15% FBS and 50 IU/mL IL 2 (Miltenyi Biotec). All remaining cell lines were cultured in RPMI 1640 medium (Life Technologies) with 10% FBS and 100 IU/mL IL‐2. Ten percent HS was included in the NK92, SNK1, and SNK6 growth media. All culture media were supplemented with 1% penicillin‒streptomycin (P/S, Gibco BRL). All cultures were routinely assessed for mycoplasma contamination.

Peripheral blood samples were obtained from healthy donors. Informed consent was provided according to the Declaration of Helsinki. The PBMCs were isolated by density gradient centrifugation while using Lymphocyte Separation Media (Biosera, Monza, Italy). The DCs were prepared, as previously described [29 (link)], with minor modifications. Briefly, monocyte purification was obtained by positive selection, while using anti-CD14 conjugated magnetic microbeads (Miltenyi Biotec, Bologna, Italy). Cell purity, as assessed by flow cytometry (FACS), was always ≥ 95%. Freshly purified monocytes were cultured for five days at the density of 350,000 cells/ml in complete medium (RPMI 1640, 10% endotoxin-free FBS, 2 mM L-Glutamine, and 1% Penicillin-Streptomycin) supplemented with 20 ng/mL of recombinant human IL-4 and 50 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) both purchased from Miltenyi Biotec to obtain immature DCs (immDCs).

Single-cell suspensions of spleen and iliac lymph nodes (pooled within a group) were prepared by homogenizing organs through a metal mesh, washed with RPMI 1640 (Gibco, Invitrogen), and counted. Cells from lungs and GT’s including the vagina and uterine horns were isolated and pooled within groups to have enough cells to analyze. Cells were transferred to gentleMACS C tubes (Miltenyi Biotec GmbH, Germany) with 1.6 mg/ml collagenase in RPMI 1640 and dissociated into 1–2 mm pieces using the gentleMACS Octo dissociator. Cells were then incubated for 1 h at 37°C and returned to the gentleMACS, dissociated, and centrifuged. Supernatants were collected for Ab measurements and pellets homogenized through cell strainer, washed in RPMI 1640, and counted.