Lgm 3 medium

Human DCs were generated from monocytes isolated from the PBMCs of healthy HLA-A2 donors using Histopaque^®-1077 (Sigma-Aldrich). The PBMCs were resuspended in x-Vivo medium (Lonza, Walkersville, MD, USA) containing 1% human serum (Sigma-Aldrich) at a cell density of 1×10⁷ cells/ml and plated in T75 flasks. The flasks were incubated in 5% CO₂ at 37°C for 90 min, and nonadherent cells were then gently resuspended and removed. The adherent cells were then cultured in LGM-3 medium (Lonza, Walkersville, MD, USA) containing 1% human serum, recombinant human GM-CSF (1,000 units/ml, R&D Systems, USA) and IL-4 (1,000 units/ml, R&D Systems, USA) for 5 days. Next, the adherent cells were harvested, counted, and resuspended in culture medium. The phenotypes of iDCs were determined by flow cytometry using PE-conjugated anti-CD83 (1:20; IM2218U; Beckman Coulter, Marseille, France), anti-HLA-ABC (1:20; IM1838U, Beckman Coulter) and PE-conjugated anti-HLA-DR (1:20; IM1639, Beckman Coulter) antibodies. The expression of cell surface markers was then determined by flow cytometry using the Cytomics FC 500 instrument (Beckman Coulter, Fullerton, CA, USA).

HEK293 (#CRL-1573), HeLa (#CCL-2), HCT116 (#CCL-247), A549 (#CCL-185, obtained from American Type Culture Collection; ATCC) and TZM-bl (#8129, from NIH AIDS Reagent Program; ARP) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). SW480 (#CCL-228), Jurkat (#TIB-152, from ATCC) and A3.01 (#166, from ARP) cells were cultured in RPMI containing 10% FBS. ACH-2 (#349) and LuSIV (#5460, from ARP) cells were cultured in RPMI containing 10% FBS, 2 mM sodium pyruvate and 10 mM HEPES. Primary human CD4⁺ T cells (#2W-200, purchased from Lonza, Basel, Switzerland) were maintained in LGM-3 medium (Lonza) supplemented with 10% FBS. These cells were confirmed to be free of mycoplasma using a MycoAlert mycoplasma detection kit (Lonza). Replication-competent HIV-1 stocks were produced by transient transfection of HEK293 cells with the pNL4-3 proviral plasmid (#114, from ARP)25 (link). Culture supernatants containing virus were collected 48 h after transfection and filtered through a 0.45 μm Millex-HV filter (Millipore, Billerica, MA).

Isogenic CD8⁺ T and monocytes, negatively isolated from two separate donors were purchased from Astarte Biologics, Bothell, WA, USA. To generate DCs, 0.5 × 10⁶ monocytes were cultured using ImmunoCult Dendritic Cell Culture Kit (Stemcell Technologies, Vancouver, Canada) in 24 well plates for 2 days, followed by medium supplementation with differentiation cocktail and incubation for 2 days, then a maturation cocktail supplied in the kit, following instructions provided by the manufacturer. Tumor priming was performed using irradiated lymphoma tumor cells (SUDHL10, derived from a germinal center DLBCL; or Raji, derived from a Burkitt lymphoma) prepared by exposing the tumor cells to 30 Gy γ-radiation using Cs-137 Shepherd irradiator (Tufts University) and culturing for an additional 6 hours. On day 7, 2.5 × 10⁵ DCs were co-cultured with irradiated tumor cells at a 1:1 ratio and maintained for 16 hours, followed by supplementation with maturation cocktail for an additional 24 hours. For T-cell stimulation, antigen-pulsed adherent DCs were rinsed with medium to remove irradiated tumor cell suspension and co-cultured with T cells at a 1:10 ratio in LGM-3 medium (Lonza, Walkersville, MD, USA) containing 5% human serum and IL-2 (10 U/ml). IL-2 was supplemented on days 12, 15 and 19, followed by harvesting T cells to perform cell mediated cytotoxicity assay on day 20.

B220⁺CD11c⁺NK1.1⁺ cells were stained using an anti-mouse IFN-γ antibody 16 to 24 h after incubation with 10 ng/mL mIL1β-RNA. Human CD56⁺CD3^-NK cells were stained with an anti-human IFN-γ antibody 48 h after priming with 50 ng/mL hIL1β-RNA in an LGM-3 medium (Lonza) supplemented with 200 IU/mL IL2. For the positive control of IFN-γ induction, cells were incubated with 40 ng/mL hIL-12 for 48 h. The Zombie Green Fixable Viability Kit (BioLegend) was used to detect the dead PKH26-stained tumor cells 24 h after incubation with B220⁺CD11c⁺NK1.1⁺ cells that were primed with 10 ng/mL mIL1β-RNA for 16–18 h.

T cell cytotoxicity is presented as the percentage of target cells that were lysed. The target cells were labeled with 5 μM Carboxifluorescein diacetate N-succinimidyl ester (CSFE) (eBioscience, USA). After labeling, the cells were washed and resuspended at 1×10⁵ cells/ml in LGM-3 medium (Lonza) supplemented with 1% human serum, GM-CSF, IL-4 and IL-2 (100 units/ml). The T cells were incubated with 1×10⁴ target cells in each FACS tube at various culture ratios (T cell: target cell = 10:1, 20:1 and 40:1), and the FACS tubes were incubated at 37°C for 6 h in a humidified, 5% CO₂ incubator. After incubation, the cells were stained with 50 μg/ml propidium iodide (PI) (Sigma-Aldrich), a red dye that penetrates the membranes of dying cells, prior to analysis by flow cytometry (Beckman Coulter FC500, USA). The cytotoxicity was calculated as the percentage of PI-positive cells among the CFSE-positive target cells.

Human peripheral blood mononuclear cells (PBMCs) were prepared from the blood of healthy donors by the standard procedure using Ficoll-Paque centrifugation (22 (link)). This research was carried out on humans following the international and national regulations. Written informed consent was obtained from the subjects. For some experiments, human CD14-positive monocytes were purchased from Lonza (Walkersville, MD, USA). Mouse blood was obtained from the inferior vena cava, and the mononuclear cells were prepared by Ficoll-Paque centrifugation. The blood mononuclear cells and CD14-positive monocytes were cultured either in LGM-3 medium (Lonza) alone or in the presence of recombinant PAP-fused cytokines under the indicated conditions. As a positive control for the differentiation of human dendritic cells, the PBMCs and CD14-positive monocytes were cultured in LGM-3 medium supplemented with human granulocyte-macrophage colony-stimulating factor (hGMCSF; 2 ng/ml) and human interleukin-4 (hIL4; 2 ng/ml) (both from R&D Systems, Minneapolis, MN, USA) (17 (link),22 (link)). The cells were cultured at 37°C in humidified incubators containing air with 5% CO₂.