Rhgm csf

In vitro priming of T cell responses and evaluation of peptide immunogenicity was carried out as described. Briefly, monocytes were purified from PBMC obtained from HLA-A*02:01 healthy donors by using CD14 microbeads (Miltenyi) and cultured for one day in complete RPMI medium containing rhGM-CSF (Miltenyi; 10 ng/ml) and rhIL-4 (Inmunotools; 2 ng/ml) for their differentiation into dendritic cells (DC). Next, rhTNF (Miltenyi; 10 ng/ml), IL-1β (Immunotools; 10 ng/ml) and PGE2 (Sigma-Aldrich; 1 µM) were added for DC maturation. One day later, cells were collected, loaded with peptide (10 µg/mL) for 1 hour and washed 3 times. DC were co-cultured (ratio 1:30) with CD8 T cells purified from the CD14^- fraction using CD8 microbeads (Miltenyi), in complete medium containing anti-human CD28 mAb (Biolegend; 0.5 µg/mL). Cells were fed on days 3, 7, 10, 14 and 17 with culture medium containing IL-2 (Proleukin, 20U/ml).
NeoAg-specific responses were evaluated at day 21 of co-culture by using a human IFN-γ ELISPOT set (BD-Biosciences) following manufacturer’s instructions. Expanded CD8 T cells (5 x 10⁴/well) were stimulated with peptides (10 µg/mL) and DC (10⁴/well) for 24 h. Next, wells were washed, incubated with conjugate antibody and developed. Spot forming cells were counted as described above.

Human monocyte-derived dendritic cells (moDCs) were derived from monocytes isolated from human peripheral blood supplied by the French Blood Bank (Établissement Français du Sang, Rungis, France). Healthy donors gave their written consent for the use of blood donation for research purposes. Peripheral blood mononuclear cells (PBMCs) were sorted from buffy coats by density centrifugation on a Ficoll gradient. Monocytes were then isolated through positive magnetic selection using MidiMacs separation columns and anti-CD14 antibodies coated on magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, CD14⁺ cells were differentiated in immature moDCs for 4 days in RPMI 1640 supplemented with GlutaMAX (Gibco, Invitrogen), 10% heat-inactivated fetal calf serum (FCS, Gibco), 550 U.mL⁻¹ interleukin-4 (rh-IL4, Miltenyi Biotec), 550 U.mL⁻¹ GM-CSF (rh-GM-CSF, Miltenyi Biotech), 1 mM sodium pyruvate (Gibco), 100 µg.mL⁻¹ streptomycin and 100 U.mL⁻¹ penicillin (Gibco) at 37 °C and 5% of CO₂. On day 4, the differentiation of moDCs was evaluated by flow cytometry. The moDCs used will show the following phenotype: CD83 < 5%, CD86 < 30%, CD1a > 80% and DC-SIGN > 90%. moDCs are then used at the density of 1 million per mL.

The human monocytic cell line THP-1 cells (TIB-202) were purchased from ATCC. Cells were cultured in 24 well plates (Corning) and differentiated into THP-1 derived macrophages 24 hours prior to infection with 40 ng/mL phorbol 12-myristate 13-acetate (PMA) in RPMI 1640 Medium GlutaMAX™ Supplement (Gibco) at 37°C and 5% CO2. Experiments were realized within 15 passages and cell viability was measured before experiments by trypan blue exclusion and was greater than 97%. CD14⁺ primary human monocytes were extracted from peripheral blood of 5 healthy donors anonymously provided by the French Blood Establishment (EFS, Lyon). CD14⁺ monocytes were purified from whole blood using an autoMACS^® Pro Separator, Whole Blood Column Kit and StraightFrom^® Whole Blood CD14 MicroBeads (Mitenyi) according to the manufacturer’s instructions. Cell viability was measured before proceeding to macrophage differentiation, by trypan blue exclusion and was always greater than 80%. CD14⁺ monocytes were then plated in 24 well plates (Corning) at a density of 400 000 cells per well and differentiated into macrophages for 5 days prior to infection with 50 µg/ml Rh-GMCSF (Miltenyi) in RPMI 1640 Medium GlutaMAX™ Supplement (Gibco) with 10% FBS (Sigma-Aldrich) at 37°C, 5% CO2.

Human peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated from whole blood by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech, Sweden). The cells were counted and plated at 2 × 10⁶ cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 µL RPMI (GibcoBRL, Invitrogen), supplemented with 10% fetal bovine serum (FBS) (GibcoBRL, Invitrogen) or 100 µL RPMI supplemented with 10% plasma from patients or controls. PBMCs were left unstimulated or were stimulated with 5 to 80 ng/mL rhGM-CSF or 100 ng/mL rhIL-3 (Miltenyi-Biotec) for 30 min at 37°C, and the cells were then fixed permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with CD14-Pacific Blue and CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific), and STAT5 phosphorylation (p-STAT5 levels) was assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD-Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).

Human moDCs were prepared from buffy coats (Blood Transfusion Service, Sheffield, UK). Use of buffy coats was approved by the Faculty of Medicine and Health Sciences Research Ethics Committee. PBMCs were isolated using Histopaque-1077 (H8889, Sigma) and monocytes were isolated using human CD14 MicroBeads (130-050-201, Miltenyi Biotec) following the manufacturer’s protocol. Purified monocytes were cultured in RPMI complete medium [RPMI-1640 (R0883, Sigma), 15% (v/v) human AB serum (H4522, Sigma), 2 mM GlutaMAX (G7513, Sigma), 10 mM HEPES (15630056, Gibco), 50 ng/ml recombinant human granulocyte macrophage colony–stimulating factor (rhGM-CSF) (130-093-865, Miltenyi Biotec), and 50 ng/ml rh interleukin (IL)-4 (130-093-921, Miltenyi Biotec)] and cultured at 37 °C, 5%CO₂ for 6 days. On Day 3, fresh RPMI complete media containing growth factors was added to each well.