Recombinant human granulocyte macrophage colony stimulating factor

The PBMCs were isolated and seeded on 6-well culture plates (Costar, USA) overnight. After discarding the cell suspension, the adherent monocytes were cultured in RPMI 1640 (Gibco, USA) complete medium containing 800 U/ml recombinant human granulocyte-macrophage colony-stimulating factor and 500 U/ml IL-4 (R&D Systems, USA) for 7 days. Culture medium and cytokines were renewed every 2 days. On day 4, antigenic peptides (B, B-T, B-2T to B-11T) were added to a final concentration of 50 μg/mL. On day 6, DCs were stimulated with 40 ng/mL recombinant human TNF-α (R&D Systems, USA). After 24 h of stimulation, mature antigen-loaded DCs were harvested carefully. Human naïve CD4⁺ T cells (purity of >95%, viability of >90%) sorted from the same donor by Human Naïve CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec, Germany) were dyed with CFSE (Thermo Fisher Scientific, USA) and then cocultured with antigen-loaded DCs for 7 days. The fluorescence of CFSE was measured by FACS to calculate the proliferation rates of naïve CD4⁺ T cells.

Blood buffy coats were obtained from healthy volunteers after informed consent was obtained. The study protocol and any amendments were reviewed and approved by an independent review board (New England IRB, Newton, MA) before the start of the study. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki.
PBMCs were freshly isolated from blood by Ficoll-Hypaque gradient centrifugation as previously described (39 (link)). Monocyte-derived dendritic cells were differentiated in vitro from freshly isolated human monocytes as previously described (39 (link)). Briefly, CD14⁺ monocytes were isolated from freshly purified PBMCs by negative selection and magnetic bead sorting (Miltenyi). Cells were then incubated in complete RPMI 1640 in the presence of 50 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor and 20 ng/ml recombinant human IL-4 (R&D Systems) for 7 days. For all experiments using human donor cells, data were generated independently with at least two donors. A representative data set was selected for incorporation into the figure.