Anti hla dr

For flow cytometry analysis of surface DC phenotype, we used the following monoclonal antibodies (mAb): FITC-labeled anti-CD1c (Biolegend, San Diego, CA, USA), anti-CD14, anti-CD16 and anti-HLA I (all from Invitrogen, Camarillo, CA, USA), Alexa Fluor 488-labeled anti-CD1a and anti-CCR7 (both Biolegend). PE-labeled antibodies included: anti-CD11b, anti-CD11c (both Biolegend), anti-CD80, anti-CD86, anti-DC-SIGN, (all Biolegend), and anti-HLA DR (Miltenyi Biotec, Bergisch Gladbach, Germany).
DCs differentiated in Cellgenix^® DC GMP medium (Cellgenix GmbH, Freiburg, Germany) were harvested and collected by centrifugation. Before staining, the cells were washed twice in DPBS in all cases. Antibody was added and the cells were incubated for 15 min in the dark, then washed twice and resuspended in 2% paraformaldehyde (PHA). Samples were analyzed on a FACSCalibur system (BD biosciences, Franklin Lakes, NJ, USA). Data were analyzed with CellQuest software (BD biosciences).

Primary human monocytes were isolated from buffy coats from healthy donors (Sanquin, Nijmegen, The Netherlands). Before sample collection, a written consent was obtained. Buffy coats were diluted 1:1 with phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS) (HyClone™ Fetal Bovine Serum, Fisher Scientific, Loughborough, UK) and loaded onto Greiner Bio-One™ LeucoSEP™ Polypropylene Tubes to obtain peripheral blood mononuclear cells (PBMCs). After being washed, the cells were diluted in MACS buffer, and a CD14 microbead kit was used to isolate the monocytes according to the manufacturer's protocol (Miltenyi Biotec, Leiden, The Netherlands). Naive CD4⁺ T-cells were isolated from the flow through using a negative selection. A naive CD4⁺ T Cell Isolation Kit, containing a cocktail of biotinylated CD45RO, CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD56, CD123, anti-TCRγ/δ, anti-HLA-DR, and CD235a (glycophorin A) antibodies (Miltenyi Biotec, Leiden, The Netherlands), was used. All cells were frozen in 1:1 FBS and FBS with 20% dimethyl sulfoxide (DMSO) and stored at −80°C until further use.

MDSCs were isolated from PBMCs by HLA-Dr negative selection and followed by CD33 positive selection, using anti-HLA-Dr and anti-CD33 antibody-coated magnetic beads according to the manufacturer’s instructions (Miltenyi Biotech, Germany). Briefly, 1.0×10⁷ cells in 80μl of PBS buffer were incubated with 20μl of HLA-Dr MicroBead Cocktail. The sample was mixed well and incubated at 2-8°C for 10min. The mixture was transferred to the separation columns (MACS MS column; Miltenyi Biotech). The effluent was collected and incubated with 20μl of CD33⁺ MicroBead Cocktail. The cells were washed with 1ml of PBS buffer and the HLA-Dr^- CD33⁺ cells were collected. Purity of the separated cells was >90% by flow cytometry. CD3⁺ T cells were also isolated as stated above using Pan T magnetic beads (Miltenyi Biotech). CD4⁺ T cells and CD8⁺ T cells were isolated from PBMCs of healthy controls using CD4⁺ and CD8⁺ MicroBeads respectively (Miltenyi Biotech, Germany).