Anti cd14 pedy594 clone mem 15

PBMCs isolated from 20 T1D patients and 10 healthy controls were stained with FITC-conjugated antibodies against lineage-specific markers (CD3 clone MEM-57, CD19 clone LT19, CD20 clone LT20, CD16 clone LNK16, and CD56 clone MEM-188), anti-CD14-PEDy594 (clone MEM-15), anti-CD11c-APC (clone BU15) (Exbio, Prague, Czech Republic), anti-CD123-PE (clone 6H6) (ThermoFisher Scientific), and anti-HLA-DR-PC7 (clone L243) (BD Biosciences, Franklin Lakes, USA), washed, filtrated through 0.2 µm pore size and sorted under low pressure to increase viability and prevent activation on a FACSAria II (BD Biosciences). Myeloid DCs were gated as Lin-HLA-DR + CD14-CD11c+ cells, and plasmacytoid DCs were defined as Lin-HLA-DR + CD14-CD123+ cells. Sorted cells were recollected in tubes containing complete RPMI.

PBMCs were cultured in complete media with or without rhIL-27 (100 ng/ml) overnight at 37 °C. Then, the cells were washed and stained with FITC-conjugated antibodies against lineage-specific markers (CD3 clone MEM-57, CD19 clone LT19, CD20 clone LT20, CD16 clone LNK16, and CD56 clone MEM-188), anti-CD14-PEDy594 (clone MEM-15), anti-CD11c-PB (clone BU15) (Exbio), anti-CD123-PerCP-Cy5.5 (clone 7G3) (BD Biosciences), anti-HLA-DR-Alexa700 (clone L243), anti-PD-L1-PC7 (clone 29E.2A3), and anti-CD86-Alexa647 (clone IT2.2) (BioLegend, San Diego, USA). The samples were acquired on a FACSAria II (BD Biosciences), and data analysis was performed using FlowJo software (TreeStar). Next, the expression of PD-L1 in PBMCs was analyzed by RT-PCR after 100 ng/ml IL-27 stimulation for 5 hours.

Whole blood was stimulated with 100 ng/ml rhIL-27 (Peprotech, New York, USA) for 15 minutes, 10 ng/ml rhIL-6, 500 ng/ml rhIFNα (both from R&D Systems) when indicated or left untreated at 37 °C. The optimal stimulation time was estimated after analyzing the effects of 5, 15 and 30 minutes of rhIL-27 stimulation on 8 healthy controls (Suppl. Fig. 2A). Intracellular signaling was prevented by using 4% paraformaldehyde without methanol (Sigma Aldrich) for 10 minutes at room temperature. Erythrocytes were lysed using 0, 1% Triton-X for 20 minutes (Sigma Aldrich) at 37 °C, leukocytes were permeabilized with ice-cold 80% methanol for 30 minutes and stained with a FITC-conjugated lineage antibody cocktail (CD3 clone MEM-57, CD19 clone LT19, CD20 clone LT20, CD16 clone LNK16, and CD56 clone MEM-188), anti-CD14-PEDy594 (clone MEM-15) (Exbio), anti-HLA-DR-Alexa700 (clone L243), anti-CD123-PC7 (clone 6H6) (BioLegend), anti-CD11c-APC (clone BU15) (Exbio), anti-phosphoSTAT1-BV421 (Tyr701) (clone 4a) and anti-phosphoSTAT3-PE (Tyr705) (clone 4/5-STAT3) (both from BD Bioscience). The samples were acquired on FACSAria II (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).