Anti cd4 pe dazzle

LN-derived cells were thawed and stained for viability using a fixable viability dye e506 (Thermo Fisher Scientific, 65-0866-14) and for different surface markers depending on the experimental set-up. The following surface antibodies were used: anti-CD3-PerCP-Cy5.5, anti-CD4-PE-Dazzle, anti-CD8-APC-Cy7, anti-CD45RA-FITC, anti-CD25-BV421, anti-CD31-BV605, anti-CXCR5-BV711, anti-TIM3-BV711, anti-CD278-BV605, anti-PD1-PE-Cy7, anti-CD69-AF700 and anti-CD244-BV421 (all BioLegend, Supplementary Table 4). For subsequent intracellular staining, cells were fixed and permeabilized with the intracellular fixation/permeabilization buffer set (Thermo Fisher Scientific, 88-8824-00) and stained with anti-Ki67-BV785, anti-FOXP3-AF647, anti-IKZF3-PE or adequate isotype controls (Thermo Fisher Scientific, BD Biosciences, Supplementary Table 4). Then, cells were analysed using an LSR Fortessa (BD Biosciences) and FACSDiva (BD Biosciences, version 8). For analysis and gating of flow cytometry data, FlowJo (v10.8.0) was used.

The potent and selective H4R antagonist JNJ 10191584 (JNJ), phorbol myristate acetate, ionomycin, and RPMI medium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Golgi-plug, anti-CD4 FITC (#100406), anti-CD4 PE (#100408), anti-CD4 APC (#100412), anti-CD4 PE/Dazzle (#100566), anti-IFN-γ APC/Cyanine7 (#505850), anti-T-bet PE (#644810), anti-IL-9 PE (#514104), anti-IRF4 Alexa Fluor^® 488 (#646406), anti-IL-17A PE/Dazzle (#506938), anti-RORγt PE (#562607), anti-TGF-β1 PE (#141404), and anti-Foxp3 PE (#320008) mouse monoclonal antibodies, as well as RBC lysing solution, permeabilization solution, and fixation buffers, were purchased from BioLegend and BD Biosciences (San Diego, CA, USA). The primers were purchased from GenScript (Piscataway, NJ, USA). TRIzol was purchased from Invitrogen (Thermo-Fisher Scientific, Carlsbad, CA, USA). High-capacity cDNA and SYBR Green kits were purchased from Applied Biosystems (Foster City, CA, USA).