Ccr4 pecy7

For immunofluorescence staining of whole blood and PBMC, antibodies included CD45-ECD (Beckman Coulter, Indianapolis IN), CD3-e780, CD4-e450, CD8-AF700, CD15-FITC (eBioscience, San Diego, CA), CD45-FITC, CD4-PerCP-Cy5-5, CD14-PECy7, HLA-DR-APC, HLA-DR-BV421, CD8-APC-H7, CCR7-BV605, CD25-BV605, CD45RA-PECy7, CD45RO-APC-H7, CCR4-PECy7, CD38-APC, CD127-BV650 (BD Bioscience, San Jose, CA), CD14-AF700, and CD3-AF700 (Biolegend, San Diego, CA). For phosphoSTAT staining, antibodies included CD3-BV785 (Biolegend), CD4-BV605, CD8-BV510, CD14-PECy7, CD19-BV421, CD16-BV650, pSTAT3-647, pSTAT5-PE (BD Biosciences), and pSTAT1-488 (Cell Signaling Technologies, Danvers, MA). Cell types were identified according to the criteria of the Human Immunology Project [19 (link)], and a full list of antibodies used to identify individual cell types including those not reported here can be found in Additional file 2: Table S1. Hematological toxicity was graded according to NCI CTCAE v4.0 guidelines and is reported in Additional files 3 and 4: Tables S2 and S3.

Freshly obtained human lymphocytes were stained with the following fluorescent antibodies against human leukocyte surface markers: CD4-peridinin-chlorophyll-cyanine 5 (PerCP-Cy5.5), CD25-phycoerythrin (PE) or -allophycocyanin (APC), CD45RA-fluorescein isothiocyanate (FITC), CCR7-PE, CCR5-FITC, CCR4-PE-Cy7, CD62L-PE, Ki67-PE or -APC from (BD Biosciences, USA), and LAP (PE and APC) from (R&D Systems, USA). Intracellular staining with Foxp3-APC or -FITC and CTLA-4-APC antibodies was performed after treatment with fixation and permeabilization buffers (eBiosciences, USA), according to the manufacturer's protocol. Intracellular staining for granzyme B, perforin, and TGF-β was performed after stimulation with PMA and ionomycin for 5 h with Golgi stop. The stimulated cells were first reacted with antibodies against the surface markers CD4 and LAP, then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences, USA), and finally stained with TGF-ß-PE, granzyme B-FITC, or perforin-PE antibodies. The fluorescence intensity was analyzed using BD FACSCalibur (BD Biosciences, USA) and FlowJo software (Tree Star, USA).

Mononuclear cells were stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’ (eBioscience, San Diego, CA) as described previously [33 (link)]. Briefly, cells were stained with surface antibodies CD8 Amcyan (BD Biosciences. Oxford, UK), CD3 APC-Cy7 (Cambridge Biosciences, Cambridge, UK), CCR4 PE-Cy7 (BD Biosciences, Oxford, UK) and CXCR3 PE (BD Biosciences. Oxford, UK) for 20 mins at 4°C. Samples were washed, re-suspended in RPMI with 10% human serum (TCS Biosciences, Buckingham, UK) and then stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’. Cells were washed, stained with a dead cell exclusion dye (Invitrogen, Oregon, USA) at 4°C for 20 mins and then washed again before fixation with 2% paraformaldehyde at room temperature for 10–15 mins. After further washing, cells were permeabilised with 0.5% saponin for 5 minutes followed by incubation with the intracellular antibodies IL-17A (BioLegend, London, UK) (2.5 ml), IFN-γ (BioLegend, London, UK) (0.5 ml) and isotype controls for 30 mins at room temperature. Samples were washed and analysed by flow cytometry using on LSR II flow cytometry. To define Th17 cells we measured the secretion of IL-17 using intracellular staining and FACS analysis.