T bet pe

Autologous T cells were co-cultured with matured DCs for 5 days at a 10:1 ratio, after being submitted to a staining protocol with carboxyfluorescein succinimidyl ester (CFSE). All co-cultures were carried out in U-bottomed 96-well plates in a final volume of 200 µL of RPMI medium. At the end of the co-culture period, cells were stained with fluorescence-conjugated antibodies, namely CD4-PerCP/Cy5.5 and CD8-APC antibodies (BioLegend) in order to evaluate the percentage of positive T cell proliferation. Type 1 T cells (Th1), type 2 T cells (Th2), and regulatory T cells (Treg) subsets were also evaluated through flow cytometry after the co-culture period with DCs for 5 days. The autologous T cells were stained using anti-CD4-PerCP/Cy5.5, anti-CD8-APC, anti-CD25-APC, anti-forkhead-box-P3 (FoxP3)-FITC, anti-GATA-binding protein 3 (GATA3)-FITC, and anti-T-box protein expressed in T cells (T-bet)-PE (Biolegend). As some markers are intracellular, the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend), a fixation and cell permeabilization kit, was used for the intracellular staining, according to the manufacturer’s instructions. Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) and the results are presented as a percentage of positive cells (%) after subtraction of isotype control values.

Lymphocytes were all stained with the anti-mouse Abs CD4 PE-F594 (BD Biosciences, San Jose, CA), CD4 FITC (BioLegend, San Diego, CA), CD11b AF-700 (BioLegend), and GL3 (γδ TCR). All cytokine staining was performed using the BD Foxp3 intranuclear transcription factor staining kit for Foxp3 PE (eBioscience), T-bet PE (BioLegend), IL-17a Rat PE-Cy7 (BioLegend), IL-17a Alexa Fluor 488 (BD Biosciences), IFN-γ PerCP-Cy5.5 (BD Biosciences), IFN-γ allophycocyanin-Cy7 (BioLegend), IL-10 allophycocyanin (BioLegend), and IL-6 allophycocyanin (BioLegend) following the manufacturer’s instructions. For phenotypic analysis, flow cytometry was performed using a BD Biosciences LSR II flow cytometer, and analysis was performed using FlowJo (Tree Star, Ashland, OR).