Pe anti cd4 clone gk1.5

The following monoclonal antibodies were used for lymphocyte surface staining: Fluorescein isothiocyanate (FITC) anti-CD3 (clone 17.12), phycoerythrin (PE) anti-CD4 (clone GK1.5), phycoerythrin–cyanine 5 (PE-Cy5) anti-CD8 (clone 53-67), PE anti-CD19 (clone ID3), peridinin–chlorophyll-protein (PerCP) anti-CD45 (clone 30-F11), PE anti-TCR γδ (clone GL3), PE-Cy5 anti-TCR αβ (clone H57-597) and anti-CD16/CD32 (clone 2.4G2) (BD Pharmingen, Milan, Italy). Each antibody was previously titrated to determine the optimal concentration for maximal staining. The IELs (1 × 106 cells) were pre-incubated for 20 min with anti-CD16/CD32 to block Fc receptors. Cells were then washed and labelled with the appropriate mixture of antibodies for 30 min, centrifuged at 650× g and resuspended in FacsFlow (BD Biosciences, Milan, Italy). Flow cytometry analysis was performed using a FACSCalibur instrument (BD Biosciences). To exclude dead/dying cells and, therefore, non-specific antibody-binding cells, lymphocytes were gated according to forward and side scatter. The percentage of B and T lymphocytes was calculated on leukocyte (CD45+) gate, whereas the CD4+, CD8+ and CD4+CD8+ subsets, as well as αβ and γδ lymphocytes, were calculated on T lymphocyte (CD3+) gate. At least 10,000 events were acquired. Data were analysed using CellQuest software (BD Biosciences).

For flow cytometry analysis, cells were treated with anti-CD16/32 (clone 2.4G2; BD) for Fc receptor blockade and then stained with PE anti-CD4 (clone GK1.5; BD), Alexa Fluor 488 anti-Foxp3 (clone MF-14; Biolegend), or isotype control Alexa Fluor 488 rat IgG2b, as described [22 (link)]. FITC anti-CD11c (clone N418; e-Bioscience) and PE anti-CD11b (clone M1/70; BD) were also used. Analysis was done with an LSRFortessa (BD BioSciences) flow cytometer and analyzed by Flowjo data analysis software (Tree Star Inc).