Flow Cytometry Assay
The following antibodies were used: CD3-PerCP (HIT3a), CD4-FITC (RPA-T4), CD8-BV510 ( RPA-T8), CD45RA-PE-Cy7 ( HI100), CD27-APC ( M-T271), TCR aβ-PE ( IP2b), TCR γδ-BV421 ( B1), CD19-APC ( HIB19), CD27-V450 ( M-T271), IgD-AF488 ( IA6-2), CD24-PE ( ML5), and CD38-PerCP ( HIT2), CD4-PE-Cy7 ( RPA-T4), CXCR5-BV421 ( J25ID4), CD25-APC ( MT271, ), CD127-PE ( A019D5, ), CCR6-PE ( G034E3, ), CD25-BV421 ( BC96, ), Helios-PerCP-cy5.5 ( 22F6, ), CD19-PerCP-Cy5.5 ( SJ25C1, ), CD27- PE-Cy7 ( MT271). All were from Biolegend. CD45RO-APC (UCHL 1), CD45RA-FITC (HI100), CXCR3-APC (1C6), and FOXP3-PE (PCH101), CD152-APC (BNI3), IgM-APC (G20-127) and IFN-γ-APC (4S.B3). All were from BD Bioscicences. Th1 was defined as CD4+CD45RA-CXCR5-CXCR3+CCR6-, Th2 was defined as CD4+CD45RA-CXCR5-CXCR3-CCR6-, Th17 cells CD4+CD45RA-CXCR5-CXCR3-CCR6+, Th1/17 cells CD4+CD45RA-CXCR5-CXCR3+CCR6+, Th1-like cells CD4+CD45RA-CXCR5+CXCR3+CCR6-, Th2-like cells CD4+CD45RA-CXCR5-CXCR3-CCR6-, Th17-like cells CD4+CD45-CXCR5+CXCR3-CCR6+, Th1/17-like cells CD4+CD45RA-CXCR5+CXCR3+CCR6+, T follicular helper cell CD3+CD4+CD45RA-CXCR5+, circulating follicular regulatory T CD3+CD4+CD45RA+CXCR5+, Treg CD3+CD4+CD25-FOXP3+, IgMhi B cells (CD19+CD27-IgM+), MZ-like B (CD19+CD27+IgM+), switched-memory B (CD19+CD27+IgM-).
The intracellular production of IFN-γ was investigated in PBMCs by flow cytometry. PBMCs (2 × 106 cells/ml) were either unstimulated or stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 5 hrs or BCG (MOI = 20) or 100 ng/ml BCG + IL-12 for 72 hrs, in 24-well plates. All the samples were treated with 1 μg/ml GolgiPlug (BD) for the last 2 or 6 hrs of culture.
The staining was performed according to manufacturers’ guides. The samples were acquired on a FACSCanto II flow cytometer, and the data were analyzed using FlowJo.