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.