Fig 1.
Annexin V binding is increased in IL-10 producing B cells (B10 cells) compared to IL-10 non-producing B cells and to TNFα producing B cells.
PBMCs were stimulated for 24 hr with CpG/ionomycin/PMA and BFA as described previously and analyzed by FACS for annexin V (AnV) binding and IL-10 and TNFα production in 21 subjects. Cytometry gating is shown in A. Results are presented in percentage of positive cells in B and median of fluorescence (MFI) in C. Wilcoxon’s matched pairs signed rank tests were used. Top of the bar represents the median, and whiskers are IQR 25–75.
Fig 2.
Annexin V binding is increased among CD5+ and CD24hiCD27+ B10 precursors.
A representative plot is presented (A). PBMCs from 12 subjects were analyzed by FACS for B10 precursors phenotype (i.e CD19+CD5+ (B), CD19+CD24hiCD27+ (C) and CD19+CD24hiCD38hi, (D)), and for annexin V binding. Wilcoxon’s matched pairs signed rank tests were used. Data are median (IQR 25–75).
Fig 3.
Annexin V positive B cells differentiate more frequently into B10 cells than Annexin V negative B cells.
PBMCs from 6 healthy subjects were sorted by flow cytometry (FACSARIA) according to annexin V (AnV) staining: AnV positive (AnV+) and AnV negative B cells (AnVneg) after exclusion of dead cells (DAPI+)(A). Each B cell subpopulation was then stimulated for B10 generation and analyzed by flow cytometry as previously described (B). (C) shows the % of cytokine-positive cells (IL-10, IL-6, GM-CSF and TNF-a) in AnV+ and AnV AnVneg sorted cells. (D) shows IL-10 and IL-6 concentrations assessed by ELISA in supernatant of AnV+ and AnV AnVneg sorted cells.
Fig 4.
Annexin V positive B cells are not apoptotic cells.
PBMCs were stained with APC anti-CD19 and FITC conjugated Annexin V, sorted by FACSARIA according to annexin V (AnV) staining: high AnV B cells (AnVhi); low AnV B cells (AnVlow) and AnV negative B cells (AnVneg). Cell death was analyzed at 72 hr using DAPI staining for necrosis (A) and DIOC6 (B) for mitochondrial apoptosis (n = 4). Additionally, cell cycle was analyzed using propidium iodide (PI) staining at 72 hr (C), showing the proportion of cells in the subG1 phase (apoptotic cells) and in the S phase (proliferating cells) in AnV+ (AnVhi and AnVlow) and AnVneg B cells (n = 5). Data are median (IQR25-75).
Fig 5.
Phosphatidylserine blockage alters B10 cell differentiation.
PBMCs from 6 healthy subjects were pretreated with biotinylated Annexin V (biot AnV) (n = 8) or with glyburide (Gly) (n = 8) and then stimulated for 24 hr according to the protocol for B10 assessment previously described. A shows representative plots and B shows the results as median (IQR25-75).