Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

<div><p>B cells can have a regulatory role, mainly mediated by interleukin 10 (IL-10). IL-10 producing B cells (B10 cells) cells remain to be better characterized. Annexin V binds phosphatidylserine (PS), which is externalized during apoptosis. Previous works suggested that B10 cells are apoptotic cells since they bind Annexin V. Others showed that Annexin V binding could also be expressed on viable B cells. We aimed to explore if PS exposure can be a marker of B10 cells and if PS exposure has a functional role on B cell IL-10 production in healthy subjects. We found that B10 cells were significantly more often Annexin V⁺ than IL-10 non-producing B cells. After CpG activation, Annexin V⁺ B cells differentiated more often into B10 cells than Annexin V^neg B cells. Cell death and early apoptosis were similar between Annexin V⁺ and Annexin V^neg B cells. PS blockage, using biotinylated AnV and glyburide, decreased B10 cell differentiation. This study showed that B10 cells have an increased PS exposure independently of any apoptotic state. B cells exposing PS differentiate more into B10 cells whereas PS blockage inhibits B10 cells generation. These results strongly suggest a link between PS exposure and B10 cells.</p></div

Annexin V positive B cells are not apoptotic cells.

<p>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 (AnV^hi); low AnV B cells (AnV^low) and AnV negative B cells (AnV^neg). 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+ (AnV^hi and AnV^low) and AnV^neg B cells (n = 5). Data are median (IQR25-75).</p

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.

<p>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.</p

Annexin V binding is increased among CD5+ and CD24hiCD27+ B10 precursors.

<p>A representative plot is presented (A). PBMCs from 12 subjects were analyzed by FACS for B10 precursors phenotype (i.e CD19+CD5+ (B), CD19+CD24^hiCD27⁺ (C) and CD19+CD24^hiCD38^hi, (D)), and for annexin V binding. Wilcoxon’s matched pairs signed rank tests were used. Data are median (IQR 25–75).</p

Phosphatidylserine blockage alters B10 cell differentiation.

<p>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).</p

Annexin V positive B cells differentiate more frequently into B10 cells than Annexin V negative B cells.

<p>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 (AnV^neg) 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 AnV^neg sorted cells. (D) shows IL-10 and IL-6 concentrations assessed by ELISA in supernatant of AnV⁺ and AnV AnV^neg sorted cells.</p