(455) The regulatory subunit ppp2r5a of protein phosphatase 2A selectively regulates spleen B-1 cell proliferation and accumulation

Introduction/Rationale: B-1 cells are a fetal/neonatal-derived B cell population located in small frequencies in the spleen. They are also the predominant B cell subset in the body cavities of common laboratory mice. B-1 cells differ from conventional (B-2) cells in that they maintain their numbers throughout life not by bone marrow replenishment but rather by “self-renewal”, a poorly understood process in which B-1 cells divide following unknown stimuli. Using cell population data obtained in an unbiased phenotypic screen, conducted by flow cytometry of 56 cell populations in 640 mouse lines each carrying a single gene deletion, we identified ppp2r5a as a candidate gene required for the selective control of B-1 cell maintenance and/or generation. Ppp2r5a encodes a regulatory subunit of protein phosphatase PP2A; a phosphase known to participate in cell activation, proliferation and cell death through its function in the WNT/Beta-catenin pathway. Here we tested the hypothesis that PP2A regulation is necessary for B-1 cell self-renewal.

Methods: Flow cytometry, in vitro cell culture, ELISA assy.

Results: Ppp2r5a-/- mice showed significantly lower frequencies of B-1 cells and B-1 derived plasma cells (PC) in spleen and BM, respectively, but not the body cavities. Consistent with B-1 cell’s role as producers of natural antibodies, mice had lower concentrations of serum IgM, IgG and IgG3 compared to wild type controls. Compared to B-1 cells from wild type mice, Ppp2r5a-/- B-1 cells showed increased proliferation in response to TLR4 agonist stimulation, but also increased cell death, as assessed by flow cytometric staining for Caspase 3 and 7. In addition, Ppp2r5a-/- B-1 cells showed dysregulated expression of mitochondria and Beta-catenin.

Conclusion: Together, these data implicate the WNT/ Beta-catenin pathway as an important regulator of B-1 cell dynamics.