Effects of rictor gene deletion on pten-deleted GC B cells
PI3K/AKT/mTOR/PTEN signaling pathway is well known to regulate metabolism, cell growth and cell survival in all kinds of cells. PI3K phosphorylates PIP2(phosphoinositol-2 phosphate) into PIP3(phosphoinositol-3 phosphate) which acts as docking site for signaling components and activates Akt and mTOR signaling, whereas, Pten is a phosphatase which removes the phosphate from PIP3 and deactivates the singnaling. mTOR signaling encompasses two components namely mTORC1 and mTORC2 complexes and activity of these complexes orchestrated the various biological functions. Several studies have shown that loss of Pten in B cells disrupt the antibody class switching reaction and mTORC1 and mTORC2 has distinct role on the class switching reaction. However, the precise role of mTORC1 and mTORC2 conplexes in the Pten deficient condition is not yet studied. Notably, class switching reaction disrupted by loss of Pten, and that was restored only upon mTORC2 inactivation but not by mTORC1 inactivation in both of in vitro induced-germinal center B cells and plasmablasts culture condition. Overall design: In order to identify the mechanistic involved in the PI3K/PTEN/mTOR dependent antibody response, we performed the RNA seqeuncing using the in vitro generated germinal center B cells. We isolated Naive B cells(CD43 negative population) from mouse spleen using CD43 (Ly-48) microbeads. Naive B cells from tamoxifen administered Rosa26CreERT2, Rosa26CreERT2:Ptenfl/fl, and Rosa26CreERT2:Ptenfl/fl,Rictorfl/fl mice perfomed in vitro induced-germinal center B cells culture. To obtain the in vitro induced-germinal center B cells, we used CD40LB co-culture system. CD40LB cells that co-express mouse CD40L and BAFF were cultured in 60 mm dish to confluency and treated with 7 µM of mitomycin C for 3 h and washed several times to completely removed the traces of mitomycin C. 2 x 105 purified Naive B cells were co-cultivated with mitomycin C-treated CD40LB cells in with 2 nM IL-4 for 4 day to obtain in vitro induced-germinal center B cells. 4 day cultured sample were colleted and harvested for RNA-seq.