Transcriptomic analysis of in-vitro plasma cell generation from follicular B cells lacking Raptor [Raptor_PC_stim]

Early UPR-affiliated gene expression occurs before Blimp1 upregulation and independent of canonical ER-stress activated Xbp1. In linked companion studies we detailed how Xbp1-independent activation of early plasma cell specific UPR-affiliated gene expression occurs prior to Blimp1 upregulation. Having observed that these genes overlapped with canonical mTORC1 signature genes and that PC-poised marginal zone B cells have higher base line mTORC1 signaling we designed this experiment to assess the dependence of early ER-remodeling on mTORC1 signaling in plasma cell differentiation. To this end we mated mice harboring a floxed allele for the mTORC1 adapter Raptor (B6.Rptor-flox) to mice expressing a tamoxifen-inducible cre recombinase under the control of the human CD20 promoter (B6.hCD20-TamCre). These mice and their hCD20-TamCre-Rptor-wt litermates were fed oral tamoxifen in their diet for two weeks and follicular B cells were prepared for in-vitro plasma cell differentiation studies. We report here that as early as 24 hours of stimulation in culture we see a marked defect in the ability of Rptor null B cells to upregulate UPR-affiliated genes associated with plasma cell differentiation. Overall design: Samples are follicular B cells from Rptor-floxed (n=3) and Rptor-wildtype (n=3) mice expressing tamoxifen inducible Cre under the CD20 promoter after tamoxinen treatment, stimulated in culture to produce plasma cells. cDNA was prepared from RNA with 3' polyA capture method. Transcript abundance was measured by Kallisto psuedo-alignment and normalized (voom) and differential expression was assessed using limma.