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. 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.

早期与未折叠蛋白反应（Unfolded Protein Response, UPR）相关的基因表达发生于Blimp1 (B淋巴细胞诱导成熟蛋白1) 上调之前，且不依赖经典内质网应激激活的XBP1 (X盒结合蛋白1) 通路。在相关配套研究中，我们详细阐释了早期浆细胞特异性UPR相关基因表达的XBP1非依赖激活机制，该过程同样发生于Blimp1上调之前。我们观察到此类基因与经典的雷帕霉素靶蛋白复合物1（mammalian target of rapamycin complex 1, mTORC1）特征基因存在重叠，且浆细胞预激活的边缘区B细胞具有更高的基线mTORC1信号水平，因此设计本实验以评估浆细胞分化过程中早期内质网重塑对mTORC1信号的依赖程度。为此，我们将携带mTORC1衔接蛋白Raptor的flox等位基因的小鼠（B6.Rptor-flox）与在人CD20启动子调控下表达他莫昔芬诱导型Cre重组酶 (Cre recombinase) 的小鼠（B6.hCD20-TamCre）进行杂交。将此类杂交小鼠及其同窝的hCD20-TamCre-Rptor-wt小鼠通过饮食口服给予他莫昔芬，持续两周，随后分离滤泡B细胞用于体外浆细胞分化实验。本研究结果显示，在体外培养刺激仅24小时时，Raptor缺失的B细胞在上调与浆细胞分化相关的UPR相关基因方面存在显著缺陷。实验样本为经他莫昔芬处理后、在CD20启动子调控下表达他莫昔芬诱导型Cre重组酶的Raptor flox等位基因小鼠（n=3）与Raptor野生型小鼠（n=3）的滤泡B细胞，此类细胞经体外培养诱导为浆细胞。采用3'端polyA捕获法从RNA中制备cDNA；通过Kallisto伪比对技术检测转录本丰度，并使用voom方法进行归一化处理；随后借助limma软件包评估差异表达情况。