Regulation of plasma cell homeostasis and antibody secretion through modulation of autophagy and endoplasmic reticulum stress by Trk-fused-gene (TFG)

抗体是由次级淋巴器官中产生的浆母细胞和浆细胞分泌的。内质网(ER)质量控制和自噬对于浆细胞的分化和维持、抗体的组装和分泌是必不可少的。我们先前已经证明，原肌球蛋白受体激酶融合基因(TFG)是一种在浆细胞中上调的支架蛋白，可以防止内质网应激并支持CH12B细胞的自噬通量。对CH12B细胞的蛋白质组学分析表明，TFG在调节细胞内转运、线粒体代谢和JCHAIN丰度方面发挥了作用。因此，我们假设TFG对体内浆细胞内稳态很重要。为了验证这一假设，我们产生了携带TFG等位基因(tfgfl/f1)的小鼠，并将其与MB1-CRE小鼠(TFG_BKO)杂交。未免疫的B小鼠的初步数据显示浆细胞数量减少。血清Ig M和Ig G降低，而Ig A无变化。而稳态B小鼠血清中无二聚体存在，粪便中的IgA明显降低。在本项目中，我们将通过诱导T细胞依赖和独立免疫来扩展这些初步数据，并探索TFG_BKO小鼠的IgM、Ig G和Ig A反应。由于TFG对JCHAIN和IgA的影响，我们将深入分析脾、骨髓和肠道相关淋巴组织(GALT)中的B细胞和浆细胞亚群，并对GALT结构进行解析。为了探索TFG依赖的JCHAIN的数量，我们将在稳态、免疫后和过表达实验中对TFG_BKO小鼠的浆细胞亚群中的JCHAIN进行定量。此外，我们还将解决Ig组装和动态平衡问题。TfG基因缺失对B小鼠原代激活的B细胞的内质网稳态、自噬和代谢信号的影响将被确定。阐明TFG在B细胞和浆细胞中的作用将有助于理解浆细胞的细胞生物学，这是支持抗体合成和体液免疫所必需的。

Antibodies (Ab) are secreted by plasma blasts and plasma cells that are generated in secondary lymphatic organs. Endoplasmic reticulum (ER) quality control and autophagy are essential for plasma cell differentiation and maintenance, Ab assembly and secretion. We have previously demonstrated that TFG (Tropomyosin receptor kinase fused gene), a scaffold protein that is up-regulated in plasma cells, prevents ER stress and supports autophagy flux in CH12 B cells. Proteomic analyses of CH12 B cells suggest a role for TFG in the regulation of intracellular transport, mitochondrial metabolism and JCHAIN abundance. We therefore hypothesize that TFG is important for plasma cell homeostasis in vivo. To test this hypothesis, we have generated mice carrying a floxed tfg allele (tfgfl/fl) that were crossed to mb1-Cre mice (TFG_BKO). Preliminary data from non-immunized tfgB mice show reduced plasma cell numbers. Serum IgM and IgG were reduced while IgA was not. However, there is no dimeric serum IgA in tfgB mice in steady state and IgA in feces is strongly diminished. In this project we will extend these preliminary data by eliciting T cell dependent and independent immunity and explore the IgM, IgG and IgA responses in TFG_BKO mice. Owing to the effects of TFG on JCHAIN and IgA we will specifically analyze B cell and plasma cell subsets in spleen, bone marrow as well as gut associated lymphoid tissue (GALT) in depth, along with resolution of GALT structure. To explore the TFG-dependent JCHAIN amount, JCHAIN will be quantified in plasma cell subsets of TFG_BKO mice in steady state, after immunization and in overexpression experiments. Furthermore, we will resolve Ig assembly and homeostasis. The effect of tfg-deletion on ER homeostasis, autophagy and metabolic signaling will be determined in primary activated B cells from tfgB mice. Elucidation of the role of TFG in B cells and plasma cells will help to understand the cell biology of plasma cells that is required to support antibody synthesis and humoral immunity.