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

抗体（Ab）是由次级淋巴器官产生的浆母细胞和浆细胞分泌的。内质网质量控制和自噬对浆细胞的分化和维持、Ab的组装和分泌至关重要。我们之前已经证明TFG（原肌球蛋白受体激酶融合基因）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CH12 B细胞的自噬通量。CH12 B细胞的蛋白质组学分析表明，TFG在调节细胞内运输、线粒体代谢和JCHAIN丰度方面发挥作用。因此，我们假设TFG对体内浆细胞稳态很重要。为了验证这一假设，我们产生了携带一个floxed tfg等位基因（tfgfl/fl）的小鼠，并将其与mb1-Cre小鼠（TFG_BKO）杂交。未免疫tfgB小鼠的初步数据显示浆细胞数量减少。血清IgM和IgG降低，而IgA未降低。而稳定状态下tfgB小鼠血清中不存在二聚体IgA，且粪便中IgA明显减少。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， IgG和IgA反应。由于TFG对JCHAIN和IgA的影响，我们将专门深入分析脾脏、骨髓和肠道相关淋巴组织（GALT）中的B细胞和浆细胞亚群，并解析GALT的结构。为了探索TFG_BKO小鼠在稳态、免疫后和过表达实验中浆细胞亚群中JCHAIN的依赖性，我们将对JCHAIN进行定量分析。此外，我们将解决Ig组装和稳态。tfg缺失对内质网稳态、自噬和代谢信号的影响将在tfgB小鼠原代活化B细胞中确定。阐明TFG在B细胞和浆细胞中的作用将有助于理解支持抗体合成和体液免疫所需的浆细胞生物学。

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.