Elucidation of the molecular mechanisms of negative regulation of autoantibody production by novel lgM receptor

用igm免疫复合物培养np特异性BCR敲入小鼠（QM小鼠）的B细胞，分析其上调CD86激活标志物的情况。虽然单独用抗原刺激B细胞以抗原剂量依赖的方式上调CD86的表达，但与单独抗原诱导相比，IgM IC下调了CD86的表达。用能够阻断IgM结合的抗fc α□μR^□mAb预处理B细胞，可以消除这种下调。因此，IgM-IC通过BCR和Fcα□μR的共连接下调BCR介导的B细胞活化。接下来我们生成A20的转染体。2J， B细胞淋巴瘤细胞系，稳定表达WT Fcα□μR （A20-WT）或缺乏细胞质部分的突变型Fcα□μR (A20-Δ Cyt)，并通过流式细胞术检测bcr介导的Ca^<2□>内流。交联BCR诱导A20- wt中Ca^<2□>内流，而BCR与Fcα□μR的共连接可下调A20- wt中Ca^<2□>内流，而在A20-Δ细胞中未观察到这种抑制作用。这些结果表明，Fcα□μR抑制bcr介导的信号通路，其胞质区是抑制bcr介导信号通路不可或缺的区域。为了研究Fcα□μR介导的信号通路对B细胞的负调控作用，我们检测了在BCR和Fcα□μR共连接后，在A20-WT中Fcα□μR胞质区域的苏氨酸残基是否被磷酸化，在这种情况下Ca^<2□>内流被抑制。我们观察到BCR与Fcα□μR共联抗体后1分钟苏氨酸磷酸化增强，但单独交联BCR后未观察到苏氨酸磷酸化增强。这些结果表明，苏氨酸残基可能参与了Fcα□μR对B细胞的负调控。

B cells obtained from NP-specific BCR knock-in mice (QM mice) were cultured with IgM-immunecomplex and analyzed for their upregulation of CD86 activation marker. While stimulation of B cells with the antigen alone up-regulated the expression of CD86 in an antigen dose-dependent manner, the IgM IC down-modulated the CD86 expression compared with that induced with antigen alone. This down-modulation was abrogated by pre-treatment of B cells with anti-Fcα□μR^□ mAb that was able to block IgM binding. Thus, IgM-IC down-regulates BCR-mediated activation of B cells by co-ligation of BCR and Fcα□μR. Next we generated transfectants of A20. 2J, a B cell lymphoma cell line, stably expressing WT Fcα□μR (A20-WT) or mutant Fcα□μR lacking the cytoplasmic portion (A20-Δ Cyt), and examined BCR-mediated Ca^<2□> influx by flow cytometry. Cross-linking BCR induced Ca^<2□> influx in A20-WT, However, co-ligation of BCR and Fcα□μR downregulated Ca^<2□> influx in A20-WT, In contrast, this inhibition was not observed in A20-Δ Cyt. These results suggested that Fcα□μR suppressed BCR-mediated signaling and their cytoplasmic region was indispensable for this suppression. To investigate Fcα□μR-mediated signaling for negative regulation of B cells, we examined whether the threonine residue in the cytoplasmic region of Fcα□μR was phosphorylated in A20-WT after co-ligation of BCR and Fcα□μR, as Ca^<2□> influx was suppressed in this case. We observed enhanced threonine phosphorylation 1 min after co-ligation of BCR and Fcα□μR by antibodies, although we did not observe such an enhanced threonine phosphorylation after cross-linking BCR alone. These results suggest that the threonine residue might be involved in a negative regulation of B cells by Fcα□μR.