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

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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.