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  • Targeting the BCR is the ideal strategy to identify the

    2023-09-18

    Targeting the BCR is the ideal strategy to identify the antigen reactive B cells. However, there is concern that B cells could be activated when BCR are bound and cross-linked by antigens, a possibility that is obviously negative for the treatment of autoimmune disease. Proby et al. (2000) tried to overcome this problem by fusing autoantigens with toxins. They used a recombinant Dsg3DN1-toxin chimera to target the autoantibody-producing B cells in Pemphigus vulgaris (PV) and found that the hybridoma cells expressing Dsg3DN1-specific antibody were killed, and the number of Dsg3DN1-specific B cells from Dsg3DN1-immunized mice decreased after treatment with the fusion protein. Reiners et al. treated Wegener's granulomatosis (WG) with a similar strategy, and the results demonstrated that autoantigen-toxins induced apoptosis in proteinase 3-specific hybridoma cells (Reiners et al., 2004). However, the toxin dosages they used were too high and apparently not suitable for clinical usage. Instead, we chose to fuse the inhibitory ligand-Fc part of human IgG1. The aim of our strategy was to trigger the inhibitory receptor, FcγRIIB, on B cells and the BCR, at the same time using an autoantigen-Fc fusion protein in order to ultimately specifically inhibit and eliminate target B cells. In our experiments, Hα1-210 rather than complete AChR molecules was fused as an autoantigen, because the majority of pathogenic anti-AChR APY29 in the sera of MG patients were directed against a part of the extracellular region of the α-subunit of the AChR called the main immunogenic region (MIR) (Tzartos et al., 1982, Tzartos et al., 1985). Thus, the majority of the AChR-reactive B cells could be eliminated by our fusion protein. Our data showed that AChR-Fc treatment could inhibit proliferation and induce apoptosis of B cell hybridomas, and eliminated 60% AChR-reactive B cells at the highest concentration in vitro. To further explore the cytotoxicity of AChR-Fc on AChR-reactive B cells, we employed splenocytes from AChR-immunized rats as an ex vivo model. The ELISPOT results showed that the reduction of AChR-reactive B cells occurred in a dose-dependent manner. An annexin V binding assay indicated AChR-Fc can induce CD19+ B cells apoptosis. These findings validate the hypothesis we proposed and suggest that AChR-reactive B cells can be targeted and effectively eliminated by AChR-Fc fusion protein. FcγRIIB is a member of FcγR family that plays an important role in mediating inhibitory signals and maintaining humoral tolerance (Nimmerjahn and Ravetch, 2008). It is expressed broadly on all leukocytes with the exception of NK cells and T cells. FcγRIIB is the only Fcγ receptor expressed by B lymphocytes. Mice deficient for it were susceptible to autoimmune diseases (Fukuyama et al., 2005) and, conversely, overexpression of FcγIIB on B cells can result in the suppression of autoimmune disease (Baerenwaldt and Nimmerjahn, 2008). Negative regulation of FcγRIIB makes it an ideal target for immunotherapy. Moreover, cross-linking FcγRIIB-FcγRIIB or FcγRIIB-BCR can cause B cell apoptosis (Nimmerjahn and Ravetch, 2008). Recently Mihaylova et al. constructed chimeric molecules coupling an immunodominant mouse histone 1 peptide, which can be recognized by MRL/lpr mice, to a rat monoclonal anti-mouse CD32 antibody (Mihaylova et al., 2008). They found that the number of anti-histone 1 antibody-producing cells, the level of IgG anti-histone 1 antibodies, the level of albuminuria and the size of lymphoid organs decreased, and that skin lesions developed slower. These findings indicate that FcγRIIB can be used as a target to eliminate autoreactive B cells (Tchorbanov et al., 2007). In our study, we fused the Hα1-210 with a Fc fragment directly, hoping that AChR-Fc could not only crosslink BCR with FcγRIIB on autoreactive B cells in MG patients, but could also crosslink BCR with FcγR+ immune effector cells and complement, leading to immune effector cell-mediate cytotoxicity and complement-dependent target cells lysis. To further verify the cytotoxicity and cell lysis function of the Fc fragment by binding the Fc receptor, we employed human PBMCs as effector cells and guinea pig serum as a complement source. We observed significant apoptosis of hybridoma cells in the AChR-Fc treatment group indicating that AChR-Fc can induce target cell apoptosis by mediating immune effector cells and complements.