The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.
The leukemogenic effects of Myc drive recurrent trisomy in a mouse model of acute myeloid leukemia.
Long-term humoral immunity is maintained by the formation of high-affinity class-switched memory B cells and long-lived antibody-secreting plasma cells. In healthy humans, a substantial fraction of IgG-positive memory B cells express self-reactive and polyreactive IgG antibodies that frequently develop by somatic mutations. Whether self-and polyreactive IgG-secreting B cells are also tolerated in the long-lived plasma cell pool is not known. To address this question, we cloned and expressed the Ig genes from 177 IgG-producing bone marrow plasma cells of four healthy donors. All antibodies were highly mutated but the frequency of self-and polyreactive IgG antibodies was significantly lower than that found in circulating memory B cells. The data suggest that in contrast to the development of memory B cells, entry into the bone marrow plasma cell compartment requires previously unappreciated selective regulation by mechanisms that limit the production of self-and polyreactive serum IgG antibodies. N ewly developing self-reactive B cells are efficiently counterselected at two tolerance checkpoints before becoming circulating naive B cells (1, 2). Antigen-mediated activation of naive B cells in the presence of T-cell help induces germinal centers and the development of memory B cells and long-lived bone marrow plasma cells that express high affinity, isotype class-switched antibodies (3, 4). Surprisingly, compared with naive B cells, circulating IgG-positive memory B cells are significantly enriched for self-reactive and polyreactive antibodies (5). These antibodies develop in the germinal center from nonself-reactive or polyreactive precursors by somatic mutations and appear in the serum of healthy humans in low amounts (6). Whether cells producing self-reactive and polyreactive IgG can enter the bone marrow plasma cell compartment has not been determined.To address this question, we produced recombinant monoclonal antibodies from isolated IgG-secreting plasma cells from bone marrow of four healthy donors (HDs) and compared the Ig gene features and antibody reactivity profiles to historic data obtained from IgG-positive memory B cells (5, 7). Ig gene sequence analysis showed that bone marrow plasma cells carry significantly higher numbers of somatic mutations than circulating memory B cells and that the two compartments differ in their Ig light (IgL) chain variable (V) gene use and IgG isotype subclass distribution. Further, the frequency of polyreactive and self-reactive IgG-positive antibodies was significantly lower in bone marrow plasma cells than in the memory B-cell compartment. In summary, the data suggest that entry into the bone marrow plasma cell compartment is selective for B cells producing highly mutated antibodies that are not self-or polyreactive.
The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.
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