B细胞骨髓免疫耐受机制－抗原非依赖性IgH和Igkappa 的V(D)J基因重排

Receptor editing作为骨髓B细胞免疫耐受的主要机制，被认为是自身抗原识别未成熟B细胞，诱导重链Igh和轻链Igκ的二次V(D)J基因重排。然而有些实验论文不支持此理论。我们提出新的B细胞骨髓耐受假说，即Ig基因自发随机的重排和选择。我们拟作下面的工作。在IgH editing中，我们测定在MT小鼠骨髓B细胞中的VH cRS cleavage，来决定其是否需要功能性的preBCR和BCR信号。在Igκ editing中，我们在103/bcl2 前B细胞系，JCκ+/-小鼠，携带无害IgH转基因H50G小鼠，和携带自身抗原IgH转基因小鼠anti-DNA 3H9小鼠未成熟B细胞中，扩增和分析VJ二次重组的中间产物。我们使用BrdU测定immature B细胞是否由于自身抗原逆向分化。这些结果将更新B细胞骨髓免疫耐受机制，即不依赖于preBCR和BCR诱导的Ig基因自发随机的重组。

Receptor editing is believed to play the major role in purging newly formed B cell compartments of autoreactivity by the induction of secondary V(D)J rearrangements. In the process of immunoglobulin heavy (H) chain editing, these secondary rearrangements are mediated by direct VH -to-JH joining or cryptic recombination signals (cRSs) within VH gene segments. Using a statistical model of RS, we have identified potential cRSs within VH gene segments at conserved sites flanking complementarity-determining regions 1 and 2. Cleavage of multiple VH cRSs was observed in the bone marrow of C57BL/6 and ?MT congenic animals, and we determined that cRS cleavage efficiencies are 30 - 50-fold lower than a physiological RS. cRS signal ends are abundant in pro-B cells, including those recovered from ?MT mice, but undetectable in pre- or immature B cells. Thus, VH cRS cleavage regularly occurs before the generation of functional preBCR and BCR. .For B-cell tolerance through immunoglobulin light-chain, receptor editing by secondary Vκ-to-Jκ rearrangements is thought to be driven by autoreactivity. However, the evidence supporting antigen-driven editing is open to other interpretations; analysis of Igκ excision circles from birds and mice indicates continuing V(D)J recombination in cis after both functional (F) and non-functional (nF) rearrangements. We have determined the extent and nature of secondary Vκ-to-Jκ rearrangements by analyzing their intermediate cleavage products in the 103/bcl2 cell line and in purified compartments of developing B cells from normal, JCκ+/-, and IgH transgenic mice. In all cases, we find that the ratio of F:nF Vκ-Jκ joints replaced by secondary rearrangement does not significantly differ from the 1:2 ratio expected for rearrangement without feedback of any kind. Secondary Ig? rearrangement is initiated in small pre-B cells and in BrdU pulse-labeling studies, we detected no evidence for immature to pre-B "retrograde" differentiation. Thus, "receptor editing" occurs in B cells that do not express surface Ig. Significantly, in 3H9 IgH transgenic mice, replacement rearrangements exhibited F:nF ratios of 1:2, regardless of whether the initial rearrangements were permissive for autoreactivity. Our results demonstrate continuing Vκ-to-Jκ rearrangements on a single chromosome without feedback and imply that "receptor editing" may be an illusion of antigen-driven selection.