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 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.

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。

在自身免疫性红斑狼疮小鼠B6.MRL-Faslpr和MRL/MpJ-Faslpr中，能对DNA和DNA/组氨酸复合物反应的自身反应性抗体是其小鼠免疫球蛋白抗体库的特征。这些小鼠Fas受体基因缺陷，因而自身反应性B细胞不能通过外源性途径凋亡。在本项目研究中，我们探讨了B6.MRL-Faslpr和MRL/MpJ-Faslpr小鼠的骨髓小前B细胞和未成熟B细胞包干自身反应性B细胞受体（BCR）并呈现自身反应性综合征，表现增强的受体编辑（receptor editing)模式。研究表明，Faslpr的前B和未成熟B细胞表现出具有更多的非功能性（nP）二次VkJk基因重排。更重要的是，这些被替换的初次轻链基因重排表现P:nP为1：2，因此提示k轻链重排看上去不能抑制二次基因重排。并且，我们发现了多个非典型VDJ基因重排，比如Vk cRS（秘密重排信号）的断端。这些结果表明轻链二次基因重排在狼疮小鼠和对照的骨髓B细胞的特定发育阶段表现为非选择性模式并且独立于BCR的自身反应性。因此我们提出二次Igk基因重排的主要功能是增加BCR抗体库的多样性。