Lantelme Erica, Orlando Luca, Porcedda Paola, Turinetto Valentina, De Marchi Mario, Amoroso Antonio, Mantovani Stefania, Giachino Claudia
Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Italy.
Mol Immunol. 2008 Jan;45(2):328-37. doi: 10.1016/j.molimm.2007.06.153. Epub 2007 Jul 30.
V(D)J recombination is a mechanism peculiar to the somatic rearrangement of antigen receptor genes. It requires both expression of the RAG-1 and RAG-2 recombinases and accessibility of the substrate to its recombinase and post-cleavage/DNA repair stage. TCR revision is a genetic correction mechanism that changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. This process is now well described in both normal or pathological murine and human settings. Many of its features, such as the question of whether it occurs in truly mature T cells, remain to be elucidated. Its occurrence in human CD8+ T cells is also an open question. We have therefore established an in vitro model of TCR revision in mature human CD8+ T cells to determine whether down-regulation of the TCR/CD3 complex from the cell surface in the presence of IL7 as a factor favouring chromatin remodelling initiates a TCR revision pathway. Only mature CD8+ T cells carrying already-formed antigen receptors were used. CD8+ T cells treated with anti-CD3 and IL7 showed rearrangement intermediates and expressed new Vbeta-chains on their surface. Investigation of the molecular pathway thus induced disclosed up-regulation of the RAG-2 transcript, but absence of the 'canonical' RAG-1 mRNA. A surprising finding was the demonstration of alternative splice forms of this mRNA, already expressed in untreated CD8+ T cells, encoding for the full-length RAG-1 protein, which was increased three-fold in the treated cells. All the V(D)J requirements were thus fulfilled when mature human CD8+ T cells were stimulated with anti-CD3 and IL7. Induction of TCR revision in vitro in mature T cells is an easily controllable system that could be employed in further studies to elucidate the molecular pathways involved in secondary V(D)J rearrangements in peripheral cells.
V(D)J重组是抗原受体基因体细胞重排所特有的一种机制。它既需要RAG-1和RAG-2重组酶的表达，也需要底物对其重组酶的可及性以及切割后/DNA修复阶段。TCR修正 是一种基因校正机制，通过在外周T细胞中重新激活V(D)J重组来改变T细胞特异性。目前，这一过程在正常或病理状态的小鼠和人类环境中均有详细描述。其许多特征，如是否发生在真正成熟的T细胞中这一问题，仍有待阐明。它在人类CD8+ T细胞中的发生也是一个悬而未决的问题。因此，我们建立了一个成熟人类CD8+ T细胞中TCR修正的体外模型，以确定在作为有利于染色质重塑的因子的IL7存在的情况下，TCR/CD3复合物从细胞表面下调是否会启动TCR修正途径。只使用携带已形成抗原受体的成熟CD8+ T细胞。用抗CD3和IL7处理的CD8+ T细胞显示出重排中间体，并在其表面表达新的Vβ链。对由此诱导的分子途径的研究揭示了RAG-2转录本的上调，但不存在“经典”的RAG-1 mRNA。一个惊人的发现是，在未处理的CD8+ T细胞中已经表达的该mRNA的可变剪接形式的证明，其编码全长RAG-1蛋白，在处理过的细胞中增加了三倍。因此，当用抗CD3和IL7刺激成熟人类CD8+ T细胞时，所有V(D)J要求均得到满足。在成熟T细胞中体外诱导TCR修正是一个易于控制的系统，可用于进一步研究以阐明外周细胞中继发性V(D)J重排所涉及的分子途径。
