An in vitro model of T cell receptor revision in mature human CD8+ T cells

Abstract

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.