An in vitro model of T cell receptor revision in mature human CD8+ T cells.
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.
Lantelme
Erica
E
Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano, Italy. erica.lantelme@unito.it
Orlando
Luca
L
Porcedda
Paola
P
Turinetto
Valentina
V
De Marchi
Mario
M
Amoroso
Antonio
A
Mantovani
Stefania
S
Giachino
Claudia
C
D000906:Antibodies / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D002460:Cell Line; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004768:Enterotoxins / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015329:Gene Rearrangement, T-Lymphocyte / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015851:Interleukin-7 / Q000494:pharmacology; D018448:Models, Immunological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology
D000906:Antibodies; D017252:CD3 Complex; D004268:DNA-Binding Proteins; D004768:Enterotoxins; D018398:Homeodomain Proteins; D015851:Interleukin-7; D009687:Nuclear Proteins; D020033:Protein Isoforms; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; C064658:RAG-1 protein; C031818:enterotoxin B, staphylococcal