THYMOCYTE DIFFERENTIATION IN GAMMA-IRRADIATED SEVERE-COMBINED IMMUNODEFICIENT MICE - CHARACTERIZATION OF INTERMEDIATES AND PRODUCTS OF V(D)J RECOMBINATION AT THE T-CELL RECEPTOR-ALPHA LOCUS

Treatment with DNA-damaging agents promotes rescue of V(D)J recombinat ion, limited thymocyte differentiation, and development of thymic lymp homas in severe-combined immunodeficient (SCID) mice. One intriguing a spect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recomb inationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermedi ates, hairpin coding ends, at the TCR alpha locus. We found that J alp ha-coding ends are undetectable in unirradiated SCID thymocytes, but a ccumulate after irradiation at times coincident with the emergence of a CD4(+) CD8(+) thymocyte population. Coding joints produced by joinin g of these ends, however, are extremely rare. To test whether the pres ence of hairpin coding ends at TCR alpha is sufficient for irradiation -mediated rescue of coding joint formation, we administered a second d ose of gamma-irradiation after abundant CD4(+) CD8(+) thymocytes and h airpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of ha irpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results s upport a refined model for irradiation-mediated rescue of TCR rearrang ements in SCID mice.