Timing of rearrangement of multiple T‐cell receptor loci in individual thymocyte precursors

Four T‐cell receptor (TCR) loci undergo rearrangement in thymic T‐cell precursors. Three of these loci, TCRβ, γ and δ, rearrange, in part, simultaneously in CD4/CD8 double negative, stage 3 (DN3) thymocytes. DN3 cells are precursors to most mature T‐cells which have rearrangements of all TCR loci. It is not known, however, whether these rearrangements occur simultaneously, or consecutively, in individual cells. We have addressed this question by analyzing VDJ recombination‐associated DNA breaks in individual DN3 cells using combined fluorescent in situ hybridization and immunofluorescence (immunoFISH) staining. The rational of this approach is that even transient DNA double strand breaks induce accumulation of DNA damage sensing factors, such as γ‐phosphorylated histone‐2AX (γ‐H2AX) and 53BP1. Such foci have been shown to associate with the TCRα locus in cells where TCRα recombination occurs. We have performed multiple FISH analysis of TCRβ, γ and δ loci using fluorescent‐labeled BAC probes and studied their co‐localization with γ‐H2AX and 53BP1 foci. These studies illuminate the spatial and temporal control of gene rearrangement and provide insight into the mechanisms that protect lymphoid precursor cells from the potentially damaging consequences of recombination during normal lymphocyte development. Supported in part from funds from the National Institute of Health. 1