Abstract
Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4−CD8− double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4+CD8+ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4+CD8+ thymocytes, resulting in reduced numbers of CD4+CD8+ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4+CD8+ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.
Highlights
Antigen-specific receptors expressed by T and B cells are heterodimers encoded by genes that must first be assembled from variable (V), diversity (D), and (J) joining gene segments by recombination
In ATMWT and KO DN2/3 cells that are RAG1-deficient there were no detectable 53 BP1 foci co-localizing with TCRb in 0/370 ATMWT or 0/396 ATMKO cells examined, reinforcing the conclusion that even when ataxia telangiectasia-mutated (ATM) is deficient, 53 BP1 foci are primarily restricted to rearranging TCR loci
Since persistent DNA damage foci in irradiated ATMKO cells have been shown to correlate with persistence of DNA damage [29], the increase in 53 BP1 foci at TCRb loci reported here may reflect a requirement for ATM for efficient resolution of RAG-induced double-strand breaks (DSB)
Summary
Antigen-specific receptors expressed by T and B cells are heterodimers encoded by genes that must first be assembled from variable (V), diversity (D), and (J) joining gene segments by recombination. The protein product of the ataxia telangiectasia-mutated (ATM) gene is a kinase critical for sensing and responding to DNA DSB in a variety of circumstances, including V(D)J recombination [13,14,15,16,17,18,19]. Phenotypes of both AT patients and ATMKO mice as well as other experimental approaches have demonstrated the importance of ATM in facilitating V(D)J recombination. Expression of a TCRb transgene (TG) in the ATMKO substantially reverses this defect in DP cell numbers, directly linking these TCRb-dependent developmental defects to the requirement for ATM during endogenous TCRb rearrangement
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