T-Cell Development Failure At β-Selection Checkpoint and TCRα/δ Locus Break Formation Associated with Chromosome 14 Translocation in Ataxia-Telangiectagia Mutated Deficient Mice

Abstract 184^FN2

Ataxia Telangiectagia (AT) is an autosomal recessive immunodeficiency, caused by mutation of ataxia telangiectagia mutated gene (ATM). ATM plays a crucial role for responding to DNA damages by extrinsic and intrinsic factors, and is a master regulator for maintaining DNA integrity. VDJ recombination and class switch recombination during lymphocyte maturation are the steps of intrinsic DNA damage response where ATM stabilizes DNA ends during recombination. ATM deficiency (ATM−/−) is known to predispose to T-cell lymphopenia and T-lineage lymphoma development. ATM−/− mouse has been shown to have a failure of T-cell development at the stage from double positive (DP) to single positive (SP) differentiation, which is due to a failure of T-cell receptor a (TCRa) recombination. Thymic lymphomas in ATM−/− mice have recently been shown to have a chromosome 14 translocation involving TCRd locus, suggesting that the first event for translocation arises during TCRd locus recombination at double negative (DN) stage. However, phenotypic features of T-cell development at DN phase and the timing of chromosome 14 translocation formation in ATM−/− are not fully elucidated.

Here we demonstrate that T cells of ATM−/− mice show a failure at the transition from DN3a to DN3b at b and gd-selection checkpoints due to multiple TCR recombination failure in-vivo. Consistent with in-vivo developmental profiles of ATM−/− mice thymocytes, long term hematopoietic stem cells (LTR-HSCs) of ATM−/− mice cultured with OP9-DLL1 show a delay at b-selection checkpoint in chronological order. In this culture system, failures in gd-T-cell development are also observed in ATM−/− LTR-HSCs. Involvement of thymic stromas in the failure of this transition was ruled out by bone-marrow transplantation (BMT) of ATM−/− donor to WT recipient mice, where thymocytes reconstitution showed the same transition failure at b-selection checkpoint. Thymocytes in RAG2−/− mice are arrested at DN3 stage by a failure of cleavage of TCR genes, but the arrested thymocytes are known to progress to DP phase by anti-CD3e antibody stimulation. This experiment enables to analyze pre-TCR dependent differentiation signal machinery. Then anti-CD3e antibody was injected into RAG2−/−ATM−/− mouse and DN3 cells were shown to be led to DP phase, indicating that ATM itself is not involved in the differentiation program during DN to DP phase. These results suggested loss of ATM attenuates T cell differentiation at DN3a to DN3b transition due to inefficient TCRg, d and b locus recombination. Thus differentiation failure from DN3a to DN3b in ATM deficiency is presumably the primary cause of T cell lymphopenia at the stage prior to positive-selection.

We next investigated when of the differentiation stages chromosome 14 translocation involving TCRa/d locus monitored. When the LTR-HSCs is cultured on the OP9-DLL1 cells with high-dose cytokine including 10 ng/ml of Flt3-L, IL-7 and SCF, differentiation of LTR-HSCs to T cells halt at DN2-3a phase before b-selection. Then, by reducing the Flt3-L and IL7 to 5 ng/ml and 1 ng/ml, respectively, the differentiation arrest is released and Tcell differentiation progresses from DN3a to DN3b. No detectable chromosome break at TCRad locus was observed at DN2-3a in wild type, while 5% of ATM−/− cells carried TCRad break, associated with chromosome 14 translocation in approximately 0.8 % of DN2-3a cells. After progression to DN3b-4 phase, TCRad locus break was still observed in AT cells at the frequency of 1%, and chromosome 14 translocations involving TCRad locus was observed in 12% of ATM−/− cells, which was in contrast to none in wild type cell. Mono- or bi-allelic TCRa/d breaks, chromosome 14 dicentric, and t (12:14) were also observed in minor population of ATM−/− cells. These results suggest that critical point for generation of chromosome 14 translocations involving TCRa/d locus lies at DN2-3a to 3b stages corresponding during b and gd selection checkpoint in ATM deficient thymocytes.

Our findings revealed that developmental failure of T-cells in AT arises during b and gd–selection checkpoint, which leads to the breaks of TCRa/d locus and subsequent chromosome 14 translocation formation. Thus we propose T-lymphopenia and predisposition to T cell leukemia/lymphoma are tightly connected in ATM deficient condition.