ADA-deficient murine FTOC was used to assess the biochemical mechanism(s) by which a loss of ADA enzyme activity blocks the development of T cells. Previous work from our group (12
) demonstrated that the earliest effects of ADA deficiency on thymocyte development were caused by the accumulation of ADA substrates derived from thymocytes undergoing apoptosis. The pan-caspase inhibitor, z-VADfmk, both corrected differentiation and prevented the accumulation of dATP, presumably by inhibiting the death of thymocytes that failed β selection. This hypothesis was further corroborated in the present study by the ability of an in-frame TCRβ
chain transgene to partially correct thymocyte differentiation in short-term ADA-deficient FTOCs. The transgene failed to provide substantial protection in cultures longer than 2 days, however, probably because of the accumulation of ADA substrates derived from thymocytes failing positive selection, because a transgenic TCRβ
chain does not assure TCRs competent to recognize self peptide plus MHC when paired with endogenous rearranged TCRα
chains. Indeed, dATP was quite elevated in 3.5-day ADA-inhibited FTOCs with TCRβ
chain transgenic thymuses (data not shown).
We next employed 5′A5′dAdo, a potent inhibitor of adenosine kinase, the primary enzyme responsible for the phosphorylation of both adenosine and deoxyadenosine in murine thymus (21
), to address the question of whether toxicity is mediated directly by ADA substrates or by phosphorylated derivatives. Correction of thymocyte differentiation with a concomitant 90% reduction in cellular dATP levels argues strongly against mechanisms of toxicity mediated by adenosine and deoxyadenosine and suggests that the culprit is dATP. To strengthen this assertion, various proposed mechanisms of toxicity due to adenosine and deoxyadenosine or their metabolites were evaluated.
One proposed mechanism of toxicity was adenosine receptor signaling, because engagement of these receptors induces apoptosis in thymocyte suspensions (25
). However, ADA-deficient FTOCs performed with thymuses from A2aR
knockout mice revealed no protective effect. Since the absence of a single adenosine receptor might not prevent toxicity, the general adenosine receptor antagonist XAC was tested for its ability to protect ADA-deficient FTOCs. Prevention of adenosine receptor engagement did not affect normal thymocyte differentiation nor did it protect developing thymocytes from the effects of ADA deficiency. In a parallel approach, FTOCs were performed with NECA, a general adenosine receptor agonist, to see if this agent would mimic the consequences of ADA deficiency. NECA, used at concentrations up to 1,000-fold higher than necessary for inducing apoptosis of thymocytes in suspension cultures, had no observable deleterious effects on thymocyte differentiation or proliferation. The reason why NECA induces apoptosis in thymocyte suspensions, but not in FTOC, is unknown, but may be related to protective signals from thymic stromal cells or some other component of the thymic microenvironment. These results suggest that aberrant adenosine receptor engagement is not the mechanism responsible for thymocyte depletion due to ADA deficiency. Our conclusions are thus different from those of Apasov et al. (29
) who studied the toxicity of adenosine on thymocyte suspensions in the presence of an ADA inhibitor. However, it is important to note that they used adenosine at 100 μM, a concentration that is probably never achieved in our FTOCs, and that the consequences of adenosine exposure may be different in isolated thymocytes compared with those in the thymic microenvironment as stated above.
Inhibition of SAH hydrolase was another potential mechanism of toxicity mediated by adenosine and deoxyadenosine. This enzyme degrades SAH, a product of transmethylation reactions where S-adenosylmethionine (SAM) is the methyl donor. Elevated adenosine can force reversal of the hydrolytic reaction to form SAH from adenosine and homocysteine (22
). Deoxyadenosine acts as a “suicide” inhibitor, forming a covalent bond within the active site of the enzyme (23
). Elevated levels of SAH, relative to SAM, as a consequence of SAH hydrolase inhibition, can lead to inhibition of multiple cellular methylation reactions (24
). Our observation that SAH hydrolase enzyme activity remained inhibited in ADA-deficient cultures rescued by 5′A5′dAdo argues strongly against SAH hydrolase inhibition as the mechanism by which ADA deficiency inhibits thymocyte development in FTOC.
With experimental evidence from our lab (12
) and others (2
) indicating that toxicity associated with ADA deficiency correlated with elevations in dATP, we next explored potential routes of toxicity associated with this molecule. There are two major mechanisms by which dATP could deleteriously affect developing thymocytes: allosteric inhibition of ribonucleotide reductase (30
), the enzyme that generates deoxyribonucleotides needed for DNA synthesis, and induction of apoptosis (11
). Our data indicate that the dATP levels attained in ADA-inhibited FTOCs must be insufficient to inhibit the generation of deoxyribonucleotides, because thymocyte proliferation is normal in dCF-treated FTOCs with Bcl-2
transgenic thymocytes where dATP levels are hyperelevated.
dATP has the potential to promote apoptosis at two points. First, dATP can induce the release of cytochrome c
from isolated mitochondria (11
). Cytochrome c
release correlates with mitochondrial membrane changes and the release of other proapoptogenic factors, initiating the irreversible effector phase of apoptosis and making this event the “point of no return” in the execution of cell suicide programs (32
). Second, dATP interacts with Apaf-1, cytochrome c
, and procaspase-9 to form the apoptosome (34
), resulting in activation of caspase-9 and triggering of the apoptotic cascade. We favor the more proximal point of dATP action because overexpression of Bcl-2, a protein that regulates cytochrome c
efflux to the cytoplasm, provides more protection for ADA-deficient FTOCs than deletion of Apaf-1 (12
). This would be expected if cytochrome c
release were the critical event, given that Bcl-2 is upstream of Apaf-1. If the primary role of dATP were to contribute to apoptosome formation, removal of Apaf-1 or other components of the apoptosome should be equally effective in affording protection to ADA-deficient FTOCs. This is an important distinction, because a deoxyadenosine analogue, 2-chlorodeoxyadenosine, has been proposed to trigger apoptosis of leukemic cells via the interaction of its triphosphorylated derivative, 2-Cl-dATP, with subapoptogenic levels of cytochrome c
present in the cytoplasm (35
). Further experimentation will be necessary to prove directly that dATP causes cytochrome c
release from mitochondria in ADA-deficient FTOCs. Our data do not rule out the possibility that dATP could act indirectly by interfering with some crucial event in thymocyte development such that apoptosis is induced in a mitochondrial-dependent fashion. However, this unspecified event is unlikely to be TCR gene rearrangement, because this was found to be normal in ADA-deficient FTOCs (12
Our experiments also provide a new perspective on the routes by which thymocytes die by apoptosis during development. The observation that dATP is hyperelevated in ADA-deficient FTOCs with thymuses from Bcl-2
transgenic mice argues that Bcl-2 does not prevent the death of thymocytes that fail positive/negative selection — otherwise, dATP levels would have normalized. Furthermore, the fact that dATP is hyperelevated relative to dCF-treated FTOCs with wild-type thymuses suggests that larger quantities of ADA substrates are generated from cells failing positive/negative selection than from those failing β selection. This is not surprising given the high degree of cellular expansion when thymocytes pass the β selection checkpoint (36
). Experiments are underway to determine whether thymocytes at later stages of development are also sensitive to the consequences of elevated dATP, and if so, by what mechanism. It will also be important to determine whether our conclusions are applicable to human thymocyte development by using a model system, such as chimeric human/mouse FTOC (37
), where human thymocyte development takes place in vitro. These studies are also in progress. We anticipate that continued use of in vitro models of thymocyte differentiation to study ADA deficiency will not only shed additional light on the pathogenesis of this immunodeficiency but will also further our understanding of normal programs of T cell development.