Deoxyadenosine Inhibits T-Cell Activation in ADA-SCID Patients through a cAMP/PKA-Dependent Pathway.

Adenosine deaminase (ADA) is a key enzyme of the purine salvage pathway, metabolizing adenosine (Ado) and deoxyadenosine (dAdo). ADA deficiency results in increased levels of Ado and dAdo in plasma and cells, leading to severe combined immunodeficiency (SCID). However, the role of intracellular accumulation of dAdo and/or an aberrant extracellular signaling, mediated by G-coupled adenosine receptors, in the pathogenesis of the disease remains to be elucidated. Retroviral-mediated gene transfer of ADA into hematopoietic stem/progenitor cells has been recently shown to be an effective treatment for ADA-SCID children, thus providing a unique model to investigate the influence of purine metabolism in the survival and functions of T lymphocytes. Using untransformed bulk or CD4+ T-cell lines established from ADA-SCID patients before and after gene therapy, we investigated the biochemical pathways responsible for the pathogenesis of the disease and the ability of gene therapy to restore normal metabolic and immunological functions. We found that the expression of functional ADA in gene corrected T cells resulted in the restoration of SAHH activity and in the normalization of apoptosis induced by exposure to Ado/dAdo in defective cells. Interestingly, while a nucleoside transporter inhibitor could prevent Ado-induced cell toxicity, dAdo exerted its cytotoxic effect via the accumulation of intracellular cAMP, mediated by the engagement of Gs-protein coupled adenosine receptors. Functional studies revealed that both proliferative responses and Th1/Th2 cytokine production were impaired in ADA-deficient cells, but were restored in T cells generated after gene therapy. Such impairment was consequent to a defective TCR signalling, as indicated by the intrinsically reduced activation of p44/p42 MAPK pathway after anti-CD3/anti-CD28 mAb stimulation. Furthermore, a reduced transcription of cytokine genes was observed in ADA−/− CD4+ T cells, which was associated to a defective activation of early transcription factor CREB and possibly to an altered nuclear recruitment of NF-kB, as predicted by the decreased phosphorylation of IkBa. Remarkably, in ADA-deficient T cells, but not in gene corrected or healthy donor cells, exposure to non-toxic concentrations of dAdo resulted in a strong inhibition of p44/p42 MAPK activation and complete abrogation of TCR/CD28-driven proliferation and cytokine production. The effects of dAdo were reverted in the presence of a cAMP/PKA inhibitor, highlighting a key role for PKA hyperactivation and involvement of A2 receptors in causing T-cell dysfunctions. Collectively, these findings provide a more clear understanding of the physiopathology of ADA-SCID and confirm the efficacy of ADA-gene transfer in restoring normal metabolic pathways and immunological functions.