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To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alpha and beta TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocytes response.