Adenosine deaminase modulation of telomerase activity and replicative senescence in human CD8 T lymphocytes

Abstract

Increased proportions of CD8 T lymphocytes lacking expression of the CD28 costimulatory receptor have been documented during both aging and chronic infection with HIV-1, and their abundance correlates with numerous deleterious clinical outcomes. CD28-negative cells also arise in cell cultures of CD8(+)CD28(+) following multiple rounds of Ag-driven proliferation, reaching the end stage of replicative senescence. The present study investigates the role of a second T cell costimulatory receptor component, adenosine deaminase (ADA), on the process of replicative senescence. We had previously reported that CD28 signaling is required for optimal telomerase upregulation. In this study, we show that the CD8(+)CD28(+) T lymphocytes that are ADA(+) have significantly greater telomerase activity than those that do not express ADA and that ADA is progressively lost as cultures progress to senescence. Because ADA converts adenosine to inosine, cells lacking this enzyme might be subject to prolonged exposure to adenosine, which has immunosuppressive effects. Indeed, we show that chronic exposure of CD8 T lymphocytes to exogenous adenosine accelerates the process of replicative senescence, causing a reduction in overall proliferative potential, reduced telomerase activity, and blunted IL-2 gene transcription. The loss of CD28 expression was accelerated, in part due to adenosine-induced increases in constitutive caspase-3, known to act on the CD28 promoter. These findings provide the first evidence for a role of ADA in modulating the process of replicative senescence and suggest that strategies to enhance this enzyme may lead to novel therapeutic approaches for pathologies associated with increases in senescent CD8 T lymphocytes.

FIGURE 1

Higher telomerase activity in CD8⁺CD28⁺ T cells that express ADA. A, Kinetics of ADA cell surface expression following stimulation with Ab-coated microbeads. Gated populations of CD3⁺CD8⁺CD28⁺ versus CD3⁺CD8⁺CD28⁻ T lymphocytes were evaluated from four individual cultures. B, Representative gel showing that four different cell equivalents of CD8⁺ T cells (10,000, 5,000, 2,500, 1,250 cell equivalents) fall in the linear range of telomerase activity. Cultures were stimulated with irradiated allogeneic B lymphoblastoid cells, and, on day 4, cell lysates were prepared, and telomerase activity was analyzed using the modified TRAP assay as described in Materials and Methods. C, TRAP assay gel from a representative culture after separation of CD8⁺CD28⁺ subset into ADA⁺ and ADA⁻ using 2500 cell equivalents per lane.

FIGURE 2

Intracellular ADA activity decreases with culture age. Long-term T cultures were established by activation with Ab-coated microbeads. CD8⁺ T lymphocytes were purified 4 d postsimulation, and cell lysates were assessed for ADA activity using the colorimetric assay described by Vielh and Castellazzi (30). Intracellular ADA activity for five individual cultures, all at equivalent PDs, is presented. **p , 0.01.

FIGURE 3

Surface ADA expression is reduced as human CD8 T cells approach replicative senescence. Cultures were established by activation with Ab-coated microbeads, and ADA expression was determined by flow cytometry 4 d after each stimulation (i.e., every ∼14 d). A, Histogram showing expression of ecto-ADA (determined by flow cytometry) on CD3⁺CD8⁺ cells from a representative culture at 8 PDs and 25 PDs. B, Mean (±SD) percentage of the CD8⁺ T cells that express ecto-ADA at early (8 PDs) and late (25 PDs) time points; the lines connect data from four individual cultures. *p < 0.05. C, ADA tran-scriptional expression (n = 4) normalized against GAPDH. **p < 0.01.

FIGURE 4

CD8 T lymphocytes exposed to adenosine have lower CD28 expression. Long-term cultures were established by stimulation with Ab-coated microbeads. DMSO, the ADA inhibitor (10 µM),oradenosine (10µM) plus ADA inhibitor (10 µM) were added on day0 and every 3 to 4 d thereafter. A, CD28 expression on CD3⁺CD8⁺ T cells in a long-term culture exposure to DMSO, ADA inhibitor (10 µM), or adenosine (10 µM) with ADA inhibitor (10µM).B,CD28messagewascomparedwithGAPDHbyQT-PCRatday70. n = 4. *p < 0.05.

FIGURE 5

Adenosine downregulation of CD28 involves activated caspase-3. T lymphocytes were stimulated with Ab-coated microbeads. DMSO, the ADA inhibitor (10 µM), or adenosine (10 µM) plus ADA inhibitor (10 ) were added at day 0 and every 3 to 4 d thereafter. Constitutive caspase-3 levels in CD8⁺ T lymphocytes that were purified on day 14 were determined using Apo-Alert commercial kit (Calbiochem). A, Fold increase in caspase-3 level for 11 individual cultures treated with adenosine/ADA inhibitor versus DMSO and mean fold increase for all cultures. B, CD28 surface expression at day 14 in CD3⁺CD8⁺ T cell cultures treated with DMSO, ADA inhibitor (10 ), adenosine (10 )/ ADA inhibitor (10 ), or adenosine/ADA inhibitor/caspase-3 inhibitor. ***p < 0.001.

FIGURE 6

Adenosine accelerates replicative senescence in human CD8 T lymphocytes. A, Total PD for DMSO, EHNA, and adenosine/EHNA-treated cultures when replicative senescence was reached; n = 4, mean value shown with horizontal line. B, IL-2 mRNA was analyzed by QT-PCR and results expressed relative to GAPDH. *p < 0.05; **p < 0.01.