Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients

Abstract

Mutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2'-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4(+) T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca(2+) flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-kappaB. Moreover, exposure to 2'-deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A(2A) adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials.gov as #NCT00598481 and #NCT0059978.

Trial registration: ClinicalTrials.gov NCT00598481 NCT00599781.

Figure 1

Characterization of the functional defect in CD4⁺ADA-deficient T cells. (A) Purified CD4⁺ T-cell lines from 5 patients and 5 NDs were stimulated with immobilized anti-CD3 mAb at the indicated concentrations, and [³H]thymidine incorporation was measured after 48 hours. One representative experiment of 3 is shown. (B) Cells were stimulated with 1 μg/mL anti-CD3, and culture supernatants were collected after 18 (IL-2) or 48 hours (all other cytokines). Cytokine concentrations were quantified by capture ELISA. Each value represents the mean of cytokine concentration measured in triplicate cultures for patients (n = 5; ADA^−/− and GT) and healthy controls (n = 5). Median values are also reported. Background levels were subtracted. One determination of 3 is shown. (C) Production of IFN-γ vs either IL-2, or IL-4, was analyzed by intracytoplasmic staining in CD4⁺ T-cell lines as described in “Cytokine detection.” The experiment shown is representative of 5 independent experiments. Two ADA-SCID patients, before and after GT, and 2 NDs are reported. Range values corresponding to 5 patients and 7 NDs are indicated in Table 1.

Figure 2

A defective transcription of cytokine genes in ADA-deficient T cells associates with reduced phosphorylation of CREB. (A) Cytokine gene expression after TCR/CD28-mediated stimulation of CD4⁺ T-cell lines from 5 patients (ADA^−/− and GT) and 4 NDs. Total RNA was prepared from either resting or stimulated cells (1 μg/mL anti-CD3 plus 10 μg/mL anti-CD28 mAbs) and gene expression were measured by semiquantitative RT-PCR. Values were normalized for the expression of HPRT and correspond to duplicate samples. Results are expressed as n-fold difference relative to resting condition. One representative experiment of 3 is shown. **P < .01; *P < .05. (B) Immunoblots depicting phosphorylated CREB, total CREB, and GAPDH. CD4⁺ T cells were stimulated with cross-linked (1 μg/mL), anti-CD3 plus (10 μg/mL), anti-CD28 mAbs for the indicated time points. The same membranes were sequentially probed for the different Abs. Upper panel: a representative experiment in Pt3 (ADA^−/− and GT) and one ND is shown. Lower panel: densitometric analysis and statistical significance (**P < .01) on 5 patients (ADA^−/− and GT) and 5 NDs are shown. Values corresponding to unstimulated cells were subtracted.

Figure 3

Defective proximal TCR signaling in ADA-deficient T cells. Intracytoplasmic analysis of phosphorylated ZAP-70 (Y319) in response to cross-linked (1 μg/mL), anti-CD3 plus (10 μg/mL), anti–CD28-mediated activation of CD4⁺ T cells. Dot plots from 2 ADA-SCID patients, before and after GT, and 2 NDs are reported. Results corresponding to frequency of positive cells after 40 seconds of stimulation, in one of 3 independent determinations, from 5 patients and 4 NDs are indicated in the graph. Values corresponding to basal phosphorylation were subtracted (*P < .05; **P < .01).

Figure 4

dAdo strongly inhibits ERKs activation and effector functions in ADA-deficient T cells activating A_2A AR signaling. (A) ERK activation was analyzed in CD4⁺ cells stimulated with cross-linked (1 μg/mL), anti-CD3 plus (10 μg/mL), anti-CD28 mAbs in the absence or presence of dAdo (500 μM). Samples of untreated and dAdo-treated stimulated cells were prepared in parallel, run on different gels but probed with the different Abs at the same time. (B) CFSE-labeled CD4⁺ cells were cultured in plain medium or stimulated with immobilized anti-CD3 mAb (1 μg/mL) in the absence or presence of dAdo (500 μM). After 64 hours, cells were analyzed by flow cytometry. TO-PRO-3 was added at the time of FACS analysis. Histograms depicting the CFSE content of TO-PRO-3⁻ viable cells are shown from one representative patient and one ND. The percentage of CFSE^dim cells is indicated in each plot. Background levels corresponding to unstimulated cells were subtracted. The total number of trypan blue–negative viable cells and the frequency of TO-PRO-3–positive cells from CFSE/TO-PRO-3 dot plots of total events are shown for 5 patients and 4 NDs. (C) CD4⁺ cells stimulated with 1 μg/mL coated anti-CD3 mAb in the absence or presence of dAdo (500 μM) or (D) CGS21680 (100 μM). The specified compounds (SCH58261 or dipyridamole) were included 1 hour before the addition of dAdo. After 48 hours, cell proliferation was assessed by [³H]thymidine incorporation. The result of a representative experiment including 5 patients (ADA^−/− and GT) and 5 NDs is reported. Percentages of inhibition with respect to the untreated condition are indicated. (E) Intracellular cAMP levels were determined in CD4⁺ T cells incubated for 15 minutes with culture medium containing 5 mM dAdo or 1 mM CGS21680. The inhibitor of adenylate cyclase (AC, 1 mM) was added 30 minutes before the other drugs. The data represent the mean plus or minus SD from 5 patients (ADA^−/− and GT) and 4 NDs in one of 3 independent experiments. Statistics: **P < .01; *P < .05.

Figure 5

PKA hyperactivation is responsible for dAdo-mediated suppression of T-cell function in ADA-deficient cells. CD4⁺ T cells were stimulated with 1 μg/mL coated anti-CD3 mAb in the presence or absence of dAdo (500 μM) with or without the inhibitor of cAMP-dependent protein kinase A, Rp-8-Br-cAMPS (150 μM). (A) Rate of proliferation was evaluated by [³H]thymidine incorporation after 48 hours. (B) Culture supernatants were collected for cytokine production quantification by ELISA after 18 (IL-2) or 48 (all other cytokine) hours. Percentages of inhibition with respect to the untreated condition are indicated. The data represent the mean plus or minus SD of 5 patients (ADA^−/− and GT) and 5 NDs from 1 of 3 independent experiments.

Figure 6

Dose- and time-dependent apoptosis induced in ADA-deficient T cells by ADA substrates. (A) Bulk T-cell lines were incubated with the indicated concentrations of purine metabolites and cultured for 15 hours (dAdo) or 6 hours (Ado). In time course experiments, T cells were incubated with dAdo (5 mM) or Ado (1 mM) for the indicated period. The percentages of early apoptotic cells (annexin V⁺/PI⁻) are shown for Pt3 (ADA^−/− and GT) and one ND as representative experiment. (B) Bulk T-cell lines from 5 patients (ADA^−/− and GT) and 6 NDs were cultured for 15 hours with dAdo (5 mM) or 6 hours with Ado (1 mM) in the absence or presence of the inhibitor of nucleoside transporter, dipyridamole (1 μM). The percentages of annexin V⁺/PI⁻ cells as mean plus or minus SD are shown. Data are representative of at least 5 independent experiments (**P < .01).