Ectonucleotidase activity of CD39⁺ and CD73⁺ T cells from patients with SS, and functional implications of CD39^high or CD73^high T cells. (A and B) Flow cytometry dot plots showing CD73 and CD39 expression in CD4⁺ T cells from a representative CD39^high patient with SS (A), a representative CD73^high patient with SS (B), and 2 different HDs. Panels on the right show high-performance liquid chromatography (HPLC) profile of the peaks of extracellular ATP and AMP (A) or AMP and ADO (B) generated by CD4⁺ T cells on ATP or AMP supply, respectively. Cells were treated with or without 100 μM sodium polyoxotungstate (POM-1, a CD39 inhibitor) or 10 μM adenosine 5′-(α,β-methylene)diphosphate (APCP, a CD73 inhibitor) for 1 hour, before incubation for 3 hours with 200 μM ATP or 100 μM AMP, as indicated. (C) Quantification of AMP concentrations in the supernatant of CD4⁺ T cells from 3 patients with SS with different levels of CD39 expression and from HDs (n = 3), under the indicated experimental conditions. (D) Quantification of ADO concentration in the supernatant of CD4⁺ T cells from 2 patients with SS with different levels of CD73 expression and from HDs (n = 3) under the indicated experimental conditions. Results are expressed as micromoles of AMP per 10⁶ cells or of ADO per 10⁶ cells. In (C) and (D), data are expressed as mean ± SD. (E) Time-dependent increase in ADO concentration in the supernatant of CD4⁺ T cells from patient SS06 (CD73⁺). Histograms indicate ADO concentrations on AMP supply for 30 minutes, 1 hour, and 3 hours. Data are shown as the mean of technical replicates. (F) Schematic representation of a coculture system comprising human pulmonary microvascular endothelial cells (HPMECs) and peripheral blood mononuclear cells (PBMCs) used as an in vitro model to evaluate the degradation of ATP to ADO by CD39 and CD73 expressed by different cell types in the system. (G) HPLC profiles showing ATP, AMP, and ADO peaks on ATP addition. CD4⁺ T cells from a representative CD39^high patient with SS (top) or from a representative CD73^high patient with SS (bottom) were seeded onto HPMEC monolayers and exposed to extracellular ATP (200 μM) for 3 hours in the presence or absence of the indicated doses of POM-1 or APCP inhibitor. (H) Quantification of ADO concentration in the coculture system under the indicated experimental conditions. Results from 1 CD39^high patient (SS10) and 1 CD73^high patient (SS06) are shown. (I) CFSE-labeled PBMCs were activated for 5 days with CD3/CD28 antibodies in the presence of 500 μM ATP alone or combined with CPI-444 (10 μM) or istradefylline (0.1 μM). After immunolabeling, proliferation of CD8⁺ T cells was determined by flow cytometry. Data are shown for 1 representative CD39^high patient with SS of 3 analyzed (mean of technical replicates), 1 representative CD73^high patient with SS of 2 analyzed (mean of technical replicates), and for HDs (n = 8; mean ± SD). A two-way analysis of variance with the Šidák multiple comparisons test was done to compare untreated with ATP-treated cells under the indicated conditions in HDs: ∗∗∗∗P < .0001; ∗∗∗P < .001. CFSE, carboxyfluorescein diacetate succinimidyl ester; FITC, fluorescein isothiocyanate; Rt, retention time.