Impact of CD39/CD73 overexpression on malignant and nonmalignant T- and NK-cell functions. (A) Quantification of adenosine 5′-monophosphate in the plasma of HD and patients with SS (n = 10 per group). (B) Quantification of cell-associated CD39/CD73 enzymatic activity using purified CD4⁺ T cells from HD or patients with SS (n = 4 per group). (C) T-cell proliferation assay. Carboxyfluorescein diacetate succinimidyl ester- or CellTrace Violet-labeled PBMC from HD or patients with SS (n = 6 per group) were either left untreated or activated for 4 days with CD3/CD28 beads in the presence of ATP alone or combined with istradefylline (50 mM), A438079 (A438; 20 μM), or a blocking anti-CD73 mAb (10 μg/mL). Following immunolabeling, proliferation of the CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. (D) ADCC assay. PBMC from HD or patients with SS were incubated with interleukin-15 in the presence of ATP alone or combined to istradefylline or A438. After 4 days, cells were mixed with Raji target cells at an E/T ratio of 20/1 and rituximab (10 μg/mL). Target cells apoptosis was monitored by 7-AAD labeling. Results are expressed as the mean ± standard deviation of the % of 7AAD⁺ Raji cells obtained for the indicated conditions with HD (left) and SS (right) PBMC (n = 4 per group). (A-D) Statistical analysis was performed using a Mann-Whitney t test. **P < .01, ***P < .001, ****P < .0001; ns, not significant.