Figure 4.

T-cell proliferation in the presence and absence of CADO, ZM241385, AZD4635, POM-1, anti-CD73, and supernatants from cell culture. MM patients’ T cells were isolated from BMMC (A) or from peripheral blood mononuclear cells (PBMC) (B–E), labeled with CFSE, and cultured in the presence of IL-2 and anti-CD3/CD28 as described in online supplementary materials and methods. Division index was determined on day 5 of culture. (A) A2AR antagonist ZM241385 restored T-cell proliferation abolished by CADO. (CFSE histogram is shown in online supplementary figure S3A). T cells were cultured as described with and without 10 µM CADO and 0.3 µM ZM2413850 for 5 days. (B) A2AR antagonist ZM241385 restored T-cell proliferation inhibited by adenosine from cocultures of JJN3 and HS-5. (A representative CFSE histogram is shown in online supplementary figure S3B.) Cells were cultured as described, in the presence or absence of 0.3 µM ZM241385. (C) POM-1 and anti-CD73 restored T-cell proliferation inhibited by adenosine from cocultures. JJN-3 cells were pretreated with or without 100 µM POM-1 and cultured for 1 hour in the presence of 100 µM ATP. T cells were cultured together with HS-5 in the presence of 150 µg/mL anti-CD73 or isotype controls as described before. Supernatants containing AMP from JJN-3 cells (CM) were added to the culture. (D) CFSE histogram of T cells from a representative T-cell culture from (C). (E) AZD4635 restored proliferation in T cells inhibited by CADO. T cells were cultured as described in the presence of 15 µM CADO and 3 µM AZD4635. Division index was determined on day 5 of culture. Data were means with SD. Statistical differences were calculated with Student’s t-test. Three independent experiments were performed. A2AR, adenosine receptor A2A; BMMC, BMmononuclear cells; CFSE, carboxyfluoresceinN-succinimidyl ester.