Pseudohypoxia induces extracellular adenosine production and purinergic signaling in an autocrine manner, thereby inducing T-cell dysfunction. (A) Normalized counts indicating the expression of genes ENTPD1 (CD39), NT5E (CD73), ADORA2A (A_2AR), ADORA2B (A_2BR), and MMP-9. (B) PBMCs from HDs were thawed and cultured in the presence or absence of PGA-1, CII, Mec-1, LME-1, U-266, RPMI-8226, and Nalm-6 cells and stimulated with anti-CD3/CD28 antibodies. CD4⁺ T cells were analyzed 48 hours after stimulation by multiparameter flow cytometry for the expression of CD39, CD73, and A2_A/BR expression on the cell surface. (C) Percentage of FOXP3⁺CD25⁺ and CD25⁺CD127^– cells within the CD4 compartment after 72 hours coculture with the cell lines, in absence of stimulation. (D) Deconvolution of RNA sequencing data with CIBERSORTx analysis; fractional abundance of indicated immune cell subsets is plotted. (E) Supernatants from the coculture stimulated with anti-CD3/CD28 antibodies were analyzed for adenosine concentration; (F) p-S6 and CD25 expression was assessed in presence of the A2_A/BR antagonist AB928 (10-30 μM). Panel D represents the same data points as that of Figure 3D in absence of AB928. Data are presented as mean ± SEM. ∗∗∗P < .001; ∗∗P < .01; ∗P < .05; ns (1-way ANOVA followed by Tukey multiple comparison test). ND, not detected; U, unstim.