Abstract

T-cell exhaustion is a state of functional decline in T cells, driven by chronic antigen exposure and inhibitory signals within the tumor microenvironment. Exhausted CD8+ T cells (Tex) derive from precursor exhausted T cells (Tpex), a self-renewing population responsible for the proliferative burst in anti-PD-1 therapies. Exhausted T cells are exposed to adenosine in the tumor, yet the role of the CD73/adenosine axis and Tpex/Tex differentiation remains unclear. Using an in vitro model for CD8+ T-cell exhaustion, we found that CD73 expression increased during Tpex formation and that its expression was negatively correlated with Tex generation. CD73-deficient OT-I cells (OT-I/CD73KO) showed impaired activation and reduced progression to Tex. Moreover, we demonstrated that CD73 promotes transcriptional expression of the adenosine receptor A2A (A2AR). RNA-seq analysis of exhausted OT-I/CD73KO cells revealed a more stem-like transcriptomic profile and enrichment in genes associated with the immune response compared to their OT-I counterparts. In vivo, the cotransfer of naïve OT-I/CD73KO and OT-I antigen-specific CD8⁺ T cells into tumor-bearing mice resulted in increased Tpex frequency and numbers among OT-I/CD73KO cells in tumors. Conversely, in vitro exhaustion in the presence of A2AR agonists decreased Tex frequency and activation/exhaustion markers, while increasing CD73 and CD62L, which are markers associated with stemness. Supporting this, A2AR blockade with SYN115 in tumor-bearing mice reduced Tpex markers and increased Tex differentiation. Altogether, our data suggest that CD73 promotes Tpex-to-Tex differentiation, whereas adenosine A2AR signaling supports Tpex maintenance in the tumor microenvironment.