腺苷信号在CD8 T细胞淋巴细胞减少的条件下促进双阴性T细胞的抗癌反应

CD8 T-cells lymphopenia is a feature of several pathological conditions and is associated with decreased survival and increased risk of developing cancer and infections. Increase in Double-Negative T-cells (DNTs), a subset of T-cells which lack CD8 and CD4 receptors, has been found in both human patients and mice with lymphopenia. DNTs could be either suppressive or immunoprotective. Generating immunoprotective DNTs could be useful to compensate for the lack of CD8 T-cells, but how to stimulate them remains unclear. Forodesine (Foro) is a nucleoside analog which has known antileukemic activity. Foro can also stimulate T-cell function, but the mechanism behind this effect and what type of T-cells is targeted by the drug is still unclear. In this study, we investigated strategies to promote immunoprotective antitumor DNTs in conditions of CD8 T-cells lymphopenia using the antileukemic drug Foro. To determine whether and how Foro stimulates T-cells, we tested Foro in an orthotopic syngeneic triple-negative breast cancer model (C0321-FVB mice). Foro significantly reduced tumor growth and stimulated tumor-infiltrating CD8 T-cells, suggesting that CD8 T-cells modulation mediates its effect. However, when CD8 T-cells were depleted to mimic CD8 T-cells lymphopenia, Foro showed an even stronger anticancer effect. Interestingly, Foro increased CD69+ DNTs in tumors and in circulation, in blood, and this increase correlated with smaller tumors, suggesting that Foro stimulates anticancer DNTs responses when there is a lack of CD8 T-cells. We next studied how Foro could promote antitumor DNTs. Our previous work showed that nucleoside analogs, like Foro, can modulate immunity by blocking the A2A receptor (A2AR). Consistently, blocking A2AR with the antagonist ZM-241385 (ZM) also increased CD69+ DNTs in immunocompetent mice, mimicking Foro treatment. To confirm whether the immunomodulatory effect of Foro in DNTs is based on its capacity to modulate A2AR, we: (i) predicted the interaction between A2AR and Foro using our validated molecular docking modeling; (ii) found that Foro reduced adenosine-induced cyclic AMP (cAMP), the second mediator of A2AR, in A2AR-expressing cells, like ZM; (iii) observed increased Notch1 activation, which is downstream of A2AR, in CD69+ DNTs in Foro-treated mice. Finally, we found that CRISPR-KO of Notch1 significantly reduced CD69+ DNTs, suggesting that Notch1 is critical for the generation and function of DNTs, in response to Foro. Our results indicate that blocking the A2AR signaling promotes Notch1 activation leading to the generation of anticancer CD69+ DNTs, a T-cell subpopulation which could compensate for the lack of CD8 T-cells when lymphopenia occurs. This can be achieved pharmacologically using compounds with A2AR antagonistic properties like Foro. Our work put forward a new rationale for the treatment of cancer patients affected by lymphopenia.