Flow-cytometry and functional evaluation of the CD39/CD73 adenosinergic immunosuppressive axis in patients with Sézary syndrome

Sézary syndrome (SS) is a primary cutaneous T-cell lymphoma characterized by measurable levels of malignant lymphocytes in the blood, and progressive impairment of the immune response. Among the mechanisms of immune modulation, adenosine can impair anti-tumor immunity, through the attenuation of protective effector cells, including T and NK cells, and by enhancing the suppressive capacity of T regulatory cells. CD39 and CD73 nucleotide-metabolizing enzymes are involved in the adenosine-generating pathway: CD39 cleaves ATP and ADP down into AMP, which is converted into adenosine by CD73. The extracellular adenosine deaminase/CD26 complex catalyzes the deamination of adenosine to inosine, thus reducing the adenosine levels. The objective of this study were: to investigate the expression of the of CD39 and CD73 nucleotide-metabolizing ectoenzymes in peripheral blood from SS patients; to define the contribution of the CD39/CD73 adenosinergic immunosuppressive pathway to tumor escape from immune response and immune dysfunctions in patients with SS. CD39 and CD73 expression analyzed in whole blood with multiparametric flow-cytometry from SS patients (n=10) and healthy donors (HD, n=11) revealed altered expression of CD39 and CD73 in SS patients compared to HD. Two subgroups of patients can be identified based on the mutually exclusive overexpression of CD39 or CD73 in CD4+ T cells. Indeed, 7 out of 10 patients had high CD39, while 3 had high CD73. CD4+T cells with high CD39 have low CD73, vice versa those with high CD73 have low CD39. To define whether CD39 and CD73 were biologically active, we compared the ability of peripheral blood CD4+T cells from SS patients and HD to hydrolyze ATP and to convert AMP into adenosine, in vitro. Briefly, CD4+ Tcells from SS/HD were seeded in 48-well plates in HBSS, pretreated or not with specific inhibitors for 1h and then incubated with exogenous (e) eATP (patients with high CD39) or eAMP (patients with high CD73) at 37°C. After 1 h incubation, analyses of the supernatant were performed with an RP-HPLC. Results indicated that CD4+ T cells from SS patients with high levels of CD39 showed an increased ability to hydrolyze ATP with increased generation of AMP compared to normal control cells. In parallel, CD4+ T cells with high levels of CD73 showed increased conversion of AMP into ADO, respect to normal control cells. The aberrant expression of CD39 and CD73 along with loss of CD26 expression in circulating Sézary cells suggest that the sequential activity of CD39 and CD73 ectoenzymes scavenges ATP and generates immunosuppressive adenosine in the tumor microenvironment contributing to tumor immune escape. The results inferred from this study are the starting point for more comprehensive studies towards the development of new therapies targeting the CD39/CD73 adenosinergic axis in order to overcome tumor immunosuppression, allowing the induction of effective anti-tumor immune response. Sézary syndrome (SS) is a primary cutaneous T-cell lymphoma characterized by measurable levels of malignant lymphocytes in the blood, and progressive impairment of the immune response. Among the mechanisms of immune modulation, adenosine can impair anti-tumor immunity, through the attenuation of protective effector cells, including T and NK cells, and by enhancing the suppressive capacity of T regulatory cells. CD39 and CD73 nucleotide-metabolizing enzymes are involved in the adenosine-generating pathway: CD39 cleaves ATP and ADP down into AMP, which is converted into adenosine by CD73. The extracellular adenosine deaminase/CD26 complex catalyzes the deamination of adenosine to inosine, thus reducing the adenosine levels. The objective of this study were: to investigate the expression of the of CD39 and CD73 nucleotide-metabolizing ectoenzymes in peripheral blood from SS patients; to define the contribution of the CD39/CD73 adenosinergic immunosuppressive pathway to tumor escape from immune response and immune dysfunctions in patients with SS. CD39 and CD73 expression analyzed in whole blood with multiparametric flow-cytometry from SS patients (n=10) and healthy donors (HD, n=11) revealed altered expression of CD39 and CD73 in SS patients compared to HD. Two subgroups of patients can be identified based on the mutually exclusive overexpression of CD39 or CD73 in CD4+ T cells. Indeed, 7 out of 10 patients had high CD39, while 3 had high CD73. CD4+T cells with high CD39 have low CD73, vice versa those with high CD73 have low CD39. To define whether CD39 and CD73 were biologically active, we compared the ability of peripheral blood CD4+T cells from SS patients and HD to hydrolyze ATP and to convert AMP into adenosine, in vitro. Briefly, CD4+ Tcells from SS/HD were seeded in 48-well plates in HBSS, pretreated or not with specific inhibitors for 1h and then incubated with exogenous (e) eATP (patients with high CD39) or eAMP (patients with high CD73) at 37°C. After 1 h incubation, analyses of the supernatant were performed with an RP-HPLC. Results indicated that CD4+ T cells from SS patients with high levels of CD39 showed an increased ability to hydrolyze ATP with increased generation of AMP compared to normal control cells. In parallel, CD4+ T cells with high levels of CD73 showed increased conversion of AMP into ADO, respect to normal control cells. The aberrant expression of CD39 and CD73 along with loss of CD26 expression in circulating Sézary cells suggest that the sequential activity of CD39 and CD73 ectoenzymes scavenges ATP and generates immunosuppressive adenosine in the tumor microenvironment contributing to tumor immune escape. The results inferred from this study are the starting point for more comprehensive studies towards the development of new therapies targeting the CD39/CD73 adenosinergic axis in order to overcome tumor immunosuppression, allowing the induction of effective anti-tumor immune response.