In Vivo Immunological Effects of CD73 Deficiency.

BACKGROUND/AIMS: The extracellular ecto-5'-nucleotidase (CD73) is involved in the production of immunosuppressive adenosin (Ado), which can influence different immune cells through the specific adenosine receptors. The main aim of this work was to characterize immune cell populations as well as serum cytokine level in healthy CD73-deficient mice compared to healthy wild-type animals. METHODS: Profound immnophenotyping of splenocytes from healthy CD73-deficient and wild-type mice was done using flow cytometry (FACS analysis). Cytokine measurement in the serum of the animals was carried out with a Bio-Plex assay. RESULTS: The CD73-deficience leads to an increase in a percentage of NK cells and pDC, as well as influences expression of the costimulatory molecules CD80 and CD86. The knockout mice in opposite to wild-type animals show high amount of effector CD4+ T-cells in the spleens. No changes have been found in the subpopulations of CD8+ T-cells. Besides, CD73-deficience leads to a decrease in the percentage of regulatory T cells. Compared with the wild-type animals we found that CD73 knockout mice possess low serum concentration of IL-6. CONCLUSION: This in vivo study clear demonstrated certain immunological changes in the CD73-deficient mice and thus immunoregulatory potential of CD73 molecule. This makes this extracellular enzyme to a real immune check point molecule, attractive for further investigations and clinical studies.

Enhanced expression of CD39 and CD73 on T cells in the regulation of anti-tumor immune responses.

Synthesis of extracellular adenosine by the ectonucleotidases CD39 and CD73 represents an important pathway of immune suppression in the tumor microenvironment. Using two mouse models (RET transgenic melanoma and Panc02 orthotopic pancreatic adenocarcinoma), we identified an elevated frequency of ectonucleotidase-expressing T cells in tumors and spleens. Importantly, these ectonucleotidase-positive T cells also showed a pronounced expression of PD-1. Conversely, the PD-1+ T cell subsets in tumors contained substantially larger proportions of ectonucleotidase-expressing cells compared to their counterparts lacking PD-1 expression. Our in vitro experiments showed that the activation of normal T cells resulted in an increase in the CD39 expression. CD39+ and CD73+ T cells displayed effector or memory phenotypes and produced IFN-γ, thereby linking ectonucleotidase expression to T cell effector functions. An accumulation of conventional and regulatory T cells expressing CD39 and/or CD73 was also detected in the peripheral blood of patients with melanoma and pancreatic cancer. Moreover, we demonstrated a significant association between low frequencies of circulating CD73+CD8+ T cells and CD73+CD4+ regulatory T cells and better overall survival of melanoma patients. Tumor-derived soluble factors (in particular, TGF-ß) significantly enhanced the frequencies of ectonucleotidase-expressing cells in mice. Our findings suggest that the upregulation of ectonucleotidase expression in T cells promotes extracellular adenosine accumulation and represents an important mechanism of homeostatic immune auto-regulation, which could be hijacked by tumors to evade anti-cancer immunity. Targeting CD39 and CD73 can open new avenues for cancer immunotherapy.

Characterization of myeloid leukocytes and soluble mediators in pancreatic cancer: importance of myeloid-derived suppressor cells.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. PDAC cells activate tumor-specific immune responses but simultaneously trigger a strong immunosuppression. We showed that PDAC cells produce high amount of chronic inflammatory mediators and PDAC tumors build an immunosuppressive cytokine milieu, which correlates with tumor progression. We observed a low frequency of dendritic cells (DC) and a pronounced accumulation of macrophages and myeloid-derived suppressor cells (MDSC) in murine PDAC tumors. A strong accumulation of MDSC has also been demonstrated in the peripheral blood of resected PDAC patients. While DC and macrophages seem not to play a significant role in this PDAC model in the context of immunosuppression, MDSC are highly suppressive, and their accumulation is associated with an increase in intratumoral VEGF concentration during the PDAC progression. Application of the phosphodiesterase-5 inhibitor sildenafil led to a prolonged survival of PDAC-bearing female mice, which was due to the decrease in MDSC frequencies and in the systemic VEGF level. This led to a restoration of anticancer immune responses, manifested in the recovery of T lymphocyte functions and in an increase in the frequency of conventional CD4+ T cells in tumors and IFNγ level in serum of PDAC-bearing mice. Thus, MDSC are strongly involved in the PDAC-associated immunosuppression and that their depletion could create new approaches for therapy of PDAC.