Abstract

Background. The tumor microenvironment is typically an immunosuppressive niche often characterized by low oxygen tension, representing an intrinsic limitation to the success of immunotherapeutic approaches. Several lines of evidence indicate hypoxia is a master regulator of the adenosinergic axis, up-regulating on one side expression of CD39 and CD73, the two enzymes that generate adenosine starting from ATP/ADP and on the other side the adenosine receptors, which are powerful inhibitors of immune responses. Our previous studies using primary samples indicate that CLL cells can produce extracellular adenosine and that the CLL microenvironment is rich in expression of the A2A receptor, which potently inhibits T cell responses and skews macrophages towards type 2 responses^{(1, 2)}.

Aim of the work. The main aims of this work are i) to dynamically study expression and activity of the adenosinergic axis using an in vivo model of CLL, and ii) to investigate whether its targeting may restore immune responses.

Results. We exploited the TCL1 mouse model of CLL by adoptively transferring different leukemias into immunocompetent wild-type C57BL/6 mice and following the re-organization of the microenvironment and the reshaping of immune responses during disease progression. The first observation is that the appearance of large leukemic nodules that subvert normal splenic architecture is accompanied by increased staining with the hypoxia marker pimonidazole and by lactic acidosis, as witnessed by progressive increase in LDH activity within the tissue. The second observation concerns the onset of progressive immunosuppression with appearance of terminally differentiated and dysfunctional T lymphocytes and skewing of classical inflammatory monocytes to anti-inflammatory patrolling monocytes.

Leukemia development is also accompanied by increased adenosine bio-synthetic potential due to the up-regulation of CD39 and CD73 and to the marked and generalized up-regulation of the A2A adenosine receptor, which is evident in all cells of the microenvironment, including leukemic cells, T lymphocytes and macrophages, as documented by real-time PCR studies on purified populations and by flow cytometry and immunohistochemistry analyses. This finding suggests that A2A may be a common mediator of immune suppression and that it may be a suitable therapeutic target. To test this hypothesis, we adoptively transferred different TCL1 leukemias in naïve C57BL/6 mice, allowed engraftment for 10 days and then treated with the commercially available SCH58621 A2A inhibitor every other day for two weeks (1mg/kg, intra-peritoneally). At the end of treatment, mice were euthanized and immune features examined as above. Results very consistently showed that A2A inhibition increased the naïve component of both CD4⁺ and CD8⁺ subsets, with a concomitant partial reduction in effector T lymphocytes and in regulatory T cells. Moreover, both CD4⁺ and CD8⁺ subsets recovered their cytotoxic functions as indicated by the production of IFN-γ and IL-2. When examining the macrophage compartment, SCH58261 repolarized monocytes, by increasing the inflammatory subset at the expense of patrolling monocytes.

Conclusions. Taken together, these results highlight the relevance of the adenosinergic axis in the creation and maintenance of a tumor microenvironment that favors immune escape. This could be due, at least in part, to the presence of a highly hypoxic leukemic niche. Interrupting this network using an A2A receptor antagonist restores the functions of the different immune cell subsets. Therefore, these data suggest that the adenosinergic axis may represent a good target in treatment strategies that combine anti-leukemic drugs with agents able to repolarize the CLL environment towards immune competence.

1. Serra S, et al. (2016) Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment. 1(1).doi:10.1182/bloodadvances.2016000984.The.

2. Serra S, et al. (2011) CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death. 118(23):6141-6153.