Up-Regulation of iNOS in AML Blasts Creates an Immunosuppressive Microenvironment, Inhibits T-Cell Proliferation and Transforms T-Cells Towards a Tumor-Tolerating Phenotype

Similar to solid tumors, AML cells are capable of creating an immunosuppressive microenvironment by reducing the function and cytotoxicity of T and natural killer (NK) cells and hence deregulate the innate and adaptive immune responses. This evasion of immune surveillance is based on several mechanisms, including the expansion of regulatory immune cells, such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs) and macrophages; the production of immunosuppressive cytokines such as IL-10 and TGF-β; and the expression of immune checkpoint proteins that act as co-inhibitory factors such as programmed death ligand 1 (PD-L1) on leukemia cells and other cells of the microenvironment like MSC and Treg. In addition, the expression of immunosuppressive factors like indoleamine 2,3-dioxygenase (lDO), inducible nitric oxide synthase (iNOS; also known as NOS2) and arginase (arginase I and II) in MDSC, tumor-associated macrophages (TAMs) and tumor-infiltrating dendritic cells may contribute to this evasion process. It has recently been demonstrated that the majority of blasts in patients with AML express arginase II suggesting an important contribution of this enzyme to the suppression of the patients' immune response (Mussai et al., Blood 2013). However, little is known about the expression of other immunosuppressive factors in AML and how they cooperate in the suppression of the immune system.

In this study, we sought to determine the expression of arginase (I and II), iNOS and IDO in blasts from AML patients and to establish a correlation with their immunosuppressive features. The expression of these immunosuppressive molecules was assessed by intracellular flow cytometry (FC) in AML blasts (n=20) compared to healthy donors PBMC (n=3). We found the expression of arginase II and iNOS to be significantly up-regulated in AML blasts (P=0.016 and P=0.006, respectively) while the expression of arginase I and IDO was not significantly different from healthy donor PBMC. There was a strong correlation between the expression of arginase I and IDO in AML samples (R²=0.58; P=0.006) although correlations between iNOS-arginase I and iNOS-IDO were weak. We next examined the immunosuppressive potential of AML samples in relation to their expression of immunosuppressive molecules. T-cells were isolated from healthy donors, labeled with CSFE, activated with immobilized anti-CD3/CD28 antibodies and co-cultured with AML blast or left alone with IL-2 (50 ng/ml) for 5 days. The proliferation of T-cells was measured by FC at day 5 after stimulation and results were analyzed using ModFit software. The immunosuppressive effect of AML samples on T-cells was variable and sample-dependent. Interestingly, the immunosuppressive potential of AML samples was positively correlated to their expression of iNOS (R²=0.58; P=0.004), while the expression of arginase I and II correlated with a modest inhibition of T-cell proliferation. In order to elucidate if AML cells can transform T-cells towards a tumor-tolerating phenotype, we analyzed the composition of T-cells at the end of the T-cell proliferation assay and found an increase in the percentage of CD4+,CD25+,FoxP3+ T-reg in activated T-cells and a decrease in the frequency of CD3+CD8+CD56+ Natural Killer T-Cells (NKTs) during co-culture with AML blasts. Such effect was not observed when T-cells were challenged with allogeneic healthy donor PBMC.

Conclusions: AML blasts express immune-modulatory molecules and their expression correlates with their immunosuppressive potential. iNOS seems to be the main contributing factor although the expression of arginase I, arginase II and IDO was also detected in AML blasts and contributed partially to this suppressive phenotype. The immunosuppressive capacity of AML not only affects the proliferation of activated T-cells but can also have a direct effect on T-cells skewing them into a T-reg phenotype and decreasing the number of NKT cells. Upfront characterization of these molecules in AML blasts can potentially improve treatment outcome by combining inhibitors of these immune-modulatory factors with existing immune-therapy.