Monocytic Myeloid Derived Suppressor CELLS (M-MDSC) As Prognostic Factor in Chronic Myeloid Leukemia Patients Treated with Dasatinib

INTRODUCTION. Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in most cancer patients. Recently, we and another group demonstrated that MDSC play an important role of immune escape in chronic myeloid leukemia (CML) patients inducing T cell tolerance. The aim of this study was to investigate the effect of the tyrosine kinase inhibitors (TKI) therapy on MDSC and possible correlation with clinical response.

METHODS. MDSCs were analyzed in peripheral blood of 20 healthy donors (HD) and 30 CML patients at diagnosis. MDSCs were also measured during TKI treatment (18 patients treated with imatinib and 10 patients with dasatinib). Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (M-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of MDSCs with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. Exosomes were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation.

RESULTS. G-MDSCs and M-MDSCs percentages in CML patients were greater than HD (respectively 82.5±9.6% vs 56.2±5.4% and 33.6±19% vs 5.9±4%, p<0.0001). Both isolated subpopulations showed expression of BCR/ABL and were able to inhibit T cells proliferation in comparison to positive control (from 48±7.6% to 25±5% for G-MDSC, p=0.0057 and 16.7±0.6% for M-MDSC, p<0.0001). No suppressive effect was observed in co-cultures with G-MDSC and M-MDSC obtained from HD. In addition, M-MDSC percentage correlated with BCR-ABLABL transcript levels in patients at diagnosis (r=0.5816, p=0.0006). Evaluating the effect of TKI therapy on MDSC levels, we found that both imatinib (IM) and dasatinib (DAS) induced a significant reduction of G-MDSC percentage at 6 months (from 82.5±9.6% to 55±17.3% after IM and 48.7±13% after DAS, p<0.0001) and 12 months (61.2±9.7% after IM and 33.4±14% after DAS, p<0.0001) of treatment. The levels of M-MDSCs significantly decreased only after DAS therapy (from 33.6±19% to 6.8±12.6% at 6 months, p=0.014 and 11.4±12.3% at 12 months, p=0.008); while there was a mild reduction after IM treatment (22.2±24.5% and 22.3±21.7% respectively at 6 and 12 months) although a great variability was observed among patients. Subsequently, correlation of MDSC reduction and clinical response to TKI therapy was investigated. We found that in DAS, but not in IM treated patients, a correlation between percentage of Major Molecular Response (MMR) and number of persistent M-MDSCs was found.. In fact, a significant difference was recorded by comparing M-MDSC levels in the MMR group (n=6) versus no MMR (n=4) at 6 or 12 months (p=0.0034).

In addition, to evaluate if leukemic cells are able to expand MDSC by releasing soluble factors or exosomes, we incubated monocytes obtained from HD with sera or exosomes from CML patients at diagnosis or healthy subjects. M-MDSCs percentage significantly increased only in conditions with CML serum ( 29±13%; p=0.0006) or exosomes (8 ±2,8%; p=0.01) while no effect was observed on G-MDSC percentage.

CONCLUSION: Therapy with TKI reduces the percentage of MDSCs and levels of the monocytic subset correlates with MMR in patients treated with dasatinib, suggesting their importance in clinical investigation as prognostic factor. Moreover, our data suggest the possible development in CML patients of a circuit primed by tumor cells that, through the release of soluble factors and exosomes, are able to expand M-MDSCs, creating an immunotolerant environment that results in T cell anergy and facilitates tumor growth.