Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity.
Authors: Giallongo Cesarina , Parrinello Nunziatina , Tibullo Daniele , La Cava Piera , Cupri Alessandra , Chiarenza Annalisa , Stagno Fabio , Palumbo Giuseppe A , Raimondo Francesco Di 
Published: October 2019

Abstract
Abstract 2779

Background:
Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity.

Aim:
The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients.

Methods:
MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation.

Results:
CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum.

Conclusions:
Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion.

Disclosures:
Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).