Analysis of NK Cells Receptors and NK Cells Ligands in Acute Myeloid Leukemia: Comparisons Between the Blood and the Bone Marrow.

The significant role of NK cells in the control of acute myeloid leukemia (AML) has been demonstrated in the setting of allogeneic stem cell transplantation. However, the implication of NK cells against autologous leukemic cells needs to be clarified.

We have previously described deficient expression of NK activatory receptors in AML at diagnosis, in particular the natural cytotoxicity receptors (NCR) namely NKp30 and NKp46. So, defective cytotoxicity of AML cells can be explained by abnormalities of activating NK-receptor expression allowing immune escape from NK cells. However, immune escape can be also due to defective activating NK receptor-ligand interactions due to abnormal expression of their ligands on blasts cells. These defects have been observed in particular on NK cells or blasts cells isolated from the blood. Few studies have analysed the bone marrow component although blasts cells concentrate here. We postulated that abnormalities of NK cells receptors or ligands expression are more severe in bone marrow, in near contact with the blasts, compared with blood. We sought to identify disparities between deficient expression of NK or ligands in the bone marrow in comparison with the blood.

We realized a phenotypic analysis of NK cells and blasts cells at the diagnosis of AML. The level of activatory NK receptors (NKRa) knew to mediate NK cell recognition and lysis of AML blasts cells (NCR (NKp30 and NKp46) and DNAM-1) was investigated by flow cytometry. The expression of NKG2D ligands (MICA/B and ULBP1-3) and DNAM-1 ligands (Nectin-2 and PVR) receptors were also analysed. These analyses were realised with coupled specimens obtained in the same patient at diagnosis of AML (n=19), peripheral blood and bone marrow samples in order to detect discrepancies between these two sites. A control group (age-matched; n=15) for blood samples was included for this study. All biological samples were obtained from patients and healthy volunteers after informed consent.

A total of 19 patients were included in this study. We included 6 cases of AML 5, 4 cases of AML 4, 4 cases of AML 2, 4 cases of AML 1 and one case of AML 0. Flow cytometry data for NKRa were only available for 11 patients. We confirmed the deficient expression of NKp30 and NKp46 receptors (as determined by MFI) on NK cells from blood of AML patients. In AML patient, the ratio MFI (MFI receptor/MFI control isotype) of NKp30 (4.27 +/- 2.97; p<0.0001) and of NKp46 (5.96 +/- 5.67; p<0.0001) significantly differed from healthy volunteers (NKp30 26.65 +/- 6.12; NKp46 39.73 +/- 9.66). Moreover, the deficient expression of these receptors was also observed on NK cells from the bone marrow (NKp30 3.66 +/- 2.22, p<0.0001; NKp46 6.71+/- 6.42, p<0.0001). However, we can not demonstrated significant differences between the NKRa expression on NK cells from blood versus from bone marrow (NKp30 p= 0.8438; NKp46 p= 0.9476 and DNAM-1 p= 0.3579).

The expression of the ligands for NKRa was analysed to compare the expression on blasts cells isolated from the blood compared to blasts cells isolated from the bone marrow. Flow cytometry data for ligands were only available for 17 patients. We observed a strong expression of HLA class I molecules on blasts cells that was equivalent in the blood and in bone marrow. DNAM-1 ligands (PVR, Nectin 2) were expressed on blasts cells (see figure). NKG2D ligands were also expressed but to a lesser extent with predominant ULBP1 expression. However, we can not observed significant differences in the expression of ligands between the blood and the bone marrow.

The deficiency of activating NK cells receptors expression at AML diagnosis is significant, present in a majority of patients, and consistent across the 2 components, ie blood and bone marrow. These defects are one component of the immune escape from NK cells. We have speculated that these abnormalities were more pronounced in bone marrow, near blasts cells, because these abnormalities are in part induced by blasts cells. However, we can not demonstrated significant differences in the expression of activating receptors or ligands between blood and bone marrow. We are accumulating more data in order to detect differences between sub-groups of AML.

No relevant conflicts of interest to declare.