Genetic modification and effector functions of natural killer cells in acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by a poor long-term outcome in the majority of patients following conventional treatment with chemotherapy. Even after allogeneic or autologous hematopoietic stem cell transplantation (HSCT) the patients are prone to relapse, indicating that the leukemic blasts escape elimination by the immune system. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of AML and were identified to participate in the graft versus leukemia effect following allogeneic HSCT. The effector functions of NK cells are regulated by the balanced engagement of activating receptors and inhibitory receptors. Triggering of activating receptors by the corresponding ligands on target cells counteracts the signalling pathways of inhibitory receptors and thereby elicits target cell lysis. In AML the leukemic blasts were shown to express low amounts of ligands for the NK cell activating receptor NKG2D and the natural cytotoxicity receptors (NCRs), while the expression of HLA class I molecules, the ligands for inhibitory receptors, is mostly retained at normal levels. This predominance of inhibitory signalling together with a putative deficiency in the expression of NK cell activating receptors may result in an insufficient stimulation of cytolytic NK cell responses against leukemic blasts. To investigate the mechanisms of impaired recognition and lysis of leukemic cells, we evaluated the phenotypic and functional properties of NK cells from AML patients (AML-NK cells). We examined the cytolytic activity against the autologous leukemic blasts in vitro and in vivo in the NOD/SCID transplantation mouse model, in order to exploit their potential in cellular immunotherapy of leukemia. Further we explored the feasibility to overexpress the NCR NKp46 in NK cells by lentivirus-mediated gene transfer. This approach was intended to test the hypothesis of shifting the receptor balance in AML-NK cells towards a status that favours NK cell activation and thereby increases the anti-tumor activity. The results demonstrated a significant, about ten-fold, reduction in the content of NK cells from patients with newly diagnosed or relapsed AML as compared to healthy individuals (donor-NK cells). Nevertheless, AML-NK cells retained a high proliferative capacity and could be efficiently expanded in vivo in response to NK cell specific growth factors. Also, the expression pattern of NK cell receptors and activation markers by AML-NK cells did not differ from donor-NK cells. AMLNK cells were fully functional in terms of IFN-γ production in response to the activation with IL-12
and IL-18 and displayed a high cytolytic activity against the NK cell sensitive erythroleukemia cell
line K562 in vitro. Also in vivo, the adoptive transfer of AML-NK cells to NOD/SCID mice
engrafted with K562 cells lead to an efficient suppression of tumor formation. The cytolytic activity
of AML-NK cells against autologous leukemic blasts in vitro was in general below 10% at the E:T
ratio of 10:1. The antibody-mediated block of inhibitory interactions could enhance the killing
responses to about 70%, indicating that AML-NK cells are able to recognize autologous blasts
through activating receptors. Cytolytic activity of AML-NK cells was also seen in NOD/SCID mice
engrafted with human leukemia. Adoptive NK cell transfer resulted in reduction of tumor load from
31% to an average of about 10% of human blasts in the BM of treated mice. This high in vivo
activity of AML-NK cells might be due to an increased expression of the ligands for NKG2D and
the NCRs.
Taken together, our results showed that AML-NK cells do not differ from healthy donor derived
NK cells; they can be isolated and efficiently expanded to high cell numbers in vitro and display the
same expression pattern of the major activating receptors. AML-NK cells have a normal cytokine
producing ability, preserve their cytolytic activity throughout the process of in vitro expansion and
display a strong anti-leukemic effect against autologous blasts in vivo in NOD/SCID mice
repopulated with human leukemia. These results suggest that escape of AML blasts from the
immunosurveillance by NK cells may be due to the reduction of the NK cell compartment and the
predominance of signals elicited by the inhibitory receptors.
We used HIV-derived lentiviral vectors for the gene transfer of the GFP marker and the NKp46
receptor to NK cell lines, primary NK cells and NK cells generated in vitro from CD34+
hematopoietic progenitor cells. Both single-gene and bicistronic vectors expressing these transgenes
were prepared. Through the FACS sorter based enrichment of transduced cells 100% transgenic
lines and primary NK cell populations were generated with a transgene expression that remained
stable during in vitro expansion. We demonstrated that GFP+ NK cells can be generated by the in
vitro differentiation of lentiviral transduced CD34+ progenitors, representing a highly efficient
approach to produce large amounts of modified NK cells.
However, a sustained expression of the exogenous NKp46 receptor was only achieved in NK cell
lines. Except for a high pseudotransduction that resulted in the transient expression of NKp46, no
stable expression of transgenic NKp46 was obtained in primary NK cells, neither in cells generated
from the progenitors nor in peripheral blood-derived mature NK cells.
Moreover, exogenous NKp46 in NK cell lines and transiently transduced primary cells had no
ability to trigger the cytokine release or cytotoxic responses, and further studies are required to
achieve the overexpression of functional NKp46.
Our results demonstrated that lentiviral vectors are suitable to obtain genetically modified NK cell
lines and primary NK cells. Transgenic NK cells can be expanded to high numbers without loosing
the transgene expression, thus indicating the possibility to use genetically modified and expanded
patient-derived NK cells for the adoptive transfer in the immunotherapy of AML. So far, the
lentivirus-based approach was successful with the GFP marker transgene, but requires further
optimisation for transfer of the NKp46-encoding gene.
The over-expression of tumor-specific activatory receptors would be of importance in an
immunotherapeutic approach to direct NK cell effector functions specifically towards the diseased
cells, thereby contributing to a graft-versus-leukemia activity against residual malignant cells.