Impairment of NK Cell Mediated Immune Surveillance Against Acute Myeloid Leukemia
Mohammed TAHA1
1Department of Pharmacology and Medical Sciences, Al-Azhar University of Gaza, Faculty of Pharmacy, Gaza-Palestine DOI : 10.5505/tjo.2022.2883


Natural killer (NK) cells are cytotoxic lymphocytes contributing in innate immune responses that recognize and kill virus-infected and tumor cells without prior stimulation. Several clinical trials have indicated that NK cell-based immunotherapy represents a promising antitumor immunotherapeutic approach due to their key role in mediating graft versus leukemic effect against hematological malignancies, particularly acute myeloid leukemia. However, the antitumor activity of NK cells is inhibited as a result to immune-evasion mechanisms developed by malignant cells through alterations in the expression of activating and inhibitory receptors and their ligands, as well as secretion of soluble NK-inhibitory mediators. Until now, the exact molecular mechanisms involved in these alterations are still not defined.


Natural killer (NK) cells are lymphocytes of the innate immune system which have the ability to recognize tumors and virus-infected cells without prior specific sensitization.[1,2] NK cell functions are regulated by the expression of numerous inhibitory and activating receptors which bind to ligands on healthy or transformed cells.[1-3] The antitumor activity of NK cells is mediated through direct cytotoxic function as well as regulation of other immune cells by cytokine-secreting function. NK cells play a key role in mediating graft versus leukemic (GvL) effect against hematological malignancies, particularly acute myeloid leukemia (AML).[4,5] However, tumor cells can develop immunosuppressive mechanisms to escape NK cell-mediated immunity. Hence, maintaining or improving NK cell performance is considered a major challenge. In this review, we focus on the different mechanisms involved in the evasion of hematological malignancies from NK cells surveillance. Futhermore, we will mention the different approaches used to restore and improve the efficacy of anti-tumor function of NK cells against hematological malignancies.

NK Cell Biology
NK cells are lymphocytes arising from the lymphoid origin which are considered as the third largest population of lymphocytes following T and B cells encompassing approximately 10-15% of all peripheral blood lymphocytes.[6] However, NK cells are considered as critical cells of the innate immune system due to their ability to kill the target cells directly without specific immunization.[3,7] Based on the expression of CD56 molecule, NK cells are defined as CD56+ lymphocytes. [6] Phenotypically, NK cells can be divided into many subsets based on the surface expression of CD56, CD16, inhibitory receptors and/or activating receptors. [6] In general, the major subpopulations of NK cells are CD56bright CD16-/+ (5-10% of NK cells) and CD56dim CD16+ NK cells (90-95% of NK cells).[6,8]

NK Cell Cytotoxicity
NK cells play a key role in immuno-surveillance and host defense against certain virus-infected or transformed cells mediated by direct cytolysis as well as regulation of the effector functions of other cytotoxic immune cells.[9-11] NK cell functions are controlled by a balance between activating and inhibitory signals provided by a varied group of activating receptors as (NKG2D, DNAM-1, NKp30, NKp44, and NKp46) and inhibitory receptors as NKG2A.[12] In general, NK cells can recognize abnormal cells through two models: Missing-self recognition and stress-induced recognition because abnormal cells as tumor cells can change their surface phenotype by losing the expression of human leukocyte antigen (HLA) class I molecules and/or upregulating damage-associated proteins. [13] Numerous damage-associated proteins have been expressed by tumor cells such as MICA and MICB binding with NKG2D, ligands of NKp30 as B7-H6, a mixed-lineage leukemia protein which is a ligand of NKp44, and CD155 and CD112 which interact with DNAM-1.[14] Consequently, NK cell activating receptors bind with their specific ligands expressed on the target cells resulting in lysis of their target cells. Alongside their activation by tumor cells and pathogens, NK cells can be directly or indirectly regulated through signals from other immune cells such as dendritic cells (DCs), macrophages, CD4+ T cells during the immune response.[14] Then, activated NK cells have the ability to kill their target cells though a variety of mechanisms, including cytolytic granule-dependent exocytosis pathway, signaling through the tumor necrosis factor (TNF) death receptor family members such as FAS (CD95) and TNF-related apoptosis-inducing ligand, the release of cytokines IFN-γ and TNF-α, and antibody dependent cell cytotoxicity (ADCC) via CD16. [15] Besides the cytotoxicity mediated by NK cells, immune response can be regulated by NK cells through the recruitment of other immune cells.

NK Cells and AML
Leukemias are cancers starting in cells that would normally develop into different types of blood cells. AML, one type of leukemias, is a heterogeneous clonal disorder of HSC characterized by accumulation of immature, non-functional myeloid precursors (blasts) in the bone marrow and blood without the ability to differentiate normally and to respond to normal regulators of proliferation.[16] In general, patients with newly diagnosed AML, cytotoxic chemotherapy results in CR in approximately 60-80%.[17] However, most of them relapse and about 40-45% of patients achieving CR remain alive at 5 years. To minimize the relapse and improve the survival rate of in patients, immunotherapy is used in combination with standard therapy.[18,19] NK cells play a principal role in the immunosurveillance of hematological malignancies by direct and indirect killing of tumor cells. Several clinical trials have been used NK cells as adoptive immunotherapy based on the alloreactivity of donor's NK cells to treat the hematological malignancies and solid tumors.[20-25] However, tumor cells can develop various mechanisms to escape immunosurveillance of NK cell and other effector cells.[26,27]

Mechanisms of AML Escape from NK Cell Immunity
Although the cytotoxic activity of NK cells against leukemia cells and their beneficial role in immunotherapy, many tumors including AML can evade the immunosurveillance of NK cell by destroying the precise balance between inhibitory and activating signals.[28- 32] Commonly, AML cells are able to escape NK cell immunosurveillance through various mechanisms: i) alteration of NK cells, ii) immunosuppressive properties of AML cells, and iii) interactions with other immune cells (Fig. 1).

Fig. 1. AML cells are able to escape from NK cell immunosurveillance through various mechanisms: i) alteration of NK cells, ii) immunosuppressive properties of AML cells, and iii) interactions with other immune cells.
IL-10: interleukin 10; TGF-β: Transforming growth factor beta; IDC: immature dendritic cell; Treg: T regulatory cells; AR: Activating receptors; IR: Inhibitory receptors; NK: Natural killer.

Alterations of NK Cell By AML
AML cells are capable to alter the expression of NK cell receptors and their ligands, resulting in a significant impairment of NK cell functions, however, the molecular mechanisms responsible for these alterations are still unknown.

Alterations of the Expression of NK Cell Receptors
Several reports have shown a clear decrease in the expression of NK activating receptors on circulating NK cells of AML patients such as natural cytotoxic receptors, NCRs (NKp30, NKp44 and NKp46), NKG2D, and DNAM-1.[28,30,31,33-35] These alterations are associated with impaired anti-leukemic activity of NK cells, a decreased cytokine production, and risk of tumor relapse. Notably, Fauriat et al.,[30] (2007) showed that NCRs downregulation on NK cells was associated with poor prognosis for AML patients, and significantly lower 5-year survival rates than their NCRbright counterparts. Interestingly, the phenotypic and functional abnormalities of NK cells are partially or totally restored in patients achieving remission, which suggest that the presence of AML cells is responsible for NK cells abnormalities.[30] Moreover, Olive's team in 2017 showed a strong correlation between NKp46 expression on NK cells of AML patients at diagnosis and the clinical outcomes after allogeneic stem cell transplantation. They found that patients with high expression of NKp46 at diagnosis had better progression-free survival and overall survival (OS) than patients with low expression of NKp46.[36] Regarding the expression of inhibitory receptors, it is clear that failure to achieve remission in AML patients is strongly associated with overexpression of NKG2A and inhibitory KIRs.[31,33]

Alterations of the Expression of NK Cell Receptors Ligands
Another strategy by which AML can escape from NK cell immunosurveillance is decreasing the expression and shedding of surface ligands for various NK cell activating receptors on AML cells themselves.[34,37-40] For example, leukemic blasts are characterized by a sharp decrease in the expression of MICA/B and ULBPs (ligands of NKG2D), CD48 (a ligand for 2B4), NCR-specific ligands, and DNAM-1 ligands (CD112/ CD155).[34,38,39,41,42] These alterations were associated with reduction of the effector functions of NK cells and OS among those patients. On other hand, DNAM- 1 ligands (CD112 and CD155) are highly expressed on AML blasts of patients younger than 65 years. However, NK recognition and killing of leukemic blasts is reduced due to downregulation of DNAM-1 on NK cells of AML patients, hypothesizing a converse relationship between DNAM-1 ligands expression on leukemic blasts and DNAM-1 expression on NK cells. [43] Besides the classical alterations of the expression of NK receptors and their ligands in AML, Olive's team reported alteration in the maturation profile of NK cells in AML patients. They found three different groups of AML patients based on NK maturation profile: hypomaturation, intermediate maturation, and hypermaturation. Interestingly, the findings revealed that patients with hypomaturation profile have decreased OS and relapse-free survival compared to patients with intermediate and hypermaturation.[44]

Alterations of NK Cell at Genetic Level
Some attempts were performed at gene level to try to identify the molecular mechanisms of NK functions defect in hematological malignancies. In this context, Costello's team aimed to realize the mechanisms underlying NCRs down-regulation in NK cells from AML patients.[32] They found that AML-NK cells showed a specific transcriptomic signature compared to NK cells from healthy volunteers, disappeared by NK cells expansion. Although the gene expression of E26 transformation- specific 1 (ETS-1) transcription factor (a potential regulatory element of NCRs expression) was decreased in presence of AML blasts, the expression of ETS-1 and NCRs was restored following AML-NK cells expansion. This proposes that ETS-1 may regulate NCRs expression.[32] In addition, miRNAs, which play an important role in fundamental NK cell biology,[45] can be well accepted to participate in many aspects of AML, including proliferation, differentiation, survival, apoptosis and invasion by targeting oncogenes or tumor suppressors.[46,47] A study showed a selective loss of immature NK cells subset and a clear reduction in the cytolytic granules containing perforin and granzyme B among NK cells in leukemic mice and the NK cells in leukemic mice showed lower levels of T-bet and Eomes, critical transcription factors for terminal NK cell differentiation. They demonstrated that these results are related to miR-29b overproduction, a negative regulator of T-bet and Eomes, in NK cells of leukemic mice because deletion of miR-29b in NK cells reversed these alterations.[48] A recent study found that the expression of CD48 molecule is down-regulated on the surface of NK cells in AML patients.[49] CD48 is expressed on the surface of lymphocytes including NK cells and participates in activation and differentiation pathways in these cells. The results showed that down-regulation of CD48 in AML patients was associated with decreasing the cytotoxic activity of NK cells.[49]

Immunosuppressive Properties of AML Blasts
Besides the phenotypic and functional alterations of NK cells, AML blasts are able to diminish NK cell immunosurveillance by other immunosuppressive factors. For example, a study found an overexpression of CD137 ligand (CD137L) and glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein ligand (GITRL) on AML blasts, where these molecules are ligands for TNFR family.[50,51] Engagement of CD137L and GITRL with their receptors on human NK cells is directly associated with impairing NK cell-mediated killing and IFN-γ secretion or indirectly via secretion of IL-10 by AML cells. Oppositely, they showed that both CD137 and GITR mediate a stimulatory signal in mouse NK cells after their binding with CD137L and GITRL, respectively.[50,51] Cytotoxic functions of NK cells are also impaired by TGF-β, which is secreted by AML blast as well as by regulatory T (Treg) cells, myeloid derived suppressor cells and other stromal cells in the tumor microenvironment.[52,53]

Alterations of Interaction Between NK Cells and Other Immune Cells
Cellular interactions between NK cells and other immune cells are also altered in AML patients resulting in more possibilities of immune escape. NK cells play a key role in regulation of DCs by killing immature DCs to limit inflammation and inappropriate T cell tolerization. Fauriat and his colleagues (2005) have noticed the inability of NK cells from AML patients to kill immature DCs which might result in an abnormal interaction between T cells and immature DCs and induction of tolerogenic T cells.[54] Further, the number of Treg cells, which are the predominant immune suppressor cells, are increased among AML patients compared to healthy donors, while their numbers are reduced in patients with complete remission (CR).[55]

Restoration of NK Cell Cytotoxic Functions
Whereas impaired NK cells are associated with the progression of AML, recovery and boosting the effector functions of NK cells are essential for the control and eradication of AML. In general, there are different approaches used to restore and enhance the effectiveness of anti-tumor function of NK cells, including cytokines, monoclonal antibodies (mAbs), and adoptive transfer of NK cells.[14,26,56-59]

Several cytokines have been confirmed to enhance NK cell proliferation and/or cytotoxicity against several types of tumors. Cytokines are used for this purpose either by direct infusion of cytokines in vivo to boost the autologous NK cell numbers and functions or by in vitro incubation of allogeneic NK cells with cytokines before adoptive NK cell immunotherapy. IL-2 is the first cytokine approved for use in patients to improve NK cells activity, where it restored NK cell receptor expression and increased NK cell activity against autologous AML cells in vitro.[60] However, infusion of IL-2 into patients was accompanied by limited clinical outcomes because IL-2 activates not only NK cells but also Treg cells, which express CD25 (high affinity receptor for IL-2) that can compete with NK cells for IL-2, and subsequently suppress their effector function.[61] In addition, IL-15 was used to activate NK cells with less toxicity.[62] IL-15 has been reported to control development, homeostasis and cytotoxicity of NK cells, which can be presented to NK cells in vivo through several cell types, including monocytes, macrophages and DCs.[63] Infusion of IL-15 into patients increased the cell numbers of circulating NK cells and upregulated the expression of the activating NK cell receptor NKp30, which augmented NK cell cytotoxicity in AML patients.[35,64]

mAbs and Checkpoint Inhibitors
mAbs can realize anti-tumor effects by modifying the activity of their target proteins and by redirecting the effector immune cells to cancer cells. mAbs treatment based on NK cells includes mAbs which target tumorassociated antigens to induce NK cell, and mAbs which target and block immune checkpoint proteins to enhance NK cell cytotoxicity.[14,26,57] By targeting tumor- associated antigens, a specific immune response can be achieved against the tumor cells. In the case of NK cells, these antibodies directly target tumor-associated antigens and also bind to Fc?RIIIA (CD16a) receptor expressed by NK cells to induce NK-mediated ADCC. Consistent with the meaning, studies showed that monoclonal anti-CD123 antibody (CD123 is overexpressed on AML stem cells showing anti-leukemic activity) improved the binding to CD16a and enhanced the anti-leukemic activity of NK cells against AML xenograft models.[65,66] Further, Koerner et al.[67] (2017) found that an Fc-optimized CD133 antibody had a greater affinity to NK cells and more cytotoxic activity for NK cells without relevant toxicity to hematopoietic progenitors in a human AML xenograft model.

The second strategy based on mAbs to recover NK cell effector functions is using targeted mAbs to block specific immune checkpoint proteins to enhance the cytotoxic activity of NK cells. One of these inhibitory proteins is NKG2A, an inhibitory receptor expressed in NK cells and binds with HLA-E ligand. The expression of HLA-E is often upregulated in some cancer cells to escape from NK cell cytotoxicity. In addition, NKG2A expression on tumor-infiltrating NK cells has increased in cancer cells.[68,69] As a result, blocking NKG2A by a humanized antibody called monalizumab improves the cytotoxic activity of NK cells in mice engrafted with primary leukemia cells as well as against HLA-E+ target cells.[70,71] Another checkpoint affecting the functional activity of NK cells is programmed cell death protein 1 (PD-1) which was recently discovered in a mature CD56dim NK cells where its expression significantly suppresses NK function against PD-1 ligand expressing tumor targets.[72,73] In addition, the expression of programmed cell death ligand-1 (PD-L1) is observed in AML blasts.[74] PD-1 antibodies such as pembrolizumab and nivolumab, preventing PD-1/ PD-L1 interaction, have been developed and their effect to enhance endogenous NK cell cytotoxic function remains attractive.[75] Interestingly, blocking PD-1 or PD-L1 increases the cytotoxic activity of NK cells, and decreases the growth of some tumors in xenograft models.[76,77] In AML, nivolumab in combination with idarubicin and cytarabine decreased the progression of AML in patients with newly diagnosed AML and also increased the OS.[78]

NK Cell-Based Adoptive Immunotherapy
,The strategy of using NK cells as adoptive immunotherapy depends on the valuable effects of NK cell alloreactivity which is induced by the mismatch between HLA class I molecules on recipient cells and KIRs on donor NK cells. Several clinical reports have shown that donor NK cell alloreactivity is a key therapeutic element in the success of transplant and killing leukemia through GvL effect without development of graft versus host disease as well as controlling infections.[79,80] In general, donor-derived NK cells are mainly obtained from donor PBMCs by separating protocols; however, obtaining sufficient numbers of NK cells from PBMCs to achieve a therapeutic effect has been a major limitation.[81] Therapeutically, a phase I clinical trial showed that infusion of IL-15 plus IL-21 stimulated NK cells which were given after hematopoietic stem cell transplantation (HSCT) reduced progression of leukemia compared with patients who have subjected to HSCT without NK cell infusion.[82] Moreover, infusion of multiple doses of NK cells expanded ex vivo with feeder cells was effective in minimizing leukemia relapse. A present study showed that infusion of IL-2 activated NK cells into patients with hematologic malignancies 2 months following HSCT was associated with increasing the expression of activating receptors on the reconstituting NK cells as well as increasing degranulation and cytokine production.[83] After follow-up, a CR of hematologic malignancies was observed in 11 patients out of 16 treated patients.


NK cells are a specific group of lymphocytes playing a key role in the innate immune responses against virus-infected cells as well as different types of cancer. Although NK cells play a major role in immunosurveillance against AML cells, it was demonstrated that NK cells in AML patients have impaired anti-leukemic activity due to multiple mechanisms of innate immune escape, including down-regulation of activating receptors expression, up-regulation of inhibitory NKG2A expression, down-regulation of NK-activating ligands, and secretion of soluble NK-inhibitory factors as well as other immunosuppressant mechanisms. However, the specific molecular mechanisms involved in these alterations are still not well defined. Hence, there is a persistent need for complete understanding of how AML escapes the natural defenses of immune system.

Peer-review: Externally peer-reviewed.

Conflict of Interest: The authors declare no conflicts of interest.

Financial Support: The authors received no financial support for the publication of this article.


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