Abstract
Purpose of review-
Natural killer (NK) cells are a type of immune cell that play a crucial role in the defense against cancer and viral infections. The development and maturation of NK cells is a complex process, involving the coordination of various signaling pathways, transcription factors, and epigenetic modifications. In recent years, there has been a growing interest in studying the development of NK cells. In this review, we discuss the field’s current understanding of the journey a hematopoietic stem cell takes to become a fully mature NK cell and detail the sequential steps and regulation of conventional NK leukopoiesis in both mice and humans.
Recent Findings-
Recent studies have highlighted the significance of defining NK development stages. Several groups report differing schema to identify NK cell development and new findings demonstrate novel ways to classify NK cells. Further investigation of NK cell biology and development is needed as multi-omic analysis reveals a large diversity in NK cell development pathways.
Summary-
We provide an overview of current knowledge on the development of NK cells, including the various stages of differentiation, the regulation of development, and the maturation of NK cells in both mice and humans. A deeper understanding of NK cell development has the potential to provide insights into new therapeutic strategies for the treatment of diseases such as cancer and viral infections.
Keywords: Natural Killer Cells, Differentiation, Development, Regulation, Adoptive Cellular Therapy
Introduction
Natural killer (NK) cells are a part of the innate immune system and play significant roles in immune-surveillance and host defense. Next only to T and B cells, NK cells are the third largest lymphocyte population, representing between 5 to 20 percent of circulating lymphocytes in humans [1■■] and 2 to 6 percent in mice [2]. Ever since their first discovery in 1975 by Herberman et al. as “naturally” cytotoxic cells with the ability to recognize and kill tumor cells without requiring prior activation, NK cells and their functions have been studied intensively [3]. The first characterization of NK cells was performed by Lanier and Perussia groups by presence of the CD16 (FcγRIIIa) surface receptor [4,5]. While NK cells have analogous effector functions to cytotoxic CD8+ T cells, many researchers have shown that these cells differ significantly in terms of their recognition, specificity, sensitivity, and memory mechanisms [6]. In addition to their ability to kill by cell-to-cell mediated apoptosis, NK cells can release cytotoxic granules, death receptor ligands (FasL), and cytokines to induce apoptosis in target cells.
Of interest to many researchers and physicians is the unique ability of NK cells to detect and kill cells with decreased major histocompatibility complex class 1 (MHC-I) expression; a process that often occurs during viral infection or tumorigenesis [7]. Unlike T and B cells, NK cells do not express clonotypic receptors and instead become activated after cellular recognition using germline-encoded receptors for natural cytotoxicity, cytokines, and/or antibody-dependent cellular cytotoxicity (ADCC). NK cells are generally considered to be a member of the innate immune response thus a member of the innate lymphoid cell (ILC) populations. These are distinct from adaptive immune cells due to their lack of rearranged antigen receptors. In 2013, the nomenclature for ILCs was once characterized by three subgroups (ILC1, ILC2, ILC3). NK cells were initially included in with the ILC1 subgroup due to their similarities with T-bet transcriptional regulated cell populations and their production of interferon γ (IFN-γ) in response to cytokines (specifically IL-12, IL-21, IL-15, and IL-18) [7–9]. After further investigation, however, ILCs have now been divided into five subgroups (ILC1, ILC2, ILC3, lymphoid tissue inducer cells (LTi), and NK cells) [10].
Our current understanding of NK development and differentiation is heavily built from early murine models studying immune leukopoiesis. While differences do exist between human and mice NK leukopoiesis, murine NK development follows a sequence of steps similar to human NK development (Fig. 1). In this review, we will discuss the field’s current understanding of the development of NK cells from hematopoietic stem cells (HSCs) in both mice and humans. We present a comprehensive summary of each stage of NK differentiation with notable surface markers that can be used to define and identify distinct NK cell populations in both mice (Table 1) and humans (Table 2). We will also discuss the factors that regulate NK development and our understanding of the cellular mechanisms that underlie each signaling pathway.
Fig 1: Distinctive Markers of NK cell Differentiation in Humans and Mice.
NK cell development in human and mouse with distinct markers listed to delineate stages of progression from HSC to terminal mature NK cell.
Table 1. Murine NK Cell Differentiation Markers.
List of commonly used murine NK cell differentiation markers reported in literature [22].
| HSC | MPP | CLP | Pre-NKPs | rNKPs | iNK Stage A | iNK Stage B | iNK Stage C | Early mNK Stage D | Terminal mNK Stage E | Terminal mNK Stage F | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Lin | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) |
| CD34 | CD34 (−) | CD34 (+) | CD34 (+) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) |
| CD244 (2B4) |
CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) |
| CD312 (IL-2Rγc) |
CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) | CD314 (+) |
| CD27 | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (−) |
| CD117 (c-Kit) |
CD117 (+) | CD117 (−) | CD117 (−) | CD117 (−) | CD117 (−) | CD117 (−) | CD117 (−) | CD117 (−) | CD117 (−) | ||
| CD27 | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (+) | CD27 (−) | |||
| CD244 | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (002B) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | |||
| CD127 (IL-7Rα) |
CD127 (+) | CD127 (+) | CD127 (+/−) | CD127 (+/−) | CD127 (+/−) | CD127 (−) | CD127 (−) | CD127 (−) | |||
| CD122 (IL-2Rα) |
CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | ||||
| CD25 (IL-2Rα) |
CD25 (+) | CD25 (−) | CD25 (−) | CD25 (−) | CD25 (−) | CD25 (−) | |||||
| NKG2D (CD314) |
NKG2D (+) | NKG2D (+) | NKG2D (+) | NKG2D (+) | NKG2D (+) | NKG2D (+) | |||||
| NK1.1 | NK1.1 (+) | NK1.1 (+) | NK1.1 (+) | NK1.1 (+) | NK1.1 (+) | ||||||
| NK2GA/C | NKG2A/C (+) | NKG2A/C (+) | NKG2A/C (+) | NKG2A/C (+) | NKG2A/C (+) | ||||||
| CD43 (Leukosialin) |
CD43 (+) | CD43 (+) | CD43 (+) | CD43 (−) | CD43 (−) | ||||||
| CD62L | CD62L (+) | CD62L (+) | CD62L (+) | CD62L (+) | CD62L (+) | ||||||
| CD226 (DNAM1) |
CD226 (+) | CD226 (+) | CD226 (+) | CD226 (+) | CD226 (+) | ||||||
| NKp46 (CD335) |
NKp46 (+/−) | NKp46 (+) | NKp46 (+) | NKp46 (+) | NKp46 (+) | ||||||
| NCR1 | NCR1 (+) | NCR1 (+) | NCR1 (+) | NCR1 (+) | |||||||
| LY49 | Ly49 (+) | Ly49 (+) | Ly49 (+) | ||||||||
| CD49b | CD49b (+) | CD49b (+) | CD49b (+) | ||||||||
| CD11b (Mac-1) |
CD11b (−) | CD11b (+) | CD11b (+) | ||||||||
| KLRG1 | KLRG1 (+) |
Table 2: Human NK Cell Differentiation Markers.
| HSC | MPP | CLP | NKP | Pre-NK Stage 2a | Pre-NK Stage 2b | iNK | mNK Stage 4a | mNK Stage 4b | mNK Stage 5 | mNK Stage 6 | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Lin | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) | Lin (−) |
| CD34 | CD34 (+) | CD34 (low) | CD34 (+) | CD34 (+) | CD34 (+) | CD34 (+) | CD34 (+/−) | CD34 (−) | CD34 (−) | CD34 (−) | CD34 (−) |
| c-Kit | c-Kit (+) | c-Kit (low) | c-Kit (+) | c-Kit (+) | c-Kit (+) | c-Kit (+) | c-Kit (+) | c-Kit (+/low) | c-Kit (low/−) | c-Kit (low/−) | c-Kit (−) |
| CD150 | CD150 (+) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) | CD150 (−) |
| CD244 | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | CD244 (+) | |
| CD45RA | CD45RA (+) | CD45RA (+) | CD45RA (+) | CD45RA (+) | CD45RA (+) | CD45RA (+) | CD45RA (+/−) | CD45RA (+/−) | CD45RA (−) | CD45RA (−) | |
| CD127 | CD127 (+) | CD127 (+) | CD127 (+) | CD127 (+) | CD127 (+/−) | CD127 (−) | CD127 (−) | CD127 (−) | CD127 (−) | ||
| CD7 | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | CD7 (+) | ||
| CD10 | CD10 (+) | CD10 (+) | CD10 (+/−) | CD10 (−) | CD10 (−) | CD10 (−) | CD10 (−) | CD10 (−) | CD10 (−) | ||
| ILR1 | ILR1 (low) | ILR1 (−) | ILR1 (+) | ILR1 (+) | ILR1 (+/low) | ILR1 (low/−) | ILR1 (low/−) | ILR1 (low/−) | |||
| CD122 | CD122 (+) | CD122 (−) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | CD122 (+) | |||
| HLA-DR | HLA-DR (+) | HLA-DR (+) | HLA-DR (+) | HLA-DR (−) | HLA-DR (−) | HLA-DR (−) | HLA-DR (−) | HLA-DR (−) | |||
| NKp46 | NKp46 (+/−) | NKp46 (+) | NKp46 (+) | NKp46 (+) | NKp46 (+) | ||||||
| NKp30 | NKp30 (+/−) | NKp30 (+) | NKp30 (+) | NKp30 (+) | NKp30 (+) | ||||||
| CD161 | CD161 (+/−) | CD161 (+) | CD161 (+) | CD161 (+) | CD161 (+) | ||||||
| NKG2D | NKG2D (+/−) | NKG2D (+) | NKG2D (+) | NKG2D (+) | NKG2D (+) | ||||||
| NKG2A | NKG2A (+) | NKG2A (+) | NKG2A (low/−) | NKG2A (low/−) | |||||||
| NKp80 | CD56 (++) | CD56 (++) | CD56 (low) | CD56 (low) | |||||||
| CD56 | NKp80 (+) | NKp80 (+) | NKp80 (+) | ||||||||
| CD16 | CD16 (+) | CD16 (+) | |||||||||
| KIR | KIR (+/−) | KIR (+) | |||||||||
| NKG2C | NKG2C (+) | NKG2C (+) | |||||||||
| CD57 | CD57 (+) |
Conventional Murine NK Cell Development
NK cells are distributed throughout the body and have been shown to reside in both lymphoid and non-lymphoid tissues such as the bone marrow (BM), spleen, lymph nodes (LNs), gut, tonsils, liver, and lungs [11]. While the development towards mature NK cells occurs outside of the BM, NK differentiation begins primarily in the BM niche from HSCs [12]. Interestingly, unlike T lymphocytes, NK cells do not appear to require thymic or splenic training as their persistence and their functions are still present without the thymus and spleen in vivo [13,14].
Hematopoietic Stem Cells, Multipotent Progenitors, & Common Lymphoid Progenitors
In mice, the BM niche maintains populations of long term (LT-HSCs) and short-term HSCs (ST-HSCs) that can differentiate into multipotent progenitor (MPP) cells and further into common lymphoid progenitor (CLP) cells through cytokine signaling from Flt3L, SCF, and IL-7. Both Flt3L and SCF are required to drive NK cell expansion and differentiation from HSCs through Flt3 and c-Kit cell receptors respectively. Mice deficient in Flt3 or c-Kit have reduced CLP numbers [15,16]. While not necessary for conventional bone marrow NK cell development, IL-7 appears to be essential for thymic CLP commitment to natural killer progenitor (NKP) cells. In the thymus, secreted IL-7 interacts with the IL-7Rα/CD127 receptor on CLPs to promote differentiation towards the lymphoid lineage (Pro-B, Pre-T, ILCs, LTis, and NKPs).
Natural Killer Progenitors
While often reported as a singular group, NKPs can be classified into two distinct cell populations: Pre-NKPs and refined NKPs (rNKPs). Pre-NKPs, also commonly termed common innate lymphoid progenitor (CILP) cells, serve as a common precursor for the innate lymphoid family (ILCs, LTis, and NK cells). Pre-NKPs can be identified by their acquisition of CD244, CD127, and CD27 receptors. Binding of CD27 to its ligand, CD70, plays a key role in regulating B-cell activation and immunoglobulin synthesis. While CD27 has been shown to predict responsiveness in function and NK migration of mature NK cells, little is currently known about how CD27 promotes pre-NKP differentiation in mice [17]. Pre-NKPs become committed to the NK lineage after transition to refined-natural killer progenitor (rNKP) cells. rNKPs are distinguished by expression of CD122 [18]. CD122 is activated by IL-15 and serves as a critical component of NK cell development and differentiation [19]. Antigen-presenting cells (APCs; i.e., dendritic cells, macrophages) in the bone marrow can present IL-15 through a unique process called trans-presentation. During development, APCs take up extracellular IL-15, which is then loaded onto the macrophage’s IL-15 receptor (IL-15R). The complex of IL-15 and IL-15R is then transferred to nearby natural killer cell progenitors, where it activates signaling pathways that promote the maturation and proliferation of the progenitors into functional natural killer cells. NKP maintenance and progression to immature NK cells (iNKs) requires activation of several NK-specific transcription factors, such as inhibitor of DNA binding 2 (Id2) and E4-binding protein 4. Id2 is a helix-loop-helix transcription factor that regulates the differentiation of multiple cell types, including NK cells. It has been shown to be involved in the expansion and survival of NK cell progenitors and the maintenance of their differentiation state. E4BP4, also known as NFIL3, is a transcription factor that regulates the expression of multiple genes involved in immune cell development and function; modulating the expression of genes involved in cytokine signaling, cytotoxicity, and the regulation of cell death.
Immature NK Cells
After migrating from the bone marrow to peripheral blood, conventional NKPs mature and gain effector functions through the expression of cellular markers. NK cell activation receptors (NKRs) are acquired germline-encoded surface molecules that can recognize self or pathogen-derived ligands and promote NK cell activation. While NKRs signal distinct stages of NK cell development, the exact mechanisms that regulate NKR expression during iNK cell differentiation are still largely unknown and require further investigation. The first acquisition of DAP10 (NKG2D/DNAX-activating protein of 10 kDa), an NKR, marks the transition of rNKPs into iNKs [20]. iNK differentiation into terminally mature NK cells is thought to occur in six stages (stages A through E) as described with stages A-C and stages D-E representing iNK cells and mature NK cells (mNKs) respectively [21]. iNK stages A, B, and C are identified by contemporaneous expression of NKG2A and DNAM-1, NK1.1, and NCR1. Additionally, increased expression of cell adhesion molecules, CD62L & CD43 (Leukosialin), occurs throughout these iNK maturation cell stages [22].
Mature NK Cells: Early and Terminal
Within peripheral blood, iNKs differentiate into mNKs. While many groups only report a single group of mature NK cells, many define mNK cells into either early or terminal mNK cell populations. Early mNK cells (stage D) can be identified by their co-expression of CD51 (integrin Vα) and CD49b (DX5) [23]. In contrast, terminally mature NK cells can be identified by their expression of CD43 (Leukosialin) and CD11b (Mac −1). Terminally mature mNK cells are subdivided by their activation status: stage E and stage F. Stage E NK cells are terminally mature mNK cells that express Ly49 which is an important mediator for NK cell activation. The Ly49 family of surface receptors enables NK cells to either become activated (Ly49D, Ly49H) or inhibited (Ly49A, Ly49C, Ly49I, Ly49G). These receptors, analogous to the killer-cell Ig-like receptors (KIRs) in humans, enable the immune surveillance and cytotoxic functions of mature NK cells [24]. The “licensing” theory proposes that a balance of activating and inhibitory signaling of these receptors can elicit a cellular activation threshold and regulate NK cell functionality. Stage F mNKs migrate into secondary lymphoid organs after expression of KLRG1 (killer cell lectin like receptor G1). KLRG1 is a surface receptor that is upregulated following infection with virus or parasites.
Many groups are currently investigating phenotypic markers typical of mature NK cells and methods to effectively enhance NK cell functionality. Several NK cell developmental models have been proposed. The first three-stage NK developmental model (using NK1.1 and CD49b) was made by Rosmaraki et al. in 2001 [20]. The most recent model, published by Ma et. al, uses differential surface expression of CD49 and NKp46 to measure NK cell maturation in a four-stage developmental process [25■]. Based on their model, NKP cells can be identified by lack of expression of both CD49 and NKp46 (termed double negative (DN)). Immature NKs are negative for NKp46 but show CD49−/+ and CD49+ iNK-a and iNK-b cells. Lastly, mNK cells are identified by co-expression of both CD49 and NKp46 (termed double-positive (DP)). All these cell populations are phenotypically distinct based on surface markers, transcription factors, and effector molecules.
Conventional Human NK Cell Development
Although several differences exist between murine and human NK cells, the fundamental principles of NK cell biology and function are similar. In humans, NK cells have been found in various stages of development in locations outside of the bone marrow and thymus. Intermediate NK cells have been shown to be present in the liver, uterus, and secondary lymph nodes; suggesting that NK cells may be able to develop in tissues outside of bone medulla. In this review, we will discuss the field’s current understanding of conventional NK leukopoiesis in humans.
Hematopoietic Stem Cells, Multi-potent Progenitor, Common Lymphoid Progenitors
The BM niche hosts and maintains a population of CD34+CD133+CD244+ multipotent HSCs that can differentiate into CD45RA+ CD133+ lymphoid primed multi-potent progenitor (MPPs) cells. MPP cells have the ability to become either common myeloid progenitor (CMP) or common lymphoid progenitor (CLP) cells, the latter of which gives rise to Pro-B, Pre-T, ILCs, LTi cells, and NKP cells and express CD38, CD7, CD10, and CD127 markers. Human HSCs have been shown to respond to SCF and Flt3L in a similar fashion to murine HSCs. Synergy between SCF and Flt3L has been reported to enhance the development and maturation of human CLPs and in result NKPs through the induction of key signaling molecules CD122 and CD215 (IL-15Ra) [26]. IL-3 and IL-7 have also shown to promote differentiation of HSCs towards the CLPs in humans [27]. Stage 1 NKPs are defined by their up-regulation of CD122, a signal of irreversible fate into the NK cell lineage. IL-15 has been shown to be an indispensable factor for NK development and homeostasis and important in both MPP to CLP and CLP to NKP differentiation. Disruptions in the downstream signaling of IL-15 with STAT5b has shown to have a severe reduction in patient NK cell numbers [28].
Natural Killer Progenitors, Pre-NK cells, & iNK cells
Within the bone marrow, conventional NKPs transition into pre-NK cells, the later of which are identified by their marked expression of CD7+ CD127+ and down-regulation of CD122. Stage 2 Pre-NK cells can be subdivided into two substages, a and b, with lack of or expression of IL-1R1 respectively. Transition of human CLPs to Pre-NKPs has been shown to regulated by RUNX3 and ID2 transcription factors. RUX3 is known to enhance the expression of NK cell receptors KIR and NKp46 as well as ID2 transcriptional regulation. Differentiation of pre-NKPs to iNKs occurs with the acquisition of several NK receptors including NKG2D/CD314, NKp46/CD335, NKp30/C337, and NK1.1/CD161. Following Stage 3, iNK cells can develop into mature CD56+ NK cells in the peripheral blood and later migrate into various tissues including the spleen, lymph nodes, and the liver.
Mature NK cells
Mature NK cells can be characterized by maximal expression of the newly acquired NK cell receptors in the preceding stage including NKG2D, NKp46, NKp30 and NK1.1, with concurrent bright expression of CD56. CD56/ NCAM expression is a pathogen recognition receptor and plays a functional role in NK cell cytotoxicity [18]. While a unifying implication for CD56 does not exist, its expression is useful for distinguishing stage 4 (CD56bright) from stage 5/6 (CD56dim) mature NK cells. It is wide belief that CD56dim NK cells have greater cytotoxic activity than CD56bright NK cells, however recent work suggests that CD56bright NK cells may just respond better to soluble factors while CD56dim NK cells respond better to receptors binding ligands present on other cells [29]. Stage 4 NK cells can be divided into both stage 4a and 4b, the latter of which expresses NKp80. Stage 4 NK cells eventually develop into stage 5 NK cells with gradual increased expression of CD94 and CD16 (FcγRIII) with down-regulation of CD56, c-Kit, and CD94. Stage 6 NK cells are marked with expression of CD57+ and killer cell immunoglobulin like receptors (KIRs). KIRs are functionally equivalent to the murine Ly49 receptor family and the acquisition of KIRs signals the complete maturation of NK cells. Stage 6 NK cells typically reside within peripheral tissues and their activity is influenced by cumulative activating and inhibitory KIR signaling upon engagement of MHC-1 present on other cells [21]. Terminally differentiated stages 5 and 6 NK cells are the predominant NK populations in the peripheral blood, compromising approximately 90% of NK cells [30,31].
Conclusion
The distinct stages through which human NK cells develop are less understood compared to murine NK cells. Here, we summarize the most recent models of NK leukopoiesis in mice and humans. While the conventional model of NK leukopoiesis has been elucidated over the last two decades, there is much work to be done by the field to understand NK development outside the bone marrow niche. NK biologists are beginning to recognize the growing diversity of NK cells and heterogeneity of NK cells resident within tissues. The emergence of single-cell sequencing technologies including transcriptomics and epigenetics will further our insight of the physiology of NK cell development and how certain stimuli promote NK leukopoiesis [32■■].
Identifying subsets of NK cells during their development pathway may lead to better understanding of their functions and cultivate more unique approaches to clinical utilization of NK cells. Thus far, several ex-vivo methods to differentiate NK cells from HSCs have already been employed, including the use of cytokines, small molecule inhibitors, and gene editing [33]. For example, the addition of IL-15 and IL-21 to cultures of HSCs has been shown to enhance the expansion and functional activity of NK cells. Several small molecules such as Lenalidomide, FLT3/KIT inhibitors, and Azacitidine are being investigated to enhance the differentiation and function of natural killer (NK) cells ex-vivo. Lenalidomide, a derivative of thalidomide, promotes the differentiation of NK cells from hematopoietic stem cells [34,35]. PLX3397 is a small molecule inhibitor that targets the kinase activity of FLT3 and KIT and enhances the expansion and function of ex-vivo differentiated NK cells. Azacitidine, a DNA methyltransferase inhibitor, is also being investigated for its ability to enhance the expansion and function of ex-vivo differentiated NK cells. Furthermore, recent advances in gene editing techniques, such as CRISPR/Cas9, have allowed for the efficient and specific modification of NK cell developmental genes to enhance their functionality.
Currently, there are 22 clinical trials that have been completed at all phases of trial development which have reported data on the toxicity profiles as well as safety and efficacy of these NK cell therapeutics (Table 3). Seldom these NK cell therapies have been applied to solid tumor treatment regimens. Most notably, these include NK cells extracted from cadaveric liver grafts to potentially prevent hepatocellular carcinoma recurrence or allogeneic NK cell transplant used in combination with T cell depletion therapy for gynecological malignancies; however, the most current trials have investigated the effects in malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia, or non-Hodgkin lymphoma among other hematological malignancies wherein there are immense unmet needs in the therapeutic landscape [36]. Many of these trials assess the effects of autologous or haploidentical/allogeneic NK cell transplants with differing regulations on HLA matched donor cells supplemented with chemotherapeutic regimens and stem cell transplants based on disease burden. A more thorough elucidation of the stages of NK cell development may lead to increased proliferation and function of ex-vivo produced NK therapies [37]. At the moment, ex-vivo derived NK cells may display different transcriptomic signatures and activity compared to primary NK cells from patients [38–40]. These potentially significant differences highlight the importance of understanding the exact steps and signals that regulate NK cell development and maturation. To better harness the full potential of NK cells as an adoptive cellular therapy and provide an “off-the-shelf” method for cancer and other therapies, more investigation and optimization of the steps regulating NK cell ontogenesis is critical. This can be achieved through the systematic characterization of NK cell development and the identification of key signaling pathways and transcriptional regulators [41]. Ultimately, a better understanding of NK cell development and the mechanisms regulating ex-vivo differentiation will provide a foundation for the clinical translation of NK cell-based therapies. The potential applications of NK cells in medicine are vast, and the future of research in this field promises to bring new and potentially exciting developments in the field of immunotherapy.
Table 3: Completed clinical trials utilizing NK cell therapeutics.
The following table contains the details of 22 clinical trials that have been completed and are publicly available on clinicaltrials.gov utilizing NK cell therapeutics. The type of trial, summary description, disease states, endpoints, location, and NCT of the trials are included. NK cell therapeutics predominately been applied to hematological diseases and disorders; however, NK therapeutics are now being tested for use in solid tumors. Safety, toxicity, and efficacy profiles of these NK cell therapeutics are included when available.
| NCT | ||||
|---|---|---|---|---|
| Expanded and Activated Autologous NK Cells + Chemotherapy (Lenalidomideor Bortezomib) | Multiple Myeloma | Toxicity and Efficacy, n=5 patients with 40% mortality | Hospital 12 de Octubre in Madrid | NCT02481934 |
| Phase 2 Haploidentical Donor NK Cells with IL-2, Rabbit anti-thymocyte globulin, and filgrastim mobilized CD34+ stem cell graft from same donor | High Risk Acute Myeloid Diseases: Acute Myeloid Leukemia, Myelodysplastic Syndrome | Number of Participants with Donor Neutrophil Engraftment; Number of Participants with Disease Free Survival | Masonic Cancer Center, University of Minnesota | NCT01370213 |
| Phase 2 Haploidentical donor HCT with Additional Natural Killer (NK) cells | Leukemia, Lymphoma | Transplant Recipients with Successful Engraftment; Number of Transplant Recipients with Malignant Relapse; Event-free Survival; Overall Survival; Transplant Recipients With Acute and/or Chronic Graft Versus Host Disease (GVHD); Transplant Recipients With Transplant-related Mortality (TRM) | St. Jude Children’s Research Hospital | NCT01807611 |
| Phase 1 NK Cell Infusion Following Allogeneic Peripheral Blood Stem Cell Transplantation from Related or Matched Unrelated Donors | Leukemia, Lymphoma | Participants With Mild, Moderate and/or Severe Chronic Graft Versus Host Disease (cGVHD); Disease-free Survival; Overall Survival Since Date of Transplant; Viral Infection and/or Reactivation in Allogeneic Peripheral Blood Stem Cell Transplant (PBSCT) Followed by Natural Killer-donor Lymphocyte Infusion (NK-DLI) | National Cancer Institute | NCT01287104 |
| Phase 3 haploidentical NK cell transplantation in addition to testing efficacy of clofarabine + cytarabine (Clo/AraC | Acute Myeloid Leukemia | Day 22 Minimal Residual Disease (MRD) Measured by Flow Cytometry; Event-free Survival of Standard Risk Patients Who Receive Chemotherapy Alone; Event-free Survival of Standard Risk Patients Who Receive Chemotherapy Followed by Natural Killer Cell Transplantation. | Genzyme, a Sanofi Company.National Cancer Institute (NCI) St. Jude Children’s Research Hospital | NCT00703820 |
| Phase 2 Combination of Decitabine with oral Vorinostat followed by a single infusion of CD3-/CD19- enriched donor natural killer (NK) cells | Myelodysplastic Syndrome | The Number of Patients Who Achieved a Clinical Response; Number of Patients Who Experienced Grade 3 or Higher Non-hematologic Adverse Events; Number of Patients Who Became Transfusion Independent; Number of Patients Who Had Natural Killer (NK) Cell Expansion; Overall Survival | Mayo Clinic; Masonic Cancer Center, University of Minnesota | NCT01593670 |
| Phase 2 Autologous Expanded NK Cell Infusion | Asymptomatic Multiple Myeloma | Increase in ENK (Expanded Natural Killer Cells) Cells 7 Days After Treatment | Millennium Pharmaceuticals, Inc.; University of Arkansas | NCT01884688 |
| Non-selected NK cell donor lymphocyte infusions 3–5/6 Human Leukocyte Antigen (HLA) Matched Family Member after Nonmyeloablative Allogeneic Stem Cell Transplantation (ASCT) | Lymphoma | Toxicity; Efficacy (Overall Survival) | Duke University | NCT00586703 |
| Expanded Natural Killer Cell Therapy with bortezomib | Multiple Myeloma | Efficacy | University of Arkansas; NIH | NCT01313897 |
| Phase 2 Autologous Natural Killer Cells Plus Aldesleukin (IL-2) Following a Lymphodepleting Chemotherapy | Metastatic Melanoma, Metastatic Kidney Cancer | Objective Response; Safety | National Institutes of Health Clinical Center (CC) | NCT00328861 |
| Phase 1 IL-2 Stimulated Natural Killer Cell Therapy from cadaveric donor liver graft exudates. | Liver Cirrhosis, Hepatocellular Carcinoma (HCC), Evidence of Liver Transplantation | Side Effect of Cadaveric Donor Liver NK Cell Infusion; NK Cell Infusion-related Toxicity; Anti-HCC Effect of This Treatment; Anti-HCV Effect of This Treatment (if applicable) | Florida Department of Health; University of Miami | NCT01147380 |
| HLA-Nonidentical Stem Cell and Natural Killer Cell Transplantation for Children Less the Two Years of Age with Hematologic Malignancies | Acute Myeloid Leukemia, Acute Lymphocytic Leukemia (ALL), Myelodysplasia, Chronic Myeloid Leukemia, Histiocytosis | One-year Survival; Number of Transplant-Related Adverse Outcomes: Regimen-Related Mortality; Number of Transplant-Related Adverse Outcomes: Engraftment Failure; Number of Transplant-Related Adverse Outcomes: Fatal Acute Graft-Versus Host Disease (GVHD); Number of Incidences of Chronic GVHD | St. Jude Children’s Research Hospital | NCT00145626 |
| Phase 2 Trial of HLA Haploidentical Natural Killer Cell Infusion for Treatment of Relapsed or Persistent Leukemia Following Allogeneic Hematopoietic Stem Cell Transplant | Leukemia, Myelodysplastic Syndromes | Treatment Efficacy as Defined by Complete or Partial Remission | Memorial Sloan Kettering Cancer Center | NCT00526292 |
| A Phase 1/2 Study Evaluating the Safety and Efficacy of Adding a Single Prophylactic Donor Lymphocyte Infusion (DLI) of Natural Killer Cells Early After Nonmyeloablative, HLA-Haploidentical Hematopoietic Cell Transplantation | *Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Aggressive Non-Hodgkin Lymphoma, Diffuse Large B-Cell Lymphoma, Previously Treated Myelodysplastic Syndrome, Recurrent Chronic Lymphocytic Leukemia | Max dose of NK cells infused a week after HCT; Incidence of relapse; Incidence of grades III-IV acute graft-versus-host disease (GVHD); Incidence of non-relapse mortality | Fred Hutchinson Cancer Center | NCT00789776 |
| Reduced Intensity Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Supplemented with Donor Natural Killer (NK) Cell Infusions in Patients with High Risk Myeloid Malignancies Who Are Unsuitable for Fully Myeloablative Transplantation | Acute Myelogenous Leukemia | Disease-free survival at 6 months and 1 year in patients with high-risk myeloid malignancies; incidence of disease relapse at 12 months; incidence of chronic GVHD at 12 months; rate of treatment-related mortality at day 100; incidence of disease relapse at 12 months; incidence of grade III-IV acute graft-versus-host disease (GVHD) at 6 months; in vivo expansion of a donor CD3- CD19-selected NK cell product; rate of graft failure defined by absolute neutrophil count (ANC) | Masonic Cancer Center, University of Minnesota | NCT00303667 |
| Phase 2 Lymphodepleting Chemotherapy with Rituximab and Allogeneic Natural Killer Cells for Patients with Refractory Lymphoid Malignancies | Non-Hodgkin Lymphoma, Chronic Lymphocytic Leukemia | Number of Patients with an Objective Response; Serious Adverse Events; Time to Disease Progression; Patients with Expansion of NK Cells | Masonic Cancer Center, University of Minnesota | NCT01181258 |
| Pilot Study of Haplo-Identical Natural Killer Cell Transplantation for Acute Myeloid Leukemia | Acute Myeloid Leukemia | Number of Patients Experiencing Grade 3 or 4 Toxicities; Duration of Engraftment of Natural Killer (NK) Cells; Relapse-free Survival; Overall Survival | St. Jude Children’s Research Hospital | NCT00187096 |
| Allogeneic Natural Killer Cells in Patients with Relapsed Acute Myelogenous Leukemia | Leukemia | Patients With Natural Killer (NK) Cell Expansion; Patients with Complete Remission; Median Time to Disease Relapse (Months); Overall Survival Time of Patients with Complete Remission; Patients with Complete Remission and Natural Killer Cell Expansion | Masonic Cancer Center, University of Minnesota | NCT00274846 |
| Phase 2 Lymphodepleting Chemotherapy and T-Cell Suppression Followed by Allogeneic Natural Killer Cells and IL-2 | Ovarian Cancer, Fallopian Tube Cancer, Primary Peritoneal Cancer, Breast Cancer | Response Rate; Time to Disease Progression; Number of Participants with Progressive Disease at One Year; Overall Survival | Masonic Cancer Center, University of Minnesota | NCT01105650 |
| Natural Killer (NK) Cells and Nonmyeloablative Stem Cell Transplantation for Chronic Myelogenous Leukemia (CML) | Leukemia, Chronic Myelogenous Leukemia | Number of Participants with Molecular Complete Remission at 3 Month Post Transplant | M.D. Anderson Cancer Center | NCT01390402 |
| T-Cell or Natural Killer (NK) Cell Adback in Patients with Lymphoid Malignancies Receiving Allogeneic Stem Cell Transplantation with Campath-IH Containing Conditioning Regimens | Lymphoma, Leukemia | 6-month Treatment Related Mortality (TRM); One-year Disease-free Survival (DFS) | M.D. Anderson Cancer Center | NCT00536978 |
| Phase 1 Safety Trial of Natural Killer (NK) Cell Donor Lymphocyte Infusions (DLI) From 6/6 Human Leukocyte Antigen (HLA) Matched Family Member Following Nonmyeloablative Allogeneic Stem Cell Transplantation (ASCT) | Lymphoma | Toxicity; Efficacy - Progression Free Survival; Efficacy - Overall Survival; Efficacy - Disease Progression | Duke University | NCT00586690 |
Key Points.
Distinct stages of conventional natural killer cell development have been proposed in both mice and human.
Natural killer cell differentiation at extramedullary sites remains not well understood despite the identification of tissue-specific resident natural killer cell markers.
Understanding the steps to differentiate natural killer cells into fully functional mature natural killer cells may further progress adoptive cellular therapies for human diseases.
Acknowledgements
This group would like to acknowledge the assistance of Jim Ropa and Lindsay Beasley in proofreading the manuscript.
Financial Support and Sponsorship
The writing of this review was supported by Indiana Clinical and Translational Sciences Institute (CTSI) Biomedical Research Grant (BRG) and US Public Heath Grant from the National Institutes of Health U54 DK106846.
Footnotes
Conflict of Interests
No conflict of interests exists.
References and Recommended Reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
■ of special interest
■ ■ of outstanding interest
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