Natural Killer Cells: Development, Maturation, and Clinical Utilization

Abstract

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses, and therefore possess promising clinical utilization. NK cells do not express polymorphic clonotypic receptors and utilize inhibitory receptors (killer immunoglobulin-like receptor and Ly49) to develop, mature, and recognize "self" from "non-self." The essential roles of common gamma cytokines such as interleukin (IL)-2, IL-7, and IL-15 in the commitment and development of NK cells are well established. However, the critical functions of pro-inflammatory cytokines IL-12, IL-18, IL-27, and IL-35 in the transcriptional-priming of NK cells are only starting to emerge. Recent studies have highlighted multiple shared characteristics between NK cells the adaptive immune lymphocytes. NK cells utilize unique signaling pathways that offer exclusive ways to genetically manipulate to improve their effector functions. Here, we summarize the recent advances made in the understanding of how NK cells develop, mature, and their potential translational use in the clinic.

Keywords: developmental stages; effector functions; human; mouse; natural killer cells.

Figure 1

Murine bone marrow niche where natural killer (NK) cells develop. Quiescent hematopoietic stem cells (HSCs) from a hypoxic microenvironment, within the perivascular region proximal to sinusoidal vessels, are induced by hormonal and cytokine cues. Upon unique stimulations [such as stem cell factor (SCF); Fms-like tyrosine kinase-3 ligand (Flt3L)], the self-renewing multipotent HSCs commit to becoming common lymphoid progenitors (CLPs). Non-hematopoietic stromal cells [mesenchymal stromal cells (MSCs), fibroblastic reticular cells] that produce interleukin (IL)-7 or IL-15 play pleiotropic roles in programming CLPs into distinct lymphoid lineages including NK cell progenitors (NKPs). MSCs also produce another common gamma chain receptor (γ_cR)-binding cytokine, IL-21 that may help with the expansion of the NKPs. CXCL12-abundant reticular (CAR) cells generate CXCL12, which stimulates NKPs via CXCR4 to functionally mature the NKPs or immature NK cells (iNKs) into established Mature NK (mNK) cells subsets. mNK cells traffic to secondary lymphoid organs via the sinusoidal blood vessels. Other cell types, pericytes, megakaryocytes, adipocytes, canopy cells, osteoblasts, osteoclasts, and osteocytes help form the niche and other supporting systems.

Figure 2

Developmental origin of murine natural killer (NK) cells in the bone marrow (BM). Murine NK cells develop in the BM. A subset of multipotent HSCs commits to becoming oligopotent common lymphoid progenitors (CLPs). CLPs give rise to Pro-B, Pre-T, innate lymphoid cells (ILCs), lymphoid tissue inducers, and CD122⁺ Pre-T/early NK cell progenitor (NKP) lineages. Expression of NKG2D by the CD122⁺ NKPs mark the earliest transition of NKPs into committed immature NK cells (iNK, Stage A). This is followed by the expression of NK1.1 and NCR1 (Stages B and C). Expression of CD51 (Integrin αV) and CD49b (DX5, Integrin VLA-2α) defines the initial stage of mature NK (mNK) cells. Expression of CD43 (Leukosialin), CD11b (Mac-1), and the acquisition of distinct sets of Ly49s define the terminal stage of mNK cells (Stage E). mNK cells migrate into secondary lymphoid organs following the expression of Killer cell Lectin-like Receptor G1 (KLRG1) (Stage F) at least in part by a subset. Additional functional classifications of mNK cells are made using CD27 and CD11b.

Figure 3

Distinct developmental stages of murine NK cell progenitors (NKPs), immature NK cells (iNKs), and mature NKs (mNKs). Lineage negative (Lin⁻) Sca⁺CD117⁺ hematopoietic stem cells (HSCs) differentiate into common lymphoid progenitors (CLPs) (Lin⁻Sca^LowCD117^LowFlt3⁺). Expression of IL-7 receptor-alpha (IL-7Rα) (CD127), CD27, and CD244 mark the full commitment of CLPs into pre-NK cell precursors (Pre-NKPs). Committed NKPs transition from Pre-NKPs to refined-NKPs (rNKPs) by expressing IL-2Rβ (CD122). Expression of NKG2D marks the conversion of rNKPs into iNK cells. Natural killer (NK) cells progressing through the iNK stages express NK1.1 and NKG2A/C followed by NCR1 (Stage A through C). Terminal maturation of iNK cells into mNK cells is defined by the acquisition of distinct sets of Ly49s that help to identify distinct subsets (Stage D). NK cells that have reached terminal maturation downregulate CD27 and express CD11b (Stage E) followed by Killer cell Lectin-like Receptor G1 (KLRG1) (Stage F) by a subset of matured NK cells.

Figure 4

Developmental origin of human natural killer (NK) cells. In human, the primary organ where NK cells mature is still under active investigation. There is ample evidence that NK cells can mature from the lymph nodes (LNs). Lin⁻CD34⁺ hematopoietic stem cells (HSCs) differentiate into CD45RA⁺ lymphoid-primed multipotential progenitor (LMPP). By expressing CD38, CD7, CD10, and the cytokine receptor CD127 (IL-7 receptor-alpha), LMPPs transition into common lymphoid progenitors (CLPs) that have the potential to make lineage commitment into Pro-B, Pre-T, NK cell progenitors (NKPs), or other innate innate lymphoid cells. Expression of CD122 (IL-2Rβ) marks the irreversible fate decision of CLPs into NK lineage. The appearance of CD56 (neural cell adhesion molecule) indicates a final transition of immature NK cell (iNK) into mature NK cells. Most of the iNK cells transition into a minor CD56^bright population (~5%) that convert into major CD56^dim (>90%) population. It is also suggested that iNK cells can directly give rise to CD56^dim population (dotted arrow) that is yet to be validated.

Figure 5

A common schema of human natural killer (NK) cell development in the bone marrow and lymph nodes. A total of six distinct developmental stages have been described with Stages 2 and 4 having additional bifurcations. Similar to the mouse, human NK cells express CD244 (2B4) throughout the developmental process starting at Stage 1 (pre-NK cell precursors). CD117 (c-Kit) and the low levels of interleukin (IL)-1R1 expressions define the Stage 2a and Stage 2b, respectively (NK cell progenitors). A higher expression of IL-1R1 defines the Stage 3 [immature NK cell (iNK)], and the expressions of NKG2D, CD335 (NKp46), CD337 (NKp30), and CD161 (NK1.1) are initiated. Stages 4a and 4b defines an entry of iNKs into mature nks, and are differentiated by the expression of NKp80 at the Stage 4b. Expressions of NKG2D, CD335, CD337, and CD161 reach their maximal levels at Stage 4. Most important of all, CD56 expression peaks (CD56^bright). Significant differences between Stage 4b and Stage 5 are defined by a decrease in the expression of CD56 (CD56^dim) in most and initiation of the expression of CD16 (FcγRIIIA) and killer immunoglobulin-like receptor (KIR) (CD158) in a subset of NK cells. Stage 6 defines the generation of “adaptive” or “memory-like” NK cells following “antigen” exposure, and it is identified by the high levels of NKG2C.

Figure 6

Role of common gamma-containing receptors in natural killer (NK) cell development. The common gamma chain-containing receptor family consists of six members, interleukin (IL)-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R. Each one of them is distinguished from others by their unique α-chains. IL-2Rβ is shared by IL-2R and IL-15R complexes. In mouse, NK cell progenitors (NKPs) utilize IL-7R early during their transition from Pre-NKPs into refined-NKPs. In human, apart from its role in the early development, IL-7 also regulates the survival and expansion of mature CD56^bright NK cells. IL-15R and IL-21R are required by NK cells to initiate and sustain their proliferation. Although it has been widely used to expand human and mouse NK cells ex vivo, the in vivo role of IL-2 that is primarily produced by CD4⁺ T cells is yet to be better understood. Role of IL-4 and IL-9 in NK cell development is less explored. Distinct sets of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) associate and transmit the signaling from the common gamma chain-associated cytokine receptors.

Figure 7

Role of a “third signal” in natural killer (NK) cell activation. (A) A brief description of the significant interactions between NK and myeloid cells. NK cells possess inherent abilities to mediate cytotoxicity and produce inflammatory cytokines and chemokines. Myeloid cell-derived cytokines play a central role in regulating the effector functions of NK cells. Interactions between the innate NK cells and the primary arms of the adaptive immunity (T and B cells) are less explored. Stimulation through activation receptors (i.e., NKG2D or Ly49H) help recognize tumor (H60) or infected target cells (murine cytomegalovirus-derived m157). (B) A summary of major soluble factors produced by NK cells and their intended functions.

Figure 8

Mechanisms of target cell recognition by natural killer (NK) cells. NK cells lack clonotypic receptors and rely on germline-encoded activation and inhibitory receptors to recognize other cells around them. The following are some of the primary mechanisms by which NK cells perceive target cells. (A) “Immunological Self”: recognition of autologous MHC class I (MHC-I) (human leukocyte antigen (HLA)) or histocompatibility antigen-2 (H2, mouse) by inhibitory receptors [killer cell immunoglobulin-like receptor (KIR) or Ly49] let the NK cells know that they are interacting with normal cells and contain their activation. (B) “Missing-self”: recognition of target cells that either does not express MHC-I or reduce them below optimal levels can induce NK cell activation. (C) “Induced-self”: recognition of activating ligands that are expressed on target cells by the germline-encoded receptors such as NKG2D (H60, mouse; MIC-A/B, human), Ly49H (murine cytomegalovirus-derived m157, mouse), NCR1 (a number of viral proteins) can overcome MHC-I-mediated inhibitory signaling resulting in NK cell activation. (D) “Non-self”: recognition of transplanted tissue by NK cells, where the donor tissue expresses either allogeneic or haploidentical MHC-I.

Figure 9

Natural killer (NK) cells in health and disease. As the largest lymphocyte population representing innate immunity, NK cells perform diverse functions. Through their ability to mediate killing and to produce soluble factors, NK cells perform multitudes of immunological functions. Counter-clockwise: bidirectional interactions between NK cell and dendritic cells (DCs)/macrophages result in priming. Activated DCs and macrophages generate interleukin (IL)-15, IL-12, IL-18, IL-35, IFN-α, IFN-β, IL-27, IL-1β, and IL-23. These, in turn, activate NK cells to be primed, proliferate, and to produce inflammatory factors and chemokines such as interferon-gamma (IFN-γ), granulocyte/monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, CCL3, CCL4, and CCL5. In addition, IFN-γ from NK cells can increase the MHC class I expression and the transcription of genes encoding immuno-proteasomal subunits in these professional antigen-presenting cells and thereby augmenting T cell priming and activation. Similarly, virus-infected cells produce IFN-α, IFN-β, and IL-1β and present either “stress-induced” self-ligands or viral proteins on the cell surface that activate NK cells. A reduction in graft-versus-host disease (GvHD) is mediated through the production of IL-10 by the CD56^brightCD16^Neg NK cell subset and augmentation of GvT is potentiated via direct tumor killing by CD56^dimCD16^Pos NK cell subset. In addition, production of IL-22 by NK subsets may help the regeneration of epithelial cells in the mucosal tissues. Irrespective of these observations, the mechanisms by which NK cells are activated to respond during active GvHD/GvT is not fully understood. Genetic manipulation of NK cells has helped to improve the effector functionality and the longevity of human NK cells in vivo. Stable integration of gene encoding IL-15 into the genome of NK cells promotes sustained proliferation via an artificial autocrine loop. Similarly, integration of gene encoding IL-12 makes this cytokine abundantly available within the microenvironmental milieu and thereby augment the effector functions of NK cells, specifically, the production of IFN-γ. Augmented expression of NK cell activation receptors (NKRs) including NKG2D and NCR1 by genetic engineering increases the anti-tumor cytotoxicity of NK cells. Other studies have shown the expression of single chain variable fragment that forms the core ectodomain of chimeric antigen receptor (CAR) to augments the tumor-targeted killing of NK cells. These genetically modified NK cells provide exciting newer opportunities for cell-based therapies. The bidirectional interaction between NK and T cells results in the regulation of adaptive immunity. IL-2 produced by CD4⁺ Th1 cells play a vital role in the proliferation and expansion of NK cells. Although in vitro experiments consistently have provided support toward this notion, the in vivo evidence is far from convincing. However, the inflammatory factors produced by NK cells have a significant impact on both CD8⁺ and CD4⁺ T cells. Expression of “self” ligands for NKG2D by T cells results in the recognition and killing of T cells by NK cells during GvHD and anti-viral responses. In addition, a cleaved soluble form of these ligands (MIC-A/B) is present in the serum of cancer patients. This, in turn, plays an important role in containing the effector functions of T cells via direct binding to the NKG2D receptor expressed on T cells. NK cells recognize bacteria-infected cells (such as epithelial cells) either using toll-like receptors (TLR) or by activated through soluble factors including aryl hydrocarbon receptor (Ahr). This results in the production of IFN-γ and IL-22 that helps with the reduction in bacterial load and regeneration of epithelial cells, respectively. NK cells can also directly mediate the lysis of bacteria using granzymes and perforin.