Introduction

Natural killer or NK cells are a sub-population of Large Granular Lymphocytes (LGLs) which arise from a common NK/T-cell progenitor. Accumulating evidence indicates that NK cells can develop and mature both in the bone marrow and secondary lymphoid tissues (SLTs) including tonsils, spleen, and Lymph nodes (LNs)[1]. In the innate immune system, NK cells participate in early cytotoxic responses against viruses, parasites, microbial pathogens, as well as tumor immunosurveillance. Also, their protective and pathogenic roles have been more defined in the development of autoimmune diseases such as systemic lupus erythematosus, type I diabetes mellitus, and autoimmune liver disease[2]. NK cell's function is regulated by various activating or inhibitory receptors. These germ-line encoded immunoreceptors on the surface of NK cells sense nonspecific alterations of self cells caused by cellular stress, infections, or malignant transformation[3]. In contrast, B and T lymphocytes in the adaptive arm, undergo gene rearrangement to generate surface receptors and mediate the antigen-specific immune responses [4][5]. 

While encountering normal cells, inhibitory receptors override activating receptors. However, molecular changes on the cell surface due to viral infection or tumor formation can stimulate the activating receptors. Upon activation, NK cells release granzyme, perforin, effector molecules of the TNF family, and Fas-ligand to induce apoptosis of the target cells. In addition, NK cells synthesize and release other cytokines and chemokines such as Il-10, gamma-interferon, GM-CSF to recruit other immune effector cells to the target site. NK cells also exhibit antibody-dependent cell cytotoxicity against cells or target antigens which are marked with specific antibodies. Recently, therapeutic applications modulating the inhibition-activation dynamic of NK cells have been developed for multiple conditions including cancer, autoimmune disease, diabetes, etc.[6]

Issues of Concern

Natural Killer cells represent the third lymphocyte population (Following T and B cells), and they play essential roles in the frontline's defense of the innate immune system. The term natural killer suggests that these cells can kill the target cells without pre-stimulation. In contrast to other immune cells, which demand a considerable period of time to induce their cytolytic activity, NK cells provide ready-to-kill machinery in the immune system.  

These cells play various roles in several organ systems; for instance, there is emerging evidence linking NK cells with the development of autism spectrum disorder. The lower degrees of NK cells infiltration in solid tumors correlate with the favorable prognosis. In addition, excessive stimulation of NK cells promotes cell senescence in tissues, laying the foundations for aging. NK cells are important for the body's salutogenic response in dealing with various physiological and non-physiological stimuli throughout life. 

Structure

Morphologically, NK cells are large, granular lymphocytes that are defined by expression of CD56 and CD16 and lack the common T cell marker, CD3, and T cell receptor (TCR) in humans. They constitute 5-15% of the total population of peripheral mononuclear circulating lymphocytes [7]. NK cells, dendritic cells, B and T lymphocytes are derived from single lymphoid progenitor cells. 

A variety of activating or inhibitory receptors have been demonstrated on the surface of NK cells as their immunoreceptor. Inhibitory receptors have cytoplasmic tyrosine-based inhibition motifs and recognize self major histocompatibility complex class I (MHC I) molecules expressed by most of the nucleated cells. However, activating receptors recognize the ligands exhibited by pathological cells and interact with cell signaling pathways to initiate effector activities. Examples of their activating receptors are CD16, NKp30, NKp44, NKp46, NKG2C/CD94, and NKG2D/CD94. NK cells have distinct cytoplasmic granules that are structurally and functionally similar to lysosomes, hence called “secretory lysosomes.” These granules have a bilayered membrane that separates their lytic content from the cytoplasm. [8]

Function

Natural killer cells' effector functions are tightly regulated by the balance of inhibitory and activating receptors and downstream intracellular signaling cascades of these receptors. NK cells were originally identified in the 1970s[9][10], although the mechanism by which they distinguish healthy cells from virus-infected or tumor cells was not fully known for years. First, the results of research on murine NK cells demonstrated that NK cells are able to detect and eliminate lymphoma cells that lost the expression of MHC I, whereas MHC I expressing tumor cells are unaffected by NK cells[3]. This led to the idea of missing self in the activation of NK cells, particularly in their anti-tumor activity. In physiological conditions, most of the circulating NK cells are in the resting state as their inhibitory receptors interact with MHC I on the healthy cells and this accounts for exhibiting self-tolerance (Fig.1a). Almost every healthy cell expresses MHC I molecules, a potential ligand for inhibitory NK cell receptors,  However, MHC I molecules are downregulated upon viral infection and tumor transformation (missing self), exerting subthreshold inhibitory signaling to the NK cells, moving their balance toward the activated state (Fig.1b). In addition, upregulation of NK cells activating receptors occurs in stressed or damaged cells resulting in targeting these cells for the cytolytic activity of NK cells (Fig.1c). Interaction between CD16 on NK cells with antigens expressed on tumor cells causes antibody-dependent cell-mediated cytotoxicity (ADCC) tumor cell lysis by NK cells(Fig.1d). [11][12][13][14][15]

NK cells can kill tumor cells without previous sensitization. They directly kill tumor cells by releasing lytic granules that contain granzyme and perforin. NK cells, together with cytotoxic CD8+ T lymphocytes, generate immunity against virus-infected and tumor cells. In most circumstances, tumor cells downregulate their MHC I to escape from cytotoxic T cells. However, the lower expression of MHC I make tumor cells susceptible to lysis by NK cell.[16]

Histochemistry and Cytochemistry

NK cells are considered the equivalent of CD8 positive T lymphocytes in the innate immune system and multiple cell markers are used for their characterization in immunocytochemistry staining. Natural killer cells are usually classified as CD3 negative and CD56 positive cells and subdivided into CD56dim and CD56bright groups. CD56dim and CD56bright subsets are defined based on the density of CD56 and have distinct functional attributes.

About 90% of circulating and spleen natural killer cells are CD56dim, CD16 positive, and express perforin. CD56dim cells mainly perform a cytotoxic action and CD16 mediates ADCC. In comparison, NK cells in lymph nodes (LNs) and peripheral lymphoid organs (e.g., tonsils) are CD56bright and CD16 negative. CD56 bright cells are mainly cytokine secreting cells and have the capacity to produce several cytokines, including IFN-γ (gamma-interferon) upon stimulation.[16][17][18]

Microscopy, Light

Natural killer cells present a distinct group of circulating lymphocytes on the peripheral blood smear. On Wright-Giemsa-staining, they are visible as large granular lymphocyte morphology. They possess large nuclei containing coarse chromatin and prominent nuclei. Their abundant basophilic cytoplasm contains plenty of azurophilic granules that are negative for peroxidase staining.[19]

Microscopy, Electron

NK cells are large and exhibit various shapes and express microvilli more prominently in the area of effector-to-target contact. At the same time, microvilli are scant cell surface in the absence of target cells. Membranes are not extensively fused in the region of target and effector cell contact.

NK cells have structurally distinct granules that consist of two different compartments. The outer compartment includes the lysosome-associated phosphatase acid enzymes and trimetaphosphatase. No enzymatic activities associated with the inner compartment have been identified. At the time of degranulation, a limited area of the cytoplasm exhibits vacuole-like areas possessing granules and apparent granular debris. Degranulation appears to be implicated in NK cells' cytotoxic activity. Convoluted nuclei with distinctive polarity against the dense granules and pseudopodia are typical. In the cytoplasm, both Mitochondria and polysomes are observed.[19][20]

Pathophysiology

In addition to NK cell roles in defense against viral infections and tumor immunosurveillance, they have been linked to several other diseases including asthma [21], inflammatory bowel disease [22], and autoimmune diseases [23]. Advancements in understanding the pathophysiology of NK cells resulted in the development of new therapeutic options and treatments. For instance, NK cells are found in the pancreas of patients with diabetes type I, and they enhance the disease development. Reducing the NK cell activation using specific blocking antibodies will prevent further disease progression.[6]

Clinical Significance

NK cells are essential components of innate immunity. Although their function may be compromised in many primary immune deficiency disorders, rare isolated Natural Killer cell deficiency syndromes may also occur. There are two potential varieties of NK cell deficiency: (1) the classical natural killer cell deficiency (CNKD) and (2) functional natural killer cell deficiency(FNKD).[24][25] 

CNKD reflects the significant absence of NK cells among peripheral blood mononuclear cells and fewer than 1% of NK cells in peripheral blood should be present in a minimum of three independent tests separated by one month to establish this diagnosis. Unlike CNKD, patients with FNKD have NK cells found in their peripheral blood within the spectrum of age-specific average values; however, permanently deficient in their functions. Both CNKD and FNKD patients are highly susceptible to human papillomavirus (HPV), herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV) infection.[24][26][27]

The prognostic value of NK cells infiltrating solid tumors has attracted considerable attention in clinical practice. Several independent studies have demonstrated that higher levels of NK cell markers such as CD56 and NKp46 in solid tumors correlate with favorable prognosis and overall survival rate [28][29][30]. However, further research is necessary to establish a scoring system including information from tumor type, clinical staging, pathological grading, etc. The clinical development of NK cell-based cancer therapy provided promising results recently. The NK cell-based clinical trials presented a desirable safety profile and efficacy in hematologic malignancies and solid tumors. [31][32][33]