11.3F: Natural Killer Cells (NK Cells) and Invariant Natural Killer T-Lymphocytes (iNKT Cells) - Biology

11.3F: Natural Killer Cells (NK Cells) and Invariant Natural Killer T-Lymphocytes (iNKT Cells) - Biology

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Learning Objectives

  1. Describe how NK cells are able to recognize and kill infected cells and cancer cells lacking MHC-I molecules.
  2. State two factors that can result in a nucleated human cell not producing MHC-I molecules.
  3. State how iNKT cells recognize glycolipids in order to become activated.
  4. Describe the overall function of iNKT cells in terms how they promote both innate and adaptive immunity and may also help to regulate the immune responses.

We will now take a closer look at natural killer (NK) cells and invariant natural killer T-lymphocytes (iNKT cells).

Natural Killer Cells (NK Cells)

NK cells are important in innate immunity because they are able to recognize infected cells, cancer cells, and stressed cells and kill them. In addition, they produce a variety of cytokines, including proinflammatory cytokines, chemokines, colony-stimulating factors, and other cytokines that function as regulators of body defenses. For example, through cytokine production NK cells also suppress and/or activate macrophages , suppress and/or activate the antigen-presenting capabilities of dendritic cells, and suppress and/or activate T-lymphocyte responses.

NK cells use a dual receptor system in determining whether to kill or not kill human cells. When cells are either under stress, are turning into tumors, or are infected, various stress-induced molecules such as MHC class I polypeptide-related sequence A (MICA) and MHC class I polypeptide-related sequence B (MICB) are produced and are put on the surface of that cell.

The first receptor, called the killer-activating receptor, can bind to these stress-induced molecules, and this sends a positive signal that enables the NK cell to kill the cell to which it has bound unless the second receptor cancels that signal.

This second receptor, called the killer-ihibitory receptor, recognizes MHC-I molecules that are usually present on all nucleated human cells. MHC-I molecules, produced by all nucleated cells in the body, possess a deep groove that can bind peptides from proteins found within the cytosol of human cells, transport them to the surface of that cell, and display the MHC-!/peptide complex to receptors on cytotoxic T-lymphocytes or CTLs. If the MHC-I molecules have peptides from the body's own proteins bound to them, CTLs do not recognize those cells as foreign and the cell is not killed. If, on the other hand, the MHC-I molecules have peptides from viral, bacterial, or mutant proteins bound to them, CTLs recognize that cell as foreign and kill that cell. (CTLs will be discussed in greater detail in Unit 6.)

If MHC-I molecules/self peptide complexes are expressed on the cell, the killer-inhibitory receptors on the NK cell recognize this MHC-I/peptide complex and sends a negative signal that overrides the original kill signal and prevents the NK cell from killing the cell to which it has bound (see Figure (PageIndex{3})).

Viruses, stress, and malignant transformation, however, can often interfere with the ability of the infected cell or tumor cell to express MHC-I molecules. Without the signal from the killer-inhibitory receptor, the kill signal from the killer-activating signal is not overridden and the NK cell kills the cell to which it has bound (see Figure (PageIndex{4})).

The NK cell then releases pore-forming proteins called perforins, proteolytic enzymes called granzymes, and chemokines. Granzymes pass through the pores and activate the enzymes that lead to apoptosis of the infected cell by means of destruction of its structural cytoskeleton proteins and by chromosomal degradation. As a result, the cell breaks into fragments that are subsequently removed by phagocytes (see Figure (PageIndex{5})). Perforins can also sometimes result in cell lysis.

Cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) produced by TH1 lymphocytes activate NK cells.

NK cells also play a role in adaptive immune responses. As will be seen in Unit 6, NK cells are also capable of antibody-dependent cellular cytotoxicity or ADCC where they kill cells to which antibody molecules have bound.

Invariant Natural Killer T-Lymphocytes (iNKT Cells)

iNKT cells are a subset of lymphocytes that bridge the gap between innate and adaptive immunity. They have T-cell receptors (TCRs) on their surface for glycolipid antigen recognition. They also have natural killer (NK) cell receptors.

Through the cytokines they produce once activated, iNKT cells are essential in both innate and adaptive immune protection against pathogens and tumors. They also play a regulatory role in the development of autoimmune diseases, asthma, and transplantation tolerance. It has been shown that iNKT cell deficiency or disfunction can lead to the development of autoimmune diseases, human asthma, and cancers.

Pathogens may not directly activate iNKT cells. The TCR of iNKT cells recognize exogenous glycolipid antigens , as well as endogenous self glycolipid antigens presented by MHC-I-like CD1d molecules on antigen presenting dendritic cells. iNKT cells can also be activated by the cytokine interleukin-12 (IL-12) produced by dendritic cells that have themselves become activated by pathogen-associated molecular patterns (PAMPs) of microbes binding to the pattern-recognition receptors (PRRs) of the dendritic cell.

Once activated, the iNKT cells rapidly produce large quantities of cytokines, including interferon-gamma (IFN-?), interleukin-4 (IL-4), interleukin-2 (IL-2), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-a), interleukin-13 (IL-13), and chemokines. Through the rapid productions of such cytokines, iNKT cells are able to promote and suppress different innate and adaptive immune responses. For example, large amounts of IFN-? are produced by activated iNKT cells. IFN-? activates NK cells and macrophages as a part of innate immunity.

It has been proposed that if the iNKT cell is repeatedly stimulated by the body's own glycolipids in the absence of microbes that this might stimulate the iNKT cell /dendritic cell interaction to produce tolerizing signals that inhibit the TH1 cell response and possibly stimulate the production of regulatory T-lymphocytes (Treg cells). In this way it might suppress autoimmune responses and prevent tissue damage.

There is also growing evidence that early childhood exposure to microbes is associated with protection against allergic diseases, asthma, and inflammatory diseases such as ulcerative colitis. It has been found that germ-free mice have large accumulations of mucosal iNKT cells in the lungs and intestines and increased morbidity from allergic asthma and inflammatory bowel disease. However, colonization of neonatal germ-free mice with normal microbiota resulted in mucosal iNKT cell tolerance to these diseases. It has been proposed that microbes the human body has been traditionally exposed to from early childhood throughout most of human history might play a role in developing normal iNKT cell numbers and iNKT cell responses.

iNKT cells will be discussed in further detail in Unit 6.


  1. Natural Killer (NK) cells are able to recognize infected cells, cancer cells, and stressed cells and kill them. In addition, they produce a variety of cytokines, including proinflammatory cytokines, chemokines, colony-stimulating factors, and other cytokines that function as regulators of body defenses.
  2. When body cells are either under stress, are turning into tumors, or are infected, various stress-induced molecules are produced and are put on the surface of that cell.
  3. NK cells use a dual receptor system in determining whether to kill or not kill human cells.
  4. The first receptor, called the killer-activating receptor, can bind to these stress-induced molecules, and this sends a positive signal that enables the NK cell to kill the cell to which it has bound unless the second receptor cancels that signal.
  5. The second receptor, called the killer-ihibitory receptor, recognizes MHC-I molecules that are usually present on all nucleated human cells. If MHC-I molecules/self peptide complexes are expressed on the cell, the killer-inhibitory receptors on the NK cell recognize this MHC-I/peptide complex and sends a negative signal that overrides the original kill signal and prevents the NK cell from killing the cell to which it has bound.
  6. Viruses, stress, and malignant transformation can often interfere with the ability of the infected cell or tumor cell to express MHC-I molecules. Without the signal from the killer-inhibitory receptor, the kill signal from the killer-activating signal is not overridden and the NK cell kills the cell to which it has bound.
  7. NK cells kill their target cells by inducing apoptosis, a programmed cell suicide.
  8. NK cells also play a role in adaptive immune responses by way of antibody-dependent cellular cytotoxicity or ADCC where they bind to and kill cells to which antibody molecules have bound.
  9. Invariant natural killer T-lymphocytes (iNKT cells) are a subset of lymphocytes that have T-cell receptors on their surface for glycolipid antigen recognition. They also have natural killer (NK) cell receptors.
  10. Through the cytokines they produce, iNKT cells are able to promote and suppress different innate and adaptive immune responses. iNKT cell deficiency or disfunction can lead to the development of autoimmune diseases, human asthma, and cancers.

Natural killer cells: a review of biology, therapeutic potential and challenges in treatment of solid tumors

Natural killer (NK) cells lead immune surveillance against cancer and early elimination of small tumors. Owing to their ability to engage tumor targets without the need of specific antigen, the therapeutic potential of NK cells has been extensively explored in hematological malignancies. In solid tumors, however, their role in the clinical arena remains poorly exploited despite a broad accumulation of preclinical data. In this article, we review our current knowledge of NK cells' biology, and highlight the challenges facing NK cell antitumor strategies in solid tumors. We further summarize the abundant preclinical attempts at overcoming these challenges, present past and ongoing clinical trial data and finally discuss the potential impact of novel insights on the development of NK cell-based therapies.

Keywords: CAR-NK NK cells adoptive transfer cancer stem cells clinical trials immune escape immune surveillance receptor signaling modulation solid tumors.

Irradiated Tumor Fibroblasts Avoid Immune Recognition and Retain Immunosuppressive Functions Over Natural Killer Cells

Recent research have demonstrated that radiotherapy is ready to induce anti-tumor immune responses along with mediating direct cytotoxic results. Cancer-associated fibroblasts (CAFs) are central constituents of the tumor stroma and take part actively in tumor immunoregulation. However, the capability of CAFs to affect immune responses within the context of radiotherapy continues to be poorly understood. This research was undertaken to find out whether or not ionizing radiation alters the CAF-mediated immunoregulatory results on pure killer (NK) cells. CAFs had been remoted from freshly resected non-small cell lung most cancers tissues, whereas NK cells had been ready from peripheral blood of wholesome donors.

Functional assays to review NK cell immune activation included proliferation charges, expression of cell floor markers, secretion of immunomodulators, cytotoxic assays, in addition to manufacturing of intracellular activation markers reminiscent of perforin and granzyme B. Our information present that CAFs inhibit NK cell activation by lowering their proliferation charges, the cytotoxic capability, the extent of degranulation, and the floor expression of stimulatory receptors, whereas concomitantly enhancing floor expression of inhibitory receptors.

Radiation delivered as single high-dose or in fractioned regimens didn’t reverse the immunosuppressive options exerted by CAFs over NK cells in vitro, regardless of triggering enhanced floor expression of a number of checkpoint ligands on irradiated CAFs. In abstract, CAFs mediate noticeable immune inhibitory results on cytokine-activated NK cells throughout co-culture in a donor-independent method. However, ionizing radiation doesn’t intervene with the CAF-mediated immunosuppressive results.

To study the cross-talk between NK cells and DCs in HCV an infection, we remoted monocytes and NK cells from 20 persistent HCV sufferers and 20 wholesome controls. Monocytes had been used to generate immature DCs which had been pulsed with HCV peptides (core, NS3-NS4 and NS5). Four totally different co-cultures had been carried out: E1: each DCs and NK cells had been from a persistent HCV affected person, E2: NK cells from a wholesome management co-cultured with DCs from a persistent HCV affected person, E3:

NK cells from a persistent HCV affected person co-cultured with DCs cells from a wholesome management and E4: each DCs and NK cells had been from a wholesome management. Using stream cytometry, we assessed the impact of those totally different co-cultures on ranges of maturation markers on DCs and ranges of activation/inhibition markers on NK cells. Results confirmed that peptide pulsed HCV DCs confirmed a maturation defect within the type of decreased HLA-DR, decreased CD86 and elevated CD83 expression particularly when co-cultured with HCV NK.

This was primarily as a result of core peptide pulsing and to a lesser extent as a result of NS5 pulsing whereas there was no impact with NS3-NS4 pulsing. Alternatively, HCV NK cells upregulated each activation and inhibition markers particularly when co-cultured with wholesome DCs. Compared to E2, E1 resulted in greater apoptosis of each NK cells and DCs with the proportion of NK apoptosis greater than that of DCs. Taken collectively, the information point out that HCV an infection impairs NK-DC cross-talk which can be a number one trigger in viral persistence and chronicity. This article is protected by copyright. All rights reserved.


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The diversity of infiltrating stromal cells occurring in human cancers exceeds 30 distinct subgroups, reflecting the huge complexity of the tumor microenvironment (TME), thereby deeply affecting the treatment option for each patient [1]. Attempts have been made to distill this out-of-order situation into a unifying method to better describe actual composition of the TME using both multi-omics and experimental technologies, shedding light on cancer biology. This trend led to a transition in cancer treatment from only targeting tumor cells (like traditional chemotherapy and radiotherapy) to a new generation of approaches emphasizing the modulation of endogenous immune response toward cancer.

The immune system can be generally divided into the innate and adaptive immune systems, both contributing to the recognition and removal of foreign pathogens as well as tumors [2]. Adaptive immunity is mainly composed of cells represented by T and B lymphocytes, which harbor an enormous repertoire of T-cell and B-cell receptors, respectively, that can respond specifically to different antigens in the body. Current immunotherapeutic methods mainly focus on T lymphocytes, especially restoring exhausted CD8 + cytotoxic T cells (CTLs). An example of such approach is immune-checkpoint blockade, with blocking of receptors or ligands that inhibit the activation of CTLs, including programmed cell death protein 1 (PD-1), its main ligand PD-L1, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and lymphocyte-activation gene-3 (LAG-3), by monoclonal neutralizing antibodies [3, 4]. In recent years, the rapid and potent anti-tumor function of innate immunity, which even occurs at a very early stage of tumor progression, has attracted increasing attention. As a subset of whole innate lymphoid cells, natural killer (NK) cells, defined by Herberman in 1976 [5] and often considered a part of type 1 innate-like cells (ILC1s), are currently defined as effector cells similar to CTLs, exerting natural cytotoxicity against primary tumor cells and metastasis by inhibiting proliferation, migration and colonization to distant tissues [6]. Beside their cytotoxic role, NK cells have been reported to produce a large number of cytokines, mainly interferon-γ (IFN-γ), to modulate adaptive immune responses and participate in other related pathways [7, 8]. In addition, as documented in multiple models and experiments, NK cells could distinguish abnormal cells from healthy ones, leading to more specific anti-tumor cytotoxicity and reduced off-target complications [9, 10].

Considering the pivotal role of NK cells in cancer biology, they naturally emerged as a prospective target for cancer therapy, and a growing number of studies and multiple therapeutic agents inhibiting cancer target NK cell-related pathways. In this review, we will review the fundamental characteristics and emerging subpopulations of NK cells. Next, we will mainly use breast cancer (BC) to discuss the plasticity of NK cells in cancer biology and metabolism, as well as current therapeutic regimens, including ongoing clinical trials and FDA-approved therapies targeting NK cells, and future possible approaches for improving cancer treatment.

Development of NK cells

NK cells possess cytotoxic abilities similar to CD8 + T cells functioning in the adaptive immunity but lack CD3 and the T cell receptors (TCRs). Largely circulating in blood and counting for about 5–10% of peripheral blood mononuclear cells (PBMCs), NK cells are found in bone marrow and lymphoid tissues such as the spleen [11, 12]. Similar to other ILCs, NK cells are originated from common lymphoid progenitor (CLP) cells in bone marrow (Fig. 1) with an average renewal cycle of about 2 weeks [12]. During development, a process termed education, which describes the interaction of NK cells expressing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) with major histocompatibility complex-I (MHC-I), helps NK cells become licensed and avoid attacking healthy normal cells [6, 9]. Interestingly, tumor cells always lack or only express low levels of MHC-I to evade CD8 + T cell-mediated cytotoxicity, whereas licensed NK cells are fully activated. However, tumor cells also express molecules that activate NK cells, e.g., MHC class I polypeptide-related sequence A (MICA) and MICB [13, 14], supporting the use of NK cells as anti-cancer agents. In addition, unlicensed NK cells also play important roles in the body, e.g., eliminating murine cytomegalovirus (MCMV) infection and MHC-I + cells [15].

Development and subgroups of NK cells. In bone marrow, NK cells develop from hematopoietic stem cells (HSCs) through common lymphoid progenitors (CLPs) and NK cell precursors (NKPs), and then migrate to peripheral blood (cNK cells) or tissue (trNK cells). The differentiation of trNK-cells occurs in distinct tissue sites, including the lung, thymus, liver, uterus, skin, subcutaneous adipose tissue, and kidney. In these sites, NK cells have different phenotypic features and functions, which constitute the circulation of NK cells at different stages of maturation. CLA, cutaneous lymphocyte-associated antigen CCR8, C-C motif chemokine receptor 8 GATA3, GATA binding protein 3 CXCR6, C-X-C motif chemokine receptor 6 KIR, killer cell immunoglobulin-like receptor CILCP, common innate-like cell precursor

To date, NK cell survival and development is thought to mainly rely on cytokines (especially IL-2 and IL-15) [16,17,18,19] and transcription factors (Nfil3, Id2 and Tox for development, and EOMES and T-bet for maturation) [16, 20]. GRB2-associated binding protein 3 (GAB3) is essential for IL-2 and IL-15-mediated, and its deficiency leads to impaired NK cell expansion [21]. In addition, targeting related signals is a potential option for promoting NK cell-induced cytotoxicity toward cancer. As reported previously, ablation of cytokine-inducible SH2-containg protein (CIS), which negatively regulates IL-15 to restrict NK cell function, could prevent metastasis and also potentiate CTLA-4 and PD-1 blockade therapy in vivo [22].

Identification and molecular features of NK cells

Surface molecules of NK cells

Due to variable expression of surface markers on NK cells, it is hard to use one or two simple molecules or traditional immunohistochemistry to accurately identify this cell type and more importantly, their functional status. However, in humans, in both clinical and research settings, CD3 − CD56 + cells are commonly identified as NK cells and can be further divided into the CD56 bright and CD56 dim subgroups. CD56 is not only a marker but also plays an important role in the terminal differentiation of NK cells since its blockade by monoclonal antibodies obviously inhibits the transition from CD56 bright to CD56 dim , thus limiting the cytotoxic ability [23]. Consistently, CD3 − NK1.1 + and CD3 − CD49b + cells are defined as NK cells in mice. In recent studies, the notion that natural cytotoxicity receptor 46 (NKp46), belonging to natural cytotoxicity receptors (NCRs), should also be included in this panel has been proposed based on the consensus of adding more functional proteins rather than surface molecules into the classification system of NK cells [24, 25].

Activating and inhibitory signals in NK cells

As the main effector cell type in innate immunity, NK cells are capable of killing tumor cells and virus-infected cells at a very early stage. Due to the lack of abundant production of receptors for distinguishing incalculable antigens in the body specifically, they rely on the “missing self” and “induced self” modes to identify target cells by maintaining a precise balance between activating co-stimulatory and inhibitory signals (mainly by functional receptors). Those interacting signals finally decide the activation and functional status of NK cells.

Activating signals include cytokine-binding receptors, integrins, killing-receptors (CD16, NKp40, NKp30 and NKp44), receptors recognizing non-self-antigens (Ly49H) and other receptors (e.g., NKp80, SLAMs, CD18, CD2 and TLR3/9) [26, 27]. In total, the activating receptors of NK cells can be divided into at least three types according to the respective ligands, including MHC-I specific, MHC-I related and MHC-I non-related receptors (Table 1) [13, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43]. To emphasize, NCRs, which belong to the third group, include three molecules (NKp30, NKp44 and NKp46), and NKp30 was shown to be capable of recognizing B7-H6 expressed on tumor cells, and could be used as a novel treatment option in the future [35].

Inhibitory signals mainly comprise receptors recognizing MHC-I, such as Ly49s, NKG2A and LLT1, as well as some MHC-I non-related receptors (Table 1) [20, 44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60]. Moreover, MHC-I specific inhibitory receptors can be generally divided into three types according to structure and function: killer cell immunoglobulin-like receptors (KIRs), killer lectin-like receptors (KLRs) and leukocyte immunoglobulin-like receptors (LILRs).

NK cell subpopulations according to site of maturation

Conventional NK (cNK) cells are mainly found in peripheral blood and migrate to a specific location to exert their effects. NK cells also include tissue-resident NK (trNK) cells. The complex process of NK-cell differentiation occurs in several distinct tissues, including bone marrow, liver, thymus, spleen and lymph nodes, and may involve cell circulation at different stages of maturation among these tissues [61]. In bone marrow, blood, spleen and lungs, NK cells are fully differentiated, while that in lymph nodes and intestines are immature and precursory [62]. Single-cell transcriptome ananlysis of bone marrow and blood NK cells helps to illustrate the changes of their characteristics during development. For example, high expreesion of TIM-3, CX3CR1 and ZEB2 represents a more mature status [63]. Many hypotheses have been proposed to describe the motivation of their migration and different biological behaviors of identically originated NK cells in different tissues. The first question could be partly explained by the multi-direction differentiation induced by heterogeneous microenvironments in different tissues, or more straightforward, different phenotypes originated from similar chemokine-recruited peripheral cNK cells.

To conclude, NK cells in various tissues have diverse features, possessing different functions and forming a close relationship with other stromal cells (Fig. 1). In the lung, trNK cells show a different phenotype from that of circulating NK cells (mainly CD56 dim ) and are considered to express different levels of CD16, CD49a and CD69, with CD56 dim CD16 + cells representing the majority of the whole NK family there [64, 65]. Of note, CD69 + cells are the main type of CD56 bright CD16 − NK cells. However, in the thymus, most NK cells are CD56 high CD16 − CD127 + , highly relying on GATA3 compared with the CD56 + CD16 + subgroup [66]. Besides, they produce more effector molecules, including TNF-α and IFN-γ [66, 67]. Similar to the phenotypic features in humans, skin NK cells in the mouse can be generally divided into two types: CD49a + DX5 − and CD49a − DX5 + [68, 69]. Similarly, hepatic trNK cells can be classified into two groups, including CD56 bright CD16 +/− and CD56 dim CD16 + , both lacking CD3 and CD19 [8]. In addition, CD49a + CD56 + CD3 − CD19 − NK cells have been identified in liver biopsies [70]. Besides, hepatic NK cells can develop memory for structurally diverse antigens, dependent on the surface molecule CXCR6 [71]. In the uterus, most NK cells are CD56 bright CD16 − , expressing high levels of KIRs [72]. Decidual NK cells are also CD49a + . For skin NK cells, it is intriguing that only few CD56 + CD16 + can be detected, which are common in peripheral blood [73]. Interestingly, trNK cells are distinct between subcutaneous (CD56 dim ) and visceral (CD56 bright ) adipose tissues, and can be generally divided into three groups according to CD49b and Eomes, showing different expression levels of CD49a (CD49b + Eomes − subgroup) and CD69 (CD49b − Eomes + subgroup) [74, 75].

Besides different tissue types, NK cells are also highly heterogeneous even in the same organ and the same tissue [61]. Through high-dimensional single-cell RNA-seq, Crinier et al. revealed the heterogeneity of human and mouse NK cells in spleen and blood and identified several subpopulations of NK cells, respectively [76]. As mentioned above, NK cells are considered a subgroup of ILC1s [6]. Although ILC1s are not detectable in many tissues, intra-epithelial ILC1 (ieILC1)-like cells, which highly express IFN-γ, integrins and other cytotoxic molecules similar to the ieILC1s previously described by Fuchs except for different NKp44 expression, could represent the majority of NK cells in the mucosal tissue [61, 77]. Due to their unique features, this cell type represents a subgroup of NK cells other than conventional ILCs. Unlike other trNK cells, DX5 − CD11c hi liver-resident NK cells participate in autoimmune cholangitis, negatively regulating immune responses, especially by inhibiting the proliferation and function of CD4 + T cells in vivo, which was validated by severer biliary disease in NK-depleted mice resulting from Nfil3 knockdown or treatment with neutralizing antibodies [78].

NK cell subpopulations according to functional molecules

According to surface CD56 expression, NK cells can be divided into CD56 bright and CD56 dim . CD56 dim NK cells are mainly found in peripheral blood, and are always also CD16-positive, expressing high levels of KIR and LFA-1 and showing cell killing ability. CD16 is a key receptor mediating antibody-dependent cell cytotoxicity (ADCC), inducing the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) [40, 79, 80]. According to a time-resolved single-cell assay, the cytotoxicity of NK cells is repressed through both necrosis and apoptosis. As a result, FasL/FasR interaction, perforin/granzyme release and Ca2 + influx are all important for NK cell function [81]. However, CD56 bright NK cells are similar to helper cells, which mainly secrete cytokines such as IFN-γ, TNF-β and GM-CSF [23]. Researchers even further subgroup these cells into the NK1 and NK2 categories, consistent with Th1 and Th2, mainly secreting IFN-γ and IL-5, respectively [82].

Besides established cytotoxic cNK cells, it has been demonstrated that NK cells could differentiate into antigen-presenting NK (AP-NK) cells [83], helper NK (NKh) cells [84] and regulatory NK (NKreg) cells, each defined by surface molecules and individual functions. A new CD8αα + MHC-II + phenotype with professional APC capacity was considered to represent unusual AP-NK cells, recognizing and eliminating autoreactive T cells and finally killing them like cNK cells [85]. Human plasmacytoid dendritic cells (DCs) activated by the preventive vaccine FSME upregulate CD56 expression on their surface [86] in mice, B220 + CD11c int NK1.1 + cells have antigen-presenting capacity like DC, hence their name interferon-producing killer DC [87, 88].

Invariant natural killer T cells (iNKT) constitute a subgroup of T cells expressing NK cell-markers. Activated by CD1d-presenting antigens, NKT could secrete not only Th1-type but also Th2-type cytokines to participate in immunity [89, 90]. Th1-polarized iNKT cells exhibit a tumor-depletion phenotype, and Th2-polarized iNKT cells contribute to tumor progression, similar to polarized T cells [91, 92]. Recent studies also highlighted new functional subtypes of iNKT cells. However, in recent years, due to their close relationship with innate immunity, iNKT cells are potentially defined as a special subgroup of ILCs.

NK cells in the tumor microenvironment

Conventional roles of NK cells in immunity

Detection of aberrant cells by NK cells is determined by the intergradation of complex signals such as IL-12, IL-15 and IL-18 [93, 94], and the balance between activating and inhibitory signals interacting with MHC-I on the surface of target cells (Fig. 2). During infection and inflammation, NK cells are recruited and activated in a short period of time, proliferate quickly and contribute largely to both innate and adaptive immune responses [8, 95]. Except for their newly proven regulatory effects, NK cells were first found to directly target infected cells or foreign pathogens therefore, deficiency in NK cells in both mice and humans results in susceptibility to many viral infections and adverse clinical outcomes, validated by clinicians and researchers.

The complex interaction between NK cells and the extracellular matrix. Exposure of NK cells to the adjacent cells, molecules and metabolites in the extracellular matrix affects their development, maturation, activation and functions. CXCR3, C-X-C motif chemokine receptor 3 NKG2D, nature-killer group 2, member D IFN-γ, interferon γ TNF-α, tumor necrosis factor α IDO, indoleamine 2,3-dioxygenase MICA, MHC class I polypeptide-related sequence A PGE2, prostaglandin E2 HCC, hepatocellular carcinoma CIS, cytokine-inducible SH2-containg protein TGF-β, transforming growth factor-β HMGB1, high-mobility group box 1 HIF-1α, hypoxia inducible factor-1α 27HC, 27-hydroxycholesterol iNKT, invariant natural killer T GM2, β-N-acetylhexosaminidase TCR, T cell receptor

Similar to other innate immune cells that are unable to accurately recognize target cells, NK cells rely on other stromal cells, including DC, which trans-presents IL-15 for NK cell activation [96], and MICA-expressing monocytes, which bind to Fc receptor to enhance antitumor function [97], to fully differentiate and induce effector responses, but surprisingly possess the ability to form immunological memory, termed “trained immunity”. Once considered as a hallmark of adaptive immunity, in recent years, the phenomenon of immunological memory has also been found in innate immune cells, especially in the myeloid lineage, e.g., monocytes and macrophages. In addition, mounting evidence indicates that in humans, NK cells can remember previous exposure to inflammatory microenvironment, and occurrence of similar cytokines could induce trans-differentiation from normal NK cells to memory NK (NKm) cells [98,99,100,101]. This was evidently observed in response to viral infection in humans, prompting the development of NK cell-based vaccines to generate potent effects toward diseases [102, 103]. A large number of NKm cells are observed in the tumor microenvironment, producing high levels of IFN-γ, perforin and granzyme family molecules after re-stimulation [104]. However, concerning to tumors, dysfunction of NK and NKm cells is emerging as an indispensable and undeniable event, leading to not only proliferation of tumor cells but also the formation of distant metastases [101]. It was observed that repeated exposure of NK cells to NK receptor ligand-expressing tumor cells (e.g. NKG2D) finally results in NK cell dysfunction, and effector responses cannot be stimulated in vivo [105, 106]. These results indicate that the formation of NKm cells may not just depend on target cell recognition through surface receptors, and certain cytokines (including IL-12, IL-15 and IL-18) could be key to this process.

Though the half-life of normal NK cells is only for 1–2 weeks, NKm cells can live for 3–4 weeks [107]. This long-term effect truly helps researchers better modulate the function of NK cells in protecting against tumors, and emerging results suggest that NK cells not only rely on MHC-I recognition but also depend on many other signals, shedding light on the use of NK cells and related signaling pathways as future treatment options.

Infiltration of NK cells with cancer genotypes and phenotypes

In 2000, an 11-year follow-up study of the Japanese general population, with rigorous use of various related biochemical and immunological markers, indicated that elevated cytotoxic activity of peripheral NK cells is positively associated with reduced cancer risk and vice versa, suggesting the certain importance of natural immune response toward tumors [108]. However, the specific role of NK cells remains controversial and largely depends on distinct cancer types [109]. Even in the same type of cancer, NK cells are highly heterogeneous, characterized by the abundance of surface receptors and the complexity of tumor intrinsic signaling pathways [95, 110]. In the CIBERSORT analysis, NK cells were thoroughly divided into resting and activated subtypes, each contributing to the formation of the tumor microenvironment [111]. In preclinical studies, NK cells were shown to indicate survival and thus therapeutic response in different types of cancer, as detected by immunohistochemistry, immunofluorescence or flow cytometry using different surface and functional markers (Table 2) [46, 56, 112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129]. Although CD3 − CD16 + CD56 + cells reflect different clinical outcomes in different cancers, functional molecule-positive NK cells, including NKp30 + and NKp46 + , indicate favorable survival, pointing out the fact that full activation but not only infiltration density finally determines NK cell-associated immune response.

In BC, besides the ability of total NK cells to reflect favorable survival, peritumoral abundance of NK cells also correlates with elevated pCR rate of neoadjuvant chemotherapy in large and locally advanced breast cancer [130], and vice versa. To address this, it is currently well accepted that tumors are highly heterogeneous, and even one histologic type can be separated into several molecular subtypes. BC can be thoroughly clustered into luminal, HER2-enriched and triple-negative types according to the expression of surface molecules, and both infiltration and activation status of NK cells vary by cluster, e.g., obviously elevated NKG2D in luminal tumors [131]. However, due to the complex and variable functional status of NK cells, their actual role in the TME still awaits further elucidation.

Underlying mechanisms of immune escape and metastasis of cancer

As noted above, NK cells rely on the balance between activating and inhibitory receptors to exert their killing effects, and perforin and the granzyme family of proteins are the main effector molecules. Consistent with cNK cells, as a newly-found subgroup of NK cells, activated NKT cells directly detect and kill CD1d + tumor cells in several types of cancer [132, 133]. In addition, by expressing high levels of CD40, NKT cells induce DC maturation, thus activating CTL and cNK cells to enhance their anti-tumor effects [134, 135].

Apart from primary tumor proliferation, metastasis remains lethal, accounting for the majority of cancer-associated deaths, and invasion-metastasis cascade is largely attributable to the immune escape [136]. With such rapid and effective ability to target tumor cells directly and indirectly, NK cells are suppressed by tumor-derived molecules, tumor-educated stromal cells (Fig. 2) and tumor cells (Fig. 3), eventually contributing to the progression and multi-step metastatic process of cancer. For example, single-cell analyses found that in lung adenocarcinoma, CD16 + NK cells are hardly infiltrated and present lower granzyme B and CD57 expression compared with normal lung tissue, appearing to form NK cell-excluded TME [137].

Interplay between cancer cells and NK cells during tumorigenesis. The interaction between tumor cells and NK cells changes continuously with NK cell development, tumor progression and metastasis. During the stage of tumorigenesis (a), NK cells recognize tumor cells through various surface molecules and switch to the active status. In the immune control stage (b), NK cells exert killing effects by ADCC, secreting cytokines and generating memory NK cells. Meanwhile, changes in the surface molecules of tumor cells also promote anti-tumor metabolic responses. However, long-term exposure of NK cells to tumor cells, tumor- derived molecules and tumor-educated stromal cells, including fibroblast, monocyte and macrophage, causes NK cells to be in an immunosuppressive state, thereby promoting tumor immune escape and metastasis (c). MHC- I, major histocompatibility complex-I MICA, MHC class I polypeptide-related sequence A MICB, MHC class I polypeptide-related sequence B NCR, natural cytotoxicity receptor Nfil3, nuclear factor interleukin-3-regulated protein Id2, inhibitor of DNA binding 2 Tox, thymocyte selection associated high mobility group box EOMES, eomesodermin T-bet, T-box transcription factor 21 ADCC, antibody-dependent cell-mediated cytotoxicity GM-CSF, granulocyte-macrophage colony stimulating factor PRF1, perforin 1 GZMB, granzyme B PD-L1, programmed cell death ligand 1 PGE-2, prostaglandin E2 HCC, Hepatocellular Carcinoma IFN, interfron TNFα, Tumor Necrosis Factor αPI3K, Class IA phosphatidylinositol 3 kinase

Class IA phosphatidylinositol 3 kinases (PI3Ks) are involved in growth and survival of normal cells, and mutation of the PI3KCA isoform is commonly found in the genomic landscape of many cancers. Inhibiting abnormally activating signals of PI3KCB with a tested inhibitor called P110β in hematologic malignancies obviously enhances susceptibility of tumors to NK cell activity in vitro, probably through the regulation of MHC-I [138]. Tumor-derived prostaglandin E2 [139], extracellular adenosine [140], fragmented mitochondria in the cytoplasm of tumor-infiltrating NK cells [141] and actin cytoskeleton remodeling [142] also lead to immunosuppression and help potentially metastatic cancer cells to avoid NK cell elimination. Interestingly, in a breast cancer cell line, CDC42 or WASP knockdown does not change the activation status of NK cells but obviously increases the expression of effective granzyme B and overcomes resistance to NK cell-mediated attack [142]. Further investigation in this field might help identify a new signaling pathway or a new marker of NK cell-activation and provide new insights into NK cell-therapy. Similar to the inhibitory role of CD73 in T cell-immunity, tumors can train normal NK cells into CD73 + NK cells which express high levels of checkpoint molecules, including LAG-3, PD-1, and PD-L1, finally resulting in immune escape [57]. As mentioned above, Th2-polarized iNKT cells in the TME contribute to tumor progression through immunosuppressive effects [91], and continuous exposure to ligands expressed on the surface of tumor cells induces the dysfunction of NKm cells that are engaged in long-term anti-tumor immunity [106]. Moreover, absence of NKG2D is a common feature of functionally suppressed NK cells, which is achieved through many different pathways [105, 143], thus could be used as a marker to guide NK cell-related therapies [144].

In addition, NK cell-associated cytotoxicity can also be impaired by stromal cells, for example cancer-associated fibroblasts, monocytes, macrophages and other immune cells. Fibroblasts in TME suppress function of NK cells through downregulating ligands of NK cell activating receptors [145], enhancing tumor-associated macrophages enrichment [146], and producing extracelluar matrix components such as IDO and PGE2 [147]. In human gastric cancer, tumor infiltrating monoctyes/macrophages can reduce IFNγ, TNFα, and Ki-67 expression of NK cells via TGFβ1, thus impairing NK cell function [148]. Meanwhile, the interaction of PD-1 + NK cells and PD-L1 + monoctyes/macrophages in Hodgkin lymphoma results in immune evasion, which can be reversed by PD-1 blockade [149]. The monocytes from hepatocellular carcinoma express CD48, which could block 2B4 on NK cells and induce NK cell dysfunction [150].

Consistent with CD8 + T cells, activated PD-1 + NK cells are inhibited by elevated PD-L1 expression in the TME [58]. Dysfunction of NK cell following surgery has been regarded as a risk factor of metastasis and can be partly explained by disturbing the balance between activating and inhibitory signals. NK cell-mediated metastasis control was found dependent on dectin-1-mediated activation of macrophages, especially the plasma membrane tetraspan molecule MS4A4A [151]. In colorectal cancer, lipid accumulation is common for postoperative patients and facilitates the formation of metastasis by impairing NK cell function, by elevating CD36 expression [152]. Hence, NK cells can be rarely seen in metastatic melanoma and are mainly TIGIT − CD226 − which are deprived of cytotoxicity toward MHC-I-defient malignant cells [153]. Interestingly, proved by convincing genetically engineered models, it is the absence of NK cells but not CD8 + T cells that evidently leads to the metastatic dissemination of small cell lung cancer, pushing us to better define the pivotal role of NK cells in both initial progression and later metastasis of cancer [154].

Relationship between NK cell-based and CD8 + T cell-based immunity

NK cells, though belonging to innate immunity, have characteristics similar to cytotoxic CD8 + T cells [95]. Originally proven important in the two-signal activation model of T cells, CD28 is also necessary for optimal cytokine secretion and proliferation of NK cells both in vitro and in vivo [155]. It was shown that IL-21 is important for the maturation and activation of NK cells [156,157,158]. However, data also uncovered the double-sided function of IL-21 in the development of NK cells and surprisingly, its positive role in T-cell-based immune response [159, 160], for example inducing KLRG1 + CD8 + T cells during acute intracellular infection [161]. Besides IL-21, NK cells produce multiple other cytokines during their proliferation, maturation and function similar to T cells, including IL-2 [162], IL-7 [163] and IL-15 [164], validating the close relationship between these two types of effector cells in the body’s immune system.

Although sharing many similarities, compared with effector T cells, NK cells are more cytotoxic to tumors and possess lower immunogenicity [10]. As mentioned above, NK cells respond to target cells more quickly and do not need extra ligation of activating receptors [95]. On the contrary, NK cells have the ability of suppressing the function of CD8 + T cells via NKG2D in severe aplastic anemia [165]. It has been underlined that during infection with chronic lymphocytic choriomeningitis virus, NK cell-intrinsic FcRγ signaling could inhibit the expansion of CD8 + T cells [166]. Interestingly, tumor cells that develop checkpoint blockade resistance to CTLs, especially through suppressed MHC-I expression, are more vulnerable to NK cell-based immunity thus, combination immunotherapy utilizing both NK cells and CD8 + T cells can constitute a future strategy in terms of tumor immune escape [167]. Besides, in tumors that lack MHC-I-related molecules, elevated amounts of HLA-E and HLA-G were observed, indicating the possibility for NK cells to harness this unclassical pathway [168]. More basic researches are urgently needed for better understanding of the complex relationship between these two major effector cells, as NK cells-based treatment is currently highly underestimated.

Crosstalk of NK cells and metabolic signaling in cancer

Immunometabolic disorder as a hallmark of cancer

Similar to high blood pressure and diabetes, cancer is currently regarded as not only a process of pathogenesis but also a social issue. Lipid accumulation in the liver, abnormal glucose metabolism and irregular lifestyle all contribute to tumorigenesis and cancer progression, which eventually prompt research about the underlying mechanisms of this phenomenon. It is admitted that the metabolic competition between tumor and stromal cells largely affects the process of tumorigenesis and cancer progression. In the TME, NK cell function is impaired not only by suppressive cytokines but is also attributable to inappropriate metabolic conditions, including hypoxia, lack of nutrition and abnormal concentrations of tumor-derived products such as lactate, which induces unfavorable acidic condition, hindering the proliferation and cytokine production of CTLs as well (Fig. 2) [169]. As metabolic disorder is currently considered a hallmark of cancer, which shares a close relationship with the microenvironment, the idea of harnessing immunometabolism attracts increasing attention to improve the efficacy of NK cell-dependent anti-tumor therapy.

The normal breast tissue is surrounded by adipose tissue, and obesity is considered a potential risk factor for breast cancer, which is supported by population-based studies, together with high mental pressure, evidently affecting lipid and glucose metabolic pathways. Obesity-induced inflammation in adipose tissue could result in the recruitment of M1-polarized macrophages, neutrophils, NK cells and CD8 + T cells, and higher expression levels of pro-inflammatory cytokines, as well as obvious exclusion of Treg and invariant NKT (iNKT) cells [170]. In 2011, aware of the importance of tumor-promoting inflammation in the TME, Weinberg et al. included this phenomenon into the hallmarks of cancer and particularly highlighted inflammation induced by the innate immune response [1]. Thus, improved understanding of the mechanism by which metabolic activity affects the function of tumor-infiltrating stromal cells, finally resulting in cancer progression and immune escape, would provide clues for developing novel therapeutics for immunometabolic targets.

Metabolic disorder of conventional NK cells in the TME

NK cell function can be altered by different components in the TME. Breast cancer metabolomics data overtly show that lipid and glucose metabolic pathways are highly activated, especially the fatty acid synthase glycolysis pathway, compared with paired peritumoral tissue.

Similar to other lymphocytes, NK cells require energy to survive, and glucose consumption is evidently increased after full activation, while the competition between NK cells and tumor cells could disturb such need. It has been shown that surface transporters, especially glucotransporter 1 (GLUT1), help NK cells utilize glucose to generate ATP and pyruvate, contributing to glycolysis and oxidative phosphorylation [171, 172]. Several studies have pointed out the importance of sufficient glucose supply for NK cell activities, including proliferative capacity, activation status, cytokine production and direct cytotoxicity [173, 174]. Glycolysis and oxidative phosphorylation contribute to maintain the cytotoxic ability of NK cells, as their inhibition highly decreases the expression levels of IFN-γ and Fas ligand [175]. NKG2D is essential for the activation of NK cells, which relies on glycolysis. Researchers have identified several pathways pertaining to the interlinked metabolic activity and NK cell function. Obesity-related inflammation is dependent on the IL-6/Stat-dependent pathway, thereby resulting in a distinct functional status of NK cells [176]. In addition, Assmann et al. highlighted that sterol regulatory element-binding proteins (SREBP) transcription factor-controlled glucose metabolism is essential for metabolic reprogramming in activated NK cells, providing new insights into this process [177]. Accordingly, SREBP inhibitors such as 27-hydroxycholesterol (27HC) are accumulated in the TME, partly affecting SREBP-related glycolysis in ER-positive BC [178]. However, as most studies only focus on GLUT1, other transporters and unclassical pathways should also be paid attention to, paving the way for deeper understanding of the complex relationship between glucose metabolism and NK cell function.

Hypercholesterolemia remains a risk factor for ER-positive BC. In 2013, 27HC, which negatively regulates SREBP, was also found by Nelson et al. to be a bridge linking hypercholesterolemia and BC [179]. It was also found that treating mice submitted to high-cholesterol feed with an inhibitor of CYP27A1, an enzyme important in 27HC biosynthesis, obviously decreases the number of metastases in mice, which reverses immune suppressive environment [180]. Therefore, using drugs designed to decrease blood cholesterol or directly inhibiting the formation of 27HC could be a potential strategy for patients with ER-positive BC. Surprisingly, a recent study suggested that high serum cholesterol and cholesterol accumulation in NK cells increase their anti-tumor ability by facilitating the formation of lipid rafts in the liver-tumor-bearing murine model [181], highlighting the heterogeneous functions of lipid metabolism in cancer.

Hypoxia is also a common feature of cancer, and often mentioned concurrently with low pH in the TME. Previous findings indicated that besides promoting tumorigenesis and cancer progression, hypoxia also stimulates NK cell formation via HIF-1α, initiating a conflict between suppressing and activating this signal [182]. Studies have also shown that low O2 in TME harms the function of NK cells by downregulating activating signals such as NKG2D, NKp30 and CD16, thereby limiting cytokine production and cytotoxicity and resulting in metastasis [183, 184]. In addition to regulating intracellular signals directly, the hypoxic microenvironment could degrade NK cell-secreted functional molecules such as granzyme B [185], together with CTL-based immunity, which is partly rescued by IL-2 [186, 187]. Considering the pivotal roles of interleukin family members in the maintenance of NK cells, we paid more attention to these cytokine-primed metabolic pathways and found more clues under hypoxic conditions.

Besides, tumor cells can directly alter the metabolic status of NK cells. This can be positive as CD25 expression on NK cells is overexpressed after interaction with tumor cells, inducing long-term anti-tumor metabolic responses by promoting glycolysis and NK cell survival, supported by mTORC1/cMYC signaling activation. However, worsening occurs later as the harmful effects overcome the positive impact. For instance, glutamine addiction and high consumption of nutrients remain common in BC. In vitro studies showed arginine deficiency inhibits IFN-γ production by primary human NK cells [188]. Additionally, mTOR signaling within NK cells can be largely suppressed under low-arginine or glutamine conditions, which also affect IL-2-related stimulation process via cMYC [189]. Upon direct contact with tumor cells to form an immune synapse in response to local energy consumption, mitochondria of NK cells are depolarized and lose metabolic energy [190]. Inhibiting PPARα/δ or blocking the transport of lipids into mitochondria reverses NK cell metabolic incapability and restores cytotoxicity [191].

Indeed, previous studies have reported that anti-PD-L1 therapy could reshape metabolic pathways in the tumor microenvironment and re-stimulate exhausted CD8 + T cells for cytotoxicity [192]. Interestingly, NK cells also express the ligands of these checkpoints. Glycoengineering of NK cells enhances their killing ability toward CD22 + lymphoma in a CD22-dependent manner [193]. Blockade of monocarboxylate transporter 1, which regulates cell metabolism, using AZD3965 also potentiates NK cell activity [194]. Studies that focus on translating mature theories into the practical use of NK cells are promising.

Aberrant metabolic features of iNKT cells

As mentioned above, iNKT cells can be polarized into different properties, each possessing distinct functions. The normal breast tissue is surrounded by adipose tissue. Different from conventional T cells, iNKT cells comprise large amounts of stromal cells in adipose tissue, whose infiltration decreases apparently in high-BMI individuals [195, 196].

With invariant TCR on the surface, iNKT cells are termed innate-like T lymphocytes and act on the front line of the immunity battle against cancer [197, 198]. iNKT cells recognize glycolipid signals but not peptides via semi-invariant TCR, and are restricted to glycolipid antigens presented via CD1d-related molecules, which are MHC-like and highly enriched, especially in adipocytes and hepatocytes, linking innate and adaptive immune responses. This process can be altered by metabolic activity. GM2 is a glycosphingolipid that binds the CD1d molecule. Pereira et al. pointed out that GM2 inhibits the activation of iNKT cells in a dose-dependent manner, which might result from its competition with α-GalCer for binding CD1d [199]. Compared with T lymphocytes, iNKT cells show much higher capacity of glycolysis but reduced mitochondrial respiratory activity, resulting in particular molecular features. Under hypoxia, widespread RNA editing can be induced by mitochondrial respiratory inhibition via APOBEC3G, an endogenous RNA editing enzyme [200]. Fu et al. demonstrated that aerobic glycolysis in iNKT cells is highly increased after TCR engagement, which is essential for the production of IFN-γ [90]. This process can also be inhibited by the lack of glucose. Hence, reduced expression of IFN-γ in iNKT cells compared with the normal tissue was confirmed in several tumor types, predicting patient response to the therapy of PD-1 blockade [201,202,203]. In humanized mouse models undergoing PD-1 blockade and CAR-T (with different costimulatory molecules) combination therapy, only those with Δ-CD28 CAR control tumor growth, and in vitro analysis showed that these cells exhibit elevated glycolysis, fatty acid oxidation and oxidative phosphorylation [204]. Overcoming the impaired metabolic function combined with immune checkpoint blockade would be a potential strategy in future researches and clinical practice.

However, what is currently known about iNKT cells is just the tip of the iceberg. Compared with T lymphocytes, it remains unclear how intracellular metabolic signals influence the survival and function of iNKT cells, which deserves further investigation, as this may be the next potential target of cancer immunometabolic therapy after CD8 + T cells and NK cells.

NK cells in cancer therapy

As an important effector of innate immunity, though suffering a resistance evolved by tumor cells, NK cells show their potential to be used in clinical practice [205,206,207]. In the past few years, researches about NK cell-related immunotherapy have flourished and the latest development mainly focused on cytokine supplement, monoclonal antibody, modification of internal signal pathway, adoptive transfer and genetic engineering of NK cells. Besides, NK cell-based therapy has achieved favorable results used either alone or in combination with other therapies, which suggests a wide and effective use in malignancies.

Cytokine supplement

IL-15 promotes the development and cytotoxic ability of NK cells, and several clinical trials have illustrated the safety profile of recombinant human IL-15 (rhIL-15) in multiple tumors [208, 209] as well as its agonist, ALT-803, in metastatic lung cancer and post-transplantation patients (Fig. 4) [210, 211]. In an open-label, phase Ib trial, ALT-803 showed a fantastic potential when combined with anti-PD-1 monoclonal antibody (nivolumab) without increasing the incidence of very severe grade 4 or 5 adverse events [211], which evidently could be the future way to enhance rhIL-15 treatment. In addition to soluble IL-15 in the microenvironment, it has been revealed that in the mouse, direct contact with membrane-bound IL-15 on adjacent stromal cells could induce stronger cytotoxic effects in NK cells [212]. Heterodimeric IL-15 can also increase intratumoral NK cell and CD8 + T cell infiltration, elevating the effective rate of current immunotherapy [213].

Possible targets harnessing NK cells in cancer therapy. In order to obtain better clinical efficacy and reduced severe adverse events, the development of NK cell-based therapies that support NK cell maintenance (a), enhance NK cell function (b) and harness abnormal immunometabolic and intracellular microenvironment (c) is essential. rhIL-12/15/18, recombinant human interleukin-12/15/18 CAR-iPS, chimeric antigen receptor-induced pluripotent stem cell MIC: MHC I chain related molecule MICA, MHC class I polypeptide-related sequence A MICB, MHC class I polypeptide-related sequence B PD-L1, programmed cell death-ligand 1scFv, single-chain variable fragment TSA, tumor specific antigen BiKE, bispecific killer cell engager TriKE, trispecific killer engager CAR-NK, chimeric antigen receptor-nature kill PD-1, programmed cell death protein 1 MerTK, MER proto-oncogene, tyrosine kinase 27HC, 27-hydroxycholesterol GLUT1, glucotransporter 1 MCT1, monocarboxylate transporter 1

Apart from IL-15, other interleukins are also synergetic to this process (Table 3). IL-21 enhances tumor rejection in mice via NKG2D-dependent NK cell activity, suggesting IL-21 to be a possible target for immune escape induced by NKG2D elicitation [214]. However, IL-15-dependent expansion of resting NK cells can be suppressed by IL-21, while on the other hand adaptive immune response is enhanced [159], providing substantial insights into this complex network in clinical use. Besides, blocking CIS could promote IL-15-type cytotoxicity and thus results in increased production of IFN-γ [22]. NK cells pre-exposed to IL-12, IL-15 and IL-18 accumulate in the tumor tissue and retain their anti-tumor function both in vitro and in vivo. However, IL-15 alone does not exert such effects [104]. In addition to the inner pathway, treatment with IL-2 and IL-15 obviously enhances glycolysis and oxidative phosphorylation of NK cells, thus promoting the killing ability. In a first-in-human phase I multicenter study, NKTR-214, a novel IL-2 pathway agonist, was found promoting proliferation and activation of NK cells without expansion of Treg cells [215]. For metastatic melanoma refractory to CD8 + T cell cytotoxicity due to the lack of MHC-I, combination of IL-15 and TIGIT blockade shall be effective by stimulating NK cell-mediated immunity [153].

As mentioned above, cytokines (especially IL-12, IL-15 and IL-18) are critical to the formation of NKm cells. Memory-like NK cells supplemented with IL-12, IL-15 and IL-18 also show enhanced responses against acute myeloid leukemia both in vitro and in vivo [107], and are currently assessed in a first-in-human clinical trial. However, studies also pointed out that IL-12 could increase NKG2A expression and inhibit the activation of NK cells [216].

Monoclonal antibodies

As shown previously, in parallel with CD8 + T cells, NK cells can also be suppressed by immune checkpoint molecules. After cetuximab treatment, PD-1 + NK cells are more enriched in the TME and are correlated with favorable clinical outcome in head and neck cancer patients, which was further demonstrated by in vivo experiments and a stage III/IVA clinical trial assessing neoadjuvant cetuximab (NCT01218048) [58]. With PD-1 blockade (nivolumab), cetuximab-induced NK cell activation and function are remarkably enhanced in PD-L1 high tumors.

In addition to already-well-defined PD-1 and PD-L1, NK cells with reduced amounts of T-cell immunoglobulins and ITIM domain (TIGIT) show higher levels of cytokine secretion, degranulation activity and cytotoxicity [217], and blockade of TIGIT could prevent exhaustion in NK cells [20]. A recent study also highlighted that TIGIT-depleted NK cells are highly sensitized [218] and develop resistance to MDSC-mediated immunosuppression [219]. Meanwhile, CD96, which shares the same ligand CD155 with CD226 and TIGIT, negatively controls the immune response by NK cells [220] and predicts adverse survival in human hepatocellular carcinoma [56]. Single use of CD96 antibody promotes NK-cell-induced anti-metastatic ability [221], and such effect is largely increased when combined with anti-CTLA-4, anti-PD-1 or doxorubicin chemotherapy [222].

Though prospective, unexpected biological events have been observed in a single-arm phase II study showing that intravenous infusion of 1 mg/kg IPH2101 (a human monoclonal antibody against KIRs) results in severe contraction and obvious inhibition of NK cells in myeloma patients [223]. Lirilumab, a 2nd generation antibody targeting KIR, had encouraging results in a phase I trial, which demonstrated its safety [224] however, the subsequent phase II trial in AML patients showed no clinical effects. Combination of CIS inhibition with CTLA-4 and PD-1 blockade exerts even greater effects in reducing melanoma metastasis compared with either of these treatments administered alone thus, CIS inhibition could offer an alternative therapeutic option for patients not responding to other immune checkpoint inhibitors [22].

As an essential receptor for the activation of NK cells, NKG2D is blocked by many ligands (e.g., MICA, MICB, and ULBP1–6) upregulated in tumor cells as a result of abnormal cellular stress in the TME. A recent study demonstrated that antibodies targeting MICA and MICB can prevent NK cell recognition and tumor cell binding, inhibiting tumor growth in fully immunocompetent mouse models as well as humanized mouse models [129]. Furthermore, combination treatment targeting soluble MIC, e.g., MICA and MICB, and PD-L1 shows better effect than monotherapy in vivo [225]. Moreover, soluble MULT1, a high affinity mouse NKG2D ligand stimulates NKG2D in distant NK cells and enhances NK cell tumor immunity [106]. Hence, a clinically used antibody, monalizumab, has been developed targeting NKG2A, an inhibitory checkpoint of NK cells, which not only promotes NK cell function in various preclinical models, as previously characrerized, but also potentiates anti-PD-1 [226] and anti-EGFR (cetuximab) therapy [227]. In addition to antibody, NKG2A null NK cells, constructed through retroviral transduction of NKG2A blocker which inhibits de novo NKG2A expression, present increased anti-tumor activity in pre-clinical model [228].

In summary, besides the targets close to T cells, including FDA-approved anti-CTLA-4 (ipilimumab) and anti-PD-1 (nivolumab, pembrolizumab) antibodies, others designed specifically for NK cells are also under clinical trials, e.g., anti-KIR (IPH2101, lirilumab) and anti-NKG2A (monalizumab) (Table 3) (Fig. 4).

Cell adoptive therapy and newly arising genetic modification of NK cells

As an applicable option for enhancing autologous immunity, adoptive transfer of NK cells has been implemented to treat certain types of cancer in the past few years [229]. Previous studies of NK adoptive transfer in acute myeloid leukemia patients have presented slightly beneficial effects in controlling disease [230, 231], and a phase II clinical trial in patients with recurrent ovarian or breast cancer showed that adoptive transfer of haploidentical NK cells after lymphodepleting chemotherapy leads to a temporary benefit but its clinical value remains controversial, and is partly limited by recipient reconstitution of regulatory T cells [232]. Apart from adults, a phase I clinical trial applied autologous ex-vivo-expanded NK cells to children with recurrent medulloblastoma and ependymoma and obtained good safety and therapitic efficacy [233]. Interestingly, transfer of NK cells along with CD34 + hematopoietic stem cells shows no added adverse effects but potential therapy response in older patients with acute myeloid leukemia [234]. Combinational application of NK cell infusion with monoclonal antibody provides a new direction of combinational immunotherapy. In a phase I trial, activated autologous NK cell were infused into patients with HER2-positive solid tumor undergoing trastuzmab and showed prelimitary anti-tumor phenotype [235].

In 2009, Fujisaki et al. found that overexpression of telomerase reverse transcriptase could lead to over 100 additional doubling cycles in NK cells, revealing a potential way to overcome the limitation of NK cell amplification in vitro [236]. However, though the adoption of NK cells seems promising in preclinical and clinical researches, many questions exist, e.g., the undeniable fact that NK cells gain self-renewal ability following infusion. Originally designed and considered a one-time-use therapeutic process, a small part of NK cells surprisingly remain alive and proliferate in the human body for months, mediating continuous surveillance against tumor [237]. However, in addition to beating tumor cells, the risk of long-lived NK cells should not be ignored where their “brake” is lost and they might also kill normal cells, even worse, finally increasing the possibilities of NK lymphoma [95, 238]. Hence, the ability of transferred NK cells may be limited by inappropriate persistence or expansion in vitro, and biology-driven methods are commonly used to overcome this issue before adoptive transfer. A recent study indicated that NK cells pre-activated by IL-12, IL-15 and IL-18 suppress graft-versus-host disease but obviously inhibit the generation and function of CD8 + T cells, which could result from mutual competition of IL-2 [239], limiting its value in the field of cancer treatment. The use of mouse models to unveil the detailed features of adoptive NK cells following in vitro proliferation would actually provide deeper insights into this treatment strategy, shedding light on the future implementation of NK cell-based therapy toward cancer.

Genetic modification of immune cells by chimeric antigen receptors (CARs) to target tumor cells directly is a promising therapeutic option in cancer therapy. Kymriah (CTL019, a CAR-T product) by Novartis was approved by the FDA for treating recurrent and refractory acute lymphoblastic leukemia in 2017, and remains under investigation for other indications in several clinical studies [240,241,242]. Two months later, Yescarta by Kite Pharma was approved for diffuse large B cell lymphoma [243]. Due to serious adverse effects induced by CARs, especially cytokine releasing storm (CRS) and neurotoxicity, Actemra (tocilizumab, anti-IL-6 monoclonal antibody) was then approved for CRS, with a further study also showing potential application of Anakinra (an antagonist IL-1 receptor) in such case [244, 245]. In NK cells, antibody engineering approaches optimize NK cell-mediated ADCC to tumor cells through the bispecific killer cell engager (BiKEs) or trispecific killer engager (TriKEs) antibodies (Fig. 4). BiKE connects a single-chain variable fragment (scFv) to the anti-CD16 recognition site with the scFv of a tumor specific antigen, such as CD19/CD20 for non-Hodgkin lymphomas, CD33/CD123 for acute myelogenous leukemia/AML and CD30 for Hodgkin lymphoma, to enhance NK cell recognition of tumor cells [246]. TriKE consists of a BiKE and cytokine IL-15, which boost NK cell function and survival. It was shown that CD19-CD16 BiKE engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies [247]. Meanwhile, CD16-IL15-CD3 TriKE can activate suppressed NK cells and induce NK cell-mediated control of MDS and AML [248]. The advantages of CAR-NK therapy are obvious, including higher possibility of recognizing tumors (including cytokines and apoptosis) and lower incidence of CRS compared with CAR-T (Table 3) [249, 250]. In 2018, Enli Liu et al. transduced cord blood-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, inducible caspase-9-based suicide gene (iC9) and IL-15, and demonstrated the efficacy and safety in cell lines and the murine model. In the engineered NK cells, CAR-CD19 redirected the specificity of NK cells against leukemia, IL-15 promoted NK cell proliferation, and iC9 allowed NK cells to initiate suicide after killing the target cells [251]. Since CAR-NK cells exhibit striking efficacy and limited toxicity, clinical trials assessing these CAR-NK cells have been launched. In recent phase I and II trials, iC9/CAR-CD19/IL-15 NK cells were prepared ex vivo and infused into patients with relapsed or refractory CD19-positive cancers after lymphodepleting chemotherapy. Among the 11 treated patients, 8 had an objective response, including 7 with complete remission, without major toxic effects [250]. Apart from engaging NK cell with CAR, geneic modification involves deleting surface molecules, e.g., CD38, on NK cells, which evidently elimates fratricide and enhances cytotoxic ability [252].

However, limitations should be mentioned. Due to the complex process of producing CAR-NK cells, the current procedure is too expensive and the effects on solid tumors are far from being satisfactory. CAR-iPS might be a future direction, which can grow in vitro and differentiate into CAR-NK cells in vivo to directly enhance anti-tumor immunotherapy [253]. Recently, Zhu H et al. reprogramed NK cell metabolism by depleting CISH in human iPSC-derived NK cells and obtained satisfactory persistence and anti-tumor activity in vivo, which could be a novel method to generate CAR-NK from CAR-iPS [254].

Refinement of the established therapies

As an important immunosuppressive cytokine that promotes tumor progression, TGF-β and its pathway represent potential opportunities for anti-tumor drug development. The clinical modulation of TGF-β, which is achieved through small-molecule inhibitors and antibodies, is being investigated in a number of clinical trials [255]. As the safety and efficacy of TGF-β blockade therapy have been demonstrated, two studies independently showed that combination treatments of TGF-β blockade with anti-PD-1/PD-L1 therapies have synergistic effects on murine EMT6 breast mammary carcinoma and colorectal cancer [256]. It is admitted that TGF-β inhibits NK cell metabolism, proliferation, cytokine production, cytotoxicity and anti-metastatic functions through mTOR signaling in multiple cancers [257]. Activin-A, a member of the TGF-β superfamily, increases ILC1-like tissue residency features, reduces cytokine production, and suppresses proliferative and metabolic functions in both human and murine NK cells through an alternative SMAD2/3-related pathway with effects similar to those of conventional TGF-β pathway [258]. Therefore, targeting TGF-β, TGF-β superfamily, and their downstream pathways in NK cells may be promising treatment options that enhance the efficacy of current immunotherapies (Fig. 4). Apart from blockage of immunosuppressive cytokine, immunostimulatory agonist have been tested in clinical trials. In a recent phase I/IIa study, BMS-986156, a human glucocorticoid-induced TNF receptor-related protein agonist, appears to increase NK cell proliferation no matter applied with or without nivolumab, and has an adorable safety and efficacy profile [259].

Rapamycin, an inhibitor of the mTOR pathway, and its derivative everolimus are effective for treating breast cancer in clinical trials, including BOLERO-6 and PrE0102 studies, either alone or in combination with endocrine and chemotherapy [260, 261]. However, NK cells highly rely on PI3K-mTOR signaling pathway-dependent metabolic reprogramming to exert their anti-tumor effects. Therefore, in patients with both PI3K-mTOR pathway activation and NK cell-based microenvironment, such inhibitors should be cautiously employed.

In HER2-enriched breast cancer, besides HER2-targeted therapies, anti-GD2 also appears to be promising in preclinical studies. However, GD2-related pathways are essential for ADCC [262], also reducing the effectiveness of NK cells, which provides insights into the complex relationships among such networks to minimize off-target effects exerted by established therapies on immunotherapy.

Radiotherapy was found to kill cancer cells via inducing DNA damage, but recent studies found its ability to cause immunogenic cell death, named immunogenic radiotherapy [263]. There is a growing interest to combine immunogenic radiotherapy and immunotherapy, plus chemotherapy or target therapy. A triple-combination therapy which inhibits PD-1 and MER proto-oncogene tyrosine kinase plus radiotherapy, increases NK cells infiltration in abscopal TME [264]. Adding indoximod, an inhibitor of IDO pathway, to radiotherapy and PD-1 blockade also enhances NK cell activity and shows great clinical response [265]. Another representative drug of triple-combination is selenium-containing nanoparticles,which delivers doxorubicin to the tumor site and releases high energy rays. The rays not only kill tumor cells, promote doxorubicin release, but also produce seleninic acid which enhances NK cell function [266].

Natural Killer Cells

Natural Killer (NK) Cells are lymphocytes in the same family as T and B cells, coming from a common progenitor. However, as cells of the innate immune system, NK cells are classified as group I Innate Lymphocytes (ILCs) and respond quickly to a wide variety of pathological challenges. NK cells are best known for killing virally infected cells, and detecting and controlling early signs of cancer. As well as protecting against disease, specialized NK cells are also found in the placenta and may play an important role in pregnancy.

NK cells were first noticed for their ability to kill tumour cells without any priming or prior activation (in contrast to cytotoxic T cells, which need priming by antigen presenting cells). They are named for this ‘natural’ killing. Additionally, NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells like Macrophage and Dendritic cells to enhance the immune response.

While on patrol NK cells constantly contact other cells. Whether or not the NK cell kills these cells depends on a balance of signals from activating receptors and inhibitory receptors on the NK cell surface. Activating receptors recognise molecules that are expressed on the surface of cancer cells and infected
cells, and ‘switch on’ the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHC I receptors which mark these cells as ‘self’. Inhibitory receptors on the surface of the NK cell recognise cognate MHC I, and this ‘switches off’ the NK cell, preventing it from killing. Cancer cells and infected cells often lose their MHC I, leaving them vulnerable to NK cell killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell.

The genes for both MHC I and the NK cell inhibitory receptors which recognise them vary a lot between individuals. The versions (or alleles) of these genes a person carries have been linked to their ability to fight HIV infection and their risk of some autoimmune diseases. NK cell varieties also change with age and are affected by chronic viral infections such as cytomegalovirus (CMV).

Because of their ability to kill tumour cells, NK cells are an attractive target for cancer immunotherapies. Some therapeutic monoclonal antibodies rely on NK cell killing. Researchers are also developing treatments to activate NK cells using small molecules or cytokines, and even testing genetically modified living NK cells as therapies.

Researchers engineer cells to destroy malignant tumor cells but leave the rest alone

HAMILTON, ON June 28, 2021 -- Researchers at McMaster University have developed a promising new cancer immunotherapy that uses cancer-killing cells genetically engineered outside the body to find and destroy malignant tumors.

The modified "natural killer" cells can differentiate between cancer cells and healthy cells that are often intermingled in and around tumors, destroying only the targeted cells.

The natural killer cells' ability to distinguish the target cells, even from healthy cells that bear similar markers, brings new promise to this branch of immunotherapy, say members of the research team behind a paper published in the current issue of the journal iScience, newly posted on the PubMed database.

The experimental treatment is an alternative to chimeric antigen receptor T-cell therapy, or CAR-T, which received FDA approval in 2017. The engineered T-cells used in CAR-T therapy are highly effective against some blood-borne cancers but cannot distinguish between cancerous and non-cancerous cells, so while they offer important benefits, they are not uniformly applicable to all forms of cancer. In patients with solid tumors, the T-cells can cause devastating, even lethal side effects.

The team behind the research wanted a treatment with the same power as CAR-T, but which could be used safely against solid-tumor cancers. They first propagated natural killer cells taken from the blood of patients with breast cancer. Such cells perform a similar function to T-cells in the immune system.

The researchers then genetically modified them to target specific receptors on cancer cells, successfully testing the CAR-NK cells in the laboratory on tumor cells derived from breast cancer patients

"We want to be able to attack these malignancies that have been so resistant to other treatments," says lead author Ana Portillo, a PhD candidate in the Department of Medicine. "The efficacy we see with CAR-NK cells in the laboratory is very promising and seeing that this technology is feasible is very important. Now, we have much better and safer options for solid tumors."

"These CAR-NK cells are a little bit smarter, in a way, in that they only kill the enemy cells and not good cells that happen to have the same marker," says Ashkar, Portillo's supervisor and a Professor of Medicine at McMaster. "These cells have a sober second thought that says, 'I recognize this target, but is this target part of a healthy cell or a cancer cell?' They are able to leave the healthy cells alone and kill the cancer cells."

Portillo and Ashkar's 12 co-authors, most associated with McMaster's Department of Medicine and its Immunology Research Centre, include McMaster's Bindi Dhesy-Thind (Associate Professor, Oncology), who provided blood samples from patients being treated for breast cancer in her clinical practice at Hamilton Health Sciences' Juravinski Cancer Centre.

"These are very exciting results, as to date the benefits of immunotherapy in breast cancer have lagged behind that of other malignancies," she said. "These engineered CAR-NK cells are an important step towards having a viable immunotherapy option in this large group of patients."

Ashkar says there is good reason to believe the technology would have a similar effect on solid tumors associated with lung, ovarian and other cancers.

The next step in moving the therapy toward clinical use is to conduct human trials, which the researchers are now arranging.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

Similarities Between NK Cells and NKT Cells

  • Both NK cells and NKT cells are two types of cytotoxic cells, which trigger innate immune responses.
  • Both NK cells and NKT cells consist of a lymphoid origin.
  • Both NK cells and NKT cells provide the first level of defense against infected cells and tumor cells.
  • Both NK cells and NKT cells produce cytokines.
  • Both NK cells and NKT cells trigger immune responses without a prior sensitization by the immune system.
  • Both NK cells and NKT cells enhance the antigen presentation by cytotoxic T cells.

Equipping Natural Killer Cells with Specific Targeting and Checkpoint Blocking Aptamers for Enhanced Adoptive Immunotherapy in Solid Tumors

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

These authors contributed equally to this work.

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

These authors contributed equally to this work.

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022 China

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022 China

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

These authors contributed equally to this work.

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

These authors contributed equally to this work.

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025 P. R. China

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022 China

MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116 P. R. China

Institute of Cancer and Basic Medicine (ICBM), Chinese Academy of Sciences, The Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, Zhejiang, 310022 China


Herein, we propose an aptamer-equipping strategy to generate specific, universal and permeable (SUPER) NK cells for enhanced immunotherapy in solid tumors. NK cells were chemically equipped with TLS11a aptamer targeting HepG2 cells and PDL1-specific aptamer without genetic alteration. The dual aptamer-equipped NK cells exhibited high specificity to tumor cells, resulting in higher cytokine secretion and apoptosis/necrosis compared to parental or single aptamer-equipped NK cells. Interestingly, dual aptamer-equipped NK cells induced remarkable upregulation of PDL1 expression in HepG2 cells, enhancing checkpoint blockade. Furthermore, in vivo intravital imaging demonstrated high infiltration of aptamer-equipped NK cells into deep tumor region, leading to enhanced therapeutic efficacy in solid tumors. This work offers a straightforward chemical strategy to equip NK cells with aptamers, holding considerable potential for enhanced adoptive immunotherapy in solid tumors.

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