Contributor Lotze, Michael T. Thomson, Angus W. ScienceDirect Online service. Bibliography Includes bibliographical references and index. Contents I. Summary The natural killer [NK] cell plays a critical role in regulating the innate and adaptive immune response to pathogens, injury and stress. It has emerged as a cell capable of helper function, expansion, contraction, and accelerated memory responses - features similar to other adaptive immune cells.
It is a professional accelerator of immunity, mediating dendritic cell maturation and its precursors critical for the origin and development of secondary lymph node structures. These characteristics place the NK cell in a unique position, with a major role in sculpting the host response to damage and injury. Natural Killer Cells details NK cell biology, the role of NK cells in regulating immunity through interactions with other cells and tissues, the participation of NK cells in disease and special topics in NK biology.
Subject Killer cells. Bibliographic information. For example, in one trial, the adoptive transfer of human haploidentical NK cells in AML patients induced complete hematologic remission in five out of 19 poor-prognosis patients [ 39 ]. In contrast to the haploidentical transplant setting, the use of allogeneic NK cells in unrelated HSCT is less clear and a matter of controversy [ 19 ]. In a phase I clinical study which evaluated allogeneic NK cells from random healthy donors in 17 patients with malignant lymphoma or advanced or recurrent solid tumors, no serious adverse event occurred [ 51 ].
This corroborates the data of other studies that immunotherapy with NK cells is usually safe and well tolerated, and only temporary side effects such as fever, weight gain, or neurotoxicity are observed [ 52 ]. Notably, in a phase II study which enrolled 20 patients with recurrent ovarian and breast cancer, one ovarian cancer patient developed tumor lysis syndrome within 6 h of the initial NK cell infusion. The timing of the event, together with the detection of NK cells in the necrotic liver specimen on autopsy, suggests that NK cells could have contributed to the tumor lysis [ 53 ].
There is a growing body of evidence that NK cells play a major role in the host response against various pathogens. For example, a number of studies have demonstrated that genetic mutations may lead to reduced NK cell numbers or functional NK cell impairment such as mutations affecting genes IL2RG , JAK3 , and ADA which cause severe combined immunodeficiency syndromes [ 54 , 55 ] or a mutation in the ITGB2 gene associated with leukocyte adhesion deficiency [ 56 ].
These immunocompromised patients have an increased susceptibility to viral infections, such as infections with herpes simplex virus HSV , Varicella Zoster virus VZV , Cytomegalovirus CMV , and with human papilloma virus [ 22 , 41 , 57 ]. However, as these patients display multiple defects of the immune system, the exact role of NK cells in the increases risk of viral infection remains unclear. An early report described a young girl who experienced a series of recurrent and severe viral infections during childhood and adolescence, including infections by multiple herpes viruses, which was thought to be the result of non-functional NK cells [ 58 ].
The clinical condition of these children significantly improved with acyclovir prophylaxis. Recently, it has been shown that decidua NK cells inhibit human immunodeficiency virus HIV -1 infection in pregnancy [ 61 ]. Similar to the fight against cancer cells, NK cells limit viral burden not only by killing of infected cells [ 38 ], but also by modulating the cytokine milieu, which in turn influences other immune cells such as T cells. Importantly, recent data of animal and human studies indicate that NK can develop long-lasting antigen specific memory cells [ 38 ].
Much work has been performed on the evaluation of the importance of NK cells in the host response against influenza virus. It has become clear that the severity of influenza disease is not uniform, with a severe clinical course being associated with transient T and NK cell deficiency [ 66 ] and with specific haplotypes of killer-immunoglobulin-like receptors KIRs [ 67 ]. Importantly, the latter could be reversed by the adoptive transfer of spleen NK cells harvested from low-dose-infected mice [ 68 ]. During influenza infection, NK cells are activated by different mechanisms, such as by influenza nucleoprotein NP and matrix 1 M1 antibodies [ 69 ], and CD16 seems to play an important role in the early activation of NK cells after vaccination against influenza [ 70 ].
In addition to the killing of virus-infected cells, NK cells provide vital cytokines for tissue regeneration, such as IL [ 71 ]. However, it is important to note that in mouse models, NK cells might mediate pathology as the depletion of NK cells in vivo reduced mortality from influenza infection, whereas the adoptive transfer of NK cells from influenza-infected lung, but not from uninfected lung resulted in increased mortality in influenza-infected mice, probably due to a deleterious NK cell-dependent alteration of T cell responses [ 72 ].
Compared to the antiviral activity of NK cells, considerably less data are available for the interaction of NK cells with bacteria and fungi. NK cells exhibit direct activity against a variety of Gram-positive and Gram-negative bacteria such as Mycobacterium tuberculosis, Bacillus anthracis , Escherichia coli or Salmonella typhi by the secretion of the soluble molecules perforin and granulysin [ 73 — 76 ]. In addition, NK cells have an antibacterial effect against intracellular bacterial pathogens by using death inducing receptor pathways such as Fas-FasL and TNF-related apoptosis-inducing ligand TRAIL pathways [ 77 , 78 ], which ultimately induce caspase-dependent apoptosis of the target cell [ 21 , 77 , 79 ].
The important role of the antibacterial activity of NK cells in vivo is demonstrated by animal models which showed higher survival rates and lower bacterial titers during infection with Shigella flexneri in mice lacking B and T cells but having NK cells as compared to mice which lack all three cell types [ 80 ]. Similar to the antibacterial activity, NK cells exhibit in vitro antifungal activity against a number of pathogenic fungi such as Aspergillus fumigatus , Candida albicans , Cryptococcus neoformans , or different species of mucormycetes [ 81 — 86 ].
Cytotoxic molecules including NK cell derived perforin seem to be important in the antifungal activity.
In addition, upon stimulation by fungi, NK cells release a number of cytokines, which modulate both innate and adaptive immune responses [ 87 ]. The in vitro data of the antifungal activity of NK cells are supported by observations made in animal studies. For example, it has been shown that NK cells proliferate in mice experimentally infected with Aspergillus niger , and this proliferation was associated with an inhibition of the fungal growth [ 88 ]. Antibody mediated depletion of NK cells in mice inoculated with C.
Although these data clearly demonstrate that NK cells exhibit important activities in the host immune response to different viral, bacterial and fungal pathogens, many questions have to be resolved. For example, further studies have to evaluate how and to which extent a pathogen may exert an immunosuppressive effect on NK cells as well as on other cells of the immune system, which has been shown for bacteria such as Pseudomonas aeruginosa and for fungi such as A.
This knowledge may be important in the clinical setting for specifically strengthening the host response during infection. Both severity and duration of a defect in the immune system are associated with risk and outcome of an infectious complication [ 4 ].
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A prospective, non-randomized study evaluated transfusions of granulocytes in order to control acute life-threatening infections or to prevent recurrence of severe fungal infections during HSCT or intensive chemotherapy [ 6 ]. In patients suffering from invasive fungal infections, a recent review of available data did not find strong evidence of a benefit of granulocyte transfusions, but it is to hope that ongoing randomized controlled studies such as the GRANITE study German Clinical Trials Register number DRKS will provide helpful results [ 98 ].
Notably, granulocyte transfusions are often associated with febrile transfusion reactions and pulmonary complications, including transfusion-related acute lung injury TRALI , and a monocenter retrospective analysis of patients with a hematological malignancy, prolonged neutropenia and invasive aspergillosis suggested that patients receiving granulocyte transfusions had a worse outcome [ 99 ]. Another approach which aims to reconstitute the long-lasting impairment of cellular immunity of allogeneic HSCT recipients became possible with the development of techniques to isolate and to generate pathogen-specific T cells.
Although immunotherapy with adoptively transferred pathogen specific T cells seems to be a promising strategy, the use of T cells may be associated with the risk of graft-versus-host disease GvHD [ ]. As compared to studies investigating NK cells as immunotherapeutic tool in patients with an underlying malignancy, relatively little is known regarding the in vivo effect of adoptively transferred NK cells into a host suffering from an infectious complication. In Aspergillus infected mice, the depletion of NK cells resulted in a higher fungal load and lower survival, whereas the transfer of activated NK cells to these mice led to greater pathogen clearance from the lungs [ 93 ].
Similarly, cyclophosphamide pretreated mice suffering from cryptococcosis showed an enhanced clearance of the fungus when they had received an NK cell-enriched graft as compared to mice which had received an NK cell-depleted graft [ , ]. Although these results suggest that adoptively transferred NK cells may be a potential tool in patients suffering from fungal infections, no study to date has proven this concept in the clinical setting.
Importantly, there may be a major difference of the adoptive transfer of NK cells between immunocompromised and immunocompetent patients. Whereas safety data in immunocompromised patients with cancer are promising [ , ], results on the adoptive transfer of NK cells into immunocompetent human individuals are lacking. However, it has been demonstrated that NK cells infused in mice with polymicrobial intra-abdominal bacterial infection contributed to an excessive induction of pro-inflammatory cytokines which ultimately resulted in a lethal septic shock [ 10 , 11 , — ].
On the other hand, as compared to pathogen-specific T cells which have already been evaluated in the clinical setting, NK cells may be of advantage since they are active against a broad range of pathogens including viruses, bacteria, or fungi. When employing NK cells to fight against a malignancy or to combat infectious complications, only functional active NK cells administered at a sufficient effector-to-target E:T ratio might result in a beneficial effect.
Studies addressing both mouse and human NK cell immunity have shown that NK cells are a heterogeneous population consisting of not only phenotypically but also functionally distinct subsets, and cytokine-stimulation induces significant changes not only in proliferation, maturation status and finally in the subset composition of the ex vivo expanded NK cell product [ 45 , ]. Therefore, knowledge on mouse NK cells cannot directly be transferred to the human system, and expansion and activation studies are mainly performed on human NK cell preparations from peripheral blood or umbilical cord blood leading to first preclinical data evaluating the cytotoxic capacity of the final NK cell product in vitro and in vivo in xenograft NSG NOD scid gamma mouse models against various human tumors [ ].
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Most clinical trials with NK cells in the autologous or allogeneic setting administer ex vivo expanded NK cells [ , ]. Of note, the expansion of NK cells usually needs several days to weeks, depending on the protocols used [ , ]. In order to improve the rapid expansion of isolated NK cells, cytokines like IL-2 have been investigated, which can be added either alone or in combination with an anti-CD3 antibody or additional cytokines such as IL and IL [ — ].
Although the exact role of anti-CD3 antibodies is unclear, it has been suggested that OKT-3 leads to a profound outgrowth of NK cells, which is probably due to the activation of T cells [ ]. Other studies suggest that co-culture of NK cells with stimulatory cells such as EBV-transformed lymphoblastoid cells or a Wilms tumor-derived cell line also enhances the proliferation of NK cells [ , ]. In an elegant approach, HLA-negative K cells were genetically modified to express membrane-bound IL and BB Ligand BBL , which specifically activate NK cells and promote their proliferation and survival, and this strategy resulted in a dramatic enhancement of NK cell expansion and activation [ , ].
After further improvement, this method has been adapted to large-scale Good Manufacturing Practices GMP conditions [ , ].
Cytokine-induced killer (CIK) cells: from basic research to clinical translation
In addition to proliferation, cytokine stimulation may also improve the functional activity of NK cells. For example, when NK cells are resting, lytic granules are distributed randomly or diffuse, whereas after exposure to IL-2, granules congregate to the microtubule-organizing center [ ]. Thus, IL-2 stimulation not only activates NK cells, but also accelerates their transition into NK cells which are ready to exhibit their cytotoxic function.
Even if there are known differences in the biology of murine and human NK cells, this observation suggests that both murine and human NK cells receive functional memory-like properties after cytokine activation, which may provide a novel rationale for integrating cytokine preactivation into NK cell immunotherapeutic strategies. However, at the same time, the use of cytokines may alter the phenotype of the NK cell and result in a potential loss of responsiveness to some stimuli [ , ].
For example, IL-2 stimulation of NK cells decreased CD16 [ ], and NK cell activation by intramuscular influenza vaccination and HIV-positive plasma induced a matrix metalloproteinase-mediated cleavage of cell surface CD16, whereas inhibition of CD16 shedding potentiated NK cell cytotoxic function [ 70 , ]. In addition to the exposure to various cytokines, improvement of NK cell activity can be achieved by the manipulation of NK cell receptors. Another interesting approach is the transduction of chimeric antigen receptors CARs into immune cells in order to improve their activity.
CARs are engineered receptors with the ability to bind to specific antigens which are expressed on the surface of tumor cells or on the surface of a pathogen. Of note, CAR constructs expressed in the NK cell line NK92 [ ], primary NK cells [ , ] or cord blood derived NK cells [ ] have demonstrated efficient killing in preclinical settings [ ]. In contrast, studies on NK cells with modified receptors or CAR-NK cells against infections are lacking, and challenges of this strategy include the relatively long time to generate these cells and the complex antigenic properties of many pathogens, which is seen in particular in fungi [ ].
The functional activity of NK cells can also be enhanced by the forced expression of the high-affinity CDV HA-CD16 Fc receptor, for which the minority of patients is homozygous [ , ]. Rituximab is used as therapeutic compound in many patients suffering from B cell non-Hodgkin lymphoma.
Similarly, in the setting of an infection, blocking the interaction between NK cell inhibitory receptors [e.
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In particular in immunocompromised patients, infections often have a sudden onset with a rapid clinical course. Therefore, an immunotherapeutic tool in this setting has to be quickly available, which might be different to the setting of immunotherapy used against an underlying malignancy. In other words, the timely access to a suitable NK cell product is crucial when planning clinical studies evaluating NK cells in patients with viral, bacterial, or fungal infections Figure 2.
In this respect, NK cell products which are standardized, well characterized and cryopreserved would be ideal and open new perspectives in this emerging field. However, the long-term storage of NK cell products remains controversial. Whereas it has been demonstrated that NK cells maintain their cytotoxic activity against the leukemia cell line K after cryopreservation [ , ], standard methods of cryopreservation seem to have a negative impact on cell expansion in vivo [ ].
Interestingly, in one clinical trial NK cells were expanded upon medical need from aliquots of individual cryopreserved leukapharesis cryopreserved peripheral blood mononuclear cells [ ]. Figure 2: Potential strategies of generating Natural Killer NK cells as an immunotherapeutic tool for patients suffering from infectious complications.
Both the infectious complication e. Having identified the challenges in derivation, activation and expansion of NK cells directly from patients, NK cell lines may be considered as an ideal source for cell-based immunotherapy. Advantages using NK cell lines for immunotherapy would include 1 the possibility to establish a master cell bank, and 2 the fact that the cell source would be extremely well standardized and characterized.
As one example, the permanent NK cell line is cryopreserved in GMP-compliant master cell bank, from which it can be easily and reproducibly expanded [ ]. The cell line exhibits cytotoxicity against a broad spectrum of tumor targets in vitro such as various leukemia, lymphoma and myeloma cell lines as well as against primary leukemic blasts [ — ]. When used as immunotherapy against cancer, preliminary data demonstrated the safety and tolerability of NK cells in the clinical setting [ , ].
A number of groups are exploring the use of CAR-modification to enhance the antitumor activity of these cells in preclinical and clinical studies [ , ] for review see: [ , ]. However, to date, relatively little is known about the activity of the NK cell line against infectious pathogens. Other drawbacks in the clinical use of NK cells include the fact that the NK cell line is derived from an NK cell lymphoma and therefore has the potential risk for uncontrolled proliferation, in particular if cells are resistant or not fully hit by prior irradiation. In turn, irradiation, which is mandatory prior to infusion, severely limits the survival and function of the transferred cells.
Whether this potential toxicity can be abrogated by genetic modification of the cells leading to constitutive expression of IL-2 and resulting in auto-activated and auto-proliferating cells is unclear to date [ , ]. Of note, in a promising preclinical study, transduction of clinically applicable NK cells with lentiviral vectors encoding human IL resulted in predominantly intracellular expression of the cytokine, proliferation and cytotoxicity of the producer cells in the absence of IL-2 [ ].
Based on these experiences, further studies evaluating the activity of the NK cell line against viral, bacterial, and fungal pathogens are urgently needed. It is important to mention that in addition to the NK cell line, there are other cell lines which potentially could be used as adoptive NK cell-based immunotherapy, both in patients with cancer and in patients with infectious complications. In addition, irradiation of these cells does not abrogate their cytotoxicity towards tumor targets. Similarly, the cell line NKL, which is biologically and functionally very similar to primary NK cells, exhibits enhanced cytotoxicity against certain tumor cells as compared to NK [ ].
Again, the antimicrobial activity of these cells lines is unclear and needs further evaluation. Whereas the anticancer effect of NK cells is currently investigated in multiple clinical trials, little is known about the potential of adoptively transferred NK cells in patients suffering from infectious complications. In vitro data clearly demonstrate that NK cells are active against viral, bacterial, and fungal pathogens, and animal studies suggest that NK cells could be a promising tool in the antimicrobial immunotherapy.
Current investigation focuses on the optimal and rapid generation of high numbers of functionally active NK cells and includes strategies such as genetic modification of the cell and its receptors and the use of NK cell lines for treatment of hemato-oncological diseases. However, before testing adoptively transferred NK cells in the clinical setting of infectious complications, a number of questions have to be resolved.
For example, it is well known that activation of the host immune system e. Therefore, both the patient population immunocompromised versus immunocompetent and the optimal time point of the adoptive transfer of NK cells are unknown to date, or, in other words, it is unclear when and whom adoptively transferred NK cells will help to overcome an infectious complication or will ultimately harm. Based on the promising results in animal studies and due to the facts that HSCT recipients suffering from fungal infections lack a sufficient immune response and outcome of this patient population is extremely poor, first clinical trials might focus on invasive fungal disease in this patient population.
Although it will be necessary to fully characterize the optimal patient population, the best time point of therapy as well as the best approach to generate NK cells for immunotherapy in infectious complication, this strategy might become important in this setting, in particular since antimicrobial compounds have limited activity and we witness emerging resistance of pathogens all over the world. All authors were involved in designing and writing the manuscript, and all authors read and approved the final version of the manuscript.
A review of infectious complications after haploidentical hematopoietic stem cell transplantations. Microbiologically documented infections and infection-related mortality in children with acute myeloid leukemia. Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia. Ann Intern Med. J Infect Dis. Prophylactic and interventional granulocyte transfusions in patients with haematological malignancies and life-threatening infections during neutropenia. Ann Hematol.
Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. It discusses how these cells mature and develop, and it covers the different isolation, culture, and propagation methods of these cells. Furthermore, it focuses on the different killer cells that are present in various parts of the human body.
The book concludes by explaining that natural killer cells are utilized for clinical therapy of malignancies, and that they have led to positive outcomes in the field of biology and medicine. Michael T. Lotze , Angus W.