Role of NK Cell Subsets in Organ-Specific Murine Melanoma Metastasis

Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b–, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.     

Natural killer cells utilize both perforin and gamma interferon to regulate murine cytomegalovirus infection in the spleen and liver

Natural killer (NK) cells are critical for innate regulation of the acute phase of murine cytomegalovirus (MCMV) infection and have been reported to utilize perforin (Pfp)- and gamma interferon (IFN-gamma)-dependent effector mechanisms in an organ-specific manner to regulate MCMV infection in the spleen and liver. In this study, we further examined the roles of NK cells, Pfp, and IFN-gamma in innate immunity to MCMV infection. With the recently described NK cell-deficient (NKD) mouse, we confirmed previous findings that NK cells, but not NKT cells, are required for control of the acute phase of MCMV infection in spleen and liver cells. Interestingly, we found that Pfp and IFN-gamma are each important for regulating MCMV replication in both the spleen and the liver. Moreover, NK cells can regulate MCMV infection in the spleens and livers of Pfp(-/-) mice in a Pfp-independent manner and can use an IFN-gamma-independent mechanism to control MCMV infection in IFN-gamma(-/-) mice. Thus, contrary to previous reports, NK cells utilize both Pfp and IFN-gamma to control MCMV infection in the spleen and liver.     

Natural killer cells and autoimmunity

Autoimmune diseases are often characterized as clinical syndromes caused by the inappropriate activation of T or B cells resulting in systemic or organ-specific damage. However, studies support a role for the innate immune system, and in particular natural killer (NK) cells, in stimulating or suppressing autoimmunity. This review focuses on recent research elucidating a potential immunoregulatory role for NK cells in modulating T and B cell-mediated autoimmunity.     

Natural killer cells and autoimmunity

Autoimmune diseases are often characterized as clinical syndromes caused by the inappropriate activation of T or B cells resulting in systemic or organ-specific damage. However, studies support a role for the innate immune system, and in particular natural killer (NK) cells, in stimulating or suppressing autoimmunity. This review focuses on recent research elucidating a potential immunoregulatory role for NK cells in modulating T and B cell-mediated autoimmunity.     

Natural killer cells can enhance the proliferative responses of B lymphocytes

In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggested that natural killer (NK) cells can modulate immune activities such as the suppression of B cell responses through noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responses of B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This "enhancer" activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as 0.05. Peak blastogenic responses of B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responses of B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggesting that cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lytic activities.     

NKT cells mediate organ-specific resistance against Leishmania major infection

Whereas the acquired T cell-mediated protection against intracellular pathogens such as Leishmania major has been well studied in the past, the cells and mechanisms involved in their innate control are still poorly understood. Here, we investigated the role of natural killer T (NKT) cells in a high dose L. major mouse infection model. In vitro, L. major only weakly stimulated NKT cells and antagonized their response to the prototypic NKT cell ligand alpha-galactosylceramide, indicating that L. major partially escapes the activation of NKT cells. NKT cell deficiency as analyzed by subcutaneous infection of Jalpha281-/- mice (lacking invariant CD1d-restricted NKT cells) and CD1-/- mice (lacking all CD1d-restricted NKT cells) led to a transient increase in skin lesions, but did not impair the clinical cure of the infection, NK cell cytotoxicity, the production of IFN-gamma, the expression of inducible nitric oxide synthase, and the control of the parasites in the lymph node. In the spleen, however, NKT cells were required for NK cell cytotoxicity and early IFN-gamma production, they lowered the parasite burden, and exerted bystander effects on Leishmania antigen-specific T cell responses, most notably after systemic infection. Thus, NKT cells fulfill organ-specific protective functions during infection with L. major, but are not essential for parasite control.     

TLR-dependent activation stimuli associated with Th1 responses confer NK cell stimulatory capacity to mouse dendritic cells

Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated DCs to elicit IFN-gamma production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas stimuli associated with Th2 responses do not have this property.     

Human NK cells: From development to effector functions

NK cells are the major lymphocyte subset of the innate immune system that mediates antiviral and anti-tumor responses. It is well established that they develop mechanisms to distinguish self from non-self during the process of NK cell education. Unlike T and B cells, natural killer cells lack clonotypic receptors and are activated after recognizing their target via germline-encoded receptors through natural cytotoxicity, cytokine stimulation, and Ab-dependent cellular cytotoxicity. Subsequently, they utilize cytotoxic granules, death receptor ligands, and cytokines to perform their effector functions. In this review, we provide a general overview of human NK cells, as opposed to murine NK cells, discussing their ontogeny, maturation, receptor diversity, types of responses, and effector functions. Furthermore, we also describe recent advances in human NK cell biology, including tissue-resident NK cell populations, NK cell memory, and novel approaches used to target NK cells in cancer immunotherapy.     

Natural killer cells promote early CD8 T cell responses against cytomegalovirus

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.     

NK cells inhibit T cell proliferation via p21-mediated cell cycle arrest

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.     

Viral modulation of NK cell immunity

Natural killer (NK) cells have been implicated in innate immune responses against viruses such as herpesviruses, which cause persistent infections in the host. In response to the selective pressure that is exerted by NK cells, many viruses have evolved strategies either to evade detection by NK cells or to modulate the activity of NK cells. Here, we review the unique relationship that exists between NK cells and viruses, with a focus on herpesviruses.     

NK cell TRAIL eliminates immature dendritic cells in vivo and limits dendritic cell vaccination efficacy

Recent studies have implicated a possible role for NK cells in regulating dendritic cells (DC) in vitro. In the present study, we demonstrate that immature DC are rapidly eliminated by NK cells in vivo via a pathway dependent on the TNF-related apoptosis-inducing ligand (TRAIL). Elimination of NK cells and/or neutralization of TRAIL function during immunization with immature DC loaded with nonself or tumor Ags significantly enhanced T cell responses to these Ags and Ag-specific tumor immunity. These data suggested that NK cell TRAIL might regulate responses to vaccination by controlling the survival of Ag-loaded DC.     

NK and CD4+ T Cell Cooperative Immune Responses Correlate with Control of Disease in a Macaque Simian Immunodeficiency Virus Infection Model

Control of infectious disease may be accomplished by successful vaccination or by complex immunologic and genetic factors favoring Ag-specific multicellular immune responses. Using a rhesus macaque model, we evaluated Ag-specific T cell-dependent NK cell immune responses in SIV-infected macaques, designated "controlling" or "noncontrolling" based on long-term chronic viremia levels, to determine whether NK cell effector functions contribute to control of SIV infection. We observed that Gag stimulation of macaque PBMCs induced subset-specific NK cell responses in SIV-controlling but not SIV-noncontrolling animals, as well as that circulatory NK cell responses were dependent on Ag-specific IL-2 production by CD4(+) central memory T cells. NK cell activation was blocked by anti-IL-2-neutralizing Ab and by CD4(+) T cell depletion, which abrogated the Gag-specific responses. Among tissue-resident cells, splenic and circulatory NK cells displayed similar activation profiles, whereas liver and mucosal NK cells displayed a decreased activation profile, similar in SIV-controlling and -noncontrolling macaques. Lack of T cell-dependent NK cell function was rescued in SIV-noncontrolling macaques through drug-mediated control of viremia. Our results indicate that control of disease progression in SIV-controlling macaques is associated with cooperation between Ag-specific CD4(+) T cells and NK cell effector function, which highlight the importance of such cell-to-cell cooperativity in adaptive immunity and suggest that this interaction should be further investigated in HIV vaccine development and other prophylactic vaccine approaches.     

NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis

We previously found that human NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages and upregulate CD8(+) T cell responses. We also found that human NK cells produce IL-22, which inhibits intracellular growth of M. tuberculosis, and that NK cells lyse M. tuberculosis-expanded CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs). To determine the role of NK cells during the protective immune response to vaccination in vivo, we studied the NK cell and T cell responses in a mouse model of vaccination with bacillus Calmette-Guérin (BCG), followed by challenge with virulent M. tuberculosis H37Rv. BCG vaccination enhanced the number of IFN-γ-producing and IL-22-producing NK cells. Depletion of NK1.1(+) cells at the time of BCG vaccination increased the number of immunosuppressive Tregs (CD4(+)CD25(hi), 95% Foxp3(+)) after challenge with M. tuberculosis H37Rv, and NK1.1(+) cells lysed expanded but not natural Tregs in BCG-vaccinated mice. Depletion of NK1.1(+) cells at the time of BCG vaccination also increased the bacillary burden and reduced T cell responses after challenge with M. tuberculosis H37Rv. IL-22 at the time of vaccination reversed these effects and enhanced Ag-specific CD4(+) cell responses in BCG-vaccinated mice after challenge with M. tuberculosis H37Rv. Our study provides evidence that NK1.1(+) cells and IL-22 contribute to the efficacy of vaccination against microbial challenge.     

Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones

Precursors of uterine NK cells home to the uterus during early pregnancy from multiple lymphohemopoietic sources. In mouse uterine tissue, pregnancy markedly up-regulates both L-selectin- and alpha(4) integrin-dependent adhesion pathways for circulating human CD56(bright) cells, the phenotype of human uterine NK cells. Based on roles for these adhesion molecules in lymphocyte homing, we examined effects of pregnancy or the steroid hormones 17beta-estradiol or progesterone on lymphocyte-endothelial interactions in secondary lymphoid tissues and in uterus. From preimplantation gestation day 3, specialized high endothelial venules in peripheral lymph nodes and Peyer's patches supported elevated L-selectin and alpha(4)beta(7) integrin-dependent lymphocyte adhesion under shear throughout pregnancy, as compared with high endothelial venules of virgin or postpartum donors. Squamous endothelium from nonlymphoid tissue was not affected. Pregnancy-equivalent endothelial responses were observed in lymph nodes and Peyer's patches from ovariectomized mice receiving 17beta-estradiol and/or progesterone replacement therapy. Adhesion of human CD56(bright) cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and alpha(4) integrin-dependent mechanisms and involved multiple vascular adhesion molecules including mucosal addressin cell adhesion molecule-1, VCAM-1, and peripheral lymph node addressin. Analysis of Tie2-green fluorescence protein transgenic mice demonstrated that CD56(bright) cells adhered primarily to vascular endothelium within the decidua basalis. Microdomain localization of adhesion involving large clusters of lymphocytes was induced on uteri from natural matings, but not pseudopregnancy. Steroid hormones also had independent effects on L-selectin function in splenic lymphocytes that mimicked physiological stimulation induced by pregnancy or fever-range temperatures. These results provide the first evidence for coordinated, organ-specific, steroid hormone-induced changes in lymphocyte homing mechanisms that could contribute to local and systemic immune responses during pregnancy.     

Advantages and clinical applications of natural killer cells in cancer immunotherapy

The past decade has witnessed a burgeoning of research and further insight into the biology and clinical applications of natural killer (NK) cells. Once thought to be simple innate cells important only as cytotoxic effector cells, our understanding of NK cells has grown to include memory-like responses, the guidance of adaptive responses, tissue repair, and a delicate paradigm for how NK cells become activated now termed “licensing” or “arming.” Although these cells were initially discovered and named for their spontaneous ability to kill tumor cells, manipulating NK cells in therapeutic settings has proved difficult and complex in part due to our emerging understanding of their biology. Therapies involving NK cells may either activate endogenous NK cells or involve transfers of exogenous cells by hematopoietic stem cell transplantation or adoptive cell therapy. Here, we review the basic biology of NK cells, highlighting characteristics which make NK cells particularly useful in cancer therapies. We also explore current treatment strategies that have been used for cancer as well as discuss potential future directions for the field.     

In vivo regulation of experimental autoimmune encephalomyelitis by NK cells: alteration of primary adaptive responses

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vbeta usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.     

Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

Compartment diversity in innate immune reprogramming

Pathogens or endogenous molecules can reprogram innate immunity. This process can take the form of priming or tolerance depending on the activating signal, and favors enhanced resistance to infection and other insults, by modulating inflammation. Similarly to their organ-specific properties, reprogramming of macrophages and NK cells, is also compartmentalized.     

Dendritic cells mediate NK cell help for Th1 and CTL responses: two-signal requirement for the induction of NK cell helper function

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other's functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized "effector/memory" DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-gamma, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, "helper" function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.