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Sample records for nk cell responses

  1. Homotypic NK cell-to-cell communication controls cytokine responsiveness of innate immune NK cells.

    PubMed

    Kim, Tae-Jin; Kim, Miju; Kim, Hye Mi; Lim, Seon Ah; Kim, Eun-Ok; Kim, Kwanghee; Song, Kwang Hoon; Kim, Jiyoung; Kumar, Vinay; Yee, Cassian; Doh, Junsang; Lee, Kyung-Mi

    2014-12-05

    While stationary organ cells are in continuous contact with neighboring cells, immune cells circulate throughout the body without an apparent requirement for cell-cell contact to persist in vivo. This study challenges current convention by demonstrating, both in vitro and in vivo, that innate immune NK cells can engage in homotypic NK-to-NK cell interactions for optimal survival, activation, and proliferation. Using a specialized cell-laden microwell approach, we discover that NK cells experiencing constant NK-to-NK contact exhibit a synergistic increase in activation status, cell proliferation, and anti-tumor function in response to IL-2 or IL-15. This effect is dependent on 2B4/CD48 ligation and an active cytoskeleton, resulting in amplification of IL-2 receptor signaling, enhanced CD122/CD132 colocalization, CD25 upregulation, and Stat3 activation. Conversely, 'orphan' NK cells demonstrate no such synergy and fail to persist. Therefore, our data uncover the existence of homotypic cell-to-cell communication among mobile innate lymphocytes, which promotes functional synergy within the cytokine-rich microenvironment.

  2. Studying NK cell responses to ectromelia virus infections in mice.

    PubMed

    Fang, Min; Sigal, Luis

    2010-01-01

    Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

  3. IL-27 stimulates human NK-cell effector functions and primes NK cells for IL-18 responsiveness.

    PubMed

    Ziblat, Andrea; Domaica, Carolina I; Spallanzani, Raúl G; Iraolagoitia, Ximena L Raffo; Rossi, Lucas E; Avila, Damián E; Torres, Nicolás I; Fuertes, Mercedes B; Zwirner, Norberto W

    2015-01-01

    IL-27, a member of the IL-12 family of cytokines, is produced by APCs, and displays pro- and anti-inflammatory effects. How IL-27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL-27 and that blockade of IL-27R (CD130) reduced the amount of IFN-γ produced by NK cells during their coculture, showing the importance of IL-27 during DC-NK-cell crosstalk. Accordingly, human rIL-27 stimulated IFN-γ secretion by NK cells in a STAT1-dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL-18. Preincubation experiments demonstrated that IL-27 primed NK cells for IL-18-induced IFN-γ secretion, which was associated with an IL-27-driven upregulation of T-bet expression. Also, IL-27 triggered NKp46-dependent NK-cell-mediated cytotoxicity against Raji, T-47D, and HCT116 cells, and IL-18 enhanced this cytotoxic response. Such NK-cell-mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL-mediated cytotoxicity but not Fas-FasL interaction. Moreover, IL-27 also potentiated Ab-dependent cell-mediated cytotoxicity against mAb-coated target cells. Taken together, IL-27 stimulates NK-cell effector functions, which might be relevant in different physiological and pathological situations.

  4. Listeria monocytogenes infection differentially affects expression of ligands for NK cells and NK cell responses, depending on the cell type infected.

    PubMed

    Shegarfi, Hamid; Rolstad, Bent; Kane, Kevin P; Nestvold, Janne

    2016-04-22

    The pivotal role of NK cells in viral infection is extensively studied, whereas the role of NK cells in bacterial infection has been poorly investigated. Here, we have examined how Listeria monocytogenes (LM) affects expression of ligands for NK cell receptors and subsequent NK cell responses, depending on the type of cell infected. LM infected rat cell lines derived from different tissues were coincubated with splenic NK cells, and NK cell proliferation and IFN-γ production were measured. In addition, expression of ligands for the NK cell receptors Ly49 and NK cell receptor protein 1 (NKR-P1), MHC class I and C-type lectin-related molecules, respectively, was assessed. Infected pleural R2 cells, but not epithelium-derived colon carcinoma cell line CC531 cells, induced proliferation of NK cells. Reporter cells expressing the inhibitory NKR-P1G receptor or the activating NKR-P1F receptor were less stimulated under incubation with infected CC531 cells versus uninfected CC531 controls, suggesting that the ligand(s) in question were down-regulated by infection. Conversely, LM infection of R2 cells did not affect reporter cell stimulation compared with uninfected R2 controls. We characterized a rat monocyte cell line, termed RmW cells. In contrast to LM infected R2 cells that up-regulate MHC class I molecules, RmW cells displayed unchanged MHC class I expression following infection. In line with MHC class I expression, more NK cells produced a higher amount of IFN-γ against infected R2 cells compared with RmW cells. Together, L. monocytogenes infection may variously regulate cellular ligands for NK cells, depending on the cell type infected, affecting the outcome of NK cell responses.

  5. Helper role of NK cells during the induction of anticancer responses by dendritic cells.

    PubMed

    Kalinski, Pawel; Giermasz, Adam; Nakamura, Yutaro; Basse, Per; Storkus, Walter J; Kirkwood, John M; Mailliard, Robbie B

    2005-02-01

    Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.

  6. Local Microenvironment Controls the Compartmentalization of NK Cell Responses during Systemic Inflammation in Mice.

    PubMed

    Rasid, Orhan; Ciulean, Ioana Sonya; Fitting, Catherine; Doyen, Noelle; Cavaillon, Jean-Marc

    2016-09-15

    Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell-intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo-transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell-intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner.

  7. Controlling NK Cell Responses: Integration of Signals for Activation and Inhibition

    PubMed Central

    Long, Eric O.; Kim, Hun Sik; Liu, Dongfang; Peterson, Mary E.; Rajagopalan, Sumati

    2013-01-01

    Understanding how signals are integrated to control NK cell responsiveness in the absence of antigen-specific receptors has been a challenge, but recent work has revealed some underlying principles that govern NK cell responses. NK cells use an array of innate receptors to sense their environment and respond to alterations caused by infections, cellular stress and transformation. No single activation receptor dominates; instead, synergistic signals from combinations of receptors are integrated to activate natural cytotoxicity and cytokine production. Inhibitory receptors for MHC class I have a critical role in controlling NK cell responses and paradoxically, in maintaining NK cells in a state of responsiveness to subsequent activation events, a process referred to as licensing. MHC-I specific inhibitory receptors both block activation signals and trigger signals to phosphorylate and inactivate the small adaptor Crk. These different facets of inhibitory signaling are incorporated into a revocable license model for the reversible tuning of NK cell responsiveness. PMID:23516982

  8. Low baseline levels of NK cells may predict a positive response to ipilimumab in melanoma therapy.

    PubMed

    Tietze, Julia K; Angelova, Daniela; Heppt, Markus V; Ruzicka, Thomas; Berking, Carola

    2016-11-28

    The introduction of immune checkpoint blockade (ICB) has been a breakthrough in the therapy of metastatic melanoma. The influence of ICB on T-cell populations has been studied extensively, but little is known about the effect on NK cells. In this study, we analysed the relative and absolute amounts of NK cells and of the subpopulations of CD56(dim) and CD56(bright) NK cells among the peripheral blood mononuclear cells (PBMCs) of 32 patients with metastatic melanoma before and under treatment with ipilimumab or pembrolizumab by flow cytometry. In 15 (47%) patients, an abnormal low amount of NK cells was found at baseline. Analysis of the subpopulations showed also low or normal baseline levels for CD56(dim) NK cells, whereas the baseline levels of CD56(bright) NK cells were either normal or abnormally high. The relative and absolute amounts of NK cells and of CD56(dim) and CD56(bright) NK cell subpopulations in patients with a normal baseline did not change under treatment. However, patients with a low baseline of NK cells and CD56(dim) NK cells showed a significant increase in these immune cell subsets, but the amounts remained to be lower than the normal baseline. The amount of CD56(bright) NK cells was unaffected by treatment. The baseline levels of NK cells were correlated with the number of metastatic organs. Their proportion increased, whereas the expression of NKG2D decreased significantly when more than one organ was affected by metastases. Low baseline levels of NK cells and CD56(dim) NK cells as well as normal baseline levels of CD56(bright) NK cells correlated significantly with a positive response to ipilimumab but not to pembrolizumab. Survival curves of patients with low amounts of CD56(dim) NK cells treated with ipilimumab showed a trend to longer survival. Normal baseline levels of CD56(bright) NK cells were significantly correlated with longer survival as compared to patients with high baseline levels. In conclusion, analysis of the amounts of total

  9. Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients.

    PubMed

    Curti, Antonio; Ruggeri, Loredana; Parisi, Sarah; Bontadini, Andrea; Dan, Elisa; Motta, Maria Rosa; Rizzi, Simonetta; Trabanelli, Sara; Ocadlikova, Darina; Lecciso, Mariangela; Giudice, Valeria; Fruet, Fiorenza; Urbani, Elena; Papayannidis, Cristina; Martinelli, Giovanni; Bandini, Giuseppe; Bonifazi, Francesca; Lewis, Russell E; Cavo, Michele; Velardi, Andrea; Lemoli, Roberto M

    2016-04-15

    In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53-73) received NK cells from haploidentical KIR-ligand-mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3-51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively;P= 0.03). Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells. ©2016 American Association for Cancer Research.

  10. Activation of Natural Killer (NK) T Cells during Murine Cytomegalovirus Infection Enhances the Antiviral Response Mediated by NK Cells

    PubMed Central

    van Dommelen, Serani L. H.; Tabarias, Hyacinth A.; Smyth, Mark J.; Degli-Esposti, Mariapia A.

    2003-01-01

    NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and α-galactosylceramide (α-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jα281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by α-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-γ), have revealed that the antiviral effects of α-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of α-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-γ. PMID:12525622

  11. NK Cells in HIV Disease.

    PubMed

    Scully, Eileen; Alter, Galit

    2016-04-01

    Natural killer (NK) cells play a critical role in viral immunity. In the setting of HIV infection, epidemiologic and functional evidence support a role for NK cells in both protection from new infection and in viral control. Specifically, NK cells directly mediate immune pressure leading to virus evolution, and NK cell receptor genotypic profiles, clonal repertoires, and functional capacity have all been implicated in virus containment. In addition, indirect NK cell-mediated antibody-dependent cellular cytotoxicity has been linked to vaccine-induced protective immunity against HIV infection. With recent advances in our understanding of NK cell deficiency, development, memory-like responses, and editing of the adaptive immune system, the opportunities to direct and exploit NK cell antiviral immunity to target HIV have exponentially grown. In this review, we seek to highlight the intersections between discoveries in basic NK cell biology and the challenges of HIV chronic infection, vaccine development, and cure/eradication strategies.

  12. Insufficient natural killer cell responses against retroviruses: how to improve NK cell killing of retrovirus-infected cells.

    PubMed

    Littwitz-Salomon, Elisabeth; Dittmer, Ulf; Sutter, Kathrin

    2016-11-08

    Natural killer (NK) cells belong to the innate immune system and protect against cancers and a variety of viruses including retroviruses by killing transformed or infected cells. They express activating and inhibitory receptors on their cell surface and often become activated after recognizing virus-infected cells. They have diverse antiviral effector functions like the release of cytotoxic granules, cytokine production and antibody dependent cellular cytotoxicity. The importance of NK cell activity in retroviral infections became evident due to the discovery of several viral strategies to escape recognition and elimination by NK cells. Mutational sequence polymorphisms as well as modulation of surface receptors and their ligands are mechanisms of the human immunodeficiency virus-1 to evade NK cell-mediated immune pressure. In Friend retrovirus infected mice the virus can manipulate molecular or cellular immune factors that in turn suppress the NK cell response. In this model NK cells lack cytokines for optimal activation and can be functionally suppressed by regulatory T cells. However, these inhibitory pathways can be overcome therapeutically to achieve full activation of NK cell responses and ultimately control dissemination of retroviral infection. One effective approach is to modulate the crosstalk between NK cells and dendritic cells, which produce NK cell-stimulating cytokines like type I interferons (IFN), IL-12, IL-15, and IL-18 upon retrovirus sensing or infection. Therapeutic administration of IFNα directly increases NK cell killing of retrovirus-infected cells. In addition, IL-2/anti-IL-2 complexes that direct IL-2 to NK cells have been shown to significantly improve control of retroviral infection by NK cells in vivo. In this review, we describe novel approaches to improve NK cell effector functions in retroviral infections. Immunotherapies that target NK cells of patients suffering from viral infections might be a promising treatment option for the

  13. CALGB 150905 (Alliance): Rituximab broadens the anti-lymphoma response by activating unlicensed NK cells

    PubMed Central

    Du, Juan; Lopez-Verges, Sandra; Pitcher, Brandelyn N.; Johnson, Jeffrey; Jung, Sin-Ho; Zhou, Lili; Hsu, Katharine; Czuczman, Myron S.; Cheson, Bruce; Kaplan, Lawrence; Lanier, Lewis L.; Venstrom, Jeffrey M.

    2014-01-01

    Natural killer (NK) cells contribute to clinical responses in patients treated with rituximab, but the rules determining NK cell responsiveness to mAb therapies are poorly defined. A deeper understanding of the mechanisms responsible for antibody-dependent cellular cytotoxicity (ADCC) could yield useful biomarkers for predicting clinical responses in patients. Unlicensed NK cells, defined as NK cells lacking expression of an inhibitory KIR for self-HLA class I ligands, are hypo-responsive in steady-state, but are potent effectors in inflammatory conditions. We hypothesized that antitumor antibodies such as rituximab can overcome NK cell dependence on licensing, making unlicensed NK cells important for clinical responses. Here we examined the influences of variations in KIR and HLA class I alleles on in vitro responses to rituximab. We tested the clinical significance in a cohort of follicular lymphoma patients treated with rituximab-containing mAb combinations and show that rituximab triggers responses from all NK cell populations regardless of licensing. Neither IL-2 nor accessory cells are required for activating unlicensed NK cells, but both can augment rituximab-mediated ADCC. Moreover, in 101 follicular lymphoma patients treated with rituximab-containing mAb combinations, a “missing ligand” genotype (predictive of unlicensed NK cells) is associated with higher progression-free survival. Our data suggest that the clinical efficacy of rituximab may be driven, in part, by its ability to broaden the NK cell repertoire to include previously hypo-responsive, unlicensed NK cells. A “missing ligand” KIR and HLA class I genotype may be predictive of this benefit, and useful for personalizing treatment decisions in lymphomas and other tumors. PMID:24958280

  14. Evaluation of Functional NK Cell Responses in Vaccinated and SIV-Infected Rhesus Macaques.

    PubMed

    Vargas-Inchaustegui, Diego A; Ying, Olivia; Demberg, Thorsten; Robert-Guroff, Marjorie

    2016-01-01

    NK cells are crucial components of the innate immune system due to their capacity to exert rapid cytotoxic and immunomodulatory function in the absence of prior sensitization. NK cells can become activated by exposure to target cells and/or by cytokines produced by antigen-presenting cells. In this study, we examined the effects of a simian immunodeficiency virus (SIV) vaccine regimen and subsequent SIV infection on the cytotoxic and immunomodulatory functions of circulatory NK cells. While vaccination did not significantly impact the capacity of NK cells to kill MHC-devoid 721.221 target cells, SIV-infection led to a significant decrease in target cell killing. NK cells from uninfected macaques were responsive to a low dose (5 ng/ml) of IL-15 pre-activation, leading to significant increases in their cytotoxic potential, however, NK cells from SIV-infected macaques required a higher dose (50 ng/ml) of IL-15 pre-activation in order to significantly increase their cytotoxic potential. By contrast, no differences were observed in the capacity of NK cells from vaccinated and SIV-infected macaques to respond to IL-12 and IL-18. Similarly, NK cells both before and after infection exhibited equivalent responses to Fc-mediated activation. Collectively, our results show that early SIV-infection impairs the natural cytotoxic capacity of circulatory NK cells without affecting Fc-mediated or cytokine-producing function.

  15. NK cells and interferons.

    PubMed

    Paolini, Rossella; Bernardini, Giovanni; Molfetta, Rosa; Santoni, Angela

    2015-04-01

    The role of Natural Killer cells in host defense against infections as well as in tumour surveillance has been widely appreciated for a number of years. Upon recognition of "altered" cells, NK cells release the content of cytolytic granules, leading to the death of target cells. Moreover, NK cells are powerful producers of chemokines and cytokines, particularly Interferon-γ (IFN-γ), of which they are the earliest source upon a variety of infections. Despite being armed to fight against pathogens, NK cells become fully functional upon an initial phase of activation that requires the action of several cytokines, including type I IFNs. Type I IFNs are now recognized as key players in antiviral defense and immune regulation, and evidences from both mouse models of disease and in vitro studies support the existence of an alliance between type I IFNs and NK cells to ensure effective protection against viral infections. This review will focus on the role of type I IFNs in regulating NK cell functions to elicit antiviral response and on NK cell-produced IFN-γ beneficial and pathological effects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells

    NASA Astrophysics Data System (ADS)

    Caccia, Michele; Gorletta, Tatiana; Sironi, Laura; Zanoni, Ivan; Salvetti, Cristina; Collini, Maddalena; Granucci, Francesca; Chirico, Giuseppe

    2010-02-01

    Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.

  17. Combination Immune Therapies to Enhance Anti-Tumor Responses by NK Cells

    PubMed Central

    Mentlik James, Ashley; Cohen, Adam D.; Campbell, Kerry S.

    2013-01-01

    Natural killer (NK) cells are critical innate immune lymphocytes capable of destroying virally infected or cancerous cells through targeted cytotoxicity and further assisting in the immune response by releasing inflammatory cytokines. NK cells are thought to contribute to the process of tumor killing by certain therapeutic monoclonal antibodies (mAb) by directing antibody-dependent cellular cytotoxicity (ADCC) through FcγRIIIA (CD16). Numerous therapeutic mAb have been developed that target distinct cancer-specific cell markers and may direct NK cell-mediated ADCC. Recent therapeutic approaches have combined some of these cancer-specific mAb with additional strategies to optimize NK cell cytotoxicity. These include agonistic mAb targeting NK cell activating receptors and mAbs blocking NK cell inhibitory receptors to enhance NK cell functions. Furthermore, several drugs that can potentiate NK cell cytotoxicity through other mechanisms are being used in combination with therapeutic mAb. In this review, we examine the mechanisms employed by several promising agents used in combination therapies that enhance natural or Ab-dependent cytotoxicity of cancer cells by NK cells, with a focus on treatments for leukemia and multiple myeloma. PMID:24391651

  18. Impaired NK cell antiviral cytokine response against influenza virus in small-for-gestational-age neonates

    PubMed Central

    Li, Jinrong; Li, Hong; Mao, Huawei; Yu, Meixing; Yang, Fan; Feng, Ting; Fan, Yingying; Lu, Qiao; Shen, Chongyang; Yin, Zhongwei; Mao, Meng; Tu, Wenwei

    2013-01-01

    The neonates, particularly small-for-gestational-age (SGA) ones, are susceptible to various microbial infections. Natural killer (NK) cells are critical components of host innate immunity system and the main source of the inflammatory cytokines, which provide critical protection during the early phase of viral infections before the development of an appropriate adaptive immune response. However, little is known about the antiviral effects of NK cells in neonates especially the SGA population. Herein, a prospective descriptive study was performed to determine the differences of NK cell immunity among adults, appropriate-for gestational-age (AGA) and SGA neonates. Adults have much higher NK cell number in peripheral blood than that in cord blood from neonates. In response to influenza virus stimulation, neonatal NK cells, especially SGA baby cells, expressed significantly lower antiviral cytokines including perforin, interferon (IFN)-γ and tumor-necrosis factor (TNF)-α responses than adult NK cells. In addition, the antiviral cytokine responses of NK cells were positively correlated with neonatal birth weight. Our data suggested that the depressed antiviral activity and less frequency of NK cells are likely to be responsible for the high susceptibility to microbial infection in neonates, at least in part. Improving the function of innate immunity may provide a new way to defend virus infection. PMID:23872919

  19. NK cells enhance dendritic cell response against parasite antigens via NKG2D pathway.

    PubMed

    Guan, Hongbing; Moretto, Magali; Bzik, David J; Gigley, Jason; Khan, Imtiaz A

    2007-07-01

    Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional, the mechanism or molecules involved in this cross-talk have not been identified. In this study, we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8+ T cell response by these cells. In vitro blockade of NKG2D, a molecule present on human and murine NK cells, neutralizes the NK cell-induced up-regulation of DC response. Moreover, treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8+ T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction, which apparently is important for the generation of robust CD8+ T cell immunity against intracellular pathogens. To the best of our knowledge, this is the first work that describes in vivo importance of NKG2D during natural infection.

  20. Expansion of CD16 positive and negative human NK cells in response to tumor stimulation.

    PubMed

    Tsukerman, Pinchas; Stern-Ginossar, Noam; Yamin, Rachel; Ophir, Yael; Stanietsky, Anna Miller Noa; Mandelboim, Ofer

    2014-05-01

    NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.

  1. NK cell responses to simian immunodeficiency virus vaginal exposure in naive and vaccinated rhesus macaques.

    PubMed

    Shang, Liang; Smith, Anthony J; Duan, Lijie; Perkey, Katherine E; Qu, Lucy; Wietgrefe, Stephen; Zupancic, Mary; Southern, Peter J; Masek-Hammerman, Katherine; Reeves, R Keith; Johnson, R Paul; Haase, Ashley T

    2014-07-01

    NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2Rβ. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge.

  2. NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

    PubMed Central

    Habib, Samar; El Andaloussi, Abdeljabar; Hisham, Ahmed; Ismail, Nahed

    2016-01-01

    Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia. PMID:27092553

  3. Chicken NK cell receptors.

    PubMed

    Straub, Christian; Neulen, Marie-Luise; Sperling, Beatrice; Windau, Katharina; Zechmann, Maria; Jansen, Christine A; Viertlboeck, Birgit C; Göbel, Thomas W

    2013-11-01

    Natural killer cells are innate immune cells that destroy virally infected or transformed cells. They recognize these altered cells by a plethora of diverse receptors and thereby differ from other lymphocytes that use clonally distributed antigen receptors. To date, several receptor families that play a role in either activating or inhibiting NK cells have been identified in mammals. In the chicken, NK cells have been functionally and morphologically defined, however, a conclusive analysis of receptors involved in NK cell mediated functions has not been available. This is partly due to the low frequencies of NK cells in blood or spleen that has hampered their intensive characterization. Here we will review recent progress regarding the diverse NK cell receptor families, with special emphasis on novel families identified in the chicken genome with potential as chicken NK cell receptors.

  4. Murine NK cell intrinsic cytokine-induced memory-like responses are maintained following homeostatic proliferation1

    PubMed Central

    Keppel, Molly P.; Yang, Liping; Cooper, Megan A.

    2013-01-01

    Several recent studies have demonstrated that innate immune NK cells exhibit memory-like properties with enhanced non-specific and specific recall responses. Cytokine activation alone of murine NK cells induces the differentiation of memory-like cells that are more likely to produce IFN-γ, a key NK cell cytokine important for activation of the immune response. Using an adoptive co-transfer system, we first show that cytokine-induced memory-like responses are NK intrinsic. However, engraftment of donor NK cells in NK-competent hosts is poor due to homeostatic control mechanisms. Therefore, we utilized alymphoid Rag- and common gamma chain (γc)-deficient mice as recipients and observed homeostatic expansion of co-transferred cytokine-activated and control donor NK cells. Despite proliferation of all cells, NK cells derived from those cells originally activated by cytokines retained an intrinsic enhanced capacity to produce IFN-γ when re-stimulated in vitro with cytokines or target cells. These NK cell memory-like responses persisted for at least 4 weeks in alymphoid hosts and 12 weeks in NK-competent hosts. These findings indicate that memory-like NK cells can readily self-renew and maintain enhanced function in a lymphopenic host for at least a month. PMID:23530145

  5. Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells.

    PubMed

    Pandey, Veethika; Oyer, Jeremiah L; Igarashi, Robert Y; Gitto, Sarah B; Copik, Alicja J; Altomare, Deborah A

    2016-02-09

    Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients.

  6. Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells

    PubMed Central

    Pandey, Veethika; Oyer, Jeremiah L.; Igarashi, Robert Y.; Gitto, Sarah B.; Copik, Alicja J.; Altomare, Deborah A.

    2016-01-01

    Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients. PMID:26802025

  7. Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

    PubMed

    Cha, Hefei; Qin, Wenjuan; Yang, Quan; Xie, Hongyan; Qu, Jiale; Wang, Mei; Chen, Daixiong; Wang, Fang; Dong, Nuo; Chen, Longhua; Huang, Jun

    2017-02-01

    Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ(+) and lower percentage of IL-5(+) pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17(+), IL-10(+), and IL-5(+) pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

  8. HIV's evasion of host's NK cell response and novel ways of its countering and boosting anti-HIV immunity.

    PubMed

    Ahmad, Ali; Ahmad, Rasheed

    2003-07-01

    NK cells were characterized by their ability to spontaneously kill certain tumor and virus-infected cells. They constitute first line of defense against invading pathogens and usually become activated in viral infections particularly in early phases. Activated NK cells play a crucial role in the induction and amplification of virus-specific immunity by providing IFN-gamma and "danger signal". The functional activities of NK cells are regulated by a balance between the engagement of their inhibitory and activating receptors. In recent years, the discovery of several MHC and non-MHC binding NK receptors has provided important insights regarding NK cell biology and its role in viral infections. These receptors are increasingly being viewed as important regulators of immune response. Like many other viruses, HIV also seems to activate NK cells. However, several studies have reported compromised NK cell functions in HIV-infected individuals. The virus employs several strategies to counter the host's NK cell response, e.g., a differential downregulation of MHC class I molecules on the surface of infected cells, a dysregulated production of NK cell function-enhancing cytokines, direct inhibitory effects of certain viral proteins on NK cell functions, and changes in the expression of NK cell receptors, etc. The individuals expressing activating NK cell receptors and their cognate MHC ligands have activated NK cells. The development of AIDS is significantly delayed in these individuals after HIV infection. The discovery of NK receptors and their ligands has opened new avenues of developing AIDS vaccine and boosting innate and adaptive antiviral immunity in HIV-infected individuals.

  9. NLRP3 suppresses NK cell-mediated responses to carcinogen-induced tumors and metastases.

    PubMed

    Chow, Melvyn T; Sceneay, Jaclyn; Paget, Christophe; Wong, Christina S F; Duret, Helene; Tschopp, Jürg; Möller, Andreas; Smyth, Mark J

    2012-11-15

    The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell-mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ-dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow-derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b(+)Gr-1(intermediate) (Gr-1(int)) myeloid cells within the lung tumor microenvironment of NLRP3(-/-) mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b(+)Gr-1(int) myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell-mediated control of carcinogenesis and metastases and identify CD11b(+)Gr-1(int) myeloid cells that promote NK cell antimetastatic function.

  10. HIV-1-Specific T Cell-Dependent Natural Killer (NK) Cell Activation: Major Contribution by NK Cells to Interferon-γ Production in Response to HIV-1 Antigens

    PubMed Central

    Loo, Christopher P.; Long, Brian R.; Hecht, Frederick M.; Nixon, Douglas F.

    2009-01-01

    Abstract Natural killer (NK) cells can directly recognize virus-infected cells. Here, we demonstrate that NK cells also produce interferon (IFN)-γ in an HIV-1-specific, T cell-dependent manner. After stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected individuals with HIV-1-derived peptides, up to half of the IFN-γ-producing PBMCs are NK cells. These results indicate that T cell-dependent NK cell IFN-γ production can be important for immune control of HIV-1, and have implications for the interpretation of data from vaccine trials using ELISPOT and ELISA. PMID:19500013

  11. Chemokine Receptor Expression on Normal Blood CD56(+) NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population.

    PubMed

    Lima, Margarida; Leander, Magdalena; Santos, Marlene; Santos, Ana Helena; Lau, Catarina; Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João; Teixeira, Maria Dos Anjos; Orfao, Alberto

    2015-01-01

    Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56(+low) CD16(+) and CD56(+high)  CD16(-/+low) NK-cells. Conventional CD56(+low) and CD56(+high) NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56(+low) NK-cells are mainly CXCR1/CXCR2(+) and CXCR3/CCR5(-/+), whereas mostly CD56(+high) NK-cells are CXCR1/CXCR2(-) and CXCR3/CCR5(+). Both NK-cell subsets have variable CXCR4 expression and are CCR4(-) and CCR6(-). The CKR repertoire of the CD56(+low) NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56(+high) NK-cells mimics that of Th1(+) T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56(+int) NK-cells. These NK-cells are CXCR3/CCR5(+), they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57(-) and CD158a(-). In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56(+high) and CD56(+low) NK-cells populations.

  12. Chemokine Receptor Expression on Normal Blood CD56+ NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

    PubMed Central

    Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João

    2015-01-01

    Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56+low CD16+ and CD56+high  CD16−/+low NK-cells. Conventional CD56+low and CD56+high NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56+low NK-cells are mainly CXCR1/CXCR2+ and CXCR3/CCR5−/+, whereas mostly CD56+high NK-cells are CXCR1/CXCR2− and CXCR3/CCR5+. Both NK-cell subsets have variable CXCR4 expression and are CCR4− and CCR6−. The CKR repertoire of the CD56+low NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56+high NK-cells mimics that of Th1+ T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56+int NK-cells. These NK-cells are CXCR3/CCR5+, they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57− and CD158a−. In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56+high and CD56+low NK-cells populations. PMID:26543875

  13. The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

    PubMed

    Melo-Silva, Carolina R; Tscharke, David C; Lobigs, Mario; Koskinen, Aulikki; Wong, Yik Chun; Buller, R Mark; Müllbacher, Arno; Regner, Matthias

    2011-11-01

    Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

  14. NK Cell Responses to SIV Vaginal Exposure in Naïve and Vaccinated Rhesus Macaques1

    PubMed Central

    Shang, Liang; Smith, Anthony J; Duan, Lijie; Perkey, Katherine E.; Qu, Lucy; Wietgrefe, Stephen; Zupancic, Mary; Southern, Peter J.; Masek-Hammerman, Katherine; Reeves, R. Keith; Johnson, R. Paul; Haase, Ashley T

    2014-01-01

    NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. Here we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at one week, and was thus in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion, and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa rapidly decreased in the second week, in an inverse relationship to the peak of local SIV RNA+ cells. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3, but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL2Rβ. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play at most a limited role in defenses in the FRT against vaginal challenge. PMID:24899503

  15. TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals.

    PubMed

    Adib-Conquy, Minou; Scott-Algara, Daniel; Cavaillon, Jean-Marc; Souza-Fonseca-Guimaraes, Fernando

    2014-03-01

    Natural killer (NK) cells are important in innate immunity, first described as guardians for the detection and clearance of transformed or virus-infected cells. Later, this cell type was revealed to be also able to recognize and respond to bacteria-infected cells. NK cells possess receptors allowing them to sense and respond to viral and bacterial patterns, including Toll-like receptors (TLRs). Initially described in other innate immune cells, particularly monocytes/macrophages, TLRs have more recently been characterized in NK cells. Controversies remain regarding the TLR expression in NK cells and their responsiveness to agonists, specifically the requirement for the presence of accessory cells, such as dendritic cells, or of accessory cytokines (IL-2, IL-12, IL-15 and IL-18) to respond to TLR agonists. Upon TLR activation, NK cells are an important source of IFN-γ and granulocyte macrophage colony-stimulating factor, cytokines necessary to fight infection but that can also contribute to deleterious inflammation if produced in excessive amounts. Here, we review the current knowledge concerning the expression of TLRs in and on NK cells and the responsiveness to their agonists and review the literature on the role of NK cells in the sensing of bacterial or viral patterns and in combatting infection.

  16. IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis.

    PubMed

    Venkatasubramanian, S; Cheekatla, S; Paidipally, P; Tripathi, D; Welch, E; Tvinnereim, A R; Nurieva, R; Vankayalapati, R

    2017-07-01

    Natural killer (NK) cells are traditionally considered as innate cells, but recent studies suggest that NK cells can distinguish antigens, and that memory NK cells expand and protect against viral pathogens. Limited information is available about the mechanisms involved in memory-like NK cell expansion, and their role in bacterial infections and vaccine-induced protective immune responses. In the current study, using a mouse model of tuberculosis (TB) infection, we found that interferon-gamma producing CD3-NKp46+CD27+KLRG1+ memory-like NK cells develop during Bacille Calmette-Guérin vaccination, expand, and provide protection against challenge with Mycobacterium tuberculosis (M. tb). Using antibodies, short interfering RNA and gene-deleted mice, we found that expansion of memory-like NK cells depends on interleukin 21 (IL-21). NKp46+CD27+KLRG1+ NK cells expanded in healthy individuals with latent TB infection in an IL-21-dependent manner. Our study provides first evidence that memory-like NK cells survive long term, expansion depends on IL-21, and involved in vaccine-induced protective immunity against a bacterial pathogen.

  17. Microfluidic-based, live-cell analysis allows assessment of NK-cell migration in response to crosstalk with dendritic cells.

    PubMed

    Mahmood, Sajid; Nandagopal, Saravanan; Sow, Ibrahim; Lin, Francis; Kung, Sam K P

    2014-09-01

    Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.

  18. NK cells influence both innate and adaptive immune responses after mucosal immunisation with antigen and mucosal adjuvant*

    PubMed Central

    Hall, Lindsay J; Clare, Simon; Dougan, Gordon

    2012-01-01

    NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice following intranasal immunisation with Ag85B-ESAT6 antigen from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and they secreted a diverse range of cytokines and other immunmodulators. Using antibody depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation and cytokine secretion of dendritic cells, macrophages and neutrophils in immunised NK cell depleted mice compared to control animals. Analysis of antigen-specific immune responses revealed an attenuated antibody and cytokine response in immunised NK cell depleted animals. Systemic administration of rIL-6 but not rIFN-γ significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell activated immune populations, plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunisation. PMID:20220095

  19. NK Cells and Cancer Immunoediting.

    PubMed

    Guillerey, Camille; Smyth, Mark J

    2016-01-01

    Natural killer (NK) cells are innate lymphoid cells (ILC) known for their ability to recognize and rapidly eliminate infected or transformed cells. Consequently, NK cells are fundamental for host protection against virus infections and malignancies. Even though the critical role of NK cells in cancer immunosurveillance was suspected years ago, the underlying mechanisms took time to be unraveled. Today, it is clear that anti-tumor functions of NK cells are tightly regulated and expand far beyond the simple killing of malignant cells. In spite of tremendous steps made in understanding the NK cell biology, further work is warranted to fully exploit the anticancer potential of these cells. Indeed, tumor-mediated immune suppression hampers NK cell activity, thus complicating their stimulation for therapeutic purposes. Herein, we review the current knowledge of NK cell functions in anti-tumor immunity . We discuss NK cell activity in the cancer immunoediting process with particular emphasis on the elimination and escape phases.

  20. Candida albicans Induces Metabolic Reprogramming in Human NK Cells and Responds to Perforin with a Zinc Depletion Response

    PubMed Central

    Hellwig, Daniela; Voigt, Jessica; Bouzani, Maria; Löffler, Jürgen; Albrecht-Eckardt, Daniela; Weber, Michael; Brunke, Sascha; Martin, Ronny; Kurzai, Oliver; Hünniger, Kerstin

    2016-01-01

    As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin. PMID:27242763

  1. Candida albicans Induces Metabolic Reprogramming in Human NK Cells and Responds to Perforin with a Zinc Depletion Response.

    PubMed

    Hellwig, Daniela; Voigt, Jessica; Bouzani, Maria; Löffler, Jürgen; Albrecht-Eckardt, Daniela; Weber, Michael; Brunke, Sascha; Martin, Ronny; Kurzai, Oliver; Hünniger, Kerstin

    2016-01-01

    As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin.

  2. A fatal cytokine-induced systemic inflammatory response reveals a critical role for NK cells.

    PubMed

    Carson, W E; Yu, H; Dierksheide, J; Pfeffer, K; Bouchard, P; Clark, R; Durbin, J; Baldwin, A S; Peschon, J; Johnson, P R; Ku, G; Baumann, H; Caligiuri, M A

    1999-04-15

    The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-alpha, IFN-gamma, macrophage-inflammatory protein-1alpha, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans.

  3. NK cells require antigen-specific memory CD4+ T cells to mediate superior effector functions during HSV-2 recall responses in vitro.

    PubMed

    Chen, Branson; Lee, Amanda J; Chew, Marianne V; Ashkar, Ali A

    2016-12-14

    Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4(+) T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4(+) T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4(+) T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4(+) T cells to produce IFN-γ.

  4. Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo

    PubMed Central

    Gill, Upkar S.; Peppa, Dimitra; Micco, Lorenzo; Singh, Harsimran D.; Carey, Ivana; Foster, Graham R.; Maini, Mala K.; Kennedy, Patrick T. F.

    2016-01-01

    NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined whether changes in the NK pool are induced when patients with eAg-positive CHB are ‘primed’ with PegIFNα and importantly, whether these changes are sustained or further modulated long-term after switching to nucleos(t)ides (sequential NUC therapy), an approach currently tested in the clinic. Longitudinal sampling of a prospectively recruited cohort of patients with eAg+CHB showed that the cumulative expansion of CD56bright NK cells driven by 48-weeks of PegIFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Unexpectedly, PegIFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors NKp30 and NKp46 during sequential NUCs. The expansion in proliferating, functional NK cells was more pronounced following sequential NUCs than in comparison cohorts of patients treated with de novo NUCs or PegIFNα only. Reduction in circulating HBsAg concentrations, a key goal in the path towards functional cure of CHB, was only achieved in those patients with enhancement of NK cell IFNγ and cytotoxicity but decrease in their expression of the death ligand TRAIL. In summary, we conclude that PegIFNα priming can expand a population of functional NK cells with an altered responsiveness to subsequent antiviral suppression by NUCs. Patients on sequential NUCs with a distinct NK cell profile show a decline in HBsAg, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses in CHB. PMID:27487232

  5. NK Cells: Uncertain Allies against Malaria

    PubMed Central

    Wolf, Asia-Sophia; Sherratt, Samuel; Riley, Eleanor M.

    2017-01-01

    Until recently, studies of natural killer (NK) cells in infection have focused almost entirely on their role in viral infections. However, there is an increasing awareness of the potential for NK cells to contribute to the control of a wider range of pathogens, including intracellular parasites such as Plasmodium spp. Given the high prevalence of parasitic diseases in the developing world and the devastating effects these pathogens have on large numbers of vulnerable people, investigating interactions between NK cells and parasitized host cells presents the opportunity to reveal novel immunological mechanisms with the potential to aid efforts to eradicate these diseases. The capacity of NK cells to produce inflammatory cytokines early after malaria infection, as well as a possible role in direct cytotoxic killing of malaria-infected cells, suggests a beneficial impact of NK cells in this disease. However, NK cells may also contribute to overproduction of pro-inflammatory cytokines and the consequent immunopathology. As comparatively little is known about the role of NK cells later in the course of infection, and growing evidence suggests that heterogeneity in NK cell responses to malaria may be influenced by KIR/HLA interactions, a better understanding of the mechanisms by which NK cells might directly interact with parasitized cells may reveal a new role for these cells in the course of malaria infection. PMID:28337195

  6. NK Cells: Uncertain Allies against Malaria.

    PubMed

    Wolf, Asia-Sophia; Sherratt, Samuel; Riley, Eleanor M

    2017-01-01

    Until recently, studies of natural killer (NK) cells in infection have focused almost entirely on their role in viral infections. However, there is an increasing awareness of the potential for NK cells to contribute to the control of a wider range of pathogens, including intracellular parasites such as Plasmodium spp. Given the high prevalence of parasitic diseases in the developing world and the devastating effects these pathogens have on large numbers of vulnerable people, investigating interactions between NK cells and parasitized host cells presents the opportunity to reveal novel immunological mechanisms with the potential to aid efforts to eradicate these diseases. The capacity of NK cells to produce inflammatory cytokines early after malaria infection, as well as a possible role in direct cytotoxic killing of malaria-infected cells, suggests a beneficial impact of NK cells in this disease. However, NK cells may also contribute to overproduction of pro-inflammatory cytokines and the consequent immunopathology. As comparatively little is known about the role of NK cells later in the course of infection, and growing evidence suggests that heterogeneity in NK cell responses to malaria may be influenced by KIR/HLA interactions, a better understanding of the mechanisms by which NK cells might directly interact with parasitized cells may reveal a new role for these cells in the course of malaria infection.

  7. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function.

    PubMed

    Krug, Anne; French, Anthony R; Barchet, Winfried; Fischer, Jens A A; Dzionek, Andrzej; Pingel, Jeanette T; Orihuela, Michael M; Akira, Shizuo; Yokoyama, Wayne M; Colonna, Marco

    2004-07-01

    Natural interferon-producing cells (IPC) respond to viruses by secreting type I interferon (IFN) and interleukin-12 (IL-12). Toll-like receptor (TLR) 9 mediates IPC recognition of some of these viruses in vitro. However, whether TLR9-induced activation of IPC is necessary for an effective antiviral response in vivo is not clear. Here, we demonstrate that IPC and dendritic cells (DC) recognize murine cytomegalovirus (MCMV) through TLR9. TLR9-mediated cytokine secretion promotes viral clearance by NK cells that express the MCMV-specific receptor Ly49H. Although depletion of IPC leads to a drastic reduction of the IFN-alpha response, this allows other cell types to secrete IL-12, ensuring normal IFN-gamma and NK cell responses to MCMV. We conclude that the TLR9/MyD88 pathway mediates antiviral cytokine responses by IPC, DC, and possibly other cell types, which are coordinated to promote effective NK cell function and MCMV clearance.

  8. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection

    PubMed Central

    Müller, Anna A.; Dolowschiak, Tamas; Sellin, Mikael E.; Felmy, Boas; Verbree, Carolin; Gadient, Sandra; Westermann, Alexander J.; Vogel, Jörg; LeibundGut-Landmann, Salome; Hardt, Wolf-Dietrich

    2016-01-01

    Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and –injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens. PMID:27341123

  9. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

    PubMed

    Müller, Anna A; Dolowschiak, Tamas; Sellin, Mikael E; Felmy, Boas; Verbree, Carolin; Gadient, Sandra; Westermann, Alexander J; Vogel, Jörg; LeibundGut-Landmann, Salome; Hardt, Wolf-Dietrich

    2016-06-01

    Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

  10. Higher Frequency of NK and CD4+ T-Cells in Mucosa and Potent Cytotoxic Response in HIV Controllers

    PubMed Central

    Taborda, Natalia Andrea; González, Sandra Milena; Alvarez, Cristiam Mauricio; Correa, Luis Alfonso; Montoya, Carlos Julio; Rugeles, María Teresa

    2015-01-01

    HIV infection induces immune alterations, mainly in gut mucosa, where the main target cells reside. However, the evolution of the infection is variable among infected individuals, as evidenced by HIV controllers who exhibit low or undetectable viral load in the absence of treatment. The aim of this study was to evaluate the frequency, phenotype and activity of T and NK cells in peripheral blood and gut mucosa in a cohort of Colombian HIV controllers. Blood and gut biopsies were included. The frequency and the activation status of T and NK cells were performed by flow cytometry. In addition, Gag-stimulated CD8+ T-cells and cytokine-stimulated NK cells were tested for cytotoxic activity. Finally, microbial translocation was measured by plasma lipopolysaccharide quantification. Compared with HIV-progressors, HIV controllers exhibited higher frequency of CD4+ T and NK cells, and lower expression of activation molecules in blood and mucosal immune cells, as well as lower microbial translocation. An increased production of molecules associated with cytotoxic activity of CD8+ T-cells in blood and mucosa and a higher percentage of polyfunctional CD8+ T cells in blood were also observed in HIV controllers. In addition, an increased activity of NK cells was observed in blood. These findings suggest that HIV controllers have a potent immune response, mainly mediated by cytotoxic cells that control HIV replication, which contribute to reducing alterations at the gut mucosa. PMID:26291824

  11. NK cell-based immunotherapy for malignant diseases

    PubMed Central

    Cheng, Min; Chen, Yongyan; Xiao, Weihua; Sun, Rui; Tian, Zhigang

    2013-01-01

    Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy by in vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced. PMID:23604045

  12. Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

    PubMed

    Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

    2015-09-01

    There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8(+) T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

  13. Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

    PubMed Central

    Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

    2015-01-01

    There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses. PMID:26405606

  14. "Natural Regulators": NK Cells as Modulators of T Cell Immunity.

    PubMed

    Schuster, Iona S; Coudert, Jerome D; Andoniou, Christopher E; Degli-Esposti, Mariapia A

    2016-01-01

    Natural killer (NK) cells are known as frontline responders capable of rapidly mediating a response upon encountering transformed or infected cells. Recent findings indicate that NK cells, in addition to acting as innate effectors, can also regulate adaptive immune responses. Here, we review recent studies on the immunoregulatory function of NK cells with a specific focus on their ability to affect the generation of early, as well as long-term antiviral T cell responses, and their role in modulating immune pathology and disease. In addition, we summarize the current knowledge of the factors governing regulatory NK cell responses and discuss origin, tissue specificity, and open questions about the classification of regulatory NK cells as classical NK cells versus group 1 innate lymphoid cells.

  15. Lenalidomide Induces Immunomodulation in Chronic Lymphocytic Leukemia and Enhances Antitumor Immune Responses Mediated by NK and CD4 T Cells

    PubMed Central

    Acebes-Huerta, Andrea; Huergo-Zapico, Leticia; Gonzalez-Rodriguez, Ana Pilar; Fernandez-Guizan, Azahara; Payer, Angel R.; Gonzalez, Segundo

    2014-01-01

    Lenalidomide is an immunomodulatory drug with therapeutic activity in chronic lymphocytic leukemia (CLL). However, it has pleiotropic effects, and the mechanism of action responsible for its therapeutic activity has not been well defined yet. Herein, we show that lenalidomide treatment does not have an effect on the proliferation of leukemia cells, but it increases the proliferation of B cells from healthy donors. Lenalidomide did not exert a direct effect on the apoptosis of leukemia cells obtained from CLL patients, although it indirectly induced their apoptosis through the activation of nonmalignant immune cells. Thus, lenalidomide markedly increased the proliferation of NK and CD4 T cells. The effect of lenalidomide on NK cells was secondary to the induction of IL-2 production by CD4 T cells. Accordingly, depletion of T cells or blockade of IL-2 activity completely abrogated the proliferation of NK cells. Additionally, lenalidomide enhanced NK and NKT-like cell-mediated natural cytotoxicity against leukemia cells from CLL patients. Lenalidomide also upregulated CD20 expression on leukemia cells and, accordingly, it had a synergistic effect with rituximab on promoting antibody-dependent cell-mediated cytotoxicity against primary leukemia cells. Overall, these observations provide a support for combining lenalidomide with rituximab as a treatment in CLL. PMID:25313353

  16. A phenotypic and functional characterization of NK cells in adenoids.

    PubMed

    Mizrahi, Sa'ar; Yefenof, Eitan; Gross, Menahem; Attal, Pierre; Ben Yaakov, Avraham; Goldman-Wohl, Debra; Maly, Bella; Stern, Noam; Katz, Gil; Gazit, Roi; Sionov, Ronit Vogt; Mandelboim, Ofer; Chaushu, Stella

    2007-11-01

    Adenoids are part of the MALT. In the present study, we analyzed cell surface markers and cytolytic activity of adenoidal NK (A-NK) cells and compared them with NK cells derived from blood of the same donors (B-NK). NK cells comprised 0.67% (0.4-1.2%) of the total lymphoid population isolated from adenoids. The majority (median=92%) of the A-NK cells was CD56(bright)CD16(-). A-NK cells were characterized by the increased expression of activation-induced receptors. NKp44 was detected on >60%, CD25 on >40%, and HLA-DR on >50% of freshly isolated A-NK cells. Functional assays indicated that the cytotoxic machinery of A-NK is intact, and sensitive target cells are killed via natural cytotoxicity receptors, such as NKG2D. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66) expression was up-regulated in 23% (median) of the A-NK cells by IL-2 activation but unchanged in B-NK cells. CEACAM1 inhibited the A-NK killing of target cells. CXCR4 was expressed on more than 40% A-NK cells prior to activation. Its ligand, CXCL12, was found in endothelial cells of the capillaries within the adenoid and in cells of the epithelial lining. In addition, A-NK cells migrated in vitro toward a gradient of CXCL12 in a dose-responsive manner, suggesting a role for this chemokine in A-NK cell recruitment and trafficking. We conclude that the A-NK cells are unique in that they display an activated-like phenotype and are different from their CD16(-) B-NK cell counterparts. This phenotype presumably reflects the chronic interaction of A-NK cells with antigens penetrating the body through the nasal route.

  17. The IL-15-based ALT-803 complex enhances FcγRIIIa-triggered NK cell responses and in vivo clearance of B cell lymphomas

    PubMed Central

    Rosario, Maximillian; Bai, Liu; Kong, Lin; Collins, Lynne I.; Schneider, Stephanie E.; Chen, Xiaoyue; Han, Kaiping; Jeng, Emily K.; Rhode, Peter R.; Leong, Jeffrey W.; Schappe, Timothy; Jewell, Brea A.; Keppel, Catherine R.; Shah, Keval; Hess, Brian; Romee, Rizwan; Piwnica-Worms, David R.; Cashen, Amanda F.; Bartlett, Nancy L.; Wong, Hing C.; Fehniger, Todd A.

    2015-01-01

    PURPOSE Anti-CD20 monoclonal antibodies (mAbs) are an important immunotherapy for B cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a super-agonist IL-15 mutant and IL-15Rα–Fc fusion complex that activates the IL-15 receptor constitutively expressed on NK cells. We hypothesized that ALT-803 would enhance anti-CD20 mAb-directed NK cell responses and antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK cell xenografts in NOD-SCID-γc−/− mice. REULTS We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFN-γ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK cell subset changes with preserved ADCC. CONCLUSIONS ALT-803 represents a novel immunostimulatory drug that enhances NK cell anti-lymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B cell lymphoma. PMID:26423796

  18. Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens.

    PubMed

    Fu, Xiaoying; Yu, Sifei; Yang, Binyan; Lao, Suihua; Li, Baiqing; Wu, Changyou

    2016-01-01

    Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.

  19. Ionomycin Treatment Renders NK Cells Hyporesponsive

    PubMed Central

    Romera-Cárdenas, Gema; Thomas, L. Michael; Lopez-Cobo, Sheila; García-Cuesta, Eva M.; Long, Eric O.; Reyburn, Hugh T.

    2016-01-01

    Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes. PMID:27007115

  20. Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition

    PubMed Central

    Kwon, Hyung-Joon; Choi, Go-Eun; Ryu, Sangryeol; Kwon, Soon Jae; Kim, Sun Chang; Booth, Claire; Nichols, Kim E.; Kim, Hun Sik

    2016-01-01

    NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses. PMID:27221592

  1. IL-15 complexes induce NK cell and T cell responses independent of type I IFN signalling during rhinovirus infection

    PubMed Central

    Jayaraman, Annabelle; Jackson, David J.; Message, Simon D.; Pearson, Rebecca M.; Aniscenko, Julia; Caramori, Gaetano; Mallia, Patrick; Papi, Alberto; Shamji, Betty; Edwards, Matt; Westwick, John; Hansel, Trevor; Stanciu, Luminita A.; Johnston, Sebastian L.; Bartlett, Nathan W.

    2014-01-01

    Rhinoviruses are the most common virus to infect man causing a range of serious respiratory diseases including exacerbations of asthma and COPD. Type I IFN and IL-15 are thought to be required for antiviral immunity however their function during rhinovirus infection in vivo is undefined. In RV infected human volunteers, IL-15 protein expression in fluid from the nasal mucosa and in bronchial biopsies was increased. In mice, RV induced type I IFN-dependent expression of IL-15 and IL-15Rα which in turn were required for NK- and CD8+ T-cell responses. Treatment with IL-15-IL-15Rα complexes (IL-15c) boosted RV-induced expression of IL-15, IL-15Rα, IFN-γ, CXCL9 and CXCL10 followed by recruitment of activated, IFN-γ expressing NK, CD8+ and CD4+ T cells. Treating infected IFNAR1−/− mice with IL-15c similarly increased IL-15, IL-15Rα, IFN-γ and CXCL9 (but not CXCL10) expression also followed by NK-, CD8+- and CD4+-T cell recruitment and activation. We have demonstrated that type I IFN induced IFN-γ and cellular immunity to RV was mediated by IL-15 and IL-15Rα. Importantly we also show that IL-15 could be induced via a type I IFN-independent mechanism by IL-15 complex treatment which in turn was sufficient to drive IFN-γ expression and lymphocyte responses. PMID:24472849

  2. The NK Cell Response to Mouse Cytomegalovirus Infection Affects the Level and Kinetics of the Early CD8+ T-Cell Response

    PubMed Central

    Mitrović, Maja; Arapović, Jurica; Jordan, Stefan; Fodil-Cornu, Nassima; Ebert, Stefan; Vidal, Silvia M.; Krmpotić, Astrid; Reddehase, Matthias J.

    2012-01-01

    Natural killer (NK) cells and CD8+ T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8+ T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8+ T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8+ T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8+ T cells has only a minor effect on the early control of wild-type MCMV, CD8+ T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8+ T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8+ T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction. PMID:22156533

  3. Equipping NK Cells with CARs.

    PubMed

    2017-09-06

    Adding a chimeric antigen receptor (CAR) to natural killer (NK) cells is garnering interest as a therapeutic strategy because this immune cell type doesn't cause graft-versus-host disease, making its widespread, off-the-shelf use feasible. Based on promising preclinical data, a phase I/II trial of one such CAR NK-cell therapy is under way, targeting CD19 in hematologic malignancies. ©2017 American Association for Cancer Research.

  4. The Tyrosine Kinase Pyk-2/Raftk Regulates Natural Killer (Nk) Cell Cytotoxic Response, and Is Translocated and Activated upon Specific Target Cell Recognition and Killing

    PubMed Central

    Sancho, David; Nieto, Marta; Llano, Manuel; Rodríguez-Fernández, José L.; Tejedor, Reyes; Avraham, Shalom; Cabañas, Carlos; López-Botet, Miguel; Sánchez-Madrid, Francisco

    2000-01-01

    The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK–target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK–target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response. PMID:10851022

  5. Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

    USDA-ARS?s Scientific Manuscript database

    Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFNgamma). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a dis...

  6. Location and cellular stages of NK cell development

    PubMed Central

    Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A

    2013-01-01

    The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to better harness NK cell functions in multiple clinical settings as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice. PMID:24055329

  7. Bystander cells enhance NK cytotoxic efficiency by reducing search time

    PubMed Central

    Zhou, Xiao; Zhao, Renping; Schwarz, Karsten; Mangeat, Matthieu; Schwarz, Eva C.; Hamed, Mohamed; Bogeski, Ivan; Helms, Volkhard; Rieger, Heiko; Qu, Bin

    2017-01-01

    Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H2O2 was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H2O2-producing bystander cells reduces target cell search time and enhances NK killing efficiency. PMID:28287155

  8. Bystander cells enhance NK cytotoxic efficiency by reducing search time.

    PubMed

    Zhou, Xiao; Zhao, Renping; Schwarz, Karsten; Mangeat, Matthieu; Schwarz, Eva C; Hamed, Mohamed; Bogeski, Ivan; Helms, Volkhard; Rieger, Heiko; Qu, Bin

    2017-03-13

    Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H2O2 was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H2O2-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

  9. Depletion of NK cells in a murine polytrauma model is associated with improved outcome and a modulation of the inflammatory response.

    PubMed

    Barkhausen, Tanja; Frerker, Christian; Pütz, Claudia; Pape, Hans-Christoph; Krettek, Christian; van Griensven, Martijn

    2008-10-01

    Sepsis and associated diseases such as systemic inflammatory response syndrome and multiple organ dysfunction syndrome represent common posttraumatic complications on intensive care units induced by a variety of body defense mechanisms. Natural killer (NK) cells are part of the innate immune system. They are thought to play an important role in the development of such syndromes by interplay with other immune cell types and subsequent activation of the inflammatory cascade. To test this hypothesis, NK cells were depleted by administration of antimouse asialo-GM1 antibody in a murine polytrauma model consisting of femur fracture, hemorrhagic shock, and subsequent sepsis. Mortality and immune parameters such as cytokine expression in lung and liver, lymphocyte phenotyping, lymphocyte apoptosis, and organ pathology were determined 96 h after sepsis induction. Survival values showed 50% in the control sepsis group and 100% after NK cell depletion. Thus, NK cell depletion resulted in 50% mortality reduction. Furthermore, we found reductions in the inflammatory response, represented by IL-6 expression in liver, and a decrease in infiltrating neutrophils in the liver and lung. In addition, lymphocyte apoptosis in spleen was decreased by depletion of NK cells. Taken together, these data demonstrate that NK cells contribute to the pathogenetic pathways in a murine polytrauma model. One main mechanism of action seems to be the induction of systemic inflammatory events. Thus, depletion of NK cells results in attenuated inflammation and an overall improvement in outcome. Therefore, NK cells can be considered as important targets for therapeutic strategies.

  10. Interferon response factor 3 is crucial to poly-I:C induced NK cell activity and control of B16 melanoma growth.

    PubMed

    Moore, Tyler C; Kumm, Phyllis M; Brown, Deborah M; Petro, Thomas M

    2014-04-28

    Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3-dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.

  11. Tricking the balance: NK cells in anti-cancer immunity.

    PubMed

    Pahl, Jens; Cerwenka, Adelheid

    2017-01-01

    Natural Killer (NK) cells are classically considered innate immune effector cells involved in the first line of defense against infected and malignant cells. More recently, NK cells have emerged to acquire properties of adaptive immunity in response to certain viral infections such as expansion of specific NK cell subsets and long-lasting virus-specific responses to secondary challenges. NK cells distinguish healthy cells from abnormal cells by measuring the net input of activating and inhibitory signals perceived from target cells through NK cell surface receptors. Acquisition of activating ligands in combination with reduced expression of MHC class I molecules on virus-infected and cancer cells activates NK cell cytotoxicity and release of immunostimulatory cytokines like IFN-γ. In the cancer microenvironment however, NK cells become functionally impaired by inhibitory factors produced by immunosuppressive immune cells and cancer cells. Here we review recent progress on the role of NK cells in cancer immunity. We describe regulatory factors of the tumor microenvironment on NK cell function which determine cancer cell destruction or escape from immune recognition. Finally, recent strategies that focus on exploiting NK cell anti-cancer responses for immunotherapeutic approaches are outlined. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Increased NK activity is responsible for higher cytotoxicity to HEF cells by lymphocytes of women with threatened preterm delivery.

    PubMed

    Szekeres-Bartho, J; Hadnagy, J; Csernus, V; Balázs, L; Magyarlaki, T; Pacsa, A S

    1985-01-01

    Recently we have shown that lymphocytes of pregnant women with threatened preterm delivery (risk group) exerted significantly higher cytotoxic activity to human embryonic fibroblast (HEF) cells than those of healthy pregnant women. The purpose of this study was to get information on the mechanism of this cytotoxicity. The possibility of prior sensitization to embryonic antigen was excluded, since no difference could be demonstrated between cytotoxic activity of lymphocytes obtained from women with two or more previous pregnancies and that of lymphocytes from never-pregnant women. For determining the effector cell type responsible for cytotoxicity, lymphocytes of 50 healthy pregnant women and those of 50 risk patients were tested in different cytotoxicity tests using HEF and K-562 target cells. The proportion of NK cells among lymphocytes was determined by counting large granular lymphocytes (LGL), IgG Fc receptor bearing cells, and cells positively stained by NK specific monoclonal antibody. Though no difference in the proportion of NK cells between the two groups was found, risk patients' lymphocytes were significantly more cytotoxic to K-562 target cells than those of healthy pregnant women. Investigations at the single-cell level made it obvious that this higher cytotoxic activity originated from increased target cell lysing ability of their lymphocytes, while their conjugating capacity did not differ significantly from that of lymphocytes obtained from healthy pregnant women.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Innate immunity regulates adaptive immune response: lessons learned from studying the interplay between NK and CD8+ T cells during MCMV infection.

    PubMed

    Mitrović, Maja; Arapović, Jurica; Traven, Luka; Krmpotić, Astrid; Jonjić, Stipan

    2012-11-01

    Natural killer (NK) cells play a crucial role in early immune response against cytomegalovirus infection. A large and mounting body of data indicate that these cells are involved in the regulation of the adaptive immune response as well. By using mouse cytomegalovirus (MCMV) as a model, several groups provided novel insights into the role of NK cells in the development and kinetics of antiviral CD8(+) T cell response. Depending on infection conditions, virus strain and the genetic background of mice used, NK cells are either positive or negative regulators of the CD8(+) T cell response. At present, there is no unique explanation for the observed differences between various experimental systems used. In this review we discuss the mechanisms involved in the interplay between NK and CD8(+) T cells in the early control of MCMV infection.

  14. Innate immunity regulates adaptive immune response: lessons learned from studying the interplay between NK and CD8+ T cells during MCMV infection

    PubMed Central

    Mitrović, Maja; Arapović, Jurica; Traven, Luka; Krmpotić, Astrid; Jonjić, Stipan

    2012-01-01

    Natural killer (NK) cells play a crucial role in early immune response against cytomegalovirus infection. A large and mounting body of data indicate that these cells are involved in the regulation of the adaptive immune response as well. By using mouse cytomegalovirus (MCMV) as a model, several groups provided novel insights into the role of NK cells in the development and kinetics of antiviral CD8+ T cell response. Depending on infection conditions, virus strain and the genetic background of mice used, NK cells are either positive or negative regulators of the CD8+ T cell response. At present, there is no unique explanation for the observed differences between various experimental systems used. In this review we discuss the mechanisms involved in the interplay between NK and CD8+ T cells in the early control of MCMV infection. PMID:22965169

  15. Models to Study NK Cell Biology and Possible Clinical Application.

    PubMed

    Zamora, Anthony E; Grossenbacher, Steven K; Aguilar, Ethan G; Murphy, William J

    2015-08-03

    Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes that is due to the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been described as natural killers that provide a first line of defense prior to the induction of adaptive immunity, a more complex view of NK cells is beginning to emerge, indicating they may also function in various immunoregulatory roles and have the capacity to shape adaptive immune responses. With the growing appreciation for the diverse functions of NK cells, and recent technological advancements that allow for a more in-depth understanding of NK cell biology, we can now begin to explore new ways to manipulate NK cells to increase their clinical utility. In this overview unit, we introduce the reader to various aspects of NK cell biology by reviewing topics ranging from NK cell diversity and function, mouse models, and the roles of NK cells in health and disease, to potential clinical applications. © 2015 by John Wiley & Sons, Inc.

  16. PDK1 orchestrates early NK cell development through induction of E4BP4 expression and maintenance of IL-15 responsiveness

    PubMed Central

    Yang, Meixiang; Li, Dan; Chang, Zai; Yang, Zhongzhou; Tian, Zhigang

    2015-01-01

    E4BP4, a circadian protein, is indispensable for NK cell development. It remains largely unknown which signal is required to induce E4BP4 expression and what effects it has during NK cell differentiation. Here, we reveal that PDK1, a kinase upstream of mTOR, connects IL-15 signaling to E4BP4. Early deletion of PDK1 caused a severe loss of NK cells and compromised antitumor activity in vivo. PDK1-deficient NK cells displayed much weaker IL-15–induced mTOR activation and E4BP4 induction, as well as remarkable reduction in CD122, a receptor subunit specifying NK cell responsiveness to IL-15. The phenotypes were partially reversible by ectopic expression of E4BP4 or bypassed activation of mTOR. We also determined that PDK1-mediated metabolic signaling was dispensable for NK cell terminal maturation and survival. Thus, we identify a role for PDK1 signaling as a key mediator in regulating E4BP4 expression during early NK cell development. Our findings underscore the importance of IL-15 self-responsiveness through a positive feedback loop that involves PDK1–mTOR–E4BP4–CD122 signaling. PMID:25624444

  17. IFN-γ, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses1

    PubMed Central

    Obara, Hideaki; Nagasaki, Kazuhito; Hsieh, Christine L.; Ogura, Yasuhiro; Esquivel, Carlos O.; Martinez, Olivia M.; Krams, Sheri M.

    2005-01-01

    The role of NK cells following solid organ transplantation remains unclear. We examined NK cells in acute allograft rejection using a high responder model (DA → Lewis) of rat orthotopic liver transplantation. Recipient-derived NK cells infiltrated liver allografts early after transplantation. Since chemokines are important in the trafficking of cells to areas of inflammation, we determined the intragraft expression of chemokines known to attract NK cells. CCL3 was significantly increased in allografts at 6 h post-transplant as compared to syngeneic grafts whereas CCL2 and CXCL10 were elevated in both syngeneic and allogeneic grafts. CXCL10 and CX3CL1 were significantly upregulated in allografts by day three post-transplant as compared to syngeneic grafts suggesting a role for these chemokines in the recruitment of effector cells to allografts. Graft-infiltrating NK cells were shown to be a major source of IFNγ and IFNγ levels in the serum were markedly increased, specifically in allograft recipients, by day three post-transplant. Accordingly, in the absence of NK cells the levels of IFNγ were significantly decreased. Furthermore, graft survival was significantly prolonged. These data suggest that IFNγ-producing NK cells are an important link between the innate and adaptive immune responses early after transplantation. PMID:16095488

  18. In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

    PubMed

    Kupz, Andreas; Curtiss, Roy; Bedoui, Sammy; Strugnell, Richard A

    2014-01-01

    Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

  19. Deletion of Slam locus in mice reveals inhibitory role of SLAM family in NK cell responses regulated by cytokines and LFA-1

    PubMed Central

    Guo, Huaijian; Lu, Yan; Zhang, Shaohua; Chen, Jun; Wu, Ning; Davidson, Dominique; Waggoner, Stephen N.

    2016-01-01

    Signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) can mediate either activating or inhibitory effects during natural killer cell (NK cell) activation. In this study, we addressed the global role, regulation, and mechanism of action of the SLAM family in NK cells by analyzing a mouse lacking the entire ∼400-kilobase Slam locus, which encodes all six SFRs and CD48, the ligand of SFR 2B4. This mouse displayed enhanced NK cell activation responses toward hematopoietic target cells. Analyses of mice lacking individual SFRs showed that the inhibitory function of the Slam locus was due solely to 2B4 and was not influenced positively or negatively by other SFRs. Differences in NK cell responses between recognition of targets expressing or lacking ligands for SFRs were enhanced by IL-12 but suppressed by type I interferon. Cytokines also changed the levels of SLAM-associated protein adaptors, which prevent the inhibitory function of SFRs. The enhanced activation responses of SFR-deficient NK cells were dependent on integrin LFA-1 but not on DNAM-1 or NKG2D. SFR-mediated inhibition prevented the generation of activated forms of LFA-1. Hence, the Slam locus has an overall inhibitory role during NK cell activation that is solely dependent on 2B4. This effect is influenced by cytokines and leads to suppression of LFA-1 activity. PMID:27573813

  20. Natural killer cell (NK) subsets and NK-like T-cell populations in acute myeloid leukemias and myelodysplastic syndromes.

    PubMed

    Aggarwal, N; Swerdlow, S H; TenEyck, S P; Boyiadzis, M; Felgar, R E

    2016-07-01

    The impact of the immune microenvironment on the behavior and therapeutic strategies for hematopoietic and lymphoid neoplasms is being increasingly recognized. Many functional studies of natural killer (NK) cell cytotoxic responses in myelodysplasia (MDS) and acute myeloid leukemia (AML) exist, but with limited data on these lymphocyte proportions and related T-cell subsets. The proportions of these cells and their prognostic implications were therefore investigated in 89 AML, 51 MDS, and 20 control marrows by flow cytometry. The median proportion of NK cells (relative to the total lymphocytes) was lower in AML versus controls (P = 0.01). Among AML, a lower proportion of NK cells predicted better survival, whereas a higher NK cell proportion was associated with the poor prognostic AML category (P = 0.002). NK cell proportions were similar in MDS, MDS subgroups, and control marrows. The relative proportion of the mature NK cell subset (CD56(dim) CD16/57(bright) ) was lower in AML and MDS versus controls (P = 0.006, P = 0.0002, respectively). The proportion of mature NK cells was not a prognostic indicator although fewer were seen in poor prognosis AML. In contrast, a lower proportion of mature NK cells correlated with worse survival in MDS (P = 0.027). A higher proportion of NK-like T-cells (of total lymphoid cells) was found in MDS compared to controls (P = 0.01). A lower proportion of NK-like T-cells predicted better survival in AML but not in MDS. Thus, the proportions of NK, NK-cell subsets, and NK-like T-cells vary in myeloid neoplasms, may potentially impact immunomodulatory therapies, and may impact outcome. © 2016 International Clinical Cytometry Society.

  1. NK cell-derived IL-10 is critical for DC-NK cell dialogue at the maternal-fetal interface.

    PubMed

    Blois, Sandra M; Freitag, Nancy; Tirado-González, Irene; Cheng, Shi-Bin; Heimesaat, Markus M; Bereswill, Stefan; Rose, Matthias; Conrad, Melanie L; Barrientos, Gabriela; Sharma, Surendra

    2017-05-19

    DC-NK cell interactions are thought to influence the development of maternal tolerance and de novo angiogenesis during early gestation. However, it is unclear which mechanism ensures the cooperative dialogue between DC and NK cells at the feto-maternal interface. In this article, we show that uterine NK cells are the key source of IL-10 that is required to regulate DC phenotype and pregnancy success. Upon in vivo expansion of DC during early gestation, NK cells expressed increased levels of IL-10. Exogenous administration of IL-10 was sufficient to overcome early pregnancy failure in dams treated to achieve simultaneous DC expansion and NK cell depletion. Remarkably, DC expansion in IL-10(-/-) dams provoked pregnancy loss, which could be abrogated by the adoptive transfer of IL-10(+/+) NK cells and not by IL-10(-/-) NK cells. Furthermore, the IL-10 expressing NK cells markedly enhanced angiogenic responses and placental development in DC expanded IL-10(-/-) dams. Thus, the capacity of NK cells to secrete IL-10 plays a unique role facilitating the DC-NK cell dialogue during the establishment of a healthy gestation.

  2. Redefining Memory: Building the Case for Adaptive NK Cells.

    PubMed

    Paust, Silke; Blish, Catherine A; Reeves, R Keith

    2017-10-15

    Classically, natural killer (NK) cells have been defined by nonspecific innate killing of virus-infected and tumor cells. However, burgeoning evidence suggests that the functional repertoire of NK cells is far more diverse than has been previously appreciated, thus raising the possibility that there may be unexpected functional specialization and even adaptive capabilities among NK cell subpopulations. Some of the first evidence that NK cells respond in an antigen-specific fashion came from experiments revealing that subpopulations of murine NK cells were able to respond to a specific murine cytomegalovirus (MCMV) protein and that in the absence of T and B cells, murine NK cells also mediated adaptive immune responses to a secondary challenge with specific haptens. These data have been followed by demonstrations of NK cell memory of viruses and viral antigens in mice and primates. Herein, we discuss different forms of NK cell antigen specificity and how these responses may be tuned to specific viral pathogens, and we provide assessment of the current literature that may explain molecular mechanisms of the novel phenomenon of NK cell memory. Copyright © 2017 American Society for Microbiology.

  3. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

    PubMed

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-11-03

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity.

  4. Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells

    PubMed Central

    Morelli, Maria Beatrice; Amantini, Consuelo; Santoni, Matteo; Soriani, Alessandra; Nabissi, Massimo; Cardinali, Claudio; Santoni, Angela; Santoni, Giorgio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC. Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy. On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner. Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity. PMID:26474283

  5. NK Cells, Tumor Cell Transition, and Tumor Progression in Solid Malignancies: New Hints for NK-Based Immunotherapy?

    PubMed

    Cantoni, Claudia; Huergo-Zapico, Leticia; Parodi, Monica; Pedrazzi, Marco; Mingari, Maria Cristina; Moretta, Alessandro; Sparatore, Bianca; Gonzalez, Segundo; Olive, Daniel; Bottino, Cristina; Castriconi, Roberta; Vitale, Massimo

    2016-01-01

    Several evidences suggest that NK cells can patrol the body and eliminate tumors in their initial phases but may hardly control established solid tumors. Multiple factors, including the transition of tumor cells towards a proinvasive/prometastatic phenotype, the immunosuppressive effect of the tumor microenvironment, and the tumor structure complexity, may account for limited NK cell efficacy. Several putative mechanisms of NK cell suppression have been defined in these last years; conversely, the cross talk between NK cells and tumor cells undergoing different transitional phases remains poorly explored. Nevertheless, recent in vitro studies and immunohistochemical analyses on tumor biopsies suggest that NK cells could not only kill tumor cells but also influence their evolution. Indeed, NK cells may induce tumor cells to change the expression of HLA-I, PD-L1, or NKG2D-L and modulate their susceptibility to the immune response. Moreover, NK cells may be preferentially located in the borders of tumor masses, where, indeed, tumor cells can undergo Epithelial-to-Mesenchymal Transition (EMT) acquiring prometastatic phenotype. Finally, the recently highlighted role of HMGB1 both in EMT and in amplifying the recruitment of NK cells provides further hints on a possible effect of NK cells on tumor progression and fosters new studies on this issue.

  6. NK Cells, Tumor Cell Transition, and Tumor Progression in Solid Malignancies: New Hints for NK-Based Immunotherapy?

    PubMed Central

    Huergo-Zapico, Leticia; Parodi, Monica; Pedrazzi, Marco; Mingari, Maria Cristina; Sparatore, Bianca; Gonzalez, Segundo; Olive, Daniel; Bottino, Cristina

    2016-01-01

    Several evidences suggest that NK cells can patrol the body and eliminate tumors in their initial phases but may hardly control established solid tumors. Multiple factors, including the transition of tumor cells towards a proinvasive/prometastatic phenotype, the immunosuppressive effect of the tumor microenvironment, and the tumor structure complexity, may account for limited NK cell efficacy. Several putative mechanisms of NK cell suppression have been defined in these last years; conversely, the cross talk between NK cells and tumor cells undergoing different transitional phases remains poorly explored. Nevertheless, recent in vitro studies and immunohistochemical analyses on tumor biopsies suggest that NK cells could not only kill tumor cells but also influence their evolution. Indeed, NK cells may induce tumor cells to change the expression of HLA-I, PD-L1, or NKG2D-L and modulate their susceptibility to the immune response. Moreover, NK cells may be preferentially located in the borders of tumor masses, where, indeed, tumor cells can undergo Epithelial-to-Mesenchymal Transition (EMT) acquiring prometastatic phenotype. Finally, the recently highlighted role of HMGB1 both in EMT and in amplifying the recruitment of NK cells provides further hints on a possible effect of NK cells on tumor progression and fosters new studies on this issue. PMID:27294158

  7. The role of natural killer (NK) cells and NK cell receptor polymorphisms in the assessment of HIV-1 neutralization.

    PubMed

    Brown, Bruce K; Wieczorek, Lindsay; Kijak, Gustavo; Lombardi, Kara; Currier, Jeffrey; Wesberry, Maggie; Kappes, John C; Ngauy, Viseth; Marovich, Mary; Michael, Nelson; Ochsenbauer, Christina; Montefiori, David C; Polonis, Victoria R

    2012-01-01

    The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses.

  8. The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

    PubMed Central

    Brown, Bruce K.; Wieczorek, Lindsay; Kijak, Gustavo; Lombardi, Kara; Currier, Jeffrey; Wesberry, Maggie; Kappes, John C.; Ngauy, Viseth; Marovich, Mary; Michael, Nelson; Ochsenbauer, Christina; Montefiori, David C.; Polonis, Victoria R.

    2012-01-01

    The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses. PMID:22509241

  9. Cytokine-Mediated Activation of NK Cells during Viral Infection

    PubMed Central

    Freeman, Bailey E.; Raué, Hans-Peter; Hill, Ann B.

    2015-01-01

    ABSTRACT Natural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections. IMPORTANCE Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+ NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated

  10. Cytomegalovirus Infection Drives Adaptive Epigenetic Diversification of NK Cells with Altered Signaling and Effector Function

    PubMed Central

    Schlums, Heinrich; Cichocki, Frank; Tesi, Bianca; Theorell, Jakob; Beziat, Vivien; Holmes, Tim D.; Han, Hongya; Chiang, Samuel C.C.; Foley, Bree; Mattsson, Kristin; Larsson, Stella; Schaffer, Marie; Malmberg, Karl-Johan; Ljunggren, Hans-Gustaf; Miller, Jeffrey S.; Bryceson, Yenan T.

    2015-01-01

    SUMMARY The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hyperme-thylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-associated adaptive NK cells and cytotoxic effector T cells but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets. PMID:25786176

  11. Involvement of NK cells against tumors and parasites.

    PubMed

    Papazahariadou, M; Athanasiadis, G I; Papadopoulos, E; Symeonidou, I; Hatzistilianou, M; Castellani, M L; Bhattacharya, K; Shanmugham, L N; Conti, P; Frydas, S

    2007-01-01

    Host resistance against pathogens depends on a complex interplay of innate and adaptive immune mechanisms. Acting as an early line of defence, the immune system includes activation of neutrophils, tissue macrophages, monocytes, dendritic cells, eosinophils and natural killer (NK) cells. NK cells are lymphoid cells that can be activated without previous stimulation and are therefore like macrophages in the first line of defence against tumor cells and a diverse range of pathogens. NK cells mediate significant activity and produce high levels of proinflammatory cytokines in response to infection. Their cytotoxicity production is induced principally by monocyte-, macrophage- and dendritic cell-derived cytokines, but their activation is also believed to be cytokine-mediated. Recognition of infection by NK cells is accomplished by numerous activating and inhibitory receptors on the NK cells' surface that selectively trigger the cytolytic activity in a major histocompability complex-independent manner. NK cells have trypanocidal activity of fibroblast cells and mediate direct destruction of extracellular epimastigote and trypomastigote forms of T. cruzi and T. lewisi in vitro; moreover, they kill plasmodia-infected erythrocytes directly through cell-cell interaction. This review provides a more detailed analysis of how NK cells recognize and respond to parasites and how they mediate cytotoxicity against tumor cells. Also the unique role of NK cells in innate immunity to infection and the relationship between parasites and carcinogenesis are discussed.

  12. Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

    PubMed Central

    Asea, A; Stein-Streilein, J

    1998-01-01

    In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4. Images Figure 4 PMID:9616382

  13. Molecular characterization of HCMV-specific immune responses: Parallels between CD8(+) T cells, CD4(+) T cells, and NK cells.

    PubMed

    Vieira Braga, Felipe A; Hertoghs, Kirsten M L; van Lier, René A W; van Gisbergen, Klaas P J M

    2015-09-01

    CD8(+) T cells are important for immunity against human cytomegalovirus (HCMV). The HCMV-specific CD8(+) T-cell response is characterized by the accumulation of terminally differentiated effector cells that have downregulated the costimulatory molecules CD27 and CD28. These HCMV-specific CD8(+) T cells maintain high levels of cytotoxic molecules such as granzyme B and rapidly produce the inflammatory cytokine IFN-γ upon activation. Remarkably, HCMV-specific CD8(+) T cells are able to persist long term as fully functional effector cells, suggesting a unique differentiation pathway that is distinct from the formation of memory CD8(+) T cells after infection with acute viruses. In this review, we aim to highlight the most recent developments in HCMV-specific CD8(+) T-cell differentiation, maintenance, tissue distribution, metabolism and function. HCMV also induces the differentiation of effector CD4(+) T cells and NK cells, which share characteristics with HCMV-specific CD8(+) T cells. We propose that the overlap in differentiation of NK cells, CD4(+) and CD8(+) T cells after HCMV infection may be regulated by a shared transcriptional machinery. A better understanding of the molecular framework of HCMV-specific CD8(+) T-cell responses may benefit vaccine design, as these cells uniquely combine the capacity to rapidly respond to infection with long-term survival. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

    PubMed Central

    Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

    2014-01-01

    Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

  15. Hematopoietic stem cell-derived myeloid and plasmacytoid DC-based vaccines are highly potent inducers of tumor-reactive T cell and NK cell responses ex vivo.

    PubMed

    Thordardottir, Soley; Schaap, Nicolaas; Louer, Elja; Kester, Michel G D; Falkenburg, J H Frederik; Jansen, Joop; Radstake, Timothy R D; Hobo, Willemijn; Dolstra, Harry

    2017-01-01

    Because of the potent graft-versus-tumor (GVT) effect, allogeneic stem cell transplantation (alloSCT) can be a curative therapy for hematological malignancies. However, relapse remains the most frequent cause of treatment failure, illustrating the necessity for development of adjuvant post-transplant therapies to boost GVT immunity. Dendritic cell (DC) vaccination is a promising strategy in this respect, in particular, where distinct biologic functions of naturally occurring DC subsets, i.e. myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), are harnessed. However, it is challenging to obtain high enough numbers of primary DC subsets from blood for immunotherapy due to their low frequencies. Therefore, we present here an ex vivo GMP-compliant cell culture protocol for generating different DC subsets from CD34(+) hematopoietic stem and progenitor cells (HSPCs) of alloSCT donor origin. High numbers of BDCA1(+) mDCs and pDCs could be generated, sufficient for multiple vaccination cycles. These HSPC-derived DC subsets were highly potent in inducing antitumor immune responses in vitro. Notably, HSPC-derived BDCA1(+) mDCs were superior in eliciting T cell responses. They efficiently primed naïve T cells and robustly expanded patient-derived minor histocompatibility antigen (MiHA)-specific T cells. Though the HSPC-pDCs also efficiently induced T cell responses, they exhibited superior capacity in activating NK cells. pDC-primed NK cells highly upregulated TRAIL and possessed strong cytolytic capacity against tumor cells. Collectively, these findings indicate that HSPC-derived DC vaccines, comprising both mDCs and pDCs, may possess superior potential to boost antitumor immunity post alloSCT, due to their exceptional T cell and NK cell stimulatory capacity.

  16. Recruitment and Activation of Natural Killer (Nk) Cells in Vivo Determined by the Target Cell Phenotype

    PubMed Central

    Glas, Rickard; Franksson, Lars; Une, Clas; Eloranta, Maija-Leena; Öhlén, Claes; Örn, Anders; Kärre, Klas

    2000-01-01

    Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon γ in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I–deficient tumor cells were ∼10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I–deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize. PMID:10620611

  17. Distinct phenotype and function of NK cells in the pancreas of nonobese diabetic mice.

    PubMed

    Brauner, Hanna; Elemans, Marjet; Lemos, Sara; Broberger, Christian; Holmberg, Dan; Flodström-Tullberg, Malin; Kärre, Klas; Höglund, Petter

    2010-03-01

    Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-gamma spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

  18. NK cell subsets in autoimmune diseases.

    PubMed

    Zhang, Cai; Tian, Zhigang

    2017-03-09

    Natural killer (NK) cells are lymphocytes of the innate immune system. They not only exert cell-mediated cytotoxicity against tumor cells or infected cells, but also play regulatory role through promoting or suppressing functions of other immune cells by secretion of cytokines and chemokines. However, overactivation or dysfunction of NK cells may be associated with pathogenesis of some diseases. NK cells are found to act as a two edged weapon and play opposite roles with both regulatory and inducer activity in autoimmune diseases. Though the precise mechanisms for the opposite effects of NK cells has not been fully elucidated, the importance of NK cells in autoimmune diseases might be associated with different NK cell subsets, different tissue microenvironment and different stages of corresponding diseases. The local tissue microenvironment, unique cellular interactions and different stages of corresponding diseases shape the properties and function of NK cells. In this review, we focus on recent research on the features and function of different NK cell subsets, particularly tissue-resident NK cells in different tissues, and their potential role in autoimmune diseases.

  19. Identification of Human Memory-Like NK Cells.

    PubMed

    Kovalenko, Elena I; Streltsova, Maria A; Kanevskiy, Leonid M; Erokhina, Sophia A; Telford, William G

    2017-01-05

    Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57-positive cells with high expression of activating-receptor NKG2C, increased expression of KIR receptors, lack of co-expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow-cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.

  20. Differentiation and functional regulation of human fetal NK cells.

    PubMed

    Ivarsson, Martin A; Loh, Liyen; Marquardt, Nicole; Kekäläinen, Eliisa; Berglin, Lena; Björkström, Niklas K; Westgren, Magnus; Nixon, Douglas F; Michaëlsson, Jakob

    2013-09-01

    The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I-negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β-mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β-mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I-negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD-induced fetal anemia.

  1. Differentiation and functional regulation of human fetal NK cells

    PubMed Central

    Ivarsson, Martin A.; Loh, Liyen; Marquardt, Nicole; Kekäläinen, Eliisa; Berglin, Lena; Björkström, Niklas K.; Westgren, Magnus; Nixon, Douglas F.; Michaëlsson, Jakob

    2013-01-01

    The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I–negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β–mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β–mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I–negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD–induced fetal anemia. PMID:23945237

  2. NK Cells and Their Ability to Modulate T Cells during Virus Infections

    PubMed Central

    Cook, Kevin D.; Waggoner, Stephen N.; Whitmire, Jason K.

    2014-01-01

    Natural killer (NK) cells are important in protection against virus infections, and many viruses have evolved mechanisms to thwart NK cell activity. NK cells respond to inflammatory signals at an early stage of virus infection, resulting in proliferation, cytokine production, and cytolytic activity that can reduce virus loads. Moreover, the rapid kinetics of the NK cell response enables NK cells to influence other populations of innate immune cells, affect the inflammatory milieu, and guide adaptive immune responses to infection. Early NK cell interactions with other leukocytes can have long-lasting effects on the number and quality of memory T cells, as well as impact the exhaustion of T cells during chronic infections. The ability of NK cells to modulate T cell responses can be mediated through direct T-NK interactions, cytokine production, or indirectly through dendritic cells and other cell types. Herein, we summarize our current understanding of how NK cells interact with T cells, dendritic cells, B cells, and other cell types involved in adaptive immune responses to virus infection. We outline several mechanisms by which NK cells enhance or suppress adaptive immune response and long-lived immunological memory. PMID:25404045

  3. Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4.

    PubMed

    Sayós, J; Nguyen, K B; Wu, C; Stepp, S E; Howie, D; Schatzle, J D; Kumar, V; Biron, C A; Terhorst, C

    2000-12-01

    SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4-SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAM-SAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of Epstein-Barr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAM-SLAM and 2B4-CD48 interactions.

  4. Involvement of autophagy in NK cell development and function.

    PubMed

    López-Soto, Alejandro; Bravo-San Pedro, José Manuel; Kroemer, Guido; Galluzzi, Lorenzo; Gonzalez, Segundo

    2017-03-04

    Natural killer (NK) cells are the prototypical members of the recently identified family of innate lymphoid cells (ILCs). Thanks to their cytotoxic and secretory functions, NK cells play a key role in the immune response to cells experiencing various forms of stress, including viral infection and malignant transformation. Autophagy is a highly conserved network of degradative pathways that participate in the maintenance of cellular and organismal homeostasis as they promote adaptation to adverse microenvironmental conditions. The relevance of autophagy in the development and functionality of cellular components of the adaptive immune system is well established. Conversely, whether autophagy also plays an important role in the biology of ILC populations such as NK cells has long remained elusive. Recent experimental evidence shows that ablating Atg5 (autophagy-related 5, an essential component of the autophagic machinery) in NK cells and other specific ILC populations results in progressive mitochondrial damage, reactive oxygen species (ROS) overgeneration, and regulated cell death, hence interrupting ILC development. Moreover, disrupting the interaction of ATG7 with phosphorylated FOXO1 (forkhead box O1) in the cytosol of immature NK cells prevents autophagic responses that are essential for NK cell maturation. These findings suggest that activating autophagy may support the maturation of NK cells and other ILCs that manifest antiviral and anticancer activity.

  5. Immunoregulatory Role of NK Cells in Tissue Inflammation and Regeneration

    PubMed Central

    Tosello-Trampont, Annie; Surette, Fionna A.; Ewald, Sarah E.; Hahn, Young S.

    2017-01-01

    NK cells represent an important first line of defense against viral infection and cancer and are also involved in tissue homeostasis. Studies of NK cell activation in the last decade have revealed that they are able to respond to the inflammatory stimuli evoked by tissue damage and contribute to both progression and resolution of diseases. Exacerbation of the inflammatory response through interactions between immune effector cells facilitates the progression of non-alcoholic fatty liver disease (NAFLD) into steatosis, cirrhosis, and hepatocellular carcinoma (HCC). When hepatic damage is incurred, macrophage activation is crucial for initiating cross talk with neighboring cells present in the liver, including hepatocytes and NK cells, and the importance of this interaction in shaping the immune response in liver disease is increasingly recognized. Inflicted structural damage can be in part regenerated via the process of self-limiting fibrosis, though persistent hepatic damage will lead to chronic fibrosis and loss of tissue organization and function. The cytotoxic activity of NK cells plays an important role in inducing hepatic stellate cell apoptosis and thus curtailing the progression of fibrosis. Alternatively, in some diseases, such as HCC, NK cells may become dysregulated, promoting an immunosuppressive state where tumors are able to escape immune surveillance. This review describes the current understanding of the contributions of NK cells to tissue inflammation and metabolic liver diseases and the ongoing effort to develop therapeutics that target the immunoregulatory function of NK cells. PMID:28373874

  6. Effects of Simulated Microgravity on Primary Human NK Cells

    PubMed Central

    Li, Qi; Mei, Qibing; Huyan, Ting; Xie, Li; Che, Su; Yang, Hui; Zhang, Mingjie

    2013-01-01

    Abstract The deleterious effects of microgravity on lymphocytes have been demonstrated in previous studies. However, research on the effects of microgravity on human natural killer (NK) cells remains exceedingly limited. In this study, we demonstrated that NK cell cytotoxicity was significantly decreased under simulated microgravity (SMG) conditions (p<0.05). Several processes, including apoptosis, receptor expression, and cytokine secretion, were investigated in human NK cells under SMG. We observed decreased cytotoxicity, concurrent with increased apoptosis and necrosis, in NK cells after exposure to SMG (p<0.05). Additionally, interferon (IFN)-γ and perforin expression decreased significantly, and the expression of granzyme-B was only slightly reduced. Meanwhile, SMG selectively inhibited the expression of certain surface receptors on NK cells. Specifically, the expression of NKG2A and NKG2D were significantly downregulated under SMG, but the expression of NKp30 and NKp44 was not affected. We also found that interleukin (IL)–15 alone or in combination with IL-12 could counteract the inhibition of NK cell cytotoxicity under SMG. Our findings indicate that human NK cells were sensitive to SMG, as reflected by their decreased cytotoxicity. Factors such as increased early apoptosis and late apoptosis/necrosis and the decreased expression of INF-γ, cytolytic proteins, and cell surface receptors may be responsible for the loss of cytotoxicity in human NK cells under SMG. A combination of IL-12 and IL-15 may be useful as a therapeutic strategy for overcoming the effects of microgravity on human NK cells during long space missions. Key Words: Simulated microgravity (SMG)—Natural killer (NK) cells—Cytotoxicity. Astrobiology 13, 703–714. PMID:23919749

  7. Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

    PubMed

    Gabrielli, Sara; Sun, Mengxi; Bell, April; Zook, Erin C; de Pooter, Renee F; Zamai, Loris; Kee, Barbara L

    2017-03-09

    Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells. This article is protected by copyright. All rights reserved.

  8. Human NK Cell Diversity in Viral Infection: Ramifications of Ramification

    PubMed Central

    Strauss-Albee, Dara M.; Blish, Catherine A.

    2016-01-01

    Natural killer (NK) cells are a unique lymphocyte lineage with remarkable agility in the rapid destruction of virus-infected cells. They are also the most poorly understood class of lymphocyte. A spectrum of activating and inhibitory receptors at the NK cell surface leads to an unusual and difficult-to-study mechanism of cellular recognition, as well as a very high capacity for diversity at the single-cell level. Here, we review the evidence for the role of NK cells in the earliest stage of human viral infection, and in its prevention. We argue that single-cell diversity is a logical evolutionary adaptation for their position in the immune response and contributes to their ability to kill virus-infected cells. Finally, we look to the future, where emerging single-cell technologies will enable a new generation of rigorous and clinically relevant studies on NK cells accounting for all of their unique and diverse characteristics. PMID:26973646

  9. ACTIVATED NOTCH SUPPORTS DEVELOPMENT OF CYTOKINE PRODUCING NK CELLS WHICH ARE HYPORESPONSIVE AND FAIL TO ACQUIRE NK CELL EFFECTOR FUNCTIONS

    PubMed Central

    Bachanova, Veronika; McCullar, Valarie; Lenvik, Todd; Wangen, Rosanna; Peterson, Karen A.; Ankarlo, Dave EM.; Panoskaltsis-Mortari, Angela; Wagner, John E.; Miller, Jeffrey S.

    2009-01-01

    Natural Killer (NK) cells are powerful effectors of cytotoxicity against “stressed” cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34+/ICN+ sorted cells resulted in a population of CD7+ early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN+ precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as IL-13, GM-CSF, TNF-α, yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN+ precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 γ, ε, δ chains, RAG1/2 and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7+ lymphoid precursor from UCB CD34+ progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive and poorly acquire NK cell receptors involved in self tolerance and effector function. PMID:19167678

  10. Persistence of Pathological Distribution of NK Cells in HIV-Infected Patients with Prolonged Use of HAART and a Sustained Immune Response

    PubMed Central

    Frias, Mario; Rivero-Juarez, Antonio; Gordon, Ana; Camacho, Angela; Cantisan, Sara; Cuenca-Lopez, Francisca; Torre-Cisneros, Julian; Peña, Jose; Rivero, Antonio

    2015-01-01

    Objective A prospective analysis of the distribution of NK subsets and natural cytotoxicity receptors (NKp30/NKp46) in HIV patients with long-term HAART use and sustained virological and immunological response. Methods The main inclusion criteria were: at least 3 years’ receipt of HAART; current CD4+ count ≥ 500 cells/mm3; undetectable viral load for at least 24 months; no hepatotropic virus co-infection. Percentages of CD56dim, CD56bright NK cells and CD56neg CD16+ cells were obtained. Expression of the NCRs, NKp30 and NKp46 was analysed in CD56+ cells. Thirty-nine infected patients and sixteen healthy donors were included in the study. Results The percentages of total CD56+ and CD56dim NK cells were significantly lower in HIV-infected patients than in healthy donors (70.4 vs. 50.3 and 80.9 vs. 66.1 respectively). The percentage of total CD56+ NK cells expressing NCR receptors was lower in HIV patients than in healthy donors (NKp30: 25.20 vs. 58.63; NKp46: 24.8 vs. 50.59). This was also observed for CD56dim and CD56bright NK cells. Length of time with undetectable HIV viral load was identified as an independent factor associated with higher expression of NKp30 and NKp46. Conclusion Despite the prolonged and effective use of HAART, HIV-infected patients do not fully reconstitute the distribution of NK cells. Length of time with an undetectable viral load was related to greater recovery of NKp30/NKp46 receptors. PMID:25811634

  11. Pegylated Interferon α-2a Triggers NK-Cell Functionality and Specific T-Cell Responses in Patients with Chronic HBV Infection without HBsAg Seroconversion.

    PubMed

    Bruder Costa, Juliana; Dufeu-Duchesne, Tania; Leroy, Vincent; Bertucci, Inga; Bouvier-Alias, Magali; Pouget, Noelle; Brevot-Lutton, Ophelie; Bourliere, Marc; Zoulim, Fabien; Plumas, Joel; Aspord, Caroline

    2016-01-01

    Pegylated interferon α-2a (Peg-IFN-α) represents a therapeutic alternative to the prolonged use of nucleos(t)ide analog (NA) in chronic hepatitis B (CHB) infection. The mechanisms leading to a positive clinical outcome remain unclear. As immune responses are critical for virus control, we investigated the effects of Peg-IFN-α on both innate and adaptive immunity, and related it to the clinical evolution. The phenotypic and functional features of the dendritic cells (DCs), natural killer (NK) cells and HBV-specific CD4/CD8 T cells were analyzed in HBeAg-negative CHB patients treated for 48-weeks with NA alone or together with Peg-IFN-α, before, during and up to 2-years after therapy. Peg-IFN-α induced an early activation of DCs, a potent expansion of the CD56bright NK subset, and enhanced the activation and functionality of the CD56dim NK subset. Peg-IFN-α triggered an increase in the frequencies of Th1- and Th17-oriented HBV-specific CD4/CD8 T cells. Peg-IFN-α reversed the unresponsiveness of patients to a specific stimulation. Most of the parameters returned to baseline after the stop of Peg-IFN-α therapy. Peg-IFN-α impacts both innate and adaptive immunity, overcoming dysfunctional immune responses in CHB patients. These modulations were not associated with seroconversion, which questioned the benefit of the add-on Peg-IFN-α treatment.

  12. Pegylated Interferon α-2a Triggers NK-Cell Functionality and Specific T-Cell Responses in Patients with Chronic HBV Infection without HBsAg Seroconversion

    PubMed Central

    Bruder Costa, Juliana; Dufeu-Duchesne, Tania; Leroy, Vincent; Bertucci, Inga; Bouvier-Alias, Magali; Pouget, Noelle; Brevot-Lutton, Ophelie; Bourliere, Marc; Zoulim, Fabien

    2016-01-01

    Pegylated interferon α-2a (Peg-IFN-α) represents a therapeutic alternative to the prolonged use of nucleos(t)ide analog (NA) in chronic hepatitis B (CHB) infection. The mechanisms leading to a positive clinical outcome remain unclear. As immune responses are critical for virus control, we investigated the effects of Peg-IFN-α on both innate and adaptive immunity, and related it to the clinical evolution. The phenotypic and functional features of the dendritic cells (DCs), natural killer (NK) cells and HBV-specific CD4/CD8 T cells were analyzed in HBeAg-negative CHB patients treated for 48-weeks with NA alone or together with Peg-IFN-α, before, during and up to 2-years after therapy. Peg-IFN-α induced an early activation of DCs, a potent expansion of the CD56bright NK subset, and enhanced the activation and functionality of the CD56dim NK subset. Peg-IFN-α triggered an increase in the frequencies of Th1- and Th17-oriented HBV-specific CD4/CD8 T cells. Peg-IFN-α reversed the unresponsiveness of patients to a specific stimulation. Most of the parameters returned to baseline after the stop of Peg-IFN-α therapy. Peg-IFN-α impacts both innate and adaptive immunity, overcoming dysfunctional immune responses in CHB patients. These modulations were not associated with seroconversion, which questioned the benefit of the add-on Peg-IFN-α treatment. PMID:27348813

  13. Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets

    PubMed Central

    Mace, Emily M.; Orange, Jordan S.

    2016-01-01

    Human NK cells play critical roles in human host defense, particularly the control of viral infection and malignancy, and patients with congenital immunodeficiency affecting NK cell function or number can suffer from severe illness. The importance of NK cell function is particularly underscored in patients with primary immunodeficiency in which NK cells are the primary or sole affected population (NK cell deficiency, NKD). While NKD may lead to the absence of NK cells, we are also gaining an increasing appreciation of the effect that NKD may have on the generation of specific NK cell subsets. In turn, this leads to improved insights into the requirements for human NK cell subset generation, as well as their importance in immune homeostasis. The presence of inherently abnormally developed or functionally impaired NK cells, in particular, appears to be problematic in the way of interfering with normal human host defense and may be more impactful than low numbers of NK cells alone. Here, we review the known genetic causes of NKD and the insight that is derived by these into the requirements for human subset generation and, by extension, for NK cell-mediated immunity. PMID:27994588

  14. Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets.

    PubMed

    Mace, Emily M; Orange, Jordan S

    2016-01-01

    Human NK cells play critical roles in human host defense, particularly the control of viral infection and malignancy, and patients with congenital immunodeficiency affecting NK cell function or number can suffer from severe illness. The importance of NK cell function is particularly underscored in patients with primary immunodeficiency in which NK cells are the primary or sole affected population (NK cell deficiency, NKD). While NKD may lead to the absence of NK cells, we are also gaining an increasing appreciation of the effect that NKD may have on the generation of specific NK cell subsets. In turn, this leads to improved insights into the requirements for human NK cell subset generation, as well as their importance in immune homeostasis. The presence of inherently abnormally developed or functionally impaired NK cells, in particular, appears to be problematic in the way of interfering with normal human host defense and may be more impactful than low numbers of NK cells alone. Here, we review the known genetic causes of NKD and the insight that is derived by these into the requirements for human subset generation and, by extension, for NK cell-mediated immunity.

  15. Bacillus Calmette-Guérin (BCG) Revaccination of Adults with Latent Mycobacterium tuberculosis Infection Induces Long-Lived BCG-Reactive NK Cell Responses.

    PubMed

    Suliman, Sara; Geldenhuys, Hennie; Johnson, John L; Hughes, Jane E; Smit, Erica; Murphy, Melissa; Toefy, Asma; Lerumo, Lesedi; Hopley, Christiaan; Pienaar, Bernadette; Chheng, Phalkun; Nemes, Elisa; Hoft, Daniel F; Hanekom, Willem A; Boom, W Henry; Hatherill, Mark; Scriba, Thomas J

    2016-08-15

    One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette-Guérin (BCG) in healthy, tuberculin skin test-positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ-expressing CD8 T, γδ T, CD3(+)CD56(+) NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis- or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ-expressing BCG-reactive CD3(+)CD56(+) NKT-like cells and CD3(-)CD56(dim) and CD3(-)CD56(hi) NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

  16. Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells.

    PubMed

    Liu, Lisa L; Béziat, Vivien; Oei, Vincent Y S; Pfefferle, Aline; Schaffer, Marie; Lehmann, Sören; Hellström-Lindberg, Eva; Söderhäll, Stefan; Heyman, Mats; Grandér, Dan; Malmberg, Karl-Johan

    2017-08-01

    Manipulation of human natural killer (NK) cell repertoires promises more effective strategies for NK cell-based cancer immunotherapy. A subset of highly differentiated NK cells, termed adaptive NK cells, expands naturally in vivo in response to human cytomegalovirus (HCMV) infection, carries unique repertoires of inhibitory killer cell immunoglobulin-like receptors (KIR), and displays strong cytotoxicity against tumor cells. Here, we established a robust and scalable protocol for ex vivo generation and expansion of adaptive NK cells for cell therapy against pediatric acute lymphoblastic leukemia (ALL). Culture of polyclonal NK cells together with feeder cells expressing HLA-E, the ligand for the activating NKG2C receptor, led to selective expansion of adaptive NK cells with enhanced alloreactivity against HLA-mismatched targets. The ex vivo expanded adaptive NK cells gradually obtained a more differentiated phenotype and were specific and highly efficient killers of allogeneic pediatric T- and precursor B-cell acute lymphoblastic leukemia (ALL) blasts, previously shown to be refractory to killing by autologous NK cells and the NK-cell line NK92 currently in clinical testing. Selective expansion of NK cells that express one single inhibitory KIR for self-HLA class I would allow exploitation of the full potential of NK-cell alloreactivity in cancer immunotherapy. In summary, our data suggest that adaptive NK cells may hold utility for therapy of refractory ALL, either as a bridge to transplant or for patients that lack stem cell donors. Cancer Immunol Res; 5(8); 654-65. ©2017 AACR. ©2017 American Association for Cancer Research.

  17. Tissue resident NK cells mediate ischemic kidney injury and are not depleted by anti-Asialo GM1 antibody

    PubMed Central

    Victorino, Francisco; Sojka, Dorothy K.; Brodsky, Kelley S.; McNamee, Eoin N.; Masterson, Joanne C.; Homann, Dirk; Yokoyama, Wayne M.; Eltzschig, Holger K.; Clambey, Eric T.

    2015-01-01

    NK cells are innate lymphoid cells important for immune surveillance, identifying and responding to stress, infection, and/or transformation. While conventional NK (cNK) cells circulate systemically, many NK cells reside in tissues where they appear to be poised to locally regulate tissue function. Here we tested the contribution of tissue-resident NK (trNK) cells to tissue homeostasis by studying ischemic injury in the mouse kidney. Parabiosis experiments demonstrate that the kidney contains a significant fraction of trNK cells under homeostatic conditions. Kidney trNK cells developed independent of NFIL3 and Tbet, and expressed a distinct cell surface phenotype as compared to cNK cells. Among these, trNK cells had reduced asialo-GM1 (AsGM1) expression relative to cNK cells, a phenotype observed in trNK cells across multiple organs and mouse strains. Strikingly, anti-AsGM1 antibody treatment, commonly used as NK cell-depleting regimen, resulted in a robust and selective depletion of cNKs, leaving trNKs largely intact. Using this differential depletion, we tested the relative contribution of cNK and trNK cells in ischemic kidney injury. Whereas anti-NK1.1 antibody effectively depleted both trNK and cNK cells and protected against ischemic-reperfusion injury, anti-AsGM1 antibody preferentially depleted cNK cells and failed to protect against injury. These data demonstrate unanticipated specificity of anti-AsGM1 antibody depletion on NK cell subsets and reveal a new approach to study the contributions of cNK and trNK cells in vivo. In total, these data demonstrate that trNK cells play a key role in modulating local responses to ischemic tissue injury in the kidney and potentially other organs. PMID:26453755

  18. Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment.

    PubMed

    Alvarez, Maite; Sun, Kai; Murphy, William J

    2016-03-03

    Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as "licensed" and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively.

  19. No monkey business: why studying NK cells in non-human primates pays off.

    PubMed

    Hong, Henoch S; Rajakumar, Premeela A; Billingsley, James M; Reeves, R Keith; Johnson, R Paul

    2013-01-01

    Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56(bright) and CD56(dim) NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.

  20. Natural Killer Cells for Immunotherapy - Advantages of the NK-92 Cell Line over Blood NK Cells.

    PubMed

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

  1. DOCK8 Drives Src-Dependent NK Cell Effector Function.

    PubMed

    Kearney, Conor J; Vervoort, Stephin J; Ramsbottom, Kelly M; Freeman, Andrew J; Michie, Jessica; Peake, Jane; Casanova, Jean-Laurent; Picard, Capucine; Tangye, Stuart G; Ma, Cindy S; Johnstone, Ricky W; Randall, Katrina L; Oliaro, Jane

    2017-08-09

    Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause an autosomal recessive form of hyper-IgE syndrome, characterized by chronic immunodeficiency with persistent microbial infection and increased incidence of malignancy. These manifestations suggest a defect in cytotoxic lymphocyte function and immune surveillance. However, how DOCK8 regulates NK cell-driven immune responses remains unclear. In this article, we demonstrate that DOCK8 regulates NK cell cytotoxicity and cytokine production in response to target cell engagement or receptor ligation. Genetic ablation of DOCK8 in human NK cells attenuated cytokine transcription and secretion through inhibition of Src family kinase activation, particularly Lck, downstream of target cell engagement or NKp30 ligation. PMA/Ionomycin treatment of DOCK8-deficient NK cells rescued cytokine production, indicating a defect proximal to receptor ligation. Importantly, NK cells from DOCK8-deficient patients had attenuated production of IFN-γ and TNF-α upon NKp30 stimulation. Taken together, we reveal a novel molecular mechanism by which DOCK8 regulates NK cell-driven immunity. Copyright © 2017 by The American Association of Immunologists, Inc.

  2. Influenza vaccine induces intracellular immune memory of human NK cells.

    PubMed

    Dou, Yaling; Fu, Binqing; Sun, Rui; Li, Wenting; Hu, Wanfu; Tian, Zhigang; Wei, Haiming

    2015-01-01

    Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)+CD56dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.

  3. Epigenetic regulation of NK cell differentiation and effector functions

    PubMed Central

    Cichocki, Frank; Miller, Jeffrey S.; Anderson, Stephen K.; Bryceson, Yenan T.

    2013-01-01

    Upon maturation, natural killer (NK) cells acquire effector functions and regulatory receptors. New insights suggest a considerable functional heterogeneity and dynamic regulation of receptor expression in mature human NK cell subsets based on different developmental axes. Such processes include acquisition of lytic granules as well as regulation of cytokine production in response to exogenous cytokine stimulation or target cell interactions. One axis is regulated by expression of inhibitory receptors for self-MHC class I molecules, whereas other axes are less well defined but likely are driven by different activating receptor engagements or cytokines. Moreover, the recent identification of long-lived NK cell subsets in mice that are able to expand and respond rapidly following a secondary viral challenge suggest previously unappreciated plasticity in the programming of NK cell differentiation. Here, we review advances in our understanding of mature NK cell development and plasticity with regards to regulation of cellular function. Furthermore, we highlight some of the major questions that remain pertaining to the epigenetic changes that underlie the differentiation and functional specialization of NK cells and the regulation of their responses. PMID:23450696

  4. NK cells activated by Interleukin-4 in cooperation with Interleukin-15 exhibit distinctive characteristics

    PubMed Central

    Kiniwa, Tsuyoshi; Enomoto, Yutaka; Terazawa, Natsumi; Omi, Ai; Miyata, Naoko; Ishiwata, Kenji; Miyajima, Atsushi

    2016-01-01

    Natural killer (NK) cells are known to be activated by Th1-type cytokines, such as IL-2, -12, or -18, and they secrete a large amount of IFN-γ that accelerates Th1-type responses. However, the roles of NK cells in Th2-type responses have remained unclear. Because IL-4 acts as an initiator of Th2-type responses, we examined the characteristics of NK cells in mice overexpressing IL-4. In this study, we report that IL-4 overexpression induces distinctive characteristics of NK cells (B220high/CD11blow/IL-18Rαlow), which are different from mature conventional NK (cNK) cells (B220low/CD11bhigh/IL-18Rαhigh). IL-4 overexpression induces proliferation of tissue-resident macrophages, which contributes to NK cell proliferation via production of IL-15. These IL-4–induced NK cells (IL4-NK cells) produce higher levels of IFN-γ, IL-10, and GM-CSF, and exhibit high cytotoxicity compared with cNK cells. Furthermore, incubation of cNK cells with IL-15 and IL-4 alters their phenotype to that similar to IL4-NK cells. Finally, parasitic infection, which typically causes strong Th2-type responses, induces the development of NK cells with characteristics similar to IL4-NK cells. These IL4-NK–like cells do not develop in IL-4Rα KO mice by parasitic infection. Collectively, these results suggest a novel role of IL-4 in immune responses through the induction of the unique NK cells. PMID:27551096

  5. Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide.

    PubMed

    Madera, Sharline; Rapp, Moritz; Firth, Matthew A; Beilke, Joshua N; Lanier, Lewis L; Sun, Joseph C

    2016-02-08

    Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar(-/-)) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar(-/-) NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar(-/-) NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell-mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar(-/-) NK cells into NK cell-deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN-dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.

  6. Acute response of peripheral CCr5 chemoreceptor and NK cells in individuals submitted to a single session of low-intensity strength exercise with blood flow restriction.

    PubMed

    Dorneles, Gilson Pires; Colato, Alana Schraiber; Galvão, Simone Lunelli; Ramis, Thiago Rozales; Ribeiro, Jerri Luiz; Romão, Pedro Roosevelt; Peres, Alessandra

    2016-07-01

    The purpose of this study was to compare the peripheral expression of natural killers and CCR5 in a session of low-intensity strength training with vascular occlusion and in high-intensity training. Young males were randomized into session groups of a high-intensity strength training (HI) and a session group of low-intensity strength training with vascular occlusion (LI-BFR). The exercise session consisted in knee extension and bicep curl in 80% 1RM (HI) and 30% 1RM (LI-BFR) with equalized volumes. Blood collection was made before, immediately after and 24 h after each training session. Immunophenotyping was carried out through CD195+ (CCR5) e CD3-CD16+CD56+ (NK) in peripheral blood and analysed by flow cytometry and presented in frequency (%). Peripheral frequency of NK cells showed no significant difference in LI-BFR group in time effect, while a gradual reduction of NK cells was identified in HI group in before-24 h postexercise and after-24 h postexercise comparison. However, significant differences have been found in relative change of NK cells immediately after exercise between sessions. In addition, HI and LI-BFR groups showed a significant reduction in the cells expressed CCR5 during 24 h postsession compared to the postsession, but CCR5 also differed when comparing before-24 h after session in the HI group. No differences were observed amongst the groups. LIO induced CCR5 response similar to the HI session, while the NK cells remained in similar frequency during the studied moments in LI-BFR, but not in HI group, suggesting that local hypoxia created by the blood flow restriction was able to prevent a change in the frequency of peripheral cells and a possible immunosuppression.

  7. Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide

    PubMed Central

    Madera, Sharline; Rapp, Moritz; Firth, Matthew A.; Beilke, Joshua N.; Lanier, Lewis L.

    2016-01-01

    Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar−/−) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar−/− NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar−/− NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell–mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar−/− NK cells into NK cell–deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN–dependent mechanism by which NK cells evade mechanisms of cell death after viral infection. PMID:26755706

  8. NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade.

    PubMed

    Kohlhapp, Frederick J; Broucek, Joseph R; Hughes, Tasha; Huelsmann, Erica J; Lusciks, Jevgenijs; Zayas, Janet P; Dolubizno, Hubert; Fleetwood, Vidyaratna A; Grin, Alisa; Hill, Graham E; Poshepny, Joseph L; Nabatiyan, Arman; Ruby, Carl E; Snook, Joshua D; Rudra, Jai S; Schenkel, Jason M; Masopust, David; Zloza, Andrew; Kaufman, Howard L

    2015-01-01

    Melanoma is one of the few types of cancer with an increasing annual incidence. While a number of immunotherapies for melanoma have been associated with significant clinical benefit, including high-dose IL-2 and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade, clinical response to either of these single agents has been limited to 11-20% of treated patients. Therefore, in this study, we sought to test the hypothesis that the combination of IL-2 and CTLA-4 blockade could mediate a more profound therapeutic response. Here, B6 mice were challenged with poorly immunogenic B16 melanoma on day 0, and treated with CTLA-4 blocking antibody (100 μg/mouse) on days 3, 6, and 9, and IL-2 (100,000 units) twice daily on days 4-8, or both. A highly significant synergistic effect that delayed tumor growth and prolonged survival was demonstrated with the combination immunotherapy compared to either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma.

  9. Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

    PubMed

    Vlasova, Anastasia N; Shao, Lulu; Kandasamy, Sukumar; Fischer, David D; Rauf, Abdul; Langel, Stephanie N; Chattha, Kuldeep S; Kumar, Anand; Huang, Huang-Chi; Rajashekara, Gireesh; Saif, Linda J

    2016-10-01

    Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4(+) mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172(+) MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis.

  10. NK cells and T cells cooperate during the clinical course of colorectal cancer

    PubMed Central

    Sconocchia, Giuseppe; Eppenberger, Serenella; Spagnoli, Giulio C; Tornillo, Luigi; Droeser, Raoul; Caratelli, Sara; Ferrelli, Francesca; Coppola, Andrea; Arriga, Roberto; Lauro, Davide; Iezzi, Giandomenica; Terracciano, Luigi; Ferrone, Soldano

    2014-01-01

    Recent evidence suggests that natural killer (NK) cells are typically defective in infiltrating solid tumors, with the exception of gastrointestinal stromal tumors (GIST). Interestingly, however, infrequently infiltrating NK cells do not appear to have a direct effect on tumor progression. Here, prompted by the recent evidence that NK cell and T cell crosstalk may trigger, or enhance, tumor antigen-specific immune responses, we have tested the clinical significance of this reciprocal signaling. To this end, a tissue microarray constructed with 1410 colorectal carcinoma (CRC) patient specimens was stained with NK and T cell antigen-specific monoclonal antibodies, utilizing the immunoperoxidase staining technique. Cut-off scores for positive (>4 NK cells) and negative (≤4 NK cells) NK cell CRC patient samples were determined using receiver operating characteristic curve analysis. Using this approach, NK cells were detected in 423 (30%) of the 1410 CRC specimens evaluated. The number of NK cells was >4 in only 132 (9%) of CRC samples. Correlation of the immunohistochemical staining results together with analysis of the clinical course of the disease revealed that the infiltration of colorectal tumors with both NK cells and CD8+ T cells is associated with prolonged patient survival. In contrast, infiltration of tumors with NK cells in combination with CD3+ and CD4+ T lymphocytes had no detectable effect on the clinical course of the disease. These results suggest that NK cell and CD8+ T cell crosstalk in the tumor microenvironment may benefit patient outcome and further, that the enumeration of infiltrating NK and CD8+ T cells in CRC tumors may provide useful prognostic information. PMID:25610741

  11. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

    PubMed

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W; Olofsson, Per E; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  12. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    PubMed Central

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  13. Functional Assessment of NK and LAK Cells Following Space Flight

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Space flight associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. PBMCs were collected from 10-ml blood specimens from 5 astronauts 10 days before launch, immediately after landing, and again at 3 days after landing and stored at -80 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4 hour Cr-51 release assay. K562 cells were used to assess NK cell cytotoxicity. Following 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 cells and Daudi cells as the target cells. NK cell cytotoxicity decreased at landing (p<.05) in 3/5 astronauts, and recovered to preflight levels by 3 days following landing; NK cell cytotoxicity was increased (p=0.1) in the remaining 2 astronauts at landing. In 4/5 astronauts, LAK cytotoxic activity was decreased at landing against K562 cells (p = 0.13) and Daudi cells (p = 0.08). Phenotyping of PBMC's and LAK cells showed alterations in some surface markers and adhesion molecules (CD11b, CD11c, CD11a, CD16, L-selectin, and CD3).

  14. Functional Assessment of NK and LAK Cells Following Space Flight

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Space flight associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. PBMCs were collected from 10-ml blood specimens from 5 astronauts 10 days before launch, immediately after landing, and again at 3 days after landing and stored at -80 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4 hour Cr-51 release assay. K562 cells were used to assess NK cell cytotoxicity. Following 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 cells and Daudi cells as the target cells. NK cell cytotoxicity decreased at landing (p<.05) in 3/5 astronauts, and recovered to preflight levels by 3 days following landing; NK cell cytotoxicity was increased (p=0.1) in the remaining 2 astronauts at landing. In 4/5 astronauts, LAK cytotoxic activity was decreased at landing against K562 cells (p = 0.13) and Daudi cells (p = 0.08). Phenotyping of PBMC's and LAK cells showed alterations in some surface markers and adhesion molecules (CD11b, CD11c, CD11a, CD16, L-selectin, and CD3).

  15. NK Cells Help Induce Anti-Hepatitis B Virus CD8+ T Cell Immunity in Mice.

    PubMed

    Zheng, Meijuan; Sun, Rui; Wei, Haiming; Tian, Zhigang

    2016-05-15

    Although recent clinical studies demonstrate that NK cell function is impaired in hepatitis B virus (HBV)-persistent patients, whether or how NK cells play a role in anti-HBV adaptive immunity remains to be explored. Using a mouse model mimicking acute HBV infection by hydrodynamic injection of an HBV plasmid, we observed that although serum hepatitis B surface Ag and hepatitis B envelope Ag were eliminated within 3 to 4 wk, HBV might persist for >8 wk in CD8(-/-) mice and that adoptive transfer of anti-HBV CD8(+) T cells restored the ability to clear HBV in HBV-carrier Rag1(-/-) mice. These results indicate that CD8(+) T cells are critical in HBV elimination. Furthermore, NK cells increased IFN-γ production after HBV plasmid injection, and NK cell depletion led to significantly increased HBV persistence along with reduced frequency of hepatitis B core Ag-specific CD8(+) T cells. Adoptive transfer of IFN-γ-sufficient NK cells restored donor CD8(+) T cell function, indicating that NK cells positively regulated CD8(+) T cells via secreting IFN-γ. We also observed that NK cell depletion correlated with decreased effector memory CD8(+) T cell frequencies. Importantly, adoptive transfer experiments showed that NK cells were involved in anti-HBV CD8(+) T cell recall responses. Moreover, DX5(+)CD49a(-) conventional, but not DX5(-)CD49a(+) liver-resident, NK cells were involved in improving CD8(+) T cell responses against HBV. Overall, the current study reveals that NK cells, especially DX5(+)CD49a(-) conventional NK cells, promote the antiviral activity of CD8(+) T cell responses via secreting IFN-γ in a mouse model mimicking acute HBV infection.

  16. Beyond NK Cells: The Expanding Universe of Innate Lymphoid Cells

    PubMed Central

    Cella, Marina; Miller, Hannah; Song, Christina

    2014-01-01

    For a long time, natural killer (NK) cells were thought to be the only innate immune lymphoid population capable of responding to invading pathogens under the influence of changing environmental cues. In the last few years, an increasing amount of evidence has shown that a number of different innate lymphoid cell (ILC) populations found at mucosal sites rapidly respond to locally produced cytokines in order to establish or maintain homeostasis. These ILC populations closely mirror the phenotype of adaptive T helper subsets in their repertoire of secreted soluble factors. Early in the immune response, ILCs are responsible for setting the stage to mount an adaptive T cell response that is appropriate for the incoming insult. Here, we review the diversity of ILC subsets and discuss similarities and differences between ILCs and NK cells in function and key transcriptional factors required for their development. PMID:24982658

  17. Beyond NK cells: the expanding universe of innate lymphoid cells.

    PubMed

    Cella, Marina; Miller, Hannah; Song, Christina

    2014-01-01

    For a long time, natural killer (NK) cells were thought to be the only innate immune lymphoid population capable of responding to invading pathogens under the influence of changing environmental cues. In the last few years, an increasing amount of evidence has shown that a number of different innate lymphoid cell (ILC) populations found at mucosal sites rapidly respond to locally produced cytokines in order to establish or maintain homeostasis. These ILC populations closely mirror the phenotype of adaptive T helper subsets in their repertoire of secreted soluble factors. Early in the immune response, ILCs are responsible for setting the stage to mount an adaptive T cell response that is appropriate for the incoming insult. Here, we review the diversity of ILC subsets and discuss similarities and differences between ILCs and NK cells in function and key transcriptional factors required for their development.

  18. Peripheral NK cell phenotypes: multiple changing of faces of an adapting, developing cell.

    PubMed

    Perussia, Bice; Chen, Yingying; Loza, Matthew J

    2005-02-01

    We have defined the existence of developmental relationships among human peripheral NK cells with distinct phenotypic and functional characteristics. These findings closely parallel the changes that occur in vivo during NK cell development, and in vitro in experimental culture systems supporting NK cell generation from hematopoietic progenitors. These new insights provide a simplified framework to understand NK cell immunobiology and the cellular bases for their roles in innate immunity, initiation and maintenance of immune responses via regulation of adaptive and accessory cell functions, and immune pathologies.

  19. Targeting Stat3 induces senescence in tumor cells and elicits prophylactic and therapeutic immune responses against breast cancer growth mediated by NK cells and CD4+ T cells.

    PubMed

    Tkach, Mercedes; Coria, Lorena; Rosemblit, Cinthia; Rivas, Martín A; Proietti, Cecilia J; Díaz Flaqué, María Celeste; Beguelin, Wendy; Frahm, Isabel; Charreau, Eduardo H; Cassataro, Juliana; Elizalde, Patricia V; Schillaci, Roxana

    2012-08-01

    Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4(+) T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.

  20. Murine peripheral NK-cell populations originate from site-specific immature NK cells more than from BM-derived NK cells.

    PubMed

    Pinhas, Nissim; Sternberg-Simon, Michal; Chiossone, Laura; Shahaf, Gitit; Walzer, Thierry; Vivier, Eric; Mehr, Ramit

    2016-05-01

    Murine NK cells can be divided by the expression of two cell surface markers, CD27 and Mac-1 (a.k.a. CD11b), into four separate subsets. These subsets suggest a linear development model: CD27(-) Mac-1(-) → CD27(+) Mac-1(-) → CD27(+) Mac-1(+) → CD27(-) Mac-1(+) . Here, we used a combination of BrdU labeling experiments and mathematical modeling to gain insights regarding NK-cell development in mouse bone marrow (BM), spleen and liver. The modeling results that best fit the experimental data show that the majority of NK cells already express CD27 upon entering the NK-cell developmental pathway. Additionally, only a small fraction of NK cells exit the BM to other sites, suggesting that peripheral NK-cell populations originate from site-specific immature NK cells more than from BM-derived mature NK cells.

  1. NK Cell-derived Exosomes From NK Cells Previously Exposed to Neuroblastoma Cells Augment the Antitumor Activity of Cytokine-activated NK Cells.

    PubMed

    Shoae-Hassani, Alireza; Hamidieh, Amir Ali; Behfar, Maryam; Mohseni, Rashin; Mortazavi-Tabatabaei, Seyed A; Asgharzadeh, Shahab

    2017-09-01

    Immune cell-derived exosomes can increase immunity against tumors. In contrast, tumor-derived exosomes can reduce the immunity and can change the tumor microenvironment to further develop and provide metastasis. These effects take place by an alteration in the innate and adaptive immune cell functions. In this experiment, we studied the natural killer (NK) cells' effectiveness on tumor cells after expansion and thereafter incubated it with exosomes. The exosomes were derived from 2 populations of NK cells: (1) naive NK cells and, (2) NK cells previously exposed to neuroblastoma (NB) cells. Moreover, we have studied the NB-derived exosomes on NK cell function. The molecular load of the characterized exosomes (by means of nanoparticle-tracking analysis, flow cytometry, scanning electron microscopy, and western blot) from NK cells exposed to the NB cell revealed their expression of natural killer cell receptors in addition to CD56, NKG2D, and KIR2DL2 receptors. These exosomes were used to treat NK cells and thereafter administered to NB tumor cells both in vitro and in vivo. Our results showed some kind of NK cells' education by the exosomes. This education from NK cells previously exposed to NB cell-derived exosomes caused efficient and greater cytotoxicity against NB tumors, but NB-derived exosomes act as tumor promoters by providing a tumor supporting niche. Hence, this method of preparing the exosomes has a dramatic effect on activation of anti-NK cells against NB cells.

  2. NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing

    USDA-ARS?s Scientific Manuscript database

    Natural killer cells are lymphocytes of the innate immune system that have crucial cytotoxic and regulatory roles in adaptive immunity and inflammation. Herein, we consider a role for these cells in corneal wound healing. After a 2-mm central epithelial abrasion of the mouse cornea, a subset of clas...

  3. Licensed and Unlicensed NK Cells: Differential Roles in Cancer and Viral Control.

    PubMed

    Tu, Megan M; Mahmoud, Ahmad Bakur; Makrigiannis, Andrew P

    2016-01-01

    Natural killer (NK) cells are known for their well characterized ability to control viral infections and eliminate tumor cells. Through their repertoire of activating and inhibitory receptors, NK cells are able to survey different potential target cells for various surface markers, such as MHC-I - which signals to the NK cell that the target is healthy - as well as stress ligands or viral proteins, which alert the NK cell to the aberrant state of the target and initiate a response. According to the "licensing" hypothesis, interactions between self-specific MHC-I receptors - Ly49 in mice and KIR in humans - and self-MHC-I molecules during NK cell development is crucial for NK cell functionality. However, there also exists a large proportion of NK cells in mice and humans, which lack self-specific MHC-I receptors and are consequentially "unlicensed." While the licensed NK cell subset plays a major role in the control of MHC-I-deficient tumors, this review will go on to highlight the important role of the unlicensed NK cell subset in the control of MHC-I-expressing tumors, as well as in viral control. Unlike the licensed NK cells, unlicensed NK cells seem to benefit from the lack of self-specific inhibitory receptors, which could otherwise be exploited by some aberrant cells for immunoevasion by upregulating the expression of ligands or mimic ligands for these receptors.

  4. NK cells: walking three paths down memory lane

    PubMed Central

    Min-Oo, Gundula; Kamimura, Yosuke; Hendricks, Deborah W.; Nabekura, Tsukasa; Lanier, Lewis L.

    2013-01-01

    Immunological memory has traditionally been regarded as a unique feature of the adaptive immune response, mediated in an antigen-specific manner by T and B lymphocytes. All other hematopoietic cells, including natural killer (NK) cells, are classified as innate immune cells, which have been considered short-lived but can respond rapidly against pathogens in a manner not thought to be driven by antigen. Interestingly, NK cells have recently been shown to survive long term after antigen exposure and subsequently mediate antigen-specific recall responses. In this review, we will address the similarities between, and the controversies surrounding, three major viewpoints of NK memory that have arisen from these recent studies: (i) MCMV (mouse cytomegalovirus)-induced memory; (ii) cytokine-induced memory; (iii) liver-restricted memory cells. PMID:23499559

  5. Adaptive NK cell and KIR-expressing T cell responses are induced by CMV and are associated with protection against CMV reactivation after allogeneic donor hematopoietic cell transplantation1

    PubMed Central

    Davis, Zachary B.; Cooley, Sarah A.; Cichocki, Frank; Felices, Martin; Wangen, Rose; Luo, Xianghua; DeFor, Todd E.; Bryceson, Yenan T.; Diamond, Don J.; Brunstein, Claudio; Blazar, Bruce R.; Wagner, John E.; Weisdorf, Daniel J.; Horowitz, Amir; Guethlein, Lisbeth A.; Parham, Peter; Verneris, Michael R.; Miller, Jeffrey S.

    2015-01-01

    Cytomegalovirus (CMV) reactivates in >30% of CMV seropositive patients after allogeneic hematopoietic cell transplantation (HCT). Previously, we reported an increase of NK cells expressing NKG2C, CD57 and inhibitory killer-cell immunoglobulin-like receptors (KIRs) in response to CMV reactivation post-HCT. These NK cells persist after the resolution of infection and display ‘adaptive’ or memory properties. Despite these findings, the differential impact of persistent/inactive vs. reactivated CMV on NK vs. T cell maturation following HCT from different graft sources has not been defined. We compared the phenotype of NK and T cells from 292 recipients of allogeneic sibling (n = 118) or umbilical cord blood (UCB; n = 174) grafts based on recipient pre-transplant CMV serostatus and post-HCT CMV reactivation. This cohort was utilized to evaluate CMV-dependent increases in KIR-expressing NK cells exhibiting an ‘adaptive’ phenotype (NKG2C+CD57+). Compared to CMV seronegative recipients, those who reactivated CMV (React+) had the highest adaptive cell frequencies, while intermediate frequencies were observed in CMV seropositive recipients harboring persistent/non-replicating CMV. The same effect was observed in T cells and CD56+ T cells. These adaptive lymphocyte subsets were increased in CMV seropositive recipients of sibling, but not UCB grafts, and correlated with lower rates of CMV reactivation (sibling 33% vs. UCB 51%; p<0.01). These data suggest that persistent/non-replicating recipient CMV induces rapid production of adaptive NK and T cells from mature cells from sibling, but not UCB grafts. These adaptive lymphocytes are associated with protection from CMV reactivation. PMID:26055301

  6. Polyclonal Expansion of NKG2C+ NK Cells in TAP-Deficient Patients

    PubMed Central

    Béziat, Vivien; Sleiman, Marwan; Goodridge, Jodie P.; Kaarbø, Mari; Liu, Lisa L.; Rollag, Halvor; Ljunggren, Hans-Gustaf; Zimmer, Jacques; Malmberg, Karl-Johan

    2015-01-01

    Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections. PMID:26500647

  7. Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

    PubMed

    Kremer, Veronika; Ligtenberg, Maarten; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

    2017-09-19

    Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK

  8. Novel APC-like properties of human NK cells directly regulate T cell activation

    PubMed Central

    Hanna, Jacob; Gonen-Gross, Tsufit; Fitchett, Jonathan; Rowe, Tony; Daniels, Mark; Arnon, Tal I.; Gazit, Roi; Joseph, Aviva; Schjetne, Karoline W.; Steinle, Alexander; Porgador, Angel; Mevorach, Dror; Goldman-Wohl, Debra; Yagel, Simcha; LaBarre, Michael J.; Buckner, Jane H.; Mandelboim, Ofer

    2004-01-01

    Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane–enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell–mediated cytotoxicity and specific ligand recognition by cell surface–activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell–activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2–deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell–activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells. PMID:15578093

  9. Inflammasome-Dependent Induction of Adaptive NK Cell Memory.

    PubMed

    van den Boorn, Jasper G; Jakobs, Christopher; Hagen, Christian; Renn, Marcel; Luiten, Rosalie M; Melief, Cornelis J M; Tüting, Thomas; Garbi, Natalio; Hartmann, Gunther; Hornung, Veit

    2016-06-21

    Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

  10. Radiation dose reduction for patients with extranodal NK/T-cell lymphoma with complete response after initial induction chemotherapy

    PubMed Central

    Wang, Liang; Bi, Xi-wen; Xia, Zhong-jun; Huang, Hui-qiang; Jiang, Wen-qi; Zhang, Yu-jing

    2016-01-01

    Previous studies have found that radiotherapy (RT) dose less than 50 Gy resulted in inferior outcomes for early stage extranodal NK/T-cell lymphoma (ENKTL). Nowadays, induction chemotherapy (CT) followed by RT consolidation is often used. For patients who get complete response (CR) after CT, whether RT dose can be safely reduced or not remains unknown. This retrospective study compared the survival outcomes between patients who received higher dose (>50 Gy) and lower dose (≤50 Gy) RT after CR was attained by CT. One hundred and forty four patients of early stage ENKTL got CR after induction CT and received RT consolidation. Thirty-one patients received lower dose RT (median 46 Gy, range, 36–50 Gy), and 113 patients received higher dose RT (median 56 Gy, range, 52–66 Gy). In univariate survival analysis, age >60, local tumor invasion, and non-asparaginase-based CT were associated with inferior progression-free survival (PFS) and overall survival (OS). However, there were no differences in PFS and OS between patients treated with higher and lower dose RT, which was confirmed in the multivariate survival analysis. Furthermore, reduced dose RT did not affect local control rate. Most common RT-related side effects were grade 1/2 mucositis and dermatitis, and the incidence rate of grade 3 mucositis or dermatitis was lower in patients treated with reduced dose RT (9.7% vs 15.0% for mucositis, and 6.5% vs 17.7% for dermatitis). In conclusion, this study found that RT dose could be safely reduced without compromising survival outcomes and further improved RT-related side effects. Prospective randomized controlled trials are warranted to validate our findings. PMID:27713641

  11. Exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions.

    PubMed

    Montenegro, Mary Lourdes; Ferriani, Rui Alberto; Basse, Per H

    2015-08-29

    Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35%-50%, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions. As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200μl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200μl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2). Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis. For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track

  12. NK cell trafficking in health and autoimmunity:a comprehensive review.

    PubMed

    Peng, Hui; Tian, Zhigang

    2014-10-01

    Natural killer (NK) cells represent an important subpopulation of lymphocytes that are distributed throughout the body. The development of NK cells primarily occurs in the bone marrow during adult life, involving several putative intermediate stages that finally result in functional maturation. At steady state, NK cell egress from the bone marrow to various peripheral areas is controlled by a network of adhesion molecules, including integrins, selectins, and chemokine receptors and their corresponding ligands. NK cells at different developmental stages express distinct repertoire of adhesion molecules and can therefore be recruited to different sites of the body, including lymphoid and non-lymphoid tissues, and NK cells undergo further differentiation driven by local microenvironmental signals, resulting in unique tissue-specific NK cells. Through their abilities of cytotoxicity and cytokine production, NK cells not only play key roles in the innate immune system, but also participate in shaping adaptive immune responses. On the basis of their heterogeneity in phenotype, function, and tissue distribution, NK cells can be further subdivided into distinct subsets. Under pathological conditions, such as in autoimmune, inflammatory, and infectious diseases, as local microenvironment changes, NK cell subsets would redistribute between tissues and organs and rapidly accumulate at the local pathological sites to exert their effector functions. Here, we describe the development and tissue distribution of NK cell subsets in mice and humans. We focus on the trafficking of NK cell subsets within the bone marrow and emigration into periphery at steady state, and molecular mechanisms involved in their trafficking in autoimmune diseases.

  13. Reversal of tumor acidosis by systemic buffering reactivates NK cells to express IFN-γ and induces NK cell-dependent lymphoma control without other immunotherapies.

    PubMed

    Pötzl, Johann; Roser, David; Bankel, Lorenz; Hömberg, Nadine; Geishauser, Albert; Brenner, Christoph D; Weigand, Michael; Röcken, Martin; Mocikat, Ralph

    2017-05-01

    Like other immune cells, natural killer (NK) cells show impaired effector functions in the microenvironment of tumors, but little is known on the underlying mechanisms. Since lactate acidosis, a hallmark of malignant tissue, was shown to contribute to suppression of effective antitumor immune responses, we investigated the impact of tissue pH and lactate concentration on NK-cell functions in an aggressive model of endogenously arising B-cell lymphoma. The progressive loss of IFN-γ production by NK cells observed during development of this disease could be ascribed to decreased pH values and lactate accumulation in the microenvironment of growing tumors. Interestingly, IFN-γ expression by lymphoma-derived NK cells could be restored by transfer of these cells into a normal micromilieu. Likewise, systemic alkalization by oral delivery of bicarbonate to lymphoma-developing mice was capable of enhancing IFN-γ expression in NK cells and increasing the NK-cell numbers in the lymphoid organs where tumors were growing. By contrast, NK-cell cytotoxicity was dampened in vivo by tumor-dependent mechanisms that seemed to be different from lactate acidosis and could not be restored in a normal milieu. Most importantly, alkalization and the concomitant IFN-γ upregulation in NK cells were sufficient to significantly delay tumor growth without any other immunotherapy. This effect was strictly dependent on NK cells.

  14. CD4+ T-cell help enhances NK cell function following therapeutic HIV-1 vaccination.

    PubMed

    Jost, Stephanie; Tomezsko, Phillip John; Rands, Keith; Toth, Ildiko; Lichterfeld, Mathias; Gandhi, Rajesh Tim; Altfeld, Marcus

    2014-08-01

    Increasing data suggest that NK cells can mediate antiviral activity in HIV-1-infected humans, and as such, novel approaches harnessing the anti-HIV-1 function of both T cells and NK cells represent attractive options to improve future HIV-1 immunotherapies. Chronic progressive HIV-1 infection has been associated with a loss of CD4(+) T helper cell function and with the accumulation of anergic NK cells. As several studies have suggested that cytokines produced by CD4(+) T cells are required to enhance NK cell function in various infection models, we hypothesized that reconstitution of HIV-1-specific CD4(+) T-cell responses by therapeutic immunization would restore NK cell activity in infected individuals. Using flow cytometry, we examined the function of CD4(+) T cells and NK cells in response to HIV-1 in subjects with treated chronic HIV-1 infection before and after immunization with an adjuvanted HIV-1 Gp120/NefTat subunit protein vaccine candidate provided by GlaxoSmithKline. Vaccination induced an increased expression of interleukin-2 (IL-2) by Gp120-specific CD4(+) T cells in response to HIV-1 peptides ex vivo, which was associated with enhanced production of gamma interferon (IFN-γ) by NK cells. Our data show that reconstitution of HIV-1-specific CD4(+) T-cell function by therapeutic immunization can enhance NK cell activity in HIV-1-infected individuals. NK cells are effector cells of the innate immune system and are important in the control of viral infection. Recent studies have demonstrated the crucial role played by NK cells in controlling and/or limiting acquisition of HIV-1 infection. However, NK cell function is impaired during progressive HIV-1 infection. We recently showed that therapeutic immunization of treated HIV-1-infected individuals reconstituted strong T-cell responses, measured notably by their production of IL-2, a cytokine that can activate NK cells. The current study suggests that reconstitution of T-cell function by therapeutic

  15. T and NK cells: two sides of tumor immunoevasion.

    PubMed

    Fruci, Doriana; Lo Monaco, Elisa; Cifaldi, Loredana; Locatelli, Franco; Tremante, Elisa; Benevolo, Maria; Giacomini, Patrizio

    2013-02-04

    Natural Killer (NK) cells are known to reject several experimental murine tumors, but their antineoplastic activity in humans is not generally agreed upon, as exemplified by an interesting correspondence recently appeared in Cancer Research. In the present commentary, we join the discussion and bring to the attention of the readers of the Journal of Translational Medicine a set of recent, related reports. These studies demonstrate that effectors of the adaptive and innate immunity need to actively cooperate in order to reject tumors and, conversely, tumors protect themselves by dampening both T and NK cell responses. The recently reported ability of indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) expressed by melanoma cells to down-regulate activating NK receptors is yet another piece of evidence supporting combined and highly effective T/NK cell disabling. Major Histocompatibility Complex class I (MHC-I) molecules, including Human Leukocyte Antigen E (HLA-E), represent another class of shared activating/inhibitory ligands. Ongoing clinical trials with small molecules interfering with IDO and PGE2 may be exploiting an immune bonus to control cancer. Conversely, failure to simultaneously engage effectors of both the innate and the adaptive immunity may contribute to explain the limited clinical efficacy of T cell-only vaccination trials. Shared (T/NK cells) natural immunosuppressants and activating/inhibitory ligands expressed by tumor cells may provide mechanistic insight into impaired gathering and function of immune effectors at the tumor site.

  16. T and NK cells: two sides of tumor immunoevasion

    PubMed Central

    2013-01-01

    Natural Killer (NK) cells are known to reject several experimental murine tumors, but their antineoplastic activity in humans is not generally agreed upon, as exemplified by an interesting correspondence recently appeared in Cancer Research. In the present commentary, we join the discussion and bring to the attention of the readers of the Journal of Translational Medicine a set of recent, related reports. These studies demonstrate that effectors of the adaptive and innate immunity need to actively cooperate in order to reject tumors and, conversely, tumors protect themselves by dampening both T and NK cell responses. The recently reported ability of indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) expressed by melanoma cells to down-regulate activating NK receptors is yet another piece of evidence supporting combined and highly effective T/NK cell disabling. Major Histocompatibility Complex class I (MHC-I) molecules, including Human Leukocyte Antigen E (HLA-E), represent another class of shared activating/inhibitory ligands. Ongoing clinical trials with small molecules interfering with IDO and PGE2 may be exploiting an immune bonus to control cancer. Conversely, failure to simultaneously engage effectors of both the innate and the adaptive immunity may contribute to explain the limited clinical efficacy of T cell-only vaccination trials. Shared (T/NK cells) natural immunosuppressants and activating/inhibitory ligands expressed by tumor cells may provide mechanistic insight into impaired gathering and function of immune effectors at the tumor site. PMID:23379575

  17. Respiratory Influenza Virus Infection Induces Memory-like Liver NK Cells in Mice.

    PubMed

    Li, Tingting; Wang, Jian; Wang, Yanshi; Chen, Yongyan; Wei, Haiming; Sun, Rui; Tian, Zhigang

    2017-02-01

    Although NK cells are classified as innate immune cells, recent studies have demonstrated the transformation of NK cells into long-lived memory cells that contribute to secondary immune responses in certain mouse models. However, whether NK cells mount an Ag-specific memory response to acute influenza virus infection has not yet been examined. Here, we show that, consistent with previous studies, lung NK cells play an important role in controlling viral proliferation after primary influenza virus infection. However, although lung NK cells display a memory phenotype at the late stage of infection, these cells do not protect mice against secondary influenza virus infection. Interestingly, liver NK cells from influenza virus-infected mice possess a memory phenotype and protect mice against secondary influenza virus infection. Memory-like liver NK cells display a CD49a(+)DX5(-) phenotype, and the adoptive transfer of purified liver CD49a(+)DX5(-) NK cells into naive mice followed by viral infection results in protective immunity and decreased viral titer. Moreover, we demonstrate that primary inactivated influenza virus induces memory NK cells residing in the liver of Rag1(-/-) mice. Collectively, these data suggest that liver CD49a(+)DX5(-) NK cells remember encountered Ag from influenza virus after primary infection and are more protective upon subsequent infection. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies.

    PubMed

    Romanski, Annette; Uherek, Christoph; Bug, Gesine; Seifried, Erhard; Klingemann, Hans; Wels, Winfried S; Ottmann, Oliver G; Tonn, Torsten

    2016-07-01

    Many B-cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK-92 cells, a continuously growing NK cell line, showing that anti-CD19-CAR (αCD19-CAR) engineered NK-92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK-92 cells. The 'first generation' CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc-tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK-92 and transduced αCD19-CAR NK-92 cells towards CD19 negative targets was found. However, αCD19-CAR NK-92 cells specifically and efficiently lysed CD19 expressing B-precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK-92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK-92 should be considered a treatment option for patients with lymphoid malignancies.

  19. Epirubicin pretreatment enhances NK cell-mediated cytotoxicity against breast cancer cells in vitro.

    PubMed

    Feng, Hui; Dong, Ying; Wu, Jing; Qiao, Yuan; Zhu, Ge; Jin, Haofan; Cui, Jiuwei; Li, Wei; Liu, Yong-Jun; Chen, Jingtao; Song, Yanqiu

    2016-01-01

    Anthracycline-based chemotherapy is a conventional treatment for breast cancer. However, it can negatively affect host immune function and thereby impair patients' quality of life. Boosting the host immune system and reducing the adverse effect of chemotherapy are important for effective cancer treatment. Natural killer (NK) cells stimulate immune responses against cancer; autologous immune enhancement therapy with NK cells prolongs patient survival without significant adverse effects. This study investigated the effects of combined treatment with the anthracycline agent epirubicin (EPI) and NK cells on human breast cancer cells. NK cells were obtained by autologous adoptive cell transfer from breast cancer patients and amplified for 14 days in vitro. The cytotoxicity of NK cells against breast cancer cells was higher following EPI (5.0 μg/ml) pretreatment than without EPI pretreatment or application of EPI alone. The expression of NKG2D ligands [unique long 16-binding protein (ULBP) 1, ULBP2, and major histocompatibility complex class I-related chain A] in breast cancer cells was upregulated by pretreatment with EPI, which also increased the secretion of interferon-γ and tumor necrosis factor-α and expression of perforin and granzyme B in NK cells. These results indicate that EPI-NK cell treatment has synergistic cytotoxic effects against breast cancer cells, and suggest that anthracycline-based chemotherapy and NK cell-based immunotherapy can be combined for more effective breast cancer treatment.

  20. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    NASA Astrophysics Data System (ADS)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  1. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    PubMed Central

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited. PMID:28074895

  2. Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion.

    PubMed

    He, Li-Xia; Ren, Jin-Wei; Liu, Rui; Chen, Qi-He; Zhao, Jian; Wu, Xin; Zhang, Zhao-Feng; Wang, Jun-Bo; Pettinato, Giuseppe; Li, Yong

    2017-09-06

    Traditionally used as a restorative medicine, ginseng (Panax ginseng Meyer) has been the most widely used and acclaimed herb in Chinese communities for thousands of years. To investigate the immune-modulating activity of ginseng oligopeptides (GOP), 420 healthy female BALB/c mice were intragastrically administered distilled water (control), whey protein (0.15 g per kg body weight (BW)), and GOP 0.0375, 0.075, 0.15, 0.3 and 0.6 g per kg BW for 30 days. Blood samples from mice were collected from the ophthalmic venous plexus and then sacrificed by cervical dislocation. Seven assays were conducted to determine the immunomodulatory effects of GOP on innate and adaptive immune responses, followed by flow cytometry to investigate spleen T lymphocyte sub-populations, multiplex sandwich immunoassays to investigate serum cytokine and immunoglobulin levels, and ELISA to investigate intestinally secreted immunoglobulin to study the mechanism of GOP affecting the immune system. Our results showed that GOP was able to enhance innate and adaptive immune responses in mice by improving cell-mediated and humoral immunity, macrophage phagocytosis capacity and NK cell activity. Notably, the use of GOP revealed a better immune-modulating activity compared to whey protein. We conclude that the immune-modulating activity might be due to the increased macrophage phagocytosis capacity and NK cell activity, and the enhancement of T and Th cells, as well as IL-2, IL-6 and IL-12 secretion and IgA, IgG1 and IgG2b production. These results indicate that GOP could be considered a good candidate that may improve immune functions if used as a dietary supplement, with a dosage that ranges from 0.3 to 0.6 g per kg BW.

  3. NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3.

    PubMed

    Hamblet, Corinne E; Makowski, Stefanie L; Tritapoe, Julia M; Pomerantz, Joel L

    2016-03-15

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. In this article, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27(+)CD11b(+) and CD27(-)CD11b(+) NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knockin mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

  4. NK Cell Maturation and Cytotoxicity are Controlled by the Intramembrane Aspartyl Protease SPPL31

    PubMed Central

    Hamblet, Corinne E.; Makowski, Stefanie L.; Tritapoe, Julia M.; Pomerantz, Joel L.

    2016-01-01

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27+CD11b+ and CD27−CD11b+ NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knock-in mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. PMID:26851218

  5. Transcription factor KLF2 regulates homeostatic NK cell proliferation and survival.

    PubMed

    Rabacal, Whitney; Pabbisetty, Sudheer K; Hoek, Kristen L; Cendron, Delphine; Guo, Yin; Maseda, Damian; Sebzda, Eric

    2016-05-10

    Natural killer (NK) cells are innate lymphocytes that recognize and lyse virally infected or transformed cells. This latter property is being pursued in clinics to treat leukemia with the hope that further breakthroughs in NK cell biology can extend treatments to other cancers. At issue is the ability to expand transferred NK cells and prolong their functionality within the context of a tumor. In terms of NK cell expansion and survival, we now report that Kruppel-like factor 2 (KLF2) is a key transcription factor that underpins both of these events. Excision of Klf2 using gene-targeted mouse models promotes spontaneous proliferation of immature NK cells in peripheral tissues, a phenotype that is replicated under ex vivo conditions. Moreover, KLF2 imprints a homeostatic migration pattern on mature NK cells that allows these cells to access IL-15-rich microenvironments. KLF2 accomplishes this feat within the mature NK cell lineage via regulation of a subset of homing receptors that respond to homeostatic ligands while leaving constitutively expressed receptors that recognize inflammatory cytokines unperturbed. Under steady-state conditions, KLF2-deficient NK cells alter their expression of homeostatic homing receptors and subsequently undergo apoptosis due to IL-15 starvation. This novel mechanism has implications regarding NK cell contraction following the termination of immune responses including the possibility that retention of an IL-15 transpresenting support system is key to extending NK cell activity in a tumor environment.

  6. The utility of flow cytometry in differentiating NK/T cell lymphoma from indolent and reactive NK cell proliferations.

    PubMed

    de Mel, Sanjay; Li, Jenny Bei; Abid, Muhammad Bilal; Tang, Tiffany; Tay, Hui Ming; Ting, Wen Chang; Poon, Li Mei; Chung, Tae Hoon; Mow, Benjamin; Tso, Allison; Ong, Kiat Hoe; Chng, Wee Joo; Liu, Te Chih

    2017-04-21

    The WHO defines three categories of NK cell malignancies; extra nodal NK/T cell lymphoma (NKTCL), aggressive NK cell leukemia, and the provisional entity chronic lymphoproliferative disorder of NK cells (CLPD-NK). Although the flow cytometric (FC) phenotype of CLPD-NK has been described, studies on FC phenotype of NKTCL are limited. To the best of our knowledge ours is the first study to compare the phenotype of NKTCL, CLPD-NK, reactive NK lymphocytosis (RNKL), and normal NK cells using eight color (8C) FC. Specimens analyzed using the Euroflow8C NK Lymphoproliferative Disorder (NKLPD) panel between 2011 and 2014 were identified from our database. All samples were analyzed on the FACSCantoII cytometer. NK cells were identified as CD45+, smCD3-, CD19-, CD56+ and normal T-cells served as internal controls. The majority of NKTCL were CD56 bright, CD16 dim, CD57-, and CD94+. CLPD-NK and RNKL were predominantly CD56+ or dim with positive expression of CD16 and CD57 and weak CD94 expression. Antigen based statistical analyses showed robust division of samples along the NKTCL/normal CD56 bright NK cell and CLPD-NK/RNKL/normal CD56 positive NK cell groups. It was concluded that FC can reliably distinguish NKTCL from CLPD-NK, normal NK cells of CD56+ phenotype, and RNKL. It was proposed that the typical phenotype for NKTCL is: CD56 bright, CD16 dim with positive CD2, CD7, CD94, HLADR, CD25, CD26, and absent CD57. This resembles the phenotype of the CD56 bright immunoregulatory subset of NK cells which we therefore hypothesize is the cell of origin of NKTCL. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  7. NK Cells Alleviate Lung Inflammation by Negatively Regulating Group 2 Innate Lymphoid Cells.

    PubMed

    Bi, Jiacheng; Cui, Lulu; Yu, Guang; Yang, Xiaolu; Chen, Youhai; Wan, Xiaochun

    2017-04-15

    Group 2 innate lymphoid cells (ILC2s) play an important role in orchestrating type II immune responses. However, the cellular mechanisms of group 2 innate lymphoid cell regulation remain poorly understood. In this study, we found that activated NK cells inhibited the proliferation of, as well as IL-5 and IL-13 production by, ILC2s in vitro via IFN-γ. In addition, in a murine model of ILC2 expansion in the liver, polyinosinic-polycytidylic acid, an NK cell-activating agent, inhibited ILC2 proliferation, IL-5 and IL-13 production, and eosinophil recruitment. Such effects of polyinosinic-polycytidylic acid were abrogated in NK cell-depleted mice and in IFN-γ-deficient mice. Adoptively transferring wild-type NK cells into NK cell-depleted mice resulted in fewer ILC2s induced by IL-33 compared with the transfer of IFN-γ-deficient NK cells. Importantly, during the early stage of papain- or bleomycin-induced lung inflammation, depletion of NK cells resulted in increased ILC2 numbers and enhanced cytokine production by ILC2s, as well as aggravated eosinophilia and goblet cell hyperplasia. Collectively, these data show that NK cells negatively regulate ILC2s during the early stage of lung inflammation, which represents the novel cellular interaction between two family members of ILCs. Copyright © 2017 by The American Association of Immunologists, Inc.

  8. Natural Killer (NK) Cell Education Differentially Influences HIV Antibody-Dependent NK Cell Activation and Antibody-Dependent Cellular Cytotoxicity.

    PubMed

    Bernard, Nicole F; Kiani, Zahra; Tremblay-McLean, Alexandra; Kant, Sanket A; Leeks, Christopher E; Dupuy, Franck P

    2017-01-01

    Immunotherapy using broadly neutralizing antibodies (bNAbs) endowed with Fc-mediated effector functions has been shown to be critical for protecting or controlling viral replication in animal models. In human, the RV144 Thai trial was the first trial to demonstrate a significant protection against HIV infection following vaccination. Analysis of the correlates of immune protection in this trial identified an association between the presence of antibody-dependent cellular cytotoxicity (ADCC) mediated by immunoglobulin G (IgG) antibodies (Abs) to HIV envelope (Env) V1/V2 loop structures and protection from infection, provided IgA Abs with competing specificity were not present. Systems serology analyses implicated a broader range of Ab-dependent functions in protection from HIV infection, including but not limited to ADCC and Ab-dependent NK cell activation (ADNKA) for secretion of IFN-γ and CCL4 and expression of the degranulation marker CD107a. The existence of such correlations in the absence of bNAbs in the RV144 trial suggest that NK cells could be instrumental in protecting against HIV infection by limiting viral spread through Fc-mediated functions such as ADCC and the production of antiviral cytokines/chemokines. Beside the engagement of FcγRIIIa or CD16 by the Fc portion of anti-Env IgG1 and IgG3 Abs, natural killer (NK) cells are also able to directly kill infected cells and produce cytokines/chemokines in an Ab-independent manner. Responsiveness of NK cells depends on the integration of activating and inhibitory signals through NK receptors, which is determined by a process during their development known as education. NK cell education requires the engagement of inhibitory NK receptors by their human leukocyte antigen ligands to establish tolerance to self while allowing NK cells to respond to self cells altered by virus infection, transformation, stress, and to allogeneic cells. Here, we review recent findings regarding the impact of inter

  9. NK cells lacking FcεRIγ are associated with reduced liver damage in chronic hepatitis C virus infection.

    PubMed

    Oh, Jun S; Ali, Alaa K; Kim, Sungjin; Corsi, Daniel J; Cooper, Curtis L; Lee, Seung-Hwan

    2016-04-01

    A novel subset of human natural killer (NK) cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently uncovered in cytomegalovirus (CMV)+ individuals. This NK-cell subset (g-NK) was characterized by a deficiency in the expression of FcεRIγ adaptor protein and the long-lasting memory-like NK-cell phenotype, suggesting a role in chronic infections. This study investigates whether the g-NK-cell subset is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK-cell proportions and function in the PBMCs of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of the g-NK-cell subset having similarly enhanced antibody-dependent cellular cytotoxicity responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56(neg) NK cell population often found in chronic HCV patients, suggesting their involvement in immune response during HCV infection. For the first time, our findings indicate that the presence of the g-NK cells in CMV+ individuals is associated with amelioration of liver disease in chronic HCV infection, suggesting the beneficial roles of g-NK cells during a chronic infection.

  10. TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

    PubMed

    Wang, Feng; Hou, Hongyan; Wu, Shiji; Tang, Qing; Liu, Weiyong; Huang, Min; Yin, Botao; Huang, Jing; Mao, Lie; Lu, Yanfang; Sun, Ziyong

    2015-10-01

    Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T-cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK-cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN-γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK-cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

  11. Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function.

    PubMed

    Schlums, Heinrich; Cichocki, Frank; Tesi, Bianca; Theorell, Jakob; Beziat, Vivien; Holmes, Tim D; Han, Hongya; Chiang, Samuel C C; Foley, Bree; Mattsson, Kristin; Larsson, Stella; Schaffer, Marie; Malmberg, Karl-Johan; Ljunggren, Hans-Gustaf; Miller, Jeffrey S; Bryceson, Yenan T

    2015-03-17

    The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-associated adaptive NK cells and cytotoxic effector T cells but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The Past, Present, and Future of NK Cells in Hematopoietic Cell Transplantation and Adoptive Transfer.

    PubMed

    Cichocki, Frank; Verneris, Michael R; Cooley, Sarah; Bachanova, Veronika; Brunstein, Claudio G; Blazar, Bruce R; Wagner, John; Schlums, Heinrich; Bryceson, Yenan T; Weisdorf, Daniel J; Miller, Jeffrey S

    2016-01-01

    Hematopoietic cell transplantation (HCT) has been used as a part of cancer therapy for over half a decade. Beyond the necessity for donor-derived cells to reconstitute hematopoiesis after radiation and chemotherapy, immunologic reconstitution from allogeneic cells is important for the elimination of residual tumor cells. Natural killer (NK) cells are first among lymphocytes to reconstitute post-transplant and protect against cancer relapse. In this review, we provide a historical perspective on the role of NK cells in cancer control in the transplant setting and focus on current research aimed at improving NK cell responses for therapeutic benefit.

  13. Hidden talents of natural killers: NK cells in innate and adaptive immunity.

    PubMed

    Cooper, Megan A; Colonna, Marco; Yokoyama, Wayne M

    2009-10-01

    Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells and producing immunoregulatory cytokines. Herein, we discuss recent studies that indicate that NK cells span the conventional boundaries between innate and adaptive immunity. For example, it was recently discovered that NK cells have the capacity for memory-like responses, a property that was previously thought to be limited to adaptive immunity. NK cells have also been identified in multiple tissues, and a subset of cells that specialize in the production of the T(H)17 cytokine IL-22, NK-22s, was recently described in mucosal-associated lymphoid tissue. Finally, we review work that shows that NK cells develop at sites that were traditionally thought to be occupied only by adaptive immune cells, including the thymus and lymph nodes.

  14. Control of Metastasis by NK Cells.

    PubMed

    López-Soto, Alejandro; Gonzalez, Segundo; Smyth, Mark J; Galluzzi, Lorenzo

    2017-08-14

    The metastatic spread of malignant cells to distant anatomical locations is a prominent cause of cancer-related death. Metastasis is governed by cancer-cell-intrinsic mechanisms that enable neoplastic cells to invade the local microenvironment, reach the circulation, and colonize distant sites, including the so-called epithelial-to-mesenchymal transition. Moreover, metastasis is regulated by microenvironmental and systemic processes, such as immunosurveillance. Here, we outline the cancer-cell-intrinsic and -extrinsic factors that regulate metastasis, discuss the key role of natural killer (NK) cells in the control of metastatic dissemination, and present potential therapeutic approaches to prevent or target metastatic disease by harnessing NK cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Caribbean and La Réunion Chikungunya Virus Isolates Differ in Their Capacity To Induce Proinflammatory Th1 and NK Cell Responses and Acute Joint Pathology

    PubMed Central

    Teo, Teck-Hui; Her, Zhisheng; Tan, Jeslin J. L.; Lum, Fok-Moon; Lee, Wendy W. L.; Chan, Yi-Hao; Ong, Ruo-Yan; Kam, Yiu-Wing; Leparc-Goffart, Isabelle; Gallian, Pierre; Rénia, Laurent; de Lamballerie, Xavier

    2015-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host. IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint

  16. Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds

    PubMed Central

    Lagrue, Kathryn; Carisey, Alex; Morgan, David J.; Chopra, Rajesh

    2015-01-01

    As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)–mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds. PMID:26002964

  17. Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds.

    PubMed

    Lagrue, Kathryn; Carisey, Alex; Morgan, David J; Chopra, Rajesh; Davis, Daniel M

    2015-07-02

    As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)-mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds.

  18. Proapoptotic Bim regulates antigen-specific NK cell contraction and the generation of the memory NK cell pool after cytomegalovirus infection

    PubMed Central

    Min-Oo, Gundula; Bezman, Natalie A.; Madera, Sharline; Sun, Joseph C.

    2014-01-01

    Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim (Bcl2l11) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11−/− Ly49H+ NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11−/− NK memory subset, which displays a less mature phenotype (CD11blo, CD27+) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11−/− memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells. PMID:24958849

  19. Proapoptotic Bim regulates antigen-specific NK cell contraction and the generation of the memory NK cell pool after cytomegalovirus infection.

    PubMed

    Min-Oo, Gundula; Bezman, Natalie A; Madera, Sharline; Sun, Joseph C; Lanier, Lewis L

    2014-06-30

    Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim (Bcl2l11) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11(-/-) Ly49H(+) NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11(-/-) NK memory subset, which displays a less mature phenotype (CD11b(lo), CD27(+)) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11(-/-) memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells. © 2014 Min-Oo et al.

  20. Functional analysis of NK cell subsets activated by 721.221 and K562 HLA-null cells.

    PubMed

    Lisovsky, Irene; Isitman, Gamze; Bruneau, Julie; Bernard, Nicole F

    2015-04-01

    HLA-null cell lines [721.221 (henceforth, 721) and K562] are often used to study NK cell activation. NK cells are innate immune lymphocytes that express a variety of stochastically expressed inhibitory and activating receptors. Although it is known that 721 and K562 have divergent origins, they have been used interchangeably to stimulate NK cells in many studies. We hypothesized that the differences between 721 and K562 cells may result in differential NK cell-activation patterns. In this report, we assessed all possible combinations of CD107a expression and IFN-γ and CCL4 secretion in total NK and 3DL1(+/-) NK cell populations induced by these 2 cell lines. 721 activates a significantly higher frequency of NK cells and 3DL1(+) NK cells than K562. The NK cell functional subsets that are stimulated to a higher degree by 721 than K562 include those secreting IFN-γ and/or CCL4. On the other hand, the functional subsets that include CD107 expression contribute to a higher proportion of the total NK cell response following stimulation with K562 than 721. These results have implications for the selection of HLA-null cell lines to use as NK cell stimuli in investigations of their role in infectious diseases, cancer, and transplantation.

  1. Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality.

    PubMed

    Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W; Böttcher, Sebastian; van Dongen, Jacques J M; Orfao, Alberto; Almeida, Julia

    2015-12-15

    Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

  2. Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

    PubMed Central

    Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C.; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W.; Böttcher, Sebastian; van Dongen, Jacques J.M.

    2015-01-01

    Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56low NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56low NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94hi/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality. PMID:26556869

  3. Cognate HLA absence in trans diminishes human NK cell education

    PubMed Central

    Landtwing, Vanessa; Raykova, Ana; Pezzino, Gaetana; Béziat, Vivien; Graf, Claudine; Moretta, Alessandro; Capaul, Riccarda; Zbinden, Andrea; Malmberg, Karl-Johan; Chijioke, Obinna; Münz, Christian

    2016-01-01

    NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA– tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand–mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor–reconstituted mice improved NK cell–mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA– tumor cell recognition but allows for improved NK cell–mediated immune control of a human γ-herpesvirus. PMID:27571408

  4. A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

    PubMed

    Wu, Ning; Zhong, Ming-Chao; Roncagalli, Romain; Pérez-Quintero, Luis-Alberto; Guo, Huaijian; Zhang, Zhanguang; Lenoir, Christelle; Dong, Zhongjun; Latour, Sylvain; Veillette, André

    2016-04-01

    Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

  5. PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma.

    PubMed

    Beldi-Ferchiou, Asma; Lambert, Marion; Dogniaux, Stéphanie; Vély, Frédéric; Vivier, Eric; Olive, Daniel; Dupuy, Stéphanie; Levasseur, Frank; Zucman, David; Lebbé, Céleste; Sène, Damien; Hivroz, Claire; Caillat-Zucman, Sophie

    2016-11-08

    Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance.

  6. PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma

    PubMed Central

    Beldi-Ferchiou, Asma; Lambert, Marion; Dogniaux, Stéphanie; Vély, Frédéric; Vivier, Eric; Olive, Daniel; Dupuy, Stéphanie; Levasseur, Frank; Zucman, David; Lebbé, Céleste; Sène, Damien; Hivroz, Claire; Caillat-Zucman, Sophie

    2016-01-01

    Programmed Death-1 (PD-1), an inhibitory receptor expressed by activated lymphocytes, is involved in regulating T- and B-cell responses. PD-1 and its ligands are exploited by a variety of cancers to facilitate tumor escape through PD-1-mediated functional exhaustion of effector T cells. Here, we report that PD-1 is upregulated on Natural Killer (NK) cells from patients with Kaposi sarcoma (KS). PD-1 was expressed in a sub-population of activated, mature CD56dimCD16pos NK cells with otherwise normal expression of NK surface receptors. PD-1pos NK cells from KS patients were hyporesponsive ex vivo following direct triggering of NKp30, NKp46 or CD16 activating receptors, or short stimulation with NK cell targets. PD-1pos NK cells failed to degranulate and release IFNγ, but exogenous IL-2 or IL-15 restored this defect. That PD-1 contributed to NK cell functional impairment and was not simply a marker of dysfunctional NK cells was confirmed in PD-1-transduced NKL cells. In vitro, PD-1 was induced at the surface of healthy control NK cells upon prolonged contact with cells expressing activating ligands, i.e. a condition mimicking persistent stimulation by tumor cells. Thus, PD-1 appears to plays a critical role in mediating NK cell exhaustion. The existence of this negative checkpoint fine-tuning NK activation highlights the possibility that manipulation of the PD-1 pathway may be a strategy for circumventing tumor escape not only from the T cell-, but also the NK-cell mediated immune surveillance. PMID:27662664

  7. Maternal obesity drives functional alterations in uterine NK cells

    PubMed Central

    Perdu, Sofie; Castellana, Barbara; Kim, Yoona; Chan, Kathy; DeLuca, Lauren; Beristain, Alexander G.

    2016-01-01

    Over one-fifth of North American women of childbearing age are obese, putting these women at risk for a variety of detrimental chronic diseases. In addition, obesity increases the risk for developing major complications during pregnancy. The mechanisms by which obesity contributes to pregnancy complications and loss remain unknown. Increasing evidence indicates that obesity results in major changes to adipose tissue immune cell composition and function; whether or not obesity also affects immune function in the uterus has not been explored. Here we investigated the effect of obesity on uterine natural killer (uNK) cells, which are essential for uterine artery remodeling and placental development. Using a cohort of obese or lean women, we found that obesity led to a significant reduction in uNK cell numbers accompanied with impaired uterine artery remodeling. uNK cells isolated from obese women had altered expression of genes and pathways associated with extracellular matrix remodeling and growth factor signaling. Specifically, uNK cells were hyper-responsive to PDGF, resulting in overexpression of decorin. Functionally, decorin strongly inhibited placental development by limiting trophoblast survival. Together, these findings establish a potentially new link between obesity and poor pregnancy outcomes, and indicate that obesity-driven changes to uterine-resident immune cells critically impair placental development. PMID:27699222

  8. Comparative Analysis of Activation Phenotype, Proliferation, and IFN-γ Production by Spleen NK1.1+ and NK1.1− T Cells During Plasmodium chabaudi AS Malaria

    PubMed Central

    Freitas do Rosário, Ana Paula; Sardinha, Luiz Roberto; Castillo-Méndez, Sheyla Inés; Zago, Cláudia Augusta; Rodriguez-Málaga, Sérgio Marcelo; Álvarez Mosig, José Maria; D'Império Lima, Maria Regina

    2010-01-01

    The NK1.1 molecule participates in NK, NKT, and T-cell activation, contributing to IFN-γ production and cytotoxicity. To characterize the early immune response to Plasmodium chabaudi AS, spleen NK1.1+ and NK1.1− T cells were compared in acutely infected C57BL/6 mice. The first parasitemia peak in C57BL/6 mice correlated with increase in CD4+NK1.1+TCR-αβ+, CD8+NK1.1+TCR-αβ+, and CD4+NK1.1−TCR-αβ+ cell numbers per spleen, where a higher increment was observed for NK1.1+ T cells compared to NK1.1− T cells. According to the ability to recognize the CD1d-α-GalCer tetramer, CD4+NK1.1+ cells in 7-day infected mice were not predominantly invariant NKT cells. At that time, nearly all NK1.1+ T cells and around 30% of NK1.1− T cells showed an experienced/activated (CD44HICD69HICD122HI) cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity. Moreover, whereas IFN-γ production by CD4+NK1.1+ cells peaked at day 4 p.i., the IFN-γ response of CD4+NK1.1− cells continued to increase at day 5 of infection. We also observed, at day 7 p.i., 2-fold higher percentages of perforin+ cells in CD8+NK1.1+ cells compared to CD8+NK1.1− cells. These results indicate that spleen NK1.1+ and NK1.1− T cells respond to acute P. chabaudi malaria with different kinetics in terms of activation, proliferation, and IFN-γ production. PMID:20187775

  9. Beta 2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses

    PubMed Central

    1996-01-01

    A number of investigations have established the critical role of interleukin 4 (IL-4) in mediating the development of T helper (Th)2 effector cells in vitro and in vivo. Despite intensive study, the origin of the IL-4 required for Th2 priming and differentiation remains unclear. Natural killer (NK)1.1+ alpha/beta T cell receptor+ T(NT) cells, a unique lineage of cells capable of producing large amounts of IL-4 after activation in vivo, are important candidates for directing Th2 priming. These cells are selected by the nonpolymorphic major histocompatibility complex (MHC) class I molecule, CD1, and are deficient in beta 2-microglobulin (beta 2m)-null mice. We used beta 2m- deficient mice on both BALB/c and C57BL/6 backgrounds to examine their capacity to mount Th2 immune responses after challenge with a number of well-characterized antigens administered by a variety of routes. As assessed by immunization with protein antigen, infection with Leishmania major, embolization with eggs of Schistosoma mansoni, intestinal infection with Nippostrongylus brasiliensis, or induction of airway hyperreactivity to aerosolized antigen, beta 2m-deficient mice developed functional type 2 immune responses that were not substantially different than those in wild-type mice. Production of IL- 4 and the generation of immunoglobulin E (IgE) and eosinophil responses were preserved as assessed by a variety of assays. Collectively, these results present a comprehensive analysis of type 2 immune responses in beta 2m-deficient mice, and indicate that beta 2m-dependent NT cells are not required for Th2 development in vivo. PMID:8879201

  10. Adaptive NK Cells with Low TIGIT Expression Are Inherently Resistant to Myeloid-Derived Suppressor Cells.

    PubMed

    Sarhan, Dhifaf; Cichocki, Frank; Zhang, Bin; Yingst, Ashley; Spellman, Stephen R; Cooley, Sarah; Verneris, Michael R; Blazar, Bruce R; Miller, Jeffrey S

    2016-10-01

    Human cytomegalovirus (CMV)-induced adaptive natural killer (NK) cells display distinct phenotypic and functional characteristics, including properties of immune memory. We hypothesized that these cells may be more resistant to suppression mediated by immunoregulatory cell subsets, making them attractive for use in cancer therapy. Here we report that relative to conventional NK cells, adaptive NK cells express lower levels of the inhibitory receptor T-cell Ig and ITIM domain (TIGIT), which results in resistance to immune suppression mediated by myeloid-derived suppressor cells (MDSC), as derived from cytokine induction in normal blood or patients with myelodysplastic syndrome. In contrast, conventional NK cells were potently suppressed by MDSCs, an effect abrogated completely by TIGIT blockade. Mechanistically, TIGIT signaling in NK cells after MDSC coculture led to a decrease in the phosphorylation of ZAP70/Syk and ERK1/2. These effects were reversed by blocking TIGIT on NK cells or by inhibiting production of reactive oxygen species (ROS) by MDSCs, the latter of which upregulated the TIGIT ligand CD155 on MDSCs. Accordingly, the blunted cytotoxicity of NK cells cocultured with MDSCs against tumor cells could be reversed by blocking TIGIT or ROS production. Overall, our results show how adaptive NK cells arising in response to CMV infection can escape MDSC-mediated suppression, and defined TIGIT antagonists as a novel type of checkpoint inhibitor to enhance NK-cell-mediated responses against cancer and infection. Cancer Res; 76(19); 5696-706. ©2016 AACR. ©2016 American Association for Cancer Research.

  11. Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

    PubMed

    Kelly, Michelle N; Zheng, Mingquan; Ruan, Sanbao; Kolls, Jay; D'Souza, Alain; Shellito, Judd E

    2013-01-01

    Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

  12. Dysregulation of regulatory CD56(bright) NK cells/T cells interactions in multiple sclerosis.

    PubMed

    Laroni, Alice; Armentani, Eric; Kerlero de Rosbo, Nicole; Ivaldi, Federico; Marcenaro, Emanuela; Sivori, Simona; Gandhi, Roopali; Weiner, Howard L; Moretta, Alessandro; Mancardi, Giovanni L; Uccelli, Antonio

    2016-08-01

    Recent evidence has shown that CD56(bright) NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56(bright) NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56(bright) NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56(bright) NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56(bright) NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56(bright) NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56(bright) NK cells. The defect in controlling autologous T cells by CD56(bright) NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

    PubMed

    Wild, J; Schmiedel, B J; Maurer, A; Raab, S; Prokop, L; Stevanović, S; Dörfel, D; Schneider, P; Salih, H R

    2015-08-01

    Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

  14. Repression of GSK3 restores NK cell cytotoxicity in AML patients.

    PubMed

    Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A; Gupta, Kalpana; deLima, Marcos; Beck, Rose C; Wald, David N

    2016-04-04

    Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.

  15. Teach Your NK Cells Well.

    PubMed

    Cooper, Megan A

    2016-08-16

    Natural killer cells readily kill target cells, and education ensures tolerance to self. In this issue of Immunity, Boudreau et al. (2016) and Chen et al. (2016) report new mechanisms of human and mouse natural killer cell education by inhibitory and activating receptors.

  16. Broadly impaired NK cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro.

    PubMed

    Johansson, Sofia E; Hall, Håkan; Björklund, Jens; Höglund, Petter

    2004-01-01

    NK cells represent a link between innate and adaptive immunity, and may play a role in regulating autoimmune disorders. We have characterized the NK cell population in non-obese diabetic (NOD) mice. The percentage and absolute numbers of NK cells were similar in NOD and control MHC-matched B6.g7 mice. However, the capacity of NOD NK cells to mediate natural cytotoxicity as well as FcR- and Ly49D-mediated killing was compromised in vitro, suggesting a defect affecting multiple activation pathways. The defect was neither linked to the NK gene complex nor to the MHC, as determined by comparison with mice congenic for these regions. Introducing the beta(2)-microglobulin mutation on the NOD background further impaired NK cell function, showing that the compromised cytotoxic capacity in these two strains arises from two independent mechanisms. In vivo rejection responses against tumor cells and against MHC class I-deficient spleen cells were decreased in naive NOD recipients, but restored in mice pre-activated with tilorone, a potent activator of NK cells. In addition, killing of some tumor targets was restored in vitro after activation of NK cells with IL-12 plus IL-18 or with IFN-alpha/beta, but not with IL-2. Interestingly, natural killing of RMA-S targets by NOD NK cells could not be restored in vitro, indicating that restoration of killing capacity was only partial. Our data suggest a severe, but partially restorable, killing defect in NOD NK cells, affecting activation through several pathways.

  17. Salivary Gland NK Cells Are Phenotypically and Functionally Unique

    PubMed Central

    Brossay, Laurent

    2011-01-01

    Natural killer (NK) cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells. PMID:21249177

  18. An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele.

    PubMed

    Jochems, Caroline; Hodge, James W; Fantini, Massimo; Fujii, Rika; Morillon, Y Maurice; Greiner, John W; Padget, Michelle R; Tritsch, Sarah R; Tsang, Kwong Yok; Campbell, Kerry S; Klingemann, Hans; Boissel, Laurent; Rabizadeh, Shahrooz; Soon-Shiong, Patrick; Schlom, Jeffrey

    2016-12-27

    Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

  19. An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele

    PubMed Central

    Jochems, Caroline; Hodge, James W.; Fantini, Massimo; Fujii, Rika; Maurice, Y. Morillon; Greiner, John W.; Padget, Michelle R.; Tritsch, Sarah R.; Tsang, Kwong Yok; Campbell, Kerry S.; Klingemann, Hans; Boissel, Laurent; Rabizadeh, Shahrooz; Soon-Shiong, Patrick; Schlom, Jeffrey

    2016-01-01

    Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies. PMID:27861156

  20. Effect of tumor cells and tumor microenvironment on NK-cell function.

    PubMed

    Vitale, Massimo; Cantoni, Claudia; Pietra, Gabriella; Mingari, Maria Cristina; Moretta, Lorenzo

    2014-06-01

    The ability of tumors to manage an immune-mediated attack has been recently included in the "next generation" of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross-interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK-cell responses to tumors. Tumor cells, tumor-associated fibroblasts and tumor-induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK-cell activation pathways or the complex receptor array that regulate NK-cell activation and antitumor activity. Thus, the definition of tumor microenvironment-related immunosuppressive factors, along with the identification of new classes of tissue-residing NK-like innate lymphoid cells, represent key issues to design effective NK-cell-based therapies of solid tumors.

  1. NK cell phenotypic modulation in lung cancer environment.

    PubMed

    Jin, Shi; Deng, Yi; Hao, Jun-Wei; Li, Yang; Liu, Bin; Yu, Yan; Shi, Fu-Dong; Zhou, Qing-Hua

    2014-01-01

    Nature killer (NK) cells play an important role in anti-tumor immunotherapy. But it indicated that tumor cells impacted possibly on NK cell normal functions through some molecules mechanisms in tumor microenvironment. Our study analyzed the change about NK cells surface markers (NK cells receptors) through immunofluorescence, flow cytometry and real-time PCR, the killed function from mouse spleen NK cell and human high/low lung cancer cell line by co-culture. Furthermore we certificated the above result on the lung cancer model of SCID mouse. We showed that the infiltration of NK cells in tumor periphery was related with lung cancer patients' prognosis. And the number of NK cell infiltrating in lung cancer tissue is closely related to the pathological types, size of the primary cancer, smoking history and prognosis of the patients with lung cancer. The expression of NK cells inhibitor receptors increased remarkably in tumor micro-environment, in opposite, the expression of NK cells activated receptors decrease magnificently. The survival time of lung cancer patient was positively related to NK cell infiltration degree in lung cancer. Thus, the down-regulation of NKG2D, Ly49I and the up-regulation of NKG2A may indicate immune tolerance mechanism and facilitate metastasis in tumor environment. Our research will offer more theory for clinical strategy about tumor immunotherapy.

  2. A simple method to measure NK cell cytotoxicity in vivo.

    PubMed

    Saudemont, Aurore; Burke, Shannon; Colucci, Francesco

    2010-01-01

    Natural killer (NK) cells were discovered in the 1970 s and named after their naturally occurring cytotoxic activity against tumor cells. It has recently become clear that NK cells are not just killers and that malignancy is unlikely to be the selective pressure driving the evolution of NK cells. Indeed, NK cells secrete a host of cytokines and chemokines that contribute to tissue remodeling at the feto-maternal interface and to both innate and adaptive immunity during infection. Moreover, in certain conditions, they cannot deliver functions cell autonomously, as they require priming from other cells, namely dendritic cells. Nevertheless, natural cytotoxicity is still considered an important parameter used to evaluate NK cell biology, both in the clinic and in the research lab. In this chapter we describe a simple method to quantify spontaneous NK cell cytotoxicity in vivo.

  3. Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFβ1 in Human Gastric Cancer.

    PubMed

    Peng, Liu-Sheng; Zhang, Jin-Yu; Teng, Yong-Sheng; Zhao, Yong-Liang; Wang, Ting-Ting; Mao, Fang-Yuan; Lv, Yi-Pin; Cheng, Ping; Li, Wen-Hua; Chen, Na; Duan, Mubing; Chen, Weisan; Guo, Gang; Zou, Quan-Ming; Zhuang, Yuan

    2017-03-01

    Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infiltrating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNγ, TNFα, and Ki-67 expression. We found that tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNγ(+) and TNFα(+) NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macrophages could impair NK-cell expression of IFNγ, TNFα, and Ki-67. Blockade of TGFβ1 attenuated such monocytes/macrophages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor-associated monocytes/macrophages, and that restoring NK-cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248-56. ©2017 AACR.

  4. NK cells: immune cross-talk and therapeutic implications

    PubMed Central

    Malhotra, Anshu; Shanker, Anil

    2011-01-01

    Increased evidence of cross-talk between NK cells and other immune cells has enhanced the possibilities of exploiting the interplay between the activation and inhibition of NK cells for immunotherapeutic purposes. The battery of receptors possessed by NK cells help them to efficiently detect aberrant and infected cells and embark on the signaling pathways necessary to eliminate them. Endogenous expansion of NK cells and their effector mechanisms are under exploration for enhancing adoptive immunotherapy prospects in combination with immunostimulatory and cell-death-sensitizing treatments against cancer, viral infections and other pathophysiological autoimmune conditions. Various modes of NK cell manipulation are being undertaken to overcome issues such as relapse and graft rejections associated with adoptive immunotherapy. While tracing the remarkable properties of NK cells and the major developments in this field, we highlight the role of immune cooperativity in the betterment of current immunotherapeutic approaches. PMID:21995569

  5. Multiple effects of IL-21 on human NK cells in ex vivo expansion.

    PubMed

    Li, Qi; Ye, Lin-Jie; Ren, Hai-Long; Huyan, Ting; Li, Jing; Shi, Jun-Ling; Huang, Qing-Sheng

    2015-07-01

    Natural killer (NK) cells (CD56(+)CD3(-)) are large, granular immunocytes that play a very pivotal role in the anti-inflammatory response and tumor surveillance. As an ideal cytotoxic lymphocyte (CTL), NK cells have attracted much attention in clinical trials. However, an insufficient number and their limited life span are bottlenecks that limit the application of NK cells in adoptive immunotherapy. Interleukins such as IL-2, IL-15 and IL-18 are recognized as factors that stimulate NK cells and have been used in NK cells ex vivo expansion. Similar to IL-2 and IL-15, IL-21 is a common γ-chain cytokine that is important in NK cell activation, maturation and proliferation. The present study aims to assess the effects of membrane-bound and soluble IL-21 on primary human NK cells during ex vivo expansion. IL-21 was found to have multiple effects on NK cells, increasing their cytotoxicity in a concentration-dependent manner by up-regulating IFN-γ and Granzyme-B expression. Nevertheless, at a high concentration (50 ng/mL), IL-21 curtailed the life span of NK cells by significantly inducing apoptosis. Moreover, when treated with IL-21, the number of NKT (CD56(+)CD3(+)) cells increased among peripheral blood mononuclear cells (PBMCs) during ex vivo expansion in a concentration-dependent manner. IL-21 also promoted expanded cells to enter into S phase of the cell cycle during the first to second weeks of culture. All these results suggest that IL-21 has multiple effects on NK cell development and functions. More attention should be given to the dosage and multiple effects of IL-21 when it was applied to NK cells in ex vivo expansion.

  6. Human NK cells in acute myeloid leukaemia patients: analysis of NK cell-activating receptors and their ligands.

    PubMed

    Sanchez-Correa, Beatriz; Morgado, Sara; Gayoso, Inmaculada; Bergua, Juan M; Casado, Javier G; Arcos, Maria Jose; Bengochea, Maria Luisa; Duran, Esther; Solana, Rafael; Tarazona, Raquel

    2011-08-01

    Natural killer (NK) cell activation is strictly regulated to ensure that healthy cells are preserved, but tumour-transformed or virus-infected cells are recognized and eliminated. To carry out this selective killing, NK cells have an ample repertoire of receptors on their surface. Signalling by inhibitory and activating receptors by interaction with their ligands will determine whether the NK cell becomes activated and kills the target cell. Here, we show reduced expression of NKp46, NKp30, DNAM-1, CD244 and CD94/NKG2C activating receptors on NK cells from acute myeloid leukaemia patients. This reduction may be induced by chronic exposure to their ligands on leukaemic blasts. The analysis of ligands for NK cell-activating receptors showed that leukaemic blasts from the majority of patients express ligands for NK cell-activating receptors. DNAM-1 ligands are frequently expressed on blasts, whereas the expression of the NKG2D ligand MICA/B is found in half of the patients and CD48, a ligand for CD244, in only one-fourth of the patients. The decreased expression of NK cell-activating receptors and/or the heterogeneous expression of ligands for major receptors on leukaemic blasts can lead to an inadequate tumour immunosurveillance by NK cells. A better knowledge of the activating receptor repertoire on NK cells and their putative ligands on blasts together with the possibility to modulate their expression will open new possibilities for the use of NK cells in immunotherapy against leukaemia.

  7. Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro.

    PubMed

    Cao, Jun; Grauwet, Korneel; Vermeulen, Ben; Devriendt, Bert; Jiang, Ping; Favoreel, Herman; Nauwynck, Hans

    2013-06-28

    The adaptive immunity against PRRSV has already been studied in depth, but only limited data are available on the innate immune responses against this pathogen. In the present study, we analyzed the interaction between porcine natural killer (NK) cells and PRRSV-infected primary porcine alveolar macrophages (PAMs), since NK cells are one of the most important components of innate immunity and PAMs are primary target cells of PRRSV infection. NK cytotoxicity assays were performed using enriched NK cells as effector cells and virus-infected or mock-inoculated PAMs as target cells. The NK cytotoxicity against PRRSV-infected PAMs was decreased starting from 6h post inoculation (hpi) till the end of the experiment (12 hpi) and was significantly lower than that against pseudorabies virus (PrV)-infected PAMs. UV-inactivated PRRSV also suppressed NK activity, but much less than infectious PRRSV. Furthermore, co-incubation with PRRSV-infected PAMs inhibited degranulation of NK cells. Finally, using the supernatant of PRRSV-infected PAMs collected at 12 hpi showed that the suppressive effect of PRRSV on NK cytotoxicity was not mediated by soluble factors. In conclusion, PRRSV-infected PAMs showed a reduced susceptibility toward NK cytotoxicity, which may represent one of the multiple evasion strategies of PRRSV.

  8. Extranodal NK/T cell lymphoma and aggressive NK cell leukaemia: evidence for their origin on CD56+bright CD16-/+dim NK cells.

    PubMed

    Lima, Margarida

    2015-10-01

    Mature natural killer (NK) cell neoplasms are classified by the World Health Organization into extranodal NK/T cell lymphoma, nasal type (ENKTL) and aggressive NK cell leukaemia (ANKL). In order to propose their normal NK cell counterparts, we reviewed the literature on the phenotype of the neoplastic NK cells from five series of patients with ENKTL (n = 411) and seven series of patients with ANKL (n = 114) and compared with that of the normal and activated mature CD56 NK cell subsets. The tumour NK cells usually express brightly the CD56 adhesion molecule and CD94 lectin type killer receptor, and have an activation-related (cytoplasmic CD3ε, CD7, CD45RO, HLA-DR) phenotype; in contrast, CD16 and killer immunoglobulin-like receptors are frequently negative, and CD57 expression is almost never observed. These phenotypic features would suggest that ENKTL and ANKL cells do represent the neoplastic counterpart of the mature CD56 NK cells, which undergo activation and malignant transformation after Epstein-Barr virus infection.

  9. Effect of radiotherapy on the natural killer (NK)-cell activity of cancer patients

    SciTech Connect

    McGinnes, K.; Florence, J.; Penny, R.

    1987-05-01

    The aim of this study was to determine the effect of radiotherapy on peripheral blood natural killer (NK)-cell number and activity in 15 patients with cancer, prior to the commencement and at the completion of radiotherapy. The following observations were made. Prior to radiotherapy NK activity could not be correlated with the stage of malignancy. In all patients with advanced disease and with subnormal baseline NK activity, the outcome of radiotherapy was unfavorable. Following radiotherapy to sites including the mediastinum, patients had decreased NK activity compared with those receiving treatment to other sites. This decrease was not related to the dose of radiotherapy or stage of malignancy. The tumor response was favorable in most patients whose NK activity decreased as a result of radiotherapy. The decrease in NK activity may be associated with a decrease in the percentage of NK (N901) cells in the peripheral blood. The reduction in NK activity in those patients receiving mediastinal irradiation may be due to the large volume of blood which transits the field, so that the NK cells, or their more radiosensitive precursors, may be damaged and/or differentiation inhibited. Thus, these new observations show that radiotherapy does indeed affect the NK activity in cancer patients predominantly when the irradiation site includes the mediastinum.

  10. Intrinsic Contribution of Perforin to NK-Cell Homeostasis during Mouse Cytomegalovirus Infection.

    PubMed

    Arapović, Maja; Brizić, Ilija; Popović, Branka; Jurković, Slaven; Jordan, Stefan; Krmpotić, Astrid; Arapović, Jurica; Jonjić, Stipan

    2016-01-01

    In addition to their role as effector cells in virus control, natural killer (NK) cells have an immunoregulatory function in shaping the antiviral T-cell response. This function is further pronounced in perforin-deficient mice that show the enhanced NK-cell proliferation and cytokine secretion upon mouse cytomegalovirus (MCMV) infection. Here, we confirmed that stronger activation and maturation of NK cells in perforin-deficient mice correlates with higher MCMV load. To further characterize the immunoregulatory potential of perforin, we compared the response of NK cells that express or do not express perforin using bone-marrow chimeras. Our results demonstrated that the enhanced proliferation and maturation of NK cells in MCMV-infected bone-marrow chimeras is an intrinsic property of perforin-deficient NK cells. Thus, in addition to confirming that NK-cell proliferation is virus load dependent, our data extend this notion demonstrating that perforin plays an intrinsic role as a feedback mechanism in the regulation of NK-cell proliferation during viral infections.

  11. NK Cell Proportion and Number Are Influenced by Genetic Loci on Chromosomes 8, 9, and 17.

    PubMed

    Pelletier, Adam-Nicolas; Guilbault, Lorie; Guimont-Desrochers, Fanny; Hillhouse, Erin E; Lesage, Sylvie

    2016-03-15

    NK cells play a crucial role in innate immunity due to their direct cytotoxicity toward tumors, virally infected cells, and stressed cells, and they also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. In secondary lymphoid organs, NK cells compose the third most abundant lymphocyte subset after T cells and B cells. In this study, we perform an unbiased linkage analysis to determine the genetic loci that may limit the size of the NK cell compartment. Specifically, we exploit differences in NK cell proportion and absolute number between the C57BL/6 and the NOD mice. In addition to the previously identified linkage to chromosome 8, we find that a locus on chromosome 17, which encompasses the MHC locus, impacts NK cell number. Moreover, we identify a locus on mouse chromosome 9 that is strongly linked to the proportion and absolute number of NK cells. Using NOD congenic mice, we validate that both the MHC and the chromosome 9 loci influence the proportion and absolute number of NK cells. We have thus identified additional loci specifically linked to the proportion of NK cells and present some of the potential candidate genes comprised within these loci.

  12. T-cell and NK-cell posttransplantation lymphoproliferative disorders.

    PubMed

    Swerdlow, Steven H

    2007-06-01

    Posttransplantation lymphoproliferative disorders (PTLDs) of T-cell or natural killer (NK)-cell origin are an uncommon heterogeneous group of lymphoid proliferations that fulfill the criteria for one of the T- or NK-cell lymphomas/leukemias. This report summarizes 130 T/NK-cell PTLDs reported in the literature or presented at the Society for Hematopathology/European Association for Haematopathology Workshop on T/NK-cell malignancies. The T/NK-cell PTLDs occur at a median of 66 months following transplantation and are usually extranodal. The most common types reported are peripheral T-cell lymphoma, unspecified, and hepatosplenic T-cell lymphoma. Approximately one third are Epstein-Barr virus (EBV)+. The median survival is 6 months. EBV+ cases have a significantly longer survival than EBV- cases, even when indolent T-cell large granular lymphocytic leukemias are included among the EBV- cases. Many T/NK-cell PTLDs have been treated with chemotherapy, often together with decreased immunosuppression, but there are infrequent patients who have done well without chemotherapy or radiation.

  13. Stage-specific requirement of IL-18 for antiviral NK cell expansion

    PubMed Central

    Madera, Sharline; Sun, Joseph C.

    2014-01-01

    Although natural killer (NK) cells are considered part of the innate immune system, recent studies have demonstrated the ability of antigen-experienced NK cells to become long-lived and contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. Here, we demonstrate that NK cells require interleukin (IL)-18 signaling to generate a robust primary response during mouse cytomegalovirus (MCMV) infection, but do not require this signal for memory cell maintenance or recall responses. IL-12 signaling and STAT4 in activated NK cells increased the expression of the adaptor protein MyD88, which mediates signaling downstream of the IL-18 and IL-1 receptors. During MCMV infection, NK cells required MyD88 but not IL-1 receptor for optimal expansion. Thus, an IL-18-MyD88 signaling axis facilitates the prolific expansion of NK cells in response to primary viral infection, but not recall responses. PMID:25589075

  14. Cutting edge: stage-specific requirement of IL-18 for antiviral NK cell expansion.

    PubMed

    Madera, Sharline; Sun, Joseph C

    2015-02-15

    Although NK cells are considered part of the innate immune system, recent studies have demonstrated the ability of Ag-experienced NK cells to become long-lived and contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. In this article, we demonstrate that NK cells require IL-18 signaling to generate a robust primary response during mouse CMV (MCMV) infection but do not require this signal for memory cell maintenance or recall responses. IL-12 signaling and STAT4 in activated NK cells increased the expression of the adaptor protein MyD88, which mediates signaling downstream of the IL-18 and IL-1 receptors. During MCMV infection, NK cells required MyD88, but not IL-1R, for optimal expansion. Thus, an IL-18-MyD88 signaling axis facilitates the prolific expansion of NK cells in response to primary viral infection, but not recall responses. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. The combination of type I IFN, TNF-α, and cell surface receptor engagement with dendritic cells enables NK cells to overcome immune evasion by dengue virus.

    PubMed

    Lim, Daniel Say Liang; Yawata, Nobuyo; Selva, Kevin John; Li, Na; Tsai, Chen Yu; Yeong, Lai Han; Liong, Ka Hang; Ooi, Eng Eong; Chong, Mun Keat; Ng, Mah Lee; Leo, Yee Sin; Yawata, Makoto; Wong, Soon Boon Justin

    2014-11-15

    Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK-DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand-dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor-ligand interactions in triggering the antidengue response of primary human NK cells.

  16. NK Cell-based Immunotherapies in Pediatric Oncology

    PubMed Central

    McDowell, Kimberly A.; Hank, Jacquelyn A.; DeSantes, Kenneth B.; Capitini, Christian M.; Otto, Mario; Sondel, Paul M.

    2015-01-01

    The past decade has seen several anti-cancer immunotherapeutic strategies transition from “promising preclinical models” to treatments with proven clinical activity or benefit. In 2013, the journal Science selected the field of Cancer Immunotherapy as the overall number-1 breakthrough for the year in all of scientific research. In the setting of cancer immunotherapy for adult malignancies, many of these immunotherapy strategies have relied on the cancer patient’s endogenous anti-tumor T cell response. While much promising research in pediatric oncology is similarly focused on T cell reactivity, several pediatric malignancies themselves, or the chemo-radiotherapy used to achieve initial responses, can be associated with profound immune suppression, particularly of the T cell system. A separate component of the immune system, also able to mediate anti-tumor effects and less suppressed by conventional cancer treatment, is the NK cell system. In recent years, several distinct immunotherapeutic approaches that rely on the activity of NK cells have moved from preclinical development into clinical testing, and some have shown clear antitumor benefit. This review provides an overview of NK cell-based immunotherapy efforts that are directed towards childhood malignancies, with an emphasis on protocols that are already in clinical testing. PMID:25590232

  17. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy

    PubMed Central

    Wang, Wei; Erbe, Amy K.; Hank, Jacquelyn A.; Morris, Zachary S.; Sondel, Paul M.

    2015-01-01

    Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be “specifically activated” through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies. PMID:26284063

  18. Human Dendritic Cells Mitigate NK-Cell Dysfunction Mediated by Nonselective JAK1/2 Blockade.

    PubMed

    Curran, Shane A; Shyer, Justin A; St Angelo, Erin T; Talbot, Lillian R; Sharma, Sneh; Chung, David J; Heller, Glenn; Hsu, Katharine C; Betts, Brian C; Young, James W

    2017-01-01

    Janus kinase (JAK) inhibitors have achieved positive responses in myeloproliferative neoplasms, but at the expense of decreased natural killer (NK) cell numbers and compromised function. Selective JAK2 inhibition may also have a role in preventing and treating graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Although JAK inhibitors can impair monocyte-derived dendritic cell (moDC) activation and function and suppress effector T-cell responses, the effects on NK cells and the relevant mechanisms remain undefined. Using common γc cytokines and distinct human dendritic cell (DC) subtypes, we compared the effects of a JAK2-specific (TG101348) with a less selective JAK1/2 (ruxolitinib) inhibitor on NK-cell activation and function. Ruxolitinib treatment completely blocked IL2, IL15, and DC-mediated STAT5 phosphorylation, along with the capacity of NK cells to secrete IFNγ or lyse NK cell-sensitive targets. Only NK-cell proliferation stimulated by moDCs resisted ruxolitinib treatment. In contrast, TG101348 treatment of stimulated NK cells resulted in far less functional compromise. TG101348 completely inhibited only soluble IL15-mediated STAT5 phosphorylation, which Langerhans-type DCs (LCs), presenting membrane-bound IL15 in trans, could salvage. These results demonstrate that ruxolitinib's nonselective inhibition of JAK1/2 results in profound NK-cell dysfunction by blocking downstream pSTAT5, hence providing a persuasive rationale for the development of selective JAK2 inhibitors for immunotherapeutic applications. Cancer Immunol Res; 5(1); 52-60. ©2016 AACR.

  19. Human Cytomegalovirus Infection Enhances NK Cell Activity In Vitro

    PubMed Central

    Tschan-Plessl, Astrid; Stern, Martin; Schmied, Laurent; Retière, Christelle; Hirsch, Hans H.; Garzoni, Christian; van Delden, Christian; Boggian, Katia; Mueller, Nicolas J.; Berger, Christoph; Villard, Jean; Manuel, Oriol; Meylan, Pascal; Terszowski, Grzegorz

    2016-01-01

    Background Occurring frequently after solid organ and hematopoietic stem cell transplantation, cytomegalovirus (CMV) replication remains a relevant cause of mortality and morbidity in affected patients. Despite these adverse effects, an increased alloreactivity of natural killer (NK) cells after CMV infection has been assumed, but the underlying physiopathological mechanisms have remained elusive. Methods We used serial analyses of NK cells before and after CMV infection in kidney transplant recipients as an in vivo model for CMV primary infection to explore the imprint of CMV infection using every patient as their own control: We analyzed NK cell phenotype and function in 47 CMV seronegative recipients of CMV seropositive kidney grafts, who developed CMV primary infection posttransplant. Seronegative recipients of seronegative kidney grafts served as controls. Results We observed a significant increase of NKG2C expressing NK cells after CMV infection (mean increase, 17.5%; 95% confidence interval [95% CI], 10.2-24.9, P < 0.001), whereas cluster of differentiation (CD)57 expressing cells decreased (mean decrease, 14.1%; 95% CI, 8.0-20.2; P < 0.001). Analysis of killer immunoglobulin-like receptor (KIR) expression showed an increase of cells expressing KIR2DL1 as their only inhibitory KIR in patients carrying the cognate ligand HLA-C2 (mean increase, 10.0%; 95% CI, 1.7-18.3; P = 0.018). In C2-negative individuals, KIR2DL1 expression decreased (mean decrease, 3.9%; 95% CI, 1.6-6.2; P = 0.001). As for activating KIR, there was no conclusive change pattern. Most importantly, we observed a significantly higher NK cell degranulation and IFNγ production in response to different target cells (target K562, CD107a: mean increase, 9.9%; 95% CI, 4.8-15.0; P < 0.001; IFNγ: mean increase, 6.6%; 95% CI, 1.6-11.1; P < 0.001; target MRC-5, CD107a: mean increase, 6.9%; 95% CI, 0.7-13.1; P = 0.03; IFNγ: mean increase, 4.8%; 95% CI, 1.7-7.8; P = 0.002). Conclusions We report

  20. NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

    PubMed Central

    Grandclément, Camille; Pick, Horst; Vogel, Horst; Held, Werner

    2016-01-01

    Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca2+ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca2+-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS. PMID:26963818

  1. Impaired NK cell functionality and increased TNF-α production as biomarkers of chronic chikungunya arthritis and rheumatoid arthritis.

    PubMed

    Thanapati, Subrat; Ganu, Mohini; Giri, Prashant; Kulkarni, Shruti; Sharma, Meenal; Babar, Prasad; Ganu, Ashok; Tripathy, Anuradha S

    2017-04-01

    The chronic chikungunya arthritis symptoms closely mimic the rheumatoid arthritis (RA) symptoms, thus making it difficult to distinguish between these two clinical entities. The current comparative study characterizes NK (CD3(-)CD56(+)) and NK-like T (CD3(+)CD56(+)) cell responses in patients with chronic chikungunya arthritis and RA. Phenotype and functions of NK and NK-like T cells repertoire were assessed in 56 chronic chikungunya arthritis, 26 RA patients and 82 controls using flow cytometry. TNF-α and IFN-γ-secreting NK-like T cells were high in both chronic arthritis patients than in controls. Percentage of TNF-α(+) NK cells was higher in RA patients than in controls. Percentage of perforin(+) NK cells was low in both chronic arthritis patient groups. Among the patient groups, expressions of perforin(+) and IFN-γ(+) NK-like T cells were higher in RA. Overall, our data show reduced frequency of NK-like T cells, lower expression of perforin(+) NK, higher expression of TNF-α(+) NK-like T and IFN-γ(+) NK-like T cells as the markers of chronic arthritic diseases. In the absence of any specific treatment for chronic chikungunya induced arthritis and promising results of anti-TNF-α therapy against RA, current data may form the basis for future in vivo studies and has scope as possible therapeutics against chikungunya.

  2. Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions.

    PubMed

    Skinner, Cassandra C; McMichael, Elizabeth L; Jaime-Ramirez, Alena C; Abrams, Zachary B; Lee, Robert J; Carson, William E

    2016-08-01

    The folate receptor (FR) is overexpressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is overexpressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis using KB (human oral epithelial) and F01 (human melanoma) as a positive and a negative control, respectively. FR-positive and FR-negative cell lines were treated with F-IgG or control immunoglobulin G in the presence or absence of cytokines to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells increased following treatment with F-IgG compared with control immunoglobulin G at all effector : target (E : T) ratios (P<0.01). This trend further increased by NK cell stimulation with the activating cytokine interleukin-12. NK cell production of cytokines such as interferon-gamma, macrophage inflammatory protein 1α, and regulated on activation normal T-cell expressed and secreted (RANTES) was also significantly increased in response to costimulation with interleukin-12 stimulation and F-IgG-coated Mel 39 target cells compared with controls (P<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells, which can be further increased by the addition of cytokines.

  3. Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

    PubMed

    Mahmoud, Ahmad Bakur; Tu, Megan M; Wight, Andrew; Zein, Haggag S; Rahim, Mir Munir A; Lee, Seung-Hwan; Sekhon, Harman S; Brown, Earl G; Makrigiannis, Andrew P

    2016-02-01

    The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

  4. Differential phenotypic and functional properties of liver-resident NK cells and mucosal ILC1s.

    PubMed

    Tang, Ling; Peng, Hui; Zhou, Jing; Chen, Yongyan; Wei, Haiming; Sun, Rui; Yokoyama, Wayne M; Tian, Zhigang

    2016-02-01

    Group 1 innate lymphoid cells (ILCs) consist of conventional natural killer (cNK) cells, tissue-resident NK cells and mucosal ILC1s. Recently identified liver-resident NK cells, which can mount contact hypersensitivity responses, and mucosal ILC1s that are involved in pathogenesis of colitis are distinct from cNK cells in several aspects, but the issue of how they are related to each other has not been clearly clarified. Here, we show that liver-resident NK cells and mucosal ILC1s have different phenotypes, as evidenced by distinct expression patterns of homing-associated molecules. Moreover, mucosal ILC1s exhibit tissue residency akin to liver-resident NK cells. Importantly, liver-resident NK cells express relative high levels of cytotoxic effector molecules, which are poorly expressed by mucosal ILC1s, and exhibit stronger cytotoxic activity compared with mucosal ILC1s. These results demonstrate differential phenotypic and functional characteristics of liver-resident NK cells and mucosal ILC1s, shedding new light on the diversity of ILC family.

  5. NK cells link obesity-induced adipose stress to inflammation and insulin resistance.

    PubMed

    Wensveen, Felix M; Jelenčić, Vedrana; Valentić, Sonja; Šestan, Marko; Wensveen, Tamara Turk; Theurich, Sebastian; Glasner, Ariella; Mendrila, Davor; Štimac, Davor; Wunderlich, F Thomas; Brüning, Jens C; Mandelboim, Ofer; Polić, Bojan

    2015-04-01

    An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

  6. Role for coronin 1 in mouse NK cell function.

    PubMed

    Tchang, Vincent Sam Yong; Stiess, Michael; Siegmund, Kerstin; Karrer, Urs; Pieters, Jean

    2017-02-01

    Coronin 1, a member of the evolutionary conserved WD repeat protein family of coronin proteins is expressed in all leukocytes, but a role for coronin 1 in natural killer (NK) cell homeostasis and function remains unclear. Here, we have analyzed the number and functionality of NK cells in the presence and absence of coronin 1. In coronin 1-deficient mice, absolute NK cell numbers and phenotype were comparable to wild type mice in blood, spleen and liver. Following in vitro stimulation of the activating NK cell receptors NK1.1, NKp46, Ly49D and NKG2D, coronin 1-deficient NK cells were functional with respect to interferon-γ production, degranulation and intracellular Ca(2+) mobilization. Also, both wild type as well as coronin 1-deficient NK cells showed comparable cytotoxic activity. Furthermore, activation and functionality of NK cells following Vesicular Stomatitis Virus (VSV) infection was similar between wild type and coronin 1-deficient mice. Taken together these data suggest that coronin 1 is dispensable for mouse NK cell homeostasis and function.

  7. Know Thyself: NK-Cell Inhibitory Receptors Prompt Self-Tolerance, Education, and Viral Control

    PubMed Central

    Nash, William T.; Teoh, Jeffrey; Wei, Hairong; Gamache, Awndre; Brown, Michael G.

    2014-01-01

    Natural killer (NK) cells provide essential protection against viral infections. One of the defining features of this lymphocyte population is the expression of a wide array of variable cell surface stimulatory and inhibitory NK receptors (sNKR and iNKR, respectively). The iNKR are particularly important in terms of NK-cell education. As receptors specific for MHC class I (MHC I) molecules, they are responsible for self-tolerance and adjusting NK-cell reactivity based on the expression level of self-MHC I. The end result of this education is twofold: (1) inhibitory signaling tunes the functional capacity of the NK cell, endowing greater potency with greater education, and (2) education on self allows the NK cell to detect aberrations in MHC I expression, a common occurrence during many viral infections. Many studies have indicated an important role for iNKR and MHC I in disease, making these receptors attractive targets for manipulating NK-cell reactivity in the clinic. A greater understanding of iNKR and their ability to regulate NK cells will provide a basis for future attempts at translating their potential utility into benefits for human health. PMID:24795719

  8. Haploidentical Haematopoietic Stem Cell Transplantation: Role of NK Cells and Effect of Cytomegalovirus Infections.

    PubMed

    Della Chiesa, Mariella; Moretta, Lorenzo; Muccio, Letizia; Bertaina, Alice; Moretta, Francesca; Locatelli, Franco; Moretta, Alessandro

    2016-01-01

    Natural killer cells play an important role in the immune responses against cancer and viral infections. In addition, NK cells have been shown to exert a key role in haploidentical hematopoietic stem cell (HSC) transplantation for the therapy of high-risk leukemias. The anti-leukemia effect is mostly related to the presence of "alloreactive" NK cells, i.e., mature KIR(+) NK cells that express inhibitory KIR mismatched with HLA class I (KIR-L) of the patient. In addition, an important role is played by certain activating KIR (primarily, but not only, KIR2DS1) upon interaction with their HLA class I ligand (C2 alleles). In general, the presence of activating KIR correlates with a better prognosis. Beside the infusion of "pure" CD34(+) cells, a novel protocol has been recently developed in which depletion of αβ T cells and CD19(+) B cells makes it possible to infuse into the patient, together with donor CD34(+) HSCs, important effector cells including mature PB NK cells and γδ T cells. Recent studies revealed that cytomegalovirus (CMV) infection/reactivation may induce rapid NK cell maturation and greatly influence the NK receptor repertoire. The remarkable expansion of a subset expressing the activating receptor NKG2C, together with a more efficient virus-specific effector response after rechallenge with CMV (i.e., antigen specificity), and the longevity of the expanded population are all features consistent with an adaptive type of response and support the notion of a memory-like activity of NK cells.

  9. Characterization of the myeloid-derived suppressor cell subset regulated by NK cells in malignant lymphoma.

    PubMed

    Sato, Yusuke; Shimizu, Kanako; Shinga, Jun; Hidaka, Michihiro; Kawano, Fumio; Kakimi, Kazuhiro; Yamasaki, Satoru; Asakura, Miki; Fujii, Shin-Ichiro

    2015-03-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1(+)CD11b(+)Ly6G(med)Ly6C(med) MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27(+)CD11b(+)NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14(+)HLA-DR(-) and CD14(-) HLA-DR(-) MDSC) in NHL patients and found that higher IL-10-producing CD14(+)HLA-DR(-)MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

  10. Characterization of the myeloid-derived suppressor cell subset regulated by NK cells in malignant lymphoma

    PubMed Central

    Sato, Yusuke; Shimizu, Kanako; Shinga, Jun; Hidaka, Michihiro; Kawano, Fumio; Kakimi, Kazuhiro; Yamasaki, Satoru; Asakura, Miki; Fujii, Shin-ichiro

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1+CD11b+Ly6GmedLy6Cmed MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27+CD11b+NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14+HLA-DR− and CD14− HLA-DR− MDSC) in NHL patients and found that higher IL-10-producing CD14+HLA-DR−MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma. PMID:25949922

  11. NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease.

    PubMed

    Azeredo, E L; De Oliveira-Pinto, L M; Zagne, S M; Cerqueira, D I S; Nogueira, R M R; Kubelka, C F

    2006-02-01

    During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

  12. Do NK-cell receptors and alloreactivity affect solid organ transplantation?

    PubMed

    Vilches, Carlos; Parham, Peter

    2006-12-01

    Although natural killer cells lyse targets without pre-sensitization, and in an MHC-unrestricted manner, they can also respond to healthy allogeneic cells of different MHC type. Such alloreactivity is a consequence of NK cells using clonally distributed, inhibitory MHC class I receptors to achieve tolerance to healthy autologous cells. Absence of an appropriate MHC class I ligand on an allogeneic cell erroneously informs the NK cell that the allogeneic cell has lost MHC class I expression and should be killed. Potential NK-cell allo-reactivities are common in non-HLA-identical hematopoietic cell transplants and can have both beneficial and detrimental effects. Less is known of NK-cell allo-reactivities in solid organ transplantation. In animal models NK cells are neither necessary nor sufficient for acute transplant rejection, but they can make a contribution by helping activate T cells. Genes encoding NK-cell receptors for polymorphic MHC class I molecules are also highly polymorphic, contributing to variability of the NK-cell repertoire and response in the human population. These receptors could represent intrinsic patient factors that influence the success of their transplanted solid organs.

  13. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  14. Hepatic stellate cell interferes with NK cell regulation of fibrogenesis via curcumin induced senescence of hepatic stellate cell.

    PubMed

    Jin, Huanhuan; Jia, Yan; Yao, Zhen; Huang, Jingjing; Hao, Meng; Yao, Shunyu; Lian, Naqi; Zhang, Feng; Zhang, Chenxi; Chen, Xingran; Bian, Mianli; Shao, Jiangjuan; Wu, Li; Chen, Anping; Zheng, Shizhong

    2017-05-01

    Hepatic fibrosis, a common scarring response to various forms of chronic liver injury, is a precursor to cirrhosis and liver cancer. During liver fibrosis, hepatic stellate cells (HSCs) initially activate and proliferate, which are responsible for the secretion of extracellular matrix components. However, these cells eventually senesce and are cleared by natural killer (NK) cells. Our previous researches have shown that the natural product curcumin could promote the senescence of activated HSC. In this study, we investigated how NK cells target senescent HSC and assessed the effect of this process on liver fibrosis. We found that senescent HSC induced by curcumin are susceptible to NK cells killing, due to the increased expression of NK cell activating ligand major histocompatibility complex class I chain-related genes A (MICA) and UL16-binding proteins 2 (ULBP2), but not Poliovirus Receptor (PVR). Further studies displayed that the interaction between NK cells and senescent LX2 cells stimulated granule exocytosis. Moreover, the inhibition of granule exocytosis weakened the cytotoxicity of NK cells and promoted the accumulation of senescent LX2 cells. Therefore, these aggregated data indicated that NK cells mediated clearance of senescent LX2 cells and granule exocytosis could play a protective role in the improvement of liver fibrosis.

  15. NKp46-mediated Dicer1 inactivation results in defective NK-cell differentiation and effector functions in mice.

    PubMed

    Degouve, Sophie; Tavares, Armelle; Viel, Sébastien; Walzer, Thierry; Marçais, Antoine

    2016-08-01

    MicroRNAs control developmental pathways and effector functions in immune cells. Previous studies have studied the role of microRNAs in natural killer (NK) cells. However, the mouse models of microRNA depletion used were nonNK-specific and only partially depleting, hampering the interpretation of the data obtained. To clarify the role of microRNAs in murine NK cells, we deleted the RNase III enzyme Dicer1 in NKp46-expressing cells. We observed a drastic decrease in several microRNAs specifically in NK cells. Furthermore, the overall size of the "NK-cell" pool was severely decreased, a phenotype associated with compromised survival. Moreover, performing a broad flow cytometry profiling, we show that Dicer1-deficient NK cells failed to complete their differentiation program. In particular, several integrins were inappropriately expressed in mature NK cells. These defects coincided with decreased response to IL-15, a cytokine responsible for "NK-cell" maturation and survival. In addition, Dicer1 deletion impaired key "NK-cell" functions: target cell killing and production of IFN-γ, leading to defective control of metastasis. Dicer1 deletion thus affects "NK-cell" biology in a cell intrinsic manner at several distinct stages.

  16. PILRα binds an unknown receptor expressed primarily on CD56bright and decidual-NK cells and activates NK cell functions.

    PubMed

    Ophir, Yael; Duev-Cohen, Alexandra; Yamin, Rachel; Tsukerman, Pini; Bauman, Yoav; Gamliel, Moriya; Mandelboim, Ofer

    2016-07-05

    Natural Killer (NK) cells are innate immune lymphocytes specializing in recognition and killing of tumors and pathogens, using an array of activating and inhibitory receptors. NK inhibition is mediated by a large repertoire of inhibitory receptors, whereas a limited number of activating NK cell receptors execute NK cell activation. The ligands recognized by the activating receptors are stress-induced, pathogen derived, tumor specific and even self ligands. However, the full spectrum of NK cell receptors and ligands that control NK cell activity remains uncharacterized. Here we demonstrate that Paired Ig-Like type 2 Receptor Alpha (PILRα), binds a distinct human NK cell sub-population present in the peripheral blood and also in the decidua. We further demonstrate that the interaction of NK cells with PILRα expressing targets lead to elevated IFNγ secretion and cytotoxicity. In conclusion, we present here a novel NK activating ligand which binds and activates an unknown NK receptor expressed on a unique NK cell subset.

  17. Dysregulation of Chemokine/Chemokine Receptor Axes and NK Cell Tissue Localization during Diseases

    PubMed Central

    Bernardini, Giovanni; Antonangeli, Fabrizio; Bonanni, Valentina; Santoni, Angela

    2016-01-01

    Chemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling via their specific receptors, chemokines regulate tissue mobilization and trafficking of a wide array of immune cells, including natural killer (NK) cells. Current research is focused on analyzing changes in chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors, including CXCR4, CXCR3, and CX3CR1, are differentially expressed by NK cell subsets, and their expression levels can be modulated during NK cell activation. At first, this review will summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed. PMID:27766097

  18. Dysregulation of Chemokine/Chemokine Receptor Axes and NK Cell Tissue Localization during Diseases.

    PubMed

    Bernardini, Giovanni; Antonangeli, Fabrizio; Bonanni, Valentina; Santoni, Angela

    2016-01-01

    Chemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling via their specific receptors, chemokines regulate tissue mobilization and trafficking of a wide array of immune cells, including natural killer (NK) cells. Current research is focused on analyzing changes in chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors, including CXCR4, CXCR3, and CX3CR1, are differentially expressed by NK cell subsets, and their expression levels can be modulated during NK cell activation. At first, this review will summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed.

  19. Novel Microchip-Based Tools Facilitating Live Cell Imaging and Assessment of Functional Heterogeneity within NK Cell Populations.

    PubMed

    Forslund, Elin; Guldevall, Karolin; Olofsson, Per E; Frisk, Thomas; Christakou, Athanasia E; Wiklund, Martin; Onfelt, Björn

    2012-01-01

    Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells. This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events. The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the

  20. Novel Microchip-Based Tools Facilitating Live Cell Imaging and Assessment of Functional Heterogeneity within NK Cell Populations

    PubMed Central

    Forslund, Elin; Guldevall, Karolin; Olofsson, Per E.; Frisk, Thomas; Christakou, Athanasia E.; Wiklund, Martin; Önfelt, Björn

    2012-01-01

    Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells. This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events. The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the

  1. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells

    PubMed Central

    Frenkel, Deborah; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Magez, Stefan; Black, Samuel J.

    2016-01-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1 -/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR -/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected

  2. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

    PubMed

    Frenkel, Deborah; Zhang, Fengqiu; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Radwanska, Magdalena; Magez, Stefan; Black, Samuel J

    2016-07-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  3. Natural Killer (NK) Cells in Antibacterial Innate Immunity: Angels or Devils?

    PubMed Central

    Souza-Fonseca-Guimaraes, Fernando; Adib-Conquy, Minou; Cavaillon, Jean-Marc

    2012-01-01

    Natural killer (NK) cells were first described as immune leukocytes that could kill tumor cells and soon after were reported to kill virus-infected cells. In the mid-1980s, 10 years after their discovery, NK cells were also demonstrated to contribute to the fight against bacterial infection, particularly because of crosstalk with other leukocytes. A wide variety of immune cells are now recognized to interact with NK cells through the production of cytokines such as interleukin (IL)-2, IL-12, IL-15 and IL-18, which boost NK cell activities. The recent demonstration that NK cells express pattern recognition receptors, namely Toll-like and nucleotide oligomerization domain (NOD)-like receptors, led to the understanding that these cells are not only under the control of accessory cells, but can be directly involved in the antibacterial response thanks to their capacity to recognize pathogen-associated molecular patterns. Interferon (IFN)-γ is the predominant cytokine produced by activated NK cells. IFN-γ is a key contributor to antibacterial immune defense. However, in synergy with other inflammatory cytokines, IFN-γ can also lead to deleterious effects similar to those observed during sepsis. Accordingly, as the main source of IFN-γ in the early phase of infection, NK cells display both beneficial and deleterious effects, depending on the circumstances. PMID:22105606

  4. Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

    PubMed Central

    Kyei, Stephen K.; Ogbomo, Henry; Li, ShuShun; Timm-McCann, Martina; Xiang, Richard F.; Huston, Shaunna M.; Ganguly, Anutosh; Colarusso, Pina; Gill, M. John

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans. Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. PMID:27555306

  5. Neonatal antibody responses are attenuated by interferon-γ produced by NK and T cells during RSV infection.

    PubMed

    Tregoning, John S; Wang, Belinda Lei; McDonald, Jacqueline U; Yamaguchi, Yuko; Harker, James A; Goritzka, Michelle; Johansson, Cecilia; Bukreyev, Alexander; Collins, Peter L; Openshaw, Peter J

    2013-04-02

    Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4(+) and CD8(+) cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-γ, and blocking IFN-γ also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-γ reduced antibody titer, confirming that IFN-γ plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-γ-secreting cells might, in some situations, be less protective than those that do not.

  6. Neonatal antibody responses are attenuated by interferon-γ produced by NK and T cells during RSV infection

    PubMed Central

    Tregoning, John S.; Wang, Belinda Lei; McDonald, Jacqueline U.; Yamaguchi, Yuko; Harker, James A.; Goritzka, Michelle; Johansson, Cecilia; Bukreyev, Alexander; Collins, Peter L.; Openshaw, Peter J.

    2013-01-01

    Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4+ and CD8+ cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-γ, and blocking IFN-γ also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-γ reduced antibody titer, confirming that IFN-γ plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-γ–secreting cells might, in some situations, be less protective than those that do not. PMID:23509276

  7. Human NK cells: From surface receptors to clinical applications.

    PubMed

    Moretta, Lorenzo; Pietra, Gabriella; Vacca, Paola; Pende, Daniela; Moretta, Francesca; Bertaina, Alice; Mingari, Maria Cristina; Locatelli, Franco; Moretta, Alessandro

    2016-10-01

    Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β(+) T cells and of CD19(+) B cells, allow the infusion, together with CD34(+) HSC, of mature KIR(+) NK cells and of TCR γ/δ(+) T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR(+) NK

  8. Use of allogeneic NK cells for cancer immunotherapy

    PubMed Central

    Geller, Melissa A; Miller, Jeffrey S

    2012-01-01

    Controversy exists as to the role that the immune system plays in cancer therapy. While the immune system has been proposed to scavenge the body to prevent microscopic transformation from forming cancer, it has been difficult to mount its potential of shrinking established tumors. NK cells are components of the innate immune system. They can recognize targets without prior sensitization, making them ideal candidates to manipulate for therapeutic use against cancer. Initially, autologous NK cells were directed against tumors but it was realized that NK cells that recognize self cells are inhibited. More encouraging advances have been made with allogeneic NK cell therapy in clinical trials to overcome this limitation. In this article, we present developments in NK cell adoptive immunotherapy for hematologic and solid tumor malignancies. PMID:22091681

  9. Biallelic IRF8 Mutations Causing NK Cell Deficiency.

    PubMed

    López-Soto, Alejandro; Lorenzo-Herrero, Seila; Gonzalez, Segundo

    2017-03-01

    Human primary immunodeficiencies result in an exacerbated susceptibility to contracting infectious diseases. Recent work by Mace et al., published in the Journal of Clinical Investigation, unveils a novel genetic cause for the development of familial natural killer (NK) cell deficiency: a biallelic compound heterozygous mutation in IRF8, which leads to impaired NK cell development and cytotoxic activity.

  10. Sleep-deprivation reduces NK cell number and function mediated by β-adrenergic signalling.

    PubMed

    De Lorenzo, Beatriz H P; de Oliveira Marchioro, Laís; Greco, Carollina Ribeiro; Suchecki, Deborah

    2015-07-01

    Reduction of sleep time triggers a stress response, leading to augmented levels of glucocorticoids and adrenaline. These hormones regulate components of the innate immune system such as natural killer (NK) and NKT cells. In the present study, we sought to investigate whether and how stress hormones could alter the population and function of NK and NKT cells of mice submitted to different lengths of paradoxical sleep deprivation (PSD, from 24 to 72 h). Results showed that 72h of PSD decreased not only NK and NKT cell counts, but also their cytotoxic activity against B16F10 melanoma cells in vitro. Propranolol treatment during PSD reversed these effects, indicating a major inhibitory role of beta-adrenergic receptors (β-AR) on NK cells function. Moreover, both corticosterone plasma levels and expression of beta 2-adrenergic receptors (β2-AR) in NK cells increased by 48 h of PSD. In vitro incubation of NK cells with dexamethasone augmented the level of β2-AR in the cell surface, suggesting that glucocorticoids could induce β2-AR expression. In summary, we propose that reduction of NK and NKT cell number and cytotoxic activity appears to be mediated by glucocorticoids-induced increased expression of β2-AR in these cells.

  11. Rhizopus oryzae hyphae are damaged by human natural killer (NK) cells, but suppress NK cell mediated immunity.

    PubMed

    Schmidt, Stanislaw; Tramsen, Lars; Perkhofer, Susanne; Lass-Flörl, Cornelia; Hanisch, Mitra; Röger, Frauke; Klingebiel, Thomas; Koehl, Ulrike; Lehrnbecher, Thomas

    2013-07-01

    Mucormycosis has a high mortality and is increasingly diagnosed in hematopoietic stem cell transplant (HSCT) recipients. In this setting, there is a growing interest to restore host defense to combat infections by adoptively transferring donor-derived immunocompetent cells. Natural killer (NK) cells exhibit antitumor and antiinfective activity, but the interaction with Mucormycetes is unknown. Our data demonstrate that both unstimulated and IL-2 prestimulated human NK cells damage Rhizopus oryzae hyphae, but do not affect resting conidia. The damage of the fungus is mediated, at least in part, by perforin. R. oryzae hyphae decrease the secretion of immunoregulatory molecules by NK cells, such as IFN-γ and RANTES, indicating an immunosuppressive effect of the fungus. Our data indicate that NK cells exhibit activity against Mucormycetes and future research should evaluate NK cells as a potential tool for adoptive immunotherapy in HSCT. Copyright © 2012 Elsevier GmbH. All rights reserved.

  12. NK cells modulate the lung dendritic cell-mediated Th1/Th17 immunity during intracellular bacterial infection.

    PubMed

    Shekhar, Sudhanshu; Peng, Ying; Gao, Xiaoling; Joyee, Antony G; Wang, Shuhe; Bai, Hong; Zhao, Lei; Yang, Jie; Yang, Xi

    2015-10-01

    The impact of the interaction between NK cells and lung dendritic cells (LDCs) on the outcome of respiratory infections is poorly understood. In this study, we investigated the effect and mechanism of NK cells on the function of LDCs during intracellular bacterial lung infection of Chlamydia muridarum in mice. We found that the naive mice receiving LDCs from C. muridarum-infected NK-cell-depleted mice (NK-LDCs) showed more serious body weight loss, bacterial burden, and pathology upon chlamydial challenge when compared with the recipients of LDCs from infected sham-treated mice (NK+LDCs). Cytokine analysis of the local tissues of the former compared with the latter exhibited lower levels of Th1 (IFN-γ) and Th17 (IL-17), but higher levels of Th2 (IL-4), cytokines. Consistently, NK-LDCs were less efficient in directing C. muridarum-specific Th1 and Th17 responses than NK+LDCs when cocultured with CD4(+) T cells. In NK cell/LDC coculture experiments, the blockade of NKG2D receptor reduced the production of IL-12p70, IL-6, and IL-23 by LDCs. The neutralization of IFN-γ in the culture decreased the production of IL-12p70 by LDCs, whereas the blockade of TNF-α resulted in diminished IL-6 production. Our findings demonstrate that NK cells modulate LDC function to elicit Th1/Th17 immunity during intracellular bacterial infection.

  13. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    PubMed Central

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  14. STATs in NK-Cells: The Good, the Bad, and the Ugly

    PubMed Central

    Gotthardt, Dagmar; Sexl, Veronika

    2017-01-01

    Natural killer (NK)-cells are major players in the fight against viral infections and transformed cells, but there is increasing evidence attributing a disease-promoting role to NK-cells. Cytokines present in the tumor microenvironment shape NK-cell maturation, function, and effector responses. Many cytokines signal via the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway that is also frequently altered and constitutively active in a broad range of tumor cells. As a consequence, there are currently major efforts to develop therapeutic strategies to target this pathway. Therefore, it is of utmost importance to understand the role and contributions of JAK–STAT molecules in NK-cell biology—only this knowledge will allow us to predict effects of JAK–STAT inhibition for NK-cell functions and to successfully apply precision medicine. We will review the current knowledge on the role of JAK–STAT signaling for NK-cell functions and discuss conditions involved in the switch from NK-cell tumor surveillance to disease promotion. PMID:28149296

  15. Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction

    PubMed Central

    Yang, Yinli; Han, Qiuju; Hou, Zhaohua; Zhang, Cai; Tian, Zhigang; Zhang, Jian

    2017-01-01

    Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus (HBV) infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B (CHB) patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer (NK) cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt (DiD)-labeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon (IFN)-γ production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly (I:C) stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I (RIG-I), on NK cells, resulting in the dampening of the nuclear factor κB(NF-κB) and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection. PMID:27238466

  16. STATs in NK-Cells: The Good, the Bad, and the Ugly.

    PubMed

    Gotthardt, Dagmar; Sexl, Veronika

    2016-01-01

    Natural killer (NK)-cells are major players in the fight against viral infections and transformed cells, but there is increasing evidence attributing a disease-promoting role to NK-cells. Cytokines present in the tumor microenvironment shape NK-cell maturation, function, and effector responses. Many cytokines signal via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway that is also frequently altered and constitutively active in a broad range of tumor cells. As a consequence, there are currently major efforts to develop therapeutic strategies to target this pathway. Therefore, it is of utmost importance to understand the role and contributions of JAK-STAT molecules in NK-cell biology-only this knowledge will allow us to predict effects of JAK-STAT inhibition for NK-cell functions and to successfully apply precision medicine. We will review the current knowledge on the role of JAK-STAT signaling for NK-cell functions and discuss conditions involved in the switch from NK-cell tumor surveillance to disease promotion.

  17. Gliadin regulates the NK-dendritic cell cross-talk by HLA-E surface stabilization.

    PubMed

    Terrazzano, Giuseppe; Sica, Michela; Gianfrani, Carmen; Mazzarella, Giuseppe; Maurano, Francesco; De Giulio, Beatrice; de Saint-Mezard, Sophie; Zanzi, Delia; Maiuri, Luigi; Londei, Marco; Jabri, Bana; Troncone, Riccardo; Auricchio, Salvatore; Zappacosta, Serafino; Carbone, Ennio

    2007-07-01

    We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-gamma production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The alpha and omega fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin alpha increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.

  18. Phenotypically distinct helper NK cells are required for gp96-mediated anti-tumor immunity

    PubMed Central

    Sedlacek, Abigail L.; Kinner-Bibeau, Lauren B.; Binder, Robert J.

    2016-01-01

    A number of Heat Shock Proteins (HSPs), in the extracellular environment, are immunogenic. Following cross-presentation of HSP-chaperoned peptides by CD91+ antigen presenting cells (APCs), T cells are primed with specificity for the derivative antigen-bearing cell. Accordingly, tumor-derived HSPs are in clinical trials for cancer immunotherapy. We investigate the role of NK cells in gp96-mediated anti-tumor immune responses given their propensity to lyse tumor cells. We show that gp96-mediated rejection of tumors requires a unique and necessary helper role in NK cells. This helper role occurs during the effector phase of the anti-tumor immune response and is required for T cell and APC function. Gp96 activates NK cells indirectly via APCs to a phenotype distinct from NK cells activated by other mechanisms such as IL-2. While NK cells have both lytic and cytokine producing properties, we show that gp96 selectively activates cytokine production in NK cells, which is important in the HSP anti-tumor immune response, and leaves their cytotoxic capacity unchanged. PMID:27431727

  19. NK Cell Subtypes as Regulators of Autoimmune Liver Disease

    PubMed Central

    2016-01-01

    As major components of innate immunity, NK cells not only exert cell-mediated cytotoxicity to destroy tumors or infected cells, but also act to regulate the functions of other cells in the immune system by secreting cytokines and chemokines. Thus, NK cells provide surveillance in the early defense against viruses, intracellular bacteria, and cancer cells. However, the effecter function of NK cells must be exquisitely controlled to prevent inadvertent attack against normal “self” cells. In an organ such as the liver, where the distinction between immunotolerance and immune defense against routinely processed pathogens is critical, the plethora of NK cells has a unique role in the maintenance of homeostasis. Once self-tolerance is broken, autoimmune liver disease resulted. NK cells act as a “two-edged weapon” and even play opposite roles with both regulatory and inducer activities in the hepatic environment. That is, NK cells act not only to produce inflammatory cytokines and chemokines, but also to alter the proliferation and activation of associated lymphocytes. However, the precise regulatory mechanisms at work in autoimmune liver diseases remain to be identified. In this review, we focus on recent research with NK cells and their potential role in the development of autoimmune liver disease. PMID:27462349

  20. HCV Sensing by Human Trophoblasts Induces Innate Immune Responses and Recruitment of Maternal NK Cells: Potential Implications for Limiting Vertical Transmission

    PubMed Central

    Giugliano, Silvia; Petroff, Margaret G; Warren, Bryce D; Jasti, Susmita; Linscheid, Caitlin; Ward, Ashley; Kramer, Anita; Dobrinskikh, Evgenia; Sheiko, Melissa A; Gale, Michael; Golden-Mason, Lucy; Winn, Virginia D; Rosen, Hugo R

    2015-01-01

    Hepatitis C virus (HCV) is the world’s most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3–6% with odds 90% higher in the presence of HIV co-infection. Prevention of vertical transmission is not possible due to lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for pre-term delivery, perinatal mortality and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. Here we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV-uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust up-regulation of Type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a pro-apoptotic response within HTR8 that could affect the morphology of the placenta. For the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI, as well as novel insights into mechanisms that limit vertical transmission, but may paradoxically lead to virus-related pregnancy complications. PMID:26342030

  1. HIV-1 adaptation to NK cell mediated immune pressure

    PubMed Central

    Alter, Galit; Heckerman, David; Schneidewind, Arne; Fadda, Lena; Kadie, Carl M.; Carlson, Jonathan M.; Oniangue-Ndza, Cesar; Martin, Maureen; Li, Bin; Khakoo, Salim I.; Carrington, Mary; Allen, Todd M.; Altfeld, Marcus

    2011-01-01

    Natural Killer (NK) cells play an important role in the control of viral infections, recognizing virally infected cells through a variety of activating and inhibitory receptors1–3. Epidemiological and functional studies have recently suggested that NK cells can also contribute to the control of HIV-1 infection through recognition of virally infected cells by both activating and inhibitory Killer Immunoglobulin-like receptors (KIRs)4–7. However, it remains unknown whether NK cells can directly mediate antiviral immune pressure in vivo in humans. Here we describe KIR-associated amino acid polymorphisms in the HIV-1 sequence of chronically infected individuals on a population level. We show that these KIR-associated HIV-1 sequence polymorphisms can enhance the binding of inhibitory KIRs to HIV-1-infected CD4+ T cells, leading to reduced antiviral activity of KIR+ NK cells. These data demonstrate that KIR+ NK cells can place immunological pressure on HIV-1, and that the virus can evade such NK cell mediated immune pressure by selecting for sequence polymorphisms, as previously described for virus-specific T cells and neutralizing antibodies8. NK cells might therefore play a previously underappreciated role in contributing to viral evolution. PMID:21814282

  2. NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

    PubMed

    Diab, Mohammad; Glasner, Ariella; Isaacson, Batya; Bar-On, Yotam; Drori, Yaron; Yamin, Rachel; Duev-Cohen, Alexandra; Danziger, Oded; Zamostiano, Rachel; Mandelboim, Michal; Jonjic, Stipan; Bacharach, Eran; Mandelboim, Ofer

    2017-02-13

    Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

  3. Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients.

    PubMed

    Meazza, Raffaella; Falco, Michela; Marcenaro, Stefania; Loiacono, Fabrizio; Canevali, Paolo; Bellora, Francesca; Tuberosa, Claudia; Locatelli, Franco; Micalizzi, Concetta; Moretta, Alessandro; Mingari, Maria C; Moretta, Lorenzo; Aricò, Maurizio; Bottino, Cristina; Pende, Daniela

    2017-06-01

    X-linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)-associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV-infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig-like receptor (KIR)/KIR-L genotype and phenotypic repertoire of self-HLA class I specific inhibitory NK receptors (self-iNKRs). We also analyzed NK-cell cytotoxicity against CD48(+) or CD48(-) KIR-ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self-iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48(+) EBV-transformed B cells and M1 macrophages. Importantly, self-iNKR defective NK cells kill CD48(-) targets, such as mature DCs. Self-iNKR(-) NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK-cell maturation. Killing of autologous mature DC by self-iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients' immune defect. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer

    PubMed Central

    Veluchamy, John P.; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A. M.; Verheul, Henk M. W.; Di Santo, James P.; de Gruijl, Tanja D.; van der Vliet, Hans J.

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RASwt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR− RASwt, EGFR+ RASmut, and EGFR+ BRAFmut cells, A-PBNK were able to initiate lysis of EGFR+ colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR+ colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR−RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR+ RASmut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered. PMID:28220124

  5. Cryptococcus neoformans Directly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity▿

    PubMed Central

    Marr, Kaleb J.; Jones, Gareth J.; Zheng, Chunfu; Huston, Shaunna M.; Timm-McCann, Martina; Islam, Anowara; Berenger, Byron M.; Ma, Ling Ling; Wiseman, Jeremy C. D.; Mody, Christopher H.

    2009-01-01

    NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans. PMID:19307209

  6. Highly effective NK cells are associated with good prognosis in patients with metastatic prostate cancer

    PubMed Central

    Pasero, Christine; Gravis, Gwenaëlle; Granjeaud, Samuel; Guerin, Mathilde; Thomassin-Piana, Jeanne; Rocchi, Palma; Salem, Naji; Walz, Jochen; Moretta, Alessandro; Olive, Daniel

    2015-01-01

    Clinical outcome of patients with metastatic prostate cancer (mPC) at diagnosis is heterogeneous and unpredictable; thus alternative treatments such as immunotherapy are investigated. We retrospectively analyzed natural killer (NK) cells by flow cytometry in peripheral blood from 39 mPC patients, with 5 year-follow-up, and their correlation with time to castration resistance (TCR) and overall survival (OS). In parallel, NK functionality was carried out against prostate tumor cell lines, analyzed for the expression of NK cell ligands, to identify the receptors involved in PC recognition. NK cells from patients with longer TCR and OS displayed high expression of activating receptors and high cytotoxicity. The activating receptors NKp30 and NKp46 were the most obvious predictive markers of OS and TCR in a larger cohort of mPC patients (OS: p= 0.0018 and 0.0009; TCR: p= 0.007 and < 0.0001 respectively, log-rank test). Importantly, blocking experiments revealed that NKp46, along with NKG2D and DNAM-1 and, to a lesser extent NKp30, were involved in prostate tumor recognition by NK cells. These results identify NK cells as potential predictive biomarkers to stratify patients who are likely to have longer castration response, and pave the way to explore therapies aimed at enhancing NK cells in mPC patients. PMID:25961317

  7. NK cell-mediated immunopathology during an acute viral infection of the CNS.

    PubMed

    Alsharifi, Mohammed; Lobigs, Mario; Simon, Markus M; Kersten, Astrid; Müller, Klaus; Koskinen, Aulikki; Lee, Eva; Müllbacher, Arno

    2006-04-01

    Natural killer (NK) and cytotoxic T (Tc) cells are prime effector populations in the antiviral response of the host. Tc cells are essential for recovery from many viral diseases but may also be responsible for immunopathology. The role of NK cells in recovery from viral infections is less well established. We have studied acute virulent Semliki Forest virus (vSFV) infection of the central nervous system in C57BL/6J mice, which was mainly controlled by NK cells without marked Tc cell involvement. We show that mice with defects in the Fas and/or granule exocytosis pathways of cytotoxicity are more resistant to lethal vSFV infection than wild-type mice. On the other hand, mice defective in the IFN-gamma response are more sensitive than wild-type mice, whereas mice lacking the Tc cell compartment (beta-2 microglobulin-deficient mice) exhibit susceptibility similar to wild-type mice. The additional finding that depletion of NK cells significantly delayed the mean time to death but did not prevent mortality in SFV-infected B6 mice suggests that cytolytic activity of NK cells is detrimental, while IFN-gamma production is beneficial for recovery from SFV infection. This is the first study illustrating an NK cell-mediated immunopathological outcome to an acute viral infection.

  8. Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function.

    PubMed

    Goodier, Martin R; Lusa, Chiara; Sherratt, Sam; Rodriguez-Galan, Ana; Behrens, Ron; Riley, Eleanor M

    2016-01-01

    Cross-linking of FcγRIII (CD16) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, downregulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal) influenza vaccination. CD16 downregulation persisted for at least 12 weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal) vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long-term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16(high) CD57(+) NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression). CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25 (IL-2Rα) expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on trivalent influenza vaccine-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 downregulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways.

  9. Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function

    PubMed Central

    Goodier, Martin R.; Lusa, Chiara; Sherratt, Sam; Rodriguez-Galan, Ana; Behrens, Ron; Riley, Eleanor M.

    2016-01-01

    Cross-linking of FcγRIII (CD16) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, downregulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal) influenza vaccination. CD16 downregulation persisted for at least 12 weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal) vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long-term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16high CD57+ NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression). CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25 (IL-2Rα) expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on trivalent influenza vaccine-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 downregulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways. PMID:27725819

  10. Cord Blood Mononuclear Cells Have a Potential to Produce NK Cells Using IL2Rg Cytokines

    PubMed Central

    Khaziri, Nahid; Mohammadi, Momeneh; Aliyari, Zeinab; Soleimani Rad, Jafar; Tayefi Nasrabadi, Hamid; Nozad Charoudeh, Hojjatollah

    2016-01-01

    Purpose: Although bone marrow represents the main site for NK cell development and also distinct thymic-dependentNK cell pathway was identified, the cytokines effect on the NK cell generation from cord blood is unclear. Studies were identified the role of cytokines in the regulation of bone marrow and thymic NK cells. Previous studies reported that IL15 are critical for bone marrow dependent and IL7 is important for thymic NK cells. It is remain unclear the cytokines influence on the expantion of NK cells in cord blood mononuclear cells. Methods: We evaluated cultured cord blood mononuclear cells suplememnted with combinations of cytokines using FACS in distinct time points. In this study, we presented the role of IL2, IL7 and IL15 as members of the common gamma receptor -chain (Il2rg) on the expansion NK cells from cord blood cells. Results: By investigating cord blood mononuclear cells in vitro , we demonstrated that IL2 and IL15 are important for expansion of NK cells. IL2 in comparision with IL15 has more influences in NK cell expansion. In contrast IL-7 is dispensable for NK cell generation in cord blood. Conclusion: Thus,IL-2Rg cytokines play complementary roles and are indispensable for homeostasis of NK cell development in cord blood. Probably these cytokines could help to use NK beneficials in engrafment of transplanted cells and Anti tumor activity of NK cells. PMID:27123412

  11. Features of Memory-Like and PD-1(+) Human NK Cell Subsets.

    PubMed

    Della Chiesa, Mariella; Pesce, Silvia; Muccio, Letizia; Carlomagno, Simona; Sivori, Simona; Moretta, Alessandro; Marcenaro, Emanuela

    2016-01-01

    Human NK cells are distinguished into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56(bright) NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56(dim) NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56(dim) cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A(-)KIR(+)LIR-1(+)CD57(+)CD56(dim) NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain "memory-like" NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The "memory-like" NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.

  12. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection.

  13. Effect of boar seminal plasma immunosuppressive factor on NK cell activity and skin graft survival.

    PubMed

    Veselsky, L; Holan, V; Soucek, J; Stanek, R; Hoskova, M

    1992-01-01

    The B 10 strain of mice was used to test the effect of the boar seminal vesicle immunosuppressive factor on the female mouse response to the male-specific transplantation antigen. Influence of this factor on human natural killer (NK) cell activity was also studied. No inhibitory effect of the immunosuppressive factor on graft survival was apparent during a time of more than 200 days, nor did the factor suppress NK cell activity.

  14. The role of NK cells in HIV-1 protection: autologous, allogeneic or both?

    PubMed

    Hens, Jef; Jennes, Wim; Kestens, Luc

    2016-01-01

    Natural killer (NK) cells specialize in killing virally infected- or tumor cells and are part of the innate immune system. The activational state of NK cells is determined by the balance of incoming activating and inhibitory signals mediated by receptor-ligand binding with the target cell. These receptor-ligand bonds mainly consist of the killer immunoglobulin-like receptors (KIR), which are expressed at the cell surface of NK cells, and their ligands: the highly variable human leukocyte antigen -class I molecules (HLA). Absence of an inhibitory receptor-ligand bond lowers the NK cell activation threshold, whereas an activating receptor-ligand bond stimulates the cell, potentially overcoming this threshold and triggering NK cell activation. NK cells influence the course of infection as well as the acquisition of HIV-1. Several lines of evidence relate the activating NK cell receptor KIR3DS1, in the presence or absence of its putative ligand HLA-Bw4, with slower disease progression as well as resistance to HIV-1 infection. Overall, resistance to HIV-1 infection predominantly correlates with activating KIR/HLA profiles, consisting of e.g. activating KIRs, group B haplotypes, or inhibitory KIRs in absence of their ligands. Such a conclusion is less evident for studies of HIV-1 disease progression, with studies reporting beneficial as well as detrimental effects of activating KIR/HLA genotypes. It is likely that KIR/HLA association studies are complicated by the complexity of the KIR and HLA loci and their mutual interactions, as well as by additional factors like route of HIV exposure, immune activation, presence of co-infections, and the effect of anti-HIV-1 antibodies. One newly discovered NK cell activation pathway associated with resistance to HIV-1 infection involves the presence of an iKIR/HLA mismatch between partners. The absence of such an iKIR/HLA bond renders donor-derived allogeneic HIV-1 infected cells vulnerable to NK cell responses during HIV-1

  15. Cytokines can counteract the inhibitory effect of MEK-i on NK-cell function.

    PubMed

    Manzini, Claudia; Venè, Roberta; Cossu, Irene; Gualco, Marina; Zupo, Simonetta; Dono, Mariella; Spagnolo, Francesco; Queirolo, Paola; Moretta, Lorenzo; Mingari, Maria Cristina; Pietra, Gabriella

    2016-09-20

    Oncogene-targeted therapies based on mutated BRAF- and/or MEK-specific inhibitors have been developed for melanoma treatment. Although these drugs induce tumor regression in a high percentage of patients, clinical responses are frequently limited in time and tumors often recur. Recent studies suggested that the combination of BRAF/MEK inhibition with immunotherapy could represent a promising strategy for the cure of melanoma. NK cells are suitable effectors for tumor immunotherapy. Here we show that PLX4032 (a mutant BRAFV600 inhibitor) had no effect on the functional properties of NK cells cultured in the presence of IL-2 or IL-15. In contrast, PD0325901 (a MEK inhibitor) induced the down-regulation of the main activating NK receptors and inhibited NK cell function. Importantly, PD0325901 did not affect the anti-tumor activity of NK cells that had been exposed to a combination of IL-15 and IL-18. In addition, both PLX4032 and PD0325901 did not exert any inhibitory effect on in vitro IL-2 or IL-15 pre-activated NK cells.Our data may provide a rationale for future clinical protocols that combine IL-15/IL-18 cytokine administration with MEK inhibitors. In addition, they suggest that oncogene-targeting drugs are compatible with NK-based adoptive therapy.

  16. Cytokines can counteract the inhibitory effect of MEK-i on NK-cell function

    PubMed Central

    Manzini, Claudia; Venè, Roberta; Cossu, Irene; Gualco, Marina; Zupo, Simonetta; Dono, Mariella; Spagnolo, Francesco; Queirolo, Paola; Moretta, Lorenzo; Mingari, Maria Cristina; Pietra, Gabriella

    2016-01-01

    Oncogene-targeted therapies based on mutated BRAF- and/or MEK-specific inhibitors have been developed for melanoma treatment. Although these drugs induce tumor regression in a high percentage of patients, clinical responses are frequently limited in time and tumors often recur. Recent studies suggested that the combination of BRAF/MEK inhibition with immunotherapy could represent a promising strategy for the cure of melanoma. NK cells are suitable effectors for tumor immunotherapy. Here we show that PLX4032 (a mutant BRAFV600 inhibitor) had no effect on the functional properties of NK cells cultured in the presence of IL-2 or IL-15. In contrast, PD0325901 (a MEK inhibitor) induced the down-regulation of the main activating NK receptors and inhibited NK cell function. Importantly, PD0325901 did not affect the anti-tumor activity of NK cells that had been exposed to a combination of IL-15 and IL-18. In addition, both PLX4032 and PD0325901 did not exert any inhibitory effect on in vitro IL-2 or IL-15 pre-activated NK cells. Our data may provide a rationale for future clinical protocols that combine IL-15/IL-18 cytokine administration with MEK inhibitors. In addition, they suggest that oncogene-targeting drugs are compatible with NK-based adoptive therapy. PMID:27563819

  17. Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy.

    PubMed

    Davis, Zachary B; Vallera, Daniel A; Miller, Jeffrey S; Felices, Martin

    2017-09-04

    Natural killer (NK) cells have long been known to mediate anti-tumor responses without prior sensitization or recognition of specific tumor antigens. However, the tumor microenvironment can suppress NK cell function resulting in tumor escape and disease progression. Despite recent advances in cytokine therapy and NK cell adoptive transfer, tumor expression of ligands to NK - expressed checkpoint receptors can still suppress NK mediated tumor lysis. This review will explore many of the checkpoint receptors tumors utilize to manipulate the NK cell response as well as some of the current and upcoming pharmacological solutions to limit tumor suppression of NK cell function. Furthermore, we will discuss the potential to use these drugs in combinational therapies with novel antibody reagents such as bi- and tri-specific killer engagers (BiKEs and TriKEs) against tumor-specific antigens to enhance NK cell-mediated tumor rejection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Microchip-Based Single-Cell Imaging Reveals That CD56dimCD57-KIR-NKG2A+ NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56dimCD57-KIR-NKG2A- NK Cells.

    PubMed

    Forslund, Elin; Sohlberg, Ebba; Enqvist, Monika; Olofsson, Per E; Malmberg, Karl-Johan; Önfelt, Björn

    2015-10-01

    NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(-) (lacking inhibitory receptors; IR(-)) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR(-) NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR(-) NK cells were generally more short-lived and IR(-) NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR(-) NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

  19. Metabolic Reprogramming Supports IFN-γ Production by CD56bright NK Cells.

    PubMed

    Keating, Sinéad E; Zaiatz-Bittencourt, Vanessa; Loftus, Roisín M; Keane, Ciara; Brennan, Kiva; Finlay, David K; Gardiner, Clair M

    2016-03-15

    Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56(dim) cells are generally considered more cytotoxic, whereas the CD56(bright) cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2-induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56(bright) cells were more metabolically active compared with CD56(dim) cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56(bright) cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56(dim) cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56(bright) subset of cells. Overall, we have defined CD56(bright) NK cells to be more metabolically active than CD56(dim) cells, which supports their production of large amounts of IFN-γ during an immune response. Copyright © 2016 by The American Association of Immunologists, Inc.

  20. Protective role of NK1.1+ cells in experimental Staphylococcus aureus arthritis

    PubMed Central

    NILSSON, N; BREMELL, T; TARKOWSKI, A; CARLSTEN, H

    1999-01-01

    In a model of Staphylococcus aureus-induced septic arthritis in C57Bl/6 mice we investigated the role of natural killer (NK) cells in the development of disease. Depletion of NK1.1+ cells was achieved by repeated injections of the PK136 antibody, whereas control mice received an irrelevant monoclonal antibody, O1C5.B2. Both groups of mice then received injections intravenously with 2 × 107 live S. aureus LS-1 secreting toxic shock syndrome toxin-1 (TSST-1). The mice were evaluated for 16 days with regard to weight, mortality and arthritis. Nine days after bacterial injection, 9/19 mice depleted of NK cells had developed arthritis compared with 1/17 in the control group (P = 0.01). The experiment was repeated twice with the same outcome. NK cell-depleted and control mice displayed the same degree of histopathological signs of arthritis at day 16. Depletion of NK cells did not affect uptake of bacteria by phagocytic cells in vitro, or bacterial clearance in vivo. In NK cell-depleted mice there was a tendency to increased levels of antibodies to TSST-1, whereas total immunoglobulin levels were similar to those in controls. NK cell depletion of non-infected mice did not affect the magnitude of inflammatory response during the T cell-dependent cutaneous DTH reaction to oxazolone, or during granulocyte-mediated inflammation. However, specific antibody responses to oxazolone were greatly increased in depleted animals. In conclusion, our study demonstrates that NK cells protect against arthritis during S. aureus infection. This outcome does not seem to be due to an influence on bacterial clearance, but could be due to an interaction with the host anti-inflammatory mechanisms. PMID:10403917

  1. Changes in Response Properties of RVM Neurons during Prolonged Inflammation: Modulation by NK-1 Receptors

    PubMed Central

    Khasabov, Sergey G.; Brink, Thaddeus; Schupp, Marianna; Noack, Joseph; Simone, Donald A.

    2012-01-01

    Activation of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) can facilitate pain transmission in conditions such as inflammation, and thereby contribute to hyperalgesia. Since blockade of NK-1 receptors in the RVM can attenuate hyperalgesia produced by prolonged inflammation, we examined the role of NK-1 receptors in changes of response properties of RVM neurons following 4 days of hind paw inflammation with complete Freund’s adjuvant (CFA). Recording were made from functionally identified ON, OFF and NEUTRAL cells in the RVM. Spontaneous activity and responses evoked by a series of mechanical (10, 15, 26, 60, 100, and 180 g) and heat (34–50°C) stimuli applied to the inflamed and non-inflamed hind paws were determined before and at 15 and 60 min after injection of the NK1-antagonist L-733,060 or vehicle into the RVM. Prolonged inflammation did not alter the proportions of functionally-identified ON, OFF and NEUTRAL cells. ON cells exhibited enhanced responses to mechanical (60–100 g) and heat (48°–50°C) stimuli applied to the inflamed paw, which were attenuated by L-733,060 but not by vehicle. Inhibitory responses of OFF cells evoked by mechanical stimuli applied to the inflamed paw were also inhibited by L-733,060, but responses evoked by stimulation of the contralateral paw were increased. Heat-evoked responses of OFF cells were not altered by L-733,060. Also, neither L-733,060 nor vehicle altered spontaneous ongoing discharge rate of RVM neurons. These data indicate that NK-1 receptors modulate excitability of ON cells which contribute to both mechanical and heat hyperalgesia, whereas NK-1 modulation of OFF cells contributes to mechanical hyperalgesia during prolonged inflammation. PMID:22917610

  2. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections.

    PubMed

    Meier, Ute-Christiane; Owen, Rachel E; Taylor, Elizabeth; Worth, Andrew; Naoumov, Nikolai; Willberg, Christian; Tang, Kwok; Newton, Phillipa; Pellegrino, Pierre; Williams, Ian; Klenerman, Paul; Borrow, Persephone

    2005-10-01

    Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.

  3. Shared Alterations in NK Cell Frequency, Phenotype, and Function in Chronic Human Immunodeficiency Virus and Hepatitis C Virus Infections

    PubMed Central

    Meier, Ute-Christiane; Owen, Rachel E.; Taylor, Elizabeth; Worth, Andrew; Naoumov, Nikolai; Willberg, Christian; Tang, Kwok; Newton, Phillipa; Pellegrino, Pierre; Williams, Ian; Klenerman, Paul; Borrow, Persephone

    2005-01-01

    Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3− CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56dim cell fraction compared to CD56bright cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56dim NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections. PMID:16160163

  4. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  5. Acute Virus Control Mediated by Licensed NK Cells Sets Primary CD8+ T Cell Dependence on CD27 Costimulation.

    PubMed

    Teoh, Jeffrey J; Gamache, Awndre E; Gillespie, Alyssa L; Stadnisky, Michael D; Yagita, Hideo; Bullock, Timothy N J; Brown, Michael G

    2016-12-01

    NK cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC class I D(k) (D(k)), a major murine CMV (MCMV) resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2(+) NK cells provide essential host resistance against MCMV infection. Additionally G2(+) NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8(+) T cell effectors in response to MCMV. However, relatively little is known about the NK cell effect on costimulatory ligand patterns displayed by DCs or on ensuing effector and memory T cell responses. In this study, we found that CD27-dependent CD8(+) T cell priming and differentiation are shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in D(k)-disparate mice failed to distinguish early DC costimulatory patterns. Nonetheless, although CD27 deficiency did not impede licensed NK-mediated resistance, CD70 and CD27 were required to efficiently prime and regulate effector CD8(+) T cell differentiation in response to MCMV, which eventually resulted in biased memory T cell precursor formation in D(k) mice. In contrast, CD8(+) T cells accrued more slowly in non-D(k) mice and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC costimulatory signals needed to prime CD8(+) T cells and eventual T cell fate decisions.

  6. IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C

    PubMed Central

    Finnemann, Claudia; Sastre, Beatriz; Lutz, Philipp; Glässner, Andreas; Wolter, Franziska; Goeser, Felix; Kokordelis, Pavlos; Kaczmarek, Dominik; Nischalke, Hans-Dieter; Strassburg, Christian P.; Spengler, Ulrich; Nattermann, Jacob

    2016-01-01

    Background Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity. Methods A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used. Results Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals. Conclusions Our data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions. PMID:27583440

  7. Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.

    PubMed

    Oelsner, Sarah; Friede, Miriam E; Zhang, Congcong; Wagner, Juliane; Badura, Susanne; Bader, Peter; Ullrich, Evelyn; Ottmann, Oliver G; Klingemann, Hans; Tonn, Torsten; Wels, Winfried S

    2017-02-01

    Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ(null) mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment

    PubMed Central

    Almeida, Catarina R.; Caires, Hugo R.; Vasconcelos, Daniela P.; Barbosa, Mário A.

    2016-01-01

    Summary Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. PMID:27052313

  9. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment.

    PubMed

    Almeida, Catarina R; Caires, Hugo R; Vasconcelos, Daniela P; Barbosa, Mário A

    2016-04-12

    Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

  10. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Dixon, Amera; Orange, Jordan S.

    2016-01-01

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation. PMID:27435370

  11. Evaluation of the Functional Capacity of NK Cells of Melanoma Patients in an In Vitro Model of NK Cell Contact with K562 and FemX Tumor Cell Lines.

    PubMed

    Konjevic, Gordana; Vuletic, Ana; Mirjacic Martinovic, Katarina; Krivokuca, Ana; Jankovic, Radmila; Babovic, Nada

    2017-09-08

    NK cells of metastatic melanoma (MM) patients display impaired function, making them incapable to mount an effective antitumor response. In this study, we evaluated immunophenotypic characteristics and functional capacity of CD3(-)CD16(+) NK cells of MM patients in an in vitro model based on NK cell contact with an NK sensitive, K562, and a tumor-specific, melanoma FemX tumor cell line. Although our results indicate similar NK cell antitumor cytotoxic potential of MM patients in contact with both cell lines based on the expression of CD107a degranulation marker, there is a discrepancy in NK cell IFNγ production, as it is not significantly induced by FemX tumor cells, found to be, contrary to K562, HLA class I positive. Furthermore, we show NKG2D receptor downregulation by K562 tumor cell line, only. This may result from the obtained higher gene expression of TGFβ and VEGFA growth factors in K562 tumor cells that can negatively regulate NKG2D expression. Additionally, aside from postcontact downmodulation of activating CD16 receptor, there are no significant changes in the expression of CD161, CD158a, and CD158b NK cell receptors. Therefore, the applied in vitro model shows that, compared to the full NK cell functional capacity of MM patients displayed in a tumor-sensitive setting represented by contact with K562 cells, tumor-specific melanoma setting provided by FemX tumor cells leads to reduced NK functional potential. The obtained insight into NK cell capacity may be of use for evaluation of the state of disease and can help in selecting effective immunotherapeutic agents for MM patients.

  12. Bone marrow produces sufficient alloreactive natural killer (NK) cells in vivo to cure mice from subcutaneously and intravascularly injected 4T1 breast cancer.

    PubMed

    van Gelder, Michel; Vanclée, Ariane; van Elssen, Catharina H M J; Hupperets, Pierre; Wieten, Lotte; Bos, Gerard M

    2017-02-01

    Administration of 5 million alloreactive natural killer (NK) cells after low-dose chemo-irradiation cured mice of 4T1 breast cancer, supposedly dose dependent. We now explored the efficacy of bone marrow as alternative in vivo source of NK cells for anti-breast cancer treatment, as methods for in vitro clinical scale NK cell expansion are still in developmental phases. Progression-free survival (PFS) after treatment with different doses of spleen-derived alloreactive NK cells to 4T1-bearing Balb/c mice was measured to determine a dose-response relation. The potential of bone marrow as source of alloreactive NK cells was explored using MHC-mismatched mice as recipients of 4T1. Chemo-irradiation consisted of 2× 2 Gy total body irradiation and 200 mg/kg cyclophosphamide. Antibody-mediated in vivo NK cell depletion was applied to demonstrate the NK cell's role. Administration of 2.5 instead of 5 million alloreactive NK cells significantly reduced PFS, evidencing dose responsiveness. Compared to MHC-matched receivers of subcutaneous 4T1, fewer MHC-mismatched mice developed tumors, which was due to NK cell alloreactivity because in vivo NK cell depletion facilitated tumor growth. Application of low-dose chemo-irradiation increased plasma levels of NK cell-activating cytokines, NK cell activity and enhanced NK cell-dependent elimination of subcutaneous tumors. Intravenously injected 4T1 was eliminated by alloreactive NK cells in MHC-mismatched recipients without the need for chemo-irradiation. Bone marrow is a suitable source of sufficient alloreactive NK cells for the cure of 4T1 breast cancer. These results prompt clinical exploration of bone marrow transplantation from NK-alloreactive MHC-mismatched donors in patients with metastasized breast cancer.

  13. Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases

    PubMed Central

    Quintieri, L; Rosato, A; Amboldi, N; Vizler, C; Ballinari, D; Zanovello, P; Collavo, D

    1999-01-01

    The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 μg kg−1 MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 μg kg−1 free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection. © 1999 Cancer Research Campaign PMID:10098738

  14. Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection

    PubMed Central

    Guthrie, Brandon S.; Schmidt, Rebecca L.; Jamieson, Amanda; Merkel, Patricia; Knight, Vijaya; Cole, Caroline M.; Raulet, David H.; Lenz, Laurel L.

    2016-01-01

    Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility. PMID:27295349

  15. NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

    PubMed Central

    Zhang, Xiaobo; Volpe, Eugene A.; Gandhi, Niral B.; Schaumburg, Chris S.; Siemasko, Karyn F.; Pangelinan, Solherny B.; Kelly, Scott D.; Hayday, Adrian C.; Li, De-Quan; Stern, Michael E.; Niederkorn, Jerry Y.; Pflugfelder, Stephen C.; De Paiva, Cintia S.

    2012-01-01

    Background The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL). Methodology/Principal Findings Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3+CD103+ (∼22%), CD3+γδ+ (∼9.6%), CD3+NK+ (2%), CD3−NK+ (∼4.4%), CD3+CD8α (∼0.9%), and CD4 (∼0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17+ cells in the cervical lymph nodes and generation of less pathogenic CD4+T cells. B6.nude recipient mice of adoptively transferred CD4+T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4+T cells. Conclusions/Significance Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response. PMID:22590618

  16. Bcl10 plays a divergent role in NK cell-mediated cytotoxicity and cytokine generation.

    PubMed

    Malarkannan, Subramaniam; Regunathan, Jeyarani; Chu, Haiyan; Kutlesa, Snjezana; Chen, Yuhong; Zeng, Hu; Wen, Renren; Wang, Demin

    2007-09-15

    Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-gamma (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.

  17. Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

    PubMed

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

    2016-04-04

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice

  18. An autosomal dominant locus, Nka, mapping to the Ly-49 region of a rat natural killer (NK) gene complex, controls NK cell lysis of allogeneic lymphocytes

    PubMed Central

    1996-01-01

    Natural Killer (NK) cells can recognize and kill MHC-incompatible normal bone marrow-derived cells. Presently characterized MHC-binding receptors on NK cells, including the Ly-49 family in the mouse, transmit inhibitory signals upon binding to cognate class I MHC ligands. Here we study in vivo NK-mediated lysis of normal allogeneic lymphocytes in crosses between alloreactivity-competent PVG rats and alloreactivity-deficient DA rats. NK cells from both strains are able to lyse standard tumor targets. We identify an autosomal dominant locus, Nka, that controls NK-mediated alloreactivity. Individuals carrying the dominant PVG allele in single dose were fully competent in eliminating allogeneic target cells, suggesting that Nka encodes or regulates a gene product inducing or activating alloreactivity. By linkage analysis and pulsed field gel electrophoresis, a natural killer gene complex (NKC) on rat chromosome 4 is described that contains the rat NKR-P1 and Ly-49 multigene families plus a rat NKG2D homologue. Nka maps within the NKC, together with the most telomeric Ly-49 family members, but separate from NKG2D and the NKR-P1 family. The Nka-encoded response, moreover, correlates with the expression of transcripts for Ly-49 receptors in NK cell populations, as Northern blot analysis demonstrated low expression of Ly-49 genes in DA NK cells, in contrast to high expression in alloreactivity-competent PVG, (DA X PVG)F1, and PVG.1AVI NK cells. The low Ly-49 expression in DA is not induced by MHC haplotype, as demonstrated by high expression of Ly-49 in the DA MHC- congenic PVG.1AVI strain. Finally, we have cloned and characterized the first four members of the rat Ly-49 gene family. Their cytoplasmic domains demonstrate substantial heterogeneity, consistent with the hypothesis that different Ly-49 family members may subserve different signaling functions. PMID:8642329

  19. A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature

    PubMed Central

    Cooley, Sarah; Xiao, Feng; Pitt, Michelle; Gleason, Michelle; McCullar, Valarie; Bergemann, Tracy L.; McQueen, Karina L.; Guethlein, Lisbeth A.; Parham, Peter

    2007-01-01

    How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56bright cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56dim cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% ± 2.8% of the CD56dim NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56dim NKG2A− KIR− NK cells lack “at least one” inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-γ production. Upon culture with IL-15 and a stromal cell line, CD56dim and CD56bright KIR− NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56bright cells precede CD56dim cells. PMID:17392508

  20. α1-Antitrypsin modifies general NK cell interactions with dendritic cells and specific interactions with islet β-cells in favor of protection from autoimmune diabetes.

    PubMed

    Guttman, Ofer; Yossef, Rami; Freixo-Lima, Gabriella; Rider, Peleg; Porgador, Angel; Lewis, Eli C

    2014-10-13

    The autoimmune destruction of pancreatic β-cells is the hallmark of type 1 diabetes (T1D). Failure of anti-CD3 antibodies to provide long-lasting reversal of T1D and the expression of an NK cell ligand on β-cells suggest that NK cells play a role in disease pathogenesis. Indeed, killing of β-cells by NK cells has been shown to occur, mediated by activation of the NK cell activating receptor, NKp46. α1-antitrypsin (AAT), an anti-inflammatory and immunomodulatory glycoprotein, protects β-cells from injurious immune responses and is currently evaluated as a therapeutic for recent onset T1D. While isolated T lymphocytes are not inhibited by AAT, dendritic cells (DCs) become tolerogenic in its presence and other innate immune cells become less inflammatory. Yet a comprehensive profile of NK cell responses in the presence of AAT has yet to be described. In the present study, we demonstrate that AAT significantly reduces NK cell degranulation against β-cells, albeit in the whole animal and not in isolated NK cell cultures. AAT-treated mice, and not isolated cultured β-cells, exhibited a marked reduction in NKp46 ligand levels on β-cells. In related experiments, AAT-treated DCs exhibited reduced inducible DC-expressed IL-15 levels and evoked a weaker NK cell response. NK cell depletion in a T1D mouse model resulted in improved β-cell function and survival, similar to the effects observed by AAT treatment alone; nonetheless, the two approaches were non-synergistic. Our data suggest that AAT is a selective immunomodulator that retains pivotal NK cell responses, while diverting their activities away from islet β-cells. This article is protected by copyright. All rights reserved.

  1. Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

    PubMed Central

    Quoc Trung, Ly; Espinoza, J. Luis; Takami, Akiyoshi; Nakao, Shinji

    2013-01-01

    Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling. PMID:23372833

  2. Histaminergic regulation of interferon-gamma (IFN-γ) production by human natural killer (NK) cells

    PubMed Central

    ASEA, A; HANSSON, M; CZERKINSKY, C; HOUZE, T; HERMODSSON, S; STRANNEGÅRD, Ö; HELLSTRAND, K

    1996-01-01

    Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-γ by CD3−/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-γ production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal, whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-γ production was operating on a pre-translational level, as indicated by the inability of enriched NK cells to accumulate IFN-γ mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-γ production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-γ production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-γ in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-γ mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-γ production by preventing monocyte-induced oxidative damage to NK cells. PMID:8706348

  3. Selection and expansion of natural killer cells for NK cell-based immunotherapy.

    PubMed

    Becker, Petra S A; Suck, Garnet; Nowakowska, Paulina; Ullrich, Evelyn; Seifried, Erhard; Bader, Peter; Tonn, Torsten; Seidl, Christian

    2016-04-01

    Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

  4. PIBF positive uterine NK cells in the mouse decidua.

    PubMed

    Bogdan, Agnes; Berta, Gergely; Szekeres-Bartho, Julia

    2017-02-01

    Though uterine NK cells (u NK cells) contain cytotoxic granules, and selectively over- express the genes of perforin and granzymes, during normal pregnancy, they are not cytotoxic. Progesterone is indispensable for the establishment and maintenance of pregnancy both in humans and in mice. Mouse uterine NK cells do not express the classical progesterone receptor, yet progesterone affects the recruitment and function of uterine NK cells, the latter partly via the Progesterone-Induced Blocking Factor (PIBF). We demonstrated PIBF positive granulated cells in the mouse decidua. The aim of this study was to characterize these cells by lectin immunohistochemistry and anti-perforin reactivity. PIBF+ granulated cells were absent from the deciduae of alymphoid mice, but appeared in the decidua of those that had been reconstituted with bone marrow from male BALB/c mice. PIBF+ granulated cells bound the DBA lectin, suggesting their NK cell nature, and also contained perforin, which co-localized with PIBF in the cytoplasmic granules. In anti-progesterone treated mice all of the PIBF+ cells were perforin positive at g. d. 12.5, in contrast to the 54% perforin positivity of PIBF+ cells in untreated mice.

  5. RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

    PubMed

    Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

    2015-01-01

    Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

  6. Regulatory Dendritic Cells Restrain NK Cell IFN-γ Production through Mechanisms Involving NKp46, IL-10, and MHC Class I-Specific Inhibitory Receptors.

    PubMed

    Spallanzani, Raúl G; Torres, Nicolás I; Avila, Damián E; Ziblat, Andrea; Iraolagoitia, Ximena L Raffo; Rossi, Lucas E; Domaica, Carolina I; Fuertes, Mercedes B; Rabinovich, Gabriel A; Zwirner, Norberto W

    2015-09-01

    Cross-talk between mature dendritic cells (mDC) and NK cells through the cell surface receptors NKp30 and DNAM-1 leads to their reciprocal activation. However, the impact of regulatory dendritic cells (regDC) on NK cell function remains unknown. As regDC constrain the immune response in different physiological and pathological conditions, the aim of this work was to investigate the functional outcome of the interaction between regDC and NK cells and the associated underlying mechanisms. RegDC generated from monocyte-derived DC treated either with LPS and dexamethasone, vitamin D3, or vitamin D3 and dexamethasone instructed NK cells to secrete lower amounts of IFN-γ than NK cells exposed to mDC. Although regDC triggered upregulation of the activation markers CD69 and CD25 on NK cells, they did not induce upregulation of CD56 as mDC, and silenced IFN-γ secretion through mechanisms involving insufficient secretion of IL-18, but not IL-12 or IL-15 and/or induction of NK cell apoptosis. Blocking experiments demonstrated that regDC curb IFN-γ secretion by NK cells through a dominant suppressive mechanism involving IL-10, NK cell inhibitory receptors, and, unexpectedly, engagement of the activating receptor NKp46. Our findings unveil a previously unrecognized cross-talk through which regDC shape NK cell function toward an alternative activated phenotype unable to secrete IFN-γ, highlighting the plasticity of NK cells in response to tolerogenic stimuli. In addition, our findings contribute to identify a novel inhibitory role for NKp46 in the control of NK cell function, and have broad implications in the resolution of inflammatory responses and evasion of antitumor responses.

  7. NK cells promote neutrophil recruitment in the brain during sepsis-induced neuroinflammation

    PubMed Central

    He, Hao; Geng, Tingting; Chen, Piyun; Wang, Meixiang; Hu, Jingxia; Kang, Li; Song, Wengang; Tang, Hua

    2016-01-01

    Sepsis could affect the central nervous system and thus induces neuroinflammation, which subsequently leads to brain damage or dysfunction. However, the mechanisms of generation of neuroinflammation during sepsis remain poorly understood. By administration of lipopolysaccharides (LPS) in mice to mimic sepsis, we found that shortly after opening the blood–brain barrier, conventional CD11b+CD27+ NK subset migrated into the brain followed by subsequent neutrophil infiltration. Interestingly, depletion of NK cells prior to LPS treatment severely impaired neutrophil recruitment in the inflamed brain. By in vivo recruitment assay, we found that brain-infiltrated NK cells displayed chemotactic activity to neutrophils, which depended on the higher expression of chemokines such as CXCL2. Moreover, microglia were also responsible for neutrophil recruitment, and their chemotactic activity was significantly impaired by ablation of NK cells. Furthermore, depletion of NK cells could significantly ameliorate depression-like behavior in LPS-treated mice. These data indicated a NK cell-regulated neutrophil recruitment in the blamed brain, which also could be seen on another sepsis model, cecal ligation and puncture. So, our findings revealed an important scenario in the generation of sepsis-induced neuroinflammation. PMID:27270556

  8. Novel immune modulators used in hematology: impact on NK cells.

    PubMed

    Krieg, Stephanie; Ullrich, Evelyn

    2012-01-01

    There is a wide range of important pharmaceuticals used in treatment of cancer. Besides their known effects on tumor cells, there is growing evidence for modulation of the immune system. Immunomodulatory drugs (IMiDs(®)) play an important role in the treatment of patients with multiple myeloma or myelodysplastic syndrome and have already demonstrated antitumor, anti-angiogenic, and immunostimulating effects, in particular on natural killer (NK) cells. Tyrosine kinase inhibitors are directly targeting different kinases and are known to regulate effector NK cells and expression of NKG2D ligands (NKG2DLs) on tumor cells. Demethylating agents, histone deacetylases, and proteasome inhibitors interfere with the epigenetic regulation and protein degradation of malignant cells. There are first hints that these drugs also sensitize tumor cells to chemotherapy, radiation, and NK cell-mediated cytotoxicity by enhanced expression of TRAIL and NKG2DLs. However, these pharmaceuticals may also impair NK cell function in a dose- and time-dependent manner. In summary, this review provides an update on the effects of different novel molecules on the immune system focusing NK cells.

  9. TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.

    PubMed

    Viel, Sébastien; Marçais, Antoine; Guimaraes, Fernando Souza-Fonseca; Loftus, Roisin; Rabilloud, Jessica; Grau, Morgan; Degouve, Sophie; Djebali, Sophia; Sanlaville, Amélien; Charrier, Emily; Bienvenu, Jacques; Marie, Julien C; Caux, Christophe; Marvel, Jacqueline; Town, Liam; Huntington, Nicholas D; Bartholin, Laurent; Finlay, David; Smyth, Mark J; Walzer, Thierry

    2016-02-16

    Transforming growth factor-β (TGF-β) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-β signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-β in vitro blocked interleukin-15 (IL-15)-induced activation of mTOR. TGF-β and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-β signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-β receptor subunit TGF-βRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-β signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression.

  10. Polarization of NK cell cytoskeleton upon conjugation with sensitive target cells.

    PubMed

    Carpén, O; Virtanen, I; Lehto, V P; Saksela, E

    1983-12-01

    We studied the cytoskeletal changes in natural killer (NK) cells during conjugate formation, i.e., when NK cells make contact with sensitive vs resistant target cells. F-actin and vinculin were seen to polarize at the contact sites upon conjugation with sensitive K562 cells, whereas in conjugates with resistant Raji target cells such an orientation was an infrequent finding. Myosin and two other cytoskeletal proteins, spectrin and vimentin, on the other hand, showed a random distribution in conjugating NK cells regardless of the target cell type. Hence the cytoskeletal redistribution associated with conjugation seems to be different from the receptor capping phenomenon, which is accompanied by clustering of actin, myosin, vimentin, and spectrin. On the basis of these results it seems probable that the lytic conjugate formation in NK-mediated cytotoxicity is associated with the formation of a specific type of junction that involves actin and vinculin. This cytoskeletal reorganization precedes and could be a prerequisite for the polarization of the cellular secretory apparatus and may be functionally responsible for the required cytokinetic movements.

  11. Human mesenchymal stromal/stem cells acquire immunostimulatory capacity upon cross-talk with natural killer cells and might improve the NK cell function of immunocompromised patients.

    PubMed

    Cui, Rongtao; Rekasi, Heike; Hepner-Schefczyk, Monika; Fessmann, Kai; Petri, Robert M; Bruderek, Kirsten; Brandau, Sven; Jäger, Marcus; Flohé, Stefanie B

    2016-07-07

    The suppressive effect of mesenchymal stromal/stem cells (MSCs) on diverse immune cells is well known, but it is unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of cytokine secretion. Human MSCs were generated from bone marrow and nasal mucosa. NK cells were isolated from peripheral blood of healthy volunteers or of immunocompromised patients after severe injury. NK cells were cultured with MSCs or with MSC-derived conditioned media in the absence or presence of IL-12 and IL-18. C-C chemokine receptor (CCR) 2, C-C chemokine ligand (CCL) 2, and the interferon (IFN)-γ receptor was blocked by specific inhibitors or antibodies. The synthesis of IFN-γ and CCL2 was determined. In the absence of exogenous cytokines, trace amounts of NK cell-derived IFN-γ licensed MSCs for enhanced synthesis of CCL2. In turn, MSCs primed NK cells for increased release of IFN-γ in response to IL-12 and IL-18. Priming of NK cells by MSCs occurred in a cell-cell contact-independent manner and was impaired by inhibition of the CCR2, the receptor of CCL2, on NK cells. CD56(bright) NK cells expressed higher levels of CCR2 and were more sensitive to CCL2-mediated priming by MSCs and by recombinant CCR2 ligands than cytotoxic CD56(dim) NK cells. NK cells from severely injured patients were impaired in cytokine-induced IFN-γ synthesis. Co-culture with MSCs or with conditioned media from MSCs and MSC/NK cell co-cultures from healthy donors improved the IFN-γ production of the patients' NK cells in a CCR2-dependent manner. A positive feedback loop driven by NK cell-derived IFN-γ and MSC-derived CCL2 increases the inflammatory response of cytokine-stimulated NK cells not only from healthy donors but also from immunocompromised patients. Therapeutic application of MSCs or their soluble factors might thus improve the NK function after severe injury.

  12. Trogocytosis and killing of IL-4-polarized monocytes by autologous NK cells.

    PubMed

    Poupot, Mary; Fournié, Jean-Jacques; Poupot, Rémy

    2008-11-01

    Cross-regulations between innate immune cells have been given more and more emphasis. Here, we address the question of bidirectional interactions between activated monocytes and autologous NK cells. Classically activated monocytes (class-monocytes), obtained by priming with IFN-gamma, drive an inflammatory immune response. On the contrary, alternatively activated monocytes (alt-monocytes), obtained by stimulation with IL-4 or IL-13, engage an anti-inflammatory immune response. We show that alt-monocytes inhibit proliferation and production of IFN-gamma by autologous, IL-2-activated NK cells, whereas class-monocytes do not inhibit these NK cell functions. Reciprocally, IL-2-activated NK cells interact and undertake intensive synaptic transfer with alt-monocytes, whereas interactions with class-monocytes are weaker. This strong trogocytosis correlates with an efficient killing of alt-monocytes, mediated by natural cytotoxicity receptors and a lowered killing of class-monocytes. These results suggest that interactions between NK cells and autologous-activated monocytes modulate inflammatory responses. This might be extended further in the elimination of tumor-associated macrophages, which actively promote solid tumor progression and metastasis.

  13. Blastoid NK cell leukemia/lymphoma with cutaneous involvement.

    PubMed

    Ginarte, M; Abalde, M T; Peteiro, C; Fraga, M; Alonso, N; Toribio, J

    2000-01-01

    Malignant neoplasms from natural killer (NK) cells are characterized by their positivity for CD56 and absence of monoclonal TCR gene rearrangement. Recently, they have been classified into four main types (nasal and nasal-type NK cell lymphoma, aggressive NK cell leukemia/lymphoma, and blastoid NK cell leukemia/lymphoma), based on clinical features, racial predisposition, presence of azurophilic granules, immunophenotype and association with Epstein-Barr virus (EBV) infection. A 72-year-old Caucasian man presented with a malignant neoplasm comprised of blastoid cells without azurophilic granules in the Giemsa stain, with positivity for CD2, CD4, HLA-DR, CD45 and CD56, and negativity for CD3 (surface and cytoplasmic) and CD5. In situ hybridization for EBV and PCR analysis of rearrangement of the T cell receptor gene were negative. Based on these results, a diagnosis of blastoid NK cell lymphoma was made. In this case the first clinical manifestations were the cutaneous lesions, and, although the disease was already advanced at the diagnosis, the patient responded completely to the treatment and remains asymptomatic 14 months after diagnosis. Copyright 2000 S. Karger AG, Basel.

  14. Effects of X-ray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumor cells and lytic function of NK cells

    SciTech Connect

    Uchida, A.; Mizutani, Y.; Nagamuta, M.; Ikenaga, M. )

    1989-01-01

    The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.

  15. Uterine NK Cells Are Critical in Shaping DC Immunogenic Functions Compatible with Pregnancy Progression

    PubMed Central

    Freitag, Nancy; Otto, Teresa; Thijssen, Victor L. J. L.; Moschansky, Petra; von Kwiatkowski, Petra; Klapp, Burghard F.; Winterhager, Elke; Bauersachs, Stefan; Blois, Sandra M.

    2012-01-01

    Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression. PMID:23056436

  16. Tissue-resident Eomes(hi) T-bet(lo) CD56(bright) NK cells with reduced proinflammatory potential are enriched in the adult human liver.

    PubMed

    Harmon, Cathal; Robinson, Mark W; Fahey, Ronan; Whelan, Sarah; Houlihan, Diarmaid D; Geoghegan, Justin; O'Farrelly, Cliona

    2016-09-01

    The adult human liver is enriched with natural killer (NK) cells, accounting for 30-50% of hepatic lymphocytes, which include tissue-resident hepatic NK-cell subpopulations, distinct from peripheral blood NK cells. In murine liver, a subset of liver-resident hepatic NK cells have altered expression of the two highly related T-box transcription factors, T-bet and eomesodermin (Eomes). Here, we investigate the heterogeneity of T-bet and Eomes expression in NK cells from healthy adult human liver with a view to identifying human liver-resident populations. Hepatic NK cells were isolated from donor liver perfusates and biopsies obtained during orthotopic liver transplantation (N = 28). Hepatic CD56(bright) NK cells were Eomes(hi) T-bet(lo) , a phenotype virtually absent from peripheral blood. These NK cells express the chemokine receptor CXCR6 (chemokine (C-X-C motif) receptor 6), a marker of tissue residency, which is absent from hepatic CD56(dim) and blood NK cells. Compared to blood populations, these hepatic CD56(bright) NK cells have increased expression of activatory receptors (NKp44, NKp46, and NKG2D). They show reduced ability to produce IFN-γ but enhanced degranulation in response to challenge with target cells. This functionally distinct population of hepatic NK cells constitutes 20-30% of the total hepatic lymphocyte repertoire and represents a tissue-resident immune cell population adapted to the tolerogenic liver microenvironment.

  17. Functional CD32 molecules on human NK cells.

    PubMed

    Morel, P A; Ernst, L K; Metes, D

    1999-09-01

    Human NK cells are large granular lymphocytes that kill neoplastic or virally infected targets using perforin-dependent mechanisms. CD16 or FcgammaRIII is one of the cell surface molecules that can trigger the killing machinery following binding of the Fc portion of IgG to the receptor: a mechanism known as antibody dependent cell-mediated cytotoxicity (ADCC). We have recently shown that some individuals express an additional FcgammaR on their NK cells, CD32 or FcgammaRII. This receptor has now been characterized at the molecular, biochemical and functional level. The present review outlines our findings to date on the features of this novel receptor. These findings suggest that the presence of a functional FcgammaRII on the surface of NK cells could have important clinical consequences in both tumor immunotherapy and autoimmune disease.

  18. Targeting NK-cell checkpoints for cancer immunotherapy.

    PubMed

    Muntasell, Aura; Ochoa, Maria C; Cordeiro, Luna; Berraondo, Pedro; López-Díaz de Cerio, Ascension; Cabo, Mariona; López-Botet, Miguel; Melero, Ignacio

    2017-02-22

    Natural Killer (NK) cells are cytotoxic lymphocytes specialized in early defense against virus-infected and transformed cells. NK-cell function is regulated by activating and inhibitory surface receptors recognizing their ligands on transformed cells. Modulation of NK numbers and/or function by a variety of agents such as cytokines and monoclonal antibodies may result in enhanced anti-tumor activity. Recombinant cytokines (i.e., IL-15 and IL-2), antibodies blocking inhibitory receptors (i.e., KIR, NKG2A and TIGIT) and agonists delivering signals via CD137, NKG2D and CD16 stand out as the most suitable opportunities. These agents can be used to potentiate NKcell- mediated antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated tumor cells, offering potential for multiple combinatorial immunotherapy strategies against cancer.

  19. Natural killer (NK) cell deficit in coronary artery disease: no aberrations in phenotype but sustained reduction of NK cells is associated with low-grade inflammation.

    PubMed

    Backteman, K; Ernerudh, J; Jonasson, L

    2014-01-01

    Although reduced natural killer (NK) cell levels have been reported consistently in patients with coronary artery disease (CAD), the clinical significance and persistence of this immune perturbation is not clarified. In this study we characterized the NK cell deficit further by determining (i) differentiation surface markers and cytokine profile of NK cell subsets and (ii) ability to reconstitute NK cell levels over time. Flow cytometry was used to analyse NK cell subsets and the intracellular cytokine profile in 31 patients with non-ST elevation myocardial infarction (non-STEMI), 34 patients with stable angina (SA) and 37 healthy controls. In blood collected prior to coronary angiography, the proportions of NK cells were reduced significantly in non-STEMI and SA patients compared with controls, whereas NK cell subset analyses or cytokine profile measurements did not reveal any differences across groups. During a 12-month follow-up, the proportions of NK cells increased, although not in all patients. Failure to reconstitute NK cell levels was associated with several components of metabolic syndrome. Moreover, interleukin (IL)-6 levels remained high in patients with sustained NK cell deficit, whereas a decline in IL-6 (P < 0·001) was seen in patients with a pronounced increase in NK cells. In conclusion, we found no evidence that reduction of NK cells in CAD patients was associated with aberrations in NK cell phenotype at any clinical stage of the disease. Conversely, failure to reconstitute NK cell levels was associated with a persistent low-grade inflammation, suggesting a protective role of NK cells in CAD.

  20. Enteropathy-type T-cell lymphoma expressing NK-cell intraepithelial lymphocyte (NK-IEL) phenotype.

    PubMed

    Inagaki, Naoko; Asaoka, Daisuke; Mori, Kiyoshi L; Sohda, Naomi; Miura, Ichiro; Miwa, Hiroto; Sato, Nobuhiro; Oshimi, Kazuo

    2004-07-01

    Enteropathy-type T-cell lymphoma (ETL) is an intraepithelial T-lymphocyte (T-IEL) tumor. The tumor cells are usually CD3+, CD4-, CD8+, and contain cytotoxic granule associated proteins. We report on a CD3-negative CD56-positive enteropathy-associated lymphoma (ETL). This is the first case report of CD3-negative, CD56-positive, CD94-negative, and CD161-positive ETL. ETL cells originate from intraepithelial T-lymphocytes of the intestine. CD3-negative intraepithelial lymphocytes are known as natural killer (NK)-IELs. The phenotype of NK-IELs is also CD3-negative, CD56-positive, CD94-negative, and CD161-positive, while most normal NK cells express CD56 and CD94. CD3-negative lymphoma cells in this report also expressed CD56 and CD161, but not CD94. Because Southern blotting analysis showed a rearrangement of T-cell receptor (TCR) Cbeta in this case, the tumor is classified as an ETL. Based on the findings, NK-IELs may originate from T-cells, not NK-cells.

  1. KIR3DS1-Mediated Recognition of HLA-*B51: Modulation of KIR3DS1 Responsiveness by Self HLA-B Allotypes and Effect on NK Cell Licensing

    PubMed Central

    Carlomagno, Simona; Falco, Michela; Bono, Maria; Alicata, Claudia; Garbarino, Lucia; Mazzocco, Michela; Moretta, Lorenzo; Moretta, Alessandro; Sivori, Simona

    2017-01-01

    Several studies described an association between killer-cell immunoglobulin-like receptor (KIR)/HLA gene combinations and clinical outcomes in various diseases. In particular, an important combined role for KIR3DS1 and HLA-B Bw4-I80 in controlling viral infections and a higher protection against leukemic relapses in donor equipped with activating KIRs in haplo-HSCT has been described. Here, we show that KIR3DS1 mediates positive signals upon recognition of HLA-B*51 (Bw4-I80) surface molecules on target cells and that this activation occurs only in Bw4-I80neg individuals, including those carrying particular KIR/HLA combination settings. In addition, killing of HLA-B*51 transfected target cells mediated by KIR3DS1+/NKG2A+ natural killer (NK) cell clones from Bw4-I80neg donors could be partially inhibited by antibody-mediated masking of KIR3DS1. Interestingly, KIR3DS1-mediated recognition of HLA-B*51 could be better appreciated under experimental conditions in which the function of NKG2D was reduced by mAb-mediated blocking. This experimental approach may mimic the compromised function of NKG2D occurring in certain viral infections. We also show that, in KIR3DS1+/NKG2A+ NK cell clones derived from an HLA-B Bw4-T80 donor carrying 2 KIR3DS1 gene copy numbers, the positive signal generated by the engagement of KIR3DS1 by HLA-B*51 resulted in a more efficient killing of HLA-B*51-transfected target cells. Moreover, in these clones, a direct correlation between KIR3DS1 and NKG2D surface density was detected, while the expression of NKp46 was inversely correlated with that of KIR3DS1. Finally, we analyzed KIR3DS1+/NKG2A+ NK cell clones from a HLA-B Bw4neg donor carrying cytoplasmic KIR3DL1. Although these clones expressed lower levels of surface KIR3DS1, they displayed responses comparable to those of NK cell clones derived from HLA-B Bw4neg donors that expressed surface KIR3DL1. Altogether these data suggest that, in particular KIR/HLA combinations, KIR3DS1 may play a role

  2. Strategies to rescue mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) from NK cell mediated cytotoxicity.

    PubMed

    Jewett, Anahid; Arasteh, Aida; Tseng, Han-Ching; Behel, Armin; Arasteh, Hobie; Yang, Wendy; Cacalano, Nicholas A; Paranjpe, Avina

    2010-03-31

    The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells. We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16(+) subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-gamma by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells. By increasing the release of IFN-gamma and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells.

  3. NK cells are strongly activated by Lassa and Mopeia virus-infected human macrophages in vitro but do not mediate virus suppression.

    PubMed

    Russier, Marion; Reynard, Stéphanie; Tordo, Noël; Baize, Sylvain

    2012-07-01

    Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Arenaviruses. LASV causes hemorrhagic fever, whereas MOPV is not pathogenic. Both viruses display tropism for APCs such as DCs and macrophages. During viral infections, NK cells are involved in the clearance of infected cells and promote optimal immune responses by interacting with APCs. We used an in vitro model of human NK and APC coculture to study the role of NK cells and to characterize their interactions with APCs during LASV and MOPV infections. As expected, NK cells alone were neither infected nor activated by LASV and MOPV, and infected DCs did not activate NK cells. By contrast, LASV- and MOPV-infected macrophages activated NK cells, as shown by the upregulation of CD69, NKp30, and NKp44, the downregulation of CXCR3, and an increase in NK-cell proliferation. NK cells acquired enhanced cytotoxicity, as illustrated by the increase in granzyme B (GrzB) expression and killing of K562 targets, but did not produce IFN-γ. Contact between NK cells and infected macrophages and type I IFNs were essential for activation; however, NK cells could not kill infected cells and control infection. Overall, these findings show that MOPV- as well as pathogenic LASV-infected macrophages mediate NK-cell activation.

  4. IL-18-induced expression of high-affinity IL-2R on murine NK cells is essential for NK-cell IFN-γ production during murine Plasmodium yoelii infection.

    PubMed

    Stegmann, Kerstin A; De Souza, J Brian; Riley, Eleanor M

    2015-12-01

    Early production of pro-inflammatory cytokines, including IFN-γ, is essential for control of blood-stage malaria infections. We have shown that IFN-γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK-cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood-stage infection, expressing CD25 and producing IFN-γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN-γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL-18 signaling is essential for induction of CD25 and synergizes with IL-12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL-infected erythrocytes induced NK-cell CD25 expression and IFN-γ production in a manner that is completely IL-18- and partially IL-12-dependent, and IFN-γ production is enhanced by IL-2. These data suggest that IL-2 signaling via CD25 amplifies IL-18- and IL-12-mediated NK-cell activation during malaria infection.

  5. Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

    PubMed

    Kübler, Ayline; Woiterski, Jeanette; Witte, Kai-Erik; Bühring, Hans-Jörg; Hartwig, Udo F; Ebinger, Martin; Oevermann, Lena; Mezger, Markus; Herr, Wolfgang; Lang, Peter; Handgretinger, Rupert; Münz, Christian; André, Maya C

    2014-12-18

    Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients. © 2014 by The American Society of Hematology.

  6. NK026680 inhibits T-cell function in an IL-2-dependent manner and prolongs cardiac allograft survival in rats.

    PubMed

    Shibasaki, Susumu; Yamashita, Kenichiro; Goto, Ryoichi; Oura, Tetsu; Wakayama, Kenji; Hirokata, Gentaro; Shibata, Tomohiro; Igarashi, Rumi; Haga, Sanae; Ozaki, Michitaka; Todo, Satoru

    2012-01-01

    NK026680 is a triazolopyrimidine derivative that has been shown to inhibit dendritic cell maturation and activation. Here, we examined the immunosuppressive properties of NK026680 on T-cell function and assessed its immunosuppressive efficacy in an ACI (RT1(av1) haplotype) to Lewis (RT1(l)) rat heart transplantation model. The effects of NK026680 on T-cell proliferation, activation, and cytokine production were investigated in vitro. Heart transplant recipient rats were administered NK026680 daily for 14 days post-transplantation. In addition to graft survival time, alloimmune responses and graft histology at 4-10 days post-transplantation were assessed. NK026680 was found to inhibit proliferation, CD25 upregulation, IL-2 production, and cell cycle progression in αCD3/αCD28-stimulated murine T cells. These effects were likely due to suppression of the p38 mitogen-activated protein kinase pathway and the subsequent inhibition of p65, c-Fos, and to a lesser extent, c-Jun. Daily NK026680 treatment suppressed alloimmune responses, prevented cellular infiltration into allografts, and prolonged graft survival. The anti-rejection effects of NK026680 were enhanced by tacrolimus. In conclusion, NK026680 inhibits the activation of T cells and prolongs cardiac allograft survival in rats. These features make it a potential candidate immunosuppressant for the treatment of organ transplant patients in the future.

  7. Oncogenic stress sensed by the immune system: role of NK cell receptors

    PubMed Central

    Raulet, David H.; Guerra, Nadia

    2010-01-01

    A growing body of research addresses how pathways dysregulated during tumorigenesis are linked to innate immune responses, which can contribute to immune surveillance of cancer. Innate components of the immune system localized in tissues are believed to eliminate early neoplastic cells, thus preventing or delaying the establishment of advanced tumours. This Review addresses our current understanding of the mechanisms that detect cellular stresses that are associated with tumorigenesis, and that culminate in the recognition and, in some cases, the elimination of the tumour cells by natural killer (NK) cells and other lymphocytes that express NK cell receptors. PMID:19629084

  8. CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells.

    PubMed

    Sivori, Simona; Falco, Michela; Della Chiesa, Mariella; Carlomagno, Simona; Vitale, Massimo; Moretta, Lorenzo; Moretta, Alessandro

    2004-07-06

    Toll-like receptors (TLRs) are pattern-recognition receptors responsible for triggering cells of innate immunity. In this study we investigated the expression and function of TLRs 3 and 9 in human natural killer (NK) cells. In the presence of IL-12, freshly isolated NK cells responded to double-stranded RNA or unmethylated CpG DNA and expressed CD69 and CD25 activation markers. Because both markers were expressed by virtually all NK cells, this would suggest that most of them can be triggered by TLRs. Remarkably, NK cell stimulation also resulted in the induction of their functional program as revealed by IFN-gamma and tumor necrosis factor-alpha release and by up-regulation of cytolytic activity against tumor cells. IL-8 could efficiently substitute IL-12 in supporting NK cell responses to TLR-mediated stimulation. Importantly, freshly isolated NK cells acquired the ability to lyse immature dendritic cells after stimulation with double-stranded RNA and IL-12. However, responses to these stimuli were not restricted to fresh NK cells, because significant responses were also detected in polyclonal NK cells cultured in the presence of exogenous IL-2 for several weeks. The analysis of NK cell clones revealed some degree of heterogeneity in the ability to respond to TLR stimulation also among NK clones derived from a single donor. These data suggest that stimuli acting on TLR not only activate immature dendritic cells to release IL-12 but also render NK cells capable of receiving triggering signals from pathogen-associated molecules, thus exerting a regulatory control on the early steps of innate immune responses against infectious agents.

  9. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation.

    PubMed

    Gross, Catharina C; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G; Wiendl, Heinz

    2016-05-24

    Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56(bright) NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4(+) T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4(+) T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor's ligand CD155 on CD4(+) T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4(+) T cells and the cytolytic activity of NK cells.

  10. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

    PubMed Central

    Gross, Catharina C.; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G.; Wiendl, Heinz

    2016-01-01

    Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4+ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4+ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4+ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4+ T cells and the cytolytic activity of NK cells. PMID:27162345

  11. Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation

    PubMed Central

    Pedroza-Pacheco, Isabela; Shah, Divya; Domogala, Anna; Luevano, Martha; Blundell, Michael; Jackson, Nicola; Thrasher, Adrian; Madrigal, Alejandro; Saudemont, Aurore

    2016-01-01

    Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions. PMID:26915707

  12. Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation.

    PubMed

    Pedroza-Pacheco, Isabela; Shah, Divya; Domogala, Anna; Luevano, Martha; Blundell, Michael; Jackson, Nicola; Thrasher, Adrian; Madrigal, Alejandro; Saudemont, Aurore

    2016-02-26

    Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag(-/-) γc(-/-) mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.

  13. NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation.

    PubMed

    Jacobs, Benedikt; Tognarelli, Sara; Poller, Kerstin; Bader, Peter; Mackensen, Andreas; Ullrich, Evelyn

    2015-01-01

    High-dose chemotherapy with consecutive autologous stem cell transplantation (autoSCT) is a well-established treatment option for patients suffering from malignant lymphoma or multiple myeloma. Natural killer (NK) cells are an important part of the immune surveillance, and their cell number after autoSCT is predictive for progression-free and overall survival. To improve knowledge about the role of NK cells after autoSCT, we investigated different NK cell subgroups, their phenotype, and their functions in patients treated with autoSCT. Directly after leukocyte regeneration (>1000 leukocytes/μl) following autoSCT, CD56(++) NK cells were the major NK cell subset. Surprisingly, these cells showed unusually high surface expression levels of CD57 and killer Ig-like receptors (KIRs) compared to expression levels before or at later time points after autoSCT. Moreover, these NK cells strongly upregulated KIR2DL2/3/S2 and KIR3DL1, whereas KIR2DL1/S1 remained constant, indicating that this cell population arose from more immature NK cells instead of from activated mature ones. Remarkably, NK cells were already able to degranulate and produce IFN-γ and MIP-1β upon tumor interaction early after leukocyte regeneration. In conclusion, we describe an unusual upregulation of CD57 and KIRs on CD56(++) NK cells shortly after autoSCT. Importantly, these NK cells were functionally competent upon tumor interaction at this early time point.

  14. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    PubMed

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  15. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Sánchez-Martínez, Diego; Lanuza, Pilar M.; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A.; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. PMID:27833611

  16. Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

    USDA-ARS?s Scientific Manuscript database

    Early interactions of innate immune cell populations such as DC, monocytes/macrophages and NK cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13+ splenic DC or CD...

  17. Activated adherent large granular lymphocytes/natural killer (LGL/NK) cells change their migratory behaviour.

    PubMed Central

    Pirelli, A; Allavena, P; Mantovani, A

    1988-01-01

    Unstimulated large granular lymphocytes/natural killer (LGL/NK) cells, unlike small T lymphocytes, exhibit prompt locomotion into nitrocellulose filters in response to chemo-attactrants, but, unlike monocytes, are unable to migrate as adherent cells across polycarbonate filters. Upon activation with 4-B-phorbol 12,13 dibutyrate (PDBU), LGL/NK cells become adherent and change their migratory behaviour, having the ability to migrate as adherent cells across polycarbonate filters. PDBU-treated high-density T lymphocytes did not show, under the same conditions, locomotory activity. The change in migratory behaviour following activation may represent an important determinant of the ability of activated LGL/NK cells to adhere to vascular linings and localize in tissues. PMID:3220508

  18. Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

    PubMed

    Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2007-01-01

    The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures.

  19. CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: Induction of cytokine release and cytotoxicity against tumors and dendritic cells

    PubMed Central

    Sivori, Simona; Falco, Michela; Chiesa, Mariella Della; Carlomagno, Simona; Vitale, Massimo; Moretta, Lorenzo; Moretta, Alessandro

    2004-01-01

    Toll-like receptors (TLRs) are pattern-recognition receptors responible for triggering cells of innate immunity. In this study we investigated the expression and function of TLRs 3 and 9 in human natural killer (NK) cells. In the presence of IL-12, freshly isolated NK cells responded to double-stranded RNA or unmethylated CpG DNA and expressed CD69 and CD25 activation markers. Because both markers were expressed by virtually all NK cells, this would suggest that most of them can be triggered by TLRs. Remarkably, NK cell stimulation also resulted in the induction of their functional program as revealed by IFN-γ and tumor necrosis factor-α release and by up-regulation of cytolytic activity against tumor cells. IL-8 could efficiently substitute IL-12 in supporting NK cell responses to TLR-mediated stimulation. Importantly, freshly isolated NK cells acquired the ability to lyse immature dendritic cells after stimulation with double-stranded RNA and IL-12. However, responses to these stimuli were not restricted to fresh NK cells, because significant responses were also detected in polyclonal NK cells cultured in the presence of exogenous IL-2 for several weeks. The analysis of NK cell clones revealed some degree of heterogeneity in the ability to respond to TLR stimulation also among NK clones derived from a single donor. These data suggest that stimuli acting on TLR not only activate immature dendritic cells to release IL-12 but also render NK cells capable of receiving triggering signals from pathogen-associated molecules, thus exerting a regulatory control on the early steps of innate immune responses against infectious agents. PMID:15218108

  20. Advances in clinical NK cell studies: Donor selection, manufacturing and quality control.

    PubMed

    Koehl, U; Kalberer, C; Spanholtz, J; Lee, D A; Miller, J S; Cooley, S; Lowdell, M; Uharek, L; Klingemann, H; Curti, A; Leung, W; Alici, E

    2016-04-01

    Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013-2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies.

  1. Advances in clinical NK cell studies: Donor selection, manufacturing and quality control

    PubMed Central

    Koehl, U.; Kalberer, C.; Spanholtz, J.; Lee, D. A.; Miller, J. S.; Cooley, S.; Lowdell, M.; Uharek, L.; Klingemann, H.; Curti, A.; Leung, W.; Alici, E.

    2016-01-01

    ABSTRACT Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013–2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies. PMID:27141397

  2. Aspergillus fumigatus responds to natural killer (NK) cells with upregulation of stress related genes and inhibits the immunoregulatory function of NK cells

    PubMed Central

    Schneider, Andreas; Blatzer, Michael; Posch, Wilfried; Schubert, Ralf; Lass-Flörl, Cornelia; Schmidt, Stanislaw; Lehrnbecher, Thomas

    2016-01-01

    Natural Killer (NK) cells are active against Aspergillus fumigatus, which in turn is able to impair the host defense. Unfortunately, little is known on the mutual interaction of NK cells and A. fumigatus. We coincubated human NK cells with A. fumigatus hyphae and assessed the gene expression and protein concentration of selected molecules. We found that A. fumigatus up-regulates the gene expression of pro-inflammatory molecules in NK cells, but inhibited the release of these molecules resulting in intracellular accumulation and limited extracellular availability. A. fumigatus down-regulatedmRNA levels of perforin in NK cells, but increased its intra- and extracellular protein concentration. The gene expression of stress related molecules of A. fumigatus such as heat shock protein hsp90 was up-regulated by human NK cells. Our data characterize for the first time the immunosuppressive effect of A. fumigatus on NK cells and may help to develop new therapeutic antifungal strategies. PMID:27738337

  3. Peripheral blood CD56(bright) NK cells respond to stem cell factor and adhere to its membrane-bound form after upregulation of c-kit.

    PubMed

    Pradier, Amandine; Tabone-Eglinger, Severine; Huber, Vincent; Bosshard, Carine; Rigal, Emmanuel; Wehrle-Haller, Bernhard; Roosnek, Eddy

    2014-02-01

    CD56(bright) NK cells express receptors for IL-2, IL-7, IL-15, and SCF. We found that human peripheral blood CD56(bright) NK cells responded to IL-2, IL-7, IL-15 by phosphorylating STAT-5, ERK, and Akt but did not respond to SCF. However, CD56(bright) NK cells in culture upregulated c-kit transcription three to fourfold, which led to a steady increase in c-kit and a concomitant acquisition of responsiveness to SCF. After 44 h, CD56(bright) NK cells had upregulated c-kit approximately 20-fold and phosphorylated ERK and Akt in response to SCF concentrations well below levels present in plasma. CD56(bright) NK cells cultured in IL-15 maintained c-kit transcription/expression at ex vivo levels and did not become responsive to SCF. Furthermore, SCF-responsive, CD56(bright) c-kit(high) NK cells swiftly downregulated c-kit and stopped responding to SCF after IL-15 stimulation. However, commitment of CD56(bright) NK cells to a c-kit-negative, SCF-unresponsive state did not occur, as after 5 days of culture, withdrawal of IL-15 restored c-kit to maximal levels and reestablished SCF-responsiveness. CD56(bright) NK cells that had upregulated c-kit firmly adhered to COS cells transfected with the membrane form of SCF. Furthermore, SCF signaling significantly increased the capacity of CD56(bright) NK cells to degranulate. Collectively, our data suggest that c-kit on human CD56(bright) NK cells is a functional receptor that is downregulated in peripheral blood, possibly to render CD56(bright) NK cells unresponsive to the SCF therein.

  4. Enhanced and efficient detection of virus-driven cytokine expression by human NK and T cells.

    PubMed

    Pattacini, Laura; Murnane, Pamela M; Fluharty, Tayler R; Katabira, Elly; De Rosa, Stephen C; Baeten, Jared M; Lund, Jennifer M

    2014-04-01

    Cutting edge immune monitoring techniques increasingly measure multiple functional outputs for various cell types, such as intracellular cytokine staining (ICS) assays that measure cytokines expressed by T cells. To date, however, there is no precise method to measure virus-specific cytokine production by both T cells as well as NK cells in the same well, which is important to a greater extent given recent identification of NK cells expressing a memory phenotype. This study describes an adaptable and efficient ICS assay platform that can be used to detect antigen-driven cytokine production by human T cells and NK cells, termed "viral ICS". Importantly, this assay uses limited amount of cryopreserved PBMCs along with autologous heat-inactivated serum, thereby allowing for this assay to be performed when sample is scarce as well as geographically distant from the laboratory. Compared to a standard ICS assay that detects antigen-specific T cell cytokine expression alone, the viral ICS assay is comparable in terms of both HIV-specific CD4 and CD8T cell cytokine response rates and magnitude of response, with the added advantage of ability to detect virus-specific NK cell responses.

  5. Enhancement of anti-leukemia activity of NK cells in vitro and in vivo by inhibition of leukemia cell-induced NK cell damage.

    PubMed

    Arriga, Roberto; Caratelli, Sara; Coppola, Andrea; Spagnoli, Giulio Cesare; Venditti, Adriano; Amadori, Sergio; Lanzilli, Giulia; Lauro, Davide; Palomba, Patrizia; Sconocchia, Tommaso; Del Principe, Maria Ilaria; Maurillo, Luca; Buccisano, Francesco; Capuani, Barbara; Ferrone, Soldano; Sconocchia, Giuseppe

    2016-01-12

    Acute myeloid leukemia (AML) cells induce, in vitro, NK cell abnormalities (NKCAs) including apoptosis and activating receptor down-regulation. The potential negative impact of AML cells on the therapeutic efficacy of NK cell-based strategies prompted us to analyze the mechanisms underlying NKCAs and to develop approaches to protect NK cells from NKCAs. NKCA induction by the AML leukemia cells target a subpopulation of peripheral blood NK cells and is interleukin-2 independent but is abrogated by a long-term culture of NK (LTNK) cells at 37°C. LTNK cells displayed a significantly enhanced ability to damage AML cells in vitro and inhibited the subcutaneous growth of ML-2 cells grafted into CB17 SCID mice. Actinomycin D restored the susceptibility of LTNK cells to NKCAs while TAPI-0, a functional analog of the tissue inhibitor of metalloproteinase (TIMP) 3, inhibits ML-2 cell-induced NKCAs suggesting that the generation of NK cell resistance to NKCAs involves RNA transcription and metalloproteinase (MPP) inactivation. This conclusion is supported by the reduced susceptibility to AML cell-induced NKCAs of LTNK cells in which TIMP3 gene and protein are over-expressed. This information may contribute to the rational design of targeted strategies to enhance the efficacy of NK cell-based-immunotherapy of AML with haploidentical NK cells.

  6. Enhancement of anti-leukemia activity of NK cells in vitro and in vivo by inhibition of leukemia cell-induced NK cell damage

    PubMed Central

    Arriga, Roberto; Caratelli, Sara; Coppola, Andrea; Spagnoli, Giulio Cesare; Venditti, Adriano; Amadori, Sergio; Lanzilli, Giulia; Lauro, Davide; Palomba, Patrizia; Sconocchia, Tommaso; Del Principe, Maria Ilaria; Maurillo, Luca; Buccisano, Francesco; Capuani, Barbara; Ferrone, Soldano; Sconocchia, Giuseppe

    2016-01-01

    Acute myeloid leukemia (AML) cells induce, in vitro, NK cell abnormalities (NKCAs) including apoptosis and activating receptor down-regulation. The potential negative impact of AML cells on the therapeutic efficacy of NK cell-based strategies prompted us to analyze the mechanisms underlying NKCAs and to develop approaches to protect NK cells from NKCAs. NKCA induction by the AML leukemia cells target a subpopulation of peripheral blood NK cells and is interleukin-2 independent but is abrogated by a long-term culture of NK (LTNK) cells at 37°C. LTNK cells displayed a significantly enhanced ability to damage AML cells in vitro and inhibited the subcutaneous growth of ML-2 cells grafted into CB17 SCID mice. Actinomycin D restored the susceptibility of LTNK cells to NKCAs while TAPI-0, a functional analog of the tissue inhibitor of metalloproteinase (TIMP) 3, inhibits ML-2 cell-induced NKCAs suggesting that the generation of NK cell resistance to NKCAs involves RNA transcription and metalloproteinase (MPP) inactivation. This conclusion is supported by the reduced susceptibility to AML cell-induced NKCAs of LTNK cells in which TIMP3 gene and protein are over-expressed. This information may contribute to the rational design of targeted strategies to enhance the efficacy of NK cell-based-immunotherapy of AML with haploidentical NK cells. PMID:26655503

  7. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection.

    PubMed

    Khakoo, Salim I; Thio, Chloe L; Martin, Maureen P; Brooks, Collin R; Gao, Xiaojiang; Astemborski, Jacquie; Cheng, Jie; Goedert, James J; Vlahov, David; Hilgartner, Margaret; Cox, Steven; Little, Ann-Margeret; Alexander, Graeme J; Cramp, Matthew E; O'Brien, Stephen J; Rosenberg, William M C; Thomas, David L; Carrington, Mary

    2004-08-06

    Natural killer (NK) cells provide a central defense against viral infection by using inhibitory and activation receptors for major histocompatibility complex class I molecules as a means of controlling their activity. We show that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of hepatitis C virus (HCV) infection. This effect was observed in Caucasians and African Americans with expected low infectious doses of HCV but not in those with high-dose exposure, in whom the innate immune response is likely overwhelmed. The data strongly suggest that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV.

  8. Effect of Spaceflight on the Functions of NK and LAK Cells

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Grimm, Elizabeth A.; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Spaceflight-associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. The subjects of this study were crewmembers of two 10-day shuttle flights. Ten-ml blood specimens were obtained from ten astronauts 10 days before launch, immediately after landing, and 3 days after landing. PBMCs were separated from the blood specimens and stored at -800 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4-hour Cr-51 release assay. K562 cells were used to assess NK-cell cytotoxicity. After 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 and Daudi cells as the target cells. NK-cell cytotoxicity was decreased at landing (p less than 0.0005) in 9/10 astronauts, and in most cases recovered to preflight levels by 3 days after landing; NK-cell cytotoxicity was increased in one astronaut at landing. LAK cytotoxic activity against K562 cells was decreased at landing in 6/10 astronauts (p=0.018), and activity against Daudi cells was decreased in 7/10 astronauts (p=0.01). Phenotyping of PBMCs and LAK cells showed alterations in some surface markers and adhesion molecules (CD1 1 b, CD1 1 c, CD1 1 a, CD1 6, L-Selectin and CD3). Thus spaceflight leads to a decrease in the functions of NK and LAK cells in most astronauts.

  9. Effect of Spaceflight on the Functions of NK and LAK Cells

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Grimm, Elizabeth A.; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Spaceflight-associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. The subjects of this study were crewmembers of two 10-day shuttle flights. Ten-ml blood specimens were obtained from ten astronauts 10 days before launch, immediately after landing, and 3 days after landing. PBMCs were separated from the blood specimens and stored at -800 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4-hour Cr-51 release assay. K562 cells were used to assess NK-cell cytotoxicity. After 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 and Daudi cells as the target cells. NK-cell cytotoxicity was decreased at landing (p less than 0.0005) in 9/10 astronauts, and in most cases recovered to preflight levels by 3 days after landing; NK-cell cytotoxicity was increased in one astronaut at landing. LAK cytotoxic activity against K562 cells was decreased at landing in 6/10 astronauts (p=0.018), and activity against Daudi cells was decreased in 7/10 astronauts (p=0.01). Phenotyping of PBMCs and LAK cells showed alterations in some surface markers and adhesion molecules (CD1 1 b, CD1 1 c, CD1 1 a, CD1 6, L-Selectin and CD3). Thus spaceflight leads to a decrease in the functions of NK and LAK cells in most astronauts.

  10. Retroviral gene transfer into primary human NK cells activated by IL-2 and K562 feeder cells expressing membrane-bound IL-21.

    PubMed

    Streltsova, Maria A; Barsov, Eugene; Erokhina, Sofia A; Kovalenko, Elena I

    2017-11-01

    Natural killer (NK) cells are capable of rapidly recognizing and efficiently killing tumor cells. This makes them a potentially promising agent for cancer immunotherapy. Additional genetic modifications of NK cells may further improve their anti-tumor efficacy. Numerous technical challenges associated with gene delivery into NK cells have significantly tempered this approach. We achieved efficient retroviral vector transduction of primary human NK cells that were stimulated by a combination of IL-2 and engineered K562 cells expressing membrane-bound IL-21. The activated NK cells were in less differentiated state and expressed NK cell activation receptors NKG2D, NKp30, CD16, and were highly HLA-DR-positive. This NK cell population was highly susceptible to the transduction by both GFP- and NGFR-expressing retroviral vectors, with transduction efficiency exceeding 50%. More mature CD57(+) NK cell population was generally resistant to retroviral vector transduction because of poor response to the stimulation. Our findings may facilitate retroviral vector-mediated genetic engineering of human primary NK cells for future immunotherapies. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

    PubMed Central

    Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

    2017-01-01

    Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

  12. Loss of STAT3 in Lymphoma Relaxes NK Cell-Mediated Tumor Surveillance.

    PubMed

    Putz, Eva Maria; Hoelzl, Maria Agnes; Baeck, Julia; Bago-Horvath, Zsuzsanna; Schuster, Christian; Reichholf, Brian; Kern, Daniela; Aberger, Fritz; Sexl, Veronika; Hoelbl-Kovacic, Andrea

    2014-01-27

    The transcription factors and proto-oncogenes STAT3 and STAT5 are highly activated in hematological malignancies and represent promising therapeutic targets. Whereas the importance of STAT5 as tumor promoter is beyond doubt, the role of STAT3 in hematological cancers is less well understood. Both, enforced as well as attenuated expression of STAT3 were reported in hematopoietic malignancies. Recent evidence implicates STAT3 as key player for tumor immune surveillance as it both mediates the production of and response to inflammatory cytokines. Here we investigated the effects of STAT3 deletion in a BCR/ABL-induced lymphoma model, which is tightly controlled by natural killer (NK) cells in vivo. Upon STAT3 deletion tumor growth is significantly enhanced when compared to STAT3-expressing controls. The increased tumor size upon loss of STAT3 was accompanied by reduced NK cell infiltration and decreased levels of the cytokine IFN-γ and the chemokine RANTES. Upon transplantation into NK cell-deficient mice differences in lymphoma size were abolished indicating that STAT3 expression in the tumor cells controls NK cell-dependent tumor surveillance. Our findings indicate that STAT3 inhibition in lymphoma patients will impair NK cell-mediated tumor surveillance, which needs to be taken into account when testing STAT3 inhibitors in preclinical or clinical trials.

  13. NK Cell and CD4+FoxP3+ Regulatory T Cell Based Therapies for Hematopoietic Stem Cell Engraftment

    PubMed Central

    Pierini, Antonio; Alvarez, Maite; Negrin, Robert S.

    2016-01-01

    Allogeneic hematopoietic cell transplantation (HCT) is a powerful therapy to treat multiple hematological diseases. The intensive conditioning regimens used to allow for donor hematopoietic stem cell (HSC) engraftment are often associated with severe toxicity, delayed immune reconstitution, life-threatening infections, and thus higher relapse rates. Additionally, due to the high incidence of graft versus host disease (GvHD), HCT protocols have evolved to prevent such disease that has a detrimental impact on antitumor and antiviral responses. Here, we analyzed the role of host T and natural killer (NK) cells in the rejection of donor HSC engraftment as well as the impact of donor regulatory T cells (Treg) and NK cells on HSC engraftment. We review some of the current strategies that utilize NK or Treg to improve allogeneic HCT therapy in order to accomplish better HSC engraftment and immune reconstitution and achieve a lower incidence of cancer relapse, opportunistic infections, and GvHD. PMID:26880996

  14. Absence of circulating natural killer (NK) cells in a child with erythrophagocytic lymphohistiocytosis lacking NK cell activity

    SciTech Connect

    Kawai, H.; Komiyama, A.; Aoyama, K.; Miyagawa, Y.; Akabane, T.

    1988-06-01

    A 5-year-old girl who was diagnosed as having erythrophagocytic lymphohistiocytosis died at age 9 years. Peripheral lymphocytes from the patient persistently lacked natural killer (NK) cell activity during the 4-year observation period: the percent lysis values as measured by a 4-hr /sup 51/Cr release assay at a 40:1 effector:target ratio were below 1.0% against K562 and Molt-4 cells as compared with the normal lymphocyte value (mean +/- SD) of 46.2% +/- 5.8% and 43.9% +/- 6.7%, respectively. The patient's lymphocytes never developed NK cell activity by their incubation with target cells for longer time periods or by their stimulation with interferon-alpha, interleukin-2, or polyinosinic-polycytidilic acid. Single cell-in-agarose assay showed the absence of target-binding cells (TBCs): TBC numbers were below 0.3% as compared with the normal lymphocyte value of 8.1% +/- 1.3% (mean +/- SD). Flow cytometry showed a marked decrease in Leu-7+ cells (1.7%) and the absence of Leu-11+ cells (0.4%) in the peripheral blood. These results first demonstrate a case of erythrophagocytic lymphohistiocytosis in which there is the lack of NK cell activity due to the absence of circulating NK cells.

  15. Application of tissue-specific NK and NKT cell activity for tumor immunotherapy

    PubMed Central

    Subleski, Jeff J.; Wiltrout, Robert H.; Weiss, Jonathan M.

    2009-01-01

    Natural killer (NK) and NKT cells are a first line of defense against pathogens and transformed cells. However, dysregulation of their function can lead to autoimmune disease. A better understanding of the mechanisms controlling NK and NKT effector function should lead to the development of improved strategies for the treatment of many diseases. The site in which NK and NKT cells reside should be taken into account, because accumulating evidence suggests that the tissue microenvironment strongly influences their function. In this regard, the liver represents a unique immunologic organ in which the balance between the need for tolerance and the ability to respond rapidly to pathogens and tissue injury is tightly regulated. NK cells in the liver have augmented cytolytic activity as compared to other organs, which is consistent with a role for liver-associated NK cells in being critical effector cells for inhibiting tumor metastasis in the liver. Several studies also suggest that hepatic NKT cells have different functions than those in other organs. Whereas splenic and thymic NKT cells have been shown to suppress diabetes development, facilitate the induction of systemic tolerance and are regulated by IL-4 and other Th2 cytokines, certain subsets of NKT cells in the liver are important sources of Th1 cytokines such as Interferon gamma, and are the primary mediators of anti-tumor responses. The unique properties and roles as critical effector cells make NK and NKT cells within the liver microenvironment attractive targets of immunotherapeutic approaches that have the goal of controlling tumor metastasis in the liver. PMID:19682859

  16. NK cell immunophenotypic and genotypic analysis of infants with severe respiratory syncytial virus infection.

    PubMed

    Noyola, Daniel E; Juárez-Vega, Guillermo; Monjarás-Ávila, César; Escalante-Padrón, Francisco; Rangel-Ramírez, Verónica; Cadena-Mota, Sandra; Monsiváis-Urenda, Adriana; García-Sepúlveda, Christian A; González-Amaro, Roberto

    2015-07-01

    Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants. Reduced numbers of NK cells have been reported in infants with severe RSV infection; however, the precise role of NK cells during acute RSV infection is unclear. In this study the NK and T cell phenotypes, LILRB1 gene polymorphisms and KIR genotypes of infants hospitalized with RSV infection were analyzed. Compared to controls, infants with acute RSV infection showed a higher proportion of LILRB1+ T cells; in addition, a subgroup of infants with RSV infection showed an increase in LILRB1+ NK cells. No differences in NKG2C, NKG2A, or CD161 expression between RSV infected infants and controls were observed. LILRB1 genotype distribution of the rs3760860 A>G, and rs3760861 A>G single nucleotide polymorphisms differed between infants with RSV infection and healthy donors, whereas no differences in any of the KIR genes were observed. Our results suggest that LILRB1 participates in the pathogenesis of RSV infection. Further studies are needed to define the role of LILRB1+ NK in response to RSV and to confirm an association between LILRB1 polymorphisms and the risk of severe RSV infection.

  17. Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice

    PubMed Central

    Pernicone, Elizabeth; Korkes, Henri A.; Burke, Suzanne D.; Rajakumar, Augustine; Thadhani, Ravi I.; Roberts, Drucilla J.; Bhasin, Manoj; Karumanchi, S. Ananth

    2016-01-01

    Decidual NK (dNK) cells, a distinct type of NK cell, are thought to regulate uterine spiral artery remodeling, a process that allows for increased blood delivery to the fetal-placental unit. Impairment of uterine spiral artery remodeling is associated with decreased placental perfusion, increased uterine artery resistance, and obstetric complications such as preeclampsia and intrauterine growth restriction. Ex vivo manipulation of human peripheral blood NK (pNK) cells by a combination of hypoxia, TGFß-1 and 5-aza-2’-deoxycytidine yields cells with phenotypic and in vitro functional similarities to dNK cells, called idNK cells. Here, gene expression profiling shows that CD56Bright idNK cells derived ex vivo from human pNK cells, and to a lesser extent CD56Dim idNK cells, are enriched in the gene expression signature that distinguishes dNK cells from pNK cells. When injected into immunocompromised pregnant mice with elevated uterine artery resistance, idNK cells homed to the uterus and reduced the uterine artery resistance index, suggesting improved placental perfusion. PMID:27736914

  18. NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype.

    PubMed

    Ames, Erik; Canter, Robert J; Grossenbacher, Steven K; Mac, Stephanie; Chen, Mingyi; Smith, Rachel C; Hagino, Takeshi; Perez-Cunningham, Jessica; Sckisel, Gail D; Urayama, Shiro; Monjazeb, Arta M; Fragoso, Ruben C; Sayers, Thomas J; Murphy, William J

    2015-10-15

    Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

  19. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.

    PubMed

    Veluchamy, John P; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A M; Verheul, Henk M W; Di Santo, James P; de Gruijl, Tanja D; van der Vliet, Hans J

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RAS(wt) metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR(-) RAS(wt), EGFR(+) RAS(mut), and EGFR(+) BRAF(mut) cells, A-PBNK were able to initiate lysis of EGFR(+) colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR(+) colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR(-)RAS(wt) (42 ± 8 versus 67 ± 7%), EGFR(+) RAS(mut) (20 ± 2 versus 37 ± 6%), and EGFR(+) BRAF(mut) (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR(+) RAS(mut) colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

  20. DNAM-1 expression marks an alternative program of NK cell maturation.

    PubMed

    Martinet, Ludovic; Ferrari De Andrade, Lucas; Guillerey, Camille; Lee, Jason S; Liu, Jing; Souza-Fonseca-Guimaraes, Fernando; Hutchinson, Dana S; Kolesnik, Tatiana B; Nicholson, Sandra E; Huntington, Nicholas D; Smyth, Mark J

    2015-04-07

    Natural killer (NK) cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling and functional studies that the activating receptor DNAM-1 (CD226) identifies two distinct NK cell functional subsets: DNAM-1(+) and DNAM-1(-) NK cells. DNAM-1(+) NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1(-) NK cells that differentiate from DNAM-1(+) NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Tumour-experienced T cells promote NK cell activity through trogocytosis of NKG2D and NKp46 ligands

    PubMed Central

    Domaica, Carolina I; Fuertes, Mercedes B; Rossi, Lucas E; Girart, María V; Ávila, Damián E; Rabinovich, Gabriel A; Zwirner, Norberto W

    2009-01-01

    Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-γ secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family, such as NKp46, by ligands expressed on tumour cells. However, it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here, we investigated the early events occurring during T cell–tumour cell interactions, and their impact on NK cell functions. We observed that on co-culture with some melanomas, activated CD4+ T cells promoted degranulation, and NKG2D- and NKp46-dependent IFN-γ secretion by NK cells, probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4+, CD8+ and resting T cells, which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new, so far, unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. PMID:19498463

  2. Comparative analysis of NK-cell receptor expression and function across primate species

    PubMed Central

    Ugolotti, Elisabetta; De Maria, Andrea

    2010-01-01

    Natural killer (NK) cells are lymphoid effectors that are involved in the innate immune surveillance against infected and/or tumor cells. Their function is under the fine-tuning control of cell surface receptors that display either inhibitory or activating function and in healthy condition, mediate self-tolerance. It is known that inhibitory receptors are characterized by clonal and stochastic distribution and are extremely sensible to any modification, downregulation or loss of MHC class I surface expression that are induced in autologous cells upon viral infection or cancer transformation. This alteration of the MHC class I expression weakens the strength of the inhibitory receptor-induced interaction, thus resulting in a prompt triggering of NK cell function, which ends up in the inhibition of tumor progression and proliferation of pathogen-infected cells. Thus, the inhibitory function of NK cells is only one face of the coin, since NK-cell activation is controlled by different arrays of activating receptors that finally are involved in the induction of cytolysis and/or cytokine release. Interestingly, the inhibitory NK-cell receptors that are involved in dampening NK cell-mediated responses evolved during speciation in different, often structurally unrelated surface-expressed molecules, all using a conserved signaling pathway. In detail, during evolution, the inhibitory receptors that assure the recognition of MHC class I molecules, originate in, at least, three different ways. This ended up in multigene families showing marked structural divergences that coevolved in a convergent way with the availability of appropriate MHC ligand molecules. PMID:21487512

  3. Macrophages help NK cells to attack tumor cells by stimulatory NKG2D ligand but protect themselves from NK killing by inhibitory ligand Qa-1.

    PubMed

    Zhou, Zhixia; Zhang, Cai; Zhang, Jian; Tian, Zhigang

    2012-01-01

    Natural killer (NK) cells and their crosstalk with other immune cells are important for innate immunity against tumor. To explore the role of the interaction between NK cells and macrophages in the regulation of anti-tumor activities of NK cells, we here demonstrate that poly I:C-treated macrophages increased NK cell-mediated cytotoxicity against target tumor cells in NKG2D-dependent manner. In addition, IL-15, IL-18, and IFN-β secreted by poly I:C-treated macrophages are also involved in NKG2D expression and NK cell activation. Interestingly, the increase in expression of NKG2D ligands on macrophages induced a highly NK cell-mediated cytotoxicity against tumor cells, but not against macrophages themselves. Notably, a high expression level of Qa-1, a NKG2A ligand, on macrophages may contribute to such protection of macrophages from NK cell-mediated killing. Furthermore, Qa-1 or NKG2A knockdown and Qa-1 antibody blockade caused the macrophages to be sensitive to NK cytolysis. These results suggested that macrophages may activate NK cells to attack tumor by NKG2D recognition whereas macrophages protect themselves from NK lysis via preferential expression of Qa-1.

  4. Orbital involvement by non-Hodgkin lymphoma NK T cells.

    PubMed

    Hervás-Ontiveros, A; España-Gregori, E; Hernández-Martínez, P; Vera-Sempere, F J; Díaz-Llopis, M

    2014-11-01

    The case is presented of 37 year-old male with a history of nasal obstruction with right rhinorrhea, headache, hearing loss and right exophthalmos of 4 months progression. The MRI revealed that the ethmoidal and maxillary sinuses contained inflammatory tissue extending into the orbital region. The biopsy confirmed a non-Hodgkin lymphoma of natural killer (NK) T cells. Non-Hodgkin's T NK lymphoma is a rare tumor in the orbital area that requires an early detection and multi-disciplinary care to ensure appropriate monitoring and treatment. Copyright © 2012 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  5. The Galectin-9/Tim-3 pathway is involved in the regulation of NK cell function at the maternal-fetal interface in early pregnancy.

    PubMed

    Li, Yan-Hong; Zhou, Wen-Hui; Tao, Yu; Wang, Song-Cun; Jiang, Yun-Lan; Zhang, Di; Piao, Hai-Lan; Fu, Qiang; Li, Da-Jin; Du, Mei-Rong

    2016-01-01

    Decidual natural killer (dNK) cells actively participate in the establishment and maintenance of maternal-fetal immune tolerance and act as local guardians against infection. However, how dNK cells maintain the immune balance between tolerance and anti-infection immune responses during pregnancy remains unknown. Here, we demonstrated that the inhibitory molecule T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) are expressed on over 60% of dNK cells. Tim-3(+) dNK cells display higher interleukin (IL)-4 and lower tumor necrosis factor (TNF)-α and perforin production. Human trophoblast cells can induce the transformation of peripheral NK cells into a dNK-like phenotype via the secretion of galectin-9 (Gal-9) and the interaction between Gal-9 and Tim-3. In addition, trophoblasts inhibit lipopolysaccharide (LPS)-induced pro-inflammatory cytokine and perforin production by dNK cells, which can be attenuated by Tim-3 neutralizing antibodies. Interestingly, a decreased percentage of Tim-3-expressing dNK cells were observed in human miscarriages and murine abortion-prone models. Moreover, T helper (Th)2-type cytokines were decreased and Th1-type cytokines were increased in Tim-3(+) but not Tim-3(-) dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy.

  6. Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity.

    PubMed

    Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S

    2017-01-01

    Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR

  7. Chimeric Antigen Receptor-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Targeted Elimination of Cancer Cells and Induction of Protective Antitumor Immunity

    PubMed Central

    Zhang, Congcong; Oberoi, Pranav; Oelsner, Sarah; Waldmann, Anja; Lindner, Aline; Tonn, Torsten; Wels, Winfried S.

    2017-01-01

    Significant progress has been made in recent years toward realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also the established NK cell line NK-92 is being developed for adoptive immunotherapy, and general safety of infusion of irradiated NK-92 cells has been established in phase I clinical trials with clinical responses observed in some of the cancer patients treated. To enhance their therapeutic utility, NK-92 cells have been modified to express chimeric antigen receptors (CARs) composed of a tumor-specific single chain fragment variable antibody fragment fused via hinge and transmembrane regions to intracellular signaling moieties such as CD3ζ or composite signaling domains containing a costimulatory protein together with CD3ζ. CAR-mediated activation of NK cells then bypasses inhibitory signals and overcomes NK resistance of tumor cells. In contrast to primary NK cells, CAR-engineered NK-92 cell lines suitable for clinical development can be established from molecularly and functionally well-characterized single cell clones following good manufacturing practice-compliant procedures. In preclinical in vitro and in vivo models, potent antitumor activity of NK-92 variants targeted to differentiation antigens expressed by hematologic malignancies, and overexpressed or mutated self-antigens associated with solid tumors has been found, encouraging further development of CAR-engineered NK-92 cells. Importantly, in syngeneic mouse tumor models, induction of endogenous antitumor immunity after treatment with CAR-expressing NK-92 cells has been demonstrated, resulting in cures and long-lasting immunological memory protecting against tumor rechallenge at distant sites. Here, we summarize the current status and future prospects of CAR

  8. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

  9. Human NK Cell Subsets in Pregnancy and Disease: Toward a New Biological Complexity

    PubMed Central

    Cristiani, Costanza Maria; Palella, Eleonora; Sottile, Rosa; Tallerico, Rossana; Garofalo, Cinzia; Carbone, Ennio

    2016-01-01

    In humans, NK cells are mainly identified by the surface expression levels of CD56 and CD16, which differentiate between five functionally different NK cell subsets. However, nowadays NK cells are considered as a more heterogeneous population formed by various subsets differing in function, surface phenotype, and anatomic localization. In human CMV- and hantaviruses-infected subjects, an increased frequency of a NKG2A−CD57+NKG2C+ NK cell subset has been observed, while the phenotype of the NK cell subpopulation associated with cancer may vary according to the specific kind of tumor and its anatomical location. The healthy human lymph nodes contain mainly the CD56bright NK cell subset while in melanoma metastatic lymph nodes the CD56dimCD57+KIR+CCR7+ NK cell subpopulation prevails. The five NK cell subpopulations are found in breast cancer patients, where they differ for expression pattern of chemokine receptors, maturation stage, functional capabilities. In pregnancy, uterine NK cells show a prevalence of the CD56brightCD16− NK cell compartment, whose activity is influenced by KIRs repertoire. This NK cell subset’s super specialization could be explained by (i) the expansion of single mature CD56dim clones, (ii) the recruitment and maturation of CD56bright NK cells through specific stimuli, and (iii) the in situ development of tumor-resident NK cells from tissue-resident CD56bright NK cells independently of the circulating NK cell compartment. This new and unexpected biological feature of the NK cell compartment could be an important source of new biomarkers to improve patients’ diagnosis. PMID:28082990

  10. Human NK Cell Subsets in Pregnancy and Disease: Toward a New Biological Complexity.

    PubMed

    Cristiani, Costanza Maria; Palella, Eleonora; Sottile, Rosa; Tallerico, Rossana; Garofalo, Cinzia; Carbone, Ennio

    2016-01-01

    In humans, NK cells are mainly identified by the surface expression levels of CD56 and CD16, which differentiate between five functionally different NK cell subsets. However, nowadays NK cells are considered as a more heterogeneous population formed by various subsets differing in function, surface phenotype, and anatomic localization. In human CMV- and hantaviruses-infected subjects, an increased frequency of a NKG2A(-)CD57(+)NKG2C(+) NK cell subset has been observed, while the phenotype of the NK cell subpopulation associated with cancer may vary according to the specific kind of tumor and its anatomical location. The healthy human lymph nodes contain mainly the CD56(bright) NK cell subset while in melanoma metastatic lymph nodes the CD56(dim)CD57(+)KIR(+)CCR7(+) NK cell subpopulation prevails. The five NK cell subpopulations are found in breast cancer patients, where they differ for expression pattern of chemokine receptors, maturation stage, functional capabilities. In pregnancy, uterine NK cells show a prevalence of the CD56(bright)CD16(-) NK cell compartment, whose activity is influenced by KIRs repertoire. This NK cell subset's super specialization could be explained by (i) the expansion of single mature CD56(dim) clones, (ii) the recruitment and maturation of CD56(bright) NK cells through specific stimuli, and (iii) the in situ development of tumor-resident NK cells from tissue-resident CD56(bright) NK cells independently of the circulating NK cell compartment. This new and unexpected biological feature of the NK cell compartment could be an important source of new biomarkers to improve patients' diagnosis.

  11. Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D.

    PubMed

    Gröschel, Carina; Hübscher, Daniela; Nolte, Jessica; Monecke, Sebastian; Sasse, André; Elsner, Leslie; Paulus, Walter; Trenkwalder, Claudia; Polić, Bojan; Mansouri, Ahmed; Guan, Kaomei; Dressel, Ralf

    2017-01-01

    Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1(-)(/)(-) mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability

  12. Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27.