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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. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

    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. PMID:26543875

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

    PubMed

    Liu, Qiong; Smith, C Wayne; Zhang, Wanyu; Burns, Alan R; Li, Zhijie

    2012-08-01

    Natural killer (NK) 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 classic NK cells migrated into the limbus and corneal stroma, peaking at 24 hours with an eightfold increase over baseline. Depletion of γδ T cells significantly reduced NK cell accumulation (>70%; P < 0.01); however, in neutrophil-depleted animals, NK cell influx was normal. Isolated spleen NK cells migrated to the wounded cornea, and this migration was reduced by greater than 60% (P < 0.01) by ex vivo antibody blocking of NK cell CXCR3 or CCR2. Antibody-induced depletion of NK cells significantly altered the inflammatory reaction to corneal wounding, as evidenced by a 114% increase (P < 0.01) in neutrophil influx at a time when acute inflammation is normally waning. Functional blocking of NKG2D, an activating receptor for NK cell cytotoxicity and cytokine secretion, did not inhibit NK cell immigration, but significantly increased neutrophil influx. Consistent with excessive neutrophil accumulation, NK depletion and blocking of NKG2D also inhibited corneal nerve regeneration and epithelial healing (P < 0.01). Findings of this study suggest that NK cells are actively involved in corneal healing by limiting the innate acute inflammatory reaction to corneal wounding.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. "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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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)

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. MHC class I–specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response

    PubMed Central

    Yawata, Nobuyo; Draghi, Monia; Partheniou, Fotini; Little, Ann-Margaret; Parham, Peter

    2008-01-01

    Variegated expression of 6 inhibitory HLA class I–specific receptors on primary NK cells was studied using high-dimension flow cytometry in 58 humans to understand the structure and function of NK-cell repertoires. Sixty-four subsets expressing all possible receptor com-binations were present in each repertoire, and the frequency of receptor-null cells varied among the donors. Enhancement in missing-self response between NK subsets varied substantially where subset responses were defined by donor KIR/HLA allotypes, reflecting the differences in interaction between inhibitory receptors and their ligands. This contrasted to the enhancement conferred by NKG2A, which was constant and of intermediate strength. We infer a mechanism that modulates frequencies of the NK subsets displaying diverse levels of missing-self response, a system that reduces the presence of KIR-expressing subsets that display either too strong or too weak a response and effectively replaces them with NKG2A-expressing cells in the repertoire. Through this high-resolution analysis of inhibitory receptor expression, 5 types of NK-cell repertoire were defined by their content of NKG2A+/NKG2A− cells, frequency of receptor-null cells, and degree of KIR receptor coexpression. The analyses provide new perspective on how personalized human NK-cell repertoires are structured. PMID:18583565

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Altered distribution of mucosal NK cells during HIV infection

    PubMed Central

    Sips, Magdalena; Sciaranghella, Gaia; Diefenbach, Thomas; Dugast, Anne-Sophie; Berger, Christoph T.; Liu, Qingquan; Kwon, Douglas; Ghebremichael, Musie; Estes, Jacob D.; Carrington, Mary; Martin, Jeffrey N.; Deeks, Steven G.; Hunt, Peter W.; Alter, Galit

    2013-01-01

    The human gut mucosa is a major site of HIV infection and infection-associated pathogenesis. Increasing evidence shows that natural killer (NK) cells play an important role in control of HIV infection but the mechanism(s) by which they mediate antiviral activity in the gut is unclear. Here we show two distinct subsets of NK cells exist in the gut, one localized to intraepithelial spaces (IEL) and the other to the lamina propria (LP). The frequency of both subsets of NK cells was reduced in chronic infection, whereas IEL NK cells remained stable in spontaneous controllers with protective KIR/HLA genotypes. Both IEL and LP NK cells were significantly expanded in immunologic non-responsive (INR) patients, who incompletely recovered CD4+ T cells on HAART. These data suggest that both IEL and LP NK cells may expand in the gut in an effort to compensate for compromised CD4+ T cell recovery, but that only IEL NK cells may be involved in providing durable control of HIV in the gut, PMID:21993602

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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).

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Immunoproteasome-Deficiency Has No Effects on NK Cell Education, but Confers Lymphocytes into Targets for NK Cells in Infected Wild-Type Mice

    PubMed Central

    Bekker, Cornelis P. J.; Boog, Claire J. P.; Zaiss, Dietmar M. W.; Sijts, Alice J. A. M.

    2011-01-01

    Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells. PMID:21887316

  3. Immunoproteasome-deficiency has no effects on NK cell education, but confers lymphocytes into targets for NK cells in infected wild-type mice.

    PubMed

    van Helden, Mary J G; de Graaf, Natascha; Bekker, Cornelis P J; Boog, Claire J P; Zaiss, Dietmar M W; Sijts, Alice J A M

    2011-01-01

    Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.

  4. 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.

  5. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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

  8. 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

  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. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

    2017-03-08

    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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. [Activation of natural killer T cells by NK-4, a criptocyanine dye].

    PubMed

    Kunikata, Toshio; Kohno, Keizo; Ushio, Shimpei; Fukuda, Shigeharu

    2011-01-01

    We previously reported that oral administration of NK-4, a criptocyanine dye, enhances interleukin (IL)-12-depend- ent interferon (IFN)-γ production by lipopolysaccharide (LPS)-stimulated mouse splenocytes. These findings raised a possibility that NK-4 potentiated IFN-γ production by T cells, natural killer (NK) cells or natural killer T (NKT) cells in response to IL-12 produced by macrophage and dendritic cells. To explore this possibility, we first analyzed percentages of T, NK or NKT cells in splenocytes of mice that were administered NK-4 orally for three days. The percentage of NKT cells in splenocytes from NK-4-treated mice was significantly (p<0.05) increased compared to vehicle-treated mice. When splenocytes were stimulated with α-galactosylceramide (α-GalCer), an NKT cell ligand, IFN-γ production by splenocytes from NK-4-treated mice tended to increase, while no difference in the IL-4 production and proliferation were observed between the vehicle- and NK-4-treated mice. When IFN-γ/IL-4 ratios were calculated in individual mice, the ratios were significantly (p<0.05) elevated in NK-4-treated mice. Furthermore, IL-12 production by α-GalCer-stimulated splenocytes from NK-4-treated mice was also significantly (p<0.05) increased. These results suggest that oral administration of NK-4 increases the population of type I NKT cells with potent IFN-γ-producing activities. Since IL-12 and IFN-γ have been shown to play important roles in anti-tumor immunity as well as in the defence against bacterial infection, our results further imply that NK-4 may provide a potential therapeutic tool in cancer immunotherapy.

  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. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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 mice

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function.

    PubMed

    Pace, Matthew; Williams, James; Kurioka, Ayako; Gerry, Andrew B; Jakobsen, Bent; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Frater, John

    2016-08-01

    In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Variable NK cell Receptors Exemplified by Human KIR3DL1/S11

    PubMed Central

    Parham, Peter; Norman, Paul J.; Abi-Rached, Laurent; Guethlein, Lisbeth A.

    2011-01-01

    Variegated expression of variable NK cell receptors for polymorphic MHC class I broadens the range of an individual’s NK cell response, and the capacity for populations and species to survive disease epidemics and population bottlenecks. On evolutionary time-scales this component of immunity is exceptionally dynamic, unstable and short-lived, being dependent upon co-evolution of ligands and receptors subject to varying, competing selection pressures. Consequently these systems of variable NK cell receptors are largely species-specific and have recruited different classes of glycoprotein, even within the primate order of mammals. Such disparity helps explain substantial differences in NK cell biology between humans and animal models, for which the population genetics is largely ignored. KIR3DL1/S1, that recognizes the Bw4 epitope of HLA-A and –B and is the most extensively studied of the variable NK cell receptors, exemplifies how variation in all possible parameters of function is recruited to diversify the human NK cell response. PMID:21690332

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  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. 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.

  3. 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.

  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. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  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. 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.

  7. Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27.

    PubMed

    Zwirner, Norberto Walter; Ziblat, Andrea

    2017-01-01

    Natural killer (NK) cells are characterized by their ability to detect and induce apoptosis of susceptible target cells and by secretion of immunoregulatory cytokines such as IFN-γ. Activation of these effector functions is triggered upon recognition of tumor and pathogen (mostly virus)-infected cells and because of a bidirectional cross talk that NK cells establish with other cells of myeloid origin such as dendritic cells (DC) and macrophages. A common characteristic of these myeloid cells is their ability to secrete different members of the IL-12 family of cytokines such as IL-12, IL-23, and IL-27 and cytokines such as IL-15 and IL-18. Although the effect of IL-12, IL-15, and IL-18 has been characterized, the effect of IL-23 and IL-27 on NK cells (especially human) remains ill-defined. Particularly, IL-27 is a cytokine with dual functions as it has been described as pro- and as anti-inflammatory in different experimental settings. Recent evidence indicates that this cytokine indeed promotes human NK cell activation, IFN-γ secretion, NKp46-dependent NK cell-mediated cytotoxicity, and antibody (Ab)-dependent NK cell-mediated cytotoxicity (ADCC) against monoclonal Ab-coated tumor cells. Remarkably, IL-27 also primes NK cells for IL-18 responsiveness, enhancing these functional responses. Consequently, IL-27 acts as a pro-inflammatory cytokine that, in concert with other DC-derived cytokines, hierarchically contributes to NK cells activation and effector functions, which likely contributes to foster the adaptive immune response in different physiopathological conditions.

  8. Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27

    PubMed Central

    Zwirner, Norberto Walter; Ziblat, Andrea

    2017-01-01

    Natural killer (NK) cells are characterized by their ability to detect and induce apoptosis of susceptible target cells and by secretion of immunoregulatory cytokines such as IFN-γ. Activation of these effector functions is triggered upon recognition of tumor and pathogen (mostly virus)-infected cells and because of a bidirectional cross talk that NK cells establish with other cells of myeloid origin such as dendritic cells (DC) and macrophages. A common characteristic of these myeloid cells is their ability to secrete different members of the IL-12 family of cytokines such as IL-12, IL-23, and IL-27 and cytokines such as IL-15 and IL-18. Although the effect of IL-12, IL-15, and IL-18 has been characterized, the effect of IL-23 and IL-27 on NK cells (especially human) remains ill-defined. Particularly, IL-27 is a cytokine with dual functions as it has been described as pro- and as anti-inflammatory in different experimental settings. Recent evidence indicates that this cytokine indeed promotes human NK cell activation, IFN-γ secretion, NKp46-dependent NK cell-mediated cytotoxicity, and antibody (Ab)-dependent NK cell-mediated cytotoxicity (ADCC) against monoclonal Ab-coated tumor cells. Remarkably, IL-27 also primes NK cells for IL-18 responsiveness, enhancing these functional responses. Consequently, IL-27 acts as a pro-inflammatory cytokine that, in concert with other DC-derived cytokines, hierarchically contributes to NK cells activation and effector functions, which likely contributes to foster the adaptive immune response in different physiopathological conditions. PMID:28154569

  9. 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.

  10. 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

  11. Regulation of NK-cell function by mucins via antigen-presenting cells.

    PubMed

    Laskarin, G; Redzovic, A; Medancic, S Srsen; Rukavina, D

    2010-12-01

    Decidual antigen-presenting cells including dendritic cells (DCs) and CD14(+) macrophages, as mediators of the first encounter with fetal antigens, appear to be critically involved in the initiation of primary immune response by regulating innate- and adaptive immunity. Interleukin-15, produced by them, permits the proliferation and differentiation of CD3(-)CD16(-)CD94(+)NKG2A(+)CD56(+bright) decidual NK cells that identify trophoblast cells. These cells are able to kill them after Th1 cytokine overstimulation and by increasing the release of preformed cytotoxic mediators. Thus, the local microenvironment is a potent modulator of antigen-presenting cell functions. Tumor associated glycoprotein-72 (TAG-72) and mucine 1 (MUC-1) are glycoproteins secreted by uterine epithelial cells. Our hypothesis is that TAG-72 and MUC-1 are the natural ligands for carbohydrate recognition domains (CRDs) of endocytic mannose receptor (MR or CD206) and DC-specific ICAM non-integrin (DC-SIGN or CD209) expressed on decidual CD14(+) macrophages and CD1a(+) DCs. They might be able to condition antigen-presenting cells to produce distinct profiles of cyto/chemokines with consequential reduction in NK-cell numbers and cytotoxic potential leading to insufficient control over trophoblast growth. This hypothesis could explain the disappearance of MUC-1 beneath the attached embryo during the process of successful implantation when tight regulation of trophoblast invasion is needed. As IL-15 is the earliest and the most important factor in NK-cell proliferation, differentiation, and maturation, we expected primarily an increase of IL-15 expression in antigen-presenting cells concomitant with the disappearance of mucins and the enhancement in NK cells numbers and of cytotoxic potential after their close contact with early pregnancy decidual antigen-presenting cells. If our hypothesis is correct, it would contribute to the understanding of the role of mucins in the redirection of immune response

  12. Proteome Analysis of Distinct Developmental Stages of Human Natural Killer (NK) Cells

    PubMed Central

    Scheiter, Maxi; Lau, Ulrike; van Ham, Marco; Bulitta, Björn; Gröbe, Lothar; Garritsen, Henk; Klawonn, Frank; König, Sebastian; Jänsch, Lothar

    2013-01-01

    The recent Natural Killer (NK) cell maturation model postulates that CD34+ hematopoietic stem cells (HSC) first develop into CD56bright NK cells, then into CD56dimCD57− and finally into terminally maturated CD56dimCD57+. The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56bright NK cells versus CD56dim NK cells and CD56dimCD57− NK cells versus CD56dimCD57+ NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa. PMID:23315794

  13. The characteristics of NK cells in Schistosoma japonicum-infected mouse spleens.

    PubMed

    Li, Lu; Cha, Hefei; Yu, Xiuxue; Xie, Hongyan; Wu, Changyou; Dong, Nuo; Huang, Jun

    2015-12-01

    Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious disease. Recently, some new characteristics of NK cells were discovered. In this study, C57BL/6 mice were infected with Schistosoma japonicum for 5-6 weeks and lymphocytes were isolated from the spleen to detect some of the NK cell characteristics by multiparametric flow cytometry. The results revealed that the S. japonicum infection induced a large amount of NK cells, although the percentage of NK cells was not increased significantly. At the same time, the results showed that infected mouse splenic NK cells expressed increased levels of CD25 and CD69 and produced more IL-2, IL-4, and IL-17 and less IFN-γ after stimulation with PMA and ionomycin. This meant that NK cells played a role in S. japonicum infection. Moreover, decreased NKG2A/C/E (CD94) expression levels were detected on the surface of NK cells from infected mouse spleens, which might serve as a NK cell activation mechanism. Additionally, high levels of IL-10, but not PD-1, were expressed on the infected mouse NK cells, which implied that functional exhaustion might exist in the splenic NK cells from S. japonicum-infected mice. Collectively, our results suggest that NK cells play important roles in the course of S. japonicum infection.

  14. Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

    PubMed Central

    Sun, Jintang; Yang, Meixiang; Ban, Yanli; Gao, Wenjuan; Song, Bingfeng; Wang, Yang; Zhang, Yun; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2016-01-01

    NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3+ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI. PMID:26789128

  15. Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.

    PubMed

    Sun, Jintang; Yang, Meixiang; Ban, Yanli; Gao, Wenjuan; Song, Bingfeng; Wang, Yang; Zhang, Yun; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2016-01-01

    NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3(+) dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.

  16. Adaptive Memory of Human NK-like CD8+ T-Cells to Aging, and Viral and Tumor Antigens

    PubMed Central

    Pita-López, María Luisa; Pera, Alejandra; Solana, Rafael

    2016-01-01

    Human natural killer (NK)-like CD8+ T-cells are singular T-cells that express both T and NK cell markers such as CD56; their frequencies depend on their differentiation and activation during their lifetime. There is evidence of the presence of these innate CD8+ T-cells in the human umbilical cord, highlighting the necessity of investigating whether the NK-like CD8+ T-cells arise in the early stages of life (gestation). Based on the presence of cell surface markers, these cells have also been referred to as CD8+KIR+ T-cells, innate CD8+ T-cells, CD8+CD28−KIR+ T-cells or NKT-like CD8+CD56+ cells. However, the functional and co-signaling significance of these NK cell receptors on NK-like CD8+ T-cells is less clear. Also, the diverse array of costimulatory and co-inhibitory receptors are spatially and temporally regulated and may have distinct overlapping functions on NK-like CD8+ T-cell priming, activation, differentiation, and memory responses associated with different cell phenotypes. Currently, there is no consensus regarding the functional properties and phenotypic characterization of human NK-like CD8+ T-cells. Environmental factors, such as aging, autoimmunity, inflammation, viral antigen re-exposure, or the presence of persistent tumor antigens have been shown to allow differentiation (“adaptation”) of the NK-like CD8+ T-cells; the elucidation of this differentiation process and a greater understanding of the characteristics of these cells could be important for their eventual in potential therapeutic applications aimed at improving protective immunity. This review will attempt to elucidate an understanding of the characteristics of these cells with the goal toward their eventual use in potential therapeutic applications aimed at improving protective immunity. PMID:28066426

  17. Adaptive Memory of Human NK-like CD8(+) T-Cells to Aging, and Viral and Tumor Antigens.

    PubMed

    Pita-López, María Luisa; Pera, Alejandra; Solana, Rafael

    2016-01-01

    Human natural killer (NK)-like CD8(+) T-cells are singular T-cells that express both T and NK cell markers such as CD56; their frequencies depend on their differentiation and activation during their lifetime. There is evidence of the presence of these innate CD8(+) T-cells in the human umbilical cord, highlighting the necessity of investigating whether the NK-like CD8(+) T-cells arise in the early stages of life (gestation). Based on the presence of cell surface markers, these cells have also been referred to as CD8(+)KIR(+) T-cells, innate CD8(+) T-cells, CD8(+)CD28(-)KIR(+) T-cells or NKT-like CD8(+)CD56(+) cells. However, the functional and co-signaling significance of these NK cell receptors on NK-like CD8(+) T-cells is less clear. Also, the diverse array of costimulatory and co-inhibitory receptors are spatially and temporally regulated and may have distinct overlapping functions on NK-like CD8(+) T-cell priming, activation, differentiation, and memory responses associated with different cell phenotypes. Currently, there is no consensus regarding the functional properties and phenotypic characterization of human NK-like CD8(+) T-cells. Environmental factors, such as aging, autoimmunity, inflammation, viral antigen re-exposure, or the presence of persistent tumor antigens have been shown to allow differentiation ("adaptation") of the NK-like CD8(+) T-cells; the elucidation of this differentiation process and a greater understanding of the characteristics of these cells could be important for their eventual in potential therapeutic applications aimed at improving protective immunity. This review will attempt to elucidate an understanding of the characteristics of these cells with the goal toward their eventual use in potential therapeutic applications aimed at improving protective immunity.

  18. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

    PubMed Central

    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

    2016-01-01

    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. PMID:26941401

  19. Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

    PubMed

    Killig, Monica; Friedrichs, Birte; Meisig, Johannes; Gentilini, Chiara; Blüthgen, Nils; Loddenkemper, Christoph; Labopin, Myriam; Basara, Nadezda; Pfrepper, Christian; Niederwieser, Dietger W; Uharek, Lutz; Romagnani, Chiara

    2014-09-01

    Haploidentical stem cell transplantation (haploSCT) offers an alternative treatment option for advanced leukemia patients lacking a HLA-compatible donor. Transfer of NK cells represents a promising therapeutic option in combination with SCT, as NK cells can promote graft versus leukemia with low risk of GVH disease. In this study, we show results from a phase I/II trial in which 24 acute myeloid leukemia patients underwent haploSCT in combination with early transfer of unmodified NK cells and observed a promising 2-year overall survival rate of 37%. By performing immunomonitoring and subsequent principal component analysis, we tracked donor NK-cell dynamics in the patients and distinguished between NK cells reconstituting from CD34(+) precursors, giving rise over time to a continuum of multiple differentiation stages, and adoptively transferred NK cells. Transferred NK cells displayed a mature phenotype and proliferated in vivo during the early days after haploSCT even in the absence of exogenous IL-2 administration. Moreover, we identified the NK-cell phenotype associated with in vivo expansion. Thus, our study indicates a promising path for adoptive transfer of unmodified NK cells in the treatment of high-risk acute myeloid leukemia.

  20. Mosquito salivary gland extracts induce EBV-infected NK cell oncogenesis via CD4 T cells in patients with hypersensitivity to mosquito bites.

    PubMed

    Asada, Hideo; Saito-Katsuragi, Mamiko; Niizeki, Hironori; Yoshioka, Akira; Suguri, Setsuo; Isonokami, Masaaki; Aoki, Toshiyuki; Ishihara, Shigehiko; Tokura, Yoshiki; Iwatsuki, Keiji; Miyagawa, Sachiko

    2005-11-01

    Severe hypersensitivity to mosquito bites (HMB) is characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. Patients with HMB often have natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection. Here we investigated whether mosquito bites have any influence on the oncogenesis of EBV-infected NK cells. We examined six HMB patients with EBV-infected NK cell lymphocytosis. We first demonstrated that CD4+ T cells, but not NK cells, proliferated well in response to mosquito salivary gland extracts (SGE), especially to SGE of Aedes albopictus. When NK cells were cocultured with autologous CD4+ T cells stimulated by mosquito SGE, the expression of viral oncogene latent membrane protein 1 (LMP1) was remarkably enhanced. Next, we stimulated mononuclear cells of the patients with mosquito SGE, and NK cell counts were monitored for 28 d. The counts changed little from initial levels in the culture with mosquito SGE, whereas they decreased steadily in the culture without the extracts. Furthermore, we detected LMP1 mRNA in the skin lesion induced by mosquito SGE. These results suggest that mosquito bites can induce expression of the viral oncogene LMP1 in NK cells via mosquito antigen-specific CD4+ T cells, which is involved in the oncogenesis of NK cells in vivo.

  1. Expression and Functional Role of α7 Nicotinic Receptor in Human Cytokine-stimulated Natural Killer (NK) Cells.

    PubMed

    Zanetti, Samanta R; Ziblat, Andrea; Torres, Nicolás I; Zwirner, Norberto W; Bouzat, Cecilia

    2016-08-05

    The homomeric α7 nicotinic receptor (nAChR) is one of the most abundant nAChRs in the central nervous system where it contributes to cognition, attention, and working memory. α7 nAChR is also present in lymphocytes, dendritic cells (DCs), and macrophages and it is emerging as an important drug target for intervention in inflammation and sepsis. Natural killer (NK) cells display cytotoxic activity against susceptible target cells and modulate innate and adaptive immune responses through their interaction with DCs. We here show that human NK cells also express α7 nAChR. α7 nAChR mRNA is detected by RT-PCR and cell surface expression of α7 nAChR is detected by confocal microscopy and flow cytometry using α-bungarotoxin, a specific antagonist. Both mRNA and protein levels increase during NK stimulation with cytokines (IL-12, IL-18, and IL-15). Exposure of cytokine-stimulated NK cells to PNU-282987, a specific α7 nAChR agonist, increases intracellular calcium concentration ([Ca(2+)]i) mainly released from intracellular stores, indicating that α7 nAChR is functional. Moreover, its activation by PNU-282987 plus a specific positive allosteric modulator greatly enhances the Ca(2+) responses in NK cells. Stimulation of NK cells with cytokines and PNU-282987 decreases NF-κB levels and nuclear mobilization, down-regulates NKG2D receptors, and decreases NKG2D-dependent cell-mediated cytotoxicity and IFN-γ production. Also, such NK cells are less efficient to trigger DC maturation. Thus, our results demonstrate the anti-inflammatory role of α7 nAChR in NK cells and suggest that modulation of its activity in these cells may constitute a novel target for regulation of the immune response.

  2. The anti-tumor role of NK cells in vivo pre-activated and re-stimulated by interleukins in acute lymphoblastic leukemia

    PubMed Central

    Jin, Fengyan; Lin, Hai; Gao, Sujun; Hu, Zheng; Zuo, Song; Sun, Liguang; Jin, Chunhui; Li, Wei; Yang, Yanping

    2016-01-01

    Although natural killer cells (NK cells) were traditionally classified as members of the innate immune system, NK cells have recently been found also to be an important player in the adaptive immune systems. In this context, in vitro activation of NK cells by cytokines leads to generation of NK cells with memory-like properties characterized by increased interferon-γ (IFNγ) production. However, it remains to be defined whether these memory-like NK cells exist in vivo after cytokine activation. Furthermore, it is also unclear whether such memory-like NK cells induced in vivo by cytokines could have effective anti-leukemia response. To address these issues, we used an in vivo pre-activation and re-stimulation system that was able to produce NK cells with increased IFNγ secretion. It was found that after in vivo pre-activation and re-stimulation with interleukins (ILs), NK cells retained a state to produce increased amount of IFNγ. Of note, whereas this intrinsic capacity of enhanced IFNγ production after in vivo IL pre-activation and re-stimulation could be transferred to the next generation of NK cells and was associated with prolonged survival of the mice with acute lymphoid leukemia. Moreover, the anti-leukemia activity of these memory-like NK cells was associated with IFNγ production and up-regulation of NK cells activation receptor-NK Group 2 member D (NKG2D). Together, these findings argue strongly that in vivo IL pre-activation and re-stimulation is capable to induce memory-like NK cells as observed previously in vitro, which are effective against acute lymphoblastic leukemia, likely via NKG2D-dependent IFNγ production, in intact animals. PMID:27816971

  3. NK cells in gamma-interferon-deficient mice suppress lung innate immunity against Mycoplasma spp.

    PubMed

    Woolard, Matthew D; Hudig, Dorothy; Tabor, Leslie; Ivey, James A; Simecka, Jerry W

    2005-10-01

    The purpose of this study was to examine the 100-fold difference in mycoplasma levels in lungs of gamma interferon knockout (IFN-gamma(-/-)) mice compared to those seen with wild-type BALB/c mice at 3 days postinfection. NK cells secreted IFN-gamma; however, their cytotoxic granule extracts failed to kill mycoplasma. We found a conundrum: the clearance of organisms was as effective in NK-depleted IFN-gamma(-/-) animals as in wild-type mice (with both IFN-gamma and NK cells). NK(+) IFN-gamma(-/-) animals had high mycoplasma burdens, but, after NK-like cell depletion, mycoplasma numbers were controlled. Essentially, IFN-gamma was important in animals with NK-like cells and unimportant in animals without NK cells, suggesting that IFN-gamma counters deleterious effects of NK-like cells. Impairment of innate immunity in IFN-gamma(-/-) mice was not due to NK-like cell killing of macrophages. The increased levels of inflammatory cytokines and neutrophils in lung fluids of NK(+) IFN-gamma(-/-) mice were reduced after NK cell depletion. In summary, in the murine model that resembles chronic human disease, innate immunity to mycoplasma requires IFN-gamma when there are NK-like cells and the positive effects of IFN-gamma counteract negative effects of NK-like cells. When imbalanced, NK-like cells promote disease. Thus, it was not the lack of IFN-gamma but the presence of a previously unrecognized NK-like cell-suppressive activity that contributed to the higher mycoplasma numbers. It appears that pulmonary NK cells may contribute to the immunosuppressive environment of the lung, but when needed, these dampening effects can be counterbalanced by IFN-gamma. Furthermore, there may be instances where perturbation of this regulatory balance contributes to the susceptibility to and severity of disease.

  4. Suppressive effect of a standardized mistletoe extract on the expression of activatory NK receptors and function of human NK cells.

    PubMed

    Lee, Soo Jung; Son, Young-Ok; Kim, Hyunjin; Kim, Joo-Young; Park, Soon-Won; Bae, Jae-Ho; Kim, Hyung Hoi; Lee, Eun-Yup; Chung, Byung-Seon; Kim, Sun-Hee; Kang, Chi-Dug

    2007-09-01

    Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe.

  5. Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

    PubMed Central

    Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M.; Tinder, Teresa L.; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J.

    2013-01-01

    Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo. PMID:22543528

  6. Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis

    PubMed Central

    Liu, Gang; Lu, Shengjun; Wang, Xuanjun; Page, Stephanie T.; Higano, Celestia S.; Plymate, Stephen R.; Greenberg, Norman M.; Sun, Shaoli; Li, Zihai; Wu, Jennifer D.

    2013-01-01

    The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of “humanized” bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in “humanized” mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell–based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization. PMID:24018560

  7. The Epstein-Barr Virus (EBV) in T Cell and NK Cell Lymphomas: Time for a Reassessment

    PubMed Central

    Gru, A. A.; Haverkos, B. H.; Freud, A. G.; Hastings, J.; Nowacki, N. B.; Barrionuevo, C.; Vigil, C. E.; Rochford, R.; Natkunam, Y.; Baiocchi, R. A.

    2015-01-01

    While Epstein-Barr virus (EBV) was initially discovered and characterized as an oncogenic virus in B cell neoplasms, it also plays a complex and multifaceted role in T/NK cell lymphomas. In B cell lymphomas, EBV-encoded proteins have been shown to directly promote immortalization and proliferation through stimulation of the NF-κB pathway and increased expression of anti-apoptotic genes. In the context of mature T/NK lymphomas (MTNKL), with the possible exception on extranodal NK/T cell lymphoma (ENKTL), the virus likely plays a more diverse and nuanced role. EBV has been shown to shape the tumor microenvironment by promoting Th2-skewed T cell responses and by increasing the expression of the immune checkpoint ligand PD-L1. The type of cell infected, the amount of plasma EBV DNA, and the degree of viral lytic replication have all been proposed to have prognostic value in T/NK cell lymphomas. Latency patterns of EBV infection have been defined using EBV-infected B cell models and have not been definitively established in T/NK cell lymphomas. Identifying the expression profile of EBV lytic proteins could allow for individualized therapy with the use of antiviral medications. More work needs to be done to determine whether EBV-associated MTNKL have distinct biological and clinical features, which can be leveraged for risk stratification, disease monitoring, and therapeutic purposes. PMID:26449716

  8. Plasmid Vector-Linked Maturation of Natural Killer (NK) Cells Is Coupled to Antigen-Dependent NK Cell Activation during DNA-Based Immunization in Mice ▿

    PubMed Central

    Zhu, Ren; Mancini-Bourgine, Maryline; Zhang, Xiao Ming; Bayard, Florence; Deng, Qiang; Michel, Marie-Louise

    2011-01-01

    Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1+ CD27− NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1+ CD27+ NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity. PMID:21775455

  9. Immunological and Translational Aspects of NK Cell-Based Antitumor Immunotherapies

    PubMed Central

    Shevtsov, Maxim; Multhoff, Gabriele

    2016-01-01

    Natural killer (NK) cells play a pivotal role in the first line of defense against cancer. NK cells that are deficient in CD3 and a clonal T cell receptor (TCR) can be subdivided into two major subtypes, CD56dimCD16+ cytotoxic and CD56brightCD16− immunoregulatory NK cells. Cytotoxic NK cells not only directly kill tumor cells without previous stimulation by cytotoxic effector molecules, such as perforin and granzymes or via death receptor interactions, but also act as regulatory cells for the immune system by secreting cytokines and chemokines. The aim of this review is to highlight therapeutic strategies utilizing autologous and allogenic NK cells, combinations of NK cells with monoclonal antibodies to induce antibody-dependent cellular cytotoxicity, or immune checkpoint inhibitors. Additionally, we discuss the use of chimeric antigen receptor-engineered NK cells in cancer immunotherapy. PMID:27891129

  10. Upregulation of thioredoxin-1 in activated human NK cells confers increased tolerance to oxidative stress.

    PubMed

    Mimura, Kousaku; Kua, Ley-Fang; Shimasaki, Noriko; Shiraishi, Kensuke; Nakajima, Shotaro; Siang, Lim Kee; Shabbir, Asim; So, Jimmy; Yong, Wei-Peng; Kono, Koji

    2017-02-21

    Adoptive transfer of immune cells, such as T lymphocytes and NK cells, has potential to control cancer growth. However, this can be counteracted by immune escape mechanisms within the tumor microenvironment, including those mediated by reactive oxygen species (ROS). Here, we determined the levels of anti-oxidant molecules in NK cells and their capacity to overcome ROS-induced immune suppression. We investigated the effect of H2O2 on resting NK cells, IL-2-activated NK cells and NK cells expanded by coculture with the K562 leukemia cell line genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Expression of anti-oxidant and anti-apoptotic genes was evaluated by expression array, and protein levels of anti-oxidant molecules by Western blot. Activated NK cells, IL-2-activated NK cells and NK cells expanded by K562-mb15-41BBL were significantly more resistant to H2O2-induced cell death than resting NK. Thioredoxin-1 (TXN1) and peroxiredoxin-1 (PRDX1) were also up-regulated in activated NK cells. Moreover, H2O2-induced cell death after IL-2 activation was significantly induced in the presence of an anti-TXN1-neutralising antibody. Collectively, these data document that activated NK cells can resist to H2O2-induced cell death by up-regulation of TXN1.

  11. Effects of prolactin and cortisol on natural killer (NK) cell surface expression and function of human natural cytotoxicity receptors (NKp46, NKp44 and NKp30).

    PubMed

    Mavoungou, E; Bouyou-Akotet, M K; Kremsner, P G

    2005-02-01

    The surface density of the triggering receptors (e.g. NKp46 and NKp30) responsible for natural killer (NK) cell-mediated cytotoxicity determines the ability of NK cells to kill susceptible target cells. In this study, we show that prolactin up-regulates and cortisol down-regulates the surface expression of NKp46 and NKp30. The prolactin-mediated activation and the cortisol-mediated inhibition of natural cytotoxicity receptor (NCR) surface expression reflects gene regulation at the transcriptional level. NKp46 and NKp30 are the major receptors involved in the NK-mediated killing of K562, a human chronic myelogenous leukaemia cell line. Accordingly, the prolactin dramatically increased the NK-mediated killing of the K562 cell line, whereas cortisol abolished this activity. Our data suggest a mechanism by which prolactin activates the lytic function of NK cells, and cortisol inhibits the NK-mediated attack.

  12. Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

    NASA Astrophysics Data System (ADS)

    Orange, Jordan S.; Ramesh, Narayanaswamy; Remold-O'Donnell, Eileen; Sasahara, Yoji; Koopman, Louise; Byrne, Michael; Bonilla, Francisco A.; Rosen, Fred S.; Geha, Raif S.; Strominger, Jack L.

    2002-08-01

    The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

  13. Phenotypic and Functional Dysregulated Blood NK Cells in Colorectal Cancer Patients Can Be Activated by Cetuximab Plus IL-2 or IL-15

    PubMed Central

    Rocca, Yamila Sol; Roberti, María Paula; Juliá, Estefanía Paula; Pampena, María Betina; Bruno, Luisina; Rivero, Sergio; Huertas, Eduardo; Sánchez Loria, Fernando; Pairola, Alejandro; Caignard, Anne; Mordoh, José; Levy, Estrella Mariel

    2016-01-01

    The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells’ antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity. PMID:27777574

  14. Natural Killers Are Made Not Born: How to Exploit NK Cells in Lung Malignancies

    PubMed Central

    Carrega, Paolo; Ferlazzo, Guido

    2017-01-01

    In recent years, progress has been made in the characterization of natural killer (NK) cells in lung malignancies, and we have now gained a better understanding of the frequency, localization, phenotype, and functional status of NK cells infiltrating these tumors. NK cell subset recruited in lung cancer is mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing. Thus, the relevance of NK cells in tumor microenvironment might also go beyond the killing of tumor cells, being NK cells endowed with regulatory functions toward an ample array of immune effectors. Nevertheless, boosting their cytotoxic functions and redirecting the migration of cytotoxic NK cell subset to the tumor site might open new therapeutic avenues for lung cancer. Also, we believe that a deeper investigation into the impact of both conventional (e.g., chemotherapy) or new therapies (e.g., anti-immune checkpoints mAbs) on NK cell homeostasis in lung cancer patients is now required. PMID:28348567

  15. The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

    PubMed

    Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

    2014-03-01

    Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation.

  16. Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1-dependent NK cell-mediated lysis of infected cells.

    PubMed

    Grauwet, Korneel; Cantoni, Claudia; Parodi, Monica; De Maria, Andrea; Devriendt, Bert; Pende, Daniela; Moretta, Lorenzo; Vitale, Massimo; Favoreel, Herman W

    2014-11-11

    Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2-infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.

  17. Purification of human NK cell developmental intermediates from lymph nodes and tonsils.

    PubMed

    Freud, Aharon G; Caligiuri, Michael A

    2010-01-01

    Accumulating data indicate that human natural killer (NK) cells undergo terminal maturation in secondary lymphoid tissues (SLTs) including lymph nodes (LNs) and tonsils. In addition, recent studies have revealed that maturing NK cells progress through at least five functionally discrete stages of development within SLTs. These discoveries provide unique possibilities for researchers to investigate the natural processes governing human NK cell development, as they exist in vivo, through analysis of NK cell maturational intermediates found in situ. Herein we describe a detailed, yet simple, four-step protocol for the viable enrichment and purification of human NK cell developmental intermediates from LNs and tonsils.

  18. NK Cells Restrain Spontaneous Antitumor CD8+ T Cell Priming through PD-1/PD-L1 Interactions with Dendritic Cells.

    PubMed

    Iraolagoitia, Ximena L Raffo; Spallanzani, Raul G; Torres, Nicolás I; Araya, Romina E; Ziblat, Andrea; Domaica, Carolina I; Sierra, Jessica M; Nuñez, Sol Y; Secchiari, Florencia; Gajewski, Thomas F; Zwirner, Norberto W; Fuertes, Mercedes B

    2016-08-01

    Despite the classical function of NK cells in the elimination of tumor and of virus-infected cells, evidence for a regulatory role for NK cells has been emerging in different models of autoimmunity, transplantation, and viral infections. However, this role has not been fully explored in the context of a growing tumor. In this article, we show that NK cells can limit spontaneous cross-priming of tumor Ag-specific CD8(+) T cells, leading to reduced memory responses. After challenge with MC57 cells transduced to express the model Ag SIY (MC57.SIY), NK cell-depleted mice exhibited a significantly higher frequency of SIY-specific CD8(+) T cells, with enhanced IFN-γ production and cytotoxic capability. Depletion of NK cells resulted in a CD8(+) T cell population skewed toward an effector memory T phenotype that was associated with enhanced recall responses and delayed tumor growth after a secondary tumor challenge with B16.SIY cells. Dendritic cells (DCs) from NK cell-depleted tumor-bearing mice exhibited a more mature phenotype. Interestingly, tumor-infiltrating and tumor-draining lymph node NK cells displayed an upregulated expression of the inhibitory molecule programmed death ligand 1 that, through interaction with programmed death-1 expressed on DCs, limited DC activation, explaining their reduced ability to induce tumor-specific CD8(+) T cell priming. Our results suggest that NK cells can, in certain contexts, have an inhibitory effect on antitumor immunity, a finding with implications for immunotherapy in the clinic.

  19. The Correlation between NK Cell and Liver Function in Patients with Primary Hepatocellular Carcinoma

    PubMed Central

    Zeng, Xiao-Hui; Min, Lu

    2014-01-01

    Background/Aims This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. Methods The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. Results When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D+ NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. Conclusions The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells. PMID:24827627

  20. Biallelic mutations in IRF8 impair human NK cell maturation and function

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Chinn, Ivan K.; Angelo, Laura S.; Maisuria, Sheetal; Keller, Michael D.; Togi, Sumihito; Watkin, Levi B.; LaRosa, David F.; Jhangiani, Shalini N.; Muzny, Donna M.; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B.; Hernández-Sanabria, Mayra; Le, Duy T.; Hogg, Graham D.; Cao, Tram N.; Freud, Aharon G.; Szymanski, Eva P.; Collin, Matthew; Cant, Andrew J.; Gibbs, Richard A.; Holland, Steven M.; Caligiuri, Michael A.; Ozato, Keiko; Paust, Silke; Doody, Gina M.; Lupski, James R.; Orange, Jordan S.

    2016-01-01

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8–/–, but not Irf8+/–, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense. PMID:27893462

  1. Biallelic mutations in IRF8 impair human NK cell maturation and function.

    PubMed

    Mace, Emily M; Bigley, Venetia; Gunesch, Justin T; Chinn, Ivan K; Angelo, Laura S; Care, Matthew A; Maisuria, Sheetal; Keller, Michael D; Togi, Sumihito; Watkin, Levi B; LaRosa, David F; Jhangiani, Shalini N; Muzny, Donna M; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B; Hernández-Sanabria, Mayra; Le, Duy T; Hogg, Graham D; Cao, Tram N; Freud, Aharon G; Szymanski, Eva P; Savic, Sinisa; Collin, Matthew; Cant, Andrew J; Gibbs, Richard A; Holland, Steven M; Caligiuri, Michael A; Ozato, Keiko; Paust, Silke; Doody, Gina M; Lupski, James R; Orange, Jordan S

    2017-01-03

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

  2. The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu).

    PubMed

    Maruyama, Hiroko; Tamauchi, Hidekazu; Iizuka, Mariko; Nakano, Takahisa

    2006-12-01

    Fucoidan from Mekabu (sporophyll of Undaria pinnatifida), a dietary alga, exerts antitumor activity possibly through enhancing the immune response. The present report describes the effects of dietary Mekabu fucoidan on the tumor growth of mouse A20 leukemia cells and on T cell-mediated immune responses in T cell receptor transgenic (DO-11 - 10 - Tg) mice. The animals were fed with a diet containing 1% Mekabu fucoidan (0.034 +/- 0.003 g/mouse/day) for 10 days and subcutaneously (s. c.) inoculated with A20 leukemia cells. Thereafter, the mice were fed with the diet containing fucoidan for 40 days. Mekabu fucoidan inhibited tumors by 65.4 %. We studied how the killer activities of T cell-mediated and natural killer (NK) cells are augmented in DO-11 - 10 mice fed with Mekabu fucoidan. The cytolytic activities of ovalbumin (OVA), which is specific against OVA-transfected A20 (OVA-A20) B lymphoma cells, and NK cells against YAC-1 were significantly enhanced in the mice fed with fucoidan compared with a basic diet. Thus, these findings suggested that Mekabu fucoidan mediates tumor destruction through Th1 cell and NK cell responses.

  3. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition.

    PubMed

    Alari-Pahissa, Elisenda; Grandclément, Camille; Jeevan-Raj, Beena; Leclercq, Georges; Veillette, André; Held, Werner

    2016-01-01

    Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells' ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated "missing-self" reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.

  4. Viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC class I-independent mechanism.

    PubMed

    Mazumdar, Budhaditya; Bolanos, Fred D; Tripathy, Sandeep K

    2013-05-01

    Continuous engagement of the Ly49H activating receptor with its ligand (m157) in a transgenic mouse expressing m157 (m157-Tg) results in hyporesponsiveness of Ly49H(+) NK cells. The same interaction, during murine cytomegalovirus (MCMV) infection, leads to activation of Ly49H(+) NK cells. MCMV infection results in decreased MHC class I (MHC-I) expression on the infected cell as well as inflammatory responses, both of which do not take place in the uninfected m157-Tg mouse, potentially allowing for activation of NK cells in the context of MCMV infection. In this study, we demonstrated that viral infection transiently reverses activation receptor-mediated NK cell hyporesponsiveness in an MHC-I-independent mechanism. Furthermore, Ly49H(+) NK cells in an MHC-I-deficient environment remained hyporesponsive in the context of m157 expression, even when mature WT splenocytes were transferred into m157-Tg mice in an MHC-I-deficient environment. However, the administration of cytokines TNF-α, IL-12, and IFN-β resulted in a partial recovery from activation receptor-induced hyporesponsiveness. Thus, the release of the aforementioned cytokines during MCMV infection and not the downregulation of MHC-I expression appears to be responsible for partial resolution of Ly49H receptor-induced NK cell hyporesponsiveness.

  5. Voluntary Running Suppresses Tumor Growth through Epinephrine- and IL-6-Dependent NK Cell Mobilization and Redistribution.

    PubMed

    Pedersen, Line; Idorn, Manja; Olofsson, Gitte H; Lauenborg, Britt; Nookaew, Intawat; Hansen, Rasmus Hvass; Johannesen, Helle Hjorth; Becker, Jürgen C; Pedersen, Katrine S; Dethlefsen, Christine; Nielsen, Jens; Gehl, Julie; Pedersen, Bente K; Thor Straten, Per; Hojman, Pernille

    2016-03-08

    Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed over 60% reduction in tumor incidence and growth across five different tumor models. Microarray analysis revealed training-induced upregulation of pathways associated with immune function. NK cell infiltration was significantly increased in tumors from running mice, whereas depletion of NK cells enhanced tumor growth and blunted the beneficial effects of exercise. Mechanistic analyses showed that NK cells were mobilized by epinephrine, and blockade of β-adrenergic signaling blunted training-dependent tumor inhibition. Moreover, epinephrine induced a selective mobilization of IL-6-sensitive NK cells, and IL-6-blocking antibodies blunted training-induced tumor suppression, intratumoral NK cell infiltration, and NK cell activation. Together, these results link exercise, epinephrine, and IL-6 to NK cell mobilization and redistribution, and ultimately to control of tumor growth.

  6. Dynamic contrast enhanced MRI detects early response to adoptive NK cellular immunotherapy targeting the NG2 proteoglycan in a rat model of glioblastoma.

    PubMed

    Rygh, Cecilie Brekke; Wang, Jian; Thuen, Marte; Gras Navarro, Andrea; Huuse, Else Marie; Thorsen, Frits; Poli, Aurelie; Zimmer, Jacques; Haraldseth, Olav; Lie, Stein Atle; Enger, Per Øyvind; Chekenya, Martha

    2014-01-01

    There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In

  7. Dynamic Contrast Enhanced MRI Detects Early Response to Adoptive NK Cellular Immunotherapy Targeting the NG2 Proteoglycan in a Rat Model of Glioblastoma

    PubMed Central

    Thuen, Marte; Gras Navarro, Andrea; Huuse, Else Marie; Thorsen, Frits; Poli, Aurelie; Zimmer, Jacques; Haraldseth, Olav; Lie, Stein Atle; Enger, Per Øyvind; Chekenya, Martha

    2014-01-01

    There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In

  8. Hepatitis C Virus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γ Production by Altering Cellular Metabolism via Arginase-1.

    PubMed

    Goh, Celeste C; Roggerson, Krystal M; Lee, Hai-Chon; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

    2016-03-01

    The hepatitis C virus (HCV) infects ∼ 200 million people worldwide. The majority of infected individuals develop persistent infection, resulting in chronic inflammation and liver disease, including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms, including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular, IFN-γ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However, NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study, we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-γ, with no effect on granzyme B production or cell viability. Importantly, this suppression of NK cell-derived IFN-γ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-γ production was reversed by l-arginine supplementation, consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses, further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver.

  9. MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells

    PubMed Central

    2010-01-01

    Background Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition. Results Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16low targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors. Conclusion MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal

  10. Role of NK, NKT cells and macrophages in liver transplantation

    PubMed Central

    Fahrner, René; Dondorf, Felix; Ardelt, Michael; Settmacher, Utz; Rauchfuss, Falk

    2016-01-01

    Liver transplantation has become the treatment of choice for acute or chronic liver disease. Because the liver acts as an innate immunity-dominant organ, there are immunological differences between the liver and other organs. The specific features of hepatic natural killer (NK), NKT and Kupffer cells and their role in the mechanism of liver transplant rejection, tolerance and hepatic ischemia-reperfusion injury are discussed in this review. PMID:27468206

  11. NK cell-based cancer immunotherapy: from basic biology to clinical application.

    PubMed

    Li, Yang; Yin, Jie; Li, Ting; Huang, Shan; Yan, Han; Leavenworth, JianMei; Wang, Xi

    2015-12-01

    Natural killer (NK) cells, which recognize and kill target cells independent of antigen specificity and major histocompatibility complex (MHC) matching, play pivotal roles in immune defence against tumors. However, tumor cells often acquire the ability to escape NK cell-mediated immune surveillance. Thus, understanding mechanisms underlying regulation of NK cell phenotype and function within the tumor environment is instrumental for designing new approaches to improve the current cell-based immunotherapy. In this review, we elaborate the main biological features and molecular mechanisms of NK cells that pertain to regulation of NK cell-mediated anti-tumor activity. We further overview current clinical approaches regarding NK cell-based cancer therapy, including cytokine infusion, adoptive transfer of autologous or allogeneic NK cells, applications of chimeric antigen receptor (CAR)-expressing NK cells and adoptive transfer of memory-like NK cells. With these promising clinical outcomes and fuller understanding the basic questions raised in this review, we foresee that NK cell-based approaches may hold great potential for future cancer immunotherapy.

  12. Synergy between Common γ Chain Family Cytokines and IL-18 Potentiates Innate and Adaptive Pathways of NK Cell Activation.

    PubMed

    Nielsen, Carolyn M; Wolf, Asia-Sophia; Goodier, Martin R; Riley, Eleanor M

    2016-01-01

    Studies to develop cell-based therapies for cancer and other diseases have consistently shown that purified human natural killer (NK) cells secrete cytokines and kill target cells after in vitro culture with high concentrations of cytokines. However, these assays poorly reflect the conditions that are likely to prevail in vivo in the early stages of an infection and have been carried out in a wide variety of experimental systems, which has led to contradictions within the literature. We have conducted a detailed kinetic and dose-response analysis of human NK cell responses to low concentrations of IL-12, IL-15, IL-18, IL-21, and IFN-α, alone and in combination, and their potential to synergize with IL-2. We find that very low concentrations of both innate and adaptive common γ chain cytokines synergize with equally low concentrations of IL-18 to drive rapid and potent NK cell CD25 and IFN-γ expression; IL-18 and IL-2 reciprocally sustain CD25 and IL-18Rα expression in a positive feedback loop; and IL-18 synergizes with FcγRIII (CD16) signaling to augment antibody-dependent cellular cytotoxicity. These data indicate that NK cells can be rapidly activated by very low doses of innate cytokines and that the common γ chain cytokines have overlapping but distinct functions in combination with IL-18. Importantly, synergy between multiple signaling pathways leading to rapid NK cell activation at very low cytokine concentrations has been overlooked in prior studies focusing on single cytokines or simple combinations. Moreover, although the precise common γ chain cytokines available during primary and secondary infections may differ, their synergy with both IL-18 and antigen-antibody immune complexes underscores their contribution to NK cell activation during innate and adaptive responses. IL-18 signaling potentiates NK cell effector function during innate and adaptive immune responses by synergy with IL-2, IL-15, and IL-21 and immune complexes.

  13. Myeloma cells resistance to NK cell lysis mainly involves an HLA class I-dependent mechanism.

    PubMed

    Gao, Minjie; Gao, Lu; Yang, Guang; Tao, Yi; Hou, Jun; Xu, Hongwei; Hu, Xiaojing; Han, Ying; Zhang, Qianqiao; Zhan, Fenghuang; Wu, Xiaosong; Shi, Jumei

    2014-07-01

    The anti-multiple myeloma (MM) potential of natural killer (NK) cells has been of rising interest in recent years. However, the molecular mechanism of NK cell cytotoxicity to myeloma cells remains unclear. In the present study, we investigated the expressions of human leukocyte antigen (HLA) class I and HLA-G in patient myeloma cells, and determined their relevance in patient tumor-cell susceptibility to NK cell cytotoxicity. Our results showed that patient myeloma cells (n = 12) were relatively resistant to NK-92 cell lysis, compared with myeloma cell lines (n = 7, P < 0.01). Gene expression profiling and flow cytometry analysis showed that both mRNA and protein of HLA class I were highly expressed in 12 patient myeloma cells. Interestingly, no or low HLA-G surface expression was detected, although multiple HLA-G transcripts were detected in these myeloma cells. NK cell function assay showed that down-regulating HLA class I expression on patient cells by acid treatment significantly increased the susceptibility of MM cells to NK-mediated lysis. Furthermore, we found that the blocking of membrane-bound HLA class I rather than HLA-G using antibodies on myeloma samples markedly increased their susceptibility to NK-mediated killing. These results demonstrated that the resistance of patient MM cells to NK lysis mainly involves an HLA class I-dependent mechanism, suggesting that HLA class I may be involved in protecting MM cells from NK-mediated attack and contribute to their immune escape in vivo.

  14. Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein

    PubMed Central

    Germain, Claire; Campigna, Emmanuelle; Salhi, Imed; Morisseau, Sébastien; Navarro-Teulon, Isabelle; Mach, Jean-Pierre; Pèlegrin, André; Robert, Bruno

    2008-01-01

    Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectinlike receptor NKG2D. To redirect NK cells against tumor cells we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the “knob into hole” heterodimerization strategy, in which “knob” and “hole” variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site. PMID:18790793

  15. Genetic deletion of Cxcl14 in mice alters uterine NK cells

    SciTech Connect

    Cao, Qichen; Chen, Hua; Deng, Zhili; Yue, Jingwen; Chen, Qi; Cao, Yujing; Ning, Lina; Lei, Xiaohua; Duan, Enkui

    2013-06-14

    Highlights: •We first examined the expression of Cxcl14 in MLAp and DB of uterus. •We found the uNK cells in MLAp and decidua express Cxcl14. •In Cxcl14{sup −/−} placenta, we found significantly decreased uNK cells. •We first performed microarray to compare the gene expression in MLAp and DB. -- Abstract: The uterine natural killer cells (uNK cells) are the major immune cells in pregnant uterus and the number of uNK cells is dramatically increased during placentation and embryo development. The uNK cells are necessary for the immune tolerance, cytokine secretion and angiogenesis of placenta. Former studies indicated that the population expansion of uNK cells was accomplished through recruitment of NK cell precursors from the spleen and bone marrow, but not proliferation of NK cells. However, the necessary molecules within this process were little understood. Here in our study, we found the co-localized expression of Cxcl14 protein with uNK cells in E13.5 pregnant uterus. Moreover, we used Cxcl14 knockout mice to examine uNK cells in mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) of E13.5 pregnant uterus and found significantly decreased uNK cells in Cxcl14{sup −/−} pregnant uteri compared with Cxcl14{sup +/−} pregnant uteri. To further explorer the molecular change in MLAp and DB after Cxcl14 knockout, we isolated the MLAp and DB from Cxcl14{sup +/+} and Cxcl14{sup −/−} pregnant uteri and performed microarray analysis. We found many genes were up and down regulated after Cxcl14 knockout. In conclusion, our results suggested the important function of Cxcl14 in uNK cells and the proper level of Cxcl14 protein were required to recruit NK cells to pregnant uterus.

  16. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma.

    PubMed

    Xu, Liyun; Huang, Yanyan; Tan, Linlin; Yu, Wei; Chen, Dongdong; Lu, ChangChang; He, Jianying; Wu, Guoqing; Liu, Xiaoguang; Zhang, Yongkui

    2015-12-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56+ cells (P<0.05) and CD3-CD56(dim) cells (P<0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56+ NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P<0.05) or with tumor stage T3-T4 (P<0.05). Tim-3 expression in CD56(dim) NK-cell subset was higher in patients with tumor size ≥3cm (P<0.05), or LNM (P<0.05) or with tumor stage T3-T4 (P<0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56+ NK cells and CD56(dim) NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P=0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.

  17. Prenatal exposure to cypermethrin modulates rat NK cell cytotoxic functions.

    PubMed

    Santoni, G; Cantalamessa, F; Mazzucca, L; Romagnoli, S; Piccoli, M

    1997-07-11

    The synthetic pyrethroid insecticide, cypermethrin, was given during gestation to pregnant rats by gavage in corn oil. Peripheral blood and spleen cytotoxic activity of dams and their offspring were then evaluated at different times (30, 60, 90, 120 days) after birth. Pups showed a significant increase in peripheral blood natural killer (NK) and antibody-dependent (ADCC) cytotoxic activity paralleled with a similar increase in the percentage of NK-RP1+ cells and decreased activity in the spleen. Pregnant cypermethrin-exposed dams showed no changes in peripheral blood or spleen cytotoxic function during the postnatal period. Overall, these results suggest that immunomodulation of cytotoxic activity observed in the offspring is likely attributable to a specific effect of cypermethrin administered during the prenatal period.

  18. MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution.

    PubMed

    Nagarajan, Uma M; Sikes, James; Prantner, Daniel; Andrews, Charles W; Frazer, Lauren; Goodwin, Anna; Snowden, Jessica N; Darville, Toni

    2011-01-01

    We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.

  19. NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

    PubMed

    Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

    2016-01-01

    Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

  20. Prenatal allospecific NK cell tolerance hinges on instructive allorecognition through the activating receptor during development

    PubMed Central

    Alhajjat, Amir M.; Strong, Beverly S.; Lee, Amanda E.; Turner, Lucas E.; Wadhwani, Ram K.; Ortaldo, John R.; Heusel, Jonathan W.; Shaaban, Aimen F.

    2015-01-01

    Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire towards tolerance or immunity. Specifically, the effect on NK cell education arising from developmental co-recognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically “hostile” NK cells that express an allospecific activating receptor without co-expressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual non-deleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell allospecific education. PMID:26136432

  1. A Mature NK Profile at the Time of HIV Primary Infection Is Associated with an Early Response to cART.

    PubMed

    Gondois-Rey, Françoise; Chéret, Antoine; Mallet, Françoise; Bidaut, Ghislain; Granjeaud, Samuel; Lécuroux, Camille; Ploquin, Mickaël; Müller-Trutwin, Michaela; Rouzioux, Christine; Avettand-Fenoël, Véronique; De Maria, Andrea; Pialoux, Gilles; Goujard, Cécile; Meyer, Laurence; Olive, Daniel

    2017-01-01

    Natural killer (NK) cells are major effectors of the innate immune response. Despite an overall defect in their function associated with chronic human immunodeficiency virus (HIV) infection, their role in primary HIV infection is poorly understood. We investigated the modifications of the NK cell compartment in patients from the ANRS-147-Optiprim trial, a study designed to examine the benefits of intensive combination antiretroviral therapy (cART) in patients with acute or early primary HIV infection. Multiparametric flow cytometry combined with bioinformatics analyses identified the NK phenotypes in blood samples from 30 primary HIV-infected patients collected at inclusion and after 3 months of cART. NK phenotypes were revealed by co-expression of CD56/CD16/NKG2A/NKG2C and CD57, five markers known to delineate stages of NK maturation. Three groups of patients were formed according to their distributions of the 12 NK cell phenotypes identified. Their virological and immunological characteristics were compared along with the early outcome of cART. At inclusion, HIV-infected individuals could be grouped into those with predominantly immature/early differentiated NK cells and those with predominantly mature NK cells. Several virological and immunological markers were improved in patients with mature NK profiles, including lower HIV viral loads, lower immune activation markers on NK and dendritic cell (DC), lower levels of plasma IL-6 and IP-10, and a trend to normal DC counts. Whereas all patients showed a decrease of viremia higher than 3 log10 copies/ml after 3 months of treatment, patients with a mature NK profile at inclusion reached this threshold more rapidly than patients with an immature NK profile (70 vs. 38%). In conclusion, a better early response to cART is observed in patients whose NK profile is skewed to maturation at inclusion. Whether the mature NK cells contributed directly or indirectly to HIV control through a better immune environment under

  2. A Mature NK Profile at the Time of HIV Primary Infection Is Associated with an Early Response to cART

    PubMed Central

    Gondois-Rey, Françoise; Chéret, Antoine; Mallet, Françoise; Bidaut, Ghislain; Granjeaud, Samuel; Lécuroux, Camille; Ploquin, Mickaël; Müller-Trutwin, Michaela; Rouzioux, Christine; Avettand-Fenoël, Véronique; De Maria, Andrea; Pialoux, Gilles; Goujard, Cécile; Meyer, Laurence; Olive, Daniel

    2017-01-01

    Natural killer (NK) cells are major effectors of the innate immune response. Despite an overall defect in their function associated with chronic human immunodeficiency virus (HIV) infection, their role in primary HIV infection is poorly understood. We investigated the modifications of the NK cell compartment in patients from the ANRS-147-Optiprim trial, a study designed to examine the benefits of intensive combination antiretroviral therapy (cART) in patients with acute or early primary HIV infection. Multiparametric flow cytometry combined with bioinformatics analyses identified the NK phenotypes in blood samples from 30 primary HIV-infected patients collected at inclusion and after 3 months of cART. NK phenotypes were revealed by co-expression of CD56/CD16/NKG2A/NKG2C and CD57, five markers known to delineate stages of NK maturation. Three groups of patients were formed according to their distributions of the 12 NK cell phenotypes identified. Their virological and immunological characteristics were compared along with the early outcome of cART. At inclusion, HIV-infected individuals could be grouped into those with predominantly immature/early differentiated NK cells and those with predominantly mature NK cells. Several virological and immunological markers were improved in patients with mature NK profiles, including lower HIV viral loads, lower immune activation markers on NK and dendritic cell (DC), lower levels of plasma IL-6 and IP-10, and a trend to normal DC counts. Whereas all patients showed a decrease of viremia higher than 3 log10 copies/ml after 3 months of treatment, patients with a mature NK profile at inclusion reached this threshold more rapidly than patients with an immature NK profile (70 vs. 38%). In conclusion, a better early response to cART is observed in patients whose NK profile is skewed to maturation at inclusion. Whether the mature NK cells contributed directly or indirectly to HIV control through a better immune environment under

  3. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

    PubMed

    Morandi, Fabio; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Chiesa, Sabrina; Imperatori, Andrea; Zanellato, Silvia; Mortara, Lorenzo; Gattorno, Marco; Pistoia, Vito; Malavasi, Fabio

    2015-08-01

    Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.

  4. Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry.

    PubMed

    Schönberg, Kathrin; Hejazi, Maryam; Uhrberg, Markus

    2012-01-01

    Natural killer (NK) cells provide a first line of defense against viral infections and prepare the ground for subsequent action of virus-specific T cells in a concerted way. Human NK cells use a sophisticated system of inhibitory and stimulatory receptors of the killer cell immunoglobulin-like receptor (KIR) gene family, which are expressed in a clonally distributed manner. Several studies suggest that KIR play a critical role in NK cell-mediated protection against HCV and HIV infection. As each NK cell expresses an individual set of KIR receptors that enables them to sense differences in HLA class I expression, classical measurement of NK cell function by analysis of target cell killing does not enable one to define and isolate the clinically relevant NK cell effector subsets. Here, we have developed a flow cytometry-based protocol to measure cytolytic activity together with KIR expression at a clonal level. Combined analysis of KIR expression in conjunction with cell surface mobilization of CD107 enables precise enumeration of cytolytic NK cells with defined specificity for HLA class I. Moreover, via inclusion of intracellular perforin or alternatively granzyme B, NK cells with deficient loading of cytotoxic granula can be identified. The present protocol enables identification and isolation of cytotoxic NK cells on a clonal level and enables reliable measurement in healthy as well as in pathological settings such as virus infection and hematological disease.

  5. Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.

    PubMed

    Tognarelli, Sara; Jacobs, Benedikt; Staiger, Nina; Ullrich, Evelyn

    2016-10-30

    Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

  6. Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications

    PubMed Central

    Carlsten, Mattias; Childs, Richard W.

    2015-01-01

    Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression in vivo. Improving NK cell persistence in vivo via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors in vivo, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic. PMID:26113846

  7. NK cell maturation to CD56(dim) subset associated with high levels of NCRs overrides the inhibitory effect of NKG2A and recovers impaired NK cell cytolytic potential after allogeneic hematopoietic stem cell transplantation.

    PubMed

    Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat; Schwarer, Anthony P; Pourfathollah, Ali Akbar

    2016-04-01

    NK cell cytotoxicity against residual leukemic cells is crucial for immune system reconstitution after hematopoietic stem cell transplantation (HSCT). Since immune recovery after transplant still remains a major concern, we studied the counterbalance of NK cell receptors after HSCT and its importance in NK cell functional recovery. We investigated NK cell reconstitution in 27 acute leukemia patients at different time points following HLA-matched allogeneic HSCT compared to those of donors. NK cells were evaluated for their cytotoxicity in a standard (51)Cr-release assay against target cells and also analyzed for their receptors expression using flow cytometry. Early after transplant, we found higher percentage of CD56(bright) NK cells, increased levels of NKG2A and NCRs as well as decreased levels of KIRs expression on NK cells associated with an impaired cytotoxicity of these cells. All the abnormalities were normalized by one year after HSCT when CD56(bright) NK cells gradually differentiated into CD56(dim) subset. Collectively, we confirmed a gradual increase of CD56(dim) NK cells expressing NCRs with the significant decrease in NKG2A expression on NK cells. This finding was also associated with the recovery of NK cell cytotoxicity that suggests an important role for the kinetics of NK cell receptors during cell maturation in HSCT outcome.

  8. 19F-MRI for monitoring human NK cells in vivo

    PubMed Central

    Bouchlaka, Myriam N.; Ludwig, Kai D.; Gordon, Jeremy W.; Kutz, Matthew P.; Bednarz, Bryan P.; Fain, Sean B.; Capitini, Christian M.

    2016-01-01

    ABSTRACT The availability of clinical-grade cytokines and artificial antigen-presenting cells has accelerated interest in using natural killer (NK) cells as adoptive cellular therapy (ACT) for cancer. One of the technological shortcomings of translating therapies from animal models to clinical application is the inability to effectively and non-invasively track these cells after infusion in patients. We have optimized the nonradioactive isotope fluorine-19 (19F) as a means to label and track NK cells in preclinical models using magnetic resonance imaging (MRI). Human NK cells were expanded with interleukin (IL)-2 and labeled in vitro with increasing concentrations of 19F. Doses as low as 2 mg/mL 19F were detected by MRI. NK cell viability was only decreased at 8 mg/mL 19F. No effects on NK cell cytotoxicity against K562 leukemia cells were observed with 2, 4 or 8 mg/mL 19F. Higher doses of 19F, 4 mg/mL and 8 mg/mL, led to an improved 19F signal by MRI with 3 × 1011 19F atoms per NK cell. The 4 mg/mL 19F labeling had no effect on NK cell function via secretion of granzyme B or interferon gamma (IFNγ), compared to NK cells exposed to vehicle alone. 19F-labeled NK cells were detectable immediately by MRI after intratumoral injection in NSG mice and up to day 8. When 19F-labeled NK cells were injected subcutaneously, we observed a loss of signal through time at the site of injection suggesting NK cell migration to distant organs. The 19F perfluorocarbon is a safe and effective reagent for monitoring the persistence and trafficking of NK cell infusions in vivo, and may have potential for developing novel imaging techniques to monitor ACT for cancer. PMID:27467963

  9. HIV-specific antibody immunity mediated through NK cells and monocytes.

    PubMed

    Kramski, Marit; Parsons, Matthew S; Stratov, Ivan; Kent, Stephen J

    2013-07-01

    The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

  10. Experimental study on rat NK cell activity improvement by laser acupoint irradiation

    NASA Astrophysics Data System (ADS)

    Yang, Dongxiao; Chen, Xiufeng; Ruan, Buqing; Yang, Feng

    1998-08-01

    To study the improvement of the natural killer (NK) cell activity by semiconductor laser acupoint irradiation, rats were used in this experiment and were injected immunosuppressant in their abdomen. The immunoassay was made after the surface irradiation and inner irradiation at Baihui point by semiconductor laser. The NK cell activity is an important index of immunologic function. The results showed that the NK cell activity after laser acupoint irradiation was enhanced. This enhancement is relatively important in the clinical therapy of tumor.

  11. Prospective study of percutaneous cryoablation combined with allogenic NK cell immunotherapy for advanced renal cell cancer.

    PubMed

    Lin, Mao; Xu, Kecheng; Liang, Shuzhen; Wang, Xiaohua; Liang, Yinqing; Zhang, Mingjie; Chen, Jibing; Niu, LiZhi

    2017-03-05

    In this study, the clinical efficacy of cryosurgery combined with allogenic NK cell immunotherapy for advanced renal cell cancer was evaluated. From July to December 2016, we enrolled 60 patients who met the enrollment criteria and divided them into two groups: (1) the simple cryoablation group (n=30); and (2) the cryoablation combined with allogenic NK cells group (n=30). The clinical efficacy, quality of life, immune function, and other related indicators were evaluated. Combining allogeneic NK cells with cryoablation had a synergistic effect, not only enhancing the immune function and improving the quality of life of the patients, but also significantly exhibiting good clinical efficacy of the patients. This study is the first clinical trial that has evaluated the safety and efficacy of allogenic NK cells combined with cryosurgery for the treatment of renal cell cancer.

  12. IL-12 Enhances the Antitumor Actions of Trastuzumab via NK Cell IFN-γ Production

    PubMed Central

    Jaime-Ramirez, Alena Cristina; Mundy-Bosse, Bethany L.; Kondadasula, SriVidya; Jones, Natalie B.; Roda, Julie M.; Mani, Aruna; Parihar, Robin; Karpa, Volodymyr; Papenfuss, Tracey L.; LaPerle, Krista M.; Biller, Elizabeth; Lehman, Amy; Chaudhury, Abhik Ray; Jarjoura, David; Burry, Richard W.; Carson, William E.

    2013-01-01

    The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26HER2/neu) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ–deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4+ or CD8+ T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26HER2/neu tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs. PMID:21321106

  13. Cannabidiol-induced lymphopenia does not involve NKT and NK cells.

    PubMed

    Ignatowska-Jankowska, B; Jankowski, M; Glac, W; Swiergel, A H

    2009-10-01

    The major non-psychoactive compound of cannabis plant, cannabidiol, has been reported to be a promising therapeutic agent for many inflammatory, autoimmune and neurodegenerative diseases. In spite of growing interest in therapeutic use of cannabidiol very little is known about its influence on the immune system. Present study aimed to evaluate lymphocyte subsets distribution in peripheral blood after repeated, systemic administration of cannabidiol. Adult male Wistar rats received intraperitoneal injections of vehicle or cannabidiol at dose of 2.5 or 5 mg/kg/day, for 14 consecutive days. Blood samples were collected one hour after the last injection. Three-color immunofluorescent antibody staining procedure (CD3-FITC/CD45RA-PC7/CD161A-APC and CD3-FITC/CD4-PC7/CD8-APC) was used for determination of T, B, NK, NKT, T helper, and T cytotoxic lymphocyte subsets. Total leukocyte number and percentage numbers of leukocyte subpopulations were also assessed. Administration of cannabidiol at dose of 5 mg/kg caused a significant decrease in total leukocyte number and a significant fall in total numbers of T, B, and both T helper and T cytotoxic lymphocyte subsets. This immunosuppressive effect did not affect the total numbers of NK and NKT cells that are responsible for the primary, nonspecific antiviral and antitumor immune response. In contrast, administration of cannabidiol at dose of 2.5 mg/kg increased the total and percentage NKT cells numbers, and the percentage number of NK cells. The results suggest that repeated treatment with cannabidiol inhibits specific immunity by reduction of T, B, T cytotoxic, and T helper cell numbers, and may enhance nonspecific antiviral and antitumor immune response related to NK and NKT cells.

  14. NK Cells: Key to Success of DC-Based Cancer Vaccines?

    PubMed Central

    Smits, Evelien L.J.M.; Berneman, Zwi N.; Van Tendeloo, Viggo F.I.

    2012-01-01

    The cytotoxic and regulatory antitumor functions of natural killer (NK) cells have become attractive targets for immunotherapy. Manipulation of specific NK cell functions and their reciprocal interactions with dendritic cells (DCs) might hold therapeutic promise. In this review, we focus on the engagement of NK cells in DC-based cancer vaccination strategies, providing a comprehensive overview of current in vivo experimental and clinical DC vaccination studies encompassing the monitoring of NK cells. From these studies, it is clear that NK cells play a key regulatory role in the generation of DC-induced antitumor immunity, favoring the concept that targeting both innate and adaptive immune mechanisms may synergistically promote clinical outcome. However, to date, DC vaccination trials are only infrequently accompanied by NK cell monitoring. Here, we discuss different strategies to improve DC vaccine preparations via exploitation of NK cells and provide a summary of relevant NK cell parameters for immune monitoring. We underscore that the design of DC-based cancer vaccines should include the evaluation of their NK cell stimulating potency both in the preclinical phase and in clinical trials. PMID:22907975

  15. Tim-3 pathway affects NK cell impairment in patients with active tuberculosis.

    PubMed

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

    2015-12-01

    Active tuberculosis (TB) patients show impaired NK cell function, and the underlying mechanism remains largely unknown. In this study, we confirmed the decrease in activation, cytokine secretion, and degranulation potential of NK cells in active TB patients. We further investigated whether coinhibitory receptor Tim-3 was involved with impairment of NK cells. Our results revealed that the expression of Tim-3 on NK cells was increased in active TB patients. Tim-3 expression was inversely correlated with IL-12-stimualted IFN-γ production. Moreover, blocking the Tim-3 pathway restored IFN-γ secretion and degranulation of NK cells. Blocking this pathway also increased NK cell cytotoxicity against K562 target cells, and improved the ability of NK cells to control Mtb growth in monocyte-derived macrophages. The Tim-3 expression on NK cells was also observed to be significantly decreased in TB patients post-treatment. In this study, we have identified that Tim-3 is involved with NK cell impairment in TB patients.

  16. Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing.

    PubMed

    Kearney, Conor J; Ramsbottom, Kelly M; Voskoboinik, Ilia; Darcy, Phillip K; Oliaro, Jane

    2016-08-01

    Acute myeloid leukemia (AML) is associated with poor natural killer (NK) cell function through aberrant expression of NK-cell-activating receptors and their ligands on tumor cells. These alterations are thought to promote formation of inhibitory NK-target cell synapses, in which killer cell degranulation is attenuated. Allogeneic stem cell transplantation can be effective in treating AML, through restoration of NK cell lytic activity. Similarly, agents that augment NK-cell-activating signals within the immunological synapse may provide some therapeutic benefit. However, the receptor-ligand interactions that critically dictate NK cell function in AML remain undefined. Here, we demonstrate that CD112/CD155 expression is required for DNAM-1-dependent killing of AML cells. Indeed, the low, or absent, expression of CD112/CD155 on multiple AML cell lines resulted in failure to stimulate optimal NK cell function. Importantly, isolated clones with low CD112/155 expression were resistant to NK cell killing while those expressing abundant levels of CD112/155 were highly susceptible. Attenuated NK cell killing in the absence of CD112/CD155 originated from decreased NK-target cell conjugation. Furthermore, we reveal by time-lapse microscopy, a significant increase in NK cell 'failed killing' in the absence of DNAM-1 ligands. Consequently, NK cells preferentially lysed ligand-expressing cells within heterogeneous populations, driving clonal selection of CD112/CD155-negative blasts upon NK cell attack. Taken together, we identify reduced CD155 expression as a major NK cell escape mechanism in AML and an opportunity for targeted immunotherapy.

  17. Activation of NK cells and disruption of PD-L1/PD-1 axis: two different ways for lenalidomide to block myeloma progression.

    PubMed

    Giuliani, Massimo; Janji, Bassam; Berchem, Guy

    2017-02-09

    Natural Killer (NK) cells play a critical role against tumor cells in hematological malignancies. Their activating receptors are essential in tumor cell killing. In Multiple Myeloma (MM) patients, NK cell differentiation, activation and cytotoxic potential are strongly impaired leading to MM escape from immune surveillance in tissues and bone marrow. Mechanisms used by MM to affect NK cell functions are mediated by the release of soluble factors, the expression of activating and inhibitory NK cell ligands, and the expression of immune check-point inhibitors. Lenalidomide represents an efficient clinical approach in MM treatment to improve patients' survival. Lenalidomide does not only promotes tumor apoptosis, but also stimulates T and NK cells, thereby facilitating NK-mediated tumor recognition and killing. This occurs since Lenalidomide acts on several critical points: stimulates T cell proliferation and cytokine secretion; decreases the expression of the immune check-point inhibitor Programmed Death-1 (PD-1) on both T and NK cells in MM patients; decreases the expression of both PD-1 and PD-L1 on MM cells; promotes MM cell death and abrogates MM/stromal microenvironment cross-talk, a process known to promote the MM cell survival and proliferation. This leads to the inhibition of the negative signal induced by PD-1/PD-L1 axis on NK cells, restoring NK cell cytotoxic functions. Given the importance of an effective immune response to counteract the MM progression and the promising approaches using anti-PD-1/PD-L1 strategies, we will discuss in this review how Lenalidomide could represent an adequate approach to re-establish the recognition against MM by exhausted NK cell.

  18. Prolonged contact with dendritic cells turns lymph node-resident NK cells into anti-tumor effectors.

    PubMed

    Mingozzi, Francesca; Spreafico, Roberto; Gorletta, Tatiana; Cigni, Clara; Di Gioia, Marco; Caccia, Michele; Sironi, Laura; Collini, Maddalena; Soncini, Matias; Rusconi, Michela; von Andrian, Ulrich H; Chirico, Giuseppe; Zanoni, Ivan; Granucci, Francesca

    2016-09-01

    Natural killer (NK) cells are critical players against tumors. The outcome of anti-tumor vaccination protocols depends on the efficiency of NK-cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK-cell activation dynamics is needed. NK-cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti-tumor functions. NK-cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two-photon microscopy. Close DC and NK-cell contacts are essential for the localized delivery of DC-derived IL-18 to NK cells, a strict requirement in NK-cell activation.

  19. Activation of NK cell cytotoxicity by aerosolized CpG-ODN/poly(I:C) against lung melanoma metastases is mediated by alveolar macrophages.

    PubMed

    Sommariva, Michele; Le Noci, Valentino; Storti, Chiara; Bianchi, Francesca; Tagliabue, Elda; Balsari, Andrea; Sfondrini, Lucia

    2017-03-01

    Controversies remain about NK cells direct responsiveness to Toll-like receptor (TLR) agonists or dependence on macrophages. In a melanoma lung metastasis model, aerosolized TLR9 and TLR3 agonists have been reported to induce antitumor immunity through NK cells activation. In the current study, we demonstrated that in vitro TLR9/TLR3 stimulation induced IFN-γ secretion by NK cells, but an increase in their cytotoxicity was detected only after NK cells co-culture with in vitro TLR9/TLR3 agonists pretreated alveolar macrophages. Alveolar macrophages from melanoma lung metastases-bearing mice, treated with aerosolized TLR agonists, also promoted NK cell cytotoxicity. Activated NK cells from lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists were able to polarize naive alveolar macrophages toward a M1-like phenotype. Our results demonstrate that activation of NK cells in the lung after TLR engagement is mediated by alveolar macrophages and that activated NK cells shape macrophage behavior.

  20. Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

    PubMed Central

    Oykhman, Paul; Timm-McCann, Martina; Xiang, Richard F.; Islam, Anowara; Li, Shu Shun; Stack, Danuta; Huston, Shaunna M.; Ma, Ling Ling

    2013-01-01

    Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse. PMID:23918783

  1. Accumulation of Intrahepatic TNF-α-Producing NKp44+ NK Cells Correlates With Liver Fibrosis and Viral Load in Chronic HCV Infection.

    PubMed

    Nel, Isabelle; Lucar, Olivier; Petitdemange, Caroline; Béziat, Vivien; Lapalus, Martine; Bédossa, Pierre; Debré, Patrice; Asselah, Tarik; Marcellin, Patrick; Vieillard, Vincent

    2016-05-01

    In the setting of chronic hepatitis C virus (HCV) infection, changes in natural killer (NK) cells have been shown to reflect activation in response to virus stimulation. The contribution of individual natural cytotoxicity receptors to HCV infection remains to be clarified. NKp44 is the sole specific natural cytotoxicity receptor expressed only on activated NK cells.In this study, peripheral blood and liver NK-cell subsets were purified from 31 patients with chronic C hepatitis or nonalcoholic steatohepatitis, and then characterized by flow cytometry. Their polyfunctional activity was determined by expression of the CD107a degranulation marker, together with intracellular cytokine production.Unlike the patients with nonalcoholic steatohepatitis, patients with chronic HCV infection had a higher frequency of NKp44 NK cells in the liver than in their peripheral blood (P < 0.0001). Intrahepatic NKp44 NK cells from HCV individuals produced higher levels of tumor necrosis factor-α than did NKp44 NK cells (P = 0.0011). Importantly, the frequency of intrahepatic NKp44 NK cells was correlated with both HCV-RNA levels (P = 0.0234) and stage of fibrosis (P = 0.0003).Our findings suggest that the accumulation of intrahepatic tumor necrosis factor-α-producing NKp44 resident NK cells play a role in the liver damage associated with chronic HCV infection.

  2. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    PubMed

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2017-03-19

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56(hi) versus CD56(dim) defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  3. Intrahepatic infiltrating NK and CD8 T cells cause liver cell death in different phases of dengue virus infection.

    PubMed

    Sung, Jui-Min; Lee, Chien-Kuo; Wu-Hsieh, Betty A

    2012-01-01

    Elevated liver enzyme level is an outstanding feature in patients with dengue. However, the pathogenic mechanism of liver injury has not been clearly demonstrated. In this study, employing a mouse model we aimed to investigate the immunopathogenic mechanism of dengue liver injury. Immunocompetent C57BL/6 mice were infected intravenously with dengue virus strain 16681. Infected mice had transient viremia, detectable viral capsid gene and cleaved caspase 3 in the liver. In the mean time, NK cell and T cell infiltrations peaked at days 1 and 5, respectively. Neutralizing CXCL10 or depletion of Asialo GM1(+) cells reduced cleaved caspase 3 and TUNEL(+) cells in the liver at day 1 after infection. CD8(+) T cells infiltrated into the liver at later time point and at which time intrahepatic leukocytes (IHL) exhibited cytotoxicity against DENV-infected targets. Cleaved caspase 3 and TUNEL(+) cells were diminished in mice with TCRβ deficiency and in those depleted of CD8(+) T cells, respectively, at day 5 after infection. Moreover, intrahepatic CD8(+) T cells were like their splenic counterparts recognized DENV NS4B(99-107) peptide. Together, these results show that infiltrating NK and CD8(+) T cells cause liver cell death. While NK cells were responsible for cell death at early time point of infection, CD8(+) T cells were for later. CD8(+) T cells that recognize NS4B(99-107) constitute at least one of the major intrahepatic cytotoxic CD8(+) T cell populations.

  4. Prognostic value of tumor infiltrating NK cells and macrophages in stage II+III esophageal cancer patients

    PubMed Central

    Zheng, Xiao; Shi, Liangrong; Wu, Changping; Jiang, Jingting

    2016-01-01

    The detailed understanding of the immunobiology of tumor microenvironment has recently translated into new therapeutic approach against human cancers. Besides the role of immune cells mediating adaptive immune responses, the tumor infiltrating components of the innate immune system including, neutrophils, mast cells, NK cells, and macrophages, also role importantly in anti-tumor immunity. In our present study, we retrospectively analyzed the prognostic value of the densities of tumor infiltrating NK cells and macrophages in esophageal cancer tissues derived from stage II+III patients. Our results showed that the density of the infiltrating NK cells in tumor stroma was significantly associated with nodal status. In addition, the densities of the infiltrating NK cells in tumor nest, and the infiltrating macrophages in tumor nest as well as in tumor stroma, were significantly associated with patients' postoperative prognoses. Furthermore, the combination of infiltrating NK cells in tumor nest and stroma, or the combination of infiltrating macrophages in tumor nest and stroma, could also be used as important prognostic tool in predicting the survival of the stage II+III esophageal cancer patients. PMID:27736796

  5. Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation.

    PubMed

    Alam, Anwar; Puri, Niti; Saxena, Rajiv K

    2016-09-01

    The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1(+) cells as well as the activated subset of NK cells (NK1.1(+)CD69(+) cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1(+) phosphatidylserine(+)) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.

  6. Revisiting human natural killer cell subset function revealed cytolytic CD56dimCD16+ NK cells as rapid producers of abundant IFN-γ on activation

    PubMed Central

    De Maria, Andrea; Bozzano, Federica; Cantoni, Claudia; Moretta, Lorenzo

    2011-01-01

    The two major functions of human natural killer (NK) cells are conventionally associated with distinct cell subsets. Thus, cytolytic activity is mostly confined to the CD56dimCD16+ subset, whereas cytokine production is generally assigned to CD56brightCD16+/− cells. In this study, we reevaluated the functional capabilities of these NK subsets with regard to the production of IFN-γ at different time points after cell triggering via NKp46 and NKp30 activating receptors. Different from previous studies, cytokine production was also assessed at early intervals. We show that CD56dim NK cells produce IFN-γ already at 2 to 4 h, whereas no cytokine production is detected beyond 16 h. In contrast, CD56bright cells release IFN-γ only at late time intervals (>16 h after stimulation). The rapid IFN-γ production by CD56dim NK cells is in line with the presence of IFN-γ mRNA in freshly isolated cells. Rapid IFN-γ production was also induced by combinations of IL-2, IL-12, and IL-15. Our data indicate that not only cytolytic activity but also early IFN-γ production is a functional property of CD56dim NK cells. Thus, this subset can assure a rapid and comprehensive NK cell intervention during the early phases of innate responses. PMID:21187373

  7. A novel TLR7 agonist reverses NK cell anergy and cures RMA-S lymphoma-bearing mice.

    PubMed

    Wiedemann, Gabriela Maria; Jacobi, Severin Johannes; Chaloupka, Michael; Krächan, Angelina; Hamm, Svetlana; Strobl, Stefan; Baumgartner, Roland; Rothenfusser, Simon; Duewell, Peter; Endres, Stefan; Kobold, Sebastian

    2016-07-01

    Toll-like receptor 7 (TLR7) agonists are potent immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting systemic toxicities, only the topically applied TLR7 agonist (imiquimod) has been approved for therapy of skin tumors. There is a need for TLR7-activating compounds with equivalent efficacy but less toxicity. SC1, a novel small molecule agonist for TLR7, is a potent type-1 interferon inducer, comparable to the reference TLR7 agonist resiquimod, yet with lower induction of proinflammatory cytokines. In vivo, SC1 activates NK cells in a TLR7-dependent manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s. c. administrations of SC1 as efficiently as by the administration of resiquimod. No relevant toxicities were observed. Mechanistically, SC1 reverses NK cell anergy and restores NK cell-mediated tumor cell killing in an IFN-α-dependent manner. TLR7 targeting by SC1-based compounds may form an attractive strategy to activate NK cell responses for cancer therapy.

  8. The dual role of NK cells in antitumor reactions triggered by ionizing radiation in combination with hyperthermia.

    PubMed

    Finkel, Patrick; Frey, Benjamin; Mayer, Friederike; Bösl, Karina; Werthmöller, Nina; Mackensen, Andreas; Gaipl, Udo S; Ullrich, Evelyn

    2016-06-01

    Classical tumor therapy consists of surgery, radio(RT)- and/or chemotherapy. Additive immunotherapy has gained in impact and antitumor in situ immunization strategies are promising to strengthen innate and adaptive immune responses. Immunological effects of RT and especially in combination with immune stimulation are mostly described for melanoma. Since hyperthermia (HT) in multimodal settings is capable of rendering tumor cells immunogenic, we analyzed the in vivo immunogenic potential of RT plus HT-treated B16 melanoma cells with an immunization and therapeutic assay. We focused on the role of natural killer (NK) cells in the triggered antitumor reactions. In vitro experiments showed that RT plus HT-treated B16 melanoma cells died via apoptosis and necrosis and released especially the danger signal HMGB1. The in vivo analyses revealed that melanoma cells are rendered immunogenic by RT plus HT. Especially, the repetitive immunization with treated melanoma cells led to an increase in NK cell number in draining lymph nodes, particularly of the immune regulatory CD27(+)CD11b(-) NK cell subpopulation. While permanent NK cell depletion after immunization led to a significant acceleration of tumor outgrowth, a single NK cell depletion two days before immunization resulted in significant tumor growth retardation. The therapeutic model, a local in situ immunization closely resembling the clinical situation when solid tumors are exposed locally to RT plus HT, confirmed these effects. We conclude that a dual and time-dependent impact of NK cells on the efficacy of antitumor immune reactions induced by immunogenic tumor cells generated with RT plus HT exists.

  9. The dual role of NK cells in antitumor reactions triggered by ionizing radiation in combination with hyperthermia

    PubMed Central

    Finkel, Patrick; Frey, Benjamin; Mayer, Friederike; Bösl, Karina; Werthmöller, Nina; Mackensen, Andreas; Gaipl, Udo S.; Ullrich, Evelyn

    2016-01-01

    ABSTRACT Classical tumor therapy consists of surgery, radio(RT)- and/or chemotherapy. Additive immunotherapy has gained in impact and antitumor in situ immunization strategies are promising to strengthen innate and adaptive immune responses. Immunological effects of RT and especially in combination with immune stimulation are mostly described for melanoma. Since hyperthermia (HT) in multimodal settings is capable of rendering tumor cells immunogenic, we analyzed the in vivo immunogenic potential of RT plus HT-treated B16 melanoma cells with an immunization and therapeutic assay. We focused on the role of natural killer (NK) cells in the triggered antitumor reactions. In vitro experiments showed that RT plus HT-treated B16 melanoma cells died via apoptosis and necrosis and released especially the danger signal HMGB1. The in vivo analyses revealed that melanoma cells are rendered immunogenic by RT plus HT. Especially, the repetitive immunization with treated melanoma cells led to an increase in NK cell number in draining lymph nodes, particularly of the immune regulatory CD27+CD11b− NK cell subpopulation. While permanent NK cell depletion after immunization led to a significant acceleration of tumor outgrowth, a single NK cell depletion two days before immunization resulted in significant tumor growth retardation. The therapeutic model, a local in situ immunization closely resembling the clinical situation when solid tumors are exposed locally to RT plus HT, confirmed these effects. We conclude that a dual and time-dependent impact of NK cells on the efficacy of antitumor immune reactions induced by immunogenic tumor cells generated with RT plus HT exists. PMID:27471606

  10. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation

    PubMed Central

    Duriez, Marion; Quillay, Héloïse; Madec, Yoann; El Costa, Hicham; Cannou, Claude; Marlin, Romain; de Truchis, Claire; Rahmati, Mona; Barré-Sinoussi, Françoise; Nugeyre, Marie-Thérèse; Menu, Elisabeth

    2014-01-01

    Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface. PMID:25071732

  11. Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells.

    PubMed

    Shwetank; Date, Onkar Sanjay; Carbone, Ennio; Manjunath, Ramanathapuram

    2014-11-01

    Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-γ treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced [(3)H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses.

  12. Murine viral hepatitis involves NK cell depletion associated with virus-induced apoptosis

    PubMed Central

    LEHOUX, M; JACQUES, A; LUSIGNAN, S; LAMONTAGNE, L

    2004-01-01

    Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of immunological disorders related to acute and chronic hepatitis. In this study, we have verified if the fulminant hepatitis induced by MHV3 could be related to an impairment of innate immunity. Groups of three C57BL/6 mice were infected with the pathogenic L2-MHV3 or attenuated YAC-MHV3 viruses, and the natural killer (NK) cell populations from liver, spleen and bone marrow were analysed. The percentage of intrahepatic NK1·1+T cell receptor (TCR)− cells did not increase while NK1·1+TCRinter cells decreased in both L2-MHV3- and YAC-MHV3-infected mice. Concurrently, splenic and myeloid NK1·1+ cells decreased in L2-MHV3-infected mice. However, the cytotoxic activity of NK cells increased in liver and decreased in bone marrow from pathogenic L2-MHV3-infected mice while no modification was detected in YAC-MHV3-infected mice. Flow cytometric analysis revealed that both normal and larger splenic or myeloid NK cells decreased more in pathogenic L2-MHV3-infected mice than in attenuated YAC-MHV3-infected mice. In vitro viral infections of interleukin (IL)-15-stimulated lymphoid cells from liver and bone marrow revealed that L2-MHV3 induced higher decreases in cell viability of NK1·1+ cells than the YAC-MHV3 variant. The NK cell decreases were due to the viral permissivity leading to cytopathic effects characterized by cell rounding, syncytia formation and apoptosis. Larger NK+ syncytia were observed in L2-MHV3-infected cells than in YAC-MHV3-infected cells. These results suggest that NK cell production is impaired by viral infection favouring fulminant hepatitis. PMID:15196242

  13. Enhanced NK cell adoptive antitumor effects against breast cancer in vitro via blockade of the transforming growth factor-β signaling pathway

    PubMed Central

    Zhao, Yue; Hu, Jinyue; Li, Rongguo; Song, Jian; Kang, Yujuan; Liu, Si; Zhang, Dongwei

    2015-01-01

    Natural killer (NK) cells have great potential for improving cancer immunotherapy. Adoptive NK cell transfer, an adoptive immunotherapy, represents a promising nontoxic anticancer therapy. However, existing data indicate that tumor cells can effectively escape NK cell-mediated apoptosis through immunosuppressive effects in the tumor microenvironment, and the therapeutic activity of adoptive NK cell transfer is not as efficient as anticipated. Transforming growth factor-beta (TGF-β) is a potent immunosuppressant. Genetic and epigenetic events that occur during mammary tumorigenesis circumvent the tumor-suppressing activity of TGF-β, thereby permitting late-stage breast cancer cells to acquire an invasive and metastatic phenotype in response to TGF-β. To block the TGF-β signaling pathway, NK cells were genetically modified with a dominant-negative TGF-β type II receptor by optimizing electroporation using the Amaxa Nucleofector system. These genetically modified NK cells were insensitive to TGF-β and resisted the suppressive effect of TGF-β on MCF-7 breast cancer cells in vitro. Our results demonstrate that blocking the TGF-β signaling pathway to modulate the tumor microenvironment can improve the antitumor activity of adoptive NK cells in vitro, thereby providing a new rationale for the treatment of breast cancer. PMID:26124672

  14. Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G.

    PubMed

    Kailayangiri, Sareetha; Altvater, Bianca; Spurny, Christian; Jamitzky, Silke; Schelhaas, Sonja; Jacobs, Andreas H; Wiek, Constanze; Roellecke, Katharina; Hanenberg, Helmut; Hartmann, Wolfgang; Wiendl, Heinz; Pankratz, Susann; Meltzer, Jutta; Farwick, Nicole; Greune, Lea; Fluegge, Maike; Rossig, Claudia

    2017-01-01

    Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

  15. Human immunodeficiency-causing mutation defines CD16 in spontaneous NK cell cytotoxicity.

    PubMed

    Grier, Jennifer T; Forbes, Lisa R; Monaco-Shawver, Linda; Oshinsky, Jennifer; Atkinson, T Prescott; Moody, Curtis; Pandey, Rahul; Campbell, Kerry S; Orange, Jordan S

    2012-10-01

    The Fc receptor on NK cells, FcγRIIIA (CD16), has been extensively studied for its role in mediating antibody-dependent cellular cytotoxicity (ADCC). A homozygous missense mutation in CD16 (encoding a L66H substitution) is associated with severe herpesvirus infections in rare patients. Here, we identified a new patient with this CD16 mutation and compared the patient's NK cells to those of the originally reported patient. Patients with the L66H mutation had intact ADCC, but deficient spontaneous NK cell cytotoxicity and decreased surface expression of CD2, a coactivation receptor. Mechanistic studies in a human NK cell line, NK-92, demonstrated that CD16 expression correlated with CD2 surface levels and enabled killing of a melanoma cell line typically resistant to CD16-deficient NK-92 cells. An association between CD16 and CD2 was identified biochemically and at the immunological synapse, which elicited CD16 signaling after CD2 engagement. Stable expression of CD16 L66H in NK-92 cells recapitulated the patient phenotype, abrogating association of CD16 with CD2 as well as CD16 signaling after CD2 ligation. Thus, CD16 serves a role in NK cell-mediated spontaneous cytotoxicity through a specific association with CD2 and represents a potential mechanism underlying a human congenital immunodeficiency.

  16. Conventional NK cells can produce IL-22 and promote host defense in Klebsiella pneumoniae pneumonia.

    PubMed

    Xu, Xin; Weiss, Ido D; Zhang, Hongwei H; Singh, Satya P; Wynn, Thomas A; Wilson, Mark S; Farber, Joshua M

    2014-02-15

    It was reported that host defense against pulmonary Klebsiella pneumoniae infection requires IL-22, which was proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22(-/-) mice had decreased survival compared with wild-type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2(-/-) mice did not differ from wild-type mice in survival or levels of IL-22 in the lungs postinfection with K. pneumoniae. In contrast, K. pneumoniae-infected Rag2(-/-)Il2rg(-/-) mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells in host defense and production of IL-22. Unlike NK cell-like innate lymphoid cells that produce IL-22 and display a surface phenotype of NK1.1(-)NKp46(+)CCR6(+), lung NK cells showed the conventional phenotype, NK1.1(+)NKp46(+)CCR6(-). Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver, compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within 1 d postinfection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild-type and Rag2(-/-) mice. Our data suggest that, during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.

  17. Effects of a cloned cell line with NK activity on bone marrow transplants, tumour development and metastasis in vivo

    NASA Astrophysics Data System (ADS)

    Warner, John F.; Dennert, Gunther

    1982-11-01

    Natural killer (NK) cells cloned in vitro have been transferred into NK-deficient hosts. These cells have been shown to have a role in the rejection of allogeneic bone marrow grafts, resistance to both radiation-induced thymic leukaemia and challenge with melanoma tumour cells. It appears that NK cells have an important role in immune surveillance.

  18. Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy

    PubMed Central

    Chazara, Olympe; Gardner, Lucy; Ivarsson, Martin A.; Farrell, Lydia E.; Xiong, Shiqiu; Hiby, Susan E.; Colucci, Francesco; Sharkey, Andrew M.

    2016-01-01

    Tissue-specific NK cells are abundant in the pregnant uterus and interact with invading placental trophoblast cells that transform the maternal arteries to increase the fetoplacental blood supply. Genetic case-control studies have implicated killer cell Ig-like receptor (KIR) genes and their HLA ligands in pregnancy disorders characterized by failure of trophoblast arterial transformation. Activating KIR2DS1 or KIR2DS5 (when located in the centromeric region as in Africans) lower the risk of disorders when there is a fetal HLA-C allele carrying a C2 epitope. In this study, we investigated another activating KIR, KIR2DS4, and provide genetic evidence for a similar effect when carried with KIR2DS1. KIR2DS4 is expressed by ∼45% of uterine NK (uNK) cells. Similarly to KIR2DS1, triggering of KIR2DS4 on uNK cells led to secretion of GM-CSF and other chemokines, known to promote placental trophoblast invasion. Additionally, XCL1 and CCL1, identified in a screen of 120 different cytokines, were consistently secreted upon activation of KIR2DS4 on uNK cells. Inhibitory KIR2DL5A, carried in linkage disequilibrium with KIR2DS1, is expressed by peripheral blood NK cells but not by uNK cells, highlighting the unique phenotype of uNK cells compared with peripheral blood NK cells. That KIR2DS4, KIR2DS1, and some alleles of KIR2DS5 contribute to successful pregnancy suggests that activation of uNK cells by KIR binding to HLA-C is a generic mechanism promoting trophoblast invasion into the decidua. PMID:27815424

  19. An indirect role for NK cells in a CD4(+) T-cell-dependent mouse model of type I diabetes.

    PubMed

    Angstetra, Eveline; Graham, Kate L; Zhao, Yuxing; Irvin, Allison E; Elkerbout, Lorraine; Santamaria, Pere; Slattery, Robyn M; Kay, Thomas W; Thomas, Helen E

    2012-02-01

    CD8(+) T cells kill pancreatic β-cells in a cell-cell contact-dependent mechanism in the non-obese diabetic mouse. CD4(+) T lymphocytes are also able to kill pancreatic β-cells, but they do not directly contact β-cells and may use another cell type as the actual cytotoxic cell. Natural killer (NK) cells could have this role but it is uncertain whether they are cytotoxic towards β-cells. Therefore, the requirement for NK cells in β-cell destruction in the CD4-dependent T-cell antigen receptor transgenic NOD4.1 mice was examined. NK cells failed to kill β-cells in vitro, even in the absence of major histocompatibility complex class I. We observed only 9.7±1.1% of islet infiltrating NK cells from NOD4.1 mice expressing the degranulation marker CD107a. Diabetogenic CD4(+) T cells transferred disease to NODscid.IL2Rγ(-/-) mice lacking NK cells, indicating that NK cells do not contribute to β-cell death in vitro or in vivo. However, depletion of NK cells reduced diabetes incidence in NOD4.1 mice, suggesting that NK cells may help to maintain the right environment for cytotoxicity of effector cells.

  20. Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Bezouška, Karel; Yuen, Chun-Ting; O'Brien, Jacqui; Childs, Robert A.; Chai, Wengang; Lawson, Alexander M.; Drbal, Karel; Fišerová, Anna; Posíšil, Miloslav; Feizi, Ten

    1994-11-01

    A diversity of high-affinity Oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca2+-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

  1. Nasal NK/T-cell lymphoma: epidemiology and pathogenesis

    PubMed Central

    Takakuwa, Tetsuya; Hongyo, Tadashi; Yang, Woo-Ick

    2008-01-01

    Nasal NK/T-cell lymphoma (NKTCL) is an uncommon disease, but usually shows a highly aggressive clinical course. The disease is much more frequent in Asian and Latin American countries than in Western countries, and is universally associated with Epstein–Barr virus (EBV) infection. Analyses of gene mutations, especially p53 and c-KIT, revealed the different frequencies by district. Epidemiological studies revealed the changes of the disease frequency in Korea during the period from 1977–1989 to 1990–1996. Case-control study showed that the exposure to pesticides and chemical solvents could be causative of NKTCL. Further studies including HLA antigen typing of patients is necessary to further clarify the disease mechanism. PMID:18256789

  2. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.

    PubMed

    Miller, Jeffrey S; Soignier, Yvette; Panoskaltsis-Mortari, Angela; McNearney, Sarah A; Yun, Gong H; Fautsch, Susan K; McKenna, David; Le, Chap; Defor, Todd E; Burns, Linda J; Orchard, Paul J; Blazar, Bruce R; Wagner, John E; Slungaard, Arne; Weisdorf, Daniel J; Okazaki, Ian J; McGlave, Philip B

    2005-04-15

    We previously demonstrated that autologous natural killer (NK)-cell therapy after hematopoietic cell transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity outpatient immune suppressive regimens were tested: (1) low-dose cyclophosphamide and methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose cyclophosphamide and fludarabine (Hi-Cy/Flu) was tested in patients with poor-prognosis acute myeloid leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who received lower intensity regimens showed transient persistence but no in vivo expansion of donor cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HCT.

  3. Postnatal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia

    PubMed Central

    Miethke, Alexander G.; Saxena, Vijay; Shivakumar, Pranavkumar; Sabla, Gregg E.; Simmons, Julia; Chougnet, Claire A.

    2010-01-01

    Background & Aims Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. Methods We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. Results While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFα and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. Conclusions 1) The liver is devoid of Tregs early after perinatal RRV infection; 2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the postnatal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury. PMID:20347178

  4. High IFN-gamma and TNF production by peripheral NK cells of Colombian patients with different clinical presentation of Plasmodium falciparum

    PubMed Central

    2012-01-01

    Background In Colombia, Plasmodium falciparum infection rarely results in severe disease or mortality compared to infections in African populations. During natural infection NK cells exhibit a cytolytic effect and regulate dendritic cells, macrophages, neutrophils as well as affect antigen specific T and B cell responses. To characterize the NK cells in P. falciparum infected patients of a highly endemic region of Colombia, the degree of NK proliferation and production of IFN gamma and TNF production in these cells were explored. Methods Seventeen patients with acute and three with severe P. falciparum malaria patients from the Northwest region of the country were recruited in the study. In addition, 20 healthy controls were included: 10 from Medellin (no-transmission area) and 10 from the Uraba region (a malaria endemic area). Immunophenotypic analysis of peripheral mononuclear cells was performed by FACS to detect total number of NK cells, subtypes and intracellular IFNγ and TNF production by NK cells in the different patient groups. Results The total mean CD56+/CD3- NK cell proportions in acute and severe malaria subjects were 9.14% (7.15%CD56dim, 2.01%CD56bright) and 19.62% (16.05%CD56dim, 3.58%CD56bright), respectively, in contrast to healthy controls from endemic (total mean CD56+/CD3-1.2%) and non-endemic area (total mean CD56+/CD3- 0.67%). Analysis of basal IFNγ and TNF levels confirmed the CD56bright NK population as the main cytokine producer (p < 0.0001) in the groups affected with malaria, with the CD56dim NK cell exhibiting the highest potential of TNF production after stimulus in the acute malaria group. Conclusions The results confirm the important role of not only CD56bright but also of CD56dim NK cell populations as producers of the two cytokines in malaria patients in Colombia. PMID:22316273

  5. NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection.

    PubMed

    Kuylenstierna, C; Snyder-Cappione, J E; Loo, C P; Long, B R; Gonzalez, V D; Michaëlsson, J; Moll, M; Spotts, G; Hecht, F M; Nixon, D F; Sandberg, J K

    2011-02-01

    Cytokine immunotherapy is being evaluated as adjunct treatment in infectious diseases. The effects on innate and adaptive immunity in vivo are insufficiently known. Here, we investigate whether combination treatment with antiretroviral therapy (ART) and Interleukin-2 (IL-2) of patients with primary HIV-1 infection induces sustained increases in circulating NKT cell and NK cell numbers and effector functions and investigate how changes are coordinated in the two compartments. Patients with primary HIV-1 infection starting ART were analyzed for numbers, phenotype and function of NKT cells, NK cells and dendritic cells (DC) in peripheral blood before, during and after IL-2 treatment. NKT cells expanded during IL-2 treatment as expected from previous studies. However, their response to α-galactosyl ceramide antigen were retained but not boosted. Myeloid DC did not change their numbers or CD1d-expression during treatment. In contrast, the NK cell compartment responded with rapid expansion of the CD56(dim) effector subset and enhanced IFNγ production. Expansions of NKT cells and NK cells retracted back towards baseline values at 12 months after IL-2 treatment ended. In summary, NKT cells and NK cells respond to IL-2 treatment with different kinetics. Effects on cellular function are distinct between the cell types and the effects appear not to be sustained after IL-2 treatment ends. These results improve our understanding of the effects of cytokine immunotherapy on innate cellular immunity in early HIV-1 infection.

  6. Relation between Acute GVHD and NK Cell Subset Reconstitution Following Allogeneic Stem Cell Transplantation

    PubMed Central

    Ullrich, Evelyn; Salzmann-Manrique, Emilia; Bakhtiar, Shahrzad; Bremm, Melanie; Gerstner, Stephanie; Herrmann, Eva; Bader, Peter; Hoffmann, Petra; Holler, Ernst; Edinger, Matthias; Wolff, Daniel

    2016-01-01

    One of the major challenges of allogeneic stem cell transplantation (allo-SCT) is to reduce the risk of graft-versus-host disease (GVHD) while boosting the graft-versus-leukemia (GVL) effect. The reconstitution of natural killer (NK) cells following allo-SCT is of notable interest due to their known capability to induce GVL without GVHD. Here, in this study, we investigate the association between the incidence and severity of acute graft-versus-host disease (aGVHD) and the early reconstitution of NK cell subsets following allo-SCT. We analyzed 342 samples from 107 patients using flow cytometry, with a focus on immature CD56high and mature cytotoxic CD56dim NK cells. Longitudinal analysis of immune reconstitution after allo-SCT showed that the incidence of aGVHD was associated with a delayed expansion of the entire NK cell population, in particular the CD56high subset. Notably, the disturbed reconstitution of the CD56high NK cells also correlated with the severity of aGVHD. PMID:28066411

  7. Relation between Acute GVHD and NK Cell Subset Reconstitution Following Allogeneic Stem Cell Transplantation.

    PubMed

    Ullrich, Evelyn; Salzmann-Manrique, Emilia; Bakhtiar, Shahrzad; Bremm, Melanie; Gerstner, Stephanie; Herrmann, Eva; Bader, Peter; Hoffmann, Petra; Holler, Ernst; Edinger, Matthias; Wolff, Daniel

    2016-01-01

    One of the major challenges of allogeneic stem cell transplantation (allo-SCT) is to reduce the risk of graft-versus-host disease (GVHD) while boosting the graft-versus-leukemia (GVL) effect. The reconstitution of natural killer (NK) cells following allo-SCT is of notable interest due to their known capability to induce GVL without GVHD. Here, in this study, we investigate the association between the incidence and severity of acute graft-versus-host disease (aGVHD) and the early reconstitution of NK cell subsets following allo-SCT. We analyzed 342 samples from 107 patients using flow cytometry, with a focus on immature CD56(high) and mature cytotoxic CD56(dim) NK cells. Longitudinal analysis of immune reconstitution after allo-SCT showed that the incidence of aGVHD was associated with a delayed expansion of the entire NK cell population, in particular the CD56(high) subset. Notably, the disturbed reconstitution of the CD56(high) NK cells also correlated with the severity of aGVHD.

  8. The Memories of NK Cells: Innate-Adaptive Immune Intrinsic Crosstalk

    PubMed Central

    Ortolani, Claudio; del Zotto, Genny; Luchetti, Francesca; Canonico, Barbara; Artico, Marco; Papa, Stefano

    2016-01-01

    Although NK cells are considered part of the innate immune system, a series of evidences has demonstrated that they possess characteristics typical of the adaptive immune system. These NK adaptive features, in particular their memory-like functions, are discussed from an ontogenetic and evolutionary point of view. PMID:28078307

  9. In Vitro Generation of Human NK cells Expressing Chimeric Antigen Receptor through Differentiation of Gene-Modified Hematopoietic Stem Cells

    PubMed Central

    Lowe, Emily; Truscott, Laurel C.; De Oliveira, Satiro N.

    2016-01-01

    Summary NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells. PMID:27177671

  10. Exercise induced alterations in NK-cell cytotoxicity - methodological issues and future perspectives.

    PubMed

    Zimmer, Philipp; Schenk, Alexander; Kieven, Markus; Holthaus, Michelle; Lehmann, Jonas; Lövenich, Lukas; Bloch, Wilhelm

    2017-01-01

    With their ability to recognize and eliminate virus-infected and neoplastic cells, natural killer cells (NK-cells) represent an important part of the innate immune system. NK-cells have attracted the attention of exercise scientists for more than thirty years ago. To date, it is widely accepted that NK-cell counts in the peripheral blood are strongly influenced by acute exercise. Additionally, many studies reported effects of both, acute and chronic exercise on NK-cell cytotoxicity. However, these findings are contradictory. The inconsistence in findings may be argued with different exercise paradigms (type, duration, intensity). Moreover, strongly varying methods were used to detect NK-cell cytotoxicity. This review gives an overview of studies, investigating the impact of acute and chronic exercise on NK-cell cytotoxicity in young and old healthy adults, as well as on specific populations, such as cancer patients. Furthermore, different methodological approaches to assess NK-cell cytotoxicity are critically discussed to state on inconsistent study results and to give perspectives for further research in this field.

  11. Vanadium pentoxide prevents NK-92MI cell proliferation and IFNγ secretion through sustained JAK3 phosphorylation.

    PubMed

    Gallardo-Vera, Francisco; Diaz, Daniel; Tapia-Rodriguez, Miguel; Fortoul van der Goes, Teresa; Masso, Felipe; Rendon-Huerta, Erika; Montaño, Luis F

    2016-01-01

    Vanadium is a major air pollutant with toxic and carcinogenic effects; it also exercises immunosuppressive effects on the adaptive immune response. Its effect on the innate immune response is poorly explored. The aim of this study was to identify if vanadium pentoxide (V2O5) impairs the function of immunoregulatory NK cells and to determine possible mechanisms associated with this effect. Interleukin-2-independent NK-92MI cells were exposed to different V2O5 concentrations for 6, 12, or 24 h periods. Cell proliferation was then evaluated using CFSE staining, apoptosis by Annexin V binding, and necrosis by 7-AAD staining. The release of IL-2, -4, -6, -10, -17A, IFNγ, and TNFα by the cells were assessed using a human CBA kit. Expression of CD45, SOCS1, JAK3, pJAK3, STAT5, pSTAT5, IL-2R, IL-15R, Fas, and FasL in/on the cells was determined by flow cytometry; JAK3 and pJAK3 expression were also evaluated via confocal microscopy. The results indicated that V2O5 could inhibit NK-92MI cell proliferation and induce cell apoptosis in a dose- and time-related manner. V2O5 also inhibited IL-2, IL-10, and IFNγ secretion but mostly only after 24 h of exposure and with primarily the higher doses tested. V2O5 had no effect on expression of JAK3 and STAT5, but did cause an increase in pJAK3 and appeared to lead (trend) to reductions in levels of phosphorylated STAT5. V2O5 increased the expression of IL-2R, IL-15R, Fas, and FasL at concentrations above the 50-100 µM range. V2O5 had no effect on expression of the CD45 membrane phosphatase, but it did cause an increase in the expression of SOCS1. These results indicate that a key toxic effect of V2O5 on NK cells is a dysregulation of signaling pathways mediated by IL-2. These effects could help to explain the previously-reported deleterious effects on innate immune responses of hosts exposed to inhaled V2O5.

  12. About Training and Memory: NK-Cell Adaptation to Viral Infections.

    PubMed

    Hammer, Q; Romagnani, C

    2017-01-01

    Viral infections continuously challenge and shape our immune system. Due to their fine antigen recognition ability, adaptive lymphocytes protect against pathogen reencounter by generating specific immunological memory. Innate cells such as macrophages also adapt to pathogen challenge and mount resistance to reinfection, a phenomenon termed trained immunity. As part of the innate immunity, natural killer (NK) cells can display rapid effector functions and play a crucial role in the control of viral infections, especially by the β-herpesvirus cytomegalovirus (CMV). CMV activates the NK-cell pool by inducing proinflammatory signals, which prime NK cells, paralleling macrophage training. In addition, CMV dramatically shapes the NK-cell repertoire due to its ability to trigger specific NK cell-activating receptors, and enables the expansion and persistence of a specific NK-cell subset displaying adaptive and memory features. In this chapter, we will discuss how different signals during CMV infection contribute to NK-cell training and acquisition of classical memory properties and how these events can impact on reinfection and cross-resistance.

  13. IFN‐λ3 polymorphism indirectly influences NK cell phenotype and function during acute HCV infection

    PubMed Central

    Depla, Marion; Pelletier, Sandy; Bédard, Nathalie; Brunaud, Camille; Bruneau, Julie

    2016-01-01

    Abstract Introduction Polymorphisms in the type III interferon IFN‐λ3 and the killer cell immunoglobulin‐like receptor (KIR) genes controlling the activity of natural killer (NK) cells can predict spontaneous resolution of acute hepatitis C virus (HCV) infection. We hypothesized that IFN‐λ3 polymorphism may modulate NK cell function during acute HCV. Methods We monitored the plasma levels of type III IFNs in relation to the phenotype and the function of NK cells in a cohort of people who inject drugs (PWID) during acute HCV infection with different outcomes. Results Early acute HCV was associated with high variability in type III IFNs plasma levels and the favorable IFN‐λ3 CC genotype was associated with higher viral loads. Reduced expression of Natural Killer Group Protein 2A (NKG2A) was associated with lower IFN‐λ3 plasma levels and the CC genotype. IFN‐γ production by NK cells was higher in individuals with the CC genotype during acute infection but this did not prevent viral persistence. IFN‐λ3 plasma levels did not correlate with function of NK cells and IFN‐λ3 prestimulation did not affect NK