CD56 Dim CD16 Research Articles

Expression of interferon‐γ in decidual natural killer cells from women with hypertensive disorder complicating pregnancy

Hypertensive disorder complicating pregnancy (HDCP) is one of the most frequent and serious pregnancy-related diseases, which is closely related to disorders of the maternal immune system, especially the local immune microenvironment of the maternal-fetal interface. Uterine decidual natural killer (dNK) cells are the major immune cells in the maternal-fetal interface and they play an important role in establishing and maintaining a normal pregnancy. The aim of this study was to investigate the phenotype and function of dNK cells from women with HDCP. Decidual tissues were collected from women with normal pregnancy (normal control group, n = 15 cases) and HDCP (HDCP group, n = 20 cases), respectively. The mononuclear cells were extracted from tissues and flow cytometry (FCM) was utilized to sort out dNK cells. The phenotypes of dNK cells (CD56(bright)CD16⁻CD3⁻ vs CD56(dim)CD16⁺CD3⁻) were detected by FCM. After being co-cultured with Phorbol 12-myristate 13-acetate, ionomycin and monensin, the expression level of interferon (IFN)-γ in the dNK cells was detected by FCM. The phenotypes of dNK cells from the two groups were dominated by the CD56(bright)CD16⁻CD3⁻ subset, with no significant statistical difference (P < 0.05). The expression level of IFN-γ in the dNK cells from women with HDCP was on a lower trend than those from women with normal pregnancy, having significant statistical difference (P = 0.000 < 0.05). Our results indicated that although the phenotype of dNK cells from women with HDCP is of no difference, their functions are abnormal. Impaired cell function leads to a lower expression level of IFN-γ and this may account for one of the pathogeneses of HDCP.

Immune status of Fanconi anemia patients: decrease in T CD8 and CD56dim CD16+ NK lymphocytes

Fanconi anemia (FA), a rare genetic disease in which patients' life is compromised mainly by hematological abnormalities and cancer prone, seems to be affected by subtle immune cell irregularities. Knowing that FA presents developmental abnormalities and, based on recent reports, suggesting that natural killer (NK) CD56(dim) and NK CD56(bright) correspond to sequential differentiation pathways, we investigated if there were changes on the total number of NK cells and subsets as well as on T CD4 and T CD8 lymphocytes and their ratio. A large sample of FA patients (n = 42) was used in this work, and the results were correlated to clinical hematological status of these patients. Among FA patients, a decreased proportion of T CD8(+) and NK CD56(dim)CD16(+) cells were observed when compared to healthy controls as well as an imbalance of the subsets NK lymphocytes. Data suggest that FA patients might have a defective cytotoxic response due to the lower number of cytotoxic cells as well as impairment in the differentiation process of the NK cells subsets which may be directly related to impairment of the immune surveillance observed in these patients.

Variaciones en el número y función de los linfocitos asesinos naturales durante infecciones recurrentes o graves

The information about defects affecting natural killer cell (NK) development and activity in patients with an abnormal increase of recurrent infections is scarce.To perform a systematic analysis of NK abnormalities in patients with recurrent infections.Our study enrolled twenty patients with severe or recurrent viral infections. Natural killer cell subsets, surface receptors expression and cytotoxicity were analyzed. Results were compared with those from age- and sex-matched healthy controls.Transient alterations were observed in the percentages and absolute numbers of NK cells in patients with infection active episodes. We also described five patients with stable disturbances in the distribution of NK cell subpopulations. These defects are mainly due to a decrease in the CD56 dim CD16 bright cells in peripheral blood. In addition, NK cell function abnormalities were observed in some patients, however, those were always transient and mainly associated to active disease.These findings demonstrate transient alterations in the percentages and absolute numbers of NK cells in patients with recurrent or severe infection. Also, stable disturbances in CD56 dim CD16 bright NK cells are observed in these patients. Nevertheless, these parameters must be thoroughly studied to determine the mechanisms that entail these immune abnormalities and investigate how they alter the immune response.

Variation in NK cell number and function in individuals with recurrent or severe infections

The information about defects affecting natural killer cell (NK) development and activity in patients with an abnormal increase of recurrent infections is scarce. To perform a systematic analysis of NK abnormalities in patients with recurrent infections. Our study enrolled twenty patients with severe or recurrent viral infections. Natural killer cell subsets, surface receptors expression and cytotoxicity were analyzed. Results were compared with those from age- and sex-matched healthy controls. Transient alterations were observed in the percentages and absolute numbers of NK cells in patients with infection active episodes. We also described five patients with stable disturbances in the distribution of NK cell subpopulations. These defects are mainly due to a decrease in the CD56 dim CD16 bright cells in peripheral blood. In addition, NK cell function abnormalities were observed in some patients, however, those were always transient and mainly associated to active disease. These findings demonstrate transient alterations in the percentages and absolute numbers of NK cells in patients with recurrent or severe infection. Also, stable disturbances in CD56 dim CD16 bright NK cells are observed in these patients. Nevertheless, these parameters must be thoroughly studied to determine the mechanisms that entail these immune abnormalities and investigate how they alter the immune response.

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Aim Natural Killer (NK) cells, first in line of defense against tumors and infections are the earliest lymphocytes to recover after Hematopoietic Cell Transplantation. NK cell responses are regulated by activating and inhibitory receptors known as Killer Immunoglobulin-like Receptors (KIR). It is still not known which NK cell subsets elicit anti-cancer or anti-viral immune response and if target induced NK cell responses are influenced by KIR gene content. Here we assessed in healthy individuals whether different NK cell subsets elicit unique immune responses against different targets (leukemia cells or herpes viruses), and if this difference is a function of individual’s KIR gene content. Methods PBMNCs from 50 healthy donors were stimulated with a leukemic cell line (K562) and a herpesvirus (Epstein-Barr virus, EBV). A 5-colour flow cytometry based estimation of cytotoxicity (CD107a expression) and cytokine (IFN- γ ) production was performed for both regulatory (CD56 bright CD16 neg ) and cytolytic (CD56 dim CD16 pos ) NK cells. KIR gene content was obtained by Luminex based rSSO method. Results Unique target induced patterns of responses were observed with different NK cell subsets. K562 cells induced degranulation with or without IFN- γ production. Contrarily, EBV induced NK cells exhibited all single (IFN- γ production and degranulation alone) and bifunctional (IFN- γ production and degranulation together) responses. In contrast to cytolytic NK cells, which in general exhibited a bifunctional response against both targets, regulatory NK cells were primarily only IFN- γ producers. High number of activating KIR genes (B/x genotypes) showed significant correlation with IFN- γ production but not with degranulation. Conclusions NK cell subsets elicit unique immune responses against different targets, some of which are influenced by the KIR gene content. Assessment of post-HCT recovery of these target-specific functional NK cell subsets and their KIR content will be important for the prediction of HCT outcomes.

Cyclic Changes and Relationship between Peripheral and Endometrial NK Cells from Women with Repeated Failure after Artificial Insemination by Donor Sperm

Cyclic changes of peripheral natural killer (pNK) cells and/or endometrial NK (eNK) cells during menstrual cycle remain controversial, and their relationship remains uncertain. Peripheral blood and endometrial biopsies were simultaneously obtained from women (n = 23) undergoing artificial insemination with donor sperm (AID) for at least three cycles at both proliferative (days 9-11) and secretory phases (days 20-23) of menstrual cycle. The percentages of CD3(-) CD56(+), CD3(-) CD56(dim) CD16(+), CD3(-) CD56(bright) CD16(-) pNK, and eNK cell subsets within lymphocytes were determined by three-color flow cytometry. The correlation between the percentages of pNK and eNK cells was further analyzed by Spearman's test. The percentages of CD3(-) CD56(+), CD3(-) CD56(dim) CD16(+), and CD3(-) CD56(bright ) CD16(-) pNK cells were not statistically different between the proliferative and secretory phases (P > 0.05, respectively). However, the percentages of CD3(-) CD56(+) and CD3(-) CD56(bright ) CD16(-) eNK cells were significantly decreased at the secretory phase, compared with those in the proliferative phase (P < 0.05, respectively). No correlation between the percentages of all pNK cell parameters and those of CD3(-) CD56(bright ) CD16(-) eNK cells (the major subset of NK cells in uterus) was found in the same women throughout the menstrual cycle (P > 0.05). We found a menstrual-cycle-dependent change in the percentage of eNK cells in women undergoing AID treatment, but not pNK cells. Moreover, the percentage of pNK cells may not reflect that of eNK cells during menstrual cycle.

An evaluation of the role of NKG2C+ natural killer cells in protection from cytomegalovirus DNAemia early following allogeneic stem cell transplantation

The role of natural killer (NK) cells in affording protection against human cytomegalovirus (CMV) in allogeneic stem cell transplant recipients is largely unknown. The current study was aimed at determining whether NKG2C+ NK cells confer protection from CMV DNAemia early following transplantation in patients lacking mono and polyfunctional CMV pp65 and IE-1-specific CD4+ and CD8+ T-cell responses, as measured by flow cytometry for intracellular cytokine staining. Fourteen out of the 36 patients included in this study developed CMV DNAemia between days +30 and +60 after transplant. Three patients did so after day +60. Peripheral blood levels of CD56(bright) CD16(-/low) and CD56(dim) CD16+ NKG2C+ NK cells measured at day +30 and at day +60 in patients who had or had not subsequent CMV DNAemia did not differ significantly. In addition, no significant correlation was found between CD56(bright) CD16(-/low) (σ = -0.229; P = 0.39) and CD56(dim) CD16+ (σ = -0.285; P = 0.28) NKG2C+ NK-cell levels and initial plasma CMV DNA loads. In summary, the data presented do not support a direct implication of NKG2C+ NK cells in preventing the development of CMV DNAemia or modulating the magnitude of CMV replication at early stages during episodes of CMV DNAemia in allogeneic stem cell transplant patients with unreconstituted CMV-specific T-cell responses.

Decidual Cell Regulation of Natural Killer Cell–Recruiting Chemokines: Implications for the Pathogenesis and Prediction of Preeclampsia

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-γ and either macrophage-derived TNF-α or IL-1β synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-γ receptor 1 and 2 mediation via JAK/STAT and NFκB signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-γR1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.

The Viral KSHV Chemokine vMIP-II Inhibits the Migration of Naive and Activated Human NK Cells by Antagonizing Two Distinct Chemokine Receptors

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56Dim CD16Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56Bright CD16Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56Dim CD16Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.

Reduced frequency of CD56dim CD16pos natural killer cells in pediatric systemic inflammatory response syndrome/sepsis patients

Sepsis continues to be a leading cause of death in infants and children. Natural killer (NK) cells serve as a bridge between innate and adaptive immunity, yet their role in pediatric sepsis has not been well characterized. We tested the hypothesis that decreased NK cell cytotoxicity is a common feature of pediatric systemic inflammatory response syndrome (SIRS)/sepsis patients by measuring, using flow cytometry, NK cell cytotoxicity and cell surface phenotype in the peripheral blood of 38 pediatric intensive care patients who demonstrated signs and symptoms of SIRS and/or sepsis. NK cell cytotoxicity was significantly reduced in peripheral blood mononuclear cells (PBMCs) of pediatric SIRS/sepsis patients as compared with healthy controls, and the percentage of CD56(dim) CD16(+) cytotoxic NK cells in PBMCs was lower in patients with SIRS/sepsis than in normal donors. However, on a per cell basis, CD56(dim) CD16(+) NK cells in patients mediated cytotoxicity as well as those in normal donors. The NK cell dysfunction in pediatric SIRS/sepsis patients reflects a quantitative rather than a qualitative difference from healthy controls.

Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Popko K, Malinowska I, Rychta M, et al. Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results. Central European Journal of Immunology. 2013;38(4):562-568. doi:10.5114/ceji.2013.39776. APA Popko, K., Malinowska, I., Rychta, M., Osińska, I., Górska, E., & Kucharska, A. et al. (2013). Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results. Central European Journal of Immunology, 38(4), 562-568. https://doi.org/10.5114/ceji.2013.39776 Chicago Popko, Katarzyna, Iwona Malinowska, Marta Rychta, Iwona Osińska, Elżbieta Górska, Anna Kucharska, and Urszula Demkow. 2013. "Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results". Central European Journal of Immunology 38 (4): 562-568. doi:10.5114/ceji.2013.39776. Harvard Popko, K., Malinowska, I., Rychta, M., Osińska, I., Górska, E., Kucharska, A., and Demkow, U. (2013). Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results. Central European Journal of Immunology, 38(4), pp.562-568. https://doi.org/10.5114/ceji.2013.39776 MLA Popko, Katarzyna et al. "Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results." Central European Journal of Immunology, vol. 38, no. 4, 2013, pp. 562-568. doi:10.5114/ceji.2013.39776. Vancouver Popko K, Malinowska I, Rychta M, Osińska I, Górska E, Kucharska A et al. Frequency and spontaneous cytotoxicity of natural killer cells in healthy children: preliminary results. Central European Journal of Immunology. 2013;38(4):562-568. doi:10.5114/ceji.2013.39776.

Increase of Reactive Oxygen Species in Natural Killer cells from Pulmonary Tuberculosis patients

Event Abstract Back to Event Increase of Reactive Oxygen Species in Natural Killer cells from Pulmonary Tuberculosis patients Diogo Silva1, Mónica Abreu1, Patricia Couceiro2, Margarida F. Teixeira1, Inês Ladeira3, Filipa Viveiros3, Raquel Duarte3, Helena Sá1 and Paulo Rodrigues-Santos1* 1 Faculdade de Medicina da Universidade de Coimbra, Instituto de Imunologia, Portugal 2 Center for Neurosciences and Cell Biology, Laboratory of Immunology and Oncology, Portugal 3 Centro Diagnóstico Pneumológico of Vila Nova de Gaia, Portugal Tuberculosis (TB) remains today a threat for public health and one third of the world population is infected. Although the role of Natural Killer (NK) cells in vivo was never fully understood, their capacity to lyse not only infected macrophages, but also directly M. tuberculosis demonstrates their possible role in the immune response in addition to their regulatory function over other immune cells and their presence inside granulomas. However their number is decreased and with impaired function in TB patients Reactive oxygen spices (ROS) produced by tissue-specific cells especially alveolar macrophages can be responsible for their suppressive state as happens in other diseases. In this work we intended to demonstrate the increase of ROS in NK cells of patients with pulmonary tuberculosis. We analyzed by flow cytometry the levels of oxidative stress in cells from peripheral blood of 14 TB patients with main focus in NK cells. In our work we demonstrated that not only phagocytic cells with active NADPH oxidase have an increase of ROS, but also NK cells and NKT cells. We did not find any significant statistical difference in CD8+ T cells comparing with healthy controls. Beyond expected decrease of total NK cell number and especially CD56dim CD16+ subset, we found that almost CD56dim are CD16- corresponding these cells to an increase of oxidative stress. We also demonstrated that contrary to granulocytes and monocytes, in multi-drug resistant (MDR) TB patients the levels of oxidative stress in NK cells are equally higher to persistent TB without (MDR-TB). Keywords: Tuberculosis, Pulmonary, Natural Killer cells, Reactive Oxygen Species, NADPH Oxidase, innate immunity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Silva D, Abreu M, Couceiro P, Teixeira MF, Ladeira I, Viveiros F, Duarte R, Sá H and Rodrigues-Santos P (2013). Increase of Reactive Oxygen Species in Natural Killer cells from Pulmonary Tuberculosis patients. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00533 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Paulo Rodrigues-Santos, Faculdade de Medicina da Universidade de Coimbra, Instituto de Imunologia, Coimbra, 3004-504, Portugal, paulo.santos@fmed.uc.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Diogo Silva Mónica Abreu Patricia Couceiro Margarida F Teixeira Inês Ladeira Filipa Viveiros Raquel Duarte Helena Sá Paulo Rodrigues-Santos Google Diogo Silva Mónica Abreu Patricia Couceiro Margarida F Teixeira Inês Ladeira Filipa Viveiros Raquel Duarte Helena Sá Paulo Rodrigues-Santos Google Scholar Diogo Silva Mónica Abreu Patricia Couceiro Margarida F Teixeira Inês Ladeira Filipa Viveiros Raquel Duarte Helena Sá Paulo Rodrigues-Santos PubMed Diogo Silva Mónica Abreu Patricia Couceiro Margarida F Teixeira Inês Ladeira Filipa Viveiros Raquel Duarte Helena Sá Paulo Rodrigues-Santos Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Changes of human decidual natural killer cells cocultured with YFP-Toxoplasma gondii: implications for abnormal pregnancy

To investigate the changes of human decidual natural killer (dNK) cells cocultured with Toxoplasma gondii in vitro and to infer implications on pregnancy. Case-control study. College and hospital. Decidual tissue was obtained from 85 patients undergoing voluntary abortion during the first trimester of gestation (6-12 weeks). None. The dNK cells were isolated and infected with YFP-Toxoplasma gondii. Cells were observed by fluorescence and confocal microscopy. The CD56(bright)CD16(-)/CD56(dim)CD16(+) dNK ratio, expression of KIR2DL4, ILT-2, and NKG2D on dNK cells were analyzed by flow cytometry and the cytotoxic activity of infected dNK cells were evaluated. The CD56(dim)CD16(+)/CD56(bright)CD16(-) dNK ratio was significantly elevated at 12, 24, and 48 hours after YFP-T. gondii infection. Expression of KIR2DL4, ILT-2, and NKG2D were increased after infection, but NKG2D were significantly higher than those of KIR2DL4 and ILT-2. Both the CD56(dim)CD16(+)/CD56(bright)CD16(-) dNK ratio and NKG2D expression were correlated with dNK cytotoxic activity. Enhanced dNK cytotoxicity due to increased CD16 and NKG2D expression may contribute to abnormal pregnancy outcomes observed with maternal infection with T. gondii.

Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56dimCD16+ and CD56−CD16+ NK cells. However, the impact of HIV-infection on CD56bright NK cells is less well understood. Here we report a rise of CD56bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7−CD56bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56dimCD16+ NK cells. Furthermore, CD56bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.

Comprehensive Analysis of Peripheral Blood Lymphocytes in 76 Women with Recurrent Miscarriage before and after Lymphocyte Immunotherapy

The efficacy of lymphocyte immunotherapy (LIT) for unexplained recurrent miscarriage (uRM) remains indefinite. The objective of the present study is to comprehensively evaluate the alterations in the proportions and functions of peripheral blood lymphocytes in patients with uRM before and after LIT. Seventy-six women with uRM were included in the present study. Immunophenotype of peripheral blood lymphocytes (76 patients), NK cytotoxicity (62 patients) and Th1 (IFN-γ and TNF-α)/Th2 (IL-10) ratios (73 patients) were assessed during the luteal phase before LIT and after the third LIT by the flow cytometer. To date, 29 patients had already delivered or been pregnant more than 20 weeks (successful pregnancy group) and 5 patients experienced subsequent abortion (abortion group). The percentages of CD3(+) T cell, CD3(+) CD4(+) T cell and IL-10-producing Th1 cell were significantly increased, whereas the percentages of CD3(+) HLA-DR(+) T cell, CD56(+) CD3(-) NK cell and CD56(dim ) CD16(+) NK cell, the NK cytotoxicity and the ratios of Th1/Th2 were significantly decreased after LIT in the total patients and in the successful pregnant group. The percentages of CD56(+ ) CD3(-) NK cell and IFN-γ-producing cell, and the ratio of IFN-γ/IL-10 were significantly lower after LIT in the successful pregnant group compared to those in the abortion group. LIT alters the proportions and functions of most peripheral blood lymphocyte subsets. Some of these alterations may be beneficial for pregnancy maintenance, whereas some may be potential markers for predicting subsequent abortion.

The role of CD14dim CD16+ monocytes in regulation of endothelial function in pathogenesis of coronary artery disease

The role of CD14dim CD16+ monocytes in regulation of endothelial function in pathogenesis of coronary artery disease

Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.

Decreased NKp46 and NKG2D and elevated PD‐1 are associated with altered NK‐cell function in pediatric transplant patients with PTLD

Post-transplantation lymphoproliferative disorders (PTLD) are life-threatening complications of organ transplantation caused by EBV infection and the use of chronic immunosuppression. While T-cell impairment is known to play a critical role in the immunopathogenesis of EBV complications post-transplantation, the role of NK cells is still under investigation. Here, we have characterized NK-cell phenotype and function in peripheral blood from asymptomatic pediatric thoracic transplant patients, patients with PTLD, and healthy controls. Overall, asymptomatic pediatric solid organ transplant (Tx) patients presented significant expansion of the CD56(bright) CD16(±) subset and displayed effective NK-cell function, while PTLD patients accumulated CD56(dim) CD16(-) and CD56(-) CD16(+) NK-cell subsets. In addition, NK cells from PTLD patients down-regulated NKp46 and NKG2D, and significantly up-regulated PD-1. These phenotypic changes were associated with NK functional impairment, resembling cellular exhaustion. Disrupting PD-1 inhibitory pathway improved IFN-γ release, but did not enhance cytotoxicity in PTLD patients, suggesting that these defects were partially PD-1 independent. Our results indicate the important role of NK cells during EBV surveillance post-transplantation, with implications for the immunopathogenesis of EBV complications, and suggest that monitoring NK cells in transplant patients may hold clinical value.

IL-2 Stimulated but Not Unstimulated NK Cells Induce Selective Disappearance of Peripheral Blood Cells: Concomitant Results to a Phase I/II Study

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9–14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/−) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(−) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/−)CD69(+)NCR(high)CD62L(−) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.

Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12.

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL+IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21day) NK cell proliferation. The expanded NK cells are predominantly CD56(bright), and we show that isolated CD56(dim)CD16(+) NK cells can switch to a CD56(bright)CD16(-) phenotype and proliferate in response to 4-1BBL+IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required 'help' from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL+IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56(bright) and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.