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Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy
Abstract
The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.
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Human NK cells are usually defined as CD3⁻CD56⁺ lymphocytes. However, a CD56⁻CD16⁺ (CD56neg) lymphocyte population that displays NK-associated markers expands during chronic viral infections such as HIV-1 and HCV, and, to lesser extent, in herpesvirus infections. This CD56neg NK cell subset has been understudied because it requires the exclusion of other lymphocytes to accurately identify its presence. Many questions remain regarding the origin, development, phenotype, and function of the CD56neg NK cell population. Our objective was to determine the frequency of this NK subset in healthy controls and its alteration in viral infections by performing a meta-analysis. In addition to this, we analyzed deposited CyTOF and scRNAseq datasets to define the phenotype and subsets of the CD56neg NK cell population, as well as their functional variation. We found in 757 individuals, from a combined 28 studies and 6 datasets, that the CD56neg subset constitutes 5.67% of NK cells in healthy peripheral blood, while HIV-1 infection increases this population by a mean difference of 10.69%. Meta-analysis of surface marker expression between NK subsets showed no evidence of increased exhaustion or decreased proliferation within the CD56neg subset. CD56neg NK cells have a distinctive pattern of KIR expression, implying they have a unique potential for KIR-mediated education. A perforin⁻CD94⁻NKG2C⁻NKp30⁻ CD56neg population exhibited different gene expression and degranulation responses against K562 cells compared to other CD56neg cells. This analysis distinguishes two functionally distinct subsets of CD56neg NK cells. They are phenotypically diverse and have differing capacity for education by HLA class-I interactions with KIRs.
Background: The importance of immune checkpoint molecules is well known in tumor and transplantation immunology; however, much less information is available regarding human pregnancy. Despite the significant amount of information about the TIGIT and CD226 immune checkpoint receptors in immune therapies, very little research has been conducted to study the possible role of these surface molecules and their ligands (CD112 and CD155) during the three trimesters of pregnancy. Methods: From peripheral blood, immune cell subpopulations were studied, and the surface expression of immune checkpoint molecules was analyzed by flow cytometry. Soluble immune checkpoint molecule levels were measured by ELISA. Results: Notable changes were observed regarding the percentage of monocyte subpopulation and the expression of CD226 receptor by CD4⁺ T and NKT cells. Elevated granzyme B content by the intermediate and non-classical monocytes was assessed as pregnancy proceeded. Furthermore, we revealed an important relationship between the CD226 surface expression by NKT cells and the serum CD226 level in the third trimester of pregnancy. Conclusions: Our results confirm the importance of immune checkpoint molecules in immunoregulation during pregnancy. CD226 seems to be a significant regulator, especially in the case of CD4⁺ T and NKT cells, contributing to the maternal immune tolerance in the late phase of pregnancy.
Uterine natural killer cells (uNK) play an important role in promoting successful pregnancy by regulating trophoblast invasion and spiral artery remodelling in the first trimester. Recently, single-cell RNA sequencing (scRNAseq) on first-trimester decidua showed that uNK can be divided into three subsets, which may have different roles in pregnancy. Here we present an integration of previously published scRNAseq datasets, together with novel flow cytometry data to interrogate the frequency, phenotype, and function of uNK1–3 in seven stages of the reproductive cycle (menstrual, proliferative, secretory phases of the menstrual cycle; first, second, and third trimester; and postpartum). We found that uNK1 and uNK2 peak in the first trimester, but by the third trimester, the majority of uNK are uNK3. All three subsets are most able to degranulate and produce cytokines during the secretory phase of the menstrual cycle and express KIR2D molecules, which allow them to interact with HLA-C expressed by placental extravillous trophoblast cells, at the highest frequency during the first trimester. Taken together, our findings suggest that uNK are particularly active and able to interact with placental cells at the time of implantation and that uNK1 and uNK2 may be particularly involved in these processes. Our findings are the first to establish how uNK frequency and function change dynamically across the healthy reproductive cycle. This serves as a platform from which the relationship between uNK function and impaired implantation and placentation can be investigated. This will have important implications for the study of subfertility, recurrent miscarriage, pre-eclampsia, and pre-term labour.
During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.
The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education.
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Natural killer (NK) cells are an important component of the innate immune system for the control of intracellular pathogens and cancer cells. NK cells demonstrate heterogeneous expression of inhibitory surface receptors. Signaling through these various receptors during NK cell development promotes functionality, referred to as NK cell education. Here we investigated the impact of education on NK cell metabolism through functional assessment of critical metabolic pathways and calcium signaling. Educated NK cells had an increased uptake of the metabolic substrates 2-NBDG, a fluorescent glucose analog, and BODIPY FL C16, a fluorescent palmitate, compared to uneducated NK cells. Comparison of NK cells educated via KIRs or NKG2A showed that NKG2A-educated NK cells were the main contributor to these differences in uptake of metabolites, and that NKG2A-educated NK cells were functionally more resilient in response to metabolic blockade of oxidative phosphorylation. Furthermore, NKG2A-educated NK cells exhibited higher peak calcium concentration following stimulation, indicating stronger signaling events taking place in these educated NK cells. These results demonstrate that cellular metabolism plays an important role in the functional differences observed between educated and uneducated NK cells, and show that NKG2A-educated NK cells remain more functionally competent than KIR-educated NK cells when oxidative phosphorylation is restricted. Understanding metabolic programming during NK cell education may unveil future targets to manipulate NK cell function for use in clinical settings, such as cancer therapies.
Natural killer (NK) cells are the host's first line of defense against tumors and viral infections without prior sensitization. It is increasingly accepted that NK cells belong to the innate lymphoid cell (ILC) family. Other ILCs, comprising ILC1s, ILC2s, ILC3s and lymphoid tissue inducer (LTi) cells, are largely non-cytotoxic, tissue-resident cells, which function to protect local microenvironments against tissue insults and maintain homeostasis. Recently, evidence has accumulated that metabolism supports many aspects of the biology of NK cells and other ILCs, and that metabolic reprogramming regulates their development and function. Here, we outline the current understanding of ILC metabolism, and describe how metabolic processes are affected, and how metabolic defects are coupled to dysfunction of ILCs, in disease settings. Furthermore, we summarize the current and potential directions for immunotherapy involving targeting of ILC metabolism. Finally, we discuss the open questions in the rapidly expanding field of ILC metabolism.
NK cells expressing self-inhibitory receptors display increased functionality compared to NK cells lacking those receptors. The acquisition of functional competence in these particular NK-cell subsets is termed education. Little is known about the underlying mechanisms that lead to the functional differences between educated and uneducated NK cells. An increasing number of studies suggest that cellular metabolism is a determinant of immune cell functions. Thus, alterations in cellular metabolic pathways may play a role in the process of NK-cell education. Here, we compared the glycolytic profile of educated and uneducated primary human NK cells. KIR-educated NK cells showed significantly increased expression levels of the glucose transporter Glut1 in comparison to NKG2A-educated or uneducated NK cells with and without exposure to target cells. Subsequently, the metabolic profile of NK-cell subsets was determined using a Seahorse XF Analyzer. Educated NK cells displayed significantly higher rates of cellular glycolysis than uneducated NK cells even in a resting state. Our results indicate that educated and uneducated NK cells reside in different metabolic states prior to activation. These differences in the ability to utilize glucose may represent an underlying mechanism for the superior functionality of educated NK cells expressing self-inhibitory receptors.
The tetraspanin CD9 is expressed by all the major subsets of leukocytes (B cells, CD4⁺ T cells, CD8⁺ T cells, natural killer cells, granulocytes, monocytes and macrophages, and immature and mature dendritic cells) and also at a high level by endothelial cells. As a typical member of the tetraspanin superfamily, a prominent feature of CD9 is its propensity to engage in a multitude of interactions with other tetraspanins as well as with different transmembrane and intracellular proteins within the context of defined membranal domains termed tetraspanin-enriched microdomains (TEMs). Through these associations, CD9 influences many cellular activities in the different subtypes of leukocytes and in endothelial cells, including intracellular signaling, proliferation, activation, survival, migration, invasion, adhesion, and diapedesis. Several excellent reviews have already covered the topic of how tetraspanins, including CD9, regulate these cellular processes in the different cells of the immune system. In this mini-review, however, we will focus particularly on describing and discussing the regulatory effects exerted by CD9 on different adhesion molecules that play pivotal roles in the physiology of leukocytes and endothelial cells, with a particular emphasis in the regulation of adhesion molecules of the integrin and immunoglobulin superfamilies.
We investigated the effect of human chorionic gonadotropin, estriol, leptin, ghrelin, and kisspeptin on the microRNA expression in separated NK cells. All of these hormones are able to effectively modulate the expression of microRNAs both stimulating and suppressing the cytotoxic potential of NK cells and thereby indirectly regulate the functions of these lymphocytes.
The effects of chorionic gonadotropin, estriol (E3), leptin, ghrelin, and kisspeptin on the intracellular expression of perforin, granzyme A, and granzyme B was studied in separated NK cells. All studied hormones except E3 are could modulate the expression of cytotoxic enzymes in NK cells by suppression of the expression of the most active proapoptotic agents, resulting in increased expression of granzyme A, which is typical of the decidual subpopulation of these lymphocytes.
Natural killer (NK) cells represent a lymphocyte subpopulation which is capable of contact cytolysis of virus-infected cells and tumor cells, being a source of cytokines which stimulate other immune cells and promote immune response. NK cell differentiation is connected with a consequent acquisition of specific NK cell receptors by stem cells and formation of functional characteristics inherent to natural killer cells. The aim of this review was to describe the CD56dim and CD56bright populations of NK cells in the course of their differentiation. The authors describe NK surface receptors and expression of transcription factors at various steps of the NK differentiation. We present comparative characteristics of data concerning cytokines and cellular microenvironment influence upon NK cell differentiation, and examine a phenomenon of existing memory-like NK cells. Uterine NK cell differentiation is of special interest, since these cells represent a special NK cell population which prevails among decidual lymphocytes during pregnancy and participates in the process of placental formation and development. This review considers some features of uterine NK cell differentiation, taking into account a possibility of formation of this NK cell population from both peripheral blood NK pool, and in situ proliferation. Moreover, functional studies of the uterine NK cells allow to get closer to understanding the role of NK cells during pregnancy and abnormality of utero-placental bed regulation by NK cells in cases of pregnancy failure.
Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56(dim) cells are generally considered more cytotoxic, whereas the CD56(bright) cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2-induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56(bright) cells were more metabolically active compared with CD56(dim) cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56(bright) cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56(dim) cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56(bright) subset of cells. Overall, we have defined CD56(bright) NK cells to be more metabolically active than CD56(dim) cells, which supports their production of large amounts of IFN-γ during an immune response.
Natural killer (NK) cells are a type of lymphocyte circulating in peripheral blood named because of their effector functions in killing target cells. Immune cells that share similar phenotypic characteristics but are poor killers populate the uterine lining at implantation and during early pregnancy when the placenta is established. The functions of these uterine NK (uNK) cells are essentially unknown but available data point to a role in regulating placentation in concert with other elements of the decidua and invading trophoblast cells. Despite the lack of scientific rationale and advice from clinical governing bodies, such as the Human Fertilisation and Embryology Authority, an increasing range of tests and therapies are still offered to women undergoing IVF or attending recurrent miscarriage clinics based on the myth that uterine NK cells need suppressing to prevent damage to the embryo. New treatments can be introduced at whim with subsequent demands for expensive trials to prove/disprove their efficacy. The evidence that targeting uNK or peripheral blood NK cells assists women with recurrent pregnancy failure is lacking. Healthcare professionals and patients should very carefully evaluate the practice of immunomodulation to enhance pregnancy outcome. A discussion on how to move towards stricter regulation of immunotherapy in non-hospital settings is now needed because it is clear that the potential risks and costs of these therapies outweigh any benefits.
© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy.
57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3 by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow cytometry. Serum Galectin-9 levels were measured by ELISA.
Our results show that the numbers of peripheral NK and cytotoxic T cells and their TIM-3 expression do not change between the first, second and third trimesters of pregnancy. Compared to non-pregnant individuals, regulatory T cells show higher level of Galectin-9 expression as pregnancy proceeds, which is in line with the level of Galectin-9 in the patients sera. Cytotoxic T cells, NK cells and NK cell subsets expressing TIM-3 molecule show altered cytokine production and cytotoxicity during pregnancy compared to non-pregnant individuals.
Our results indicate that Galectin-9 expressing regulatory T cells, TIM-3+ cytotoxic T cells and NK cells could play an important role in the maintenance of healthy pregnancy.
The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.
It has been proposed that progesterone (P4) induces the suppression of immune responses, particularly during pregnancy. However, knowledge about the mechanisms involved has remained largely elusive. We demonstrate herein that peripheral blood NK (PBNK) cells express both classical progesterone receptor (PR) isoforms and are specifically affected by the actions of P4 through two apparently independent mechanisms. Progesterone induces caspase-dependent PBNK cell death, which is reversed by two different anti-progestins, ZK 98.299 and RU 486, supporting the involvement of classical PR isoforms. It was suggested that CD56(bright)CD16(-) killer Ig-like receptor (KIR)(-) NK cells might represent precursor cells, which, upon activation, acquire the features of a more mature NK subset expressing KIR receptors. The present study demonstrates that PR expression seems to be restricted to more mature KIR(+) PBNK cells. The expression of PR had a functional counterpart in the suppressive effect of P4 on IL-12-induced IFN-gamma secretion. This cytokine suppression was mainly observed in KIR(+) PBNK cells, without affecting the high secretion of IFN-gamma by CD56(bright) PBNK cells. The lack of PR expression on CD56(bright)KIR(-) PBNK cells provides an additional phenotypic marker to test the idea that they might represent the PBNK precursors selectively recruited into the endometrium where they differentiate to become the uterine NK cells. Additionally, these findings may be relevant to NK cell function in viral immunity, human reproduction, and tumor immunity.
Natural killer cells (NK) and T-lymphocytes with NK functions (NKT) are the leading effectors of the mother’s immune tolerance to a semi-allogeneic fetus and have a fetal trophic effect during physiological pregnancy. Tim-3 (T-cell Ig and mucin domain-containing protein 3) and CD9 molecules play a critical role in the immunoregulatory and fetal trophic functions of NK and NKT, but their expression in peripheral blood cells has not been studied. The aim of this work was to study the expression of Tim-3 and CD9 in peripheral blood NK and NKT during physiological pregnancy. The object of the study was the peripheral blood of healthy women in I and III trimesters of a physiological pregnancy. The control group consisted of healthy non-pregnant women in the first phase of the menstrual cycle. Expression of Tim-3, CD9 molecules was analyzed by flow cytometry on regulatory NK (CD16–CD56bright) and NKT (CD16–CD56+), cytotoxic NK (CD16+CD56dim/–) and NKT (CD16+CD56+). It was found that in the first trimester of pregnancy, the total number and subpopulation composition of NK and NKT cells did not change. Tim-3 expression increased in all NK and NKT subpopulations, except for cytotoxic CD16+CD56dimNK. CD9 expression increased in all NK subpopulations, but in NKT did not differ from non-pregnant. At the same time, a direct correlation between CD9 and Tim-3 expressions was revealed in regulatory NK and NKT in the first trimester of pregnancy. In the third trimester, the regulatory CD16–CD56brightNK number increased, while cytotoxic CD16+CD56dimNK and regulatory CD16–CD56+NKT decreased compared to non-pregnant women. The number of CD16+CD56–NK did not change in I and III trimesters of a physiological pregnancy. Tim-3 expression was upregulated in all NK and cytotoxic NKT subpopulations, while CD9 was upregulated only in regulatory NKs. Thus, Tim-3 and CD9 molecule expressions of s on different NK and NKT subpopulations changed during I and III trimesters of a physiological pregnancy, which plays an important role in the regulation of their phenotype and functions.
During pregnancy, complicated connections are formed between a mother and a fetus. In a successful pregnancy, the maternal-fetal interface is affected by dynamic changes, and the fetus is protected against the mother’s immune system. Natural killer (NK) cells are one of the immune system cells in the female reproductive system that play an essential role in the physiology of pregnancy. NK cells not only exist in peripheral blood (PB) but also can exist in the decidua. Studies have suggested multiple roles for these cells, including decidualization, control of trophoblast growth and invasion, embryo acceptance and maintenance by the mother, and facilitation of placental development during pregnancy. Natural killer T (NKT) cells are another group of NK cells that play a crucial role in the maintenance of pregnancy and regulation of the immune system during pregnancy. Studies show that NK and NKT cells are not only effective in maintaining pregnancy but also can be involved in infertility-related diseases. This review focuses on NK and NKT cells biology and provides a detailed description of the functions of these cells in implantation, placentation, and immune tolerance during pregnancy and their role in pregnancy complications.
Background & Aims
Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function, and subset distribution, including the appearance of CD56‐CD16+ (CD56‐) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56‐ NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown.
Methods
Phenotype and function of CD56‐ NK cells from patients with CHB (n=28) were assessed using flow cytometry and in vitro stimulation with HBV antigen.
Results
CHB patients had a higher frequency of CD56‐ NK cells compared to healthy controls in peripheral blood (6.2 vs 1.4%, p<0.0001). Compared to CD56+ NK cells, CD56‐ NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56‐ NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56‐ NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69, and impaired capacity of NK cells to eliminate immature DCs in co‐culture studies. Finally, frequency of CD56‐ NK cells was positively correlated with serum HBV DNA levels.
Conclusion
Chronic HBV infection induces the expansion of highly dysfunctional of CD56‐ NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.
The immunological paradox of pregnancy, when maternal immune system recognizes but do not reject the semiallogenic fetus, is not yet fully understood. The aim of the present work was to detail the mechanisms of the interaction of decidual CD56+ NK, infiltrating the maternal part of placenta, and trophoblast cells of fetal origin. Samples of the endometrial tissue from 13 healthy non-pregnant women, 37 placentas, obtained after medical abortion of viable pregnancy at 7-10 weeks of gestation, and 26 samples of placentas from first-trimester recurrent spontaneous abortion (RSA) were used as the material for investigation. Phenotype of NK was assessed by flow cytometry. The influence of trophoblast cells upon IFNγ and GrB mRNAs expression by dNK was investigated by RT-PCR. The influence of dNK upon trophoblast cells migration and invasion was studied using collagen and Matrigel systems. In RSA group comparing to the normal pregnancy the decrease of dNK with inhibitory receptors (NKG2A) and elevation of activated dNK were seen. In normal pregnancy, but not in RSA, trophoblast cells increased the expression of IFNγ and GrB mRNAs by CD56+ dNK. Both in normal and RSA pregnancy dNK inhibited the migration and invasion of trophoblast cells. Initially low invasive and migration capacities of trophoblast cells were seen during RSA. Thus, unbalanced activation of dNK can lead to the impairment of dNK and trophoblast cells interaction during RSA. This article is protected by copyright. All rights reserved.
Mitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial-specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results.
To the editor:
In April 2009, we saw a 64-year-old woman with persistent indolent proliferation of CD3−CD56+ large granular lymphocytes.[1][1] She had no other clinical symptoms and no autoimmune events, and her clinical course has remained benign over 22 months. Neither clinical nor imaging
Progesterone is crucial for the establishment and maintenance of pregnancy. Progesterone-regulated genes in the pregnant uterus control the development of endometrial receptivity as well as recruitment and differentiation of decidual NK cells, which in turn act on angiogenesis and trophoblast invasion. The link between progesterone and the immune system is established by lymphocyte progesterone receptors expressed in peripheral blood gammadelta T cells of pregnant women and in peripheral NK cells. Regulation of lymphocyte progesterone receptors is activation related, thus efficient recognition of fetal antigens is a requirement for the initiation of progesterone-dependent immunoregulatory mechanisms. Several immunological effects of progesterone are mediated by progesterone-induced blocking factor--the product of a progesterone-induced gene in lymphocytes. One part of unexplained recurrent miscarriages might have an immunological etiology. Immunization of the mothers with paternal or third-party leukocytes aims to correct the misregulated antifetal immune response. There are, however, serious concerns about this treatment, including the lack of information about the mode of action and possible adverse effects of the treatment, the failure to detect a significant effect of immunotherapy and the lack of a reliable generally accepted marker for patient selection. These concerns will be discussed in this review.
Natural killer (NK) cells are potent immune effector cells that can respond to infection and cancer, as well as allowing maternal adaptation to pregnancy. In response to malignant transformation or pathogenic invasion, NK cells can secrete cytokine and may be directly cytolytic, as well as exerting effects indirectly through other cells of the immune system. To recognize and respond to inflamed or infected tissues, NK cells express a variety of activating and inhibitory receptors including NKG2D, Ly49 or KIR, CD94-NKG2 heterodimers and natural cytotoxicity receptors, as well as co-stimulatory receptors. These receptors recognize cellular stress ligands as well as major histocompatibility complex class I and related molecules, which can lead to NK cell responses. Importantly, NK cells must remain tolerant of healthy tissue, and some of these receptors can also prevent activation of NK cells. In this review, we describe the expression of prominent NK cell receptors, as well as expression of their ligands and their role in immune responses. In addition, we describe the main signaling pathways used by NK cell receptors. Although we now appreciate that NK cell biology is more complicated than first thought, there are still facets of their biology that remain unclear. These will be highlighted and discussed in this review.
NK cells were initially identified based on their capacity to destroy susceptible target cells via granule-mediated cytotoxicity. Subsequently, NK-cell cytokine production (IFN-gamma, TNF-alpha) was shown to be critical in restricting pathogen infection, defining a non-cytotoxic role for NK cells in host defense. Recently, specialized NK-cell subsets with biased effector functions have been described in mice and man. This overview will consider the developmental origins and biological implications of this NK-cell diversification.
Fc gamma RIII (CD16) is a hetero-oligomeric receptor composed of a ligand-binding alpha subunit associated with homo- or heterodimers of the TCR zeta- and Fc epsilon RI gamma-chains. We have previously demonstrated that CD16 ligation promotes complex formation between tyrosine-phosphorylated shc and Grb2, leading to activation of ras signaling pathway in human NK cells. Here we report that CD16 engagement induces rapid shc association with the tyrosine-phosphorylated receptor complex in human NK cells. In vitro binding studies demonstrate that this interaction is mediated by the shc-SH2 domain, and immunodepletion experiments indicate that the zeta- but not the gamma-chain has the capability to mediate this association. Jurkat cell clones expressing CD16-zeta or -gamma homodimers have been used to gain more information about the mechanism of shc/CD16 association. Our data show that, while engagement of both receptors induces tyrosine phosphorylation of shc and Grb2 recruitment, shc-SH2/receptor complex association is evident only in CD16-zeta but not in CD16-gamma transfectants. Overall, our data demonstrate that the adaptor protein shc can be recruited to the activated CD16 complex by interaction with tyrosine-phosphorylated zeta-chain in a SH2-dependent manner. These results also provide further support to the notion that zeta- and gamma-chains might couple to different biochemical pathways.
Natural killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors and infections. An important limitation in the field of NK research is attributable to the deficit of assays available for the detection of the functional activity of NK cells. Recently, lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been described as a marker of CD8+ T-cell degranulation following stimulation. Here we describe CD107a as a marker of NK cell functional activity using multi-parameter flow cytometry. CD107a is significantly upregulated on the surface of NK cells following stimulation with MHC devoid targets. Additionally, CD107a expression correlates with both cytokine secretion and NK cell-mediated lysis of target cells. However, as well as being coordinately expressed on nearly all cytokine secreting cells, CD107a was also expressed on a large subset of NK cells that did not secrete cytokine following stimulation. These data suggest that employing CD107a as a marker of NK cell functional activity may allow for the identification of a large fraction of activated NK cells that may degranulate in the absence of cytokine secretion. Cumulatively, the data presented here demonstrate that CD107a is a sensitive marker of NK cell activity.
The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.
MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.
NK cells kill tumor cells and virus-infected cells as well as secrete a variety of cytokines. These effector functions are regulated by the balance between activating receptor signals and inhibitory receptor signals which are triggered by specific major histocompatibility complex (MHC) or non-MHC ligands. It is thought currently that the balance between immunostimulation and immunoregulation in T cell immunity is achieved by a Th1/Th2/Th3/Tr1 and CD4(+)CD25(+) regulatory T (Treg) cell paradigm. Here, we discuss the cytokine paradigm of NK cells in human pregnancy. During normal, intact pregnancy, peripheral blood NKr1 cells and decidual NK3 cells increase, while these NK cell populations decrease significantly in miscarriage cases, suggesting that an imbalance in NK1/NK2/NK3/NKr1 is correlated with miscarriage. Recent investigations have shown that not only Treg cells, but also regulatory NK (NK reg) cells, play very important roles in the maintenance of pregnancy. We summarize the progress in studying NK reg cells and focus on how NK reg cells and cytotoxic NK cells affect the reproductive immune response.
Recent study has suggested that innate immune system might play an important role in pregnancy progression. In this study, to investigate whether NK cells and NKT cells, instead of T cells, are the dominant populations of peripheral blood in early pregnancy, flow cytometry was used to detect the percentage and intracellular cytokine expressions of T cells, NK cells, NKT cells in peripheral blood of non-pregnant women and early pregnant women. In our result, the percentages of NK cells and NKT cells were significantly increased in pregnancy compared to non-pregnancy. However, the percentage of T cells was not changed. We did not detect the Th2-dominance of total lymphocytes or T cells in peripheral blood of early pregnant women and there were also no significant changes of type 1 and type 2 cytokines in T cells, but IFN-gamma production in both NK and NKT cells was decreased in early pregnancy. These results suggest that the innate immune system including NK cells and NKT cells should play a pivotal role in pregnancy progression. Type 1/type 2 shift mechanisms in innate immune system during the human early pregnancy should be paid more attention.
Иммунология материнско-фетальных взаимодействий. Екатеринбург. УрО РАН
- С В Ширшев
Characteristics of the subpopulation composition of immunocompetent cell placenta after a pre-existing layer
- О С Гребнева
- М Ю Зильбер
- O S Grebneva
- M U Silber