NKT cells at the maternal-fetal interface

Establishment of the maternal-fetal interface is characterized by the influx of maternal NK cells, macrophages, and T cells into the decidua. Although a great deal has been learned about the function of NK cells in the decidua, comparatively little is known of decidual T cell function. NKT cells are an unusual T cell subset capable of producing both Th1-like and Th2-like cytokines. Unlike conventional alphabeta T cells that recognize peptides in the context of MHC molecules, NKT cells recognize glycolipids presented by the MHC class I-like molecule, CD1d. Recent reports have demonstrated that NKT cells and CD1d are present at the maternal-fetal interface. Moreover, activation of NKT cells can have dramatic effects on pregnancy. In this article, we will review basic aspects of NKT cell biology and summarize the recent literature on NKT cells at the maternal-fetal interface.     

CD56+-T-cell responses to bacterial superantigens and immune recognition of attenuated vaccines

Natural killer T (NKT) cells, coexpressing natural killer (NK) and T-cell receptors (TCR), are associated with immunity to viruses, tumors, and parasites. A well-characterized subclass of these NKT cells expresses biased TCR and recognizes glycolipids such as alpha-galactoceramide, which is found naturally only in marine sponges and presented by the cell surface glycoprotein CD1d. However, a larger number of T cells present in human blood coexpress the NK marker CD56 and unbiased TCR and do not appear to require CD1 for antigen presentation. Observing high frequencies of CD4 and CD8 coreceptor expression in human CD56+ T cells, we examined the potential role of major histocompatibility complex (MHC) class II molecules in the activation of these cells. Activation of mononuclear cells with bacterial superantigens presented by MHC class II molecules resulted in increased frequency of CD56+ T cells. Primarily, CD4+ cells within the CD56+-T-cell population responded to the bacterial superantigens, and cytokine expression profiles were Th1-like. Further, increased levels of T cells expressing CD56 were observed in mononuclear cell cultures responding to a Staphylococcus aureus vaccine or tetanus toxoid. Collectively, our data suggest that a significant number of CD56+ T cells recognize pathogen-associated ligands in association with MHC class II molecules.     

The immunoregulatory role of CD1d-restricted natural killer T cells in disease

Natural killer T (NKT) cells constitute a T cell subpopulation that shares several characteristics with NK cells. NKT cells are characterized by a narrow T cell antigen receptor (TCR) repertoire, recognize glycolipid antigen in the context of the monomorphic CD1d antigen-presenting molecule, and have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines. Important roles of NKT cells have now been demonstrated in the regulation of autoimmune, allergic, antimicrobial, and antitumor immune responses. Here, we review the immunoregulatory role of NKT cells in disease and discuss NKT cell based immunotherapeutic strategies.     

Characterization of the phenotype and function of CD8(+), alpha / beta(+) NKT cells from tumor-bearing mice that show a natural killer cell activity and lyse multiple tumor targets

Natural Killer (NK) T cells are a specialized T cell population that co-expresses receptors of the NK lineage with the alpha / beta TCR receptor and other T cell surface markers. Their functions, regulation and relationship to other cells in the immune system are not fully understood. This report demonstrates that tumor-bearing C57BL / 6 mice have a population of NKT cells that co-express CD8 and CD161 (NK1.1) surface markers. These cells are maintained in long-term culture with T helper 2 (Th2) cytokine interleukin-4 (IL-4), but produce large amounts of Th1 cytokine interferon-gamma (IFN-gamma) following activation. NK1.1(+)CD8(+) T cells show a potent NK-like cytotoxic activity against multiple tumor targets, and lysis is independent of major histocompatibility complex (MHC)-class I or non-classical MHC-class I molecules (Qa, TL). The NK1.1(+)CD8(+) T cells express Vbeta14 chain of the TCR. These NKT cells are not CD1d restricted, and their cytotoxic activity is CD1d independent. Therefore, they represent a unique subset of T cells with an unknown restriction element which produce large quantities of IFN-gamma following expansion with IL-4. Furthermore, their cytotoxic activity is enhanced by B7 co-stimulatory molecules present on tumor cells. CD161(+) T cells that are expanded in tumor-bearing hosts may function as a part of the innate immune system with potential role(s) in tumor surveillance.     

Differential regulation of Ly49 expression on CD4+ and CD4-CD8- (double negative) NK1.1+ T cells

The pan-NK cell marker NK1.1, present in some mouse strains, is also found on a subset of TCRalphabeta+ lymphocytes termed NKT cells. These cells are primarily CD4+ or CD4-CD8- (double negative, DN), and both NKT subpopulations contain cells reactive with the MHC class I-like molecule CD1d. Murine NK cells express clonally distributed inhibitory receptors of the Ly49 family that bind to different alleles of MHC class I molecules and transmit negative signals regulating NK cell function. Ly49 receptors are also found on TCRalphabeta+ NK1.1+ T cells. To investigate the potential role of inhibitory Ly49 markers in the regulation of NKT cells, we have done a thorough analysis of their expression on different NKT populations. The CD4+ and DN NK1.1+ T cell subsets have traditionally been dealt with as one NK1.1+ T cell population, but we found dramatic differences between these two NKT cell subsets. We demonstrate here expression of Ly49 receptors on DN, but not on CD4+, NK1.1+ T cells in spleen and liver. Absence of the specific MHC class I ligand in the host was associated with elevated levels of expression and, to a greater extent than has been found for NK cells, increased the frequencies of Ly49-positive cells within the DN subset, while CD4+ NK1.1+ cells remained negative. In the thymus and bone marrow both NK1.1+ T cell subsets contained high frequencies of Ly49-positive cells. Results from in vitro stimulation of DN NKT cells further suggest that activation and expansion of NKT cell subsets are regulated by the Ly49 receptors.     

Regulation of immune responses by CD1d-restricted natural killer T cells

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share receptor structures and properties with conventional T lymphocytes and natural killer (NK) cells. NKT cells are specific for glycolipid antigens such as the marine sponge-derived agent α-galactosylceramide (α-GalCer) presented by the major histocompatibility complex (MHC) class I-like molcule CD1d. My laboratory has evaluated the function of NKT cells by generating and analyzing CD1d-deficient mice. These studies showed that CD1d expression is required for NKT cell development, but not absolutely necessary for the generation of polarized T helper (Th) cell responses. Further, we have studied the in vivo response of NKT cells toα-GalCer stimulation and the capacity of α-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of α-GalCer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. Our findings indicate that NKT cells play a regulatory role in the immune response and that specific activation of these cells may be exploited for therapeutic purposes.     

Intensive generation of NK1.1- extrathymic T cells in the liver by injection of bone marrow cells isolated from mice with a mutation of polymorphic major histocompatibility complex antigens

Whether intermediate TCR (TCRint) cells and natural killer T (NKT or NK1.1+TCRint) cells are extrathymically generated remains controversial. This arises from the fact that there are few of these T cells in athymic nude mice and neonatally thymectomized mice. However, when athymic mice were provided with appropriate microenvironments or stimulation, many TCRint cells (mainly NK1.1-) were found to arise in the liver. NKT cells are known to be positively selected by monomorphic major histocompatibility complex (MHC) -like antigens (e.g. CD1d). This is true even if they are CD4+. In other words, a MHC class I-like antigen is restricted to CD4 antigen. This rule is somewhat different from that seen in conventional T cells (i.e. the restriction of class II with CD4 and that of class I and CD8). In the case of NK1.1-TCRint cells, they were selected by polymorphic MHC antigens, but their MHC restriction to CD4 or CD8 antigen was incomplete. This was revealed by experiments of bone marrow transfer with class I (bm 1) or II (bm 12) disparity. Depending on the disparity, a unique cytokine profile in sera was detected. These results suggest that the development of T lineage lymphocytes and MHC restriction to CD4 and CD8 might have occurred in parallell as a phylogenic event, and that NK1.1- extrathymic T cells (i.e. NK1.1-TCRint) are at an intermediate position between NKT cells and conventional T cells in phylogeny.     

An insight into the dendritic cells at the maternal-fetal interface

The conditions that permit the genetically distinct fetus to survive and develop within the mother are among the most fascinating immunologic puzzles. The presence of dendritic cells in the maternal decidua pointed to a biologic role of antigen-presenting cells in maternal-fetal interaction. The method of study included recent findings on the lineage, maturity, phenotype and function of dendritic cells at the maternal-fetal interface. The increment of uterine dendritic cells occurs simultaneously with the decisive phase of gestation, when implantation takes place. Decidual dendritic cells of the first trimester pregnancy, with a phenotype characteristic of the mature myeloid lineage, express MHC class II, co-stimulatory and adhesion molecules, control Th1/Th2 balance and activate the proliferative response of autologous NK cells. Dendritic cells are specifically equipped to control immunity, to trigger immune response and also to maintain tolerance, avoiding the rejection of the conceptus by the maternal immune system.     

Immediate antigen-specific effector functions by TCR-transgenic CD8+ NKT cells

Only recently have natural antigens for CD1d-dependent, invariant Valpha14+ natural killer T (iNKT) cells been identified. Similar data for CD1d-independent and CD8+ NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8+ TCR-transgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8+ NK1.1+ T cells. In liver, most of NK1.1+ T cells express CD8alphaalpha homodimers. Transgenic NKT cells did not bind invariant Valpha14-to-Jalpha18 TCR rearrangement (Valpha14i)-specific CD1d/alpha-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8+ NKT cells recognized their cognate antigen in the context of H2-Kb and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8+ NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8+ NKT cell function in an antigen-specific manner.     

CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasite-specific antibody responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN-gamma and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1(-/-) mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)(-/-) mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arise from both MHCII-dependent and independent pathways.     

Differential distribution of NK cells in decidua basalis compared with decidua parietalis after uncomplicated human term pregnancy

As pregnancy progresses, a characteristic decline in the percentage of CD56bright CD16- uterine natural killer (NK) cells occurs. Studies of term decidua, however, have focused only on leukocytes derived from decidua basalis, the site of implantation. The decidua parietalis, lining the remainder of the uterine cavity is another important region of the maternal-fetal interface that forms contact with fetal tissue at the end of the first trimester. The aim of this study was to evaluate possible differences in expression of CD16 and CD56 on leukocytes from normal term decidua basalis and decidua parietalis. Decidua basalis and parietalis samples were obtained from 30 placentas collected after elective cesarean section. Percentages of leukocyte subpopulations and NK cell subsets within the CD45+ cell fraction were determined by flow cytometry. In six decidual samples, concurrent immunohistochemical staining was performed. Higher percentages of CD56dim CD16+ NK cells and CD56- CD16+ cells were found in decidua basalis in comparison to decidua parietalis. In contrast, the percentage of CD56bright CD16- uterine NK cells was significantly higher in decidua parietalis. Immunohistochemical quantification supported flow cytometric results. We conclude that significant differences exist with respect to the distribution of NK cells in term decidua basalis and parietalis. Future functional studies may improve our understanding of their role at the maternal-fetal interface.     

Application of natural killer T cells in antitumor immunotherapy

NKT cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. NKT cells have the potential to produce key cytokines of both Th1 and Th2 T cells and are involved in the control of several types of immune response. Furthermore, NKT cells perform spontaneous tumor immunosurveillance. Upon specific activation with alpha-GalCer, NKT cells show strong antitumor immune responses through direct cytotoxicity and indirect activation of a cascade of antitumor effector cells such as natural killer (NK) cells and CD8+ cytotoxic T cells. In addition to alpha-GalCer, many other CD1d ligands, including self and bacterial glycolipids and modified synthetic glycolipid antigens, have also been discovered. Structurally different glycolipid antigens have the distinct ability to activate NKT cells. Thus, it seems that we are now close to a position in which we can control the activation status of NKT cells; this makes NKT cells an ideal target of anticancer immunotherapies. Clinical trials with soluble alpha-GalCer or alpha-GalCer-pulsed dendritic cells aimed at in vivo reconstitution and activation of human NKT cells have provided both promising and challenging results. In this review, we discuss NKT-cell-mediated antitumor immune responses, as well as the early outcomes and implications of recent clinical studies.     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system

Natural Killer T cells are a distinct lymphocyte lineage that regulates a broad range of immune responses. NKT cells recognize glycolipids presented by the non-classical MHC molecule CD1d. Structural insight into the TCR/glycolipid/CD1d tri-complex has revealed an unusual and unexpected mode of recognition. Recent studies have also identified some of the signaling events during NKT cell development that give NKT cells their innate phenotype. Pathogen-derived glycolipid antigens continue to be found, and new mechanisms of NKT cell activation have been described. Finally, NKT cells have been shown to be remarkably versatile in function during various immune responses. Whether these extensive functional capacities can be attributed to a single population sensitive to environmental cues or if functionally distinct NKT cell subpopulations exist remains unresolved.     

NKT cells: T lymphocytes with innate effector functions

Natural killer T (NKT) cells are innate-like T lymphocytes that recognize glycolipid antigens in the context of the MHC class I-related glycoprotein CD1d. Recent studies have identified multiple ways in which NKT cells can become activated during microbial infection. Mechanisms of CD1d-restricted antigen presentation are being unraveled, and a surprising connection has been made to proteins that control lipid metabolism and atherosclerosis. It appears that several microorganisms have developed strategies to interfere with the CD1d antigen-presentation pathway. New studies have also provided important insight into the mechanisms that control effector cell differentiation of NKT cells and have revealed specialized functions of distinct NKT cell subsets. Finally, there is continued enthusiasm for the development of NKT cell-based therapies of human diseases.     

Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection

NKT cells are specialized cells of the immune system that bear both T cell and NK cell markers. Classical NKT cells display TCRs of restricted heterogeneity (Valpha14-Jalpha281) and recognize lipid antigens (e.g., alpha-galactosyl ceramide) presented by nonpolymorphic CD1 molecules. Recently, other nonclassical NKT subsets have been recognized, including NKT cells not reactive with CD1d-alpha-galactosyl ceramide complexes. The biological functions of these cells are unknown. Here, we show that nonclassical NKT cells that are CD1d restricted but nonreactive to alpha-GalCer are activated in response to hepatocytes expressing hepatitis B viral antigens in a transgenic mouse model of acute hepatitis B virus infection. Our results document the first in vivo function for such nonclassical NKT cells and suggest a role for these cells in the host response to HBV infection.     

Type II natural killer T cells use features of both innate-like and conventional T cells to recognize sulfatide self antigens

Glycolipids presented by the major histocompatibility complex (MHC) class I homolog CD1d are recognized by natural killer T cells (NKT cells) characterized by either a semi-invariant T cell antigen receptor (TCR) repertoire (type I NKT cells or iNKT cells) or a relatively variable TCR repertoire (type II NKT cells). Here we describe the structure of a type II NKT cell TCR in complex with CD1d-lysosulfatide. Both TCR α-chains and TCR β-chains made contact with the CD1d molecule with a diagonal footprint, typical of MHC-TCR interactions, whereas the antigen was recognized exclusively with a single TCR chain, similar to the iNKT cell TCR. Type II NKT cell TCRs, therefore, recognize CD1d-sulfatide complexes by a distinct recognition mechanism characterized by the TCR-binding features of both iNKT cells and conventional peptide-reactive T cells.     

NKT cell cytokine imbalance in murine diabetes mellitus.

A minor subset of murine MHC class I-restricted T cells which express both the alphabeta form of the T cell receptor and a NK lineage marker, termed NKT cells, is capable of secreting significant amounts of Interleukin-4 and Interferon-y upon activation. As such NKT cells may play a role in development of Th1 and Th2 cells during T cell ontogeny or expansion of T cells expressing a dominant cytokine pattern in the effector phase. We have studied the role of NKT cells in a murine model of disease multidose streptozotocin induced diabetes mellitus (MDSDM). In MDSDM thymic and splenic NKT cells are present at normal levels but have greatly reduced capacity to secrete Interleukin-4 upon stimulation with anti-TCR antibody compared to control mice; conversely, Interferon-y secretion is maintained. By analysis of cytokine RNA production we found that treatment of several strains of mice with streptozotocin changes the peripheral helper T cell phenotype elicited after immunization with Keyhole Limpet Hemocyanin from a mixed Th1- and Th2-type cytokine pattern (characterized by IFN-gamma and IL-4 and IL-5 expressions, respectively) to predominately Th1-type. Furthermore, susceptibility to MDSDM is significantly enhanced when NKT cells are selectively eliminated in vivo by administration of depleting anti-CD122 antibody TMbeta-1. In addition, antibody depletion of NKT cells from non-obese diabetic mice significantly accelerates onset of disease. Collectively these data support a model for development of murine diabetes mellitus in which NKT cell cytokine expression influences the development of Th1-type diabetogenic T cells.     

A critical role for the T cell receptor alpha-chain connecting peptide domain in positive selection of CD1-independent NKT cells.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.