Mechanism of CD1d-restricted natural killer T cell activation during microbial infection

CD1d-restricted natural killer T (NKT) cells are important for host defense against a variety of microbial pathogens. How and when these T cells become activated physiologically during infection remains unknown. Our data support a model in which NKT cells use a unique activation mechanism not requiring their recognition of microbial antigens. Instead, weak responses to CD1d-presented self antigens were amplified by interleukin 12 made by dendritic cells in response to microbial products, resulting in potent interferon-gamma secretion. NKT cells were among the first lymphocytes to respond during Salmonella typhimurium infection, and their activation in vivo also depended on interleukin 12 and CD1d recognition. We propose this mechanism of activation as a major pathway responsible for the rapid activation of NKT cells in different microbial infections.     

Lipid and glycolipid antigens of CD1d-restricted natural killer T cells

In spite of their relatively limited antigen receptor repertoire, CD1d-restricted NKT cells recognize a surprisingly diverse range of lipid and glycolipid antigens. Recent studies of natural and synthetic CD1d-presented antigens provide an increasingly detailed picture of how the specific structural features of these lipids and glycolipids influence their ability to be presented to NKT cells and stimulate their diverse immunologic functions. Particularly for synthetic analogues of α-galactosylceramides which have been the focus of intense recent investigation, it is becoming clear that the design of glycolipid antigens with the ability to precisely control the specific immunologic activities of NKT cells is likely to be feasible. The emerging details of the mechanisms underlying the structure–activity relationship of NKT cell antigens will assist greatly in the design and production of immunomodulatory agents for the precise manipulation of NKT cells and the many other components of the immune system that they influence.     

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.     

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.     

Critical proinflammatory and anti-inflammatory functions of different subsets of CD1d-restricted natural killer T cells during Trypanosoma cruzi infection

Trypanosoma cruzi infects 15 to 20 million people in Latin America and causes Chagas disease, a chronic inflammatory disease with fatal cardiac and gastrointestinal sequelae. How the immune response causes Chagas disease is not clear, but during the persistent infection both proinflammatory and anti-inflammatory responses are critical. Natural killer T (NKT) cells have been shown to regulate immune responses during infections and autoimmune diseases. We report here that during acute T. cruzi infection NKT-cell subsets provide distinct functions. CD1d(-/-) mice, which lack both invariant NKT (iNKT) cells and variant NKT (vNKT) cells, develop a mild phenotype displaying an increase in spleen and liver mononuclear cells, anti-T. cruzi antibody response, and muscle inflammation. In contrast, Jalpha18(-/-) mice, which lack iNKT cells but have vNKT cells, develop a robust phenotype involving prominent spleen, liver, and skeletal muscle inflammatory infiltrates comprised of NK, dendritic, B and T cells. The inflammatory cells display activation markers; produce more gamma interferon, tumor necrosis factor alpha, and nitric oxide; and show a diminished antibody response. Strikingly, most Jalpha18(-/-) mice die. Thus, in response to the same infection, vNKT cells appear to augment a robust proinflammatory response, whereas the iNKT cells dampen this response, possibly by regulating vNKT cells.     

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells

Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t(1/2) life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.     

Turned on by danger: activation of CD1d-restricted invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.     

Acidification-dependent activation of CD1d-restricted natural killer T cells is intact in cystic fibrosis

CD1d‐restricted natural killer T (NKT) cells are emerging as critical regulators of the immune response to infectious agents, including Pseudomonas aeruginosa; and therapies to augment NKT‐cell activation may represent a novel approach to treat chronic, antibiotic‐resistant bacterial infections. We examined the capacity of dendritic cells (DCs) from people with cystic fibrosis (CF) to activate NKT cells. Our study was motivated by three lines of evidence: (i) NKT cells play a critical role in clearing P. aeruginosa infection; (ii) activation of NKT cells requires acidification‐dependent processing of glycolipid antigens within the endolysosomal compartment; and (iii) endolysosomal acidification may be reduced in CF. We demonstrated that NKT‐cell activation was dependent upon intact organelle acidification as inhibitors of the vacuolar (H⁺)‐ATPases prevented DCs from activating NKT cells with two glycolipid antigens, α‐galactosylceramide and galactose‐galactosylceramide. In contrast, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel dysfunction had no significant biological impact on the capacity of DCs to activate NKT cells. Dendritic cells from subjects with CF and DCs treated with the thiazolidinone CFTR_inh‐172 inhibitor showed no reduction in their ability to activate NKT cells. Based on these data, we find no evidence for an inherent defect in glycolipid antigen presentation to NKT cells in CF subjects.     

Therapeutic potential of CD1d-restricted invariant natural killer T cell-based treatment for autoimmune diseases

CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of T cells that recognize glycolipid antigens presented by the CD1d molecule. iNKT cells participate in various kinds of immunoregulation due to a potent ability to produce a variety of cytokines. Recent advances in studies of novel synthetic glycolipid ligands has led to new strategies to manipulate the pleiotropic functions of iNKT cells. The molecular mechanism of selective cytokine production by glycolipid ligands will be discussed. We will also focus on the possible therapeutic application of such ligands for the clinical treatment of various autoimmune diseases.     

CD56bright natural killer (NK) cells: an important NK cell subset

Human natural killer (NK) cells can be subdivided into different populations based on the relative expression of the surface markers CD16 and CD56. The two major subsets are CD56^bright CD16^dim/− and CD56^dim CD16⁺, respectively. In this review, we will focus on the CD56^bright NK cell subset. These cells are numerically in the minority in peripheral blood but constitute the majority of NK cells in secondary lymphoid tissues. They are abundant cytokine producers but are only weakly cytotoxic before activation. Recent data suggest that under certain conditions, they have immunoregulatory properties, and that they are probably immediate precursors of CD56^dim NK cells. CD56^bright NK cell percentages are expanded or reduced in a certain number of diseases, but the significance of these variations is not yet clear.     

CD1d-restricted natural killer T cells are potent targets for human immunodeficiency virus infection

Invariant human natural killer T cells (NKT) express a restricted T-cell receptor (TCR) Valpha24Vbeta11 repertoire. These cells share both phenotypic and functional similarities between NK and T cells. Given the emerging role of NKT cells as critical cells in bridging the gap between innate and adaptive immunity, we examined their susceptibility to productive human immunodeficiency virus (HIV) infection by T-tropic, M-tropic, and primary isolates of HIV. We generated three human NKT cell clones (CA5, CA29, and CA31). Phenotypic characterization of these Valpha24+ Vbeta11+ clones indicated that they were predominately positive for CD4, CD161, HLA-DR, CD38, CD45RO, and CD95 expression. The NKT cell clones expressed significantly more surface CCR5 molecules/cell and lower CXCR4 molecules/cell than phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMC). Consistent with the surface expression of CCR5 and CXCR4, the NKT clones were also selectively susceptible to HIV M-tropic, T-tropic, and primary isolate infection, as evaluated by both HIV p24 enzyme-linked immunosorbent assay and intracellular staining of HIV proteins. The amount of p24 production was dependent on the NKT clone studied and the HIV strain used. Clones CA29 and CA31 were also susceptible to HIV IIIB infection. The virions produced by these clones were able to productively infect PHA-stimulated PBMCs with the same kinetics as for primary infection of CD4+ blast. Collectively, this data demonstrates that NKT cells can be a target for productive HIV infection but with a lag in the time to peak p24 production.     

Blastic natural killer (NK) cell leukemia (agranular CD4+CD56+ leukemia)

Blastic NK cell lymphoma is a rare hematolymphoid neoplasm. This report illustrates an unusual presentation of this entity, namely as a primary leukemia, but without skin lesions.     

CD56bright natural killer cell subsets: Characterization of distinct functional responses to interleukin-2 and the c-kit ligand

Natural killer (NK) cells are bone marrow-derived large granular lymphocytes that express the CD56 surface antigen. The CD56^bright NK subset represents approximately 10 % of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcRγIII) at high density, whereas CD56^bright NK cells either lack CD 16 (CD56^brightCD16^?) or express it at low density (CD56^brightCD16^dim). c-kit is a tyrosine kinase receptor which is expressed on both CD34⁺ hemato-poietic precursor cells and CD56^bright NK cells. In the current study, we characterize interleukin (IL)-2 receptor (IL-2R) and c-kit expression in each of the CD56^bright subsets. Both the CD56^brightCD16^? and CD56^brightCD16^dim NK subsets express the high-affinity IL-2R and the c-kit receptor when isolated from fresh blood. However, each CD56^bright NK cell subset has distinct functional responses to IL-2, the c-kit ligand (KL), or both. Activation of the high-affinity IL-2R on CD56^brightCD16^? NK cells induces a proliferative response that is significantly weaker than that observed in the CD56^brightCD16^dim NK cell subset. Incubation of the CD56^brightCD16^? NK cell subset with KL significantly enhances IL-2-induced proliferation, while KL has no such effect on the CD56^brightCD16^dim NK subset. Activation of the high-affinity IL-2R in both CD56^bright subsets induces lymphokine-activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co-stimulation of either CD56^bright subset with IL-12 and concentrations of IL-2 that only saturate the high-affinity IL-2R induces substantial interferon (IFN)-γ production. The addition of KL to this co-stimulatory signal enhances IFN-γ production in both CD56^bright NK subsets. The distinct functional responses to IL-2 and KL seen in the CD56^brightCD16^? and CD56^brightCD16^dim NK subsets provide insight into IL-2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.     

Deficiency in circulating natural killer (NK) cell subsets in common variable immunodeficiency and X-linked agammaglobulinaemia

Absolute and relative NK cell numbers were determined in peripheral whole blood by flow cytometry in patients with common variable immunodeficiency (CVID) (n = 55) and X-linked agammaglobulinaemia (XLA) (n = 19) on regular immunoglobulin (IVIG) therapy. Absolute CD3-CD16+ NK cell numbers were significantly reduced in CVID patients (median 108/microl, range 23-815), compared with normal subjects (n = 60) (289/microl, range 56-640, P < 0.001). Total lymphocyte concentrations were significantly lower in CVID (median 1587/microl, range 523-7519) compared with normal subjects (median 2019/microl, range 1124-3149, P = 0.004), with the percentage of NK cells also being significantly decreased (median 7.5%, range 3.0-33. 0%, compared with 14.2%, range 2.6-30.8%, P < 0.001). In XLA, absolute NK cell numbers (median 140/microl, range 32-551, P < 0. 001) but not relative numbers were significantly reduced compared with normal controls. We excluded the possibility that IVIG interferes with in vitro binding of CD16 MoAbs. Further analysis of NK cell subsets showed a deficiency of both CD16+ and CD56+ cells in CVID, most marked in the CD3-CD8dim subpopulation, which may be due to increased homing of these cells to the gut. Serial studies on a small number of patients suggest that IVIG therapy has no short-term effect on NK cells, although we cannot exclude an effect with prolonged use. Although there are no obvious clinical effects of the NK depletion in CVID and XLA, this may be a factor in their predisposition to cancer.     

Murine CD1d-restricted T cell recognition of cellular lipids

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.     

Intravital imaging reveals distinct dynamics for natural killer and CD8(+) T cells during tumor regression

Recognition of NKG2D ligands by natural killer (NK) cells plays an important role during antitumoral responses. To address how NKG2D engagement affects intratumoral NK cell dynamics, we performed intravital microscopy in a Rae-1β-expressing solid tumor. This NKG2D ligand drove NK cell accumulation, activation, and motility within the tumor. NK cells established mainly dynamic contacts with their targets during tumor regression. In sharp contrast, cytotoxic T lymphocytes (CTLs) formed stable contacts in tumors expressing their cognate antigen. Similar behaviors were observed during effector functions in lymph nodes. In vitro, contacts between NK cells and their targets were cytotoxic but did not elicit sustained calcium influx nor adhesion, whereas CTL contact stability was critically dependent on extracellular calcium entry. Altogether, our results offer mechanistic insight into how NK cells and CTLs can exert cytotoxic activity with remarkably different contact dynamics.     

Antigen recognition by CD1d-restricted NKT T cell receptors

αβ T cell receptors (TCRs) have traditionally been viewed as receptors for peptide antigens presented by either Major Histocompatibility Complex (MHC) class I (for CD8 T cells) or MHC class II (for CD4 T cells) antigen-presenting molecules. However, it is now clear that some T cell lineages express TCRs that are specialized for recognition of lipid-based antigens presented by the MHC class I-like CD1 family. Recently, the molecular basis for the TCR recognition of glycolipid antigens presented by CD1d has revealed an evolutionarily conserved-docking mode that is distinct from that of peptide-based recognition. T cells carrying these receptors follow a unique developmental pathway that results not only in unconventional antigen specificity, but also seemingly exaggerated functional capabilities, which makes these cells and their antigens highly attractive targets for immunotherapeutic manipulation.     

Developing understanding of the roles of CD1d-restricted T cell subsets in cancer: reversing tumor-induced defects

Invariant natural killer T-cells ('iNKT') are the best-known CD1d-restricted T-cells, with recently-defined roles in controlling adaptive immunity. CD1d-restricted T-cells can rapidly produce large amounts of Th1 and/or Th2//Treg/Th17-type cytokines, thereby regulating immunity. iNKT can stimulate potent anti-tumor immune responses via production of Th1 cytokines, direct cytotoxicity, and activation of effectors. However, Th2//Treg-type iNKT can inhibit anti-tumor activity. Furthermore, iNKT are decreased and/or reversibly functionally impaired in many advanced cancers. In some cases, CD1d-restricted T-cell cancer defects can be traced to CD1d(+) tumor interactions, since hematopoietic, prostate, and some other tumors can express CD1d. Ligand and IL-12 can reverse iNKT defects and therapeutic opportunities exist in correcting such defects alone and in combination. Early stage clinical trials have shown potential for reconstitution of iNKT IFN-gamma responses and evidence of activity in a subset of patients, with rational new approaches to capitalize on this progress ongoing, as will be discussed here.     

Decreased natural killer (NK) cell function in chronic NK cell lymphocytosis associated with decreased surface expression of CD11b

Chronic natural killer cell lymphocytosis (CNKL) is characterized by greatly increased numbers of natural killer (NK) cells and patients with this disease may survive for long periods. This is in contrast to patients with leukemic proliferations of NK cells who can have a rapidly progressive clinical course. We identified a pediatric patient who was largely healthy who had CNKL and we sought to determine if the expanded CD16(+)CD3(-) population in this patient functions differently than classical NK cells. Cytotoxic activity against NK cell-sensitive K562 target cells was present, but lower than that in control donors when calculated as lytic units per CD16(+)CD3(-) cell. This cytolytic activity was inducible in patient samples by IL-2/IL-12 stimulation proportionately to that induced in samples from control donors. Intracellular perforin was also present and induced in patient CD16(+)CD3(-) cells similarly to controls. Other presumed NK cell activities, such as IL-2/IL-12 induced IFN-gamma expression and initiation of apoptosis evidenced by annexin V binding after CD16 crosslinking were present in patient samples. Patient CD16(+)CD3(-) cells, however, differed from classical NK cells, as the majority did not express CD56, CD57, CD8, or CD11b. Most convincingly, there was a 5 log decrease in CD11b expression in patient CD16(+)CD3(-) cells compared to control as determined by mean channel fluorescence. These observed differences may explain the relatively benign phenotype of this disorder.