Natural Killer T Cell Function Research Articles

Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions

BackgroundNatural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery.ResultsWe found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed.Conclusions and SignificanceTogether, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines.

Hyporesponsiveness to Natural Killer T-Cell Ligand α-Galactosylceramide in Cancer-Bearing State Mediated by CD11b+ Gr-1+ Cells Producing Nitric Oxide

CD1d-restricted natural killer T (NKT) cells are a potential therapeutic target for cancer, for which several clinical trials have already been reported. NKT cells are specifically activated by a synthetic glycolipid, alpha-galactosylceramide (alpha-GalCer). However, it is known that, in human cancer patients, NKT cells express a degree of hyporesponsiveness to alpha-GalCer. In this study, we have examined the mechanism by which hyporesponsiveness to alpha-GalCer can be induced. In cancer-bearing mice, alpha-GalCer-induced NKT cell expansion, cytokine production, cytotoxicity, and antimetastatic effect in vivo were all significantly impaired. In fact, alpha-GalCer could eliminate metastatic disease in naive animals but failed to protect cancer-bearing mice. CD11b(+) Gr-1(+) cells were particularly increased in cancer-bearing mice and were necessary and sufficient for the suppression of the alpha-GalCer response in a nitric oxide-mediated fashion. Administration of a retinoic acid to cancer-bearing mice reduced the population of CD11b(+) Gr-1(+) cells and effectively restored alpha-GalCer-induced protection. These results show a novel feature of NKT cell function in cancer. Furthermore, our data suggest a new strategy to enhance NKT cell-mediated anticancer immune responses by suppressing CD11b(+) Gr-1(+) cell functions.

Enhancement of ligand-dependent activation of human natural killer T cells by lenalidomide: therapeutic implications

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand alpha-galactosylceramide (alpha-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-gamma. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.

Loss of Morphine–induced Suppression of NK Cell activity and T-cell Functions in μ-Opioid Receptor Knockout Mice

In vivo administration of µ-opioid receptor selective agonists to various species is known to suppress lymphocyte, NK cell, and macrophage functions, in addition to mediate pain relief and euphoria. Using a mouse model in which the µ-opioid receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in natural killer (NK) cell activity and T lymphocyte function. Following peripheral morphine administration, NK cell activity was not affected in homozygous µ-opioid receptor knockout mice, heterozygous animals marginally responded to the immunosuppressive effects of the drug, while wild-type animals were significantly suppressed. In addition, splenic T-cell proliferative responses to concanavalin A, phytohemaglutinin and an antibody to T-cell receptorabαβ (TCR) plus interleukin-2 were not affected by morphine treatment in µ-opioid receptor knockout homozygous and heterozygous mice, whereas morphine significantly suppressed T-cell proliferation in wild-type mice. Taken together, these results suggest a role of the µ-opioid receptor in immunoregulation.

The Role of Natural Killer T Cells in Atherosclerosis

Atherosclerosis is a chronic inflammatory disease characterized by the progressive growth of lesions in the artery wall with activation of both innate and adaptive immunity. Recently, a unique group of immune cells, termed natural killer T (NKT) cells, has emerged as playing a critical role in the regulation of inflammation and immunity. NKT cells share characteristics of conventional T lymphocytes and natural killer cells and express an invariant T cell receptor, which recognizes glycolipid antigen associated with the major histocompatibility complex class I-like molecule CD1d. NKT cells play a role in protection against many autoimmune, inflammatory diseases such as type 1 diabetes, multiple sclerosis and systemic lupus erythematosus. Interestingly, new data is emerging that describes a role for NKT cells in atherosclerosis. Recent studies have shown that CD1d and NKT cells are present in the atherosclerotic lesions of both humans and mice. Furthermore, we and others have demonstrated that absence of CD1d in apolipoprotein E-deficient (apoE-/-) mice ameliorates atherosclerosis and that specific activation of NKT cells is proatherogenic. In addition, hyperlipidemic apoE-/- mice exhibit defective NKT cell numbers and functions. These findings and the well-established role of NKT cells in regulating immunity make these cells attractive targets for immunotherapy of atherosclerosis. Keywords: atherosclerosis, natural killer t cells, inflammation, autoimmunity, glycolipids

Negative feedback regulation of T helper type 1 (Th1)/Th2 cytokine balance via dendritic cell and natural killer T cell interactions

AbstractThe ability of extracellular stimuli to modulate dendritic cell (DC) activation of natural killer T (NKT) cells was not well understood. We investigated the effects of the T helper type 1 (Th1)/Th2-cytokine environment on DC induction of NKT cell-mediated cytokine production in mice. Pretreatment of myeloid DCs with Th1 or Th2 cytokines, interleukin (IL)-4 or interferon (IFN)-γ, led to the enhanced production of reciprocal cytokines by NKT cells (eg, IL-4 pretreatment led to the enhanced production of Th1 cytokines) in vitro and in vivo. Thus, the recognition of Th1 or Th2 cytokines by DCs acts as a negative feedback loop to maintain Th1/Th2-cytokine balance via NKT cell functions. Using these data, we manipulated cytokine levels and innate cytolytic activity in vivo to increase an antitumor response. This is the first description of a novel regulation system governing Th1/Th2 cytokine balance involving DCs and NKT cells. (Blood. 2005;106:1685-1693)

Development and function of CD1d-restricted NKT cells: influence of sphingolipids, SAP and sex

Natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. They express an invariant and conserved T-cell receptor that recognizes glycolipids presented by CD1d. Manipulation, activation or loss of NKT cells has profound effects on diverse immune functions and can influence desirable responses to infectious organisms and tumors, as well as undesirable responses to autoantigens. Here, we discuss recent progress in the identification of natural endogenous and exogenous CD1d-presented antigens, the understanding of factors that regulate development of NKT cells in the thymus and of factors that influence their function in the periphery. Clinical implications of these findings are briefly touched on.

Application of Natural Killer T-Cells to Posttransplantation Immunotherapy

Graft-versus-host disease (GVHD) and graft-versus leukemia (GVL) effects are closely related to each other after allogeneic stem cell transplantation. This association exists because of the extensive and complicated interaction between cellular donor components and recipient components concomitant with cytokine storms. It has been demonstrated that part of this interaction may be related to the induction of a variety of regulatory cells, such as regulatory T-cells and natural killer T (NKT) cells. A lower number of NKT cells may be found in patients with autoimmune diseases, cancer, viral infection, and severe GVHD. When activated, NKT cells rapidly release suppressive cytokines, such as interleukin 4 (IL-4), IL-10, and IL-13, as well as inflammatory cytokines, such as interferon gamma and tumor necrosis factor alpha. NKT cells therefore act as a double-edged sword in their progressive or suppressive effects on diseases. Such contradictory phenomena may be related to the function or types of antigen-presenting cells (APCs) in response to their ligand. A single-dose injection of a ligand for NKT cells, alpha-galactosylceramide (alpha-GalCer), can induce immunity through fully mature dendritic cells in an antigen-specific manner. By contrast, multiple injections of alpha-GalCer would induce tolerance, which may be caused by immature APCs. This response suggests that the function of NKT cells can be determined by alpha-GalCer for controlling the immune response. Furthermore, activation of NKT cells followed by activation of APCs and IL-12 production may lead to activation of NK cells and suppress GVHD in mismatched major histocompatibility complex combinations or may induce GVL effects. Control and modification of NKT cell function may play an important role in regulating GVHD/GVL effects.

Natural killer T cells and rheumatoid arthritis: friend or foe?

Natural killer T cells and rheumatoid arthritis: friend or foe?

Osteopontin as a Mediator of NKT Cell Function in T Cell-Mediated Liver Diseases

Both osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases.

NK and NKT cell functions in immunosenescence.

Immunosenescence is defined as the state of dysregulated immune function that contributes to the increased susceptibility to infection, cancer and autoimmune diseases observed in old organisms, including humans. However, dysregulations in the immune functions are normally counterbalanced by continuous adaptation of the body to the deteriorations that occur over time. These adaptive changes are likely to occur in healthy human centenarians. Both innate (natural) and adaptive (acquired) immune responses decline with advancing age. Natural killer (NK) and natural killer T (NKT) cells represent the best model to describe innate and adaptive immune response in aging. NK and NKT cell cytotoxicity decreases in aging as well as interferon-gamma (IFN-gamma) production by both activated cell types. Their innate and acquired immune responses are preserved in very old age. However, NKT cells bearing T-cell receptor (TCR) gammadelta also display an increased cytotoxicity and IFN-gamma production in very old age. This fact suggests that NKT cells bearing TCRgammadelta are more involved in maintaining innate and adaptive immune response in aging leading to successful aging. The role played by the neuroendocrine-immune network and by nutritional factors, such as zinc, in maintaining NK and NKT cell functions in aging is discussed.

Alpha-galactosylceramide-driven expansion of human natural killer T cells is inhibited by prednisolone treatment.

The mechanisms of action of most treatments in the autoimmune disorders are unclear and steroids remain the first-line therapy in these diseases. Natural killer T (NKT) cell activity has been implicated in the autoimmune process but whether steroids act via an affect on NKT cell function, such as antigen-specific proliferative capacity, is unknown. Immune thrombocytopenia (ITP) patients were studied ex vivo for NKT cell expansion in response to the specific NKT cell antigen, alpha-galactosylceramide, before, during or after prednisolone treatment. Prednisolone inhibited antigen-specific NKT cell expansion in ITP patients in remission. Untreated ITP patients also showed reduced NKT cell proliferative capacity, although this was less marked than in treated patients. These results support a role for NKT cells in ITP and aid understanding of the immunosuppressive activities of prednisolone in autoimmune disease.

Expansion of human Vα24 + NKT cells by repeated stimulation with KRN7000

Changes in Vα24 +Vβ11 + NKT cell number and function are associated with human autoimmune diseases and cancer. Restoration of this corresponding NKT cell population in mice or in vivo activation with α-galactosylceramide (KRN7000) can prevent or reduce tumor growth and autoimmunity. Although the therapeutic value of these natural killer T (NKT) cells in man remains to be determined, large numbers of functional antigen-specific NKT cells can be expanded in vitro. We show that Vα24 +Vβ11 + human NKT cells are expanded by repeated stimulation with KRN7000, unfractionated donor peripheral blood mononuclear cells (PBMC), and recombinant human interleukin-2 (rhIL-2). NKT cells were expanded continuously for more than 2 months with a potential yield of >10 12 cells. The expanded NKT cells retained their CD4 + or CD4 − phenotype after restimulation and were functional as shown by cytokine secretion, killing of antigen-pulsed target cells, and activation of NK cell cytotoxicity. This expansion method may be useful for proof-of-concept studies involving adoptive transfer of ex vivo-expanded NKT cells as a new therapeutic option for cancer and autoimmune diseases.

Suppression of collagen-induced arthritis by natural killer T cell activation with OCH, a sphingosine-truncated analog of alpha-galactosylceramide.

OCH, a synthetic analog of alpha-galactosylceramide with a truncated sphingosine chain, stimulates natural killer T (NKT) cells to produce predominantly Th2 cytokines. Thus, OCH may be a potential agent for the treatment of Th1-mediated autoimmune diseases. This study was designed to evaluate the protective effects of OCH on collagen-induced arthritis (CIA) in mice. Mice were immunized with type II collagen (CII) and injected intraperitoneally twice per week with OCH, before or after the onset of CIA. They were monitored to assess the effect of OCH treatment on the severity of disease. Anti-CII antibodies and cytokine production were measured by enzyme-linked immunosorbent assay. Expression of cytokine genes was determined by quantitative reverse transcriptase-polymerase chain reaction. OCH inhibited CIA in wild-type C57BL/6 (B6) mice but not in NKT-deficient mice. OCH suppressed CIA in SJL mice, which are prone to autoimmune diseases and have a deficiency in the number and function of NKT cells which is similar to that in patients with autoimmune diseases, even after disease has already developed. Disease protection conferred by OCH correlated with its ability to selectively induce Th2 cytokine production mediated by NKT cells and to promote collagen-specific Th2 responses. Neutralization of interleukin-4 (IL-4) or IL-10 with monoclonal antibodies abolished disease protection by OCH, indicating a critical role for these cytokines. Taken together, our findings suggest that OCH holds possibilities as a therapeutic agent for autoimmune diseases such as rheumatoid arthritis.

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 alpha-galactosylceramide (alpha-GalCer) presented by the major histocompatibility complex (MHC) class I-like molecule 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 to alpha-GalCer stimulation and the capacity of alpha-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of alpha-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.

A reversible defect in natural killer T cell function characterizes the progression of premalignant to malignant multiple myeloma.

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM–ITAM signaling is likely to play an important role in the developmental program of NKT cells.

NKT cells protect against pre-erythocytic malaria

Natural killer T (NKT) cells are a recently recognized, unique subset of lymphocytes that coexpress NK cell receptors and a semi-invariant TCR, which recognizes the MHC class I-like molecule CD1d. In mice, NKT cells express the NK cell marker NK1.1 and an invariant Vα chain TCR, encoded by the Vα14 and Jα281 gene segments, together with a highly skewed set of Vβ chains. NKT cells have been shown to participate in the initiation of certain anti-tumour responses. In the context of CD1d molecules, the glycolipid α-galactosylceramide (α-GalCer) activates murine Vα14-expressing NKT cells, inhibiting hepatic melanoma development. NKT cells have also recently been implicated in the immune response to a number of intracellular pathogens, including Mycobacterium, Toxoplasma and Listeria, with both protective and pathogenic roles proposed. However, the function of NKT cells in immunity to Plasmodium, the protozoan parasite that causes malaria, has hitherto not been examined.

Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide.

It has been established that alpha-galactosylceramide (alpha-GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC-like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of alpha-GalCer in mice. When 2 microg (or more)/mouse of alpha(-GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT-deficient mice such as beta2-microglobulin(-/-) and CD1d(-/-) mice. These results shed further light on the in vivo interaction between NKT cells and alpha-GalCer in mice.

Dendritic cells are targets for human invariant Vα24+ natural killer T-cell cytotoxic activity: An important immune regulatory function

Objective Human invariant Vα24+ natural killer T (NKT) cells, a subpopulation of NK cell-receptor (NKR-P1A)-expressing T cells with an invariant Vα24JαQ T-cell receptor (TCR), are stimulated by the glycolipid a-galactosylceramide (KRN7000), in a CD1d-dependent, TCR-mediated fashion. Little is known about invariant Vα24+ NKT cell function or mechanisms of effector activity. Evidence suggests this cell population protects against autoimmunity and has antitumor effects against leukemia and solid tumors. Materials and Methods We compared the phenotype and function of invariant Vα24+ NKT cells, from patients with chronic myeloid leukemia (CML) and normal donors, generated by stimulation of peripheral blood mononuclear cells with α-galactosylceramide pulsed monocyte-derived dendritic cells. The CD4 − CD8 − (double negative) population was studied further. Results Activated human invariant Vα24+ NKT cells were cytotoxic against autologous and allogeneic peripheral blood dendritic cells and monocyte-derived dendritic cells but not against autologous or allogeneic T-cell PHA blasts, B-cell lymphoblastoid cell lines, monocytes, or leukemic cells from patients with CML. The findings are consistent with previous observations showing the importance of CD1d in target cell recognition. None of the Vα24+ NKT cell lines expressed the NK markers CD16, CD56, CD94, or killer inhibitory receptors, but all expressed NKR-P1A. There was no difference in phenotype, function, or ease of generation of invariant Vα24+ NKT cells between normal donors and patients with CML. Conclusion Based on our results and the previous evidence linking reduced Vα24+ NKT cells to autoimmunity, we propose that double-negative Vα24+ NKT cells have important immune regulatory functions, including contribution to the prevention of excessive antigen stimulation by virtue of cytotoxic activity against antigen presenting cells, particularly in dendritic cells.