Glycolipid alpha-C-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice

alpha-Galactosylceramide (alpha-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with alpha-GalCer. We find that a recently described analogue, alpha-C-galactosylceramide (alpha-C-GalCer), more potently induces these innate and adaptive immune responses in mice. alpha-C-GalCer acts as a more effective trigger for IL-12 and IFN-gamma production, although it minimally elicits IL-4 and TNF-alpha release into the serum. Also, alpha-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that alpha-C-GalCer binds more stably to dendritic cells than alpha-GalCer and that dendritic cells loaded with alpha-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, alpha-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, alpha-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.     

In vitro differentiation of natural killer T cells from human cord blood CD34+ cells

Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T-cell receptor Valpha24 and Vbeta11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon-gamma and interleukin 4 (IL-4) were detected in the CD56hi fractions. IL-15 was essential and, in combination with either flt3-ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA- and CD4+CD8alpha+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL-15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.     

Recent advances in the role of NKT cells in allergic diseases and asthma

Asthma is the result of chronic airway inflammation that is dominated by the presence of eosinophils and CD4⁺ T lymphocytes. CD4⁺ T cells include several subsets and play a critical role in orchestrating the inflammation, predominantly by secreting interleukin-4 and interleukin-13. Recently, research identified a new subset of T cells, natural killer T (NKT) cells, which also express the CD4 marker. In contrast to conventional CD4⁺ T cells, NKT cells do not respond to peptide antigens, but rather to glycolipids. In animal models of asthma, direct activation of NKT cells by glycolipids results in the secretion of extensive amounts of cytokines and triggers the development of airway hyperreactivity. Moreover, in patients with chronic asthma, NKT cells can be found in bronchoalveolar lavage fluids in significant amounts. These data strongly suggest that NKT cells play an important role in asthma pathogenesis.     

Lymphocytes T CD8+ : implications dans l’asthme

CD8⁺ T lymphocytes include several T cell populations with various functional properties: CD8⁺αβ T cells that produce cytokines (Tc1, Tc2), cytotoxic T cells, CD8⁺ NKT cells, and CD8⁺γδ. Their role in asthma has been assessed in a limited number of studies which are reviewed here. Evidence from animal models indicates that conventional CD8⁺αβ T cells are protective or enhance the immunoallergic immune response. While Tc2 is always an amplifier in immune reactions, the role of Tc1 is still controversial. Cytotoxic T cells, producing perforin and expressing CX3CR1⁺, are found in excess in asthma, notably during exacerbations. Bronchial infiltration by CD8⁺ T cells is associated with reduced FEV₁ in both asthma and COPD; it is also correlated with asthma mortality. During exacerbations, viruses, allergens, and/or pollutants induce CD8⁺ lymphocytes that are able to increase asthmatic inflammation, probably non-specifically, via the involvement of these bystander CD8⁺ cells. The role of the nonconventional CD8⁺γδ T cells, intermediate between innate and adaptive immunity, is likewise controversial and depends on the models studied. They recognize nonpeptidic antigens and are said to be involved in occupational asthma caused by low molecular weight agents. In conclusion, all the CD8⁺ T cell populations are implicated in asthmatic inflammation. They are involved in exacerbations of asthma and its severity. As their activation is less sensitive to corticosteroids than CD4⁺ T cells, targeting CD8⁺ T cells appears to be an important therapeutic target in severe corticosteroid-dependent asthma.     

Regulation of memory CD4 T-cell pool size and function by natural killer T cells in vivo

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4⁺ T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ–producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.     

Mouse V alpha 14i natural killer T cells are resistant to cytokine polarization in vivo

Under different circumstances, natural killer T (NKT) cells can cause a T helper (Th) 1 or a Th2 polarization of immune responses. We show here, however, that mouse NKT cells with an invariant V alpha 14 rearrangement (V alpha 14i NKT cells) rapidly produce both IL-4 and IFN-gamma, and this pattern could not be altered by methods that polarize naive CD4+ T cells. Surprisingly, although cytokine protein was detected only after activation, resting V alpha 14i NKT cells contained IL-4 and IFN-gamma mRNAs. Despite this finding, in vivo priming of mice with the glycolipid antigen recognized by V alpha 14i NKT cells resulted in a more Th2-oriented response upon antigen re-exposure. The V alpha 14i NKT cells from primed mice retain the ability to produce IL-4 and IFN-gamma, but they are less effective at activating NK cells to produce IFN-gamma. Our data therefore indicate that V alpha 14i NKT cells have a relatively inflexible immediate cytokine response, but that changes in their ability to induce IFN-gamma secretion by NK cells may determine the extent to which they promote Th1 responses.     

Inhibition of glycolipid shedding rescues recognition of a CD1+ T cell lymphoma by natural killer T (NKT) cells

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor alpha chain (Valpha14Jalpha281) associated with Vbeta chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1(+) cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Valpha14(+)) but not noncanonical (Valpha5(+)) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells with d-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the host's innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.     

CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses

A variety of tumor-derived antigens have been defined by IgG antibodies in tumor bearers' sera with serological identification of antigens by recombinant expression cloning (SEREX), a serological expression cloning method. The majority of these antigens show no structural abnormality and seem to be wild-type autoantigens. Coimmunization with DNA encoding these autoantigens and tumor-specific cytotoxic T lymphocytes epitopes heightened CD8+ T cell responses and increased resistance to tumor challenge in a CD4+ T cell-dependent manner. In contrast, immunization with these SEREX-defined autoantigens alone leads to heightened susceptibility to tumor challenge. This suppressive effect of immunization is mediated by CD4+ CD25+ T cells. In mice immunized with one of the SEREX-defined autoantigens, Dna J-like 2, the number of alpha-GalCer/CD1d tetramer+ CD3+ T cells [representing natural killer T (NKT) cells] was reduced in the pulmonary compartment, whereas no evident change in the number of other T cell subsets was observed. Experiments with Jalpha281-/- mice lacking most NKT cells indicate that NKT cells are primarily responsible for metastasis suppression and that their activity is inhibited by immunization with Dna J-like 2. We propose that SEREX identifies a pool of autoantigens that maintains and regulates immunological homeostasis via CD4+ CD25+ regulatory T cells.     

Expansion of CD1d-restricted NKT cells in patients with primary HIV-1 infection treated with interleukin-2

Innate CD1d-restricted natural killer T (NKT) cells are infected and lost in HIV-1-infected patients, and this could contribute to HIV-1 pathogenesis because NKT cells play an important role in directing both adaptive and innate immunity. Administration of interleukin-2 (IL-2) to HIV-1-infected patients leads to substantial and sustained CD4+ T-cell expansion, involving both naive and memory cells. We investigated whether IL-2 treatment could restore the NKT cell compartment in patients with primary HIV-1 infection. We show that IL-2 combined with effective antiretroviral therapy (ART) resulted in significant expansion of CD1d-restricted NKT cells. Expansion occurred in both the CD4- and CD4+ subsets of NKT cells, and expanded cells expressed the CD161 maturation marker while expression of the HIV coreceptor CCR5 was reduced. These data indicate that IL-2 treatment in combination with effective ART is beneficial for the restoration of innate NKT cell immunity in patients with primary HIV-1 infection.     

Virus-stimulated plasmacytoid dendritic cells induce CD4+ cytotoxic regulatory T cells

Immune responses to pathogens need to be maintained within appropriate levels to minimize tissue damage, whereas such controlled immunity may allow persistent infection of certain types of pathogens. Interleukin 10 (IL-10) plays an important role in such immune regulation. We previously showed that HSV-stimulated human plasmacytoid dendritic cells (pDCs) induced naive CD4+ T cells to differentiate into interferon gamma (IFN-gamma)/IL-10-producing T cells. Here we show that HSV-stimulated pDCs induce allogeneic naive CD4+ T cells to differentiate into cytotoxic regulatory T cells that poorly proliferate on restimulation and inhibit proliferation of coexisting naive CD4+ T cells. IL-3-stimulated pDCs or myeloid DCs did not induce such regulatory T cells. Both IFN-alpha and IL-10 were responsible for the induction of anergic and regulatory properties. High percentages of CD4+ T cells cocultured with HSV-stimulated pDCs, and to a lesser extent those cocultured with IL-3-stimulated pDCs, expressed granzyme B and perforin in an IL-10-dependent manner. CD4+ T cells cocultured with HSV-stimulated pDCs accordingly exhibited cytotoxic activity. The finding that virus-stimulated pDCs are capable of inducing CD4+ cytotoxic regulatory T cells suggests that this DC subset may play an important role in suppressing excessive inflammatory responses and also in inducing persistent viral infection.     

地塞米松对哮喘小鼠CD4+CD25+调节性T细胞及IL-4、IL-10水平的影响

目的 探讨地塞米松对哮喘小鼠CD4+ CD25+调节性T细胞及IL-4、IL-10水平的影响.方法 30只雄性BALB/c小鼠随机分为三组:正常对照组、哮喘组和地塞米松组.利用卵清白蛋白腹腔注射和雾化吸入制备哮喘模型;通过流式细胞仪检测各组小鼠脾脏单个核细胞CD4+ CD25+调节性T细胞占CD4+T细胞的百分比;使用免疫组织化学方法分析各组IL-4在小鼠肺组织中的表达情况;用酶联免疫吸附试验检测各组小鼠血清IL-10的水平.结果 哮喘组脾脏单个核细胞CD4+CD25+调节性T细胞百分比及IL-10的表达水平较正常对照组和地塞米松组降低(P＜0.05),哮喘组IL-4水平较正常对照组和地塞米松组增高(P＜0.05).结论 地塞米松的抗炎作用可能通过上调CD4+ CD25+调节性T细胞、调节性T细胞亚群失衡的途径来实现.     

Potential of targeting natural killer T cells for the treatment of autoimmune diseases

Natural killer (NK) T cells emerge as unique lymphocytes subsets implicated in the regulation of autoimmunity. Abnormalities in the numbers and functions of NKT cells have been observed in patients with diverse autoimmune diseases as well as in a variety of mouse strains that are genetically predisposed for the development of autoimmune diseases. Unlike conventional T cells that recognize peptides in association with major histocompatibility complex (MHC), NKT cells recognize glycolipid antigens presented by the nonpolymorphic MHC class I-like protein, CD1d. Recently, vigorous activation of NKT cells by synthetic glycolipids such as α-galactosylceramide (α-GC) or its sphingosine truncated derivative OCH have been shown to suppress autoimmune diseases such as experimental auto-immune encephalomyelitis (EAE), diabetes in nonobese diabetic (NOD) mice, and collagen-induced arthritis (CIA) by inducing T helper (Th) 2 bias of autoimmune T cells. In this review, we examine the potential roles of NKT cells in the pathogenesis of autoimmune disease regulation, and the recent advances in glycolipid therapy for autoimmune disease models. In addition, we summarize studies suggesting a role for NKT cells in human autoimmune disease, and discuss the potential of targeting NKT cells for the treatment of autoimmunity.     

Severe and selective deficiency of interferon-gamma-producing invariant natural killer T cells in patients with myelodysplastic syndromes

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.     

The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramatic in vivo expansion and surface phenotype alterations after NKT cell activation with alpha-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oral Salmonella infection. Our results indicate that in vivo activation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.     

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.     

CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.     

自然杀伤T细胞在支气管哮喘中的研究进展

自然杀伤T（NKT）细胞是一类参与先天性免疫与获得性免疫的特殊的T淋巴细胞，近年来研究发现，NKT细胞对哮喘的Th1／Th2失衡以及气道高反应性的产生具有重要作用，因此研究NKT细胞对揭示哮喘的发生机制以及治疗有着积极的意义。     

Increased interleukin-4, interleukin-5, and interferon-gamma in airway CD4+ and CD8+ T cells in atopic asthma

Increased Th2 cytokine production in asthma is widely accepted, but excess production by asthmatic human airway CD4(+) T cells has not been demonstrated, nor has a relationship with disease severity. The importance of airway CD8(+) T cell type 1 and type 2 cytokine production in asthma is unknown. We investigated frequencies of IFN-gamma, interleukin (IL)-4 and IL-5 producing CD4(+) and CD8(+) blood and sputum T cells from normal subjects and subjects with asthma and compared between cell subsets, subject groups, and body compartments with and without in vitro stimulation and investigated relationships between cytokine production and asthma severity. Production of IL-4, IL-5, and IFN-gamma by unstimulated sputum CD4(+) and CD8(+) T cells was increased in subjects with asthma and related to disease severity, more for CD8(+) than for CD4(+) T cells. Frequencies of sputum CD8(+) T cells producing type 1 and type 2 cytokines were similar to those of CD4(+) T cells. In vitro stimulation polarized peripheral blood cytokine production toward IFN-gamma production, significantly more in subjects with asthma than in normal subjects. These data demonstrate increased type 1 and 2 cytokine production in CD4(+) and CD8(+) T cells in sputum and relate production to disease severity. Findings in blood did not reflect those in airways.     

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.     

Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity

Human Valpha24+Vbeta11+ natural killer T (NKT) cells are a distinct CD1d-restricted lymphoid subset specifically and potently activated by alpha-galactosylceramide (alpha-GalCer) (KRN7000) presented by CD1d on antigen-presenting cells. Preclinical models show that activation of Valpha24+Vbeta11+ NKT cells induces effective antitumor immune responses and potentially important secondary immune effects, including activation of conventional T cells and NK cells. We describe the first clinical trial of cancer immune therapy with alpha-GalCer-pulsed CD1d-expressing dendritic cells. The results show that this therapy has substantial, rapid, and highly reproducible specific effects on Valpha24+Vbeta11+ NKT cells and provide the first human in vivo evidence that Valpha24+Vbeta11+ NKT cell stimulation leads to activation of both innate and acquired immunity, resulting in modulation of NK, T-, and B-cell numbers and increased serum interferon-gamma. We present the first clinical evidence that Valpha24+Vbeta11+ NKT cell memory produces faster, more vigorous secondary immune responses by innate and acquired immunity upon restimulation.