The NKT cell TCR repertoire can accommodate structural modifications to the lipid and orientation of the terminal carbohydrate of iGb3

Isoglobotrihexosylceramide (iGb3) is a known NKT cell agonist, however the specific interactions required to trigger NKT cell TCR activation in response to this mammalian glycolipid are not fully understood. Here we report the synthesis of 1,3-β-Gal-LacCer (βG-iGb3) that displays a β-linked terminal sugar. βG-iGb3 activated NKT cells to a similar extent as iGb3 with a terminal α-linkage, indicating that the conformation of the terminal sugar residue of iGb3 is not essential to facilitate NKT cell TCR recognition. In addition, the immunological activity of four recently described iGb3 analogues with modifications to their terminal sugar or lipid backbone were also investigated. These iGb3 analogues all induced NKT cell proliferation, with IL-13 the predominate cytokine detected. This highlights the ability of the NKT cell TCR to accommodate variations in iGb3-based glycolipids and suggests that undiscovered NKT cell ligands may exist within the lacto-series of mammalian glycosphingolipids.

The synthesised βG-iGb3 glycolipid, with a terminal 1,3-β linked galactose, induced NKT cell proliferation indicating that the α conformation of the terminal sugar residue of iGb3 is not essential for NKT cell TCR recognition.     

SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-{gamma} and IL-17A production

Regulatory T cells (T(reg) cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of T(reg) cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1(-/-) T(reg) cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2(-/-) mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1(-/-) T(reg) cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ(-/-)Socs1(-/-) T(reg) cells, the restriction of IFN-γ-STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ(-/-)Socs1(-/-) T(reg) cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ(-/-)Socs1(+/+) T(reg) cells and could not suppress colitis induced by naive T cells in Rag2(-/-) mice. In vitro experiments suggested that cytokines produced by Socs1(-/-) T(reg) cells and Ifnγ(-/-)Socs1(-/-) T(reg) cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in T(reg) cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively.     

The role of invariant natural killer T cells in microbial immunity

Invariant natural killer T cells (iNKT cells) are unique lymphocytes with characteristic features, such as expression of an invariant T-cell antigen receptor (TCR) α-chain, recognition of glycolipid antigens presented by CD1d molecules, and ability to rapidly produce large amounts of cytokines, including interferon-γ (IFN-γ) and interleukin 4 (IL-4) upon TCR stimulation. Many studies have demonstrated that iNKT cells participate in immune response against diverse microbes, including bacteria, fungi, protozoan parasites, and viruses. Generally, these cells play protective roles in host defense against infections. However, in some contexts they play pathogenic roles, by inducing or augmenting inflammation. Recent reports show that iNKT cells recognize glycolipid antigens from pathogenic bacteria including Streptococcus pneumoniae, and they contribute to host defense against infection. iNKT cell responses to these microbial glycolipid antigens are highly conserved between rodents and humans, suggesting that iNKT cells are evolutionally conserved because their invariant TCR is useful in detecting certain pathogens. Furthermore, glycolipid-mediated iNKT cell activation during immunization has adjuvant activity, enhancing humoral and cell-mediated responses. Therefore, iNKT cell activation is an attractive target for developing new vaccines for infectious diseases.     

Distinct functional lineages of human V(alpha)24 natural killer T cells

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy subjects express <1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents alpha-galactosylceramide (alphaGalCer) loaded CD1d-tetramers and anti-V(alpha)24 monoclonal antibody, to specifically identify and characterize these rare cells in fresh PBLs. We report here that CD4(+) and CD4(-)CD8(-) (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4(+) NKT cells were the exclusive producers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases.     

Dectin-1 diversifies Aspergillus fumigatus-specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus-infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus-infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus-specific CD4 T cells. Our findings indicate that Dectin-1-mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus-specific CD4 T cells and enabling Th17 differentiation.     

Glycolipid antigen induces long-term natural killer T cell anergy in mice

Natural killer T (NKT) cells recognize glycolipid antigens presented by the MHC class I-related glycoprotein CD1d. The in vivo dynamics of the NKT cell population in response to glycolipid activation remain poorly understood. Here, we show that a single administration of the synthetic glycolipid alpha-galactosylceramide (alpha-GalCer) induces long-term NKT cell unresponsiveness in mice. NKT cells failed to proliferate and produce IFN-gamma upon alpha-GalCer restimulation but retained the capacity to produce IL-4. Consequently, we found that activation of anergic NKT cells with alpha-GalCer exacerbated, rather than prevented, B16 metastasis formation, but that these cells retained their capacity to protect mice against experimental autoimmune encephalomyelitis. NKT cell anergy was induced in a thymus-independent manner and maintained in an NKT cell-autonomous manner. The anergic state could be broken by IL-2 and by stimuli that bypass proximal TCR signaling events. Collectively, the kinetics of initial NKT cell activation, expansion, and induction of anergy in response to alpha-GalCer administration resemble the responses of conventional T cells to strong stimuli such as superantigens. Our findings have important implications for the development of NKT cell-based vaccines and immunotherapies.     

Mechanisms imposing the Vbeta bias of Valpha14 natural killer T cells and consequences for microbial glycolipid recognition

Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial alpha-glycuronosylceramides. The importance of the canonical Valpha14-Jalpha18 TCR alpha chain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the Vbeta8, Vbeta7, and Vbeta2 bias in mouse have not been explored. To study the influences of thymic selection and the constraints of pairing with Valpha14-Jalpha18, we have created a population of mature T cells expressing Valpha14-Jalpha18 TCR alpha chain in CD1d-deficient mice and studied its recognition properties in vitro and in vivo. Transgenic cells expressed a diverse Vbeta repertoire but their recognition of endogenous ligands and synthetic iGb3 was restricted to the same biased Vbeta repertoire as expressed in natural NKT cells. In contrast, alpha-GalCer, a synthetic homologue of microbial alpha-glycuronosylceramides, was recognized by a broader set of Vbeta chains, including the biased NKT set but also Vbeta6, Vbeta9, Vbeta10, and Vbeta14. These surprising findings demonstrate that, whereas Vbeta8, Vbeta7, and Vbeta2 represent the optimal solution for recognition of endogenous ligand, many Vbeta chains that are potentially useful for the recognition of foreign lipids fail to be selected in the NKT cell repertoire.     

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.     

不同来源人Vα24＋NKT细胞体外扩增及细胞因子分泌

为了研究人脐血（CB）、外周血（PB）及G—CSF动员后外周血采集物单个核细胞（G—PBMNC）中Vα24＋NKT细胞的表型及体外扩增、细胞因子分泌情况,分别取来自CB和PB的单个核细胞（MNC）以及G-PBMNC,在含α-半乳糖酰基鞘胺醇（α-GalCer）及细胞因子IL-2、IL-15的体系中,体外扩增培养Vα24＋NKT细胞;采用间标磁珠分选纯化NKT细胞;流式细胞术（FCM）检测NKT细胞表型、NKT细胞含量;酶联免疫吸附试验（ELISA）测定细胞因子IL4、IFN-γ浓度。结果表明：由CB或PB或G-PBMNC诱导扩增Vα24＋NKT细胞可分别扩增221．5倍、456．5倍、756．38倍。CB或PB来源的Vα24＋NKT细胞经PMA刺激后,24小时生成IL-4、IFN-γ浓度和IL-4／IFN-γ比率分别为180．33VS190．67ng／ml、864．33vs508．49ng／ml、0．503vs0．455。由G-PBMNC诱导扩增Vα24＋NKT细胞,培养9天及12天时生成IL-4、IFN-γ浓度及IL-4／IFN-γ比率分别为139．08vs89．3ng／ml、14264．8ml、14488ng／ml、0．0531vs0．0376。结论：不同来源MNC在α-GalCer、IL-2、IL-15诱导下可以短期有效扩增Vα24＋NKT细胞,G-PBMNC诱导扩增的效果优于CB和PB。NKT细胞活化后可以分泌大量IL4及IFN-γ,但以IFN-γ为主。     

环孢菌素A对慢性再生障碍性贫血患者T-bet、GATA-3、相关信号分子、细胞因子及Th1/Th2的影响

本研究探讨转录因子T-bet、GATA-3及相关信号通路在慢性再生障碍性贫血(CAA)免疫发病中的作用,从Th细胞失衡、转录因子及相关信号通路水平研究环孢菌素(CsA)治疗慢性AA的免疫调节机理。采用实时荧光定量PCR(real-time FQ-PCR)检测慢性AA患者治疗前和CsA治疗6月后外周血单个核细胞(PBMNC)T-bet、GATA-3及STAT4、STAT6mRNA表达;采用流式细胞术、酶联免疫吸附法(ELISA)检测慢性AA患者治疗前和CsA治疗6月后外周血Th1、Th2比例及PBMNC培养上清IFN-γ、IL-12、IL-4水平,并与正常人进行比较。结果表明:慢性AA患者PBMNC T-bet、STAT4mRNA表达、T-bet/GATA-3比值、Th1比例、Th1/Th2比值、PBMNC培养上清IFN-γ、IL-12表达均明显高于正常组(p0.01),经CsA治疗6月后,患者T-bet、STAT4表达、T-bet/GATA-3比值、Th1比例、IFN-γ、IL-12表达均有所下降,但T-bet、STAT4、T-bet/GATA-3比值、Th1比例、IFN-γ表达仍未达到正常水平,GATA-3、STAT6mRNA表达、Th2比例、IL-4表达在治疗前后与正常人比较均无明显差异(p0.05)。结论:IFN-γ/T-bet、IL-12/STAT4通路的异常活化及Th平衡向Th1型偏移在AA免疫异常的发病过程中起到关键的作用;CsA能通过下调IFN-γ/T-bet、IL-12/STAT4通路的异常活化及纠正Th1过度极化而减轻AA异常亢进的细胞免疫,解除造血抑制。     

Treatment for multiple sclerosis with a synthetic glycolipid

Experimental autoimmune encephalomyelitis(EAE), regarded as a model of multiple sclerosis, is a prototype Th1-mediated autoimmune disease. Although a prototype natural killer T(NKT) cell ligand, alpha-galactosylceramide(alpha-GC), would render NKT cells produce both IFN-gamma and IL-4, this novel ligand, an analog of alpha-GC with a truncated sphingosine chain, can induce a predominant production of IL-4. Consistently, an oral administration of this glycolipid induces Th2 bias of autoimmune T cells via production of IL-4 by NKT cells, leading to suppression of EAE. The lack of polymorphism of CD1d and cross-reactive response of mouse and human NKT cells to the same ligand would indicate that targeting NKT cells with this ligand may be an attractive means for intervening in multiple sclerosis.     

再生障碍性贫血患者外周血Th 1/Th2水平测定及其临床意义

目的：探讨辅助性T(T helper,Th)细胞亚群数量和功能的变化在再生障碍性贫血(再障)患者发病机制中的作用，及这两种变化之间的关系；为再障的诊断及疗效预测提供实验依据。方法：流式细胞术检测再障患者及正常人外周血T细胞亚群、Th1和Th2细胞；RT-PCR法测定再障患者及正常人外周血中Th1，Th2型细胞因子基因表达；用ELISA法测定再障患者血浆中IFN-γ和IL-4的水平。结果：再障患者Th1/Th2显著失衡，Th1细胞显著增多，Th1型细胞因子IFN-γmRNA的阳性表达率和血浆IFN-γ浓度均较正常对照显著升高；再障患者Th1细胞百分数分别与IFN-γmRNA相对表达强度(RV)和血浆IFN-γ浓度呈正相关；Th1细胞百分比、IFN-γmRNA的RV及血浆IFN-γ的浓度在比值倒置型与比值超高型患者体内相近且均高于比值正常型患者。结论：Th细胞亚群失衡，Th1细胞数量增多及功能亢进可能是再障发病的重要免疫机制之一；Th1细胞数量及功能异常相互作用是再障患者疾病发生和发展的根本原因；CD4+T或CD8+T细胞相对变化可能是Th细胞在发挥免疫抑制作用后的外在表象。     

Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.     

The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Valpha14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-gamma in vivo and in vitro after activation with alpha-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Valpha14 hybridoma cells. Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.     

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 cell-dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs

NKT cells have pivotal roles in immune regulation and tumor immunosurveillance. We report that the G-CSF and FMS-like tyrosine kinase 3 ligand (Flt-3L) chimeric cytokine, progenipoietin-1, markedly expands the splenic and hepatic NKT cell population and enhances functional responses to alpha-galactosylceramide. In a murine model of allogeneic stem cell transplantation, donor NKT cells promoted host DC activation and enhanced perforin-restricted CD8+ T cell cytotoxicity against host-type antigens. Following leukemic challenge, donor treatment with progenipoietin-1 significantly improved overall survival when compared with G-CSF or control, attributable to reduced graft-versus-host disease mortality and paradoxical augmentation of graft-versus-leukemia (GVL) effects. Enhanced cellular cytotoxicity was dependent on donor NKT cells, and leukemia clearance was profoundly impaired in recipients of NKT cell-deficient grafts. Enhanced cytotoxicity and GVL effects were not associated with Flt-3L signaling or effects on DCs but were reproduced by prolonged G-CSF receptor engagement with pegylated G-CSF. Thus, modified G-CSF signaling during stem cell mobilization augments NKT cell-dependent CD8+ cytotoxicity, effectively separating graft-versus-host disease and GVL and greatly expanding the potential applicability of allogeneic stem cell transplantation for the therapy of malignant disease.