Structure modifications based on KRN7000 and their SARs in activating NKT cells

α-Galactosylceramides, which can be recognized by natural killer T （NKT） cells, are now attracting more and more attention due to their therapeutic potential in cancer, infection and autoimmune diseases. Advances have been achieved in discovering compounds with better activities and efforts have been made to understand the structure-activity relationships （SARs） of these NKT cell ligands. In this review, we discuss the structure modifications based on KRN7000, the principal glycolipid used in the study of NKT cell stimulation, and the SARs based on these modified structures.

NKT cells in HIV-1 infection

Natural killer T （NKT） cells are a unique T cell population that have important immunoregulatory functions and have been shown to be involved in host immunity against a range of microorganisms. It also emerges that they might play a role in HIV-1 infection, and therefore be selectively depleted during the early stages of infection. Recent studies are reviewed regarding the dynamics of NKT depletion during HIV-1 infection and their recovery under highly active antiretroviral treatment （HAART）. Possible mechanisms for these changes are proposed based on the recent developments in HIV pathogenesis. Further discussions are focused on HIV＇s disruption of NKT activation by downregulating CDld expression on antigen presentation cells （APC）. HIV-1 protein Nefis found to play the major role by interrupting the intracellular trafficking of nascent and recycling CDld molecules.

Greater Expansion of IFN-<i>γ</i>^﹣CD4⁺NKT Cells in HIV-1 Compared with HIV-2-Infected Subjects with Preserved CD4⁺T Cell Counts

Context: Human Natural Killer T cells are T lymphocytes that express an invariant αβ T cells receptors and NK cells receptors. They regulate innate and adaptive immune response but are susceptible to HIV-1 infection. Objective: We compare the frequency and the activity of NKT cells in HIV-1 and HIV-2 infected individuals with CD4+ counts greater than 500/mm3 using flow cytometry after overnight stimulation with phytohemagglutinin (PHA). Results: The frequency of NKT cells was similar between both groups and also to sero-negative control subjects. There were also no significant differences in the proportions of total NKT cells and the CD4+ NKT subset that secreted interferon gamma (IFN-γ) after polyclonal stimulation. However, there was a significantly higher frequency of IFN-γ﹣ CD4+ NKT cells in HIV-1-infected compared with HIV-2 infected subjects (p = 0.043). Conclusion: These data suggest there is no relationship between the functional activity of NKT cell subsets and the total NKT cell population in HIV infection. The expansion of IFN-γ﹣ CD4+ NKT cells in HIV-1 infection may serve as target for viral infection and may eventually result in their depletion during chronic infection.

NKT cell subsets as key participants in liver physiology and pathology

Natural killer T （NKT） cells are innate-like lymphocytes that generally recognize lipid antigens and are enriched in microvascular compartments of the liver. NKT cells can be activated by self- or microbial-lipid antigens and by signaling through toll-like receptors. Following activation, NKT cells rapidly secrete pro-inflammatory or anti- inflammatory cytokines and chemokines, and thereby determine the milieu for subsequent immunity or tolerance. It is becoming clear that two different subsets of NKT cells-type I and type II--have different modes of antigen recognition and have opposing roles in inflammatory liver diseases. Here we focus mainly on the roles of both NKT cell subsets in the maintenance of immune tolerance and inflammatory diseases in liver. Furthermore, how the differential activation of type I and type II NKT cells influences other innate cells and adaptive immune cells to result in important consequences for tissue integrity is discussed. It is crucial that better reagents, including CDld tetramers, be used in clinical studies to define the roles of NKT cells in liver diseases in patients.

Lactic acid in tumor microenvironments causes dysfunction of NKT cells by interfering with mTOR signaling

Cellular metabolism has been shown to regulate differentiation and function of immune cells. Tumor associated immune cells undergo phenotypic and functional alterations due to the change of cellular metabolism in tumor microenvironments. NKT cells are good candidates for immunotherapies against tumors and have been used in several clinical trials. However, the influences of tumor microenvironments on NKT cell functions remain unclear. In our studies, lactic acid in tumor microenvironments inhibited IFN？ and IL4 productions from NKT cells, and more profound influence on IFN？ was observed. By adjusting the pH of culture medium we fiu-ther showed that, dysfunction of NKT cells could simply be induced by low extracellular pH. Moreover, low extracellular pH inhibited NKT cell functions by inhibiting mammalian target of rapamycin （roTOR） signaling and nuclear translocation of promyelocytic leukemia zinc-finger （PLZF）. Together, our results suggest that tumor acidic microenvironments could interfere with NKT cell functions through metabolic controls.

CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells

Gut-derived bacterial products contribute to liver inflammation and injury during chronic hepatitis B virus infection;however,the underlying mechanisms remain obscure.In this study,hepatitis B surface antigen transgenic(HBs-Tg)mice and their wild-type(WT)control C57BL/6 mice were injected with CpG-oligodeoxynucleotides(ODNs)to mimic the translocation of gut microbial products into the systemic circulation.We found that,compared with the WT mice,the HBs-Tg mice were oversensitive to CpG-ODN-induced liver injury,which was dependent on natural killer T(NKT)cells.CpG-ODN injection enhanced the expression of Fas ligand(FasL)on NKT cells.In addition,hepatocytes from the HBs-Tg mice expressed higher levels of Fas than did those from the WT mice,which was further augmented by CpG-ODN.Interaction of Fas and FasL was involved in the cytotoxicity of NKT cells against hepatocytes in the HBs-Tg mice.Moreover,Kupffer cells in the HBs-Tg mice expressed higher levels of CD205 and produced greater amounts of interleukin(IL)-12 than did those in the WT mice.Finally,the depletion of Kupffer cells,neutralization of IL-12 or specific silencing of CD205 on Kupffer cells significantly inhibited CpG-ODN-induced liver injury and NKT activation in the HBs-Tg mice.Our data suggest that CD205-expressing Kupffer cells respond to CpG-ODNs and subsequently release IL-12 to promote NKT cell activation.Activated NKT cells induce liver damage through the Fas signaling pathway in HBs-Tg mice.

EBV-Induced Human CD8^＋ NKT Cells Synergise CD4^＋ NKT Cells Suppressing EBV-Associated Tumours upon Induction of Thl-Bias

CD8^＋ natural killer T （NKT） cells from EBV-associated tumour patients are quantitatively and functionally impaired. EBV-induced CD8^＋ NKT cells drive syngeneic T cells into a Thl-bias response to suppress EBV-associated malignancies. IL-4-biased CD4^＋ NKT cells do not affect either syngeneic T cell cytotoxicity or Th cytokine secretion. Circulating mDC1 cells from patients with EBV-associated malignancies impair the production of IFN-T by CD8^＋ NKT cells. In this study, we have established a human-thymus-SCID chimaera model to further investigate the underlying mechanism of EBV-induced CD8^＋ NKT cells in suppressing EBV-associated malignancies. In the human-thymus-SCID chimera, EBV-induced CD8^＋ NKT cells suppress EBV-associated malignancies in a manner dependent on the Thl-bias response and syngeneic CD3^＋ T cells. However, adoptive transfer with CD4^＋ NKT cells alone inhibits T cell immunity. Interestingly, CD4^＋ NKT cells themselves secrete high levels of IL-2, enhancing the persistence of adoptively transferred CD8^＋ NKT cells and T cells, thereby leading to a more pronounced T cell anti-tumour response in chimaeras co-transferred with CD4^＋ and CD8^＋ NKT cells. Thus, immune reconstitution with EBV-induced CD4^＋ and CD8^＋ NKT cells synergistically enhances T cell tumour immunity, providing a potential prophylactic and therapeutic treatment for EBV-associated malignancies.

M2-specific reduction of CD1d switches NKT cell-mediated immune responses and triggers metaflammation in adipose tissue

Metaflammation is responsible for several metabolic syndromes,such as type 2 diabetes.However,the mechanisms by which metabolic disorders trigger metaflammation remain unclear.We identified a cell type-specific downregulation of CD1d expression in M2 macrophages during the progression of obesity prior to the onset of inflammation in visceral adipose tissues.A reduction in CD1d expression influenced the ability of M2 macrophages to present antigens and caused a change in antigen-presenting cells from M2 macrophages to M1 macrophages.With CD1d conditional knockout(KO)mice,we further demonstrated that natural killer T(NKT)cell activation by M2 macrophages inhibited metaflammation and insulin resistance by promoting Th2 responses and M2 polarization in visceral adipose tissues of obese mice,whereas NKT cell activation by M1 macrophages exacerbated metaflammation and insulin resistance by promoting Th1 responses and inhibiting M2 polarization.Our results suggest that an M2-specific reduction of CD1d is an initiating event that switches NKT cell-mediated immune responses and disrupts the immune balance in visceral adipose tissues in obese mice.

T细胞亚群及NK细胞、NKT细胞与儿童紫癜性肾炎的相关性研究

目的探讨T淋巴细胞亚群、自然杀伤细胞(NK)、自然杀伤性T细胞(NKT)在儿童紫癜性肾炎(HSPN)患者中的临床意义。方法回顾性分析2017年7月至2022年7月在贵阳市妇幼保健院住院的HSPN患儿作为研究对象,作为HSPN组(n=116);同时以同期健康体检儿童为对照组(n=100),两组资料进行分析比较。HSPN患儿依据临床分型:孤立性血尿组(n=23),孤立性蛋白尿组(n=15),血尿与蛋白尿组(n=59),肾病综合征组(n=19);依据病理活检分型:轻度组(n=28),中度组(n=58),重度组(n=6)。比较各组患儿外周血中T淋巴细胞亚群、NK细胞、NKT细胞绝对值计数水平。结果与对照组相比,HSPN组外周血CD3^(+)、CD4^(+)、CD8^(+)T细胞、NK细胞均降低、CD4^(+)/CD8^(+)、CD19^(+)B细胞、NKT细胞均增高,差异均有统计学意义(P<0.05)。HSPN临床分型中,CD3^(+)、CD4^(+)、CD8^(+)T细胞、NK细胞随临床分型的加重而降低,肾病综合征型与各临床分型比较,CD3^(+)、CD4^(+)、CD8^(+)T细胞明显降低,血尿与蛋白尿型和肾病综合征型与孤立性血尿型比较,NK细胞明显降低,CD19^(+)B细胞、NKT细胞随临床分型的加重而增高,不同临床分型组间差异均有统计学意义(P<0.05)。随着病理分级加重,CD4^(+)、CD8^(+)T淋巴细胞绝对值呈下降趋势,其中轻度组与重度组比较,差异均有统计学意义(P<0.05);随着病理分级加重,CD19^(+)B淋巴细胞、NKT细胞呈增高趋势,不同病理分级组间比较差异有统计学意义(P<0.05)。结论HSPN患儿存在体液免疫及细胞免疫功能紊乱,通过检测外周血T细胞亚群、NK细胞、NKT细胞水平可协助HSPN临床及病理损害的评估。

Transcription factor YY1 is essential for iNKT cell development

Invariant natural killer T(iNKT)cells develop from CD4+CD8+double-positive(DP)thymocytes and express an invariant Vα14–Jα18 T-cell receptor(TCR)α-chain.Generation of these cells requires the prolonged survival of DP thymocytes to allow for Vα14–Jα18 gene rearrangements and strong TCR signaling to induce the expression of the iNKT lineage-specific transcription factor PLZF.Here,we report that the transcription factor Yin Yang 1(YY1)is essential for iNKT cell formation.Thymocytes lacking YY1 displayed a block in iNKT cell development at the earliest progenitor stage.YY1-deficient thymocytes underwent normal Vα14–Jα18 gene rearrangements,but exhibited impaired cell survival.Deletion of the apoptotic protein BIM failed to rescue the defect in iNKT cell generation.Chromatin immunoprecipitation and deep-sequencing experiments demonstrated that YY1 directly binds and activates the promoter of the Plzf gene.Thus,YY1 plays essential roles in iNKT cell development by coordinately regulating cell survival and PLZF expression.

Boosting the immune response： the use of iNKT cell ligands as vaccine adjuvants

Natural killer T （NKT） cells comprise a small, but important T cell subset and are thought to bridge the innate and adaptive immune responses. The discovery of NKT cells and extensive research on their activating ligands have paved the way for modulation of these potent immunoregulatory cells in order to improve the outcome of various clinical conditions. Efforts to modulate NKT cell effector functions have ranged from therapy for influenza to anti- tumor immunotherapy. These approaches have also led to the use of NKT cell agonists such as a-Galactosylceramide （a- GalCer） and its analogs as vaccine adjuvants, an approach that is aimed at boosting specific B and T cell responses to a vaccine candidate by concomitant activation of NKT cells. In this review we will provide a comprehensive overview of the efforts made in using a-GalCer and its analogs as vaccine adjuvants. The diverse array of vaccination strategies used, as well as the role of NKT cell activating adjuvants will be discussed, with focus on vaccines against malaria, HIV, influenza and tumor vaccines. Collectively, these studies demonstrate the efficacy of NKT cell-specific agonists as adjuvants and suggest that these compounds warrant serious consideration during the development of vaccination strategies.

过继骨髓细胞通过分化为NKT细胞并增加自身脂质含量减轻小鼠代谢功能障碍相关脂肪性肝病肝损伤

目的 探讨骨髓细胞过继对于小鼠代谢功能障碍相关脂肪性肝病(MAFLD)的治疗作用,并探索起主要作用的细胞群体。方法 HE染色检测蛋氨酸-胆碱缺乏(MCD)诱导的C57BL/6小鼠MAFLD肝组织病变并通过检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平评估骨髓细胞过继对MAFLD的治疗作用。实时定量PCR检测T细胞、自然杀伤T(NKT)细胞、 Kupffer细胞等肝脏免疫细胞的低密度脂蛋白受体(LDLR)和白细胞介素4(IL-4) mRNA表达。羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记的骨髓细胞经小鼠尾静脉注射,采用冰冻切片观察肝组织CFSE+细胞比例,流式细胞术追踪CFSE+细胞在肝、脾组织的比例。流式细胞术检测CFSE+的过继细胞CD3、 CD4、 CD8、 NK1.1、 CD11b、粒细胞受体1(Gr-1)的表达确定其分化情况。尼罗红脂质染色评估肝组织NKT细胞胞内脂质含量。结果 骨髓细胞过继输入的MAFLD小鼠肝组织损伤明显减轻、血清ALT、 AST水平降低。同时,肝脏免疫细胞上调其IL-4和LDLR的表达。LDLR敲除小鼠给予MCD饮食后引发更严重的MAFLD。骨髓细胞过继细胞治疗效果显著并分化出更多的NKT细胞定殖在肝脏,同时该群NKT细胞胞内脂质显著增多。结论骨髓细胞过继治疗通过分化更多的NKT细胞,同时通过提高该群细胞的胞内脂质含量从而减轻MAFLD小鼠肝损伤。

原发免疫性血小板减少症患者外周血淋巴细胞亚群、NKT细胞及Th1/Th2细胞因子水平分析

目的分析原发免疫性血小板减少症患者外周血淋巴细胞亚群、自然杀伤T细胞(natural killer T-cell,NKT)及Th1/Th2细胞因子水平的变化及临床意义。方法纳入2021年6月—2022年6月麻城市人民医院收治的60例原发免疫性血小板减少症患者为观察组,均施予流式细胞术检测,将血小板(platelet,PLT)<20×109/L患者分为观察1组(30例),将PLT≥20×109/L患者分为观察2组(30例);纳入同期30例健康体检者为对照组,对上述受试者的外周血淋巴细胞亚群、NKT细胞及Th1/Th2细胞因子水平进行测定。结果3组CD3^(+)、CD3^(+)CD4^(+)、CD3^(+)CD8^(+)、CD3-CD19^(+)、CD3-CD16^(+)CD56^(+)、CD4^(+)/CD8^(+)、NKT比较,差异有统计学意义(F_(CD3+)=16.806,F_(CD3+CD4+)=24.271,F_(CD3+CD8+)=4.880,F_(CD3-CD19+)=34.955,F_(CD3-CD16+CD56+)=11.051,F_(CD4+/CD8)=13.014,F_(NKT)=38.576,P<0.001);原发免疫性血小板减少症患者的CD3^(+)、CD3^(+)CD4^(+)、CD3-CD16^(+)CD56^(+)、CD4^(+)/CD8^(+)、NKT与PLT计数为正相关关系(P<0.001);CD3^(+)CD8^(+)、CD3-CD19^(+)、白细胞介素2、白细胞介素4、白细胞介素6、白细胞介素10、肿瘤坏死因素、干扰素-γ与PLT计数为负相关关系(P<0.001)。结论原发免疫性血小板减少症患者体内的淋巴细胞亚群、NKT细胞、Th1/Th2细胞因子紊乱,且病情进展与T、B淋巴细胞亚群与NKT细胞为正相关关系。

食管鳞癌患者外周血NK细胞、NKT细胞中Ku70蛋白水平检测及临床意义

目的检测食管鳞癌(esophageal squamous cell carcinoma,ESCC)患者外周血NK细胞、NKT细胞中Ku70蛋白表达水平,并探索其对ESCC的临床意义及预后价值。方法对前期ESCC患者和健康人的外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)质谱结果进行生物信息学分析,筛选出差异蛋白Ku70,流式细胞学验证Ku70在NK细胞和NKT细胞中的表达,分析两者的联系及临床意义。结果蛋白质谱发现522个差异蛋白,其中310个蛋白上调,212个蛋白下调,通过生信分析筛选出Ku70蛋白。流式细胞术分析显示,肿瘤组的PBMCs中CD3^(+)CD56^(+)NKT细胞和CD16^(+)CD56^(+)NK细胞的Ku70荧光强度和阳性表达率均低于健康对照组。结论我们获得了ESCC患者PBMCs的差异表达蛋白谱,其中Ku70在ESCC患者CD3^(+)CD56^(+)NKT细胞和CD16^(+)CD56^(+)NK细胞中表达降低显著,可能成为ESCC外周血检测的生物标志物。

NKT细胞在MRSA感染性肺炎中作用的初步研究

目的:初步探讨NKT细胞在耐甲氧西林金葡菌(MRSA)感染性肺炎模型中的作用。方法:MRSA经鼻黏膜感染C57BL/6野生型(WT)小鼠和CD1d^(-/-)小鼠。观察小鼠生存率,检测肺组织中细菌载量,ELISA检测肺组织中细胞因子TNF-α、IFN-γ、IL-17、IL-6含量,流式细胞术检测肺组织中CD3^(+)CD8^(+)T细胞、NK细胞和NKT细胞百分比。结果:MRSA致死剂量感染5 d,WT小鼠生存率显著低于CD1d^(-/-)小鼠(46.2%vs 90.0%,P<0.05)。感染12~48 h,WT小鼠肺组织中细菌载量显著高于CD1d^(-/-)小鼠(P<0.01);WT小鼠肺组织中TNF-α、IFN-γ、IL-6和IL-17含量较CD1d^(-/-)小鼠明显增高(P<0.01)。MRSA感染24 h,WT小鼠肺组织中NKT细胞、NK细胞百分比显著高于CD1d^(-/-)小鼠(P<0.01),而CD3^(+)CD8^(+)T细胞百分比低于CD1d^(-/-)小鼠(P<0.01)。结论:在MRSA肺炎中,NKT细胞被激活,从而加重肺部炎症反应,降低生存率,减缓细菌清除。

NK细胞和NKT细胞在慢性乙型肝炎中的表型和功能特征

目的检测慢性乙型肝炎(CHB)患者外周血自然杀伤(NK)细胞、自然杀伤T(NKT)细胞的比例、NKG2D/NKG2A水平和γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)的水平。方法采集并分离CHB患者外周血单个核细胞(PBMC),流式细胞术检测NK、NKT细胞占PBMC的比例,NKG2D、NKG2A水平,经佛波酯(PMA)、布雷菲德菌素A(BFA)、离子霉素(ionomycin)处理后,流式细包术检测IFN-γ、TNF-α的水平,分析各指标与乙肝病毒载量和血清丙氨酸氨基转移酶的关系,采用独立样本t检验与Pearson相关性分析。结果与正常对照者相比,CHB患者外周血NK细胞和NKT细胞比例明显降低;NKG2A阳性的NK细胞和NKT细胞明显升高;经PMA、BFA、ionomycin刺激后,IFN-γ阳性的NK细胞和NKT细胞明显降低。结论 CHB患者NK、NKT细胞存在不同程度的减少、受体表达失调和细胞因子分泌功能下降等功能紊乱。

超抗原SEB活化耐受性NKT细胞亚群的研究

目的:探讨超抗原SEB活化的效应细胞参与免疫耐受的NKT细胞亚群及分化特征。方法:采用C57BL/J小鼠脾细胞经SEB诱导,收集体外扩增10d的淋巴细胞为效应细胞,与刀豆蛋白(ConA)、脂多糖(LPS)和白介素-2(IL-2)共同培养3d,测定效应细胞对刺激剂的应答反应能力。在正常小鼠淋巴细胞与上述刺激剂反应的同时添加效应细胞,3d后测定效应细胞抑制正常淋巴细胞对刺激原的应答反应能力。用MTT染色方法记录细胞增殖的A值。以ConA活化的淋巴细胞做参照,用流式细胞术(FCM)解析了耐受性效应细胞中NKT细胞亚群并显示分化来源与功能的相关性。结果:与正常淋巴细胞对抗原应答反应能力相比,SEB活化的效应细胞对ConA、LPS和IL-2的应答反应能力明显降低,细胞生长的A值从正常组的0.80±0.04、0.60±0.03和0.55±0.07下降到0.60±0.05、0.30±0.05及0.27±0.04(P<0.01,n=3)。效应细胞抑制正常淋巴细胞对上述抗原的应答反应,尤其抑制ConA活化的T淋巴细胞的应答反应;A值分别由正常值降低到0.26±0.002、0.48±0.04及0.34±0.02(P<0.01,n=3)。效应细胞中CD4+NK1.1+、CD8+NK1.1+、TcRVβ8+/NK1.1+NKT细胞亚群显著增加(P<0.05和P<0.01,n=4)。ConA活化的淋巴细胞是T淋巴细胞并包括CD4-CD8-/CD3+NK1.1+NKT细胞。结论:超抗原SEB活化的耐受功能与CD4+NK1.1+、CD8+NK1.1+、TcRVβ8+NK1.1+NKT细胞亚群有关,它们由T细胞分化而来。CD4-CD8-/CD3+NK1.1+NKT细胞没有参与耐受性调节。

NKT细胞发育及功能研究进展

自然杀伤T细胞(Natural killer T cells,NKT细胞)是一类天然存在的介导先天性免疫和获得性免疫的重要淋巴细胞,参与机体的抗感染、抗肿瘤、移植免疫和自身免疫调节作用。NKT细胞的来源、选择分化和成熟过程及其调控研究有了很大的进展,同时在这些过程中仍存在一些备受争议和值得深入研究的问题。清楚了解NKT细胞的分化发育过程和调控机制,结合NKT细胞的生物学功能特征,将对临床预防和治疗相关的免疫性疾病具有重要的指导意义。