Mucosal-associated invariant T cells in hepatitis B virus-related liver failure

BACKGROUND Liver failure has high mortality and poor prognosis,and establishing new reliable markers for predicting its prognosis is necessary.Mucosal-associated invariant T(MAIT)cells are a novel population of innate-like lymphocytes involved in inflammatory liver disease,and their potential role in liver failure remains unclear.AIM To investigate alteration of circulating MAIT cells and assess its prognostic value in patients with hepatitis B virus(HBV)-related liver failure.METHODS We recruited 55 patients with HBV-related liver failure,48 patients with chronic hepatitis B and 40 healthy controls(HCs)from Nantong Third People’s Hospital Affiliated to Nantong University.Peripheral blood mononuclear cells were isolated,and the percentage and number of circulating MAIT cells were detected by flow cytometry.Plasma levels of interleukin(IL)-7,IL-12p70,IL-18 and interferon-αwere measured by Luminex assay.RESULTS Circulating MAIT cells were significantly decreased in HBV-related liver failure patients(percentage:2.00±1.22 vs 5.19±1.27%,P<0.0001;number:5.47±4.93 vs 84.43±19.59,P<0.0001)compared with HCs.More importantly,there was a significant reduction of MAIT cells in patients with middle/late-stage compared with early-stage liver failure.Circulating MAIT cells partially recovered after disease improvement,both in percentage(4.01±1.21 vs 2.04±0.95%,P<0.0001)and in cell count(17.24±8.56 vs 7.41±4.99,P<0.0001).The proportion(2.29±1.01 vs 1.58±1.38%,P<0.05)and number(7.30±5.70 vs 2.94±1.47,P<0.001)of circulating MAIT cells were significantly higher in the survival group than in the dead/liver transplantation group,and the Kaplan–Meier curve showed that lower expression of circulating MAIT cells(both percentage and cell count)predicted poor overall survival(P<0.01).Also,the levels of IL-12(20.26±5.42 pg/mL vs 17.76±2.79 pg/mL,P=0.01)and IL-18(1470.05±1525.38 pg/mL vs 362.99±109.64 pg/mL,P<0.0001)were dramatically increased in HBV-related liver failure patients compared with HCs.CONCLUSION Circulating MAIT cells may play an important role in the process of HBV-related liver failure and can be an important prognostic marker.

Incorporating mucosal-associated invariant T cells into the pathogenesis of chronic liver disease

Mucosal-associated invariant T(MAIT)cells have been described in liver and nonliver diseases,and they have been ascribed antimicrobial,immune regulatory,protective,and pathogenic roles.The goals of this review are to describe their biological properties,indicate their involvement in chronic liver disease,and encourage investigations that clarify their actions and therapeutic implications.English abstracts were identified in PubMed by multiple search terms,and bibliographies were developed.MAIT cells are activated by restricted non-peptides of limited diversity and by multiple inflammatory cytokines.Diverse pro-inflammatory,anti-inflammatory,and immune regulatory cytokines are released;infected cells are eliminated;and memory cells emerge.Circulating MAIT cells are hyper-activated,immune exhausted,dysfunctional,and depleted in chronic liver disease.This phenotype lacks disease-specificity,and it does not predict the biological effects.MAIT cells have presumed protective actions in chronic viral hepatitis,alcoholic hepatitis,non-alcoholic fatty liver disease,primary sclerosing cholangitis,and decompensated cirrhosis.They have pathogenic and pro-fibrotic actions in autoimmune hepatitis and mixed actions in primary biliary cholangitis.Local factors in the hepatic microenvironment(cytokines,bile acids,gut-derived bacterial antigens,and metabolic by-products)may modulate their response in individual diseases.Investigational manipulations of function are warranted to establish an association with disease severity and outcome.In conclusion,MAIT cells constitute a disease-nonspecific,immune response to chronic liver inflammation and infection.Their pathological role has been deduced from their deficiencies during active liver disease,and future investigations must clarify this role,link it to outcome,and explore therapeutic interventions.

Mucosal-associated invariant T cells from induced pluripotent stem cells:A novel approach for modeling human diseases

Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases.These models help elucidatethe mechanisms underlying the disease and in the development of novel therapies.However,if mice are deficient in certain cells and/or effectors associated with human diseases,how can their functions be investigated in this species?Mucosal-associated invariant T(MAIT)cells,a novel innate-like T cell family member,are a good example.MAIT cells are abundant in humans but scarce in laboratory mice.MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2metabolites from bacteria and yeasts.Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases.MAIT cells possess granulysin,a human-specific effector molecule,but granulysin and its homologue are absent in mice.Furthermore,MAIT cells show poor proliferation in vitro.To overcome these problems and further our knowledge of MAIT cells,we have established a method to expand MAIT cells via induced pluripotent stem cells(iP SCs).In this review,we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iP SCderived MAIT cells.

Mucosal-associated invariant T cells and oral microbiome in persistent apical periodontitis

Opportunistic bacteria in apical periodontitis (AP) may pose a risk for systemic dissemination.Mucosal-associated invariant T (MAIT) cells are innate-like T cells with a broad and potent antimicrobial activity important for gut mucosal integrity.It was recently shown that MAIT cells are present in the oral mucosal tissue,but the involvement of MAIT cells in AP is unknown.Here,comparison of surgically resected AP and gingival tissues demonstrated that AP tissues express significantly higher levels of Vα7.2-Jα33,Vα7.2- Jα20,Vα7.2-Jα12,Cα and tumour necrosis factor (TNF),interferon (IFN)-γ and interleukin (IL)-17A transcripts,resembling a MAIT cell signature.Moreover,in AP tissues the MR1-restricted MAIT cells positive for MR1–5-OP-RU tetramer staining appeared to be of similar levels as in peripheral blood but consisted mainly of CD4^+ subset.Unlike gingival tissues,the AP microbiome was quantitatively impacted by factors like fistula and high patient age and had a prominent riboflavin-expressing bacterial feature.When merged in an integrated view,the examined immune and microbiome data in the sparse partial least squares discriminant analysis could identify bacterial relative abundances that negatively correlated with Vα7.2-Jα33,Cα,and IL-17A transcript expressions in AP,implying that MAIT cells could play a role in the local defence at the oral tissue barrier.In conclusion,we describe the presence of MAIT cells at the oral site where translocation of oral microbiota could take place.These findings have implications for understanding the immune sensing of polymicrobial-related oral diseases.

Human CD4- CD8- Invariant Natural Killer T Cells Promote IgG Secretion from B Cells Stimulated by Cross-Linking of Their Antigen Receptors

Immunoglobulin (Ig) M production can be induced by the interaction of thymus-independent type-2 (TI-2) antigen (Ag) with B cell Ag receptors (BCRs) without the involvement of conventional T cells;for IgG production through the same process, however, a second signal is required. Previous studies have reported that invariant natural killer T (iNKT) cells may be responsible for the second signal involved in IgG production. In the present study, we addressed whether human iNKT cells could participate in the production of Ig against TI-2 Ag in vitro. Two major distinct subsets of human iNKT cells, CD4+ CD8β- (CD4) and CD4- CD8β- [double negative (DN)] cells, were generated from peripheral blood monocytes from a healthy volunteer. BCR engagement, triggered by anti-IgM antibody stimulation, examined here as a model of BCR engagement triggered by TI-2 Ag, induced abundant IgM production by B cells. Both CD4 and DN iNKT cells reduced IgM production and conversely enhanced IgG production in a dose-dependent manner. In addition, IgG production by CD19+CD27- (naïve) and CD19+CD27+ (memory) B cells was predominantly promoted by DNiNKT cells rather than CD4 iNKT cells;nevertheless, IgM production by both B cell subsets was similarly reduced by either subset of iNKT cells. These results suggest that the DN iNKT subsets may preferentially promote Ig class switching by B cells upon stimulation with TI-2 Ag.

Activation-induced pyroptosis contributes to the loss of MAIT cells in chronic HIV-1 infected patients

Background: Mucosal-associated invariant T(MAIT) cells are systemically depleted in human immunodeficiency virus type 1(HIV-1) infected patients and are not replenished even after successful combined antiretroviral therapy(cART).This study aimed to identify the mechanism underlying MAIT cell depletion.Methods: In the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.Results: Single-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4+ T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D(GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12(IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.Conclusions: Activation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients,which could potentiate disease progression and poor immune reconstitution.

流行性感冒患儿外周血MAIT细胞的免疫生物学特性

目的探讨流行性感冒患儿外周血黏膜相关恒定T(MAIT)细胞的变化及其临床意义。方法选取2023年1—5月就诊于某儿童医院感染科门诊和住院治疗的流行性感冒患儿,分为普通型组和重症型组,同时选择该院体检的健康儿童作为健康对照组。患儿入院24 h内抽血送检,采用流式细胞技术检测MAIT细胞(CD3^(+)CD161^(+)TCRVα7.2^(+)细胞)比例,表达PD-1、CD69、穿孔素、CD107α的MAIT细胞比例,比较各组差异。结果与对照组相比,普通型、重症型患儿外周血MAIT细胞比例逐步下降,表达CD69和穿孔素阳性的MAIT细胞比例逐步升高,三者间比较,差异均有统计学意义(均P<0.05);表达CD107α的MAIT细胞比例比较,差异无统计学意义(P>0.05);PD-1阳性的MAIT细胞比例升高(P<0.05),但普通型、重症型组间比较,差异无统计学意义(P>0.05)。结论外周血MAIT细胞减少伴免疫活化在流行性感冒发病中起一定作用。

代谢相关脂肪性肝病患儿外周血黏膜相关恒定T淋巴细胞的频率、表型及功能改变

目的探讨代谢相关脂肪性肝病(MAFLD)患儿外周血黏膜相关恒定T(MAIT)淋巴细胞的变化及其临床意义。方法选取2022年3月—2022年5月在湖南省儿童医院就诊的18例MAFLD患儿(MAFLD组)及同期20例正常对照儿童(对照组),采集外周血,流式细胞仪检测MAIT淋巴细胞(CD3^(+)CD161^(+)TCRVα7.2^(+)细胞)及其不同MAIT淋巴细胞亚型(CD4^(+)CD8-MAIT淋巴细胞、CD4-CD8-MAIT淋巴细胞、CD4-CD8^(+)MAIT淋巴细胞和CD4^(+)CD8^(+)MAIT淋巴细胞)比例,表达PD-1、CD69、穿孔素、CD107α、CXCR3、CXCR6和CCR6的MAIT淋巴细胞比例,分析MAIT淋巴细胞频率与肝脏炎症、脂肪含量和纤维化程度的相关性。符合正态分布的计量资料两组间比较采用t检验;非正态分布计量资料两组间比较采用Mann-Whitney U检验;相关性分析采用Spearman相关分析法。结果与对照组相比,MAFLD组中外周血MAIT淋巴细胞比例及表达PD-1、CD69、CD107α、CXCR3、CXCR6和CCR6的MAIT淋巴细胞比例均明显升高(P值均<0.05);MAIT淋巴细胞亚型中CD4^(+)CD8-MAIT淋巴细胞、CD4^(+)CD8^(+)MAIT淋巴细胞所占MAIT淋巴细胞比例明显升高(P值均<0.001),CD4-CD8^(+)MAIT淋巴细胞所占MAIT淋巴细胞比例降低(P<0.001);CD4^(+)CD8^(+)MAIT淋巴细胞(r=-0.474,P=0.047)和CD107α阳性MAIT淋巴细胞比例(r=-0.550,P=0.018)与ALT呈负相关。结论外周血MAIT淋巴细胞向肝脏趋化聚集,在儿童MAFLD肝脏炎症中发挥保护作用。

Evaluation of antibody-dependent cell-mediatedcy totoxicity activity and cetuximab response in KRAS wildtype metastatic colorectal cancer patients

AIM:To investigate the prognostic role of invariant natural killer T(iNKT) cells and antibody-dependent cell-mediated cytotoxicity(ADCC) in wild type KRAS metastatic colorectal cancer(mC RC) patients treated with cetuximab.METHODS: Forty-one KRAS wt mC RC patients,treated with cetuximab and irinotecan-based chemotherapy in Ⅱ and Ⅲ lines were analyzed. Genotyping of single nucleotide polymorphism(SNP)s in the FCGR2A,FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS,BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprepperipheral blood mononuclear cell and iN KT cells were defined by co-expression of CD3,TCRVα24,TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity,measuring lactate dehydrogenase release.RESULTS: At basal,mCRC patients performing ADCC activity above the median level(71%) showed an improved overall survival(OS) compared to patients with ADCC below(median 16 vs 8 mo;P=0.026). We did not find any significant correlation of iN KT cells with OS(P=0.19),albeit we observed a trend to a longer survival after 10 mo in patients with iN KT above median basal level(0.382 cells/microliter). Correlation of OS and progression-free survival(PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2 A and TT in FCGR3A presented a trend of longer PFS(median 9 vs 5 mo;P=0.064). Chemotherapy impacted both iN KT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment.CONCLUSION: Our results suggest a link between iN KT cells,basal ADCC activity,genotypes in FCGR2A and FCGR3A,and efficacy of cetuximab in KRAS wt mC RC patients.

脂肪组织黏膜相关恒定T细胞通过分泌白介素4调节小鼠脂肪棕色化

目的探究黏膜相关恒定T(MAIT)细胞与脂肪棕色化之间的关系以及调节脂肪棕色化的分子机制。方法构建MAIT细胞功能缺陷小鼠模型,对比野生型小鼠及MAIT细胞缺陷小鼠,通过Westernblot和RT-PCR检测冷刺激前后小鼠脂肪棕色化标志物的水平差异,并通过流式细胞术检测了小鼠脂肪组织内MAIT细胞在冷刺激前后数量、活化水平以及细胞因子分泌能力的差异。采用MAIT细胞与脂肪细胞共培养的方式,通过Westernblot和RT-PCR检测是否存在MAIT细胞以及是否加入白介素4(IL-4)封闭抗体的条件下脂肪细胞棕色化标志物的表达差异。利用MAIT细胞缺陷的小鼠模型,通过代谢笼、免疫组化、Westernblot和Seahorse方法检测MAIT细胞缺陷小鼠在冷刺激条件下的脂肪棕色化相关指标。结果Westernblot和RT-PCR结果显示小鼠冷刺激后脂肪组织棕色化标志物UCP-1、PGC1-α的表达水平均上调,在此情景下小鼠脂肪组织内MAIT细胞数量差异无统计学意义(P>0.05),CD69的表达水平上调(P<0.05),效应细胞因子IL-4的表达水平上调(P<0.05)。体外共培养实验显示,脂肪细胞与MAIT细胞共培养后的棕色化水平上调(P<0.05),若阻断IL-4信号则棕色化水平下调(P<0.05)。对MAIT细胞缺陷的小鼠进行冷刺激后,其能量消耗水平低于野生型小鼠(P<0.05),脂肪棕色化水平低于野生型小鼠(P<0.05),且脂肪细胞代谢水平低于野生型小鼠。结论本研究首次通过MAIT细胞功能缺陷小鼠模型验证MAIT细胞参与脂肪棕色化,动物实验及体外实验进一步显示冷刺激通过促进MAIT细胞分泌IL-4调控脂肪细胞棕色化。

腹腔注射α-半乳糖苷神经酰胺对小鼠肺恒定自然杀伤T细胞及亚群的影响

目的探讨腹腔注射α-半乳糖苷神经酰胺(α-GalCer)对小鼠肺恒定自然杀伤T细胞(iNKT)、细胞亚群及外周血细胞因子的影响,从而为治疗相关肺部疾病提供基础研究数据。方法60只6~8周龄雄性C57BL/6小鼠,采用随机数字表法分为α-GalCer干预组和正常对照组,每组30只,选择不同时间点观察干预组与对照组小鼠肺iNKT细胞、亚群及外周血细胞因子的变化。结果组间比较发现,与对照组(3.49±0.25)%相比,腹腔注射α-GalCer可引起肺脏iNKT细胞频率及亚群的变化,差异有统计学意义(P<0.05)。α-GalCer干预组肺iNKT细胞频率前2 d(6.56±0.05)%明显升高而后[d6(2.56±0.08)%、d8(2.64±0.14)%、d10(2.37±0.24)%]降低(P<0.05)。iNKT 1细胞频率低于对照组且呈降低的趋势(P<0.05)。iNKT 2细胞频率高于对照组在第4天肺部iNKT2细胞频率达到峰值,随后降低(P<0.05)。iNKT 17细胞频率略高于对照组,差异无统计学意义(P>0.05),与第2天相比,第4、6、8、10天iNKT17细胞频率均显著升高(P<0.05)。组间比较发现,干预组与对照组外周血血清IFN-γ、IL-4、IL-17A浓度比较,差异有统计学意义,α-GalCer干预组在不同时间点IFN-γ、IL-4、IL-17A浓度比较,差异有统计学意义,与第2天相比,IFN-γ、IL-4水平于第6天、第8天均降低(P<0.05);IL-17A水平于第6天降低(P<0.05)。α-GalCer干预组的IFN-γ水平与对照组相比均有明显升高,差异有统计学意义(P<0.05);除第8天外,α-GalCer干预组的IL-4水平与对照组比较,差异有统计学意义(P<0.05);第2天、第8天α-GalCer干预组的IL-17A水平均低于对照组,差异有统计学意义(P<0.05)。结论腹腔注射α-GalCer对小鼠肺组织iNKT细胞和亚群有不同的激活效应;iNKT细胞激活后,会由脉管系统迁入间质;腹腔注射α-GalCer可诱导血清细胞因子的变化,提示α-GalCer注射可影响机体免疫功能。

颅脑损伤患者术后外周血黏膜相关恒定T细胞水平与感染的关系

目的分析外周血黏膜相关恒定T细胞(MAIT细胞)水平与颅脑损伤患者术后感染的关系。方法收集南方医院增城分院自2020年1月至2021年6月收治的颅脑损伤手术患者临床资料,依据术后1个月内是否发生感染,分为感染组(35例)和非感染组(45例),对感染组的感染学特征进行分析。采集两组患者外周血进行MAIT细胞、C反应蛋白(CRP)、白细胞介素6(IL-6)水平检测,采用Pearson线性相关分析MAIT细胞水平与CRP、IL-6水平的相关性;受试者工作特征(ROC)曲线分析MAIT、CRP、IL-6指标诊断颅脑损伤患者术后并发感染的灵敏度与特异度。结果35例术后感染患者样本中共检出35株病原菌,其中革兰阴性菌的检出率高于革兰阳性菌、真菌(P<0.05);相较于非感染组,感染组患者的CRP、IL-6水平均明显升高(P<0.05);MAIT细胞数量显著下降(P<0.05)。多因素分析显示:年龄≥60岁、合并肺部基础疾病、开放性损伤、入院时格拉斯哥昏迷指数(GCS)评分<8分、卧床时间≥2周及MAIT细胞水平均为颅脑损伤患者术后感染的危险因素(P<0.05)。MAIT细胞水平与CRP、IL-6水平呈负相关(r=-0.738、-0.762,P<0.001)。血清MAIT细胞数量<46.259/μL时预测并发感染的曲线下面积(AUC)为0.793(95%CI:0.688~0.876,P<0.001),灵敏度、特异度分别为84.44%、77.14%;血清CRP≥28.765 mg/L时预测并发感染的AUC为0.878(95%CI:0.785~0.940,P<0.001),灵敏度、特异度分别为84.44%、77.14%;血清IL-6≥35.257 ng/L时诊断并发感染的AUC为0.887(95%CI:0.796~0.947,P<0.001),灵敏度、特异度分别为91.11%、74.29%。结论颅脑损伤患者术后外周血MAIT细胞数量与感染的发生存在一定联系,其水平降低提示可能有较高的术后感染风险,可辅助临床早期诊断,及早控制感染,提高患者生存率。

黏膜相关恒定T细胞在肝脏疾病中的作用研究进展

黏膜相关恒定T细胞(MAIT)是一群进化保守、非传统的先天性T细胞亚群,兼具固有免疫细胞和适应性免疫细胞的特点,具有抗菌和组织修复等作用,是构成免疫系统的重要部分。MAIT细胞在肝脏中大量存在,并在各种肝脏疾病中发挥着重要作用。研究发现,MAIT细胞数量的减少及免疫功能的异常与疾病严重程度密切相关。本文综述MAIT细胞的特点及其在慢性病毒性肝炎、自身免疫性肝病、非酒精性脂肪性肝病、酒精性肝病及原发性肝癌中作用的最新研究进展,并探讨造成MAIT细胞减少的可能机制。

MAIT细胞在肝炎发生发展中的作用研究进展

黏膜相关恒定T(MAIT)细胞是一种非传统的固有免疫样T细胞,其激活方式不同于传统T细胞,其TCR主要识别主要组织相容性复合物相关蛋白1(MR1)提呈的抗原,其激活也可通过识别腺病毒载体及IL-12、IL-18等细胞因子完成。活化后的MAIT细胞可释放多种细胞因子,直接或间接参与机体免疫应答。MAIT细胞主要分布于黏膜组织和肝脏中,在肝脏中占T细胞的比例可高达50%。MAIT细胞在治疗肝脏疾病过程中发挥着重要作用。肝炎是许多肝脏疾病的早期诱导因素,早期的肝炎得不到及时治疗就会发展为慢性肝炎甚至肝癌。就MAIT细胞应对肝炎所发挥的免疫作用展开综述。

抗感染免疫应答中黏膜相关恒定T细胞作用的研究进展

黏膜相关恒定T细胞(mucosal-associated invariant T cells,MAIT细胞)是一类进化保守的固有样淋巴细胞,通过主要组织相容性复合体Ⅰ类样蛋白识别抗原,分泌多种细胞因子,产生细胞毒性,发挥免疫学效应。MAIT细胞作为具有适应性免疫功能特点的固有免疫细胞,参与了多种感染性疾病的发生发展,有望成为潜在的免疫治疗靶点。

非酒精性脂肪性肝病患者外周血恒定自然杀伤T细胞和CD4^(+)/CD8^(+)T细胞活化差异研究

目的分析不同非酒精性脂肪性肝病(NAFLD)患者外周血恒定自然杀伤T细胞(iNKT)、CD4^(+)和CD8^(+)T细胞活化标记物(CD69、CD25、HLA-DR和NKG2D)的表达差异.方法2020年1月~2022年7月我院诊治的NAFLD患者64例和同期健康体检者50例,对NAFLD患者行肝穿刺活检,使用流式细胞仪检测外周血iNKT、CD4^(+)和CD8^(+)T细胞CD69、CD25、HLA-DR和NKG2D表达.结果在64例NAFLD患者中,经组织病理学检查,诊断NAFL 37例和NASH 27例;健康对照者、NAFL和NASH患者健康对照者、NAFL和NASH患者外周血CD69^(+)iNKT细胞百分比分别为(10.1±1.7)%、(6.1±1.3)%和(26.7±3.6)%(P<0.05),CD25^(+)iNKT细胞百分比分别为(83.0±5.9)%、(94.1±8.0)%和(90.8±7.5)%(P<0.05),HLA-DR^(+)iNKT细胞百分比分别为(15.3±1.7)%、(15.8±2.0)%和(22.3±2.0)%(P>0.05),NKG2D^(+)iNKT细胞百分比分别为(44.5±3.5)%、(59.7±4.0)%和(71.3±6.0)%(P<0.05);外周血CD69^(+)CD4^(+)T细胞百分比分别为(0.7±0.2)%、(0.4±0.1)%和(0.5±0.1)%(P>0.05),CD25^(+)CD4^(+)T细胞百分比分别为(1.4±0.6)%、(3.0±1.3)%和(1.5±0.7)%(P>0.05),HLA-DR^(+)CD4^(+)T细胞百分比分别为(2.7±0.7)%、(4.1±1.0)%和(3.9±1.0)%(P<0.05),NKG2D^(+)CD4^(+)T细胞百分比分别为(1.6±0.5)%、(0.6±0.2)%和(0.9±0.2)%(P<0.05);外周血CD69^(+)CD8^(+)T细胞百分比分别为(2.0±0.4)%、(1.6±0.3)%和(2.1±0.6)%(P>0.05),CD25^(+)CD8^(+)T细胞百分比分别为(1.3±0.3)%、(1.1±0.2)%和(1.0±0.2)%(P>0.05),HLA-DR^(+)CD8^(+)T细胞百分比分别为(5.0±0.7)%、(6.5±1.0)%和(9.6±1.4)%(P<0.05),NKG2D^(+)CD8^(+)T细胞百分比分别为(0.6±0.1)%、(0.5±0.1)%和(0.9±0.2)%(P<0.05).结论本研究发现NAFL与NASH患者可能存在外周血iNKT细胞、CD4^(+)和CD8^(+)T细胞活化的免疫表型差异,显示NASH患者CD69^(+)iNK T细胞百分比增高,可能对诊断有帮助,值得进一步研究.

iNKT细胞对哮喘小鼠肺树突状细胞表面分子和促炎性细胞因子表达水平的影响

目的:观察iNKT细胞对哮喘小鼠肺树突状细胞(LDCs)表面分子和促炎性细胞因子表达水平的影响。方法:24只野生型BALB/c小鼠随机分为正常对照组、哮喘组和过继转移组,每组8只; 12只CD1d^(-/-)-BALB/c小鼠随机分为CD1d^(-/-)对照组和CD1d^(-/-)哮喘组,每组6只。哮喘组、过继转移组和CD1d^(-/-)哮喘组小鼠以卵清白蛋白致敏和激发,正常对照组和CD1d^(-/-)对照组以等量PBS替代,其中过继转移组在第1次激发前1 h给予iNKT细胞尾静脉注射。采用流式细胞仪检测各组小鼠LDCs及表面分子MHCⅡ、CD80、CD86和CD40的表达水平; ELISA法检测LDCs体外培养上清液IL-12p70、IL-6、TNF-α和IL-10水平。结果:过继转移组小鼠LDCs数量和MHCⅡ、CD40、CD80、CD86表达水平及LDCs体外培养上清液IL-12p70、IL-6、TNF-α水平明显高于哮喘组(P<0. 05或P<0. 01); CD1d^(-/-)哮喘组小鼠LDCs数量和MHCⅡ、CD40、CD80、CD86表达水平及LDCs体外培养上清液IL-12p70、IL-6、TNF-α水平明显低于哮喘组(P<0. 05或P<0. 01),但明显高于正常对照组和CD1d^(-/-)对照组(P<0. 05或P<0. 01)。结论:iNKT细胞可以增强哮喘小鼠LDCs表面分子和促炎性细胞因子的表达水平。

重型再生障碍性贫血患儿外周血及骨髓NKT细胞数量及功能变化

目的探讨NKT细胞数量及其功能在重型再生障碍性贫血患儿外周血及骨髓中的变化。方法采用流式细胞术检测10例重型再生障碍性贫血初治患儿及10例骨科对照儿童外周血及骨髓CD3+CD1d四聚体+NKT细胞水平。利用免疫磁珠法分离纯化i NKT细胞后,在NKT细胞的配体OCH+重组人白介素-2(rh IL-2)+重组粒系集落刺激因子(rh GCSF)培养体系下进行扩增培养。测定在不同浓度OCH培养条件下,i NKT细胞的扩增倍数,并利用酶联免疫斑点技术测定i NKT细胞扩增活化后表达肿瘤坏死因子(IFN-γ)、白细胞介素(IL-4)的斑点形成细胞数。结果再生障碍性贫血患儿外周血中CD3+CD1d四聚体+NKT百分率为(0.72±0.03)%,明显低于对照组的(0.92±0.02)%,骨髓中CD3+CD1d四聚体+NKT百分率为(0.82±0.02)%,明显低于对照组的(1.05±0.05)%,差异有统计学意义(P均=0.000)。再生障碍性贫血患儿骨髓i NKT细胞体外扩增能力显著低于对照组;且中高浓度OCH条件下,i NKT细胞扩增倍数增高,生成IFN-γ降低,而生成IL-4增高,与低浓度条件下相比,差异有统计学意义(P均<0.01)。结论重型再生障碍性贫血患儿存在外周血及骨髓NKT细胞水平降低及功能异常;OCH能够促进i NKT细胞扩增,并可改善IL-4/IFN-γ的失衡,可能具有潜在的治疗价值。

含噻唑烷-4-酮的免疫刺激剂CH2a增强人iNKT细胞的功能

目的研究含噻唑烷-4-酮的糖类衍生小分子免疫刺激剂CH2a对人iNKT细胞功能的影响。方法分离健康人外周血单个核细胞(PBMC),经α-半乳糖神经酰胺(α-Galcer)和IL-2体外扩增后,利用磁珠分选分离纯化得到恒定型自然杀伤T细胞(iNKT细胞)。5(6)-羧基二乙酸荧光黄琥珀酰亚胺酯(CFDA-SE)标记的纯化iNKT细胞,与CH2a共孵育后,流式细胞术检测细胞增殖和分代,IFN-γ和IL-4的表达;MTT法定量检测细胞增殖率及对K562细胞的杀伤活性。ELISA检测CH2a处理细胞培养基中IFN-γ和IL-4水平。结果 CH2a能显著促进IL-2引起的iNKT细胞体外增殖;提高IFN-γ和IL-4的生成量及IFN-γ/IL-4的比值;并显著增强iNKT细胞的杀伤活性。结论 CH2a有可能通过诱导iNKT分泌Th1类的细胞因子,调节T细胞向Th1分化,从而增强细胞免疫功能;同时显著提高iNKT的细胞毒功能。

CD_4^+ iNKT细胞在实验性变应性鼻炎发病中的作用

目的探讨实验性变应性鼻炎发病过程中CD_4^+iNKT细胞浸润情况。方法将30只C57BL/6小鼠随机分为变应性鼻炎组(A组)和对照组(B组)。比较2组小鼠变应性鼻炎症状评分、鼻黏膜嗜酸粒细胞(EOS)计数、鼻黏膜组织形态,免疫酶标技术检测各组鼻黏膜CD_4^+ iNKT细胞的浸润,流式细胞技术(FCM)检测鼻腔黏膜中的CD_4^+ iNKT细胞。并进行比较分析。结果(1)A组症状评分(7.3±1.0)分较B组(0.7±0.6)分明显增高(P<0.01)。(2)A组EOS计数(7.5±1.2)个/HP较B组(0.4±0.5)个/HP明显增高(P<0.01)。(3)HE染色见A组纤毛结构大量脱落,组织水肿,小血管增生,黏膜上皮下固有层EOS、淋巴细胞浸润;B组纤毛结构完整,无明显EOS浸润。(4)A组鼻黏膜下有多量的CD_4和NK1.1双阳性染色细胞,即CD_4^+ iNKT细胞;B组仅见少量的CD_4^+细胞。(5)A组鼻黏膜CD_4^+ iNKT细胞阳性率(72.6%±7.9%)明显高于B组(0.49%±0.01%)(P<0.01)。结论变应性鼻炎小鼠鼻腔黏膜有大量的CD_4^+ iNKT细胞浸润,其数量远远超过CD_4^+T细胞。