HIV感染者自然杀伤样T细胞分泌的TGF-β和IL-10水平

目的探究HIV感染者自然杀伤样T细胞(NKT-like)分泌的TGF-β、IL-10水平及其与疾病进展的关系。方法提取正常对照者和HIV感染者外周血单个核细胞,加入IL-2(200 IU),IL-15(50 ng/ml)及IL-12(10 ng/ml)进行培养,共培养24 h后进行破膜胞内染色,FACS LSRII流式细胞仪检测NKT-like细胞TGF-β、IL-10水平。结果 HIV感染者NKT样细胞分泌的TGF-β明显高于正常对照者(P=0.017),分泌的IL-10有高于正常对照者的趋势;HIV感染者NKT样细胞分泌的TGF-β、IL-10与CD4+T细胞计数、病毒载量(log10VL)没有统计学上的相关性;NKT样细胞分泌的IL-10和TGF-β水平具有明显的正相关(r=0.663,P=0.001);体外加入r IL-10、r TGF-β可抑制NKT样细胞CD107a或IFN-γ的分泌。结论 HIV感染者NKT样细胞分泌的免疫抑制性细胞因子TGF-β、IL-10增多,可能对HIV感染者免疫功能紊乱起着促进作用。     

未治疗HIV 感染者NKT 样细胞活化、凋亡和增殖的相关研究

目的:研究HIV 感染后NKT 样细胞活化、凋亡和增殖的变化情况。方法:选取47 名未治疗的HIV 感染者和31 名健康对照者,提取外周血细胞,用荧光标记抗体进行染色,利用流式细胞仪检测HIV 感染者NKT 样细胞HLA-DR、Annexin-V、Ki-67 等表面分子的表达。结果:未治疗HIV 感染者NKT 样细胞百分数为(3.03±1.61)%,正常人NKT 样细胞百分数为(8.30±7.42)%,HIV 感染者NKT 样细胞百分数显著低于健康对照组(P<0.05);未治疗HIV 感染者HLA-DR 表达为(5.40±4.10)%,健康对照组HLA-DR 表达为(0.89±0.83)%,HIV 感染者活化程度明显高于健康对照组(P<0.001),且活化程度与CD4+ T 细胞计数呈负相关(r =-0.885 7,P<0.05);未治疗HIV 感染者Annexin-V 表达为(30.21±13.15)%,凋亡程度明显高于健康对照组(5.40±8.05)%,(P<0.01);未治疗HIV 感染者Ki-67 的表达为(11.15±4.76)%,增殖能力明显低于健康对照组(27.63±18.31)%,(P<0.05)。结论:HIV 感染可明显降低NKT 样细胞数量及增殖能力,而其活化及凋亡能力增加。     

中国HIV/AIDS患者NK细胞及NKT细胞变化的检测

目的　探讨HIV感染后机体NK(naturalkillercells)及NKT细胞的变化情况。方法　取外周血细胞 ,用标记荧光的抗体进行染色 ,流式细胞仪分析HIV AIDS患者NK和NKT细胞的变化。结果　HIV AIDS患者NK、NKT细胞和CD4 + T细胞显著低于正常对照 ;CD8+ T细胞显著高于正常对照。HIV AIDS患者NK百分数显著低于正常对照 ,与CD4 + T细胞数量成正比 ,r=0 .2 89,P <0 .0 1 ;NKT细胞数量与CD4 + T细胞数量成正比 ,r =0 .378,P <0 .0 1 ;与CD8+ T细胞数量成正比 ,r =0 .340 ,P <0 .0 1 ;长期不进展组NKT、NK细胞数量与正常对照组差异无显著性。结论　HIV感染可明显降低HIV AIDS患者NK和NKT细胞数量 ,NK和NKT细胞变化与疾病进展密切相关。     

抗病毒治疗后HIV 感染者NKT 样细胞衰老和体外增殖研究

目的:研究高效抗逆转录病毒治疗(Highly active antiretroviral therapy,HAART)后NKT 样细胞衰老和体外增殖情况。方法:选取未接受HAART 治疗、接受HAART 治疗的HIV 感染者以及健康人的外周血细胞,利用流式细胞仪检测接受HAARRT 治疗前后的HIV 感染者NKT 样细胞CD57 的表达情况以及体外增殖能力。结果:HIV 感染者在HAART 治疗前NKT 样细胞的百分数显著低于健康对照组(P<0.01),HAART 治疗后恢复(P<0.05);HAART 治疗前NKT 样细胞CD57 的表达明显高于健康对照组(P<0.01),HAART 治疗后恢复(P<0.05);HAART 治疗前NKT 样细胞体外增殖能力明显低于健康对照组,HAART 治疗后有所恢复。结论:经过HAART 治疗后,HIV 感染者NKT 样细胞的数量、CD57 表达以及体外增殖能力有所恢复。     

HIV慢性感染者CD3~+CD56~+NKT样细胞表面Gal-9受体表达研究

目的研究Gal-9在HIV慢性感染者NKT样细胞上的表达情况。方法选取HIV慢性感染者29例和健康人21例。对外周血单个核细胞进行荧光抗体染色,利用流式细胞仪检测CD3~+CD56~+NKT样细胞Gal-9受体表达的情况,然后分析Gal-9受体在HIV感染前后的表达差异及其与CD4~+T细胞计数和病毒载量之间的关系。结果 HIV慢性感染者CD3~+CD56~+NKT样细胞占总淋巴细胞的百分比明显低于健康对照组(P=0.020)。HIV慢性感染者Gal-9~+NKT%与健康对照组相比又明显的增加(P=0.016)。正常人和感染者的NKT样细胞上Gal-9的平均荧光强度(MFI)无明显差异,而低CD4~+T细胞组(CD4~+T350/μl)的NKT样细胞上Gal-9的MFI比正常人组显著增加(P=0.022)。Gal-9~+NKT%与CD4~+T细胞数无明显相关性,而Gal-9的MFI与CD4~+T细胞数呈负相关(P=0.025)。结论 HIV慢性感染者与正常人相比CD3~+CD56~+NKT样细胞GAL-9受体表达的百分数明显增加,Gal-9 MFI与CD4~+T细胞数成负相关,提示NKT样细胞Gal-9受体表达是HIV疾病进展的标志物之一。     

HIV原发感染者自然杀伤样T细胞NKG2A/NKG2D变化的研究

目的深入了解人类免疫缺陷病毒(human immunodeficiency virus,HIV)原发感染者(primary HIV infection,PHI)NKT样细胞表面NKG2A/NKG2D受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV原发感染者和27例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血NKT样细胞表面NKG2D和NKG2A的表达。结果 HIV原发感染者NKT样细胞绝对数和百分率显著低于健康对照(P<0.01)。HIV原发感染者NKT样细胞表面NKG2A、NKG2D受体表达与健康对照并无显著差异。HIV原发感染者病毒调定点低组NKG2A+NKT样细胞、NKG2A+NKG2D-NKT样细胞以及NKG2A+NKG2D+NKT样细胞百分率均显著低于病毒调定点高组(P<0.05);NKT细胞绝对数和百分率、NKG2D+NKT样细胞、NKG2D+NKG2A-NKT样细胞百分率在两组间相似,没有显著性差异。NKG2A+NKT细胞的百分比与病毒载量正相关(R=0.430,P=0.032)。结论 NKT样细胞数量以及其表面NKG2A受体的表达可作为HIV疾病进程的预测指标之一。     

HIV感染症状长期不进展者NK细胞变化研究

目的探讨HIV长期不进展者NK细胞的变化. 方法应用流式细胞术对HIV长期不进展者、典型进展者和HIV-抗体阴性正常对照外周血NK细胞、NKT细胞及NK细胞趋化因子受体等进行研究. 结果长期不进展者NKT细胞绝对计数与正常对照差异无统计学意义(P＝0.301),高于HIV感染者和艾滋病病人(P＝0.01, P＝0.002);长期不进展者NK细胞绝对计数低于正常对照(P＝0.03),高于HIV感染者和艾滋病病人(P＝0.005, P＜0.0001);长期不进展者NK细胞与CD4+ T淋巴细胞呈正相关(r＝0.393,P＝0.001);NKT细胞与CD8+ T淋巴细胞呈正相关(r＝0.372,P＝0.002).长期不进展者NK细胞表达的CCR5受体低于典型进展者和正常对照(P＜0.01). 结论 NK细胞的变化与HIV疾病进展相关,值得深入研究.     

CD11b与CD27定义的T细胞亚群与HIV疾病进展的相关性研究

目的探究CD11b与CD27定义的T细胞亚群与HIV疾病进展的相关性,为研究HIV感染者T细胞免疫缺陷提供新的思路。方法外周血荧光抗体染色,用流式细胞仪检测CD11b、CD27及各亚群的表达;破膜胞内染色检测各亚群分泌IFN-γ的功能;细胞因子与PBMC共培养,检测CD11b、CD27的变化。结果在HIV-1感染者,CD27~+CD11b~-、CD27~+CD11b~+和CD27~-CD11b~+的T细胞亚群明显降低(P0.05),而CD27~-CD11b~-T细胞亚群明显增多(P0.05),该亚群与CD4~+T细胞呈显著的负相关(r=-0.545,P0.001);CD27~+CD11b~-、CD27~+CD11b~+和CD11b~+CD27~-亚群分泌IFN-γ功能均强于CD27~-CD11b~-亚群;细胞因子TGF-β随着质量浓度的增加对CD11b表达的抑制也逐渐增强,而IL-12和IL-15能够刺激CD11b的增多,IL-2与IL-7能够刺激CD27增多。结论 HIV感染导致人体T细胞亚群发生紊乱。     

CD4+CD25 nt/hi CD127lo调节性T细胞对HIV感染者免疫状态及病程进展的影响

目的:探讨具有CD4 CD25nt/hi CD127lo特征的调节性T细胞频率对中国HIV-1感染者免疫状态及病程进展的影响.方法:选取100名未经治疗的HIV-1感染者和4个年龄组的312名健康人对照,采集静脉血,经三重免疫荧光染色,用流式细胞术分析CD4 CD25 Treg表型和CD4 CD25nt/hiCD127loTreg频率并进行CD4 T细胞绝对计数;应用酶联免疫斑点技术(ELISpot)在单细胞水平观察受试者的HIV-1特异性细胞免疫功能;平行检测HIV感染者的病毒载量(NASBA方法).结果:不同病程的HIV-1感染者外周血中CD4 CD25nt/hiCD127loTreg频率存在差异,进展期高于新近感染者(P＜0.001),其平均水平显著高于健康人(P＜0.001);CD4 CD25nt/hiCD127loTreg频率与HIV感染者CD4 T细胞数量呈显著负相关(r=0.354,P＜0.01),与病毒载量呈明显正相关(r=0.287,P＜0.01);高频率CD4 CD25nt/hiCD127loTreg HIV-1感染者(进展期)的外周血PBMCs经HIV-1多肽刺激后分泌IFN-γ的CD8 T细胞频率明显低于无症状的新近感染者.结论:初步证实HIV-1感染者外周血中CD4 CD25nt/hiCD127kloTreg细胞频率增加与CD4 T细胞数量降低及病程进展相关;高频率CD4 CD25nt/hiCD127lo Treg细胞对HIV-1感染者的细胞免疫功能具有抑制作用.本结果为进一步阐明HIV持续感染的免疫机制提供了新依据.     

免疫调控受体PD-1和TIGIT在HIV感染者/HIV患者CD3~+CD4~-T细胞表达研究

深入了解免疫调控受体程序性死亡分子1(programmed death 1,PD-1)和T细胞免疫球蛋白及免疫酪氨酸样抑制基序(T cell immunoglobulin and ITIM domain,TIGIT)在人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者/HIV患者外周血CD3~+CD4~-T细胞的表达情况及其与疾病进展的关系。用流式细胞仪检测24例未治疗的HIV感染者/HIV患者外周血CD3~+CD4~-T细胞表面PD-1和TIGIT的表达百分比、CD4+T细胞绝对值计数,实时荧光定量PCR检测HIV病毒载量,并与20例HIV抗体阴性者表达情况比较(HIV-negative normal control,NC)。结果发现HIV感染者/HIV患者CD3~+CD4~-T细胞表面TIGIT受体表达百分数较健康对照组明显增高(P0.000 1),与CD4+T细胞绝对计数呈负相关(r=-0.450 4,P=0.027 1),与病毒载量呈正相关(r=0.621 4,P=0.001 2);HIV感染者CD3~+CD4~-T细胞PD-1受体表达百分数较健康对照组明显增高(P0.000 1),与CD4+T细胞绝对计数呈负相关(r=-0.455,P=0.0255);PD-1和TIGIT共表达的CD3~+CD4~-T细胞百分数在HIV感染者/HIV患者中表达明显增高(P0.000 1)。HIV感染机体后,CD3~+CD4~-T细胞PD-1受体和TIGIT受体表达百分数均明显增高,且与HIV疾病进展密切相关。     

Differential modulating effect of natural killer (NK) T cells on interferon-γ production and cytotoxic function of NK cells and its relationship with NK subsets in Chlamydia muridarum infection

Natural killer T (NKT) cells are a newly identified T-cell population with potential immunomodulatory functions. Several studies have shown modulating effects of NKT cells activated by α-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on the interferon-γ (IFN-γ) production and cytolytic function of NK cells in a chlamydial infection model, using NKT-cell-deficient mice and antibody blocking (anti-CD1d monoclonal antibody) approaches. Our results showed that both NKT and NK cells became activated and produced IFN-γ following Chlamydia muridarum infection in vitro and in vivo. The NK cells in NKT-cell-deficient mice and CD1d-blocked mice showed decreased CD69 expression, cellular expansion and IFN-γ production but surprisingly showed increased cytolytic activity (degranulation) of immature and more mature NK cell subsets, suggesting an inhibitory role of NKT cells on NK cell killing activity. The results suggest that NKT cells preferentially promote IFN-γ production but are inhibitory for the cytotoxic function of NK cells in this infection model. Furthermore, the differential modulating effect of NKT cells on the IFN-γ production and cytotoxicity of NK cells was observed in immature and mature NK cell subsets, although it was more dramatic in the relatively mature CD11b(high) CD27(high) NK cell subset. This finding demonstrates the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting.     

Higher NK Cell IFN-γ Production is Associated with Delayed HIV Disease Progression in LTNPs

Natural killer (NK) cells are important effectors of the innate immune system that help control viral infections and tumorigenesis. However, the relationship between NK cell function and HIV disease progression remains poorly defined. In this study, we examined the function of NK cells in Chinese patients who were HIV-infected but treatment-naïve. These individuals include primary HIV-infected patients (PHIs), typical progressors (TPs), and long-term nonprogressors (LTNPs). We observed an increase of CD56^dim NK cells in PHIs, but the production of interferon-gamma (IFN-γ) and CD107a expression in PHIs were not altered compared with normal control subjects (NCs). However, the NK cells from LTNPs exhibited increased activities in IFN-γ production, CD107a expression and granzyme B change after K562 stimulation compared with NCs. Furthermore, the percentage of IFN-γ⁺CD107a^? NK cells in LTNPs was higher than that in TPs, PHIs and NCs; levels of IFN-γ production in LTNP NK cells exhibited an inverse correlation with viral loads. Similar correlations, however, were not observed in the PHI and TP groups. Taken together, these data demonstrate that enhanced NK cell function may contribute to the control of HIV infection, and increased IFN-γ secretion may be associated with delayed disease progression.     

IFN-γ production downstream of NKT cell activation in mice infected with influenza virus enhances the cytolytic activities of both NK cells and viral antigen-specific CD8 T cells

Natural killer T (NKT) cell activation is responsible for eliminating pathogens. However, the biological functions of NKT cells against influenza virus are not fully understood. We therefore investigated the effects of NKT cells in viral infection using CD1d knockout (KO) mice. When CD1d KO or wild-type (WT) mice were infected with a sub-lethal dosage of the influenza virus, the survival rate of CD1d KO mice was significantly lower than for WT mice in association with delayed viral clearance in the lungs. Consistently, IFN-γ production in bronchoalveolar lavage fluid of CD1d KO mice was largely reduced compared to WT mice during infection. Moreover, the cytotoxic activities of NK cells and viral antigen-specific CD8⁺ T cells were impaired in CD1d KO mice. It was concluded that activated NKT cell-induced IFN-γ release enhances both NK-cell activity and antigen-specific CD8⁺ T cells to eliminate the influenza virus, thus leading to an enhanced survival.     

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes.We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.     

HIV感染者T细胞NKG2C/NKG2A表达及与疾病进展关系研究

目的：研究HIV慢性感染者和HARRT治疗者T细胞NKG2C/NKG2A受体的表达变化,探讨其与疾病进展的关系。方法：选取HIV慢性感染者、接受HAART治疗的HIV感染者以及健康人的外周血细胞,通过荧光抗体染色,利用流式细胞仪检测T细胞上表达的NKG2C/NKG2A受体。结果：HIV慢性感染者NKG2C⁺ T细胞,NKG2A⁺ T细胞和NKG2C+NKG2A- T细胞百分比明显高于健康对照组 (P=0.025、P=0.032、P=0.029),HARRT治疗组则明显低于HIV慢性感染者(P=0.033、P=0.037、P=0.018),恢复到正常水平,与健康对照组相比无统计学差异。HIV慢性感染者外周血CD4⁺ T淋巴细胞绝对数和表达NKG2A、NKG2C⁺NKG2A⁺和NKG2C-NKG2A⁺ 的T细胞呈负相关(r=-0.697,P<0.000 1;r=-0.463,P=0.015;r=-0.693,P<0.000 1),HIV慢性感染者外周血T细胞上表达的NKG2C与NKG2A的比值与CD4⁺ T淋巴细胞绝对数呈正相关(r=0.476,P=0.012)。结论：HIV感染者T细胞表面NKG2C和NKG2A的表达研究具有重要意义,为HIV感染的临床预后评估提供科学依据。