Linker for activation of T cells contributes to airway inflammation in an asthmatic mouse model

Background Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by dysregulated production of cytokines secreted by allergen-specific helper T-type 2 （Th2） cells. The linker for activation of T cells （LAT） is a membrane-associated adaptor protein, which has been shown to take part in regulating T cell receptor （TCR） signaling and T cell homeostasis. In this study, we established an asthmatic mouse model to examine the changes in LAT levels during allergic airway disease and the effects of LAT transgenic expression on airway inflammation. Methods T cells from mouse lung tissues were isolated from allergen challenged （ovalbumin （OVA）） and control mice, and the purity of these isolated T cells was examined by fluorescence-activated cell sorter （FACS）. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of the LAT gene and LAT protein, respectively. After an intranasally administered mixture of pCMV-HA-LAT plasmid and Lipofectamine 2000, 24 hours before and 72 hours after allergen challenge, the BALF cell count and the differential cytologies were studied. In addition, IL-4 and IFN-γ levels in the BALF were determined by ELISA, and pathological changes in lung tissues were observed. Results LAT protein and mRNA expression were decreased in lung T cells in a mouse model of allergen-induced airway disease. After intranasal administration of pCMV-HA-LAT, histopathological examination of the lungs showed that intervention with LAT overexpression prevented mice from developing airway inflammation, and the number of total cells, eosinophils, neutrophils, and lymphocytes in the BALF was reduced significantly compared with the OVA sensitized and challenged group. In addition, the Th2 cytokine IL-4 decreased, while the Thl cytokine IFN-γ increased compared to the OVA sensitized and challenged group or the OVA sensitized group plus pCMV-HA treatment. Conclusion This study demonstrates that LAT might effectively diminish Th2 cytokine responses, lung histopathological changes and lung inflammation to allergen challenge in a model of expedmentally induced asthma.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

AIM:To investigate the role of nuclear factor of activated T cell 2(NFAT2),the major NFAT protein in peripheral T cells,in sustained T cell activation and intractable inflammation in human ulcerative colitis(UC). METHODS:We used two-dimensional gel-electrophoresis, immunohistochemistry,double immunohistochemical staining,and confocal microscopy to inspect the expression of NFAT2 in 107,15,48 and 5 cases of UC, Crohn's disease(CD),non-specific colitis,and 5 healthy individuals,respectively. RESULTS:Up-regulation with profound nucleo- translocation/activation of NFAT2 of lamina propria mononuclear cells(LPMC)of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC,as compared to CD or NC(P<0.001,Kruskal- Wallis test).Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T,but was less prominent in CD4+T cells or CD20+B cells.It was strongly associated with the disease activity,including endoscopic stage (τ=0.2145,P=0.0281)and histologic grade(τ=0.4167, P<0.001). CONCLUSION:We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis.Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity.Since activation of NFAT2is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways,our results not only provide new insights into the mechanism for sustained intractable inflammation,but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis.

Novel function of perforin in negatively regulating CD4^＋ T cell activation by affecting calcium signaling

Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic T lymphocytes （CTLs） and natural killer cells. However, whether it also plays a role in conventional CD4^＋ T cell function remains unclear. Here we report that in perforin-deficient （PKO） mice, CD4^＋ T cells are hyperproliferative in response to T cell receptor （TCR） stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD4^＋ T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFAT1. Our results indicate that perforin plays a negative role in regulating CD4^＋ T cell activation and immune response by affecting TCR-dependent Ca^2＋ signaling.

The phenotype and activation status of regulatory T cells during Friend retrovirus infection

The suppressive capacity of regulatory T cells （Tregs） has been extensively studied and is well established for many diseases.The expansion,accumulation,and activation of Tregs in viral infections are of major interest in order to find ways to alter Treg functions for therapeutic benefit.Tregs are able to dampen effector T cell responses to viral infections and thereby contribute to the establishment of a chronic infection.In the Friend retrovirus （FV） mouse model,Tregs are known to expand in all infected organs.To better understand the characteristics of these Treg populations,their phenotype was analyzed in detail.During acute FV-infection,Tregs became activated in the spleen and bone marrow,as indicated by various T cell activation markers,such as CD43 and CD103.Interestingly,Tregs in the bone marrow,which contains the highest viral loads during acute infection,displayed greater levels of activation than Tregs from the spleen.Treg expansion was driven by proliferation but no FV-specific Tregs could be detected.Activated Tregs in FV-infection did not produce Granzyme B （GzmB） or tumor necrosis factor α （TNFα）,which are thought to be a potential mechanism for their suppressive activity.Furthermore,Tregs expressed inhibitory markers,such as TIM3,PD-1 and PD-L1.Blocking TIM3 and PD-L1 with antibodies during chronic FV-infection increased the numbers of activated Tregs.These data may have important implications for the understanding of Treg functions during chronic viral infections.

Driving Forces of AIDS Pathogenesis:Massive CD4^+ T Lymphocyte Depletion and Abnormal Immune Activation

The occurrence of massive CD4+ T cell depletion is one of the most prominent characteristics of human immunodeficiency virus type 1 (HIV-1) infection during acute phase, resulting in unrestorable destruction to the immune system. The infected host undergoes an asymptomatic period lasting several years with low viral load and ostensibly healthy status, which is presumably due to virus-specific adaptive immune responses. In the absence of therapy, an overwhelming majority of cases develop to AIDS within 8-10 years of latent infection. In this review, we discuss the roles in AIDS pathogenesis played by massive CD4+ T lymphocytes depletion in gut-associated lymphoid tissue (GALT) during acute infection and abnormal immune activation emerging in the later part of chronic phase.

ACTIVATION OF TCELLS BY SYNTHETIC PEPTIDES OF SUPERANTIGEN TSST-1 UNDER ASSISTANCE OF ASSITANT MOLECULES

根据以往对超抗原ＴＳＳＴ－１分子的Ｔ细胞表位预测结果，从ＴＳＳＴ－１分子中选择了一段序列，合成了两个交叠肽，Ｔ３４和Ｔ５８；观察它们在辅助分子辅助下的促Ｔ细胞增殖能力。结果发现：Ｔ３４和Ｔ５８单独虽均不能活化人的ＰＢＭＣ或小鼠脾细胞，但在ＣＤ２８的共刺激或ＰＭＡ的辅助下却可活化人的ＰＢＭＣ和小鼠的脾细胞。提示：Ｔ３４和Ｔ５８不具备ＭＨＣ结合位，但含有Ｔ细胞表位，两者的Ｔ细胞表位可能位于两肽的共同序列内，即ＴＳＳＴ－１（１２５～１５８）。

BAFF调节免疫性血小板减少症模型小鼠的Th17/Treg平衡的研究

目的:探讨B细胞激活因子(BAFF)对免疫性血小板减少症模型小鼠体内辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的调节作用和潜在机制。方法:制备豚鼠抗小鼠血小板抗血清(GP-APS),并将150只无特定病原级别的成年雄性BALB/c小鼠(7~8周龄)随机分为5组,每组30只。分别为对照组(空白对照)和ITP组(GP-APS诱导),ITP+rhBAFF组(ITP组联合静脉注射50μg/kg/50μL重组人BAFF蛋白),并在ITP+rhBAFF组处理的基础上分别联合Notch1的抑制剂(DAPT)或PI3K/Akt的抑制剂Polygalacin D(PGD),设立ITP+rhBAFF+DAPT组和ITP+rhBAFF+PGD组,除对照组和ITP组外,均为静脉注射给药,DAPT注射剂量100μg/kg;PGD注射剂量25μg/kg,静脉注射总体积均为50μL,每日1次。1周后取小鼠1mL外周血并分离血清和单个核细胞。用免疫荧光化学检测单个核细胞中BAFF和Notch1的定位。对外周血中的血小板进行计数。酶联免疫吸附法(ELSIA)检测小鼠外周血血清BAFF的水平。Western blot检测小鼠外周血单个核细胞中PI3K、AKT、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的蛋白表达。流式细胞术检测单个核细胞中Th17/Treg的比例变化。结果:ITP小鼠外周血单个核细胞的BAFF与Notch1共定位在细胞膜。与对照组比较,ITP组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和Treg比例减少,Th17比例增加(P<0.05)。与ITP组比较,ITP+rhBAFF组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达增加,血小板数目和Treg比例减少,Th17比例增加(P<0.05)。与ITP+rhBAFF组比较,ITP+rhBAFF+DAPT组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和Treg比例增加,Th17比例降低(P<0.05)。与ITP+rhBAFF组比较,ITP+rhBAFF+PGD组BAFF、Notch1、p-Akt(Thr308)、p-Akt(Ser473)的表达降低,血小板数目和Treg比例增加,Th17比例降低(P<0.05)。结论:BAFF通过激活Notch1/PI3K/Akt信号通路促进免疫性血小板减少症模型小鼠体内Th17比例增加及Treg比例减少。

T细胞活化谱对脓毒症患者病原菌类型的鉴别效果

目的探讨T细胞活化谱鉴别脓毒症不同病原菌感染中的临床价值。方法选取2020年9月至2022年6月于西安交通大学第一附属医院接受治疗的100例脓毒症患者作为脓毒症组,选取同期于门诊进行体检的37例健康志愿者作为对照组。脓毒症组根据患者入院病原学检查结果分为革兰氏阴性菌组(简称阴性菌组,46例)和革兰氏阳性菌组(简称阳性菌组,54例)。通过流式细胞仪检测患者外周血中T淋巴细胞的活化状态及其表面共刺激分子的表达情况。采用受试者操作特征(ROC)曲线评估CD38^(+)HLA-DR^(+)T细胞区分革兰氏阴性菌脓毒症和革兰氏阳性菌脓毒症的能力。结果阴性菌组和阳性菌组的外周血中CD4^(+)CD38^(+)CD69^(+)T细胞和CD8^(+)CD38^(+)CD69^(+)T细胞计数比较差异无统计学意义(P>0.05),但均高于对照组(P<0.05)。与对照组相比,脓毒症各亚组外周血中CD4^(+)CD38^(+)HLA-DR^(+)、CD8^(+)CD38^(+)HLA-DR^(+)T细胞计数显著增加(P<0.05),并且阴性菌组大于阳性菌组(P<0.05)。鉴别革兰氏阴性菌脓毒症和革兰氏阳性菌脓毒症时,CD4^(+)CD38^(+)HLA-DR^(+)T细胞的ROC曲线下面积(AUC)为0.901(95%CI 0.837~0.965),特异度为0.867,敏感度为0.836;CD8^(+)CD38^(+)HLA-DR^(+)T细胞的AUC为0.927(95%CI 0.872~0.982),特异度为0.778,敏感度为0.933。结论人白细胞DR抗原(HLA-DR)参与了脓毒症患者外周血中T细胞活化的关键免疫反应,并能区分脓毒症不同病原菌感染。

川崎病患儿血清CaN、NFATc1水平与免疫球蛋白治疗反应的相关性

目的探讨川崎病患儿血清钙调神经磷酸酶(CaN)、活化T细胞核因子c1(NFATc1)水平与免疫球蛋白(IVIG)治疗反应的相关性,以期为临床改善治疗效果提供理论参考。方法采用前瞻性研究,选取平顶山市第一人民医院2018年1月至2023年1月100例川崎病患儿作为研究对象,检测入院时患儿的血清CaN、NFATc1水平,同时收集患儿的一般资料,根据IVIG治疗反应情况分为IVIG治疗敏感组与IVIG治疗无反应组。比较两组患儿的血清CaN、NFATc1水平及一般资料,采用点二列相关性检验血清CaN、NFATc1水平与IVIG治疗反应之间的关系,并采用logistic回归性检验二者对IVIG治疗反应的影响。结果100例患儿中有20例为IVIG治疗无反应,占比为20%(20/100)。IVIG治疗无反应组患儿入院时血清CaN、NFATc1及C反应蛋白(CRP)水平高于IVIG治疗敏感组(P<0.05)。经点二列相关性检验显示血清CaN、NFATc1水平与川崎病患儿IVIG治疗无反应存在正相关关系(r>0,P<0.05)。经logistic回归性分析检验显示高水平血清CaN、血清NFATc1是导致患儿IVIG治疗无反应的影响因素(P<0.05)。结论川崎病患儿IVIG治疗无反应发生风险较高,且与血清CaN、血清NFATc1存在关系,二者的高水平表达是导致IVIG治疗无反应的影响因素。

免疫检查点T细胞激活抑制物免疫球蛋白可变区结构域、人内源性逆转录病毒-H长端重复相关蛋白2在子宫内膜癌中的表达及临床意义

目的分析免疫检查点T细胞激活抑制物免疫球蛋白可变区结构域(VISTA)、人内源性逆转录病毒-H长端重复相关蛋白2(HHLA2)在子宫内膜癌(EC)中的表达及临床意义。方法选取2017年9月至2019年10月唐山市妇幼保健院收治的120例的EC患者作为研究对象,收集其的EC组织和癌旁正常组织。检测EC组织和癌旁正常组织VISTA、HHLA2的表达;采用Spearman分析免疫检查点VISTA、HHLA2的相关性;Kaplan-Meier分析VISTA、HHLA2与预后的关系;Cox回归分析影响EC患者预后的危险因素。结果EC组织中VISTA、HHLA2阳性表达率显著高于正常癌旁组织(P<0.05)。Spearman相关性结果显示EC组织中VISTA与HHLA2表达呈正相关性(r=0.587,P<0.05)。EC组织中VISTA、HHLA2表达与临床病理特征有关(P<0.05)。Kaplan-Meier曲线结果显示VISTA阳性表达患者3年生存率低于阴性表达患者(χ^(2)=11.864,P<0.05),HHLA2阳性表达患者3年生存率低于阴性表达患者(χ^(2)=4.975,P<0.05)。Cox回归分析结果显示VISTA和HHLA2是影响EC患者预后的危险因素(P<0.05)。结论免疫检查点VISTA、HHLA2在EC中高表达,其是影响EC预后的危险因素,二者可能是有价值的预后标志物。

Indoleamine 2,3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs), and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC func- tions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflam- matory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regu- lated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.

Effect of IFNα-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B

Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

Transplantation Expression of 4-1BB molecule on peripheral blood T cells in liver transplanted patients and its clinical implication

OBJECTIVE: To investigate the gene expression of 4-1BB in peripheral blood mononuclear cells (PBMCs) and its possible significance in clinical liver transplantation. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of 4-1BB in PBMCs from 22 patients receiving liver transplantation, 13 patients with primary liver carcinoma (PLC), and 12 healthy controls. To determine whether 4-1BB molecule is also expressed on the surface of CD4^+ and CD8^+ T cell, flow cytometry was used to analyse the phenotype of T cell subsets from the blood of liver transplantation patients. RESULTS: 4-1BB mRNA was detected in PBMCs from stable survivors after liver transplantation, but almost not deteeted in PBMCs from PLC patients and healthy controls. Meanwhile, 4-1BB was almost not expressed on the surface of CD4^+ and CD8^+ T cells in healthy controls and PLC patients. A low level of 4-1BB expression, however, was found on the surface of CD4^+ and CD8^+ T cells from the stable survivors after liver transplantation. CONCLUSIONS: This study demonstrates that although patients are stable after liver transplantation, effector T-cells can also be activated through the signal of 4-1BB molecule and persistent irmmune response to grafts. Blockage of 4-1BB/4-1BBL pathway may benefitially reduce the clinical dosage of immunosuppressive agents and prolong the survival of grafts.

Repression of interferon-γ expression in T cells by Prosperorelated Homeobox protein

Interferon-gamma （IFN-γ） is a major proinflammatory effector and regulatory cytokine produced by activated T cells and NK cells. IFN-γ has been shown to play pivotal roles in fundamental immunological processes such as inflammatory reactions, cell-mediated immunity and autoimmunity. A variety of human disorders have now been linked to irregular IFN-γ expression. In order to achieve proper IFN-γ-mediated immunological effects, IFN-γ expression in T cells is subject to both positive and negative regulation. In this study, we report for the first time the negative regulation of IFN-γ expression by Prospero-related Homeobox （Proxl）. In Jurkat T cells and primary human CD4＋ T cells, Proxl expression decreases quickly upon T cell activation, concurrent with a dramatic increase in IFN-γ expression. Reporter analysis and chromatin immunoprecipitation （CHIP） revealed that Proxl associates with and inhibits the transcription activity of IFN-γ promoter in activated Jurkat T cells. Co-immunoprecipitation and GST pull-down assay demonstrated a direct binding between Proxl and the nuclear receptor peroxisome proliferator-activated receptor gamma （PPARγ）, which is also an IFN-γ repressor in T cells. By introducing deletions and mutations into Proxl, we show that the repression of IFN-γ promoter by Proxl is largely dependent upon the physical interaction between Proxl and PPARγ. Furthermore, PPARγ antagonist treatment removes Proxl from IFN-γ promoter and attenuates repression of IFN-γ expression by Proxl. These findings establish Proxl as a new negative regulator of IFN-γ expression in T cells and will aid in the understanding of IFN-γ transcription regulation mechanisms.

Expression characteristics of peripheral lymphocyte programmed death 1 and FoxP3+ Tregs in gastric cancer during surgery and chemotherapy

BACKGROUND Programmed death 1(PD-1)and CD4^(+)CD25^(+)FoxP3^(+)expression in peripheral blood T-cells has been previously reported in various types of cancer.However,the specific variation tendency during surgery and chemotherapy,as well as their relationship in gastric cancer patients,still remain unclear.Understanding this aspect may provide some novel insights for future studies on tumor recurrence and tumor immune escape,and also serve as a reference for determining the optimal timing and dose of clinical anti-PD-1 antibodies.AIM To observe and analyze the expression characteristics of peripheral lymphocyte PD-1 and FoxP3^(+)regulatory T cells(FoxP3^(+)Tregs)before and after surgery or chemotherapy in gastric cancer patients.METHODS Twenty-nine stomach cancer patients undergoing chemotherapy after a D2 gastrectomy provided 10 mL peripheral blood samples at each phase of the perioperative period and during chemotherapy.This study also included 29 agematched healthy donors as a control group.PD-1 expression was detected on lymphocytes,including CD4^(+)CD8^(+)CD45RO^(+),CD4^(+)CD45RO^(+),and CD8^(+)CD45RO^(+)lymphocytes as well as regulatory T cells.RESULTS We observed a significant increase of PD-1 expression on immune subsets and a larger number of FoxP3^(+)Tregs in gastric cancer patients(P<0.05).Following D2 gastrectomy,peripheral lymphocytes PD-1 expression and the number of FoxP3^(+)Tregs notably decrease(P<0.05).However,during postoperative chemotherapy,we only observed a decrease in PD-1 expression on lymphocytes in the CD8^(+)CD45RO^(+)and CD8^(+)CD45RO^(+)populations.Additionally,linear correlation analysis indicated a positive correlation between PD-1 expression and the number of CD4^(+)CD45RO^(+)FoxP3high activated Tregs(aTregs)on the total peripheral lymphocytes(r=0.5622,P<0.0001).CONCLUSION The observed alterations in PD-1 expression and the activation of regulatory T cells during gastric cancer treatment may offer novel insights for future investigations into tumor immune evasion and the clinical application of anti-PD-1 antibodies in gastric cancer.

Downregulation of CD4+CD25+ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.

BIOLOGICAL FEATURES OF HUMAN T-ACTIVATED KILLER CELLS

Objective: To investigate the immunobiological essence of T-activated killer (T-AK) cells induced by anti-CD3 monoclonal antibody (CD3McAb) and recombinant interleukin-2 (rIL-2) co-stimulation. Methods: The cytomorphology, phenotype and cytotoxicity of T-AK cells generated from human peripheral blood mononuclear cells (PBMC) were determined. Results: T-AK cells were similar to activated lymphoblasts in morphology, more than 90% of T-AK cells expressed the phenotypes of T-lymphocytes (CD3 +, CD8 +, and 20%～50% of the cells were NK-like phenotype (CD16 +, CD56 +, some of them expressed IL-2 receptor (CD25 +), CD38 antigen (CD38 +) and MHC-II antigen (HLA-DR+) characteristic marks for the activated T lymphocytes. T-AK cells attacking targets were morphologically large volumes with granules and mainly contained CD8 + and CD56 + cells. T-AK cells possessed high tumoricidal activities against NK-sensitive K562 cells and NK-resistant Raji cells, the cytotoxicity was composed of mainly CD3McAb-activated CD3AK activity (～50%), IL-2 induced LAK activity (～30%), NK activity (～10%) and the activities of inhibitory factors in T-AK supernatant (～10%). Conclusion: T-AK cells are a heterogeneous cell population consisting of mainly activated T lymphocytes and NK-like cells, the main part of T-AK cytotoxicity is the common activities of CD3AK cells and LAK cells.

2型糖尿病合并骨质疏松患者血清miR-9-5p和NFAT5的表达及其与骨折的关系

目的:2型糖尿病(type 2 diabetes mellitus,T2DM)是一种多病因代谢性疾病,骨质疏松(osteoporosis,OP)和骨折是其常见并发症。本研究旨在探讨T2DM合并OP患者血清中微RNA(microRNA,miR)-9-5p和核转录因子5(nuclear factor of activated T-cells 5,NFAT5)的表达水平,以及其与骨折的关系。方法:收集郑州市第七人民医院就诊的T2DM合并OP患者184例(OP组),另纳入同时间段单纯T2DM患者184例(T2DM组)。实时聚合酶链反应检测血清miR-9-5p、NFAT5表达水平。随访2年,根据新发骨折情况,将T2DM合并OP患者分为骨折组(43例)与无骨折组(141例)。Pearson法分析血清miR-9-5p、NFAT5分别与空腹血糖(fasting plasma glucose,FPG)、I型前胶原N末端前肽(procollagen I N-terminal propeptide,PINP)、空腹胰岛素(fasting insulin,FINS)、胰岛素抵抗指数(insulin resistance index,HOMA-IR)、骨密度T值、I型胶原羧基端β降解产物(type I collagen hydroxy terminal peptideβdegradation products,β-CTX)相关性,以及miR-9-5p与NFAT5的相关性;采用受试者操作特征(receiver operator characteristic,ROC)曲线评估血清miR-9-5p、NFAT5对T2DM合并OP患者骨折的预测价值,多因素logistic回归分析T2DM合并OP患者骨折的影响因素。结果:OP组血清miR-9-5p水平高于T2DM组,NFAT5水平低于T2DM组(均P<0.05)。与无骨折组相比,骨折组患者糖尿病病程、FPG、HOMA-IR、β-CTX、miR-9-5p水平均升高,而PINP、NFAT5水平均降低(均P<0.05)。骨折患者血清miR-9-5p与NFAT5水平呈负相关(r=−0.716,P<0.05);miR-9-5p水平与FPG、HOMA-IR、β-CTX均呈正相关,与PINP呈负相关(均P<0.05),而血清NFAT5水平与FPG、HOMA-IR、β-CTX均呈负相关,与PINP呈正相关(均P<0.05)。血清miR-9-5p、NFAT5单一预测T2DM合并OP患者骨折风险的曲线下面积(area under curve,AUC)分别为0.878和0.868,联合预测的AUC为0.933。β-CTX、miR-9-5p为T2DM合并OP患者骨折的危险因素,PINP、NFAT5为T2DM合并OP患者骨折的保护因素(均P<0.05)。结论:T2DM合并OP患者血清miR-9-5p表达水平升高,NFAT5表达水平降低,两者与骨折发生均有一定关系,miR-9-5p联合NFAT5对骨折预测价值更高。

类风湿关节炎患者血清脂肪因子趋化素与疾病活动度和Th17/Treg的关系

目的探究类风湿关节炎(RA)患者血清脂肪因子趋化素水平与疾病活动度和辅助性T细胞17/调节性T细胞(Th17/Treg)比值的相关性。方法纳入RA患者180例为观察组,根据DAS28评分将观察组分为高活动组、中活动组和低活动组,每组60例;另选取同期体检的健康者180例作为对照组。采用酶联免疫吸附试验(ELISA)法检测血清趋化素、白细胞介素(IL)-9、IL-10、IL-17水平;流式细胞术检测Th17/Treg比例;分析RA患者血清趋化素水平与DAS28评分,Th17、Treg细胞百分比及Th17/Treg比值的相关性。结果观察组血清趋化素水平高于对照组(P<0.05)。RA患者血清趋化素水平与DAS28评分呈正相关;高、中、低活动组血清趋化素水平和DAS28评分依次降低;观察组Th17细胞百分比、Th17/Treg比值、IL-17、IL-9水平高于对照组,Treg细胞百分比、IL-10水平低于对照组(P<0.05)。RA患者血清趋化素水平与Th17细胞百分比、Th17/Treg比值呈正相关,与Treg细胞百分比呈负相关(P<0.05)。结论RA患者的血清趋化素水平升高,与疾病活动度和Th17/Treg比值具有密切关系。