T细胞耐受的诱导及其机理研究

目的　阐明抗原特异性 Ｔ 细胞无能的诱导条件、无能细胞的特性及其耐受的机理。方法　抗 Ｂ７１ 单抗与 Ｃｓ Ａ 联用诱导抗原特异性 Ｔ 细胞无能, 通过３ Ｈ Ｔｄ Ｒ 掺入法测定 Ｔ 细胞增殖和 Ｍ Ｌ Ｒ, 利用 Ｒ Ｔ Ｐ Ｃ Ｒ 检测细胞因子基因表达。结果　耐受 Ｔ 细胞与异体淋巴细胞比例为０ ．０１∶１时, 可显著抑制 Ｍ Ｌ Ｒ, 转染 Ｂ７１ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 能协同刺激 Ｃ Ｄ３ 诱导的 Ｔ 细胞增殖,不表达 Ｂ７ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 无此作用。 Ｐ Ｈ Ａ、 Ｃ Ｄ３ 单抗、 Ｐ Ｍ Ａ＋ Ａ２３１８７ 可以逆转本试验所用诱导方法所致的 Ｔ 细胞的耐受状态。无能 Ｔ 细胞 Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 不表达, 而 Ｉ Ｌ４ 和 Ｉ Ｌ１０ ｍ Ｒ Ｎ Ａ可表达。无能 Ｔ 细胞活化后, Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 能够表达。结论　抗原特异性 Ｔ 细胞耐受是可以人为诱导的, 无能 Ｔ 细胞细胞因子基因格局向 Ｔ Ｈ２ 细胞偏离。     

激活蛋白-1(c-Fos/c-Jun) mRNA水平与牙龈炎症相关性研究

目的 研究激活蛋白-1 (AP-1) (c-Fos/c-Jun) mRNA的表达水平与牙龈炎症间的相关性,探讨牙周炎的发病机理.方法 将获得的牙龈组织根据牙龈指数(GI)分为3组:GI=0组(对照组,14例)、GI=1组(15例)和GI=2组(11例).提取各组牙龈组织的总RNA,反转录合成cDNA,采用反转录PCR检测健康牙龈组织(GI=0组)中c-Fos mRNA和c-Jun mRNA的表达情况,实时定量PCR分析各组c-Fos mRNA和c-JunmRNA的表达水平.结果 c-Fos mRNA和c-Jun mRNA在健康牙龈组织中均有表达.GI=1组c-Fos mRNA和c-Jun mRNA的表达水平(15.58±.9.19、3.47±1.77)明显高于GI=0组(1.31±1.03、1.32±0.94),差异均具有统计学意义(均P＜0.05).GI=2组c-Fos mRNA的表达水平(3.01±1.48)低于GI=1组(P＜0.05),但高于GI=0组(P＜0.05);c-Jun mRNA的表达水平(1.48±0.65)低于GI=1组(P＜0.05),但与GI =0组间差异无统计学意义(P＞0.05).结论 AP-1 (c-Fos/c-Jun)与牙龈炎症程度存在相关性,提示其参与牙龈炎症的发生发展.     

T细胞无能与凋亡关系初探

目的：探讨无能Ｔ细胞与凋亡的关系。方法：利用Ｂ７－１单抗和环孢素Ａ（ＣｓＡ）联用处理ＡＰＣ即Ｔ细胞系统,在体外诱致抗原特异性Ｔ细胞无能,用ＰＩ染色法经ＦＡＣＳ分析了无能Ｔ细胞的凋亡,用ＲＴ－ＰＣＲ法检测了凋亡细胞Ｆａｓ ｍＲＮＡ的表达水平。结果：无能Ｔ细胞在１、３、５、１０ｄ细胞凋亡的百分率依次为２．１６％１１．２８％、１９．２７％、４１．２２％。当加入１００Ｕ／ｍｌ ＩＬ－２维培养,无能Ｔ细胞为     

Role of tumour necrosis factor-alpha (TNF-α) in the induction of HIV-1 gp120-mediated CD4+ T cell anergy

The HIV-1 envelope glycoprotein (gp120) is known to induce antigen-specific and non-specific CD4⁺ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G₁ (G_1A) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-α produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-α, these changes induced by gp120 were inhibited, while recombinant TNF-α induced similar abnormalities of CD4⁺ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4⁺ T cells by gp120, which may be related to gp120-mediated down-regulation of CD4 expression on T cells and activation of protein tyrosine kinase p56^lck in CD4⁺ T cells, was observed even in the absence of macrophage-derived TNF-α induced by gp120. These observations indicate that both TNF-α-dependent and independent events contribute to gp120-mediated CD4⁺ T cell anergy, and TNF-α appears to play an important role in inducing CD4⁺ T cell anergy in HIV-1 infection.     

Co-stimulation via CD28 induces activation of a refractory subset of MRL-Ipr/Ipr T lymphocytes

Peripheral lymphoid tissues of Ipr mice contain a large proportionof TCRß/CD3⁺CD4^–CD8^– T cells that lacksurface CD2 and express the B cell isoform of CD45, B220. Thissubset of T cells does not proliferate or produce IL-2 in responseto mitogenic signals or TCR–CD3 ligation. At the sametime, these abnormal T cells display several characteristicsof an activated phenotype. Collectively, these properties ofIpr CD4^–CD8^– T cells have functional parallels withanergic T cells. A critical co-stimulatory molecule implicatedin the prevention of or recovery from anergy is CD28, whichbinds the ligand BB1/B7 on certain accessory cells. Ipr CD4^–CD8^–T cells express normal levels of CD28 which is capable of transducinga strong proliferative signal to these cells in co-stimulationwith mitogens. However, proliferation of Ipr CD4^–CD8^–T cells in response to CD28 co-stimulation does not reach thelevels observed in normal T cells stimulated under similar conditions.Stimulation with anti-CD28 mAb in conjunction with phorbol myristateacetate and lonomycin promotes cell cycling in the CD2^–subset of CD4^–CD8^– T cells, and results in a slightinduction of CD2 levels during the course of the culture period.However, the majority of cells obtained at the end of the cultureperiod remain TCRß+ CD4^–CD8^–, CD2^low/–and B220^high, similar to freshly isolated CD4^–CD8^–Ipr T cells. In contrast, if IL-2 is included in the cultures,a strong shift toward a CD2⁺ phenotype is observed by a majorityof the Ipr T cells. Upon repeat stimulation, these Ipr CD4^–CD8^–T cells can now proliferate in an IL-2-dependent manner whenstimulated with only anti-CD3 mAb or mitogens, in the absenceof exogenous IL-2 or anti-CD28 mAb. These data show that thehyporesponsiveness of Ipr CD4^–CD8^– T cells doesnot result from a lack of CD28 expression, that it is not afixed state, and that it can be reversed by the induction ofcell cycling in the presence of IL-2. These observations extendthe parallels between Ipr CD4^–CD8^– T cells and anergicT cells.     

Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism

Program death-1 (PD-1) has been documented to negatively regulate immune responses. However, the cellular and molecular mechanisms for PD-1-mediated immune suppression have not been fully elucidated. In this study, we show that loss of PD-1 does not lead to defective induction of CD4(+) T cell anergy in vitro and in vivo. Rather, the absence of PD-1 inhibits the development of inducible CD4(+)Foxp3(+) regulatory T cells (iTregs) induced by TGF-β in vitro. In support of this finding, PD-1 deficiency impairs the generation of iTregs in vivo and leads to development of severe T cell-transfer-induced colitis. Mechanistically, defective iTreg generation in the absence of PD-1 was attributed to the heightened phosphorylation of Akt. Therefore, we first demonstrate that PD-1 controls peripheral T cell tolerance via an anergy-independent but iTreg-dependent mechanism.     

在单个细胞水平上分析抗原特异性Th1/Th2细胞因子产生的关联性

目的　在单个细胞水平上 ,观察抗原特异性Th1和Th2细胞因子产生的关联性 ,为进一步阐明CD4 T细胞的分化 ,细胞因子产生的相互关系及其特征提供理论依据。方法　从OVA TCR转基因小鼠的脾和淋巴结中分离CD4 T细胞 ,在体外在抗原提呈细胞存在下 ,用卵白蛋白 (OVA)抗原多肽刺激 3d后 ,再以同样的培养条件刺激 5～ 6h ,固定细胞 ,然后进行细胞表面和细胞内细胞因子染色 ,最后利用流式细胞仪在单个细胞水平上分析Th1和Th2细胞因子产生的关联性。结果　抗原特异性CD4 T细胞经抗原再一次刺激后 ,分泌Th1(IFN γ和IL 2 )和Th2 (IL 4、IL 5和IL 10 )细胞因子。IL 12促进IFN γ的表达 ,控制Th2细胞的分化。此外 ,大多数抗原特异性CD4 T细胞只产生 1种细胞因子 ,1个细胞同时产生 2种细胞因子极少见。结论　在单个细胞水平上的研究结果表明 ,经抗原短暂刺激后 (3d) ,不同的CD4 T细胞亚群只产生 1种Th1和 或Th2细胞因子 ,同时产生两种以上者占有很低的比率     

Increased immunoreactivities against endothelin-converting enzyme-1 and monocyte chemotactic protein-1 in hepatic stellate cells of rat fibrous liver induced by thioacetamide

The progression of rat liver fibrosis induced by intraperitoneal administration of thioacetamide (TAA) was evaluated by immunocytochemistry using anti-α-smooth muscle actin (α-SMA), antiendothelin-converting enzyme (ECE)-1, and anti-monocyte chemotactic protein (MCP)-1 antibodies. The fibrous septal spaces gradually increased after administration of TAA, and pseudolobules were established in the 7-week TAA-treated groups. Immunoreactivities against α-SMA were not detected in hepatic stellate cells (HSCs) of the control group without TAA treatment, although they were observed in the HSCs around the fibrous septal spaces in all TAA-treated groups, indicating that activation of HSCs occurs during the establishment of pseudolobules. Immunoreactivities against ECE-1 and MCP-1 were seen in such HSCs of the TAA-treated groups, but few or no immunoreactivities were detected in the HSCs of the control group. The most significant increase in the ECE-1 immunoreactivities was detected in the 1-week TAA-treated group, whereas that in MCP-1 was observed in the 7-week TAA-treated group. The present immunocytochemistry indicated a difference in the accelerated expression period between immunoreactivities against ECE-1 and MCP-1 in the HSCs during the progression of TAA-induced liver fibrosis, suggesting that ECE-1 is involved in the early phase of liver fibrosis and that MCP-1 plays a role during the later phase.     

Simultaneous aberrations in Mphi and T cell function adversely affect trauma patients' clinical outcome: a possible faulty IL-13 feedback loop

Possible defective trauma patients' Mphi-T-cell feedback interactions between T cell IL-13 production and IL-1beta and IL-18 Mphi secretion were assessed. Mphi produced IL-1 and IL-18 augment T cell IL-13, which in turn limits excessive macrophage activation. Immunodepressed patients' T cells (depressed proliferation to alphaCD3 + alphaCD4) had decreased IL-13 production concomitant to aberrant Mphi activities ( upward arrow mTNFalpha, downward arrow IL-10) and consequent multiple organ failure (MOF). Decreased IL-13 levels in patients' T cell and diminished Mphi supernatant augmentation of healthy controls' T cell IL-13 production appeared concomitantly, suggesting patients' aberrant monokine levels might intensify in vivo T cell dysfunction severity. Patients' Mphi supernatants, which failed to augment controls' T cell IL-13 production, had depressed IL-1beta and lower induction of IL-18 than immunocompetent patients' Mphi, but combined addition of IL-1beta and IL-18 restored these Mphis' IL-13 enhancing activity. These data suggest that immunodepressed patients' aberrant monokine and depressed T cell IL-13 production are independent but synergistic contributors to emergence of MOF.     

Renegade homeostatic cytokine responses in T1D: Drivers of regulatory/effector T cell imbalance

Homeostatic cytokines contribute to the balance between regulatory and effector T cells (Tregs and Teffs respectively) and are necessary to maintain peripheral tolerance. These cytokines include IL-2 that supports Treg and IL-7 and IL-15 that drive Teff. In overt settings of lost tolerance (i.e. graft rejection), IL-2 Treg signatures are decreased while IL-7 and IL-15 Teff signatures are often enhanced. Similar cytokine profile imbalances also occur in some autoimmune diseases. In type 1 diabetes (T1D), there are underlying defects in the IL-2 pathway and Teff cytokine blockade can prevent and treat diabetes in NOD mice. In this review, we summarize evidence of IL-2, IL-7 and IL-15 genetic and cellular alterations in T1D patients. We then discuss how the combined effect of these cytokine profiles may together contribute to altered Treg/Teff ratios and functions in T1D. Implications for combination therapies and suggestions for integrated cytokine and Treg/Teff biomarker development are then proposed.     

The ability of murine dendritic cell subsets to direct T helper cell differentiation is dependent on microbial signals

Dendritic cells (DC) initiate T cell responses and direct the class of T cell immunity through the production of Th-polarizing cytokines. In the mouse, immunization with CD8alpha(+) DC has led to Th1 priming whereas immunization with CD8alpha(-) DC has been associated with Th2 induction. Here, we use a direct T cell priming assay in vitro to re-examine the Th-directing potential of total DC or purified CD4(+) DC, CD8alpha(+) DC or CD4(-) CD8alpha(-) (double-negative; DN) DC subsets from mouse spleen. We show that the default Th effector phenotype induced by priming with DC depends on the protocol used for T cell purification, the T cell:antigen-presenting cell ratio and the antigen dose but is only marginally affected by DC subtype. All DC subsets can direct increased Th1 development in response to microbial stimuli known to elicit IL-12 production. Similarly, all subsets can suppress Th1 development and allow Th2 cellsto expand upon exposure to IL-10-inducing microbial agents. The flexibility of DC in directing Th development in function of microbial signals argues against the notion of pre-determined "DC1" and "DC2" subsets and suggests that multiple DC subtypes can direct an appropriate Th response to different classes of infectious agents.     

T1/ST2 expression on Th2 cells negatively regulates allergic pulmonary inflammation

The transmembrane form of T1/ST2 (ST2) is a specific marker on murine Th2 cells that have been generated in vitro, or isolated from sites of allergic type 2 inflammation. Despite the association of ST2 with Th2 cells, to date no obligate role for ST2 in type 2 responses in vivo has been described. We have specifically addressed the role of ST2 on T cells by generation of ST2(-/-) mice crossed with ovalbumin (OVA) T cell receptor-transgenic mice. OVA-specific ST2(-/-) cells had normal cytokine responses to T cell activation after in vitro Th2 differentiation, but OVA stimulation of IL-5 was increased. Transfer of OVA-specific ST2(-/-) Th2 cells into BALB/c mice caused exacerbated pulmonary inflammation with occluded airways, elevated airway hyper-responsiveness and increased susceptibility to methacholine challenge that was associated with mortalities of recipient mice. The increased pulmonary inflammation in OVA-specific ST2(-/-) Th2 cell recipients was associated with selective differences in pulmonary levels of Th2 cytokines compared with OVA-specific ST2(+) Th2 cell recipients. Recipients of OVA-specific ST2(-/-) Th2 cells had a significant increase in eosinophils and a significant reduction in F4/80(hi) macrophages in the lungs. This is the first demonstration of a role for ST2 expression on Th2 cells down-regulating pulmonary inflammation. These data have major implications for the targeting of ST2 as a therapy for allergic airway disorders.     

急性期川崎病患儿滤泡辅助性T细胞改变观察

目的 探讨滤泡辅助性T细胞(Tfh细胞)在儿童川崎病(Kawasaki disease,KD)中的数量变化及其可能机制.方法 急性期川崎病患儿20例,采用流式细胞术检测外周血CD4+CXCR5+ICOS+T细胞(Tfh)的比例,采用real-time PCR检测转录调节因子Bcl-6、Blimp-1 mRNA表达,酶联免疫吸附试验检测血浆中IL-4和IL-21浓度;20例同龄健康儿童作为对照组.结果 (1)急性期KD患儿Tfh细胞比例明显高于正常对照组[(2.6±0.6)％vs(1.8±0.7)％,P＜0.05];(2)Tfh细胞转录因子Bcl-6 mRNA表达较正常对照组明显增高(P＜0.05),其拮抗因子Blimp-1 mRNA表达降低(P＜0.05);(3)血浆IL-21和IL-4蛋白浓度明显高于正常对照组(P＜0.05).结论 Tfh细胞过度活化可能参与了KD免疫发病机制,Bcl-6/Blimp-1表达失衡,IL-4和IL-21细胞因子微环境改变可能与Tfh细胞异常活化有关.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

转化生长因子β和骨形成蛋白2联合诱导小鼠成骨细胞系MC3T3-E1细胞的增殖与分化

背景:生长因子是骨组织形成、改建、修复的关键要素之一,多种生长因子联合诱导骨干细胞成骨分化相较于单一生长因子具有一定的优势,但是何种类型的联合、什么样的浓度组合才能发挥最佳的诱导作用?目前该方向研究较少.目的:探索转化生长因子β和骨形成蛋白2联合诱导对小鼠MC3T3-E1细胞增殖和分化的影响.方法:将不同质量浓度的转化...