组织驻留型记忆T细胞：疫苗设计的新方向

免疫记忆的诱导是适应性免疫的核心，也是疫苗设计的基石。组织驻留型记忆T细胞(T_RM)是T细胞的一个特殊子集，驻留于非淋巴器官中，是屏障部位的第一道防线，可以在再次遭遇同源抗原后迅速激活。由于T_RM具有在外周非淋巴组织中长期稳定驻留的优势，在抗感染和癌症的局部免疫保护中起着至关重要的作用。目前诱导T_RM产生正在成为下一代疫苗设计的新目标。越来越多的案例显示疫苗接种可在局部诱导T_RM为机体提供高效的免疫保护。多种内在和外在的信号都被证明可调控T_RM的分化与发育，驱动T_RM形成的具体因素因不同组织而异。对T_RM异质性和形成机制的深入探讨对优化疫苗接种参数，开发更高效安全的疫苗具有指导意义。文章讨论了T_RM的驻留特征、分化发育需求以及在疫苗研究领域的研究进展，强调组织来源因子、局部抗原和佐剂在疫苗接种过程中对T_RM细胞生成的作用，以期为更高效的疫苗设计提供理论基础与新思路。     

“初免-吸引”免疫策略的研究进展

 “初免-吸引”免疫策略是指经胃肠外疫苗初次免疫后，通过局部使用T细胞引诱剂，在病原体进入的主要部位如生殖道、气道和肠道等招募和建立组织驻留记忆性T细胞，可应用到病毒或细菌等感染黏膜引起的疾病中，建立长期黏膜免疫。此文就该免疫策略在病毒、细菌、衣原体及螺旋体感染性疾病领域的研究进展进行综述，以期为疫苗研发提供新的思路。       

CD4+CD25+调节性T细胞在多种类型疫苗中作用的研究进展

CD4⁺CD25⁺ 调节性T细胞具有免疫抑制作用。CD4⁺CD25⁺ 调节性T细胞可由多种类型肿瘤、病原体诱导产生。一方面抑制炎症,防止机体组织损伤;另一方面阻碍机体清除肿瘤细胞和病原体,有利于肿瘤和病原体逃避宿主免疫攻击。使用抗体阻断CD4⁺CD25⁺ 调节性T细胞或干扰其抑制功能则利于机体清除肿瘤和病原体。同样,多种类型疫苗可诱导宿主产生CD4⁺CD25⁺ 调节性T细胞,因而抑制了疫苗引起的免疫反应。使用抗体如抗CD25单克隆抗体等封闭CD4⁺CD25⁺ 调节性T细胞或者抑制其分泌的细胞因子如白细胞介素-10(IL-10)、转化生长因子-β(TGF-β),可增强疫苗的免疫保护效果,从而为寻找更有效的疫苗提供新的思路。     

病毒T细胞表位和T细胞疫苗的研究进展

细胞免疫应答是机体免疫防御和免疫监视的重要途径,主要由T细胞完成。研究鉴定T细胞表位,研制T细胞疫苗,诱导机体产生细胞免疫应答,是预防病毒等病原体感染和治疗自身免疫病和肿瘤的重要途径,本文介绍了几种T细胞疫苗的研制方法。     

对禽流感H5N1灭活疫苗与不同免疫方式诱导的免疫应答及异亚型保护研究

目的  观察禽流感H5N1灭活疫苗以不同方式免疫后诱导的免疫应答及抗异亚型病毒攻击的保护作用.  方法 将H5N1灭活疫苗分别通过腹腔和滴鼻方式免疫BALB/c小鼠,同时以PBS作为对照;免疫后分别以PR8和H9N2病毒攻击,观察小鼠的体重变化和生存情况.采用ELISA对各组小鼠攻毒后不同时间的血清IgG及其亚类水平进行动态检测;流式细胞仪检测脾淋巴细胞亚群情况.采用t检验对各组数据进行比较.   结果 PR8和H9N2病毒攻击后,各组小鼠体重均下降,但疫苗组小鼠于后期体重恢复正常,存活率分别为100%和70%-80%,而PBS组小鼠则全部死亡.无论以何种方式免疫,疫苗组的特异性IgG及其亚类水平均明显升高,其中以IgG2a水平升高更为明显.攻毒后疫苗组小鼠脾CD4+与CD8+T淋巴细胞比值出现下降(t=6.8017,P<0.01);滴鼻免疫组与腹腔免疫组相比降低更明显(t=3.9701,P<0.05).    结论   H5N1疫苗免疫原性良好,可诱导较高水平的特异性抗体产生,诱导的抗体亚类以IgG2a为主.两种免疫方式均可对小鼠提供很好的异亚型保护,而滴鼻免疫能够诱导更强的CD8+T细胞应答.     

CD4 CD25 调节性T细胞在多种类型疫苗中的作用的研究进展

CD4 CD25 调节性T细胞具有免疫抑制作用，Foxp3为其特征性标志。可由多种类型肿瘤、病毒、寄生虫等诱导产生，一方面抑制炎症，防止机体组织损伤，另一方面不利于机体清除肿瘤细胞和病原体。若用抗体阻断CD4 CD25 Tregs或干扰其抑制功能则利于机体清除肿瘤和病原体。同样，多种疫苗本身能诱导CD4 CD25 Tregs的大量产生，因而抑制了疫苗引起的免疫反应。疫苗期间若采用适量的抗体如抗CD25单克隆抗体等封闭CD4 CD25 Tregs或者相应的抑制性细胞因子如IL-10、TGF-β，则可增强疫苗的免疫保护性效果。利用anti-CD25 mAb或其他阻断性抗体作为佐剂阻断CD4 CD25 Tregs，可增强多种类型疫苗的免疫保护性效果，从而为寻找更有效的疫苗提供新的策略。     

调节性T细胞与常见自身免疫性疾病

人体免疫系统可以通过杀伤受感染的细胞的方式保护不受侵入的病原体与微生物的侵害,而只对正常组织造成最小程度的损害。为了维持这种平衡,CD4＋CD25＋调节性T细胞在控制免疫应答与维持外周免疫耐受中起重要作用。这种T细胞的数量减少或功能的缺失可能导致自身免疫疾病的发生。增强调节性T细胞免疫抑制功能的治疗措施已经被证明可以改善自身免疫性疾病的病情,本文就调节性T细胞的作用机制及与常见自身免疫性疾病的关系做一综述。     

CD40配体及其应用的研究进展

CD40配体是肿瘤坏死因子超家族中的一员,主要活化T细胞表面的共刺激分子,特别是CD4＋T细胞.CD40配体通过与其受体CD40的相互作用,在诱导机体产生免疫应答中起重要作用.此文就CD40L配体的结构和功能以及在病毒DNA疫苗和肿瘤治疗中的应用作一综述.     

抗实验性利什曼病的ISCOM疫苗

疫苗接种产生的保护性免疫力取决于疫苗诱导能控制或清除病原体的免疫应答的能力。研究证实纯化的利什曼原虫抗原组分可明显保护小鼠免受实验性感染。利什曼原虫表面抗原gp63蛋白酶是一种侯选疫苗,可产生Th1 CD4~+细胞应答。作者以小鼠为模型,对gp63与免疫刺激复合物（ISCOM）构成的疫苗的免疫原性及保护效果进行了研究。     

婴儿免疫接种核酸疫苗的潜在优点与风险

婴儿的抗体应答由于细菌多糖诱导的有缺陷的B细胞活化、对蛋白抗原的缓慢的B细胞应答和辅助性T细胞活性降低而减弱。这些特征导致婴儿对有荚膜细菌的严重感染更加易感并需要反复接种疫苗。通过识别新型保护性抗原,提供缺失的细胞因子,获得持久的免疫应答,或通过研制新型联合疫苗以减少注射次数,核酸疫苗能够使婴儿的抗体应答最优化。婴儿细胞免疫应答的缺陷使得他们对胞内病原体感染易感,但可能从核酸疫苗诱发强的辅助性T细胞(TH1)样应答的潜能中受益。最后,如果证明核酸疫苗能够克服母体抗体介导的抑制婴儿对疫苗抗原的应答,那么它将对婴儿免疫作出重要贡献。     

Cyclic AMP differentially modulates CD40L expression on human nai;ve and memory CD4(+) T cells

Although differences in nai;ve and memory T cell signaling have been recognized, how these differences relate to cell regulation and function is not well understood. In this study, we investigated CD40 ligand (CD40L) regulation by cyclic AMP (cAMP) and prostaglandin E(2) (PGE(2)) and observed differential effects depending upon the cell subset and mode of activation. cAMP inhibited CD3-induced CD40L in both nai;ve and memory subsets, although greater inhibition was observed in memory cells. With CD3/CD28 costimulation, cAMP inhibited CD40L in memory cells but had a minimal effect on nai;ve cells. In primed T cells, cAMP increased CD40L on nai;ve cells but inhibited expression on memory cells. Differential cAMP effects appear interrelated to calcium signaling since the level of CD40L induced by calcium ionophore was increased by cAMP in both cell subsets, although nai;ve cells were more calcium responsive. Calcium-dependent calcineurin activity appeared necessary for CD40L expression, although no interaction of calcineurin and cAMP regulation was demonstrable. In contrast, inhibitors of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) blocked cAMP effects to increase CD40L and resulted in marked CD40L inhibition. The importance of CaMKIV in cAMP regulation was confirmed by transfection studies using a dominant negative CaMKIV construct. We conclude that cAMP differentially regulates CD40L expression in a manner that appears dependent upon CaMKIV activation. In view of the central role of CD40L expression in immunity as well as the pathophysiology of common diseases, it is of interest that cAMP can either increase or decrease CD40L expression depending upon the T cell subtype and mechanism of cell activation.     

PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8⁺ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4⁺ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4⁺ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4⁺ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4⁺ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4⁺ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.     

Inhibitory effect of Uncaria sinensis on human aortic smooth muscle cell migration is based on matrix metalloproteinase-9 inhibitory activity

Medicinal extracts of Cho-Deung-san and Uncaria sinensis Havil. (UR) have previously been shown to have inhibitory effects on migration of vascular smooth muscle cells (VSMC) and matrix metalloproteinase (MMP)-2/9 production, which play key roles in the development of atherosclerosis. In this study, we have more extensively investigated the inhibitory effect of UR on MMP-9 activity and TNF- induced human aortic smooth muscle cells (HASMC) migration. The result from gelatin zymography showed that UR inhibited MMP-9 activity in a dose-dependent manner (IC₅₀ = 55 μg/ml). In addition, UR strongly inhibited the migration of HASMC induced by TNF- treatment (IC₅₀ = 125 μg/ml), although it has very low cytotoxic effect on HASMC (IC₅₀ > 500 μg/ml). These results suggest that UR is a potential anti-atherosclerotic agent through inhibition of MMP-9 activity and VSMC migration.     

Colistin susceptibility testing by Etest and disk diffusion methods

The accuracy of disk susceptibility methods for colistin against 778 bacterial pathogens was evaluated in comparison with Etest using interpretive criteria available from the Clinical and Laboratory Standards Institute (CLSI). Colistin exhibited excellent activity against Acinetobacter baumannii and Escherichia coli isolates (minimum inhibitory concentration for 90% of the organisms (MIC₉₀) = 0.5 mg/L), whilst it was less active both against Enterobacter spp. and Klebsiella pneumoniae (MIC for 50% of the organisms (MIC₅₀) = 0.5 mg/L, MIC₉₀ = 16 mg/L). Colistin also showed good activity against Pseudomonas aeruginosa (MIC₉₀ = 2 mg/L, MIC₅₀ = 1 mg/L) but poor activity against Stenotrophomonas maltophilia (MIC₅₀ = 8 mg/L, MIC₉₀ = 128 mg/L). Only 0.8% of minor errors were observed between the studied methods for P. aeruginosa isolates when the CLSI criteria were applied. All A. baumannii isolates with a zone diameter ≤12 mm were resistant and those with a zone diameter ≥14 mm were susceptible according to MIC breakpoints established by the CLSI. Among nine isolates exhibiting a zone diameter of 13 mm, one was resistant to colistin (MIC = 8 mg/L) and eight isolates were susceptible (MIC = 0.5 mg/L). Applying a MIC breakpoint of ≤2 mg/L for susceptibility in Enterobacteriaceae, all isolates with a zone diameter ≥14 mm were susceptible, whilst all isolates with a zone diameter ≤11 mm were resistant. Among isolates with zone diameters of 12–13 mm, 59% were characterised as susceptible. Major errors were observed only in K. pneumoniae isolates at a rate of 0.8%. The poor agar diffusion characteristics of colistin limit the predictive accuracy of the disk diffusion test and consequently values of 12–13 mm should be confirmed with MIC determination by Etest or broth dilution method.     

Simultaneous approach using systemic, mucosal and transcutaneous routes of immunization for development of protective HIV-1 vaccines

Mucosal tissues are major sites of HIV entry and initial infection. Induction of a local mucosal cytotoxic T lymphocyte response is considered an important goal in developing an effective HIV vaccine. In addition, activation and recruitment of memory CD4(+) and CD8(+) T cells in systemic lymphoid circulation to mucosal effector sites might provide the firewall needed to prevent virus spread. Therefore a vaccine that generates CD4(+) and CD8(+) responses in both mucosal and systemic tissues might be required for protection against HIV. However, optimal routes and number of vaccinations required for the generation of long lasting CD4(+) and CD8(+) CTL effector and memory responses are not well understood especially for mucosal T cells. A number of studies looking at protective immune responses against diverse mucosal pathogens have shown that mucosal vaccination is necessary to induce a compartmentalized immune response including maximum levels of mucosal high-avidity CD8(+) CTL, antigen specific mucosal antibodies titers (especially sIgA), as well as induction of innate anti-viral factors in mucosa tissue. Immune responses are detectable at mucosal sites after systemic delivery of vaccine, and prime boost regimens can amplify the magnitude of immune responses in mucosal sites and in systemic lymphoid tissues. We believe that the most optimal mucosal and systemic HIV/SIV specific protective immune responses and innate factors might best be achieved by simultaneous mucosal and systemic prime and boost vaccinations. Similar principals of vaccination may be applied for vaccine development against cancer and highly invasive pathogens that lead to chronic infection.     

Oral bioavailability and intestinal secretion of amitriptyline: Role of P-glycoprotein?

The aim of the study was to evaluate the influence of quinidine, a P-glycoprotein inhibitor, on oral bioavailability and on intestinal secretion of amitriptyline, a tricyclic antidepressant. Amitriptyline was administrated intravenously (5 mg/kg) and orally (50 mg/kg) to rabbits, with and without quinidine. Jejunal segments of rats were mounted on diffusions chambers and the permeation of amitriptyline was measured across the tissue in luminal–serosal (LS) and serosal–luminal (SL) directions, with and without quinidine. Finally, an in situ recirculating intestinal perfusion model was performed in rabbits to study amitriptyline permeation in LS direction with and without quinidine. Absolute oral bioavailability (F) of amitriptyline was significantly increased more than three-fold in presence of quinidine (F = 0.6 ± 0.4% versus 1.9 ± 1.1%). The apparent permeability coefficients in SL direction were significantly higher than in LS direction (P_{app (SL)} = 6.01 ± 2.42 versus P_{app (LS)} = 4.90 ± 2.73 × 10⁻⁴ cm min⁻¹). In presence of quinidine, the intestinal absorption was increased (P_{app (LS)} = 4.02 ± 2.91 versus P_{app (LS)} = 5.99 ± 2.43 × 10⁻⁴ cm min⁻¹) and the intestinal secretion was decreased (P_{app (SL)} = 4.58 ± 0.54 versus P_{app (LS)} = 3.63 ± 1.46 × 10⁻⁴ cm min⁻¹) but not significantly. In conclusion, P-glycoprotein appears to be involved in oral amitriptyline absorption but other intestinal uptake and efflux transporters maybe implicated.     

The influence of metallothionein on exposure to metals: An in vitro study on cellular models

In the present study, the interactions between zinc (Zn) and copper (Cu) or iron (Fe) have been examined. Rat hepatoma cell line H4-II-E-C3, fibroblast cell line mutant MT−/−, and wild-type MT+/+ cells treated with ZnSO₄ or CuSO₄ or FeSO₄ or CuSO₄ + ZnSO₄ or ZnSO₄ + FeSO₄ for different times have been employed to study the effect of metallothionein (MT), glutathione (GSH) and metal (Cu, Fe and Zn) accumulation during cellular adaptation to supraphysiological metal concentrations. To investigate the different biological functions in the processes of metal homeostasis and detoxification, the levels of both MT-1 and MT-2 mRNAs have been evaluated. The three cell lines responded differently to metal treatments suggesting that the uptake and storage of these metals are affected by the specific cellular model and MT presence. In particular, Zn treatment significantly decreased Fe accumulation (p < 0.05), whereas MT induced by Zn increased intracellular Cu content (p < 0.05). Moreover, in H4-II-E-C3 cells administration of metals resulted in a rapid and transient induction of MT (p < 0.05) and in GSH accumulation (p < 0.05) suggesting synergistic interactions in which both appear essential for a protective regulatory function against the redox activity of metals. Taken together these results demonstrate that Zn affects the cellular levels of Cu and Fe by competition with the same ligand sites and/or by coordinate regulation of MT and GSH content.     

In vitro cytopathic effects of mycotoxin T-2 on human peripheral blood T lymphocytes

The trichothecene mycotoxin T-2 is reported to exhibit immunotoxic activity. The potential presence of T-2 in foods renders it as public health hazard and its toxicity needs to be better understood. We investigated the in vitro effects of T-2 at sub-toxic (0.1 ng/ml) and toxic (10 ng/ml) levels on freshly isolated human peripheral blood lymphocytes (PBLs). We observed no direct influence on untreated PBLs. The toxic dose of T-2, however, totally inhibited phytohemagglutinin-induced T lymphocyte proliferation and caused early apoptosis that peaked after 8 h of exposure. Both major T lymphocyte subsets (CD4⁺ and CD8⁺) were affected as they appeared to show a positive response to T-2 at 8 h followed by their sharp reduction after 96 h. Further investigation on the naïve (CD45RA⁺) and memory (CD45RO⁺) subpopulations confirmed these observations and indicated that T-2 affected equally all the subpopulations studied, although PHA preferentially stimulated CD45RO⁺ T lymphocytes. Sub-toxic T-2 appeared to exhibit co stimulatory properties to PHA-stimulated cells. These results support the hypothesis that T-2 affects the activation-induced cell death mechanism of T lymphocytes.     

慢性乙型肝炎病毒感染者外周血T细胞亚群变化与其血清HBV DNA的水平分析

目的:观察慢性乙型肝炎病毒(HBV)感染者细胞免疫功能的变化,探讨病毒复制程度与细胞免疫功能的关系。方法:应用流式细胞仪直接免疫荧光法检测136例乙肝病毒感染者、60例正常对照者外周血T淋巴细胞亚群百分率,用荧光定量聚合酶链式反应(PCR)法检测乙型肝炎感染者血清HBV DNA。结果:84例慢性HBV携带者(ASC)、52例慢性乙型肝炎(CHB)患者与健康对照组相比,CD3+ T淋巴细胞百分比、CD4+T淋巴细胞百分比和CD4+/CD8+ T淋巴细胞比值均显著降低(P<0.001),CD8+ T淋巴细胞百分比均显著升高(P<0.001)。慢性乙型肝炎患者与慢性HBV携带者相比,CD3+ T淋巴细胞百分比差异无统计学意义(P>0.05)、CD4+ T淋巴细胞百分比明显降低(P<0.05)、CD8+ T淋巴细胞百分比明显升高(P=0.01)、CD4+/CD8+ T淋巴细胞比值明显降低(P<0.05)。随着病情发展,从健康对照组、慢性HBV携带者到慢性乙型肝炎,CD4+T淋巴细胞百分比、CD4+/CD8+比值呈逐渐下降趋势,CD8+ T细胞百分比呈逐渐升高的趋势。慢性HBV携带者HBV DNA阳性组与HBV DNA阴性组相比,CD3+ T淋巴细胞差异均无统计学意义(P>0.05),CD4+ T淋巴细胞百分比明显下降(P<0.001),CD4+/CD8+ T淋巴细胞比值明显下降(P<0.001),CD8+ T淋巴细胞百分比明显升高(P<0.01)。结论:HBV感染可导致感染者细胞免疫功能的改变,HBA DNA复制增加进一步加重乙肝病毒感染者T细胞亚群的紊乱,CD4+/CD8+ T淋巴细胞比值的动态变化可及时提示临床HBV感染者细胞免疫功能的变化并加强临床的监测。     

Vaccines against intracellular bacterial pathogens

There is a long history of remarkable success in developing vaccines against bacteria that are extracellular pathogens. In general, the development of vaccines against intracellular bacterial pathogens has proven to be more challenging. Typically, such vaccines need to induce a range of immune responses, including antibody, CD4(+) and CD8(+) T cell responses. These responses can be induced by live attenuated vaccines, but eliciting these responses with non-living vaccines has proven to be difficult. The difficulties appear to be related partly to the problems associated with the identification of protective antigens and partly with the difficulties associated with inducing CD8(+) T cell responses.