T细胞免疫BALB/c小鼠诱导调节性体液免疫应答的研究

目的 :研究T细胞免疫后正常小鼠的调节性体液免疫应答。方法 :用经γ射线照射的体外扩增的活化OVA特异T细胞克隆免疫BALB c小鼠 ,FACS法分析血清中抗T细胞抗体水平 ,免疫共沉淀法分析靶抗原。结果 :T细胞免疫能诱导BALB c小鼠产生抑制T细胞增殖的抗T细胞抗体 ,这种抗体不是多克隆活化的结果 ,其靶抗原可能是T细胞上 93、87、5 5和 4 5kD的蛋白。结论 :T细胞免疫能诱导正常小鼠产生调节性体液免疫应答     

编码TCR可变区的DNA免疫诱导BALB/C小鼠调节性免疫应答

目的：研究编码卵清白蛋白(OVA)T细胞克隆TCR Vα5．2或Vβ2.1的DNA免疫正常小鼠诱发调节性免疫应答。方法：用重组质粒pcDNA3．1-Vα5．2或pcDNA3．1-Vβ2.1免疫BALB／C小鼠,RT-PCR证实重组质粒能在体内和体外真核细胞中转录。^3H-TdR掺入法分析细胞增殖,^3H-TdR标记的靶细胞检测杀伤T细胞的杀伤效应,免疫荧光法分析血清中特异抗体水平和Vβ2^ T细胞数量。结果：pcDNA3．1-Vα．2或pcDNA3．1-Vβ2．1能在体内和体外真核细胞中转录,重组质粒免疫BALB／C小鼠能诱导产生特异性体液免疫应答、T细胞增殖反应及对靶细胞CTL反应,但pcDNA3．1-Vβ2．1免疫没有导致Vβ2^ T克隆清除,而是细胞活性部分封闭。结论：编码TcR Vα5．2或Vβ2．1的DNA免疫能诱导正常BALB/C小鼠调节性免疫应答。     

自身反应性T细胞系免疫生物学活性研究

目的：探讨自身反应性T细胞系过继转移对受体小鼠免疫应答的影响，为在BXSB小鼠探索T细胞疫苗的可行性奠定基础。方法：应用体外扩增的自身反应性T细胞过继转移给同系小鼠体内：^3H－TdR掺入法检测细胞增殖，ELISA检测抗体细胞因子水平。结果：将自身反应性T细胞系过继转移到年轻未发病的同系小鼠体内可以诱导血清中自身抗体和外来抗原反应性抗体以及细胞因子IFN－γ水平显著升高，可以降低受体小鼠对外来抗原（OVA）体液免疫应答能力。结论：自身反应性T细胞的过继转移可以影响机体自免疫应答和对外来抗原的体液免疫应答能力。     

减毒鼠伤寒沙门氏菌运送CD8+T细胞表位的细胞免疫应答

目的： 探索减毒沙门氏菌运送CD8＋ T 细胞表位诱导机体产生特异性细胞免疫应答的规律性.方法： 通过构建融合表达OVA 257～264aa和LCMV NP 118～132aa CD8＋ T 细胞表位的原核表达质粒ptG2F, 以电穿孔法转化减毒鼠伤寒沙门氏菌SL7207, 筛选重组菌SL7207（ptG2F）.采用静脉注射免疫C57BL/6和BALB/c小鼠, 间隔2 wk, 分别于第2次和第3次免疫后, 取免疫小鼠脾细胞, 用ELISPOT法检测特异性IFN-γ分泌细胞和IL- 4分泌细胞.结果： 携带CD8＋ T细胞表位的重组菌SL7207（ptG2F）能诱导产生细胞免疫应答.在提呈OVA CD8＋ T细胞表位时, 2次免疫后, 诱导产生的细胞免疫应答趋向于Th1; 而在3次免疫后, 呈现Th1/Th2的平衡转换.在提呈LCMV NP CD8＋ T细胞表位过程中, Th2免疫应答水平高于Th1, 且有增强趋势.结论： 减毒沙门氏菌可以有效运送CD8＋ T细胞表位并诱导产生特异细胞免疫应答, 这为减毒细菌作为运送载体的研究提供了参考依据.     

卵清白蛋白特异性T细胞克隆的建立及T细胞受体使用情况分析

目的：体外建立可长期培养的抗原特异性T细胞系及克隆，并分析其T细胞受体的使用情况。方法：用卵清白蛋白（OVA）免疫BALB／c小鼠，取淋巴结细胞在体外用抗原原复刺激建立抗原特异性T细胞系，通过能限稀释法进行克隆，应用锚定PCR方法分析其特异的T细胞受体的应用。结果：建立了H－2^d限制的OVA特异性T细胞系，获得了3株特异性较高的T细胞克隆，T细胞系中TCR AV11S4和BV4S1应用频率最高，3株T细胞克隆的TCR均为AV5S2和BV2S1，这些T细胞所使用的TCRα链和β链在CDR3区有明显的共性，结论：获得了卵清白蛋白特异的T细胞系和克隆，且其T细胞受体的应用有一定的限制性。     

环孢霉素A对OVA免疫TCR转基因DO11.10小鼠CD4+CD25+T细胞的影响

目的：研究免疫抑制剂Csh对处于免疫应答状态的小鼠脾脏调节性CD4^＋CD25^＋T细胞影响。方法：采用流式细胞术检测卵白蛋白（OVA）免疫的DO11．10小鼠脾脏CD4^＋CD25^＋T细胞及其中Foxp 3^＋T细胞的变化。结果：OVA免疫后脾脏CD4^＋CD25^＋T细胞占CD4^＋T细胞百分数及Foxp 3^＋T细胞占CD4^＋CD25^＋T细胞百分数均增加，显示CsA可明显减少正常及免疫应答状态下小鼠脾脏CD4^＋CD25^＋T细胞及CD4^＋CD25^＋Foxp3^＋T细胞百分数。结论：CsA在抑制免疫应答的同时也可能抑制了免疫耐受的诱导。     

晚期肿瘤患者外周血CTL、Ts及NK细胞免疫表型的联检及临床意义

机体对肿瘤的免疫应答及其抗肿瘤免疫效应机理是肿瘤免疫学研究中的重要内容。细胞免疫在抗肿瘤免疫应答中发挥着重要作用。人类肿瘤特异性T细胞大多为CD8^＋T细胞,又可分为抑制性T细胞（Ts）和细胞毒性T细胞（CTL）,前者抑制体液和细胞免疫应答,调节维持内环境稳定,后者是直接杀伤肿瘤细胞的免疫效应细胞。NK细胞可溶解靶细胞如特异性肿瘤细胞株和受细胞内病原体感染的细胞,还可通过分泌炎性介质参与T细胞的分化,而控制T细胞反应。我们采用双色荧光标记流式细胞术分别检测了晚期肿瘤患者外周血CTL、Ts及NK细胞比例,从而评价晚期肿瘤患者的细胞免疫功能,对临床肿瘤治疗和预后判断具有重要的意义。     

双歧杆菌完整肽聚糖对重组乙肝表面抗原细胞免疫应答的影响

目的:探讨双歧杆菌完整肽聚糖(BWPG)对重组乙肝表面抗原(rHBsAg)诱导细胞免疫应答的影响。方法:以BWPG作为rHBsAg的佐剂免疫BALB/c小鼠,观察免疫小鼠的生长状态及中枢淋巴器官的发育情况,检测NK细胞和CTL杀伤活性的变化,脾淋巴细胞的增殖和细胞因子的产生。结果:免疫小鼠未出现可见的毒副反应,胸腺和脾脏组织呈增生状态,脾脏NK细胞和CTL的杀伤活性增强,脾淋巴细胞体外增殖活跃,细胞因子产生增多。结论:BWPG能促进中枢淋巴器官增生,增强机体对rHBsAg的细胞免疫应答。     

丙肝核酸疫苗免疫的CTL活性检测

目的 :探讨丙肝核酸疫苗的细胞免疫应答效果。方法 :用丙肝核酸疫苗pSVL HCV C E1 通过皮下注射途径免疫BALB C小鼠 ,以绿色荧光载体重组体pEGFP N1 HCV C E1 转染的小鼠骨髓瘤细胞SP2 0作为靶细胞 ,采用51 Cr释放法 ,以(Na) 2 51 CrO4 标记靶细胞进行标准的 4h杀伤试验检测其CTL活性。结果 :CTL杀伤率达 4 0 7%。结论 :丙肝核酸疫苗能激发强烈的细胞免疫     

人黑色素瘤抗原-3冲击的树突状细胞诱导抗肿瘤免疫应答

目的:观察经MAGE-3蛋白抗原冲击的树突状细胞在体外诱导抗肿瘤免疫应答的能力。方法:用粒-巨噬细胞集落刺激因子和白介素-4从人外周血分化、诱导树突状细胞,经MAGE-3蛋白冲击后,与T淋巴细胞共培养7d,收集T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验。结果:与MAGE-3冲击后的树突状细胞共培养的T淋巴细胞在体外能有效地杀伤MAGE-3阳性的靶细胞。结论:经MAGE-3蛋白抗原冲击的树突状细胞,在体外能有效提呈抗原,激活抗原特异性CTL,诱导特异性抗肿瘤免疫应答。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅰ.同种特异T细胞疫苗诱导的细胞免疫水平低下

在体外实验中观察了同种特异T细胞疫苗（TCV）免疫鼠T淋巴细胞对同种抗原和有丝分裂原（ConA）的反应能力。B6同种抗原特异TCV免疫的BALB/c小鼠对B6同种抗原的反应（MLR）能力明显变抑制,对无关第三者同种抗原AKR的反应能力也显著下降,表明存在抗原非特异性的抑制作用。在BALB/c同种抗原特异TCV免疫的B6小鼠中,获得一致的结果。在观察两组TCV免疫动物T细胞对Cond诱导的淋巴细胞增殖实验中,与正常小鼠 T细胞反应性比较,TCV免疫动物 T细胞的增殖能力显著下降,表现为抗原非特异作用。此与MLR中ConA-T细胞免疫动物的结果相一致。在CML实验里,同种抗原特异TCV免疫小鼠的脾细胞体外诱导同种CTL活性明显受到抑制,CTL 活性十分低下。体外实验结果表明:同种特异TCV免疫小鼠可诱发同种抗原反应和对ConA诱发的增殖反应能力显著低下,表现为抗原非特异性作用。     

移植免疫中T细胞疫苗作用机制的初步分析

为分析Ｔ细胞疫苗同种兔疫抑制作用的机制，在体外观察了Ｔ细胞疫苗免疫小鼠细胞免疫应答能力的改变，分别在ＭＩＣ实验中，观察了免疫小鼠血清和免疫小鼠脾细胞对同种应答细胞反应能力的影响。结果表明：免疫小鼠淋巴细胞ＣｏｎＡ转化反应能力及同种抗原反应能力均显著降低，免疫小鼠血清及其脾细胞不同程度地抑制了同种应答细胞的反应能力，但以上作用并未表现出抗原特异性。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅱ.抗独特型T细胞免疫反应

同种特异T细胞疫苗(TCV)免疫诱导出同种免疫反应低下,同种移植物存活时间显著延长,推测其作用机制可能是同种特异TCV免疫诱导机体抗同种特异TCV(独特型)T细胞的上调.实验证实了“抗TCV-T细胞”的存在.以同种特异TCV免疫动物可诱导出对TCV特异的细胞增殖反应.TCV免疫小鼠淋巴细胞能特异杀伤TCV细胞.将TCV免疫小鼠脾细胞作为调节细胞,观察到它能显著地抑制同种MLR,表明它是一“抑制性T细胞”.这些结果提示,同种特异TCV免疫可诱导机体免疫网络中独特型-抗独特型的上调,产生了同种反应T细胞的独特型T细胞,从而保护同种移植物免受同种反应性T细胞的攻击使同种移植物存活时间显著延长.     

Significant anti-tumour activity of adoptively transferred T cells elicited by intratumoral dendritic cell vaccine injection through enhancing the ratio of CD8+ T cell/regulatory T cells in tumour

We have shown that immunization with dendritic cells (DCs) pulsed with hepatitis B virus core antigen virus‐like particles (HBc‐VLP) packaging with cytosine–guanine dinucleotide (CpG) (HBc‐VLP/CpG) alone were able to delay melanoma growth but not able to eradicate the established tumour in mice. We tested whether, by modulating the vaccination approaches and injection times, the anti‐tumour activity could be enhanced. We used a B16‐HBc melanoma murine model not only to compare the efficacy of DC vaccine immunized via footpads, intravenously or via intratumoral injections in treating melanoma and priming tumour‐specific immune responses, but also to observe how DC vaccination could improve the efficacy of adoptively transferred T cells to induce an enhanced anti‐tumour immune response. Our results indicate that, although all vaccination approaches were able to protect mice from developing melanoma, only three intratumoral injections of DCs could induce a significant anti‐tumour response. Furthermore, the combination of intratumoral DC vaccination and adoptive T cell transfer led to a more robust anti‐tumour response than the use of each treatment individually by increasing CD8⁺ T cells or the ratio of CD8⁺ T cell/regulatory T cells in the tumour site. Moreover, the combination vaccination induced tumour‐specific immune responses that led to tumour regression and protected surviving mice from tumour rechallenge, which is attributed to an increase in CD127‐expressing and interferon‐γ‐producing CD8⁺ T cells. Taken together, these results indicate that repeated intratumoral DC vaccination not only induces expansion of antigen‐specific T cells against tumour‐associated antigens in tumour sites, but also leads to elimination of pre‐established tumours, supporting this combined approach as a potent strategy for DC‐based cancer immunotherapy.     

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2D^b. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2⁺ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2^?/? responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.     

Vaccination with T cell receptor peptides primes anti-receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL

We selected three peptides from the germ-line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative K^d- and L^d-restricted epitopes. Immunization of BALB/c (H-2^d) mice with the Vβ8.2(67–90) 23-mer peptide 1 as well as the 15-mer Vβ8.2(95–108)-peptide 2 efficiently primed specific CD8⁺ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR-Vβ8.2 epitopes. Vβ8.2⁺ T cells were not deleted in TCR peptide-immunized mice because the fractions of Vβ8.2⁺ CD4⁺ and Vβ8.2⁺ CD8⁺ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2⁺ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide-immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3-derived peptide 3 stimulated a CD8⁺ CTL response against a class I-restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele-specific motifs. The described experiments indicate that TCR peptide-primed CD8⁺ CTL recognize class I-restricted, natural Vβ/Jβ-TCR epitopes. Such anti-TCR CTL may, thus, operate in Vβ-specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.     

T细胞疫苗抗同种移植排斥反应的研究

应用Ｔ细胞疫苗于同种心脏移植的研究，用Ｃｏｎ Ａ和ＣＤ３单抗诱导受同种特异性抗原（Ｃ５７ＢＬ／６（Ｈ－２＾ｂ）小鼠脾细胞）免疫的ＢＡＬＢ／Ｃ（Ｈ－２＾ｄ）小鼠，取后者脾细胞所制备的Ｔ细胞疫苗具有延长同种心脏移植物的存活期。受Ｔ细胞疫苗接种的ＢＡＬＢ／Ｃ（Ｈ－２＾ｄ）鼠脾细胞对经Ｃｏｎ Ａ激活或未激活的Ｃ５７ＢＬ／６小鼠脾细胞皆表现出特异的强烈的增殖反应，而对同系脾细胞呈低的反应。Ｔ细胞疫苗表型分析     

Detection of primary direct and indirect human anti-porcine T cell responses using a porcine dendritic cell population

The pathways of human anti-pig T cell xenorecognition have been investigated. Freshly isolated porcine alveolar lavage (AL) cells induced primary proliferative responses by human peripheral and cord blood mononuclear cells which were inhibited by anti-HLA-DR antibody (indirect xenorecognition). Following overnight culture, the AL cells acquired the capacity to stimulate proliferation by purified human T cells which was inhibited by anti-SLA-DR antibody (direct xenorecognition). The marked increase in immunogenicity in the porcine AL cells was accompanied by a phenotypic change consistent with dendritic cell maturation. Limiting dilution assays indicate that the total anti-pig T cell response, in particular that mediated by indirect xenorecognition, is stronger than comparable alloresponses.     

A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice

DNA vaccination represents a novel therapeutic strategy for chronic hepatitis B virus (HBV) infection. Recently, some HBV DNA vaccines have been used in the preliminary clinical trials and exhibited exciting results in chronic HBV carriers. But these vaccines only encoded the single viral antigen, the S or the PreS2/S antigen. In this study, we designed a polytope DNA vaccine encoding multiple T cell epitopes. We found that it induced stronger CTL responses than the vaccine encoding the single antigen in H-2d and H-2b mice, although the CTL response to Ld-restricted epitope suppressed the CTLs to other epitopes in H-2d-restricted mice. Interestingly, heat shock protein 70 as an adjuvant not only enhanced CTL response to the viral antigen but also overcame this epitope suppression. Furthermore, the polytope DNA vaccine resulted in a long-term down-regulation of hepatitis B virus surface antigen and inhibition of HBV DNA replication in a HBV transgenic mouse model. Therefore, our research indicates that it is practicable and feasible to design a polytope DNA vaccine for chronic hepatitis B immunotherapy.