过继转输的肿瘤特异性T杀伤细胞在小鼠体内维持杀伤功能的研究

体外试验从免疫小鼠脾细胞中诱导出对ＦＢＬ－３瘤细胞具有特异性杀伤功能的Ｔ杀伤细胞，转输到同系正常小鼠体内后发现，同时给予肿瘤抗原刺激和７ｄｒＩＬ－２作用，可使受鼠脾细胞获得杀伤ＦＢＬ－３细胞的活性，其杀伤功能与转输的Ｔ杀伤细胞有关，若转输后连续２１ｄ只给予ｒＩＬ－２不能维持体内建立的杀瘤活性，而间断给予肿瘤抗原再刺激，并联合应用短程ｒＩＬ－２，以１４ｄ为１周期，则可长期维持体内的肿瘤特异性杀伤功能     

肿瘤特异性T杀伤细胞系的建立及其生物学特性

<正> 用小鼠T淋巴细胞性克隆瘤细胞LAC-1免疫同系SW_1小鼠,诱导对该瘤株特异性T杀伤细胞,并采用TCGF条件培液在体外扩大培养,建立体外长期培养肿瘤特异性T杀伤细胞系。实验结果表明:经体内3～4次免疫的小鼠脾脏、腹腔淋巴结细胞均对LAC-1癌细胞有明显的特异性杀伤效应,并随效/靶比例增高,杀     

转输肿瘤特异性T杀伤细胞对L783白血病小鼠的继承性免疫治疗作用

以小鼠L783淋巴细胞性白血病瘤株为模型,通过静脉转输对L783白血病细胞特异的Tc细胞,观察对白血病小鼠的继承性免疫治疗作用。结果表明,在接种L783白血病细胞后6、24小时转输特异Tc细胞对早期白血病小鼠呈现明显的洽疗效果,实验组小鼠均得到保护而长期存活(>6O天)。在接种L783白血病细胞后第5天单纯转输特异Tc细胞对晚期白血病小鼠进行治疗未观察到明显的疗效;联合化疗(三尖杉酯碱)转输Tc细胞使晚期白血病小鼠的存活时间显著延长,与对照组及单纯化疗组小鼠存活时间相比均有显著性差异(P<0.01)。     

用合成肽替代肿瘤抗原诱导肿瘤特异性T杀伤细胞的研究

利用海绵移植模型，将Ｆｆｉｅｎｄ病毒ｇａｇ基因编码蛋白的两个合成肽片段分别注入海绵中，植入Ｃ５７ＢＬ／６（Ｈ－２）小鼠皮下，经一定潜伏期后收集其中的浸润淋巴细胞（ｓｐｏｎｇｅｉｎｆｉｌｔｒａｔｉｎｇｌｙｍｐｈｏｃｙｔｅ，ＳＩＬｓ）体外培养扩增后，发现合成肽３号能诱导出针对该合成肽特异性的ＳＩＬｓ，并特异性杀伤用合成肽３号致鳘的Ｅ１０靶细胞，但不杀伤Ｆｒｉｅｎｄ病毒在Ｃ５７ＢＬ／６小鼠诱导的ＦＢＬ－     

抵抗LAK细胞杀伤的胃癌细胞株的建立及生物学特性的研究

人胃癌细胞系ＭＧＣ－８０３经反复与ＬＡＫ细胞共培养十个周期，获得对ＬＡＫ细胞杀伤抵抗的特性，命名该细胞变株为ＭＧＣ－８０３Ｒ。当效靶比为４０∶１时，对该细胞变株的杀伤率仅为４０％，而对亲代细胞的杀伤率为９０％。用“冷”靶细胞抑制试验、电镜、ＦＡＣＳ及荧光标记技术分析，证实ＭＧＣ－８０３Ｒ细胞膜结合识别位点有改变，ＭＨＣ－Ⅰ表达升高，ＩＣＡＭ－１表达降低，相互粘附的细胞数减少，胞内Ｆ肌动蛋白小体消失，微丝恢复有序排列，细胞生长速率减慢。上述结果提示经免疫筛选对杀伤有抵抗的ＭＧＣ－８０３Ｒ细胞在生物学特性上有所改变。     

多发性硬化症患者APL特异性T细胞系免疫生物学特性分析

本文报告采用髓鞘碱性蛋白 (MBP )的修饰性肽段配体 (APL )对多发性硬化症 (MS )患者治疗 1年后 ,在细胞克隆水平分析T细胞免疫应答的结果。从 5例患者外周血淋巴细胞中建立了 31个APL特异性T细胞系。首先测定患者和健康人体内APL特异性T细胞的反应频率 ,发现患者中平均为 31 6%± 2 3 7% ,而 10例对照者中仅为 5 %± 2 %。 31株APL反应性T细胞系对APL抗原表现出高度的增殖特异性。同时发现 ,APL特异性T细胞系与MBP优势肽段 (氨基酸 83 99)发生交叉反应的比例 ,患者中为 19 4% (6/ 31) ,而对照组中仅有 4 3% (1/ 2 3)。未发现与MBP优势肽段 83 99有交叉反应。来自MS治疗组的APL特异性T细胞系中 ,67%分泌IL 5和 /或IL 13,表明这些T细胞系具有Th2细胞特性。以上结果提示 ,这一修饰性MBP多肽作为一种治疗性疫苗 ,有可能在体内通过诱导Th2调节细胞 ,下调自身反应性T细胞介导的免疫损伤 ,发挥治疗作用。     

杂交—杂交瘤细胞系的建立及其生物学特性

研究了以产生抗人小细胞肺癌相关抗原和ＣＤ３双特异性抗体（Ｂｉｓｐｅｃｉｆｉｃａｎｔｉｂｏｄｙ）的方法及双特异性抗体的性质，两个具有不同表型（ＨＡＴ敏感／Ｎｅｏ拮抗和ＨＡＴ拮抗／Ｎｅｏ敏感）杂交瘤细胞系，经常规方法进行融合，最后选出７个分泌双特异性抗体的杂交－杂交瘤细胞系以其中一个克隆系细胞进行了测示研究，结果表明，该克隆系细胞培养上清能有效增细ＬＡＫ细胞对靶瘤细胞的杀伤活性，表明其具有临床应用前景     

基于pMHC分子靶向的清除抗原特异性T细胞的研究进展

免疫抑制剂目前仍然是自身免疫病和移植排斥反应的主要治疗方法,但是它能非特异性地抑制患者对肿瘤和感染的抵抗能力.选择性清除抗原表位特异性T细胞理论上能减缓T细胞介导的病理作用,同时能保留机体的整体免疫功能状态.近年来,MHC四聚体技术和杀伤性人工抗原递呈细胞技术被用来靶向清除抗原特异性T细胞,以pMHC复合体为靶向分子,...     

疫苗：肿瘤特异性杀伤（T淋巴）细胞CTLs的有益助手

人体自然免疫系统通过被类浆细胞化树突状细胞(pDCs)和B细胞选择性表达的Toll样受体9(TLR9)来识别在细菌或病毒DNA中的被称为CpG基序的序列，并引起适应性免疫应答的发生，这些基序因此被研究作为潜在的新的疫苗佐剂。Hartmann及其同事首次注意到CpGDNA能增强人体免疫系统中CD8^ 细胞毒性T淋巴细胞(CTL)的应答。     

Establishment and biological activity of a proliferative anti-idiotype-activated T cell line

Antisera were raised in rabbits against a monoclonal antibody (McAb 103) of C3H.SW origin which is specific to the synthetic polypeptide (T,G)-A---L and was shown to express the major idiotypic determinants of conventional anti-(T,G)-A---L antibodies. Antibodies were purified and were shown in a binding assay to recognize McAb 103 as well as C3H.SW anti-(T,G)-A---L antibodies. C57BL/6 mice were immunized with the purified rabbit anti-McAb 103 (Ra 103) and their lymph nodes were studied in a proliferation assay. Proliferation was observed in the presence of both Ra 103 and (T,G)-A---L, although the latter stimulated the cells to a lesser extent, suggesting the induction in vivo of (T,G)-A---L-specific clones in low frequency. A T cell line was established from these lymph node cells. The line is kept in continuous growth in the presence of IL-2 and periodic triggering with Ra 103. A significant proliferative response was obtained with Ra 103 only. This proliferation could be almost completely inhibited by either McAb 103 or by conventional anti-(T,G)-A---L antibodies of C3H.SW origin, indicating the cross reaction between the idiotypes expressed on the T cell line and the (T,G)-A---L-specific antibodies. No proliferation could be detected in the presence of either normal rabbit IgG or rabbit anti-mouse IgG. Thus, the T cell line TId 103 allows the analysis of the role of idiotype in T cell recognition and regulation.     

配对盒基因6/小鼠胚胎干细胞细胞系的建立及干细胞生物学特性分析

目的 建立配对盒基因6（Pax6）/小鼠胚胎干细胞（mESCs）细胞系并鉴定其干细胞生物学特性。 方法 体外培养mESCs,将重组载体pEF1α-Pax6-IRES-AcGFP和空载体pEF1α-IRES-AcGFP分别用脂质体法转染mESCs,经G418梯度及荧光蛋白双筛选后,使用细胞免疫荧光染色、免疫印迹法及RT-PCR技术检测Pax6的表达情况,流式细胞术检测Pax6/mESCs阳性细胞的比例。将获得正确的细胞系分别采用细胞免疫荧光染色对其干细胞标志物阶段特异性胚胎抗原1（SSEA1）、八聚体结合转录因子4（OCT4）进行检测,碱性磷酸酶（AP）染色法对其多能性进行检测,流式细胞术检测增殖指数Ki67。将Pax6/mESCs进行肾背囊下移植,移植物行HE染色观察其分化能力。 结果 Pax6成功在mESCs内表达,经G418筛选后,获得Pax6/mESCs细胞系,流式结果显示,Pax6阳性率为90%,免疫荧光显示,干细胞标志物SSEA1、OCT4表达阳性且AP染色阳性,并且在体内移植后能向3个胚层分化。 结论 Pax6成功在mESCs内表达,经G418筛选后,获得细胞系Pax6/mESCs并维持良好的干细胞特性。     

Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.     

感染小鼠白血病病毒的L783V／3T3细胞系的建立及鉴定

L783是上海医科大学病理生理教研室七十年代建立的一株小鼠可移植性淋巴细胞型白血病。用L783发病小鼠的血液、脾脏等组织的无细胞提取液,分别感染NIH-3T3培养细胞,建立了L783小鼠白血病病毒感染的NIH-3T3细胞系(L783V/3T3)。实验结果表明:L783V/3T3细胞的XC合胞形成试验阳性,电镜下可观察到典型的A型和C型病毒颗粒。制备L783V/3T3细胞无细胞提取液,接种新生昆明种乳鼠,能诱发淋巴细胞型白血病。     

The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation

Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft–versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201⁺ hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1_235–243, hTERT_615–624 or PRAME_100–108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting.     

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.     

稳定表达表皮生长因子受体293细胞系的建立及其生物学特性研究

目的建立稳定表达野生型表皮生长因子受体(EGFR)全长的293细胞系。方法通过脂质体瞬间转染的方法将含有EGFR全长的质粒转染293细胞系。利用抗生素筛选的方法使转染阳性的细胞扩增,单克隆化,进一步稳定传代。通过Western印迹的方法挑选高表达克隆,进而使用MTT法测细胞增殖活性。反复传代、冻存、复苏鉴定表达的稳定性。结果获得1株稳定表达EGFR的293细胞系,命名为WT293,且转染前后细胞生长速度存在差异。结论pcDNA3.1(-)载体能够作为稳定表达的载体用于筛选。稳定表达EGFR的293细胞系为配体受体相互作用及靶向EGFR的抗肿瘤药物作用机制的研究提供了实验素材和理论依据。     

抗SRBC杂交瘤细胞株的建立及其在细胞毒T细胞功能测定中的应用

本文报告用淋巴细胞杂交瘤技术建立抗SRBC的杂交瘤细胞株MX-87的方法及其应用情况。MX-87具有H-2~d表型并分泌IgM类抗SRBC单克隆抗体。用微量溶血空斑试验检查,每个MX-87杂交瘤细胞都可以形成一个清晰、透明的空斑。以这个杂交瘤细胞作为靶细胞,用空斑减少试验可以敏感地检测细胞毒T细胞(CTL)的功能。本文探讨了用溶血空斑减少试验进行CTL功能测定的最适条件。     

Establishment and epitope analysis of allo-specific cytotoxic T lymphocytes at a tumor site recognizing a spouse's HLA-A0206 molecule

PROBLEM: The molecular basis of allo-reactivity in reproductive immunity has not been fully clarified. METHOD OF STUDY: Cytotoxic T lymphocytes (CTLs) were established from tumor-infiltrating lymphocytes (TILs). The allo-reactivity of the CTLs against various tumor cell lines or human leukocyte antigen (HLA)-A allele-transfected COS-7 cells was measured by 51Cr-release or interferon-gamma production assay. RESULTS AND CONCLUSIONS: We have established CTLs reacting to an HLA-A0206 molecule that matched a spouse's HLA-A allele from the TILs of a 68-year-old multiparous patient with gastric cancer. The amino acids at positions 66 and 88 in the alpha1 domain of HLA-A0206, both of which were common in the other HLA-A2 subtypes, were involved in the recognition by the CTLs. Endogenous peptides in the groove were not involved in the recognition. These results suggest the presence of long-lasting memory CTLs raised by the reproduction process, and may facilitate a better understanding of the molecular basis of allo-recognition during reproduction.     

HLA-Dw/LD directed cytotoxic T cell clones

T lymphocyte clones were derived by micromanipulation from an MLC between a stimulator and responder matched for class I but mismatched for class II HLA antigens; among the possible stimulating antigens were DR4 and Dw4. Two of the clones, when tested for cytotoxicity on a panel of DR4 positive and negative targets, appeared to recognize a determinant closely associated with Dw4, but did not lyse, with one exception, targets expressing other DR4 associated Dw specificities or DR4 negative targets. Blocking studies, using monoclonal antibodies directed against monomorphic epitopes on class I or class II molecules, revealed that the cytotoxic activity of these clones was strongly inhibited by an anti-class II (L-243) but not by an anti-class I (w6/32) monoclonal antibody. Both clones were T3⁺, T4⁺, T8^?. These findings show that cytotoxic T cell clones, (directed against class II antigens), may have specificity that correlates with a T lymphocyte-defined LD/Dw determinant. The blocking experiments using monoclonal antibodies give further support to the idea that these clones recognize determinant(s) on a class II molecule. The cell surface phenotyping results are in agreement with previous reports that most anti-class II cytotoxic clones have a T4⁺ T8^? phenotype.