Methodological approach to the ex vivo expansion and detection of T-cruzi-specific T cells from chronic Chagas disease patients
Methodological approach to the ex vivo expansion and detection of T-cruzi-specific T cells from chronic Chagas disease patients
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DOI：
10.1371/journal.pone.0178380
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发表时间：
2017-05-26
期刊：
影响因子：
3.7
通讯作者：
Gomez, Karina A.
中科院分区：
文献类型：
--
作者：
Acevedo, Gonzalo R.;Longhi, Silvia A.;Gomez, Karina A.
T细胞表位的发现不仅对于获得关于宿主对感染性疾病的反应的知识，而且对于免疫干预策略的发展都是必不可少的。在查加斯病中，考虑到克氏锥虫蛋白质组的大小和复杂性及其与宿主免疫系统的相互作用，T细胞的精细特异性尚未得到广泛研究，对于CD 4（+）T细胞区室尤其如此。本工作的目的是优化的寄生虫特异性记忆T细胞系，其在体内的前体群体的代表，并能够响应寄生虫抗原后，长期培养产生的协议。因此，来自慢性无症状和心脏病患者以及来自未感染个体的外周血单核细胞（PBMC）经历了不同的体外培养和刺激条件。随后，测试细胞对T细胞的应答能力。cruzi裂解物通过测量[H-3]-胸苷掺入和干扰素-。和GM-CSF分泌。结果允许我们调整初始T。cruzi裂解物孵育时间以及在特异性评价之前用植物血凝素（PHA）和经辐照的同种异体PBMC扩增的次数。此外，我们的数据表明，寄生虫特异性T细胞显示出明确和强烈的激活，通过使用T。cruzi裂解物脉冲的EB病毒（EBV）转化的人B淋巴细胞（B-LCL）作为自体抗原呈递细胞。在这些培养条件下，我们从无症状患者的记忆CD 4（+）T细胞中产生了一个克隆，该细胞对上鞭毛体和锥鞭毛体蛋白裂解物有反应。我们的研究结果描述了一种分离T.来自恰加斯病患者的克氏特异性T细胞克隆，其使得能够获得关于个体T细胞的功能性和特异性的信息。
The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the host's immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4(+) T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from noninfected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [H-3]-thymidine incorporation and interferon-. and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patient's memory CD4(+) T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzi specific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells.