Evaluation of immunosuppressive function of regulatory T cells using a novel in vitro cytotoxicity assay.
Evaluation of immunosuppressive function of regulatory T cells using a novel in vitro cytotoxicity assay.
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DOI：
10.1186/2045-3701-4-51
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发表时间：
2014
影响因子：
7.5
通讯作者：
Wei CH
中科院分区：
文献类型：
--
作者：
Zhang L;Manirarora JN;Wei CH
天然存在的调节性T细胞（TCRs）由于其内在的免疫抑制活性而在维持自身耐受中起关键作用。目前，正在进行一些人体临床试验，以研究TdR在治疗各种免疫介导的疾病中的作用。传统上，使用胸苷掺入测定法（其是放射性测定法）或基于CFSE的流式细胞术测定法（其需要相对大量的细胞）来测量TcB的抑制活性。因此，越来越需要开发新的替代生物测定法，其可以在体外表征TdR的免疫抑制功能的各个方面。在这项研究中，使用小鼠克隆的CD 8 + T细胞特异性的胰岛抗原作为应答T细胞，我们首先建立了一种新的，灵敏的和定量的体外发光的细胞活力测定来测量细胞毒性。然后，我们使用该测定来测量TdR是否可以抑制CD 8效应T细胞的细胞毒性。该测定不涉及放射性同位素的使用，并且仅需要相对低数量的TdR。由于正常情况下，T淋巴细胞仅占外周CD 4 + T细胞的5-10%，因此与其他方法相比，这种优势值得注意。通过我们开发的试验，我们证明了调节性T细胞（Tcells）可以抑制CD 8+细胞毒性T细胞对粘附靶细胞单层的抗原特异性杀伤。我们观察到更多的抑制时，T淋巴细胞和CD 8杀伤T细胞在体外活化（刺激）阶段的细胞毒性T淋巴细胞（CTL）比当他们被添加后，在效应阶段的开始孵育。有趣的是，来自B6小鼠的TcR显示出比来自NOD小鼠的TcR更高的CD 8 + T细胞杀伤抑制。此外，IL-2/抗IL-2 mAb复合物在体内诱导Treg的扩增，以及增强Treg对每个细胞的抑制活性。因此，这种新的非放射性的，基于发光的细胞毒性试验介导的克隆胰岛抗原特异性的CD 8 T细胞可用于测量，表征，和定量的天然T细胞的免疫抑制活性，代表一个有用的方法来表征的功能，T细胞在自身免疫性疾病的设置，并阐明Treg细胞介导的免疫调节机制。本文的在线版本（doi：10.1186/2045-3701-4-51）包含补充材料，可供授权用户使用。
Naturally occurring regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance due to their intrinsic immunosuppressive activity. Currently, a number of human clinical trials are being conducted to investigate the roles of Tregs in treating various immune-mediated disorders. Traditionally, the suppressive activity of Tregs is measured using either a thymidine incorporation assay, which is a radioactive assay; or CFSE based flow cytometry assay, which requires a relatively large number of cells. Consequently, there is an increasing need to develop novel alternative bioassays that can characterize various aspects of the immunosuppressive function of Tregs in vitro. In this study, using murine clonal CD8+ T cells specific for an islet antigen as responder T cells, we first established a novel, sensitive and quantitative in vitro luminescence based cell viability assay to measure cytotoxicity. Then we used this assay to measure if Tregs could inhibit the cytotoxicity of CD8 effector T cells. This assay does not involve the use of radioisotopes and only needs relatively low number of Tregs. Since normally Tregs only constitute 5-10% of peripheral CD4+ T cells, this advantage is noteworthy compared with other methods. With the assay we developed, we demonstrated that regulatory T cells (Tregs) could inhibit the antigen-specific killing of an adherent target cell monolayer by the CD8+ cytotoxic T cells. We observed more inhibition when Tregs and CD8 killer T cells were incubated during the in vitro activation (stimulation) stage of the cytotoxic T lymphocytes (CTL) than when they were added later at the start of the effector phase. Interestingly, Tregs from B6 mice demonstrated higher suppression of CD8+ T cell killing than Tregs from NOD mice. Moreover, IL-2/anti-IL-2 mAb complexes induced expansion of Tregs in vivo, as well as enhancing the Treg’s suppressive activity per cell. Therefore, this novel non-radioactive, luminescence based cytotoxicity assay mediated by clonal islet antigen-specific CD8 T cells can be used to measure, characterize, and quantitate the immunosuppressive activity of natural Tregs, representing a useful approach to characterize the functions of Tregs in the setting of autoimmune diseases and to elucidate the mechanisms for Treg cell-mediated immunoregulation. The online version of this article (doi:10.1186/2045-3701-4-51) contains supplementary material, which is available to authorized users.
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DOI：
10.1073/pnas.0932778100
发表时间：
2003-07-08
影响因子：
11.1
作者：
Lieberman, SM;Evans, AM;DiLorenzo, TP
通讯作者：
DiLorenzo, TP
影响因子：
4.4
作者：
Clough, Louise E.;Wang, Chun Jing;Walker, Lucy S. K.
通讯作者：
Walker, Lucy S. K.
影响因子：
32.4
作者：
Tang, Qizhi;Adams, Jason Y.;Bluestone, Jeffrey A.
通讯作者：
Bluestone, Jeffrey A.
影响因子：
3.7
作者：
Boks, M. A.;Zwaginga, J. J.;ten Brinke, A.
通讯作者：
ten Brinke, A.
影响因子：
7.7
作者：
HAMAGUCHI, K;GASKINS, HR;LEITER, EH
通讯作者：
LEITER, EH