Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes
Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes
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DOI：
10.1016/j.humimm.2004.06.006
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发表时间：
2004-11-01
期刊：
影响因子：
2.7
通讯作者：
Tartour, E
中科院分区：
文献类型：
--
作者：
Godard, B;Gazagne, A;Tartour, E
各种争论表明，CD 8（+）T淋巴细胞在控制巨细胞病毒（CMV）感染中起主要作用。因此，CMV特异性CD 8（+）T细胞的检测可能提供有关CMV病毒检测的额外信息，以预测CMV疾病的发展风险，特别是在免疫抑制的移植受者中。我们比较并测试了各种实验条件，以优化酶联免疫斑点试验（Elispot）检测CMV特异性CD 8（+）T淋巴细胞的方法。与使用未分级外周血单核细胞或纯化的CD 8（+）T细胞的直接Elispot相比，使用一个为期6天的体外致敏步骤的间接Elispot检测是检测CMV特异性CD 8（+）T细胞的最灵敏方法。我们发现，在体外培养过程中低剂量的白细胞介素-2增强了该测试的灵敏度，并进行四聚体染色以验证该体外刺激步骤的高效率。我们在Elispot测定期间直接加载特异性CMY肽，并证明使用T2细胞不会提高其灵敏度。Elispot用于检测干扰素-γ似乎比测量肿瘤坏死因子-α或颗粒酶B更敏感和可靠。该技术已成功应用于检测人类白细胞抗原A2（HLA-A2）和HLA-137健康患者以及1例CMV血清学阳性的移植后淋巴细胞减少患者的CMV特异性CD 8（+）T细胞。这种高度敏感的测试可能是一个有用的工具，以评估针对CMV的免疫抑制患者的T细胞免疫。人类免疫学65，（C）美国组织相容性和免疫遗传学学会，2004年。爱思唯尔公司出版
Various arguments suggest that CD8(+) T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8(+) T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8(+) T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8(+) T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8(+) T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMY peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor-alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8(+) T cells in human leukocyte antigen A2 (HLA-A2) and HLA-137 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients. Human Immunology 65, (C) American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc.