Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+T Cell Responses in the Cervix
Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+T Cell Responses in the Cervix
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DOI：
10.1371/journal.pone.0013077
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发表时间：
2010-10-07
期刊：
影响因子：
3.7
通讯作者：
Rebbapragada, Anuradha
中科院分区：
文献类型：
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作者：
Chege, Duncan;Chai, Yijie;Rebbapragada, Anuradha
几种候选艾滋病毒疫苗旨在诱导病毒特异性细胞免疫，特别是在生殖道，通常是艾滋病毒的初始获得部位。然而，缺乏在生殖器水平评估艾滋病毒特异性免疫反应的标准化和敏感方法。因此，我们评估了实时定量PCR （qPCR）作为测量这些反应的潜在平台。β -肌动蛋白和GAPDH分别被确定为外周血单核细胞（PBMCs）和宫颈单核细胞（cmc）中最稳定的内参基因，并用于使转录物mRNA表达正常化。采用qPCR方法检测HIV感染的PBMC中HIV特异性细胞T细胞对优化的CD8+ HIV表位池（HIV表位池）和葡萄球菌肠毒素B （SEB）超抗原对照的免疫应答，并平行评估细胞因子蛋白的产生。IFN γ、IL-2和TNF α的hiv特异性mRNA表达分别在3、5和12小时后达到峰值。将pbmc滴定到宫颈合适的细胞数，以确定最低所需的检测输入细胞数；qPCR在输入至少2.5x10(4)个PBMCs时保持敏感性。该优化的qPCR检测随后用于评估HIV阳性个体的细胞刷衍生宫颈T细胞中的HIV特异性细胞T细胞反应。在cmc中检测到SEB诱导的IFN γ mRNA转录，并与IFNc蛋白的产生呈正相关。然而，qPCR无法检测hiv感染妇女子宫颈中hiv诱导的细胞因子mRNA的产生，尽管在pbmc中检测到了基因诱导。综上所述，尽管qPCR可用于检测血液中HIV的体外细胞免疫应答，但子宫颈中HIV特异性应答可能低于qPCR检测的阈值。尽管如此，该平台可能在测量生殖道中丝裂原诱导的免疫反应方面具有潜在的作用。
Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. beta-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFN gamma, IL-2 and TNF alpha occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5x10(4) PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFN gamma mRNA transcription was detected in CMCs and correlated positively with IFNc protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.