Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining
Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining
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DOI：
10.3791/54881
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发表时间：
2016-12-01
影响因子：
1.2
通讯作者：
Martins, Mauricio A.
中科院分区：
文献类型：
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作者：
Gonzalez-Nieto, Lucas;Domingues, Aline;Martins, Mauricio A.
肽-主要组织相容性复合物I类（pMHC-I）四聚体已成为研究CD 8（+）T细胞应答的宝贵工具。由于这些试剂直接与CD 8（+）T淋巴细胞表面的T细胞受体结合，荧光染料标记的pMHC-I四聚体能够准确检测抗原（Ag）特异性CD 8（+）T细胞，而无需体外再刺激。此外，当与多色流式细胞术结合时，pMHC-I四聚体染色可以揭示Ag特异性CD 8（+）T细胞的关键方面，包括分化阶段、记忆表型和活化状态。这些类型的分析在HIV免疫学领域特别有用，其中CD 8（+）T细胞可以影响艾滋病的进展。猴免疫缺陷病毒（SIV）感染恒河猴的实验为研究艾滋病病毒的细胞免疫提供了一个宝贵的工具。因此，在定义和表征这种动物模型中的T细胞应答方面取得了相当大的进展。在这里，我们提出了一个优化的方案，用于计数猴SIV特异性CD 8（+）T细胞的pMHC-I四聚体染色。我们的检测允许每次测试同时定量和记忆两个pMHC-I四聚体+CD 8（+）T细胞群体，这可能有助于追踪疫苗接种或SIV感染产生的SIV特异性CD 8（+）T细胞应答。考虑到非人灵长类动物在生物医学研究中的相关性，该方法适用于研究多种疾病背景下的CD 8（+）T细胞应答。
Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8(+) T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8(+) T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8(+) T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8(+) T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8(+) T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8(+) T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8(+) T-cell populations per test, which might be useful for tracking SIV-specific CD8(+) T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8(+) T-cell responses in multiple disease settings.