肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集
Summary
此协议描述了使用的肽：MHC四聚体和磁性微珠分离低频表位特异性的T细胞群体，并通过流式细胞术分析它们。这种方法能够直接研究内源性T细胞群的兴趣<em在体内</em实验系统。
Abstract
研究与体内实验模型的适应性免疫识别能力，根据他们的T细胞抗原受体（TCR）的特异性的T细胞的基本需要。许多的间接的方法是可在其中通过测量确定的官能反应，如增殖，细胞因子的产生，或活化标志物的表达的1与特定的抗原和表位的特异性T细胞在体外刺激T细胞堆积人口。然而，这些方法只能识别特定表位的T细胞表现出的许多可能的功能之一，它们是不够敏感检测表位特异性的T细胞在幼稚前体频率。一种流行的选择是TCR基因转殖继转移模型，其中单克隆T细胞的TCR转基因小鼠接种到组织相容性的主机创建一个大的前体人口的表位特异性的T细胞，可以EASI光年跟踪与一个同类系的标记抗体2,3使用。虽然功能强大，该方法遭受从实验与工件的非生理性T细胞的频率与一个单一的抗原决定簇特异性的4,5。此外，该系统不能被用于研究的功能内的异质性的表位特异性的T细胞克隆的多克隆人口。
理想的方式来学习适应性免疫应包括使用的方法，区分TCR特异性完全由结合到同源肽：MHC（住房抵押贷款公司）的直接检测表位特异性T细胞的内源性T细胞库。住房抵押贷款公司的四聚体和流式细胞仪的使用来完成这6，但被限制在检测高频只符合下列抗原诱导的克隆扩增的特定表位的T细胞群体。在这个协议中，我们描述了一种方法，协调使用的住房抵押贷款公司四和磁学抽动细胞富集技术，使极低频小鼠淋巴组织3,7的特定表位的T细胞的检测。利用这种技术，人们可以全面跟踪整个在小鼠内源性T细胞表位的特定人群的免疫反应的所有阶段。
Protocol
1。淋巴组织细胞分离加入1毫升：冰：冷cEHAA（EHAA + 10％FBS，钢笔/链球菌，庆大霉素，2mM的L-谷氨酰胺，55 mM的2 – 巯基乙醇）或其它等效的T细胞的介质，以60毫米的培养皿中含有一个小正方形100μm的尼龙网和冰。 安乐死鼠标。 取出脾和淋巴结尽可能方便。这些措施应包括至少在腹股沟，腋窝，上臂，子宫颈癌，肠系膜淋巴结肿大。将它们放置在顶部的尼龙网眼的培养皿中。 …
Representative Results
图1描述了代表性的流式细胞仪地块pMHCII四聚体富集的脾脏和淋巴结从幼稚小鼠的样品，而图2示出了有代表性的数据，先前与相关肽+ CFA免疫小鼠。串行浇注删除的分析CD4 + T细胞群的自体荧光和其他有害事件。的CD8 + T细胞群体作为一个有用的内部pMHCII四聚体染色的CD4 + T细胞的阴性对照。需要注意的是结合态的富集通常包含一个显着较高比例的自体荧光的细胞比未结合的?…
Discussion
本协议提出的住房抵押贷款公司四聚体为基础的细胞富集方法的研究表位特异性T细胞内源性T细胞剧目是一个功能强大的工具。住房抵押贷款公司四聚体的使用，可以直接基于绑定同源住房抵押贷款公司配体的能力，其电阻温度系数的表位特异性的T细胞的检测。的富集提供了一个极为罕见的群体可以检测到抗原特异性T细胞的T细胞内源性剧目没有他们的基因构成任何操作或前体频率的灵敏度水平?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者想感谢安德烈·汉和劳伦斯日元的技术援助，成员的詹金斯实验室的帮助，在此协议的发展。
Materials
| Reagent | Vendor | Catalog number |
| PE or APC conjugated pMHC tetramer (or multimer) | Made by investigator, obtained from the NIH tetramer core, or purchased from commercial sources | |
| Anti-PE conjugated magnetic microbeads | Miltenyi | 130-048-801 |
| Anti-APC conjugated magnetic microbeads | Miltenyi | 130-090-855 |
| LS magnetic columns | Miltenyi | 130-042-401 |
| MidiMACS or QuadroMACS magnet | Miltenyi | 130-042-302 or 130-090-976 |
| Cell counting beads | Life Technologies | PCB-100 |
Riferimenti
- Knutson, K. L., dela Rosa, C., Disis, M. L. Laboratory analysis of T-cell immunity. Front Biosci. 11, 1932-1944 (2006).
- Kearney, E. R., Pape, K. A., Loh, D. Y., Jenkins, M. K. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity. 1, 327-339 (1994).
- Moon, J. J. Tracking epitope-specific T cells. Nat Protoc. 4, 565-581 (2009).
- Hataye, J., Moon, J. J., Khoruts, A., Reilly, C., Jenkins, M. K. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science. 312, 114-116 (2006).
- Marzo, A. L. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat Immunol. 6, 793-799 (2005).
- Davis, M. M., Altman, J. D., Newell, E. W. Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis. Nature reviews. Immunology. 11, 551-558 (2011).
- Moon, J. J. Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity. 27, 203-213 (2007).
- Seah, S. G. The linear range for accurately quantifying antigen-specific T-cell frequencies by tetramer staining during natural immune responses. European Journal of Immunology. 41, 1499-1500 (2011).
- Obar, J. J., Khanna, K. M., Lefrancois, L. Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection. Immunity. 28, 859-869 (2008).
- Daniels, M. A., Jameson, S. C. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J Exp Med. 191, 335-346 (2000).
- Pittet, M. J. Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells. Journal of Immunology. 171, 1844-1849 (2003).
- Choi, E. M. High avidity antigen-specific CTL identified by CD8-independent tetramer staining. Journal of Immunology. 171, 5116-5123 (2003).
- Chu, H. H. Positive selection optimizes the number and function of MHCII-restricted CD4+ T cell clones in the naive polyclonal repertoire. Proc Natl Acad Sci U S A. 106, 11241-11245 (2009).
- Chu, H. H., Moon, J. J., Kruse, A. C., Pepper, M., Jenkins, M. K. Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide-MHC Class II-Specific CD4+ T Cell Populations. J Immunol. 185, 4705-4713 (2010).
- Legoux, F. Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans. J Immunol. 184, 6731-6738 (2010).
- Alanio, C., Lemaitre, F., Law, H. K., Hasan, M., Albert, M. L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood. 115, 3718-3725 (2010).
- Kwok, W. W. Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire. Journal of Immunology. 188, 2537-2544 (2012).
- Jenkins, M. K., Chu, H. H., McLachlan, J. B., Moon, J. J. On the composition of the preimmune repertoire of T cells specific for Peptide-major histocompatibility complex ligands. Annu Rev Immunol. 28, 275-294 (2010).
- Matechak, E. O., Killeen, N., Hedrick, S. M., Fowlkes, B. J. MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity. 4, 337-347 (1996).
- Burchill, M. A. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity. 28, 112-121 (2008).
- Pepper, M. Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells. Nature Immunology. 11, 83-89 (2010).
Tags
Peptide:MHC Tetramer-based EnrichmentEpitope-specific T CellsT Cell Antigen Receptor SpecificityFunctional ResponseProliferationCytokine ProductionActivation MarkersTCR Transgenic Adoptive Transfer ModelMonoclonal T CellsHistocompatible HostsCongenic Marker AntibodyExperimental ArtifactsFunctional HeterogeneityPolyclonal Population
Citazione di questo articolo
Legoux, F. P., Moon, J. J. Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells. J. Vis. Exp. (68), e4420, doi:10.3791/4420 (2012).