Abstract：
The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection. As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.
摘要：
健康和疾病过程中抗原特异性T细胞的鉴定和表征仍然是提高我们对免疫病理生理学理解的关键。通过开发肽:MHC四聚体试剂,已经大大推进了在内源性T细胞库内追踪抗原特异性T细胞群体的技术挑战。这些与抗原肽表位复合的I类或II类MHC分子的荧光标记可溶性多聚体直接与具有相应T细胞受体（TCR）特异性的T细胞结合，因此,鉴定处于其天然状态的抗原特异性T细胞群体,而不需要通过离体刺激诱导的功能性应答。对于极其罕见的人群来说，可以磁性富集四聚体结合的T细胞以提高检测的灵敏度和可靠性。随着组织驻留T细胞免疫研究的深入，迫切需要鉴定转运并驻留在非淋巴组织中的抗原特异性T细胞。在这个协议中,我们为小鼠肺内存在的抗原特异性T细胞的分离和表征提供了一套详细的说明.这涉及从消化的肺组织中分离T细胞,然后进行一般的T细胞磁性富集步骤和四聚体染色以进行流式细胞术分析和分选。本协议中强调的步骤利用常见的技术和容易获得的试剂，几乎所有从事小鼠T细胞免疫学的研究人员都可以使用它，并且高度适应于存在于肺内的任何低频率抗原特异性T细胞群的各种下游分析。
