Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia.
J Immunol Methods. 2013 Jan 31;387(1-2):181-90. doi: 10.1016/j.jim.2012.10.013. Epub 2012 Oct 30.
CD4(+) T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation of cognate B cells directly, a process that is initiated through the engagement of their T cell-receptor (TCR) with cognate peptide presented on major histocompatibility complex class II (MHC-II) molecules by B cells. Here we report a method to monitor CD4(+) T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of lymphocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4(+) T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry. We call this the "FTA T helper assay", and have found it to be a robust and sensitive assay to measure CD4(+) T cell helper activity across a multitude of peptide-pulsed B "target" cells in real time in vivo. Furthermore, the technique can be used simultaneously with the FTA killing assay that measures cytotoxic T cell function, to provide a comprehensive tool for measuring both CD4(+) and CD8(+) T cell activity during an immune response in vivo.
CD4(+) T 细胞在调节免疫反应中发挥核心作用。它们的效应功能通常通过其分泌细胞因子的能力来评估，这些细胞因子可通过 ELISPOT 和细胞内细胞因子染色检测到。然而，它们的效应功能的一个方面经常被忽视，即它们能够直接帮助激活同源 B 细胞，这一过程是通过 B 细胞上 MHC-II 分子上的同源肽与 T 细胞受体 (TCR) 的结合而启动的。在这里，我们报告了一种使用多重高通量测定法在体内监测 CD4(+) T 细胞介导的 B 细胞辅助的方法。该测定法利用荧光靶阵列 (FTA)，该阵列由用大量 (>200) 独特荧光特征标记的淋巴细胞组成，这些特征可以根据与三种重要染料羧基荧光素二乙酸琥珀酰亚胺酯 (CFSE)、CellTrace Violet (CTV) 和细胞增殖染料 eFluor 670 (CPD) 的组合标记，在单个受体内的单个动物中进行区分。通过用滴定量的同源 MHC-II 结合肽脉冲不同的 B 细胞群体，通过抗体标记和流式细胞术可以测量 B 细胞激活标志物 CD69 和 CD44 的诱导来评估 CD4(+) T 细胞辅助。我们称之为“FTA T 辅助测定法”，并发现它是一种强大而敏感的测定法，可以实时在体内测量大量肽脉冲 B“靶”细胞中的 CD4(+) T 细胞辅助活性。此外，该技术可与 FTA 杀伤测定法同时使用，该测定法可测量细胞毒性 T 细胞功能，为在体内免疫反应期间测量 CD4(+) 和 CD8(+) T 细胞活性提供了一种全面的工具。
