Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2012 Mar 6;109(10):3885-90. doi: 10.1073/pnas.1111205109. Epub 2012 Feb 21.
The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 10(6) cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 10(4)-10(5) single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins--called "microengraving"--to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8(+) T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.
某些临床样本（组织活检、体液）或研究对象本身（儿科研究对象、新生儿）的性质通常限制了评估感染或治疗干预后功能性 T 细胞反应广度的可用细胞数量。目前最常用于评估这种功能多样性的体外方法和特异性细胞的回收以进行体外扩增的方法通常需要超过 10(6)个细胞。在这里，我们介绍了一种从血液或黏膜组织中 10(4)-10(5)个单个细胞中高效鉴定抗原特异性反应的方法，该方法使用亚纳升级别的密集微井阵列。该方法将芯片上成像细胞术与一种捕获分泌蛋白的技术（称为“微刻蚀”）相结合，以类似于 ELISpot 和细胞内细胞因子染色等传统方法的方式对单个 T 细胞的抗原特异性反应进行计数。然而，与这些测定方法不同的是，可以通过微操作轻松回收鉴定的单个细胞，以便进一步进行体外鉴定。应用该方法评估 HIV 特异性 T 细胞反应表明，使用比传统方法少 100-1000 倍的细胞并无需预先进行广泛的基因分析，就有可能建立代表循环中最丰富特异性的克隆 CD8(+)T 细胞系。该快速（<24 h）、高效且廉价的方法应该会改善不同年龄和解剖部位人类 T 细胞免疫学的比较研究。
