Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Anal Chem. 2010 Jan 15;82(2):473-7. doi: 10.1021/ac9024363.
The frequencies of antigen-specific CD4+ T cells in samples of human tissue have been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNgamma and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples.
在人体组织样本中，抗原特异性 CD4+T 细胞的频率很难准确地进行体外检测，特别是对于多发性硬化症或 1 型糖尿病等自身免疫性疾病。传统方法涉及体外扩增原代 T 细胞以增加细胞数量，然后通过有限稀释或使用负载肽的主要组织相容性复合物 (MHC) 受体的荧光标记四聚体来评估扩增群体中抗原特异性 T 细胞的频率。在这里，我们描述了一种替代方法，该方法使用涂有重组肽负载 MHC 类 II 单体的亚纳升级微井阵列，以特异性方式分离和刺激单个 CD4+T 细胞。在这些实验中，通过微刻蚀来监测激活，以捕获从单个细胞释放的两种细胞因子（IFNγ和 IL-17）。这种新方法应该能够直接从临床样本中对体外抗原特异性 CD4+T 细胞进行计数。
