Tario Joseph D, Chen George L, Hahn Theresa E, Pan Dalin, Furlage Rosemary L, Zhang Yali, Brix Liselotte, Halgreen Charlotte, Jacobsen Kivin, McCarthy Philip L, Wallace Paul K
Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York, 14263.
Cytometry B Clin Cytom. 2015 Jan;88(1):6-20. doi: 10.1002/cyto.b.21196. Epub 2014 Oct 23.
The enumeration of antigen-specific T cells is increasingly relevant in clinical and research settings. This information is useful for evaluating immune responses to treatment, monitoring the efficacy of anticancer vaccines, and for detecting self-reactive T cells in autoimmune disorders. Quantifying antigen-specific T cells can be accomplished via IFNγ ELISpot assay, the measurement of intracellular cytokine production by flow cytometry, or by lymphocyte proliferation assays in response to antigen. While robust, these technologies are labor-intensive and can take several days to obtain results. New technology has led to more powerful tools for quickly and accurately measuring antigen-specific T cells by flow cytometry via fluorescently-labeled TCR-specific multimers. In this study, we evaluated the use of an assay based on Dextramer reagents for enumerating cytomegalovirus (CMV) antigen-specific T cells (CASTs). Assay performance characteristics were assessed by establishing Dextramers' sensitivity (median=0.4; range=0.1-1.4 CASTs μl(-1) ), determining their specificity (100%), evaluating assay robustness with different leukocyte sources and assay reproducibility via interlaboratory and interinstrument investigations. Furthermore, the levels of CASTs in 95 peripheral blood samples from 62 unique blood and marrow transplants recipients correlated well between Dextramers and Tetramers (R(2) =0.9042).
在临床和研究环境中，抗原特异性T细胞的计数越来越重要。这些信息对于评估治疗的免疫反应、监测抗癌疫苗的疗效以及检测自身免疫性疾病中的自身反应性T细胞很有用。可以通过IFNγ ELISpot测定、流式细胞术测量细胞内细胞因子产生或通过对抗原的淋巴细胞增殖测定来定量抗原特异性T细胞。虽然这些技术很可靠，但它们劳动强度大，可能需要几天时间才能获得结果。新技术带来了更强大的工具，可通过荧光标记的TCR特异性多聚体，利用流式细胞术快速准确地测量抗原特异性T细胞。在本研究中，我们评估了基于Dextramer试剂的检测方法用于计数巨细胞病毒（CMV）抗原特异性T细胞（CASTs）的情况。通过确定Dextramer的灵敏度（中位数=0.4；范围=0.1 - 1.4 CASTs μl(-1)）、测定其特异性（100%）、评估不同白细胞来源的检测稳健性以及通过实验室间和仪器间研究评估检测重复性，来评估检测性能特征。此外，在来自62名独特的血液和骨髓移植受者的95份外周血样本中，Dextramer和Tetramer检测出的CASTs水平相关性良好（R(2) =0.9042）。
