Chattopadhyay Pratip K, Melenhorst J Joseph, Ladell Kristin, Gostick Emma, Scheinberg Phillip, Barrett A John, Wooldridge Linda, Roederer Mario, Sewell Andrew K, Price David A
Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cytometry A. 2008 Nov;73(11):1001-9. doi: 10.1002/cyto.a.20642.
The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated peptide-major histocompatibility complex class I (pMHCI) tetramers in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where T cell receptor-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidities, minimize background noise, and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake.
在过去十年中，使用荧光染料偶联的肽 - 主要组织相容性复合体I类（pMHCI）四聚体结合流式细胞术，能够对抗原特异性CD8 + T细胞进行定量和表征，而无需考虑功能读数，这改变了我们对细胞免疫反应的理解。对于那些以高亲和力结合同源pMHCI四聚体的普遍存在的CD8 + T细胞群体，直接离体鉴定和后续数据解释相对简单。然而，低频抗原特异性CD8 + T细胞群体的准确鉴定可能会很复杂，特别是在T细胞受体介导的四聚体结合以低亲和力发生的情况下。在这里，我们重点介绍一些简单的技术，这些技术可用于通过流式细胞术提高四聚体结合CD8 + T细胞群体的视觉分辨率，从而实现准确的定量。这些方法学改进增强了信号强度，特别是在那些以低亲和力结合同源抗原的特定CD8 + T细胞群体的情况下，最大限度地减少了背景噪声，并能够更好地区分真正的pMHCI四聚体结合事件与非特异性摄取。
