Melenhorst J Joseph, Scheinberg Phillip, Chattopadhyay Pratip K, Lissina Anna, Gostick Emma, Cole David K, Wooldridge Linda, van den Berg Hugo A, Bornstein Ethan, Hensel Nancy F, Douek Daniel C, Roederer Mario, Sewell Andrew K, Barrett A John, Price David A
Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Immunol Methods. 2008 Sep 30;338(1-2):31-9. doi: 10.1016/j.jim.2008.07.008. Epub 2008 Jul 31.
The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8(+) T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8(+) T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) without impacting the TCR binding platform can detect cognate CD8(+) T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8(+) T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8(+) T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8(+) T cells.
可溶性重组肽 - 主要组织相容性复合体I类（pMHCI）分子以多聚体形式与荧光标记物偶联，使得通过流式细胞术能够对抗抗原特异性CD8(+) T细胞群体进行物理定量和表征。有几个因素决定了使用这些试剂可视化同源CD8(+) T细胞的结合阈值；这些因素包括T细胞受体（TCR）对pMHCI抗原的亲和力。在这里，我们表明，由在重链α2结构域中工程改造以增强CD8结合（解离常数K(D)约为85 microM）而不影响TCR结合平台的肽 - 人白细胞抗原（pHLA）A0201单体构建的多聚体，可以检测到带有低亲和力TCR的同源CD8(+) T细胞，而相应的野生型pHLA A0201多聚体复合物则无法检测到这些细胞。从机制上讲，这种效应是由TCR/pMHCI结合速率的不成比例增强介导的。在直接的体外应用中，这些共受体增强的多聚体表现出忠实的同源结合特性；非同源CD8(+) T细胞群体中背景染色的相应增加可以通过多色流式细胞术作为幼稚细胞和记忆细胞的混合物进行表型解析。这些发现首次验证了一种用于物理检测低亲和力抗原特异性CD8(+) T细胞群体的新方法；这种共受体增强的多聚体试剂可能在多种情况下用于检测自身免疫、肿瘤特异性和交叉反应性CD8(+) T细胞。
