Réthi Bence, Detre Cynthia, Gogolák Péter, Kolonics Attila, Magócsi Mária, Rajnavölgyi Eva
Department of Immunology, Eötvös L. University, Göd, Hungary.
Cytometry. 2002 Apr 1;47(4):207-16. doi: 10.1002/cyto.10086.
In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry.
We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels (Ca(2+)) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals.
The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-kappaB was induced.
Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells.
在本研究中，通过流式细胞术研究了抗原呈递细胞（APC）、肽浓度和CD28共刺激对抗原特异性T细胞活化诱导的钙信号的影响。
我们采用了两种实验方法，它们在时间尺度和T细胞与APC相互作用的持续时间上有所不同，以测量活化T细胞内游离钙水平（Ca(2+)）的增加：（1）用负载肽的APC共离心激活负载Fluo-3的T细胞，并连续监测荧光强度变化的动力学；（2）将负载肽的APC和T细胞混合、共培养，并在不同时间间隔测量荧光强度。
T细胞的钙信号依赖于APC，这通过高荧光强度与低荧光强度细胞的比例以及活化群体中钙信号的大小得以证明。T细胞与低效APC或在低抗原浓度下与高效APC的短期相互作用导致钙信号减弱。CD28介导的共刺激增强了细胞内钙水平的幅度并维持其升高。与强烈且持续的钙信号一致，诱导了钙依赖性转录因子NF-AT、AP-1和NF-κB的活化。
建立了流式细胞术方法，可用于快速灵活地分析抗原特异性T细胞活化后的钙信号。平均荧光强度增加的动力学反映了整个细胞群体的钙反应，而在选定时间点对荧光强度的统计分析提供了关于单细胞活化状态的信息。
