Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei.
Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei.
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DOI：
10.4049/immunohorizons.1800032
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发表时间：
2018-08-01
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影响因子：
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通讯作者：
Berg, Leslie J
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文献类型：
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作者：
Gallagher, Michael P;Conley, James M;Berg, Leslie J
循环中的幼稚T细胞处于静止状态。在TCR与次级淋巴结构中APC呈递的同源肽接触后，T细胞经历了一段快速转录变化的时期，这为决定命运的效应器或记忆编程奠定了基础。我们描述了一种新的方法来分析从原代小鼠原始T细胞中分离的TCR信号通路在天然多肽AGS刺激后的激活情况。我们用细胞跟踪染料预先标记细胞，以便于用常规的流式细胞术区分CD8+T细胞核和APC核。用这种方法，我们观察到在大量脾细胞呈递的OVA多肽刺激的OT-I T细胞中，NFAT1转录因子被明显的数字激活。OVA浓度对NFAT1转位细胞的比例有离散的控制，这表明在幼稚T细胞中需要不同的阈值TCR信号量来开启NFAT1。这种行为依赖于细胞接触，并且在质量上比离子霉素刺激的初始T细胞中的NFAT1反应更准确。这些数据有助于我们理解其他研究中记录的TCR信号组件的数字行为，并表明T细胞如何在实际感染期间区分Ag利用率的对数倍变化。总体而言，这些结果突出了这种共培养核分离方案在解决初级淋巴细胞中依赖刺激的蛋白质易位问题上的潜力。
Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8+ T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes.