Raychaudhuri Kumarkrishna, Rangu Rohita, Ma Alison, Alvinez Neriah, Tran Andy D, Pallikkuth Sandeep, McIntire Katherine M, Garvey Joseph A, Yi Jason, Samelson Lawrence E
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.
Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.
Front Immunol. 2024 Dec 24;15:1503018. doi: 10.3389/fimmu.2024.1503018. eCollection 2024.
T cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation.
We employed TIRF microscopy to establish detailed spatial and kinetic relationships among these molecules in live Jurkat and murine primary T cells. We used anti-TCR (CD3) antibodies to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Using this model, we aimed to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events, and how these effects could affect the immune response.
Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation.
These results suggest that CD28 co- stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.
T细胞活化需要其特异性配体与T细胞受体（TCR）结合。这种相互作用引发了一系列近端事件，包括CD3和TCRζ链的酪氨酸磷酸化、蛋白酪氨酸激酶Lck和ZAP70的募集与激活，随后募集衔接蛋白和信号蛋白。CD28共刺激对于产生功能性免疫反应也是必需的。目前我们对CD28激活的分子机制还缺乏全面的了解。
我们采用全内反射荧光显微镜（TIRF显微镜）来确定这些分子在活的Jurkat细胞和小鼠原代T细胞中的详细空间和动力学关系。我们使用抗TCR（CD3）抗体来触发TCR微簇（MC）的形成，TCR微簇是T细胞活化过程中形成的亚微米大小的基本信号单位。利用这个模型，我们旨在描绘CD28共刺激信号如何改变TCR近端信号事件的动力学和分子化学计量，以及这些效应如何影响免疫反应。
我们的结果表明，CD28共刺激特异性地加速了ZAP70向MC中TCRζ链的募集，并增加了ZAP70的激活。CD28介导的ZAP70募集加速是由增强的Lck向MC的募集驱动的。由于CD28共刺激，在MC中还观察到Lck的活性和非活性形式之间有更大的空间分离。
这些结果表明，CD28共刺激可能通过增强TCR微簇中Lck的激活形式来降低TCR激活阈值。
