Department of Neurology, University of Münster, Albert-Schweitzer-Campus 1, Münster 48149, Germany.
J Neuroinflammation. 2013 Oct 5;10:121. doi: 10.1186/1742-2094-10-121.
Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky' immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.
Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.
Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.
Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.
神经抗原反应性细胞毒性 CD8+T 细胞有助于多种炎症性中枢神经系统疾病中的神经元功能障碍和退化。面对大量的靶细胞，中枢神经系统浸润的 CD8+T 细胞通过向神经元有限释放细胞毒性效应分子，或者通过“渗漏”免疫突触中的细胞毒性效应分子溢出和 CD8+T 细胞自身在连续和同时杀伤少突胶质细胞或星形胶质细胞时的非限定释放，导致神经元细胞死亡。
在体外刺激野生型和 T 细胞受体转基因 CD8+T 细胞，通过流式细胞术评估其激活状态，并使用酶联测定法测定上清液谷氨酸水平。通过定量实时 PCR 检查参与囊泡谷氨酸释放的分子的表达调控，通过药理学阻断实验研究非囊泡谷氨酸释放的机制。在急性脑切片制备中研究 CD8+T 细胞介导的谷氨酸释放对神经元活力的影响。
T 细胞受体刺激后，CD8+T 细胞获得了囊泡谷氨酸释放的分子谱：（i）它们上调谷氨酰胺酶的表达，通过谷氨酰胺脱氨作用产生谷氨酸；（ii）它们上调囊泡质子-ATP 酶和囊泡谷氨酸转运体的表达，这些蛋白将谷氨酸填充到囊泡中。随后，CD8+T 细胞以严格依赖刺激的方式释放谷氨酸。在重复 T 细胞受体刺激后，CD25high CD8+T 效应细胞比 CD25low CD8+T 记忆细胞表现出更高的估计单个细胞谷氨酸释放率。此外，寡突胶质细胞反应性 CD25high CD8+T 效应细胞释放的谷氨酸能够引发完整中枢神经系统灰质中神经元的旁分泌兴奋性细胞死亡（尽管神经胶质细胞和神经元会重新摄取谷氨酸）。
谷氨酸释放可能是神经抗原反应性 CD8+T 细胞的关键效应途径，有助于中枢神经系统炎症中的兴奋性毒性。
