Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany.
Organic Chemistry, Saarland University, 66123 Saarbrücken, Germany; and.
J Immunol. 2020 May 15;204(10):2818-2828. doi: 10.4049/jimmunol.1901536. Epub 2020 Apr 8.
CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H)-adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.
CTL 会在靶细胞界面（免疫突触）通过细胞毒性颗粒（CG）融合释放细胞毒性蛋白，如颗粒酶和穿孔素，以杀死病毒感染和致癌的靶细胞。这些颗粒的一个特征是其腔内的酸性 pH 值，这是将颗粒酶和穿孔素的前体加工成熟形式所必需的。然而，酸性 pH 值在 CG 成熟、运输和融合中的作用尚不清楚。我们在原代小鼠 CTL 中证明，使用新生成的 CG 特异性 pH 指示剂 ClopHensorN(Q69M)，空泡型（H）三磷酸腺苷的 a3 亚基是在 CG 内建立 6.1 的腔内 pH 值所必需的。a3 亚基的敲低导致靶细胞的杀伤明显减少，全内反射荧光显微镜下 CG 融合减少了>50%，这是由于免疫突触处的 CG 数量减少所致。超分辨率显微镜显示，a3 亚基敲低后 CG 与微管网络的相互作用减少。最后，我们通过电子和结构照明显微镜发现，a3 亚基的敲低改变了单个 CG 的直径和密度，而 CTL 中的 CG 数量不受影响。我们得出结论，空泡三磷酸腺苷的 a3 亚基不仅负责 CG 的酸化，而且还有助于 CG 的成熟和向免疫突触的有效运输。
