PDF(Pubmed)
Abstract：
Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.
摘要：
细胞毒性T淋巴细胞（CTL）运动性是有效CTL反应的重要特征，当CTL耗尽时会受损，例如在慢性逆转录病毒感染期间。一个突出的T细胞耗尽标记是程序性细胞死亡蛋白1(PD-1),并且已知抗PD-1和PD-配体1(PD-L1)相互作用的抗体改善CTL功能。然而,抗体阻断会影响所有PD-1/PD-L1表达细胞类型，因此，观察到的效应不能选择性地归因于CTL。为了克服这个问题，我们在幼稚Friend逆转录病毒（FV）特异性CTL中对PD-1编码基因PDCD1进行了基于CRISPR/Cas9的敲除。我们将1000个这样的细胞转移到小鼠中，在FV感染后它们会增殖。使用活体双光子显微镜，我们可视化了骨髓中的CTL运动，并通过流式细胞术评估了细胞毒性分子的表达。PDCD1的敲除改善了感染后14天的CTL运动并增强了细胞毒性标志物的表达。我们的数据显示了天然抗病毒CTL的遗传调整的潜力，并且可能与改进的T细胞介导的疗法的未来设计相关。
