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中文摘要：
在這里，我們報告了 NK 細胞中免疫檢查點信號調節蛋白 α （SIRPα） 的存在和功能，并描述了如何對其進行細胞治療的調節。NK 細胞 SIRPα 在 IL-2 刺激后上調，以閾值依賴性方式與靶細胞 CD47 相互作用，并對抗其他刺激信號，包括 IL-2、CD16 或 NKG2D。CD47 表達升高保護 K562 腫瘤細胞以及小鼠和人 MHC I 類缺陷靶細胞對抗 SIRPα+ 原代 NK 細胞，但不保護 SIRPα? NKL 或 NK92 細胞。SIRPα 缺陷或抗體阻斷增加了 NK 細胞的殺傷能力。在人 MHC 缺陷細胞中過表達 Rh 猴 CD47 阻止了 Rh NK 細胞在異種環境中的細胞毒性。發現 SIRPα-CD47 軸具有高度的物種特異性。總之，結果表明，SIRPα-CD47 免疫檢查點的破壞可能會增強 NK 細胞的抗腫瘤反應，而 CD47 表達的升高可能會阻止 NK 細胞介導的同種異體和異種組織殺傷。
英文摘要：
Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I–deficient target cells against SIRPα+ primary NK cells, but not against SIRPα? NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα–CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα–CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell–mediated killing of allogeneic and xenogeneic tissues.
論文信息：
論文題目： The SIRPα–CD47 immune checkpoint in NK cells
期刊名稱：JEM- J Exp Med
時間期卷：J Exp Med (2021) 218 (3): e20200839.
在線時間：2021年1月8日
DOI： doi.org/10.1084/jem.20200839
氯膦酸鹽脂質體助力免疫檢查點在NK細胞中研究，Liposoma巨噬細胞清除劑Clodronate Liposomes見刊于JEM：
Liposoma巨噬細胞清除劑Clodronate Liposomes氯膦酸二鈉脂質體的材料和方法：
JEM期刊巨噬細胞清除解決方案
Mouse in vivo innate cytotoxicity assay
Five million WT miECs and five million B2m?/?Ciita?/? miECs or B2m?/?Ciita?/? Cd47 tg miECs were mixed and stained with 5 µM CFSE (Thermo Fisher). Cells in saline were injected i.p. into syngeneic C57BL/6 mice, CD11b-DTR mice or Sirpa?/? mice. Some mice received a coinjection i.p. with 1 µg mouse IL-2 or mouse IL-15 (PeproTech). After 48 h, cells were collected from the abdomen and stained with PerCP-eFlour710–labeled anti-MHC class I (clone AF6-88.5.5.3, mouse IgG2a,κ; eBioscience) mAb for 45 min at 4°C. The CFSE-positive and MHC class I–negative population was analyzed by flow cytometry (FACSCalibur; BD Biosciences) and compared between the WT and the engineered miEC group. All animals were pretreated 18 h with poly I:C injection (100 µg in sterile PBS i.p.; Sigma-Aldrich) before miEC injection. Some animals were pretreated with clodronate (200 µl i.p. 3 d before the experiment; Liposoma) to eliminate macrophages and make the assay more specific for NK cells. Some animals were pretreated with anti-NK1.1 (clone PK136, 200 µl i.p. 3 d before the experiment; BD Biosciences) to eliminate NK cells for macrophage-specific experiments. Some animals received clodronate and anti-NK1.1 for cell depletion. Some of the CD11b-DTR mice were pretreated with DT (Lystlab) 3 d and 1 d before the experiment at a concentration of 25 ng/g mouse weight in 100 µl saline i.p. For peritoneal transfer, 106 peritoneal cells from naive C57BL/6 mice were injected on day 0 with the target miECs. Some animals were pretreated with an anti-Cd47 blocking antibody (clone MIAP301, rat IgG2a,κ; BioXCell; 100 µg i.p., 2 d before implantation of the miEC). Some animals were pretreated with an anti-Sirpα blocking antibody (clone P84, rat IgG1,κ; BioLegend; 100 µg i.p. 2 d before implantation of the miEC). To investigate mouse in vivo innate killing of B2m?/?Ciita?/? miECs and B2m?/?Ciita?/? Cd47 tg miECs, 5 × 106 of both cells were injected after staining with DiO and DiD, respectively according to the manufacturer’s protocol (Vybrant Multicolor cell labeling kit; Invitrogen). Syngeneic C57BL/6 mice were pretreated 18 h with poly I:C (100 µg i.p.; Sigma-Aldrich) in saline before cell injection. After 48 h, cells were collected from the peritoneum and analyzed by flow cytometry (FACSCalibur; BD Bioscience).
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