中文摘要：
在這里，我們報(bào)告了 NK 細(xì)胞中免疫檢查點(diǎn)信號調(diào)節(jié)蛋白 α （SIRPα） 的存在和功能，并描述了如何對其進(jìn)行細(xì)胞治療的調(diào)節(jié)。NK 細(xì)胞 SIRPα 在 IL-2 刺激后上調(diào)，以閾值依賴性方式與靶細(xì)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(dá)升高保護(hù) K562 腫瘤細(xì)胞以及小鼠和人 MHC I 類缺陷靶細(xì)胞對抗 SIRPα+ 原代 NK 細(xì)胞，但不保護(hù) SIRPα? NKL 或 NK92 細(xì)胞。SIRPα 缺陷或抗體阻斷增加了 NK 細(xì)胞的殺傷能力。在人 MHC 缺陷細(xì)胞中過表達(dá) Rh 猴 CD47 阻止了 Rh NK 細(xì)胞在異種環(huán)境中的細(xì)胞毒性。發(fā)現(xiàn) SIRPα-CD47 軸具有高度的物種特異性。總之，結(jié)果表明，SIRPα-CD47 免疫檢查點(diǎn)的破壞可能會增強(qiáng) NK 細(xì)胞的抗腫瘤反應(yīng)，而 CD47 表達(dá)的升高可能會阻止 NK 細(xì)胞介導(dǎo)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(zhì)體助力免疫檢查點(diǎn)在NK細(xì)胞中研究，Liposoma巨噬細(xì)胞清除劑Clodronate Liposomes見刊于JEM：
Liposoma巨噬細(xì)胞清除劑Clodronate Liposomes氯膦酸二鈉脂質(zhì)體的材料和方法：
JEM期刊巨噬細(xì)胞清除解決方案
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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