自然杀伤（NK）细胞具有将细胞疗法从复杂的个体自体选项转变为通用现成式选项的潜力。尽管NK细胞在白血病治疗中已经证明了其疗效和安全性，但NK细胞免疫疗法对实体肿瘤的有限疗效仍然是一个重要的障碍。在免疫抑制性肿瘤微环境(TME)中，癌细胞和免疫细胞之间的抑制性相互作用损害了抗肿瘤免疫。KLRC1基因编码了NK细胞抑制性受体NKG2A，是一种强效的NK细胞免疫检查点。NKG2A特异地结合HLA-E，一种在肿瘤中经常过度表达的非典型HLA I类分子，导致传递抑制性信号，严重损害NK细胞功能。为了恢复对HLA-E阳性肿瘤的NK细胞细胞毒性，我们使用CRISPR介导的KLRC1基因编辑靶向了NKG2A/HLA-E免疫检查点。KLRC1基因敲除导致体外扩增的人类NK细胞中NKG2A阳性细胞频率减少了81％。体外，肿瘤细胞对HLA-E的过表达显著抑制了野生型（WT）NK细胞的细胞毒性，p值范围从0.0071到0.0473，具体取决于肿瘤细胞系。相反，与WT NK细胞相比，KLRC1 KO NK细胞对四种不同的HLA-E阳性实体肿瘤细胞系表现出显著较高的细胞毒性，p值范围从<0.0001到0.0154。有趣的是，在编辑的NK细胞群体中，43.5％至60.2％的NKG2A- NK细胞比例足以最大程度地逆转HLA-E介导的NK细胞细胞毒性抑制。KLRC1 KO NK细胞中激活受体NKG2C的表达增加，对改善针对HLA-E阳性肿瘤的NK细胞细胞毒性发挥了作用。体内，在HLA-E阳性转移性乳腺癌的异种移植小鼠模型中，人体KLRC1 KO NK细胞的移植显著延缓了肿瘤进展并增加了存活时间，与WT NK细胞相比（p = 0.0015）。我们的结果表明，KLRC1基因敲除是提高NK细胞对HLA-E阳性肿瘤抗肿瘤活性的有效策略，可以应用于实体肿瘤的NK细胞疗法的开发中。 版权所有 © 2023 Mac Donald, Guipouy, Lemieux, Harvey, Bordeleau, Guay, Roméro, Li, Dion, Béland和Haddad。

Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function.To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing.KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015).Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.Copyright © 2023 Mac Donald, Guipouy, Lemieux, Harvey, Bordeleau, Guay, Roméro, Li, Dion, Béland and Haddad.