肿瘤细胞治疗研究
英文原题：KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.
我们的结果表明，敲除 KLRC1 是提高 NK 细胞对 HLA-E⁺ 肿瘤抗肿瘤活性的有效策略，并可应用于实体瘤 NK 细胞疗法的开发。
引言：自然杀伤（NK）细胞有望推动细胞疗法从复杂的自体方案转向通用现成型疗法。尽管 NK 细胞治疗白血病已显示疗效和安全性，但 NK 细胞免疫疗法对实体瘤的疗效有限，仍是主要障碍。在免疫抑制性肿瘤微环境（TME）中，癌细胞与免疫细胞间的抑制性相互作用会削弱抗肿瘤免疫。KLRC1 基因编码 NK 细胞抑制性受体 NKG2A，后者是一种强效 NK 细胞免疫检查点。NKG2A 特异性结合常在肿瘤中过表达的非经典 HLA I 类分子 HLA-E，进而传递抑制信号并显著损害 NK 细胞功能。方法：为恢复 NK 细胞对 HLA-E⁺ 肿瘤的细胞毒性，研究人员利用 CRISPR 介导的 KLRC1 基因编辑靶向 NKG2A/HLA-E 免疫检查点。结果：KLRC1 敲除使离体扩增并经细胞分选的人 NK 细胞中 NKG2A⁺ 细胞频率降低 81%。体外实验中，肿瘤细胞 HLA-E 过表达显著抑制野生型（WT）NK 细胞的细胞毒性，具体 p 值因肿瘤细胞系不同而为 0.0071–0.0473。相比之下，KLRC1 敲除 NK 细胞对 4 种不同 HLA-E⁺ 实体瘤细胞系的细胞毒性均显著高于 WT NK 细胞，p 值范围 <0.0001–0.0154。有趣的是，编辑后的 NK 细胞群体中仅需 43.5%–60.2% 为 NKG2A⁻ 细胞，即足以最大程度逆转 HLA-E 对 NK 细胞毒性的抑制。KLRC1 敲除 NK 细胞中活化受体 NKG2C 表达增加，并促进其对 HLA-E⁺ 肿瘤的细胞毒性。体内实验中，与 WT NK 细胞相比，过继转移人 KLRC1 敲除 NK 细胞可显著延缓 HLA-E⁺ 转移性乳腺癌异种移植小鼠模型中的肿瘤进展并延长生存（p = 0.0015）。结论：结果表明，敲除 KLRC1 是提高 NK 细胞对 HLA-E⁺ 肿瘤抗肿瘤活性的有效策略，可用于开发治疗实体瘤的 NK 细胞疗法。
INTRODUCTION: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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