Department of Biochemistry and Molecular Biology, University of Georgia, 120 E. Green St., 30602, Athens, GA, Georgia.
Complex Carbohydrate Research Center, University of Georgia, Athens, GA, Georgia.
Sci Rep. 2024 Apr 4;14(1):7938. doi: 10.1038/s41598-024-58541-6.
Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.
自然杀伤 (NK) 细胞破坏被抗体调理的组织。预计生成或识别具有更高效力的细胞的策略将增强基于 NK 细胞的免疫疗法。我们之前通过用甘露糖苷酶抑制剂 kifunensine 处理生成了具有增强的抗体依赖性细胞介导的细胞毒性 (ADCC) 的 NK 细胞，甘露糖苷酶抑制剂靶向 N-糖基化加工途径早期的甘露糖苷酶。kifunensine 处理还增加了 Fc γ 受体 IIIa/CD16a 的抗体结合亲和力。在这里，我们证明抑制 NK 细胞 N-糖基化加工会增加 ADCC。我们通过 CRISPR-CAS9 敲低另一种早期 N-糖基化加工酶 MGAT1 来减少 N-糖基化加工，结果表明这些细胞同样增加了抗体结合亲和力和 ADCC。这些实验导致了这样的观察结果，即具有减弱的 N-糖基化加工能力的 NK 细胞在使用 B73.1 和 3G8 抗体结合两个不同的 CD16a 表位的流式细胞术实验中也显示出明显的表型。我们使用原代 NK 细胞评估了这种“亲和力分析”方法，并通过流式细胞术鉴定了与 ADCC 增加相关的独特偏移和分化群体。
