Kir6.1, a component of an ATP-sensitive potassium channel, regulates natural killer cell development

Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. We show that Kcnj8, which codes for the Kir6.1 subunit of a certain type of ATP-sensitive potassium (KATP) channel, is highly expressed in murine splenic and uterine NK cells compared to other K+ channels previously identified in NK cells. Kcnj8 expression is highest in the most mature subset of splenic NK cells (CD27-/CD11b+) and in NKG2A+ or Ly49C/I+ educated uterine NK cells. Using patch clamping, we show that a subset of NK cells expresses a current sensitive to the Kir6.1 blocker PNU-37883A. Kcnj8 does not participate in NK cell degranulation in response to tumor cells in vitro or rejection of tumor cells in vivo, or IFN-γ release. Transcriptomics show that genes previously implicated in NK cell development are amongst those differentially expressed in CD27-/CD11b+ NK cells deficient of Kcnj8. Indeed, we found that mice with NK-cell specific Kcnj8 gene ablation have fewer CD11b+CD27- and KLRG-1+ NK cells in the bone barrow and spleen. These results show that the KATP subunit Kir6.1 has a key role in NK-cell development. Splenocytes were isolated from wild-type mice, and Kir6.1flx/flx x NKp46-CreERT2 mice immediately following 4 days of tamoxifen induction (respectively WT and LKO; n=2 each). Cells were first gated for NK cell markers NK1.1 and NKp46, then sorted into four pools: CD27-/CD27b- (population 3), CD27+/CD27b- (population 2), CD27+/CD27b+ (population 1), and CD27-/CD27b+ (population 0).

自然杀伤（natural killer, NK）细胞参与免疫与生殖过程，为开发治疗感染、癌症或缓解妊娠并发症的新型免疫疗法提供了潜在契机。当前多数研究策略采用细胞因子或抗体增强NK细胞功能，但尚未有使用离子通道调节剂的方案——这类调节剂已广泛应用于临床，用于治疗高血压、糖尿病、癫痫等多种疾病。目前对于NK细胞中的离子通道所知甚少。
本研究证实，相较于此前在NK细胞中鉴定出的其他钾离子通道，编码某类ATP敏感性钾（ATP-sensitive potassium, KATP）通道Kir6.1亚基的Kcnj8基因，在小鼠脾脏与子宫NK细胞中呈高表达。Kcnj8的表达水平在最成熟的脾脏NK细胞亚群（CD27-/CD11b+）以及经教育的NKG2A+或Ly49C/I+子宫NK细胞中达到最高。通过膜片钳技术，我们发现部分NK细胞表达对Kir6.1阻断剂PNU-37883A敏感的电流。Kcnj8既不参与体外环境下NK细胞针对肿瘤细胞的脱颗粒反应，也不参与体内肿瘤细胞排斥过程，亦不参与γ-干扰素（IFN-γ）的释放。转录组学分析显示，此前被认为与NK细胞发育相关的基因，在Kcnj8缺陷的CD27-/CD11b+ NK细胞中存在差异表达。实际上，我们发现NK细胞特异性敲除Kcnj8基因的小鼠，其骨髓与脾脏中CD11b+CD27-及KLRG-1+ NK细胞的数量显著减少。上述结果表明，KATP通道亚基Kir6.1在NK细胞发育中发挥关键作用。
本研究从野生型小鼠以及他莫昔芬诱导4天后的Kir6.1flx/flx × NKp46-CreERT2小鼠中分离脾细胞（分别记为WT组与LKO组，每组n=2）。首先以NK细胞标志物NK1.1与NKp46进行流式门控，随后将细胞分为四个亚群：CD27-/CD27b-（群3）、CD27+/CD27b-（群2）、CD27+/CD27b+（群1）以及CD27-/CD27b+（群0）。