Defining the AHR-regulated transcriptome in NK cells reveal gene expression programs relevant to development and function

Aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays a critical role in shaping innate and adaptive immune responses. However, the molecular mechanism by which AHR regulates the function of human NK cells remains largely unknown. Here, we use RNA sequencing on IL21-expanded NK cells in the presence of AHR agonists and antagonist to demonstrate that AHR directly regulates the expression of known regulators of phenotype, development, metabolism, and function. NK cells were isolated from leukopak of healthy volunteer donors using NK cell RosetteSep. NK cells were expanded using irradiated feeder cells overexpressing membrane bound IL21 (FC21) for period of 14-21 days. NK cells from 3-donors (NK1, NK124 and NK126) were expanded using human interleukin-2 (IL2-50 IU/ml) alone, IL2+stemregenin (SR1-1uM) or IL2+kynurenine (Kyn-25uM). The media was changed every 2-3 days. The samples for RNA were collected on days 0, 7, 14 and 21 (D0, D7, D14 and D21). RNA was isolated using RNeasy kit all the samples were DNase treated and concentrated using RNA concentrator kit. We compared gene expression profiling between IL2 vs SR1 and IL2 vs Kyn on days 7 and 14.

芳烃受体（aryl hydrocarbon receptor, AHR）是一类配体激活型转录因子，在塑造先天与适应性免疫应答进程中发挥关键作用。然而，AHR调控人类自然杀伤（NK）细胞功能的分子机制仍在很大程度上尚不明确。本研究通过对经白细胞介素21（IL21）扩增、同时暴露于AHR激动剂与拮抗剂的NK细胞开展RNA测序，证实AHR可直接调控已知的与细胞表型、发育、代谢及功能相关调控因子的表达。

NK细胞从健康志愿者捐赠的白细胞分离液（leukopak）中分离，采用NK细胞RosetteSep分离试剂盒获取原代细胞；后续使用过表达膜结合型IL21（FC21）的辐照饲养细胞进行扩增，扩增周期为14至21天。另有3名供者来源的NK细胞（NK1、NK124与NK126），分别采用仅含人白细胞介素2（IL2，50 IU/ml）、IL2+ stemregenin（SR1，1μM）或IL2+犬尿氨酸（Kyn，25μM）的培养基进行扩增，培养基每2至3天更换一次。

分别在培养第0、7、14和21天（D0、D7、D14及D21）收集用于RNA测序的细胞样本。采用RNeasy试剂盒提取所有样本的RNA，经DNase处理后，使用RNA浓缩试剂盒进行浓缩。本研究对比了培养第7天与第14天时，IL2组分别与SR1组、Kyn组之间的基因表达谱差异。