人组织 CXCR6+ NK细胞的表型功能鉴定及其体外扩增

自然杀伤细胞（Natural Killer，NK），作为固有免疫防御的重要细胞，在抵御病原体入侵和肿瘤监视中发挥重要作用。根据其生物学特性，NK细胞可分为外周血NK（Peripheral blood NK, PB-NK）细胞和组织定植NK （Tissue-resident, Tr-NK）细胞。其中，定植于人体肝脏和脾脏的Tr-NK细胞对于机体抗病原和抗肿瘤免疫具有重要意义。然而，当前针对肝脏定植NK（Liver-resident NK，Lr-NK）细胞和脾脏定植NK细胞（Spleen-resident NK，Sr-NK）的功能与特性研究较少。因此，本课题在符合伦理要求的情况下，获得移植供者肝脏灌洗液及脾脏组织，经样本处理后，得到表达CXCR6的Sr-NK细胞和Lr-NK细胞，结果显示：组织定植NK细胞高表达CD69和激活受体。在细胞因子刺激下，无论健康供者还是肝硬化患者，其CXCR6⁺ Tr-NK细胞杀伤能力均强于CXCR6^- NK细胞。

不同于T细胞，NK细胞低表达HLA抗原（Human leukocyte antigen, HLA），功能发挥不依赖抗原递呈，临床应用时副作用小，是理想的“即用式（off-the-shelf）”免疫细胞过继药物。因此，本课题联合应用无血清培养基与细胞因子，体外扩增Tr-NK细胞200倍以上；并分析鉴定了培养扩增前后Tr-NK细胞表面受体及相关分子标志物的表达差异。结果表明，体外扩增后的NK细胞高表达激活受体。比较扩增前后Tr-NK细胞对靶细胞系（K562）的杀伤能力及细胞毒性标志物，发现 Tr-NK细胞在扩增后保持高水平的靶细胞杀伤功能；通过构建体外肝脏类器官模型，我们揭示Tr-NK细胞具有良好的肝脏组织趋向性。

综上所述，该课题初步探究了脾脏和肝脏定植NK细胞的表型、功能及杀伤效力。开发了一种高效体外扩增Tr-NK细胞的技术，并初步鉴定了Tr-NK细胞扩增后的表型与功能，对Tr-NK细胞的基础免疫学及细胞治疗临床研究具有重要的技术指导意义。

Natural killer(Natural killer,NK)cells play critical roles against tumors and pathogens.NK cellsare classifiedinto peripheral blood NK(PB-NK)cells and tissue resident NK(Tr-NK)cells according to theirbiological characteristics.Especially,Tr-NK cells locatedin human liver and spleenare importantforlocalimmunity,andtreatment against pathogens and cancer.But thereisrareattentionon the immune functions and biological properties of liver-resident NK(Lr-NK)cells and spleen-resident NK(Sr-NK)cells.Using healthy liver perfusate,liver and spleensamples,weisolatedSr-NK cells and Lr-NK cells expressing CXCR6fromsamples.The result showedthat Tr-NK cells express high levels ofCD69andactivating receptors.In vitrostimulation
with cytokines,CXCR6+NKcellsweremore cytotoxic than CXCR6-NK cells independent on tissues and disease status.
ComparedwithTcells,NK cellsexpress lower levels ofhuman leukocyte antigen(HLA). NK cells have less potentials to present antigen and less side effects whenappliedin the clinic.Hence,NKcellsarethe most promising "off-the-shelf" immune cells foradoptive-therapy.Therefore,basedon the biological characteristics of Tr-NK,weexpanded more than200-fold NK cellsinthe incubationwithserum-freemediumand cytokines.The expandedTr-NK cells expressedhigher levels ofactivating receptor than thoseoffresh Tr-NK cells.In addition, the expanded Tr-NK cells maintained their cytotoxic activity.Utilizing theliverorganoid modelin vitro,we found thatTr-NK cellswere morechemotaxis to liverexvivo.
In conclusion,the study investigated the phenotype, function and biological characteristics ofTr-NK cells in the spleen and liver, andalso developed an efficientexpansionprotocolof Tr-NK cellsin vitro.Using these expanded Tr-NK cells,weidentified theirphenotypesand functions.These preliminary dataprovidedinsights on thebasic immunologyandclinical applicationof Tr-NK cells.

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