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目的:探讨NKG2D配体在不同发育阶段树突状细胞(DC)表面的表达及其对自然杀伤(NK)细胞杀伤活性的影响。方法:用细胞因子(rhIL-4、rhGM-CSF、TNF-α)体外诱导培养单核细胞来源的未成熟树突状细胞(iDC)和成熟树突状细胞(mDC)并鉴定形态和表型,免疫磁珠法分离纯化NK细胞。流式细胞术(FCM)检测iDC和mDC表面NKG2D配体MICA/B、ULBP1-3的表达。用LDH释放法检测NK细胞对iDC和mDC的杀伤活性以及抗NKG2D单克隆抗体(mAb)阻断NK细胞后的杀伤活性。结果:培养的iDC和mDC具有典型的细胞形态和免疫表型特征。iDC表面表达MICA、MICB、ULBP1、ULBP3,表达率分别为(32·39±8·30)%、(17·75±3·40)%、(26·71±6·48)%、(38·37±6·89)%;mDC表面表达MICA、ULBP3,表达率分别为(7·82±2·67)%、(8·36±2·42)%,比iDC表面相应配体表达率低(P<0·01)。各效靶比NK细胞对iDC的杀伤活性均比对mDC的杀伤活性高,差异有统计学意义(P<0·01)。抗NKG2D mAb阻断NK细胞后对iDC杀伤活性比阻断前减弱(P<0·05);对mDC的杀伤活性与阻断前相比无统计学意义(P>0·05)。结论:NKG2D配体在iDC表面表达高,介导了NK细胞对iDC的杀伤,而对mDC的杀伤无影响,是NK细胞对iDC选择性高杀伤的分子机制之一。
Objective: To investigate the expression of NKG2D ligands on dendritic cells (DCs) at different developmental stages and their effects on the cytotoxic activity of natural killer (NK) cells. METHODS: Monocyte-derived immature dendritic cells (iDCs) and mature dendritic cells (mDCs) were cultured and induced in vitro by rhIL-4, rhGM-CSF and TNF-α, and the morphology and phenotype , Immunomagnetic beads method to separate and purify NK cells. Flow cytometry (FCM) was used to detect the expression of NKG2D ligands MICA / B and ULBP1-3 on the surface of iDC and mDC. The killing activity of NK cells against iDC and mDC and the killing activity of NK cells against NKG2D monoclonal antibody (mAb) were tested by LDH release assay. Results: The cultured iDC and mDC have the typical cell morphology and immunophenotypic characteristics. The expression rates of MICA, MICB, ULBP1 and ULBP3 on the surface of iDC were (32.39 ± 8.3)%, (17.53 ± 3.40)%, (26.71 ± 6.48)% and · 37 ± 6 · 89%). The expression rates of MICA and ULBP3 on mDC surface were (7 · 82 ± 2 · 67)% and (8 · 36 ± 2 · 42)%, respectively, Low (P <0.01). The cytotoxic activity of NK cells against iDC was higher than that of mDC on NK cells, the difference was statistically significant (P <0.01). The killing activity of iDC after anti-NKG2D mAb blocking NK cells was weaker than that before blocking (P <0.05). The killing activity of mDC on mDC was not statistically significant (P> 0.05). Conclusion: The high expression of NKG2D ligand on iDC surface mediates the killing of iDC by NK cells, but has no effect on the killing of mDC. It is one of the molecular mechanisms that NK cells selectively kill iDC.