Screening Cytotoxic Defects With Flow Cytometric Detection Of CD107Α On Nk Cells And Cytotoxic T Lymphoclyte

Objective:1、To establish a novel fow cytometry-based assay formeasuring the expression of Lysosomal-Associated Membrane Protein1（LAMP-1，CD107α） on the cell surface of NK cells and Cytotoxic Tlymphoclyte (CTL).2、To evaluate the screening value of this assay for cytotoxicdefects-related diseases such as familial hemophagocytic syndrome (FHL).Methods:Three suspected Chediak-Higashi Syndrome (CHS) patients,three suspected FHL patients and10healthy children were enrolled in thestudy from October2010to June2011. Their PBMCs were separated andactivated overnight with IL-2. After the granule release of NK cells activatedby phytohemagglutinin (PHA) and CD8+T cells by Anti-CD3, the CD107αexpression were analyzed by fow cytometry. The peripheral blood DNAandRNA of the patients were extracted to analyze the pathogenic genes viaDNA-PCR/RT-PCR and direct sequencing.Results: The CD107α expression on CTL in the ten healthy childrensignificant increased after activation by Anti-CD3((0.18±0.07)%vs. (4.47±2.36)%，P<0.05) and NK cells after activation by PHA (0.27±0.07%vs.5.80±2.83%，P<0.05). The frequencyof CD107α-expression NK cells inthree suspected CHS after activation was significantly elevated whencompared with the healthy control (0.5%、0.6%vs.(5.80±2.83)%) exceptpatient2. After the anti-CD3activation, the frequency of CD107αexpression on CTL cells also showed no significant difference (0.3%、0.9%、0.2%vs.(4.47±2.36)%) in three patients. All of their MFI showed the sametrend. Patient1and3were identified LYST mutations （Patient1:c.5411-5414del TTTC, L1741fsX1758and c.7975C>T, R2596X; Patient3:c.4863G>A, R1563H and c.5392-5393delAA, E1739fsX1756.） There wasno mutation identified in the LYSTgene for patient2. CD107α expression ofNK cells and CTL in the suspected FHL patients and in mirror of thesefindings, no underling gene variation of PRF, MUNC13-4and STX11wereidentified.Conclusion： We raised a method to quantitatively assess cytotoxicityof the NK cells and CTLby measuring the expression of CD107α on the cellmembrane, which appeared to be an effective and rapid screening test forcytotoxic defects-related diseases such as FHL and other HLH secondaryto primary immunodeficiency.