CFSE and AnnexinV double staining in assessing lethal effect of cytotoxic T lymphocyte

Objective To establish a novel flow cytometry assay for lethal effect of cytotoxic T lymphocyte (CTL) using CFSE and AnnexinV double staining. Methods CD8+ CTL cells and spleen dendritic cells (sDCs) of OT- Ⅰ T cell receptor (TCR) from transgenic mice were selected by magnetic beads. After these two types of cells were cultured with ovalbumin (OVA) peptide for 65 hours, cell division and proliferation, expression features of intracellular IFN-γand IL-4, perforin, granzyme effector molecules were analyzed, and CD8+ CTL cells with lethal effect were obtained. Pre-activated mice CD8+ CTL cells were used as effector cells, and pre-CFSE-labeled splenocytes derived from allogenic mice as target cells. Specific CTL effects were analyzed in vitro and in vivo by AnnexinV staining, results of which were compared with those of classic CTL lethal effect detection method, I. E. CFSEhigh and CFSElow for labeling target cells in vivo.Results At the beginning of OT- Ⅰ , 4 division peaks were observed after CD8 + CTL cells activation cultured in vitro, with 54.1% CTL cells of IFN-γexpression, and 0.78% cells of IL-4 expression. Perforin and granzyme these two CTL effector molecules were positive expression. CD8 + CTL cells had been CTL killing activity. In in vitro assay, more AnnexinV-pesitive OVA+ target cells were identified under co-culture than under separation culture [ (62.4±3.5)% vs (28.3±2.2)%, P＜0.01 ], while no statistical difference was shown in percentage of AnnexinV-positive OVA- target cells under different cultures [ (20.3±4.8)% vs ( 17.3±2.9)%, P＞0.05]; under co-culture, more AnnexinV-positive OVA+ cells were identified than AnnexinV-positive OVA- cells (P＜0.01), but no statistical difference was revealed under separation culture (P＞0.05).Antigen dependency and partial cell-to-cell contact dependency were observed in lethal effect mediated by activated CD8+ CTL cell. In the CTL experiment in vivo, the killing rate of target cell by CFSE and AnnexinV double staining was higher than that of control group, in which target cells were not incubated in OVA peptide [ (52.63±8.12)% vs (13.84±4.37) %, P＜0.01 ]. By CFSEhigh and CFSElow double staining under same condition, killing rate of 41% was detected. Conclusion CFSE and AnnexinV double labeling method has fair comparability with CFSEhigh and CFSElow labeling in detecting CTL lethal effect, and may provide a new choice for detecting cytotoxicity with flow cytometry. Key words: T- Lymphocytes, cytotoxic; Flow cytometry; CFSE; AnnexinV; Cytotoxicity tests, immunologic