Figure 2. Sequence-specific shRNA targeting LMP1 retains the nasopharyngeal carcinoma (NPC)-LMP1 immunogenicity and provokes T cell immunity. A, Schematic illustration of the generation of NPC-LMP1-antigen-specific cytotoxic T-lymphocyte (CTLs) from peripheral blood mononuclear cells (PBMCs) of healthy donors by co-culturing with the irradiated TW03-LMP1 cells under the treatment of shcontrol, LMP1-shRNA1, and LMP1-shRNA2 in interleukin (IL)-2 medium for 14 days and re-stimulated with the irradiated TW03-LMP1 cells at day 7. B, In vitro cytotoxicity measurement of NPC-LMP1-antigen-specific CTLs induced by the above indicated condition against TW03-LMP1 cells at different ratios for 6 h. The killing capacity of CTLs was determined via LDH Cytotoxicity Assay Kit. C and D, NPC-LMP1-antigen-specific CTLs induced by the above condition co-cultured with TW03-LMP1 cells for 4 h and harvested for FACS staining. The FACS plot (C) and the statistical graph (D) show the releasing level of interferon (IFN)-γ and granzyme (GrB) from CD8+ T cells. Representative FACS plots are shown from 1 of 3 independent experiments. E, Cytotoxicity measurement of NPC-LMP1-antigen-specific CTLs induced by the above condition against TW03-LMP1 cells at 50:1 and 100:1 ratios in the presence of MHC-class I blocking antibody (W6/32). F and G, The NPC-LMP1-antigen-specific CTLs induced by the above condition were co-cultured with TW03-LMP1 cells in the presence of MHC-class I blocking antibody (W6/32) for 4 h and harvested for FACS staining. The FACS plot (F) and statistical graph (G) of 1 of 3 independent experiments show the level of IFN-γ and granzyme (GrB) in CD8+ T cells. H and I, CD33+cells were isolated from healthy PBMCs using human CD33 microbeads and co-cultured with TW03-LMP1 cells under the treatment of shcontrol, LMP1-shRNA1, and LMP1-shRNA2 in a Transwell system for 48 h. The percentage of myeloid-derived suppressor cells (MDSCs) was measured by FACS staining. Representative density plots are shown as the CD33+CD11b+cells in the HLA-DR cell population (H) and statistical graph of the percentage of MDSCs induced by NPC-LMP1 cells under the indicated treatment (I) are shown from at least three independent experiments. Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test).