Figure 2.

Evaluation of the OVA-specific cellular immune response and CTL killing activity. (A) Schematic representation of OT-I/II proliferation studies protocol. Purified CD8 or CD4 T cells from transgenic OTI and OTII mice were CFSE-labeled and adoptively transferred to C57BL/6 mice. The mice were treated 2 days later with 1 µg pTOP injection into the ear Pinna, followed by electroporation and sacrificed 4 days later to collect the draining lymph nodes for preparation of single-cell suspension and FACS analysis. (B) Quantification of the divided OTI and OTII cells is shown; n=5. (C) In vivo OVA-specific cytotoxic CD8 T cell killing assay protocol. C57BL/6 mice were first vaccinated three times by intramuscular electroporation of 1 µg pTOP every 2 weeks. Three weeks after the last vaccine administration, the mice received labeled splenocytes from naive mice that were pulsed with either SIINFEKL (the OVA peptide) or an irrelevant peptide (ie, a peptide that should not induce an immune response). Two days after transfer, splenocytes were collected and analyzed by FACS to determine the antigen-specific killing; n=5. (D) Percentage of the CTL killing activity for the different groups. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001 compared with untreated or to the specified group (n=5). ANOVA, analysis of variance; CTL, cytotoxic T cell; CFSE, carboxyfluorescein diacetate succinimidyl ester; FSC, forward scatter (cell diameter); SSC, side scatter (cell granulometry); OVA, ovalbumin; pTOP, plasmid to deliver T cell epitopes; pOVA, plasmid OVA.