Proliferation of CD4 and CD8 T lymphocytes obtained after pcDNA3-HSP65 immunization. (A-B) C57BL/6 wild-type (WT) mice were immunized three times with 100 μg of naked pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-HSP65)/mouse in fifteen days interval. Fifteen (A) or thirty days (B) after the last immunization, the spleens were cultured in the presence of peritoneal macrophages and 50 μg of recombinant Hsp5 for three days to re-stimulate Hsp65 specific cells. CD8 and CD4 T cells were sorted and incubated for three days with different antigen presenting cells that had been previously electroporated with pcDNA3, pcDNA3-Hsp65 or a mock control (medium). The proliferation was measured by CFSE staining dilution by flow cytometry and the mock control values were subtracted from the mock vector and pcDNA3-Hsp65 ones. Assays were performed in triplicate and the results represent the mean ± SD of at least two independent experiments. *P < 0,05 versus other stimuli (One-way ANOVA with Tukey's post-test). (C-D) Gene profile of GATA3, T-bet and Foxp3 from total RNA of both population were evaluated by real-time PCR 15 (C) and 30 (D) days after the last dose of the vaccine and incubated for three days with B cells as previously described. The level of expression was calculated using the mock cell as an internal control. The data is representative of 2 independent experiments.