Coexpression of MAPK and IRF3 constitutive activators with an antigen transgene modulates CD4 and CD8 immune responses to lentivector vaccination. (A) Schemes of the lentivector constructs and inactive MAPK activators are shown. Activators and their inactive mutants were cloned under the control of the SFFV promoter, while the IiOVA antigen was expressed from the ubiquitin (UBIQ) promoter. Inactivating mutations are indicated within each bar. Expression levels of each constitutive activator and inactive mutants were compared by FLAG immunoblot of 293T cells transduced with the lentivectors at a multiplicity of transduction of 2. Immunoblots were also probed with an OVA-specific antibody (α-OVA). (B) Groups of 6 mice were vaccinated with lentivectors encoding only IiOVA and dual lentivectors encoding IiOVA together with the MKK6EE (MKK6) and the inactive mutant MKK6 K82A (bottom of the graph), at the indicated doses. OVA-specific CD8 T cells in spleen were evaluated by IFN-γ ELISPOT and represented as column graphs. Each column shows the mean from 3 independent experiments together with error bars (SD). Relevant statistical comparisons are shown within each graph, between indicated samples and vaccination with lentivectors encoding only IiOVA. Significant (*P < .05) and highly significant (***P < .001) differences are shown in the graphs. Ns, nonsignificant differences. The rest of the graphs show vaccination results performed with lentivectors encoding MKK7-JNK1 and MKK7-JNK1 AA, MEK1 ΔNES ED and MEK1 ΔNES AA, or IRF3 2D and IRF3ΔC. (C) Groups of 9 mice were vaccinated with the indicated dual lentivectors encoding IiOVA together with each MAPK and IRF3 activator. OVA-specific MHC class I and class II primary responses in spleen were primed with CD8 and CD4 OVA epitope peptides and evaluated by IFN-γ ELISPOT and represented as column graphs. Each column represents the mean with error bars (SD). Relevant statistical comparisons are shown within each graph between indicated samples and unprimed mice vaccinated only with OVA protein. Ns indicates nonsignficant differences; ** very significant differences (P < .01); *** highly significant (P < .001) differences. Secondary immune responses were evaluated 1 month after vaccination (lentivector vaccines, bottom of the graphs) using 5 μg of OVA per mouse in Freund incomplete adjuvant (Boost, bottom of the graphs). – indicates absence of vaccination or boosting. (D) Groups of 3 mice were vaccinated with PBS or lentivector vaccines coexpressing IiOVA and the indicated activators on top of the panels. Dot plots of CD4⁺ CD25⁺ T lymphocytes from spleen are shown, with Foxp3 fluorescence intensity represented as a function of CD4 fluorescence on a logarithmic scale. Quadrants were established from isotype controls. Percentages of CD4⁺ CD25⁺ Foxp3⁺ T lymphocytes relative to CD4⁺ CD25⁺ T lymphocytes are shown within the upper right quadrant. Below the percentages, MFIs from Foxp3⁺ cells are shown. (E) The mean percentage of Tregs from mice immunized with the lentivector vaccines (bottom of the graph) is represented as a column graph (left), together with error bars (SD). The mean of Foxp3 expression levels (MFIs) within CD4⁺ CD25⁺ Foxp3⁺ T cells from mice immunized with the lentivector vaccines is represented as a column graph (right), together with error bars (SD). Selected statistical comparisons between indicated samples and the group vaccinated with lentivectors encoding only IiOVA are shown, and were performed as described in “Statistical analysis.” *, significant differences (P < .05); **, very significant differences (P < .01).