Figure 5.

SMARRT Can Be Used to Express Cytokines, Resulting in Epitope Spreading to Weakly Immunogenic Tumor-Associated Antigens

SMARRT expressing hIL-7/15 fusion, mIL-7/15 fusion, mIL-7, mIL-15, mGM-CSF, and IL-12 were used to transfect BHK cells. (A) Supernatants were harvested 24 h post-electroporation and cytokine concentrations were measured using ELISA. SMARRT.Trp2 was used to immunize C57BL/6 mice on days 0 and 14. Spleens were harvested on day 21 and restimulated with the dominant CD8 epitope TRP2_180–188. (B and C) T cell function was measured by IFN-γ ELISpot (B) and intracellular cytokine staining (C). (D) Mice were immunized as described above with SMARRT.Trp2; in addition, mice were immunized with SMARRT.IL-12 at prime only, as indicated in the figure. The graph shows intracellular cytokine staining following stimulation with an overlapping peptide pool of TRP2 (Sub.) or TRP2_180–188 (Dom.). Graphs in (B)–(D) show mean with standard deviation, n = 5 mice per group. Statistical testing for (B) and (C) was done using Mann-Whitney U test. Statistical testing in (D) was done using ordinary one-way ANOVA to compare the total IFN-γ⁺ CD8 T cells, ∗p < 0.05; ∗∗p < 0.01.