Figure 4. Heterologous RNA prime/protein boost vaccine strategy elicits robust ID91-specific CD4⁺ T_H1 immune responses post-boost.

Splenocytes were cultured from all six groups of mice one week post boost immunization and stimulated with ID91 or ID91 components (Rv1886, Rv3619, Rv3478, Rv2389) ex vivo and evaluated for CD4⁺ T cell responses by intracellular cytokine staining flow cytometry including: (A) Percent frequency of CD4⁺CD44⁺ ID91-specific single-cytokine-producing cells; (B) ID91-specific CD4⁺CD44⁺ polyfunctional T_H1 cytokine-producing cells; (C-F) Percent frequency of CD4⁺CD44⁺ Rv1886 (C), Rv3619 (D), Rv3478 (E), and Rv2389 (F) specific single-cytokine-producing cells. Bars show mean ± SEM, dots represent individual mice, n = 4/group. Asterisks indicate statistical significance, where **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test.