T cell-mediated immune responses, and particularly activation of polyfunctional T cells that simultaneously produce multiple cytokines, are necessary for the control of Mycobacterium tuberculosis . In the present study, we examined if DNA immunization of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) elicits polyfunctional T cell responses in mice.

C57BL/6 mice were immunized intramuscularly three times, at 3-week intervals, with RpfB-expressing plasmid DNA. For comparison, protein immunization was performed with recombinant RpfB in control mice. After immunization, RpfB-specific T cell responses were assessed by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assay and intracellular cytokine staining (ICS), and T cell polyfunctionality was assessed from the ICS data.

RpfB DNA immunization induced not only humoral immune responses, but also CD8⁺ and CD4⁺ T cell responses. Immunodominant T-cell epitopes were identified within RpfB by assays with overlapping peptides. RpfB DNA immunization elicited a polyfunctional CD8⁺ T cell response that was dominated by a functional phenotype of IFN-γ⁺/TNF-α⁺/IL-2^-/CD107a⁺.

RpfB DNA immunization elicits polyfunctional CD8⁺ T cell responses, suggesting that RpfB DNA immunization might induce protective immunity against tuberculosis.