Clin Exp Vaccine Res.  2014 Jul;3(2):235-243. 10.7774/cevr.2014.3.2.235.

DNA immunization of Mycobacterium tuberculosis resuscitation-promoting factor B elicits polyfunctional CD8+ T cell responses

• Lee J
• Kim J
• Lee J
• Shin SJ
• Shin EC

• KMID: 1730629
• DOI: http://doi.org/10.7774/cevr.2014.3.2.235

Abstract

PURPOSE
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.
MATERIALS AND METHODS
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-gamma (IFN-gamma) enzyme-linked immunosorbent spot assay and intracellular cytokine staining (ICS), and T cell polyfunctionality was assessed from the ICS data.
RESULTS
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-gamma+/TNF-alpha+/IL-2-/CD107a+.
CONCLUSION
RpfB DNA immunization elicits polyfunctional CD8+ T cell responses, suggesting that RpfB DNA immunization might induce protective immunity against tuberculosis.

Keyword

Tuberculosis; Resuscitation-promoting factor B; Polyfunctional T cells; DNA vaccines

MeSH Terms

Animals
Complement Factor B*
Cytokines
DNA*
Epitopes, T-Lymphocyte
Immunity, Humoral
Immunization*
Interferon-gamma
Mice
Mycobacterium tuberculosis*
Peptides
Phenotype
Plasmids
T-Lymphocytes
Tuberculosis
Vaccines, DNA
Complement Factor B
Cytokines
DNA
Epitopes, T-Lymphocyte
Interferon-gamma
Peptides
Vaccines, DNA

Figure

• 

  Fig. 1 Resuscitation-promoting factor B (RpfB)-specific antibody response induced by RpfB immunization. Mice were immunized as described in Materials and Methods, and serum collected. The sera were serially diluted (1:5,000), and RpfB-specific Ig was measured by indirect enzyme-linked immunosorbent assay. BCG, bacillus Calmette-Guérin. The graph represents mean±SEM (n=6). Mann-Whitney U test was performed (**p<0.01).

• 

  Fig. 2 Identification of immunodominant epitope overlapping peptides (OLPs) recognized by T cells. Mice were immunized with resuscitation-promoting factor B (RpfB) plasmid DNA as described in Materials and Methods. Splenocytes were obtained from immunized mice, and CD8+ and CD4+ T cells were further isolated. Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays were performed with 500,000 total splenocytes per well (n=6) (A), 50,000 CD8+ T cells per well (n=3) (B), and 50,000 CD4+ T cells per well (n=3) (C). In IFN-γ ELISpot assays, cells were stimulated with each of 71 OLPs. SFU, spot forming units. Each bar graph represents mean±SEM.

• 

  Fig. 3 Resuscitation-promoting factor B (RpfB)-specific T cell responses induced by RpfB immunization. Mice were immunized as described in Materials and Methods, and splenocytes were obtained from immunized mice. Intracellular cytokine staining was performed for interferon-γ (IFN-γ) (A, B), tumor necrosis factor-α (TNF-α) (C, D), CD107a (E, F), and interleukin-2 (IL-2) (G, H) after stimulation with the indicated overlapping peptide (OLP) mix or epitope OLP. CD4+ (A, C, E, G) and CD8+ (B, D, F, H) T cell subsets were separately analyzed by gating on each subset during luorescence-activated cell sorting analysis. BCG, bacillus Calmette-Guérin. Each bar graph represents mean±SEM (n=6). Mann-Whitney U test was performed (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 4 Analysis of the polyfunctionality of resuscitation-promoting factor B (RpfB)-specific T cells induced by RpfB DNA immunization. Representative examples of the analysis of polyfunctionality of CD8+ T cells (A, B) and CD4+ T cells (C, D) from an RpfB DNA-immunized mouse are presented. Cells were stimulated with overlapping peptide (OLP) mix 1 for the analysis of CD8+ T cells (A, B), and with OLP mix 2 for the analysis of CD4+ T cells (C, D). Polyfunctionality for interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and CD107a was assessed. The data are represented by the pie graphs showing the fraction of T cells positive for a given number of functions (A, C). Detailed analysis of polyfunctionality is presented with every possible combination of functions (B, D).

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