Burkholderia pseudomallei proteins presented by monocyte-derived dendritic cells stimulate human memory T cells in vitro

Abstract

Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4(+) T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8(+) T cells. Activation of antigen-specific CD4(+) T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

FIG. 1.

IFN-γ production by T-cell subsets from seropositive compared to seronegative healthy donors in response to moDCs pulsed with heat-killed B. pseudomallei (HkBp). Monocyte-derived DCs were pulsed with heat-killed B. pseudomallei or medium for 18 h and then cocultured with autologous CD4⁺ and CD8⁺ T cells (ratio of moDCs to T cells of 1:10). PHA-stimulated T cells were used as a positive control. Culture supernatants were collected at 48 h, and IFN-γ levels were measured by ELISA. The controls included medium as a negative control and PHA as a positive control for mitogen stimulation. ***, P < 0.001 by Mann-Whitney test between IFN-γ production of autologous CD4⁺ T cells in response to heat-killed B. pseudomallei-pulsed moDCs from seropositive (n = 9) and seronegative (n = 8) donors and between IFN-γ production of autologous CD4⁺ T cells and CD8⁺ T cells in response to heat-killed B. pseudomallei-pulsed moDCs from seropositive donors.

FIG. 2.

IFN-γ production of CD4⁺ T cells from seropositive versus seronegative healthy donors in response to purified B. pseudomallei proteins. The moDCs of seropositive and seronegative healthy donors were pulsed with heat-killed B. pseudomallei or its proteins for 18 h and then cocultured with CD4⁺ T cells. Culture supernatants were collected at 48 h, and IFN-γ levels in culture supernatants were measured by ELISA. (A) The negative-control set consisted of moDCs plus T cells, moDCs alone, and T cells alone that were cultured in medium. Positive controls included mitogen (PHA) and heat-killed B. pseudomallei. (B) Responses to purified proteins. **, P < 0.01; *, P < 0.05 (by Mann-Whitney test between IFN-γ production of autologous CD4⁺ T cells in response to antigen-pulsed moDCs from seropositive and seronegative donors).

FIG. 3.

IFN-γ response of CD4⁺ T cells from exposed healthy donors to heat-killed B. pseudomallei- or purified B. pseudomallei protein-pulsed moDCs requires cell contact. (A) CD4⁺ T cells isolated from seropositive healthy donors were cocultured with moDCs pulsed with heat-killed B. pseudomallei, LolC, or Rp2 in the presence (white bars) or absence (black bars) of polycarbonate insert wells to separate moDCs from T cells. The culture supernatants were collected, and IFN-γ levels were measured by ELISA. (B) Phenotypic flow cytometry analysis of intracellular IFN-γ-producing CD4⁺ T cells from a single seropositive healthy donor in response to moDCs pulsed with heat-killed B. pseudomallei and LolC. CD4⁺ T cells were stained with APC-anti-IFN-γ, FITC-anti-CCR7, and PE-anti-CD45RA prior to analysis by gating on lymphocyte cells (first row) and IFN-γ⁺ cells (second row). Isotype-matched controls were also included in the analysis (third row).

FIG. 4.

Granzyme B production from purified CD8⁺ T cells in response to moDCs pulsed with heat-killed B. pseudomallei or recombinant proteins. moDCs were pulsed with heat-killed B. pseudomallei or recombinant proteins for 18 h and then cocultured for 48 h with CD8⁺ T cells (ratio of moDCs to T cells of 1:10), and GrB-producing cells were enumerated by ELISPOT assay. (A) Numbers of GrB-producing CD8⁺ T cells with medium alone or in the presence of IL-12 and IL-15. (B) Numbers of GrB-producing CD8⁺ T cells in response to heat-killed B. pseudomallei or 10 purified proteins. SFC, spot-forming cells. *, P < 0.05 by Mann-Whitney test between GrB secretion of autologous CD8⁺ T cells in response to antigen-pulsed moDCs and that for the medium negative control (moDCs plus T cells) in the same panel.

FIG. 5.

Identification of immunogenic epitopes of B. pseudomallei Rp protein. PBMC were separated from leukocyte-rich samples from 6 donors. PBMC (5 × 10⁵ cells/ml) were incubated with each stimulator for 42 h. (A) The controls included medium as a negative control, PHA as a mitogen positive control, and LolC as an antigen-specific control. (B) Rp proteins (Rp1 and Rp2) and seven Rp peptides were used as antigen stimulators. IFN-γ-secreting cells were enumerated under a stereomicroscope. Each horizontal line denotes the mean, and error bars show the standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant (by Mann-Whitney test between IFN-γ spot-forming cells of individual antigen proteins/peptides and the medium control).