Immunostimulatory activity of major membrane protein II from Mycobacterium tuberculosis

Abstract

Previously, we observed that both major membrane protein II of Mycobacterium leprae (MMP-ML) and its fusion with M. bovis BCG (BCG)-derived heat shock protein 70 (HSP70) (Fusion-ML) are immunogenic and that recombinant BCG secreting either of these proteins effectively inhibits the multiplication of M. leprae in mice. Here, we purified M. tuberculosis-derived major membrane protein II (MMP-MTB) and its fusion with HSP70 (Fusion-MTB) in a lipopolysaccharide-free condition and evaluated their immunostimulatory abilities. Both MMP-MTB and Fusion-MTB activated monocyte-derived dendritic cells (DC) in terms of phenotype and interleukin-12 (IL-12) production, but Fusion-MTB more efficiently activated them than MMP-MTB did. The IL-12 production was a consequence of the ligation of those recombinant proteins with Toll-like receptor 2. The M. tuberculosis-derived and M. leprae-derived recombinant proteins activated naïve T cells of both CD4 and CD8 subsets, but M. tuberculosis-derived proteins were superior to M. leprae-derived proteins and fusion proteins were superior to MMP, regardless of the origin of the protein. Memory-type CD4(+) T cells obtained from BCG-vaccinated healthy individuals seem to be primed with MMP-MTB by the vaccination, and both M. tuberculosis-derived recombinant proteins produced perforin-producing CD8(+) T cells from memory-type CD8(+) T cells. Further, infection of DC and macrophages with M. tuberculosis H37Ra and H37Rv induced the expression of MMP on their surface. These results indicate that M. tuberculosis-derived MMP, as a sole protein or as part of a fusion protein, may be useful for developing new vaccinating agents against tuberculosis.

FIG. 1.

Expression of APC-associated molecules and MMP on DC by stimulation with recombinant proteins. Immature DC obtained from monocytes in the presence of rGM-CSF and rIL-4 were pulsed with either MMP-MTB or Fusion-MTB at 10 μg/ml on day 4 of culture. The DC were gated and analyzed on day 6 after the start of culture. Dotted lines, isotype-matched control IgG or IgM (for MMP); solid lines, the indicated test MAb. Representative results of three separate experiments are shown. The value in the top right corner of each graph is the mean fluorescence intensity of three independent experiments with a control Ig or the test MAb ± the standard deviation. Titers were statistically compared using Student's t test.

FIG. 2.

IL-12p40 production by DC stimulated with recombinant proteins. Monocyte-derived DC from 5 days of culture in the presence of rGM-CSF and rIL-4 were stimulated with the indicated recombinant protein at 10 μg/ml for 24 h. In some cases, immature DC were pretreated with normal murine IgG or TLR2-antagonistic Ab (10 μg/ml) and subsequently stimulated with recombinant protein for 24 h. The concentration of IL-12p40 was determined by the ELISA method. A representative of three separate experiments is shown. Assays were performed in triplicate, and the results are expressed as the mean ± the standard deviation. Titers were statistically compared by Student's t test.

FIG. 3.

(A) IFN-γ production by memory-type CD4⁺ T cells stimulated with recombinant proteins. Monocyte-derived DC or macrophages were pulsed with the indicated recombinant protein at the indicated concentration and used to stimulate memory-type CD4⁺ T cells in a 4-day culture. Responder CD4⁺ T cells (1 × 10⁵) were stimulated with the indicated dose of Ag-pulsed DC or macrophages. (B) IFN-γ production by naïve CD4⁺ T cells by stimulation with recombinant protein. Monocyte-derived DC were pulsed with the indicated recombinant protein at 5 or 10 μg/ml and used to stimulate naïve CD4⁺ T cells in a 4-day culture. Responder CD4⁺ T cells (1 × 10⁵) were stimulated with the Ag-pulsed DC at a T cell/DC ratio of 10:1. (C) Inhibition of naïve CD4⁺ T cell activation by treatment of Ag-pulsed DC with MAb. Monocyte-derived DC were pulsed with Fusion-MTB at 10 μg/ml and subsequently treated at 10 μg/ml with MAb to HLA-DR, CD86, MMP, or normal murine IgG or IgM. These DC were used to stimulate naïve CD4⁺ T cells (1 × 10⁵) at a T cell/DC ratio of 10:1. IFN-γ produced from T cells was measured by the ELISA method. A representative of three separate experiments is shown. Assays were performed in triplicate, and the results are expressed as means ± standard deviations. Titers were statistically compared by Student's t test.

FIG. 4.

(A) IFN-γ production by memory-type CD8⁺ T cells by stimulation with recombinant protein. Monocyte-derived DC were pulsed with MMP-MTB or Fusion-MTB at 10 or 20 μg/ml, costimulated with or without CD40L (1.0 μg/ml), and used to stimulate memory-type CD8⁺ T cells in a 4-day culture. Responder CD8⁺ T cells (1 × 10⁵) were stimulated with the Ag-pulsed DC at a T cell/DC ratio of 10:1. (B) IFN-γ production by naïve CD8⁺ T cells stimulated with recombinant proteins. Monocyte-derived DC were pulsed with the indicated recombinant protein at 10 or 20 μg/ml, further costimulated with or without CD40L (1.0 μg/ml), and used to stimulate naïve CD8⁺ T cells in a 4-day culture. Responder CD8⁺ T cells (1 × 10⁵) were stimulated with the Ag-pulsed DC at a T cell/DC ratio of 10:1. (C) Inhibition of naïve CD8⁺ T cell activation by treatment of Fusion-MTB-pulsed DC with MAb. Monocyte-derived DC were pulsed with MMP-MTB at 20 μg/ml, costimulated with CD40L (1.0 μg/ml), and subsequently treated at 10 μg/ml with MAb to HLA-ABC, CD86, or normal murine IgG. These DC were used to stimulate naïve CD8⁺ T cells (1 × 10⁵) at a T cell/DC ratio of 10:1. IFN-γ produced by T cells was measured by the ELISA method. A representative of three separate experiments is shown. Assays were performed in triplicate, and the results are expressed as means ± standard deviations. Titers were statistically compared by Student's t test. (D) Intracellular production of perforin by CD8⁺ T cells. Monocyte-derived DC were pulsed at 10 μg/ml with either MMP-MTB or Fusion-MTB and cultured with unseparated memory-type T cells (T cell/DC ratio, 40:1) for 5 days. The stimulated CD8⁺ T cells were gated and analyzed for perforin production. Values are the mean percentages of the CD8⁺ T cell population that were perforin positive in three independent experiments and the standard deviations. Titers were statistically compared using Student's t test. A representative of three separate experiments is shown.

FIG. 5.

Expression of MMP on DC and macrophage infected with M. tuberculosis. Monocyte-derived DC or macrophages were infected with either H37Ra or H37Rv at an MOI of 1.0 and cultured for another 2 days in the presence of rGM-CSF plus rIL-4 or rM-CSF, respectively. The DC and macrophages were gated and analyzed on day 5 after the start of culture. Dotted lines, control IgM; solid lines, MMP MAb. Results representative of three separate experiments are shown. The values are the mean percentages of major membrane protein II-positive DC or macrophages in three independent experiments and the standard deviations. Titers were statistically compared using Student's t test.