Neisserial immunoglobulin A1 protease induces specific T-cell responses in humans

Abstract

We have previously shown that immunoglobulin A1 (IgA1) protease, an exoenzyme of pathogenic neisseriae, can trigger the release of proinflammatory cytokines from human monocytic subpopulations. Here, we demonstrate a dose-dependent T-cell response to recombinant gonococcal IgA1 protease (strain MS11) in healthy human blood donors. This response was delayed in comparison to the immune response against tetanus toxoid. Stimulation with IgA1 protease led to the activation of CD4(+) and CD8(+) T cells, as well as CD19(+) B cells and CD56(+) NK cells, indicated by de novo expression of CD69. Only CD4(+) T cells proliferated and stained positive for intracellular gamma interferon (IFN-gamma). Both proliferation and IFN-gamma production were dependent on antigen presentation via major histocompatibility complex class II. Peripheral blood mononuclear cells stimulated with IgA1 protease produce IFN-gamma and tumor necrosis factor alpha but no, or very low amounts of, interleukin-10 (IL-10) or IL-4, indicating a Th1-based proinflammatory immune response. These findings support the significance of IgA1 protease as a virulence determinant of bacterial meningitis and its function as a dominant proinflammatory T-cell antigen.

FIG. 1.

Proliferation of human PBMC in response to IgA1 protease (0.1, 1, 10, and 20 μg/ml), TT (2 LF/ml), and medium alone. PBMC from donors 14, 26, and 27 were cultured with the indicated stimuli for 4, 5, 7, and 10 days. Filled symbols, different concentrations of IgA1 protease used in the experiment; open symbols, controls. The results (means ± standard deviations of six determinations) shown are from donor 27 as representative of the donors tested.

FIG. 2.

Proliferative responses to IgA1 protease of donors differ. The plot shows values from all three donors tested on day 10 of the experiment for which the results are shown in Fig. 1. Proliferation in response to IgA1 protease occurred at different concentrations of enzyme used, depending on the donor. Medium alone and TT (2 LF/ml) were used as controls (data not shown). Means ± standard deviations for six determinations are shown.

FIG. 3.

Specific proliferation of PBMC in response to IgA1 protease. To determine whether the proliferative response was IgA1 protease specific, BSA (10 μg/ml) and LPS (200 pg/ml) were used as controls along with medium alone, TT (2 LF/ml), and IgA1 protease (10 μg/ml). PBMC were cultured with the respective stimuli for 3, 5, and 7 days, and proliferation of cells was monitored by incorporation of [³H]thymidine. Results (means ± standard deviations for three determinations) shown here are from donor 14 as representative of the donors tested. The filled triangle accounts for the response to IgA1 protease, whereas the open symbols represent the stimuli which were used as controls.

FIG. 4

(A) IgA1 protease is associated with human CD14⁺ cells (monocytes). After isolation, PBMC were incubated with FITC-conjugated IgA1 protease (10 μg/ml) for 30 min at 37°C. The cells were washed and stained with anti-CD3 or anti-CD14 antibody to differentiate between cell subpopulations. The results for the interaction of IgA1 protease with monocytes (CD14⁺), T cells (CD3⁺) and non-T cells (CD3⁻) from donor 26 as representative of the donors tested are shown. (B) IgA1 protease colocalizes with hLAMP1 within lysosomal compartments of CD14⁺ monocytes. After isolation of PBMC from peripheral blood, monocytes were isolated. Cells were then incubated with FITC-labeled IgA1 protease and with anti-hLAMP1 antibody to mark the lysosomal compartments. The figure shows confocal images demonstrating the localization of IgA1 protease within lysosomal compartments of CD14⁺ monocytes (shown for donor 26 as representative of the donors tested). (I) IgA1 protease is shown in green; (II) lysosomal compartments are shown in red; (III) the overlay in yellow indicates colocalization.

FIG. 5.

IgA1 protease leads to an upregulation of CD69 on the cell surfaces of lymphocytes. Human PBMC were incubated with IgA1 protease (10 μg/ml) overnight. Nonstimulated cells and TT-stimulated cells (2 LF/ml) were used as controls. Cells were harvested and stained with a monoclonal antibody against the early activation marker CD69 on the cell surface of T lymphocytes (CD4⁺ or CD8⁺), B lymphocytes (CD19⁺), or NK cells (CD56⁺) for flow cytometry. The results (donor 26 as representative of the donors tested) shown are the percentages of CD69⁺ cells of each cell subpopulation investigated.

FIG. 6.

Purified IgA1 protease induces dose-dependent release of the cytokines IFN-γ, TNF-α, IL-10, and IL-4 from PBMC. PBMC from donors 14, 26, and 27 were cultured with different concentrations of IgA1 protease. Cell-free supernatants were harvested, and cytokine levels were determined by ELISA. Filled symbols, different concentrations of IgA1 protease used in the experiment; open symbols, control nonstimulated cells (medium) and TT (2 LF/ml)-stimulated cells. Results (means ± standard deviations for six determinations) for donor 27 as representative of the donors tested are shown.

FIG. 7.

(A) IFN-γ production can be detected intracellularly by flow cytometry in CD3⁺ lymphocytes after stimulation of human PBMC with IgA1 protease. Human PBMC were cultured with IgA1 protease (10 μg/ml) for different lengths of time. Nonstimulated cells and TT-stimulated cells (2 LF/ml) were used as controls. After 1, 2, 3, and 6 days, cells were harvested and stained with monoclonal antibodies against CD3 on the cell surface and against IFN-γ intracellularly. The results (donor 27 as representative of the donors tested) are the percentage of double-positive cells. (B) CD4⁺ T cells are mainly responsible for the IFN-γ produced upon incubation of human PBMC with IgA1 protease. In the same experiment for which the results are shown in panel A, cells were harvested and stained with monoclonal antibodies against lymphocytic surface markers and IFN-γ intracellularly, of T lymphocytes (CD4⁺ or CD8⁺), B lymphocytes (CD19⁺), or NK cells (CD56⁺), respectively, for flow cytometry. The results are the percentages of double-positive cells of each cell subpopulation investigated.

FIG. 7.

(A) IFN-γ production can be detected intracellularly by flow cytometry in CD3⁺ lymphocytes after stimulation of human PBMC with IgA1 protease. Human PBMC were cultured with IgA1 protease (10 μg/ml) for different lengths of time. Nonstimulated cells and TT-stimulated cells (2 LF/ml) were used as controls. After 1, 2, 3, and 6 days, cells were harvested and stained with monoclonal antibodies against CD3 on the cell surface and against IFN-γ intracellularly. The results (donor 27 as representative of the donors tested) are the percentage of double-positive cells. (B) CD4⁺ T cells are mainly responsible for the IFN-γ produced upon incubation of human PBMC with IgA1 protease. In the same experiment for which the results are shown in panel A, cells were harvested and stained with monoclonal antibodies against lymphocytic surface markers and IFN-γ intracellularly, of T lymphocytes (CD4⁺ or CD8⁺), B lymphocytes (CD19⁺), or NK cells (CD56⁺), respectively, for flow cytometry. The results are the percentages of double-positive cells of each cell subpopulation investigated.

FIG. 8.

Production of IFN-γ by CD4⁺ T cells is inhibited by blocking antigen presentation via MHC class II molecules. Human PBMC were cultured for 2 days with IgA1 protease (10 μg/ml) in the presence or absence of the monoclonal anti-MHC class II antibody L243 (10 μg/ml) or an isotype control (10 μg/ml). Unstimulated cells and TT-stimulated cells were treated in the same manner (data not shown). The results (donor 14 as representative of the donors tested) are the means ± standard deviations representing the number of spots from three wells.