Phosphoantigen presentation by macrophages to mycobacterium tuberculosis--reactive Vgamma9Vdelta2+ T cells: modulation by chloroquine

Abstract

Vgamma9Vdelta2+ T cells (gammadelta T cells) are activated by Mycobacterium tuberculosis and recognize mycobacterial nonpeptide phosphoantigens. The role of antigen-presenting cells in the processing and presentation of phosphoantigens to Vgamma9Vdelta2+ T cells is not understood. We analyzed the role of macrophages for activation of gammadelta T cells by a new synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) and M. tuberculosis. Macrophages greatly increased gammadelta T-cell activation by both BrHPP and M. tuberculosis. Fixation of macrophages before infection demonstrated that uptake of M. tuberculosis was required for presentation to gammadelta T cells. Antigens of M. tuberculosis remained stably associated with macrophage surface and were not removed by paraformaldehyde fixation or washing. Macrophages processed M. tuberculosis for gammadelta T cells through a brefeldin A-insensitive pathway, suggesting that transport through the endoplasmic reticulum and Golgi complex of a putative presenting molecule is not important in the early processing of M. tuberculosis antigens for gammadelta T cells. Processing of M. tuberculosis was not eliminated by chloroquine, indicating that processing of gammadelta antigens is not dependent on acidic pH in the lysosomes. Chloroquine treatment of BrHPP-pulsed macrophages increased activation of gammadelta T cells. Ammonium chloride treatment of macrophages did not increase reactivity of gammadelta T cells to BrHPP, indicating that the effect of chloroquine was independent of pH changes in endosomes. Chloroquine, by inhibiting membrane traffic, may increase association and retention of phosphoantigens with cell surface membrane molecules on macrophages.

FIG. 1.

BrHPP expands γδ T cells from peripheral blood and induces proliferation and IFN-γ secretion in the presence of IL-2. (A) PBMC from PPD⁺ donors were stimulated with live M. tuberculosis bacilli (MTB) at a 10:1 ratio of bacteria to macrophage or the indicated concentrations of IPP or BrHPP in the presence of IL-2 (50 U/ml). After 10 days, viable cells were harvested, counted, and analyzed by two-color flow cytometry for CD3, CD4, CD8, and γδ TCR expression. Results (mean value + standard error of the mean [error bars]) are expressed as the absolute number of γδ TCR⁺ T cells after 10 days in 6 ml of cell culture. (B and C) PBMC were stimulated for 5 days with BrHPP or IPP in the presence or absence of exogenous IL-2. Proliferation (B) and IFN-γ secretion in culture supernatants (C) were determined. Shown are the mean values + standard errors (error bars) from triplicates in an experiment representative of three.

FIG. 2.

Accessory cells enhance γδ T-cell responses to M. tuberculosis and phosphoantigens. Prestimulated and positively selected γδ T cells (5 × 10⁴ cells/well) from different donors were restimulated with M. tuberculosis (MTB) at the indicated bacterium/cell ratio) or BrHPP (10 nM to 1 μm) in the presence (black bars) or absence (gray bars) of autologous macrophages (10⁵ cells/well). (A) Proliferation is expressed as stimulation index (counts per minute of stimulated cultures/counts per minute of unstimulated cultures). (B and C) IFN-γ was measured in 48-h culture supernatants by ELISA. One representative experiment of four is shown.

FIG. 3.

Up-regulation by macrophages of γδ T-cell responses is not MHC restricted. Prestimulated and positively selected γδ T cells (5 × 10⁴ cells/well) from different donors were restimulated with M. tuberculosis (MTB) (A), M. tuberculosis lysate (B), BrHPP (C), or IPP (D) in the presence (+ APC) or absence (No APC) of mismatched THP-1 macrophages (10⁵ cells/well). IFN-γ levels in 48-h culture supernatants were determined by ELISA. Shown are the mean values of an experiment representative of three.

FIG. 4.

Effect of fixation of macrophages on their ability to present M. tuberculosis and phosphoantigens to γδ T cells. Macrophages (1.5 × 10⁵ cells/well) were fixed before or after M. tuberculosis (MTB) infection (MOI = 10:1), treatment with M. tuberculosis lysate (dilution 1:20), or BrHPP (10 μM) pulse. Antigens were washed away, and macrophages were used to stimulate γδ T-cell lines (5 × 10⁴ cells/well). IFN-γ was measured (ELISA) in 48 h-culture supernatants. Mean values + standard errors (error bars) of one representative experiment of three are shown.

FIG. 5.

Effect of BFA and chloroquine on M. tuberculosis and phosphoantigen-processing by macrophages. Macrophages (1.5 × 10⁵ cells/well) were pretreated with BFA (5 μg/ml) or chloroquine (50 μM) for 30 min and either infected with M. tuberculosis (MTB) (MOI = 10:1), pulsed with BrHPP (1 μM), or incubated with medium (None). After 4 h, antigens were washed away, and macrophages fixed and used to stimulate γδ T cells (5 × 10⁴ cells/well). IFN-γ was measured (ELISA) in 48-h culture supernatants. Mean values + standard errors (error bars) of one representative experiment out of four are shown.

FIG. 6.

Dose dependence of chloroquine effect on phosphoantigen presentation by macrophages to γδ T cells. Macrophages (1.5 × 10⁵ cells/well) were treated with chloroquine and pulsed with BrHPP (A) or IPP (B) for 4 h. Macrophages were washed, fixed, and used to stimulate γδ T-cell lines (5 × 10⁴ cells/well). Proliferation (A) and IFN-γ production (B) were determined in 72-h cultures. Mean value ± standard errors (error bars) of a representative experiment of four is shown.

FIG. 7.

Effect of chloroquine on M. tuberculosis and soluble antigen presentation by macrophages to γδ and CD4⁺ T cells. Macrophages (1.5 × 10⁵ cells/well) were treated with indicated concentration of chloroquine and pulsed with indicated concentrations of M. tuberculosis, BrHPP or PPD for 4 h. Macrophages were washed, fixed and used to stimulate positively selected γδ (A and B) or CD4⁺ T cells (5 × 10⁴ cells/well) (C). Culture supernatants were collected after 48 h, and ELISA was used to measure IFN-γ. Shown are mean values + standard errors of a representative experiment.

FIG. 8.

Comparison of chloroquine and ammonium chloride on BrHPP presentation by macrophages to γδ T cells. Macrophages (1.5 × 10⁵ cells/well) were treated with the indicated concentration of chloroquine (A) or ammonium chloride (NH₄Cl) (B) and pulsed with different concentrations of BrHPP for 4 h. Macrophages were washed, fixed, and used to stimulate positively selected γδ T-cell lines (5 × 10⁴ cells/well). After 72 h, cultures were pulsed with [³H]thymidine and the number of counts per minute was determined by liquid scintillation counting. Shown are mean values + standard errors (error bars) for an experiment representative of three.