CD4+ CD25(high) Foxp3+ regulatory T cells downregulate human Vdelta2+ T-lymphocyte function triggered by anti-CD3 or phosphoantigen

Abstract

Vdelta2+ T cells, the major circulating T-cell receptor-gammadelta-positive (TCR-gammadelta+) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vdelta2+ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4+ T cells. CD4+ CD25(high) Foxp3+ T cells (Tregs) comprise 5-10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-alphabeta+ lymphocytes, Tregs suppress Vdelta2+ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vdelta2+ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vdelta2+ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-gamma secretion by Vdelta2+ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vdelta2+ T-cell interferon-gamma responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vdelta2+ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vdelta2+ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vdelta2+ T-cell functional deficiencies observed in TB.

Figure 1

Depletion of CD25⁺ T cells increases Vδ2⁺ T-cell expansion in response to Mycobacterium tuberculosis. Peripheral blood mononuclear cells (PBMC) isolated from tuberculin-skin-test-positive (TST⁺) donors were not depleted (a) or CD25⁺ depleted with anti-CD25-labelled magnetic beads (b). Not depleted or CD25⁺-depleted PBMC (2 × 10⁶ cells/well) were stimulated for 7 days with or without whole M. tuberculosis (H37Ra, 0.2 × 10⁵ CFU/ml) (c). Total cell numbers were determined by the trypan blue exclusion method and the percentage of Vδ2⁺ T cells was determined by flow cytometry. Means ± SEM are shown for four experiments.

Figure 2

Purification and functional characterization of CD4⁺ CD25^high Foxp3⁺ T cells. (a) Peripheral blood mononuclear cells (PBMC) were labelled with anti-CD4 and anti-CD25 monoclonal antibodies and purified by fluorescence-activated cell sorting based on the level of CD25 expression. CD25^high gate was drawn around the events with > 10² mean fluorescence on the CD25 axis. Foxp3 expression was determined by intracellular staining in the sorted populations. (b) Flow-sorted CD4⁺ CD25⁻ and CD4⁺ CD25^high T cells were stimulated with plate-bound anti-CD3 monoclonal antibody plus interleukin-2. Interferon-γ (IFN-γ) was measured by enzyme-linked immunosorbent assay in 5-day culture supernatants. One representative experiment of three is shown.

Figure 3

CD4⁺ CD25^high T cells suppress polyclonally stimulated CD4⁺ CD25⁻ and Vδ2⁺ T cells. Fluorescence-activated cell sorted CD4⁺ CD25⁻ (a) and Vδ2⁺ T cells (b) (2.5 × 10⁴ cells/well) were stimulated with plate-bound anti-CD3 monoclonal antibody (10 μg/ml) and interleukin-2 (25 U/ml) in the presence or absence of CD4⁺ CD25^high regulatory T cells (Tregs) in a 1 : 1 effector to suppressor ratio. Tregs were also stimulated without effector cells. Cell culture supernatants were harvested on day 5 and interferon-γ (IFN-γ) was determined by enzyme-linked immunosorbent assay. Shown are mean values ± SEM of three independent experiments. (c) Percentage suppression of IFN-γ production was determined as follows: (IFN-γ in cultures with CD4⁺ CD25^high)/(IFN-γ in cultures without CD4⁺ CD25^high) × 100. Means ± SEM of three independent experiments are shown.

Figure 4

CD4⁺ CD25^high T cells suppress antigen-specific CD4⁺ CD25⁻ and Vδ2⁺ T-cell responses. (a) Fluorescence-activated cell sorting (FACS) sorted CD4⁺ CD25⁻ (2.5 × 10⁴ cells) isolated from tuberculin-skin-test-positive (TST⁺) donors were stimulated with purified protein derivatve (PPD; 10 μg/ml) in the presence or absence of CD4⁺ CD25^high T cells (Tregs) at a 1 : 1 effector : suppressor ratio. Interferon-γ (IFN-γ) in 5-day culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Mean values ± SEM of four independent experiments are shown. (b) FACS-sorted Vδ2⁺ T cells (2.5 × 10⁴ cells) isolated from TST⁺ donors were stimulated with BrHPP (10 μm) plus interleukin-2 (25 U/ml) and PPD (10 μg/ml) in the presence or absence of CD4⁺ CD25^high T cells (Tregs) at 1 : 1 effector : suppressor ratio. IFN-γ in 5-day culture supernatants was determined by ELISA. Mean values ± SEM of four independent experiments are shown. (c) Percentage suppression was determined as follows: (IFN-γ in cultures with CD4⁺ CD25^high)/(IFN-γ in cultures without CD4⁺ CD25^high) × 100. Means ± SEM are shown for four independent experiments.

Figure 5

CD4⁺ CD25^high T cells can suppress Vδ2⁺ T cells in the absence of TCR-αβ triggering. (a) Fluorescence-activated cell-sorted Vδ2⁺ T cells (2.5 × 10⁴ cells) isolated from tuberculin-skin-test-positive donors were stimulated with BrHPP (10 μm) plus interleukin-2 (IL-2; 25 U/ml) with or without purified protein derivative (PPD; 10 μg/ml) in the presence or absence of CD4⁺ CD25^high T cells (Tregs) at 1 : 1 effector : suppressor ratio. Interferon-γ (IFN-γ) in 5-day culture supernatants was determined by enzyme-linked immunosorbent assay. Mean values ± SEM are shown of triplicates of one representative experiment. (b) Percentage suppression was determined as follows: (IFN-γ in cultures with CD4⁺ CD25^high)/(IFN-γ in cultures without CD4⁺CD25^high) × 100. Means ± SEM of seven independent experiments are shown.