Comparison of circulating CD4⁺ FoxP3⁺ regulatory T cell (Treg) frequencies in healthy donors and cancer patients. Peripheral blood mononuclear cells (PBMC) from healthy donors and cancer patients were stained with anti-CD4 fluorescein isothiocyanate (FITC), anti-CD3 peridinin chlorophyll protein (PerCP) and anti-FoxP3 phycoerythrin (PE) and analysed by flow cytometry. To determine the frequencies of Tregs, CD4/CD3 double-positive cells were gated and further analysed for FoxP3 expression. (a) Representative fluorescence-activated cell sorter (FACS) analysis of Tregs from one healthy donor and one cancer patient, showing 5% versus 11% Tregs in PBMC, respectively. (b) Percentages of CD4⁺ CD25⁺ FoxP3⁺ Tregs in CD4⁺ T cells from nine healthy donors (mean ratio: 5·4) and 113 cancer patients, with maintained (n= 36; mean ratio: 7·5) and impaired (n= 77; mean ratio: 7·1) phosphoantigen-induced proliferation of γδ T cells. (c) Ratio of Tregs to the phosphoantigen-sensitive Vδ2⁺ subset of γδ T cells in the peripheral blood of cancer patients with preserved (mean: 47·0) and impaired (mean: 8·3) phosphoantigen-induced γδ T-cell proliferation. Student’s t-test was used to determine statistical significance of detected differences. P<0·05 was considered significant.

Effects of purified regulatory T cells (Tregs) on γδ T-cell function. Fresh peripheral blood mononuclear cells (PBMC) were incubated with bromohydrinpyrophosphate (BrHPP, 1 μm) or medium alone and co-cultured with autologous purified interleukin (IL)-2-activated Tregs at ratio 1 : 1 for 24 hr and further analysed for (a) expression of activation marker CD69 or (b) interferon (IFN)-γ secretion. Each bar represents mean values ± standard deviation (SD) of triplicate cultures from one healthy donor and is representative of two independent experiments. (c) In a standard cytotoxicity assay, a BrHPP-induced γδ effector T-cell line was incubated with the Burkitt lymphoma cell line Daudi alone [effector: target (E:T) ratio = 25 : 1] or together with purified Tregs, or as a control with the CD4⁺ CD25⁻ fraction of PBMC (Treg or CD4⁺ CD25⁻ to PBMC ratio = 3 : 1 and E:T ratio = 25 : 1). The percentage cytotoxicity is expressed as percentage specific lysis ± SD of triplicate cultures from one representative donor.

Mechanisms of regulatory T cell (Treg)-mediated suppression of γδ T-cell proliferation. (a) Peripheral blood mononuclear cells (PBMC) were incubated with bromohydrinpyrophosphate (BrHPP, 1 μm) or medium alone and co-cultured with autologous purified Tregs separated by semi-permeable membranes (Transwells) or with supernatant (SN) from overnight-cultivated and interleukin (IL)-2-stimulated Tregs. (b) Supernatants were heated for 15 min at 56° or 99°, or treated with proteinase K (2 hr at 37°). (c) PBMC incubated with BrHPP were co-cultured with supernatant from overnight-cultivated Tregs alone or treated with 1-methyl-l-tryptophan (1·5 mm), N^G-monomethyl-l-arginine (l-NMMA; 2 mm) or N^ω-hydroxy-nor-l-arginine (nor-NOHA; 0·5 mm) at ratio 1 : 1 and proliferation of γδ T cells was analysed on day 7 by flow cytometry. Each bar represents mean values ± standard deviation of triplicate cultures from one healthy donor and is representative of four independent experiments.

Proliferative response of γδ T cells to phosphoantigen/interleukin (IL)-2. Peripheral blood mononuclear cells (PBMC) from cancer patients were incubated with phosphoantigen (1 μm bromohydrinpyrophosphate) and IL-2 (100 U/ml), harvested on day 7, stained with anti-pan γδ T-cell receptor (TCR) fluorescein isothiocyanate (FITC) and anti-CD3 phycoerythrin (PE) and analysed by flow cytometry. Fluorescence-activated cell sorter (FACS) analysis of γδ T cells from one representative cancer patient (no. 236) with maintained and one representative cancer patient (no. 004) with impaired phosphoantigen-induced proliferation showed 49% versus 1·1%γδ T cells in CD3⁺ T cells, respectively.

Inhibitory effects of purified regulatory T cells (Tregs) on phosphoantigen-induced γδ T-cell proliferation. (a) CD4⁺ CD25⁺ Tregs were isolated from peripheral blood mononuclear cells (PBMC) by using a FACSVantage cell sorter cytometer. After sorting, the CD4⁺ CD25⁺ fraction and the CD4⁺ CD25⁻ fraction were reanalysed to confirm enrichment and purity (88% versus 98%, respectively) as shown for one representative donor. (b) After depletion of αβ T cells a carboxyfluorescein succinimidyl ester (CFSE)-labelled γδ T-cell line was incubated with medium alone or bromohydrinpyrophosphate (BrHPP, 1 μm) and co-cultured with autologous purified Tregs at a ratio of 1 : 2·5. On day 6, cells were analysed by flow cytometry. γδ T-cell receptor (TCR)/CD3 double-positive T cells were gated, and loss of CFSE represented proliferation of gated cells. The percentage of proliferating cells is indicated for one representative donor. (c, d) PBMC were incubated with medium alone or BrHPP (1 μm) and co-cultured (c) with autologous purified Tregs at the indicated ratios or (d) with the CD4⁺ CD25⁻ T-cell fraction. On day 7, proliferation of γδ T cells was analysed by flow cytometry. (e) Stimulation of Tregs with phytohaemagglutinin (PHA; 2·5 μg/ml). (f) Preserved inhibition of phosphoantigen-induced γδ T-cell proliferation by Tregs after depletion of other PBMC populations [αβ T cells, B cells, natural killer (NK) cells and monocytes]. Representative results are shown after depletion of αβ T cells within PBMC. Similar results were obtained after depletion of B cells, NK cells or monocytes. After depletion of αβ T cells, 2 × 10⁴γδ T cell-enriched PBMC (25%γδ T cells in PBMC) were incubated with medium alone or BrHPP (1 μm) and co-cultured with autologous Tregs at a ratio of 1 : 1, which corresponds to a γδ T-cell:Treg ratio of 1 : 4. On day 7, proliferation of γδ T cells was analysed by flow cytometry, and absolute γδ T-cell numbers are shown. Each bar represents mean values ± standard deviation of triplicate cultures from one representative of at least three independent healthy donors.

Depletion of CD25⁺ cells restores the capacity of γδ T cells to respond to phosphoantigen. Peripheral blood mononuclear cells (PBMC) from cancer patients were stimulated with bromohydrinpyrophosphate (BrHPP; 0·014; 0·041; 0·123; 0·37 and 1·11 μm) and interleukin (IL)-2 (100 U/ml) before and after in vitro depletion of CD25⁺ T cells and proliferation of γδ T cells was analysed on day 7 by flow cytometry. Data shown represent mean values ± standard deviation of triplicate cultures from one donor representative of three cancer patients.

Effects of purified regulatory T cells (Tregs) on αβ T-cell proliferation. (a) Peripheral blood mononuclear cells (PBMC) from cytomegalovirus (CMV)-seropositive healthy donors were re-stimulated with CMV pp65-loaded mature dendritic cells (DCs) alone or co-cultured with autologous purified Tregs (ratio PBMC:Treg = 1 : 4) or supernatant (SN) from overnight-cultivated Tregs (ratio PBMC:Treg supernatant = 1 : 1). After 1 week, proliferation of CMV pp65-specific CD8⁺αβ T cells was determined by flow cytometry. Each bar represents mean values ± standard deviation of triplicate cultures from one representative donor. (b) In a mixed lymphocyte reaction (MLR), carboxyfluorescein succinimidyl ester (CFSE)-labelled PBMC were incubated with allogeneic immature dendritic cells (ratio 1 : 10) alone or together with supernatant from overnight-cultivated autologous purified Tregs (ratio PBMC:Treg supernatant = 1 : 1). On day 6, cells were analysed by flow cytometry. αβ T-cell receptor (TCR)/CD3 double-positive T cells were gated, and loss of CFSE represents proliferation of gated cells. One representative donor of three is shown.