Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta - PubMed (original) (raw)

Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta

K Nakamura et al. J Exp Med. 2001.

Abstract

CD4(+)CD25(+) T cells have been identified as a population of immunoregulatory T cells, which mediate suppression of CD4(+)CD25(-) T cells by cell-cell contact and not secretion of suppressor cytokines. In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4. As in prior studies, we found that CD4(+)CD25(+) T cells suppress proliferation of CD4(+)CD25(-) T cells; however, we observed here that such suppression is abolished by the presence of anti-TGF-beta. In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression. Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface. This, plus the fact that we could find no evidence that a soluble factor mediates suppression, strongly suggests that CD4(+)CD25(+) T cells exert immunosuppression by a cell-cell interaction involving cell surface TGF-beta1.

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Figures

Figure 1

Proliferation and cytokine production of CD4+CD25+ regulatory T cells are CD28 and IL-2 dependent. (A) 2.5 × 104 CD4+CD25+ or CD4+CD25− cells were stimulated with plate-bound anti-CD3 Ab (10 μg/ml) with or without soluble anti-CD28 Ab (2 μg/ml) and/or exogenous IL-2 (20 U/ml) in 96-well plates. Cell proliferation was measured by incorporation of [3H]thymidine after 72 h. The results shown represent the mean ± SEM of triplicate wells and representative of three independent experiments. (B–E) 105 CD4+CD25+ or CD4+CD25− cells were stimulated with plate-bound anti-CD3 Ab (10 μg/ml) with or without soluble anti-CD28 Ab (2 μg/ml) and/or exogenous IL-2 (20 U/ml) in 100 μl culture. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate, and are representative of three independent experiments. n.d., not detected, *: P < 0.00007, **: P < 0.0002, ***: P < 0.0009, ****: P < 0.0008. (F) 105 CD4+CD25+ or CD4+CD25− cells were stimulated with soluble anti-CD3 Ab (10 μg/ml), 2 × 105 irradiated non–T cells and IL-2 (20 U/ml) in 100 μl culture. 2.5% FCS/RPMI was used for culture media and TGF-β1 content in media was subtracted as a background. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate, and are representative of three independent experiments.

Figure 2

Expression of CTLA-4, CD80, and CD86 on CD4+CD25+ T cells. (A) CTLA-4 on/in splenocytes was stained as described in Materials and Methods. CD4+CD25+ and CD4+CD25− fractions were gated and expression of CTLA-4 on/in each population is shown. (B and C) Purified CD4+CD25+ and CD4+CD25− T cells were stimulated with anti-CD3 (10 μg/ml), irradiated non–T cells and IL-2 (20 U/ml) for 72 h and then stained with Cy-chrome™–conjugated anti-CD4 and either of PE-conjugated anti-CD80 or PE-conjugated anti-CD86. Expression of CD80 (B) and CD86 (C) in the CD4+ gate is shown.

Figure 3

Signaling through CTLA-4 enhances proliferation and TGF-β1 secretion of CD4+CD25+ T cells. (A) 2.5 × 104 CD4+CD25+ or CD4+CD25− T cells were stimulated with plate-bound anti-CD3 with or without soluble anti-CD28 (2 μg/ml), plate-bound anti–CTLA-4, or exogenous IL-2 (20 U/ml) in 96-well plates. Cell proliferation was measured by incorporation of [3H]thymidine after 72 h. Plates were coated either with anti-CD3 (10 μg/ml) plus anti–CTLA-4 (15 μg/ml) or anti-CD3 plus hamster IgG (15 μg/ml). The results shown are the mean ± SEM of triplicate wells and representative of three independent experiments. (B and C) 105 CD4+CD25+ or CD4+CD25− T cells were stimulated with plate-bound anti-CD3 Ab, soluble anti-CD28 Ab (2 μg/ml), and exogenous IL-2 (20 U/ml) with or without plate-bound anti–CTLA-4 in 100 μl of culture. The amount of TGF-β1 (B) and IL-10 (C) was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. Plates were coated either with anti-CD3 (10 μg/ml) plus anti–CTLA-4 (10 μg/ml) or anti-CD3 plus hamster IgG (10 μg/ml). The results shown are the mean ± SEM of triplicate wells with each well measured in duplicate, and are representative of three independent experiments. n.d., not detected, *: P < 0.005, **: P < 0.00003.

Figure 4

Suppression of T cell proliferation by CD4+CD25+ regulatory T cells is mediated by TGF-β. (A and B) 2.5 × 104 CD4+CD25− (black bars) or CD4+CD25+ T cells (hatched bars), or both (white bars) were stimulated with soluble anti-CD3 Ab (10 μg/ml) and 5 × 104 of irradiated syngenic non–T cells in 96-well plates in the presence of anti-cytokine or control IgG. (A) 50 μg/ml or 100 μg/ml of control mouse IgG or anti-TGF-β (1D11). (B) 100 μg/ml of control rat IgG, anti–IL-10, or anti–IL-10R. Cell proliferation was measured by incorporation of [3H]thymidine after 72 h. The results shown are the mean ± SEM of triplicate wells and are representative of four independent experiments. (C) 2.5 × 104 CD4+CD25− T cells were stimulated with soluble anti-CD3 Ab (10 μg/ml) and 5 × 104 of irradiated syngenic non–T cells in 96-well plates in the presence of various amounts of rTGF-β1 (active form). Cell proliferation was measured by incorporation of [3H]thymidine after 72 h. The results shown are the mean ± SEM of triplicate wells and are representative of three independent experiments.

Figure 5

CD4+CD25+ regulatory T cells suppress B cell Ig synthesis through TGF-β. (A) 5 × 104 CD4+CD25− or CD4+CD25+ T cells, plus 5 × 104 non–T cells were stimulated with 20 μg/ml of PWM and 20 U/ml of IL-2 in 200 μl culture. In some wells, CD4+CD25+ cells were added to 5 × 104 CD4+CD25− cells at the ratio indicated in the figure. Cells were cultured at 37°C for 8 d and culture supernatant was collected. The concentration of mouse IgG in the supernatant was determined by ELISA. The results shown are the mean ± SEM of triplicate wells and are representative of three independent experiments. (B) 5 × 104 CD4+CD25− (black bars), or 5 × 104 of both CD4+CD25+ and CD4+CD25− T cells (white bars) were cocultured with 5 × 104 non–T cells, and stimulated with 20 μg/ml of PWM and 20 U/ml of IL-2 in 200 μl culture. 20 μg/ml of control chicken IgG or chicken anti–TGF-β1 Ab, or 100 μg/ml of rat anti–IL-10 mAb was added to the culture. Cells were cultured at 37°C for 8 d and culture supernatant was collected. Concentration of mouse IgG in the supernatant was determined by ELISA. The results shown are the mean ± SEM of triplicate wells and representative of three independent experiments.

Figure 5

CD4+CD25+ regulatory T cells suppress B cell Ig synthesis through TGF-β. (A) 5 × 104 CD4+CD25− or CD4+CD25+ T cells, plus 5 × 104 non–T cells were stimulated with 20 μg/ml of PWM and 20 U/ml of IL-2 in 200 μl culture. In some wells, CD4+CD25+ cells were added to 5 × 104 CD4+CD25− cells at the ratio indicated in the figure. Cells were cultured at 37°C for 8 d and culture supernatant was collected. The concentration of mouse IgG in the supernatant was determined by ELISA. The results shown are the mean ± SEM of triplicate wells and are representative of three independent experiments. (B) 5 × 104 CD4+CD25− (black bars), or 5 × 104 of both CD4+CD25+ and CD4+CD25− T cells (white bars) were cocultured with 5 × 104 non–T cells, and stimulated with 20 μg/ml of PWM and 20 U/ml of IL-2 in 200 μl culture. 20 μg/ml of control chicken IgG or chicken anti–TGF-β1 Ab, or 100 μg/ml of rat anti–IL-10 mAb was added to the culture. Cells were cultured at 37°C for 8 d and culture supernatant was collected. Concentration of mouse IgG in the supernatant was determined by ELISA. The results shown are the mean ± SEM of triplicate wells and representative of three independent experiments.

Figure 6

CD4+CD25+ T cells express TGF-β1 on the cell surface. (A) Enriched CD4+ T cells were stained with FITC-conjugated anti-CD25, Cy-Chrome™–conjugated anti-CD4 and either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken IgG, washed, and stained with PE-conjugated streptavidin. CD4+CD25+ and CD4+CD25− T cells were gated and expression of cell surface-bound TGF-β1 is shown. Thick lines: anti–TGF-β, thin lines: normal chicken IgG. The results shown are the representative of three independent experiments. (B and C) Purified CD4+CD25+ and CD4+CD25− T cells were stimulated with soluble anti-CD3 (10 μg/ml) and irradiated non–T cells in the presence of IL-2 (20 U/ml) for 24 h (B) or 6 d (C). Cells were then stained with Cy-Chrome™–conjugated anti-CD4 and either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken IgG, washed and stained with PE-conjugated streptavidin. CD4+ cells were gated and expression of cell surface-bound TGF-β1 is shown. Thick lines: anti–TGF-β, thin lines: normal chicken IgG. The results shown are the representative of three independent experiments. (D) Graphic representation of the percentage of surface TGF-β1 positive cells in CD4+CD25+ (circle) and CD4+CD25− (triangle) T cells after stimulation.

Figure 7

CD4+CD25+ T cells express LAP of TGF-β1 but not IL-10 or IL-4 on the cell surface. Purified CD4+CD25+ and CD4+CD25− T cells were stimulated with soluble anti-CD3 (10 μg/ml) and irradiated non–T cells in the presence of IL-2 (20 U/ml) for 24 h. Cells were incubated with Cy-Chrome™–conjugated anti-CD4 and either anti-LAP mAB (27232.11) (A), PE-conjugated anti–IL-10 (B), or PE-conjugated anti–IL-4 (C). Incubation with isotype-matched control IgG for each anti-cytokine was performed in parallel. In panel A, after incubation with anti-LAP, cells were washed, incubated with biotin-conjugated anti–mouse IgG1, washed, and incubated with PE-conjugated streptavidin. CD4+ cells were gated and expression of LAP (A), IL-10 (B), and IL-4 (C) on the cell surface was shown. Thick lines: anti-cytokine; thin lines: isotype-matched control IgG.

Figure 8

Immunoblot analyses for the expression of TGF-β1 in CD4+CD25+ T cells. (A) Total cell lysates purified from 3 × 106 of activated CD4+CD25−, CD4+CD25+ and CD4+ T cells were run in SDS/PAGE and blotted to the membrane. 5 ng of active and latent rTGF-β1 were loaded in parallel to serve as positive controls. Membrane was first probed using chicken anti–TGF-β1, stripped, and reprobed with anti-actin Ab. (B) Lysates from membrane (derived from 2.5 × 107 cells) and cytoplasmic (derived from 1.25 × 107 cells) preparations of activated CD4+CD25− and CD4+CD25+ T cells were subjected to SDS/PAGE and immunoblot analysis. Membrane was probed using chicken anti–TGF-β1.

Figure 9

Expression of surface TGF-β1 after the stimulation with plate-bound anti-CD3 and anti–CTLA-4, soluble anti-CD28, and IL-2. (A) Purified CD4+CD25+ and CD4+CD25− T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). Cultures were carried out in 1% Nutridoma/RPMI. 24 h later, cells were incubated with Cy-Chrome™–conjugated anti-CD4 and either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken IgG, washed, and stained with PE-conjugated streptavidin. CD4+ cells were gated and expression of cell surface-bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B) Purified CD4+CD25+ T cells were stimulated with soluble anti-CD3 (10 μg/ml), non–T cells and IL-2 (20 U/ml) (left panel), or with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml) (right panel). 60 h later, cells were incubated with Cy-Chrome™–conjugated anti-CD4 and either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken IgG, washed, and stained with PE-conjugated streptavidin. CD4+ cells were gated and expression of cell surface-bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (C) Purified CD4+CD25+ T cells were stimulated with soluble anti-CD3 (10 μg/ml), non–T cells and IL-2 (20 U/ml) (condition 1) or with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml) (condition 2) for 4 d and irradiated (1,000 rad). 2.5 × 104 CD4+ T cells were mixed with 2.5 × 104 CD4+CD25+ T cells stimulated in condition 1 or condition 2, and stimulated with soluble anti-CD3 Ab (10 μg/ml) and 5 × 104 of irradiated syngenic non–T cells in 96-well plates. Cell proliferation was measured by incorporation of [3H]thymidine after 72 h. The results shown are the mean ± SEM of triplicate wells and are representative of two independent experiments.

Figure 10

Comparison of cytokine expression among CD25+, CD25−CD45RBlow and CD25−CD45RBhigh populations of CD4+ T cells. (A) Purified CD4+CD25+, CD4+CD25− CD45RBlow and CD4+CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). 24 h later, cells were incubated with either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken Ig G, washed, and stained with Cy-Chrome™–conjugated streptavidin. Expression of cell surface–bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B–E) 105 CD4+ CD25+, CD4+ CD25−CD45RBlow and CD4+ CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml) and IL-2 (20 U/ml) in 100 μl culture for 72 h. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate. n.d., not detected.

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