CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness

Abstract

CD4(+)CD25(+) regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4(+)CD25(-) T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact-dependent and cytokine independent. The role of TGF-beta1 in CD4(+)CD25(+) suppressor function remains unclear. While most studies have failed to reverse suppression with anti-transforming growth factor (TGF)-beta1 in vitro, one recent study has reported that CD4(+)CD25(+) T cells express cell surface TGF-beta1 and that suppression can be completely abrogated by high concentrations of anti-TGF-beta suggesting that cell-associated TGF-beta1 was the primary effector of CD4(+)CD25(+)-mediated suppression. Here, we have reevaluated the role of TGF-beta1 in CD4(+)CD25(+)-mediated suppression. Neutralization of TGF-beta1 with either monoclonal antibody (mAb) or soluble TGF-betaRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4(+)CD25(+) T cells. Responder T cells from Smad3(-/-) or dominant-negative TGF-beta type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-beta1-induced growth arrest, were as susceptible to CD4(+)CD25(+)-mediated suppression as T cells from wild-type mice. Furthermore, CD4(+)CD25(+) T cells from neonatal TGF-beta1(-/-) mice were as suppressive as CD4(+)CD25(+) from TGF-beta1(+/+) mice. Collectively, these results demonstrate that CD4(+)CD25(+) suppressor function can occur independently of TGF-beta1.

Figure 1.

TGF-β1 blockade does not reverse CD4⁺CD25⁺-mediated suppression of T cell proliferation. (A) WT CD4⁺ T cells (5 × 10⁴) were stimulated with irradiated T cell–depleted spleen cells (2 × 10⁵) either alone or with freshly isolated or activated (•) CD4⁺CD25⁺ T cells (1:2 suppressor/responder ratio) in the presence of titrated doses of anti–TGF-β1. Results from a representative experiment of three are shown. (B) OT-I Tg CD8⁺ T cells were stimulated with H-2K^b/OVA_257–264 soluble tetramer (0.1 μg/ml) either alone or with activated CD4⁺CD25⁺ T cells (1:2 suppressor/responder ratio) in the presence or absence of soluble TGF-βRII-Fc (10 μg/ml). In some instances, rhTGF-β1 was added as a control (5 ng/ml).

Figure 1.

TGF-β1 blockade does not reverse CD4⁺CD25⁺-mediated suppression of T cell proliferation. (A) WT CD4⁺ T cells (5 × 10⁴) were stimulated with irradiated T cell–depleted spleen cells (2 × 10⁵) either alone or with freshly isolated or activated (•) CD4⁺CD25⁺ T cells (1:2 suppressor/responder ratio) in the presence of titrated doses of anti–TGF-β1. Results from a representative experiment of three are shown. (B) OT-I Tg CD8⁺ T cells were stimulated with H-2K^b/OVA_257–264 soluble tetramer (0.1 μg/ml) either alone or with activated CD4⁺CD25⁺ T cells (1:2 suppressor/responder ratio) in the presence or absence of soluble TGF-βRII-Fc (10 μg/ml). In some instances, rhTGF-β1 was added as a control (5 ng/ml).

Figure 2.

CD4⁺ T cells from TGF-β1–insensitive Smad3^−/− mice are susceptible to suppression mediated by CD4⁺CD25⁺ T cells. (A) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous rhTGF-β1. (B) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated as described in A in the presence of titrated numbers of either activated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells. (C) Smad3-dependent signaling in CD4⁺CD25⁺ T cells is not required for suppressor effector function. WT CD4⁺ T cells were stimulated as described in A and in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from WT or Smad3^−/− mice. The final purity of sorted suppressors was >98%.

Figure 2.

CD4⁺ T cells from TGF-β1–insensitive Smad3^−/− mice are susceptible to suppression mediated by CD4⁺CD25⁺ T cells. (A) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous rhTGF-β1. (B) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated as described in A in the presence of titrated numbers of either activated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells. (C) Smad3-dependent signaling in CD4⁺CD25⁺ T cells is not required for suppressor effector function. WT CD4⁺ T cells were stimulated as described in A and in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from WT or Smad3^−/− mice. The final purity of sorted suppressors was >98%.

Figure 2.

CD4⁺ T cells from TGF-β1–insensitive Smad3^−/− mice are susceptible to suppression mediated by CD4⁺CD25⁺ T cells. (A) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous rhTGF-β1. (B) WT or Smad3^−/− CD4⁺ T cells (5 × 10⁴) were stimulated as described in A in the presence of titrated numbers of either activated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells. (C) Smad3-dependent signaling in CD4⁺CD25⁺ T cells is not required for suppressor effector function. WT CD4⁺ T cells were stimulated as described in A and in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from WT or Smad3^−/− mice. The final purity of sorted suppressors was >98%.

Figure 3.

CD4⁺CD25⁺ T cells suppress T cells from DNRIITg mice. (A) T cells from WT or DNRIITg mice (5 × 10⁴ cells) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous rhTGF-β1. (B) CD4⁺ or CD8⁺ T cells (5 × 10⁴) from WT or DNRIITg mice were stimulated as described in A in the presence or absence of either activated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells.

Figure 3.

CD4⁺CD25⁺ T cells suppress T cells from DNRIITg mice. (A) T cells from WT or DNRIITg mice (5 × 10⁴ cells) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous rhTGF-β1. (B) CD4⁺ or CD8⁺ T cells (5 × 10⁴) from WT or DNRIITg mice were stimulated as described in A in the presence or absence of either activated CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells.

Figure 4.

CD4⁺CD25⁺ regulatory T cell activity is operative in TGF-β1^−/− mice. (A) The frequency of CD4⁺CD25⁺ T cells is comparable in WT and TGF-β1^−/− neonatal mice. (B) WT or TGF-β1^−/− CD4⁺ CD25⁺ or CD4⁺CD25⁻ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous IL-2 (5 ng/ml). (C) WT CD4⁺ T cells were stimulated as described in B in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺ CD25⁻ T cells from WT or TGF-β1^−/− neonatal mice.

Figure 4.

CD4⁺CD25⁺ regulatory T cell activity is operative in TGF-β1^−/− mice. (A) The frequency of CD4⁺CD25⁺ T cells is comparable in WT and TGF-β1^−/− neonatal mice. (B) WT or TGF-β1^−/− CD4⁺ CD25⁺ or CD4⁺CD25⁻ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous IL-2 (5 ng/ml). (C) WT CD4⁺ T cells were stimulated as described in B in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺ CD25⁻ T cells from WT or TGF-β1^−/− neonatal mice.

Figure 4.

CD4⁺CD25⁺ regulatory T cell activity is operative in TGF-β1^−/− mice. (A) The frequency of CD4⁺CD25⁺ T cells is comparable in WT and TGF-β1^−/− neonatal mice. (B) WT or TGF-β1^−/− CD4⁺ CD25⁺ or CD4⁺CD25⁻ T cells (5 × 10⁴) were stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) in the presence or absence of exogenous IL-2 (5 ng/ml). (C) WT CD4⁺ T cells were stimulated as described in B in the presence of either freshly isolated CD4⁺CD25⁺ or CD4⁺ CD25⁻ T cells from WT or TGF-β1^−/− neonatal mice.

Figure 5.

In vitro depletion of CD4⁺CD25⁺ regulatory T cell activity in TGF-β1^−/− mice enhances T cell proliferation. CD25-depleted or nondepleted splenocytes/lymph node cells from either WT (A) or TGF-β1^−/− (B) neonatal mice were labeled with CFSE and stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) for a period of 48 h. Cells were then analyzed by flow cytometry for CFSE division status relative to unstimulated T cells (solid yellow line). All histograms were gated on CD4⁺ or CD8⁺ T cells. (C) CD25-depleted WT or TGF-β1^−/− cells were reconstituted with the respective nondepleted cell preparation, stimulated with soluble anti-CD3, and then examined the frequency of dividing cells based on CFSE dilution by flow cytometry. Cells were gated on CD4⁺ T cells.

Figure 5.

In vitro depletion of CD4⁺CD25⁺ regulatory T cell activity in TGF-β1^−/− mice enhances T cell proliferation. CD25-depleted or nondepleted splenocytes/lymph node cells from either WT (A) or TGF-β1^−/− (B) neonatal mice were labeled with CFSE and stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) for a period of 48 h. Cells were then analyzed by flow cytometry for CFSE division status relative to unstimulated T cells (solid yellow line). All histograms were gated on CD4⁺ or CD8⁺ T cells. (C) CD25-depleted WT or TGF-β1^−/− cells were reconstituted with the respective nondepleted cell preparation, stimulated with soluble anti-CD3, and then examined the frequency of dividing cells based on CFSE dilution by flow cytometry. Cells were gated on CD4⁺ T cells.

Figure 5.

In vitro depletion of CD4⁺CD25⁺ regulatory T cell activity in TGF-β1^−/− mice enhances T cell proliferation. CD25-depleted or nondepleted splenocytes/lymph node cells from either WT (A) or TGF-β1^−/− (B) neonatal mice were labeled with CFSE and stimulated with anti-CD3 (0.5 μg/ml) and irradiated, T cell–depleted spleen cells (2 × 10⁵) for a period of 48 h. Cells were then analyzed by flow cytometry for CFSE division status relative to unstimulated T cells (solid yellow line). All histograms were gated on CD4⁺ or CD8⁺ T cells. (C) CD25-depleted WT or TGF-β1^−/− cells were reconstituted with the respective nondepleted cell preparation, stimulated with soluble anti-CD3, and then examined the frequency of dividing cells based on CFSE dilution by flow cytometry. Cells were gated on CD4⁺ T cells.

Figure 6.

Anti–TGF-β does not reverse suppression of AIG mediated by CD4⁺CD25⁺ T cells. Nu/nu mice (6–8 wk) were injected with splenocytes depleted of CD25⁺ cells (20 × 10⁶ cells), as described in the Materials and Methods. Simultaneously, some nu/nu mice were coinjected with CD4⁺CD25⁺ T cells (2 × 10⁶ cells). Anti–TGF-β (2 mg) was injected on days –1 and 1 of cell transfer and weekly thereafter for the duration of the experiment. Stomachs were harvested 6 wk after transfer and H&E stains of formalin-fixed, paraffin-embedded sections were analyzed for gastric pathology.