Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood

Abstract

A subpopulation of peripheral human CD4(+)CD25(+) T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte-associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4(+)CD25(+) T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-gamma on either protein or mRNA levels. The anergic state of CD4(+)CD25(+) T cells is not reversible by the addition of anti-CD28, anti-CTLA-4, anti-transforming growth factor beta, or anti-IL-10 antibody. However, the refractory state of CD4(+)CD25(+) T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties.

Figure 1

Phenotype of freshly isolated and stimulated CD4⁺ and CD4⁺CD25⁺ T cells. CD4⁺ and CD4⁺CD25⁺ T cells were isolated from buffy coats of healthy volunteers by positive selection using paramagnetic beads as described in Materials and Methods. (A) The figure shows the surface expression of freshly isolated T cells, representative of 10 independent experiments. (B) Phenotype of CD4⁺CD25⁺ T cells 10 d after stimulation with allogeneic DCs.

Figure 1

Phenotype of freshly isolated and stimulated CD4⁺ and CD4⁺CD25⁺ T cells. CD4⁺ and CD4⁺CD25⁺ T cells were isolated from buffy coats of healthy volunteers by positive selection using paramagnetic beads as described in Materials and Methods. (A) The figure shows the surface expression of freshly isolated T cells, representative of 10 independent experiments. (B) Phenotype of CD4⁺CD25⁺ T cells 10 d after stimulation with allogeneic DCs.

Figure 2

A subpopulation of CD4⁺CD25⁺ human anergic T cells shows constitutive expression of CTLA-4. (A) Surface and intracellular CTLA-4 expression of freshly isolated CD4⁺ and CD4⁺CD25⁺ T cells. (B) Surface expression of CTLA-4 16 h after stimulation with allogeneic DCs. The analysis is representative of seven independent experiments. (C) Proliferation assays of CD4⁺ and CD4⁺CD25⁺ T cells stimulated with varying numbers of allogeneic mature DCs. Proliferation of T cells was determined by the addition of [³H]Tdr after 4 (primary culture) or 2 d (restimulation) of culture for 16 h. Similar results were obtained in five independent experiments. (D) Cell cycle analysis of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells 4 d after the first restimulation with allogeneic DCs. Similar results were obtained in five independent experiments.

Figure 2

A subpopulation of CD4⁺CD25⁺ human anergic T cells shows constitutive expression of CTLA-4. (A) Surface and intracellular CTLA-4 expression of freshly isolated CD4⁺ and CD4⁺CD25⁺ T cells. (B) Surface expression of CTLA-4 16 h after stimulation with allogeneic DCs. The analysis is representative of seven independent experiments. (C) Proliferation assays of CD4⁺ and CD4⁺CD25⁺ T cells stimulated with varying numbers of allogeneic mature DCs. Proliferation of T cells was determined by the addition of [³H]Tdr after 4 (primary culture) or 2 d (restimulation) of culture for 16 h. Similar results were obtained in five independent experiments. (D) Cell cycle analysis of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells 4 d after the first restimulation with allogeneic DCs. Similar results were obtained in five independent experiments.

Figure 2

A subpopulation of CD4⁺CD25⁺ human anergic T cells shows constitutive expression of CTLA-4. (A) Surface and intracellular CTLA-4 expression of freshly isolated CD4⁺ and CD4⁺CD25⁺ T cells. (B) Surface expression of CTLA-4 16 h after stimulation with allogeneic DCs. The analysis is representative of seven independent experiments. (C) Proliferation assays of CD4⁺ and CD4⁺CD25⁺ T cells stimulated with varying numbers of allogeneic mature DCs. Proliferation of T cells was determined by the addition of [³H]Tdr after 4 (primary culture) or 2 d (restimulation) of culture for 16 h. Similar results were obtained in five independent experiments. (D) Cell cycle analysis of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells 4 d after the first restimulation with allogeneic DCs. Similar results were obtained in five independent experiments.

Figure 2

A subpopulation of CD4⁺CD25⁺ human anergic T cells shows constitutive expression of CTLA-4. (A) Surface and intracellular CTLA-4 expression of freshly isolated CD4⁺ and CD4⁺CD25⁺ T cells. (B) Surface expression of CTLA-4 16 h after stimulation with allogeneic DCs. The analysis is representative of seven independent experiments. (C) Proliferation assays of CD4⁺ and CD4⁺CD25⁺ T cells stimulated with varying numbers of allogeneic mature DCs. Proliferation of T cells was determined by the addition of [³H]Tdr after 4 (primary culture) or 2 d (restimulation) of culture for 16 h. Similar results were obtained in five independent experiments. (D) Cell cycle analysis of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells 4 d after the first restimulation with allogeneic DCs. Similar results were obtained in five independent experiments.

Figure 3

Cytokine profiles of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells after the first restimulation. CD4⁺ and CD4⁺CD25⁺ T cells were primed and restimulated with allogeneic DCs from the same donor. The figure shows the cytokine profiles of T cells after the first restimulation. (A) 6 d after the first restimulation, T cells were activated with PHA/PMA in the presence of monensin. The cytokine profiles were detected by intracellular FACS^® staining 6 h later. (B) Cytokine profiles of T cells determined by ELISA 48 h after the first restimulation with allogeneic DCs. Black bars, CD4⁺ T cells; white bars, CD4⁺CD25⁺ T cells. (C) The cytokine mRNA profile of T cells 2 h after restimulation with allogeneic DCs as detected by RT-PCR. A representative result of four independent experiments is shown.

Figure 3

Cytokine profiles of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells after the first restimulation. CD4⁺ and CD4⁺CD25⁺ T cells were primed and restimulated with allogeneic DCs from the same donor. The figure shows the cytokine profiles of T cells after the first restimulation. (A) 6 d after the first restimulation, T cells were activated with PHA/PMA in the presence of monensin. The cytokine profiles were detected by intracellular FACS^® staining 6 h later. (B) Cytokine profiles of T cells determined by ELISA 48 h after the first restimulation with allogeneic DCs. Black bars, CD4⁺ T cells; white bars, CD4⁺CD25⁺ T cells. (C) The cytokine mRNA profile of T cells 2 h after restimulation with allogeneic DCs as detected by RT-PCR. A representative result of four independent experiments is shown.

Figure 3

Cytokine profiles of alloreactive CD4⁺ and CD4⁺CD25⁺ T cells after the first restimulation. CD4⁺ and CD4⁺CD25⁺ T cells were primed and restimulated with allogeneic DCs from the same donor. The figure shows the cytokine profiles of T cells after the first restimulation. (A) 6 d after the first restimulation, T cells were activated with PHA/PMA in the presence of monensin. The cytokine profiles were detected by intracellular FACS^® staining 6 h later. (B) Cytokine profiles of T cells determined by ELISA 48 h after the first restimulation with allogeneic DCs. Black bars, CD4⁺ T cells; white bars, CD4⁺CD25⁺ T cells. (C) The cytokine mRNA profile of T cells 2 h after restimulation with allogeneic DCs as detected by RT-PCR. A representative result of four independent experiments is shown.

Figure 4

The anergic state of CD4⁺CD25⁺ T cells is partially reversible by exogenous IL-2 or IL-4. CD4⁺CD25⁺ T cells were primed and restimulated with allogeneic DCs. T cell proliferation (5 × 10⁴ T cells per well plus 5 × 10³ DCs per well, first restimulation) in the presence or absence of blocking Abs (2 μg/ml anti–TGF-β mAb, 10 μg/ml anti–IL-10 mAb, 20 μg/ml anti–CTLA-4 mAb) or additional stimulation with 2 μg/ml anti-CD3, 2 μg/ml anti-CD28, or cytokines (200 U/ml IL-2, 500 U/ml IL-4). [³H]Tdr was added after 2 d of culture for the final 16 h. Proliferation of restimulated CD4⁺ T cells served as a control. Similar results were obtained in seven independent experiments.

Figure 5

CD4⁺CD25⁺ T cells inhibit dose dependently proliferation and cytokine production of conventional CD4⁺ T cells. (A, left) Freshly isolated CD4⁺ T cells (2 × 10⁵ cells per well) were stimulated with allogeneic DCs (2 × 10⁴ cells per well) in the presence of different numbers of freshly isolated CD4⁺CD25⁺ T cells from the same donor. Proliferation was determined after 4 d of culture by the addition of [³H]Tdr for the final 16 h. (Right) Coculture of separately primed CD4⁺ and CD25⁺ T cells in a secondary stimulation assay. Proliferation was determined after 2 d of culture by addition of [³H]Tdr for the final 16 h. (B) On the left, the cytokine profile of CD4⁺ T cells primed and restimulated with allogeneic DCs alone and on the right, the cytokine profile of CD4⁺ T cells restimulated with allogeneic DCs in the presence of CD4⁺CD25⁺ T cells are illustrated. A representative result of four independent experiments is shown. (C) Anti-CD3 assay. CD4⁺ and CD4⁺CD25⁺ T cells were stimulated with 2 μg/ml anti-CD3 in the presence of irradiated syngeneic PBMCs and were used for secondary anti-CD3 mAb proliferation assays 10 d after as described in Materials and Methods. The proliferative response of CD4⁺ and CD4⁺CD25⁺ T cells restimulated separately or in coculture is shown. A representative result of three independent experiments is shown.

Figure 5

CD4⁺CD25⁺ T cells inhibit dose dependently proliferation and cytokine production of conventional CD4⁺ T cells. (A, left) Freshly isolated CD4⁺ T cells (2 × 10⁵ cells per well) were stimulated with allogeneic DCs (2 × 10⁴ cells per well) in the presence of different numbers of freshly isolated CD4⁺CD25⁺ T cells from the same donor. Proliferation was determined after 4 d of culture by the addition of [³H]Tdr for the final 16 h. (Right) Coculture of separately primed CD4⁺ and CD25⁺ T cells in a secondary stimulation assay. Proliferation was determined after 2 d of culture by addition of [³H]Tdr for the final 16 h. (B) On the left, the cytokine profile of CD4⁺ T cells primed and restimulated with allogeneic DCs alone and on the right, the cytokine profile of CD4⁺ T cells restimulated with allogeneic DCs in the presence of CD4⁺CD25⁺ T cells are illustrated. A representative result of four independent experiments is shown. (C) Anti-CD3 assay. CD4⁺ and CD4⁺CD25⁺ T cells were stimulated with 2 μg/ml anti-CD3 in the presence of irradiated syngeneic PBMCs and were used for secondary anti-CD3 mAb proliferation assays 10 d after as described in Materials and Methods. The proliferative response of CD4⁺ and CD4⁺CD25⁺ T cells restimulated separately or in coculture is shown. A representative result of three independent experiments is shown.

Figure 5

CD4⁺CD25⁺ T cells inhibit dose dependently proliferation and cytokine production of conventional CD4⁺ T cells. (A, left) Freshly isolated CD4⁺ T cells (2 × 10⁵ cells per well) were stimulated with allogeneic DCs (2 × 10⁴ cells per well) in the presence of different numbers of freshly isolated CD4⁺CD25⁺ T cells from the same donor. Proliferation was determined after 4 d of culture by the addition of [³H]Tdr for the final 16 h. (Right) Coculture of separately primed CD4⁺ and CD25⁺ T cells in a secondary stimulation assay. Proliferation was determined after 2 d of culture by addition of [³H]Tdr for the final 16 h. (B) On the left, the cytokine profile of CD4⁺ T cells primed and restimulated with allogeneic DCs alone and on the right, the cytokine profile of CD4⁺ T cells restimulated with allogeneic DCs in the presence of CD4⁺CD25⁺ T cells are illustrated. A representative result of four independent experiments is shown. (C) Anti-CD3 assay. CD4⁺ and CD4⁺CD25⁺ T cells were stimulated with 2 μg/ml anti-CD3 in the presence of irradiated syngeneic PBMCs and were used for secondary anti-CD3 mAb proliferation assays 10 d after as described in Materials and Methods. The proliferative response of CD4⁺ and CD4⁺CD25⁺ T cells restimulated separately or in coculture is shown. A representative result of three independent experiments is shown.

Figure 6

The inhibitory effect of human regulatory CD4⁺CD25⁺ T cells requires cell–cell contact and is antigen nonspecific. (A) CD4⁺ and CD4⁺CD25⁺ T cells from the same donor were primed with allogeneic DCs. After 10 d, the alloreactive T cells were restimulated with mature DCs from the same allogeneic donor. CD4⁺CD25⁺ T cells (10⁶ cells per well) and CD4⁺ T cells (10⁶ cells per well) were placed and activated separately in transwell chambers in the same well. Alternatively, CD4⁺CD25⁺ T cells were added directly to the cocultures of CD4⁺ T cells plus allogeneic DCs in the presence or absence of inhibitory Abs as indicated (2 μg/ml anti–TGF-β, 10 μg/ml anti–IL-10, 20 μg/ml anti–CTLA-4/CTLA-4 F(ab′)₂ fragments or 2 μg/ml anti-CD28). Proliferation of restimulated CD4⁺ T cells served as a control. (B) Syngeneic CD4⁺ and CD4⁺CD25⁺ T cells were primed with allogeneic DCs from different donors (A or B). 10 d after, donor A–specific CD4⁺ T cells (^ACD4⁺) were restimulated in the presence of donor B–specific CD4⁺CD25⁺ T cells (^BCD4⁺CD25⁺) stimulated with allogeneic DCs (donor A and/or B) as indicated. Additionally, ^BCD4⁺CD25⁺ T cells were preactivated with 0.5 μg/ml anti-CD3 at 37°C for 30 min, washed in PBS, and added to cultures of activated ^ACD4⁺ T cells. After 2 d of culture, activated T cells were transferred in 96-well plates to measure incorporation of [³H]Tdr for the final 16 h. Results representative of five independent experiments are presented as mean cpm of triplicate determinations.

Figure 6

The inhibitory effect of human regulatory CD4⁺CD25⁺ T cells requires cell–cell contact and is antigen nonspecific. (A) CD4⁺ and CD4⁺CD25⁺ T cells from the same donor were primed with allogeneic DCs. After 10 d, the alloreactive T cells were restimulated with mature DCs from the same allogeneic donor. CD4⁺CD25⁺ T cells (10⁶ cells per well) and CD4⁺ T cells (10⁶ cells per well) were placed and activated separately in transwell chambers in the same well. Alternatively, CD4⁺CD25⁺ T cells were added directly to the cocultures of CD4⁺ T cells plus allogeneic DCs in the presence or absence of inhibitory Abs as indicated (2 μg/ml anti–TGF-β, 10 μg/ml anti–IL-10, 20 μg/ml anti–CTLA-4/CTLA-4 F(ab′)₂ fragments or 2 μg/ml anti-CD28). Proliferation of restimulated CD4⁺ T cells served as a control. (B) Syngeneic CD4⁺ and CD4⁺CD25⁺ T cells were primed with allogeneic DCs from different donors (A or B). 10 d after, donor A–specific CD4⁺ T cells (^ACD4⁺) were restimulated in the presence of donor B–specific CD4⁺CD25⁺ T cells (^BCD4⁺CD25⁺) stimulated with allogeneic DCs (donor A and/or B) as indicated. Additionally, ^BCD4⁺CD25⁺ T cells were preactivated with 0.5 μg/ml anti-CD3 at 37°C for 30 min, washed in PBS, and added to cultures of activated ^ACD4⁺ T cells. After 2 d of culture, activated T cells were transferred in 96-well plates to measure incorporation of [³H]Tdr for the final 16 h. Results representative of five independent experiments are presented as mean cpm of triplicate determinations.

Figure 7

Human CD4⁺CD25⁺ T cell populations contain regulatory and conventional T cells. Freshly isolated CD4⁺CD25⁺ T cells were separated into CD45RO⁺ and CD45RO⁻ cells using anti-CD45RO beads as described in Materials and Methods and separately primed with allogeneic DCs. 10 d after, the T cell populations were restimulated under the same conditions or in coculture with primed CD4⁺ T cells. The proliferation of restimulated T cell populations is shown (2-d culture plus an additional 16-h [³H]Tdr). The figure is representative for three independent experiments.