Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood

Abstract

It has been known for years that rodents harbor a unique population of CD4(+)CD25(+) "professional" regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4(+)CD25(+)CD45RO(+) T cells (mean 6% of CD4(+) T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4(+)CD25(+) T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte-associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4(+)CD25(+) T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4(+)CD25(+) T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4(+) and CD8(+) T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4(+)CD25(+) T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

Figure 2

CD4⁺CD25⁺ T cells are nonproliferative/anergic to both allogeneic and polyclonal stimulation, which is partially reversed by the addition of IL-2 and/or IL-15, but not by neutralizing anti–IL-10 Abs. (A) Whole CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁻ T cells were isolated from adult blood by MACS^® sorting as in Fig. 1. 10⁵ T cells per 96 well were stimulated with different numbers of mature allogeneic DCs. Proliferation of T cells (triplicate cultures) was determined by [³H]Tdr incorporation. Results were similar in five independent experiments. (B) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were primed and restimulated every week with mature allogeneic DCs from the same donor (DC/T cell ratio of 1:20). Proliferation (10⁵ T cells per 96 well) was determined by [³H]Tdr incorporation. Similar results were obtained in three independent experiments. (C) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 10 μg/ml immobilized anti-CD3 and 10 μg/ml soluble anti-CD28 (top) or with 5 × 10³ mature allogeneic DCs (bottom) as described in (A). 500 U/ml IL-2, 100 ng/ml IL-15, a mixture of 10 U/ml IL-2 plus 1 ng/ml IL-15 or 10 μg/ml anti–IL-10 were added at the onset of culture. [³H]Tdr incorporation was measured after 5 d of culture. One of three independent experiments is shown. The addition of IL-2 and/or IL-15 in the absence of a polyclonal or allogeneic T cell stimulus did not induce significant proliferation in the CD25⁺ or CD25⁻ T cell subset (data not shown).

Figure 2

CD4⁺CD25⁺ T cells are nonproliferative/anergic to both allogeneic and polyclonal stimulation, which is partially reversed by the addition of IL-2 and/or IL-15, but not by neutralizing anti–IL-10 Abs. (A) Whole CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁻ T cells were isolated from adult blood by MACS^® sorting as in Fig. 1. 10⁵ T cells per 96 well were stimulated with different numbers of mature allogeneic DCs. Proliferation of T cells (triplicate cultures) was determined by [³H]Tdr incorporation. Results were similar in five independent experiments. (B) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were primed and restimulated every week with mature allogeneic DCs from the same donor (DC/T cell ratio of 1:20). Proliferation (10⁵ T cells per 96 well) was determined by [³H]Tdr incorporation. Similar results were obtained in three independent experiments. (C) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 10 μg/ml immobilized anti-CD3 and 10 μg/ml soluble anti-CD28 (top) or with 5 × 10³ mature allogeneic DCs (bottom) as described in (A). 500 U/ml IL-2, 100 ng/ml IL-15, a mixture of 10 U/ml IL-2 plus 1 ng/ml IL-15 or 10 μg/ml anti–IL-10 were added at the onset of culture. [³H]Tdr incorporation was measured after 5 d of culture. One of three independent experiments is shown. The addition of IL-2 and/or IL-15 in the absence of a polyclonal or allogeneic T cell stimulus did not induce significant proliferation in the CD25⁺ or CD25⁻ T cell subset (data not shown).

Figure 2

CD4⁺CD25⁺ T cells are nonproliferative/anergic to both allogeneic and polyclonal stimulation, which is partially reversed by the addition of IL-2 and/or IL-15, but not by neutralizing anti–IL-10 Abs. (A) Whole CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁻ T cells were isolated from adult blood by MACS^® sorting as in Fig. 1. 10⁵ T cells per 96 well were stimulated with different numbers of mature allogeneic DCs. Proliferation of T cells (triplicate cultures) was determined by [³H]Tdr incorporation. Results were similar in five independent experiments. (B) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were primed and restimulated every week with mature allogeneic DCs from the same donor (DC/T cell ratio of 1:20). Proliferation (10⁵ T cells per 96 well) was determined by [³H]Tdr incorporation. Similar results were obtained in three independent experiments. (C) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 10 μg/ml immobilized anti-CD3 and 10 μg/ml soluble anti-CD28 (top) or with 5 × 10³ mature allogeneic DCs (bottom) as described in (A). 500 U/ml IL-2, 100 ng/ml IL-15, a mixture of 10 U/ml IL-2 plus 1 ng/ml IL-15 or 10 μg/ml anti–IL-10 were added at the onset of culture. [³H]Tdr incorporation was measured after 5 d of culture. One of three independent experiments is shown. The addition of IL-2 and/or IL-15 in the absence of a polyclonal or allogeneic T cell stimulus did not induce significant proliferation in the CD25⁺ or CD25⁻ T cell subset (data not shown).

Figure 1

CD4⁺CD25⁺ T cells exhibit distinct phenotypical differences to CD4⁺CD25⁻ T cells. CD4⁺ T cells were isolated from PBMCs by negative MACS^® sorting yielding highly purified untouched CD4⁺ T cells. These cells were labeled with anti-CD25 magnetic beads and sorted. (A) Sorting resulted in virtually pure CD25⁺ T cells. A representative result out of 20 independent standardized experiments is shown. (B) The phenotype of CD4⁺CD25⁺, CD4⁺CD25⁻, and activated CD4⁺CD25⁻ T cells was analyzed as described in Materials and Methods. In addition, CD4⁺CD25⁻ T cells were activated with immobilized anti-CD3 plus soluble anti-CD28 for 48 h. After activation cells were labeled with anti-CD25 magnetic beads and sorted. Results were similar in five independent experiments. (C) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stained with anti–CTLA-4 Ab at 37°C for 2 h. Staining was performed ex vivo and at different time points after activation with immobilized anti-CD3 plus soluble anti-CD28. One representative result of four independent experiments is shown.

Figure 3

CD4⁺CD25⁺ T cells if stimulated via the TCR suppress the activation of CD4⁺ and CD8⁺ T cells in a cell contact– and dose-dependent manner. (A and B) MACS^®-sorted whole CD4⁺ (A) and CD8⁺ (B) T cells (10⁵ T cells per 96 well) were added to CD4⁺CD25⁺ T cells at the ratios indicated and stimulated with allogeneic DCs at a DC/CD4⁺ or CD8⁺ T cells ratio of 1:20. Proliferation was determined by [³H]Tdr incorporation after 5 d. One of five independent experiments is shown. (C) DCs and CD4⁺CD25⁺ T cells were generated/ isolated from the same donor (donor I). In addition, whole CD4⁺ T cells and CD4⁺ CD25⁺ T cells were isolated from another donor (donor II). 10⁵ whole CD4⁺ T cells per 96 well were cultured with 5 × 10³ DCs per well (i.e., DC/T ratio = 1:20; results were comparable at a DC/T ratio of 1:100, data not shown). CD4⁺CD25⁺ T cells from donor I and donor II were then added, respectively. Proliferation was determined by [³H]Tdr incorporation after 5 d of culture. Results are representative of three independent experiments shown as mean cpm of triplicate cultures. (D) Whole CD4⁺ T cells or CD4⁺CD25⁺ T cells were (10⁵ T cells per 96 well) stimulated with 5 × 10³ allogeneic mature DCs (DC/T ratio = 1:20) (upper two panels). In addition, whole CD4⁺ T cells were cocultured with CD4⁺CD25⁺ T cells at a ratio of 1:1 (10⁵ T cells per 96 well each) and stimulated with allogeneic DCs again at a DC/T ratio of 1:20 in the presence or absence of 10 μg/ml anti–IL-10, 2 μg/ml anti–TGF-β, 500 U/ml IL-2, 50 ng/ml IL-15, or a mixture of 10 U/ml IL-2 and 1 ng/ml IL-15. In a parallel transwell approach the CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T cell ratio of 1:20) in a transwell chamber, and whole CD4⁺ T cell responders were put into the well together with allogeneic DCs again at a DC/T ratio of 1:20. Proliferation after 5 d of culture was determined by [³H]Tdr incorporation. One of four representative experiments is shown.

Figure 3

CD4⁺CD25⁺ T cells if stimulated via the TCR suppress the activation of CD4⁺ and CD8⁺ T cells in a cell contact– and dose-dependent manner. (A and B) MACS^®-sorted whole CD4⁺ (A) and CD8⁺ (B) T cells (10⁵ T cells per 96 well) were added to CD4⁺CD25⁺ T cells at the ratios indicated and stimulated with allogeneic DCs at a DC/CD4⁺ or CD8⁺ T cells ratio of 1:20. Proliferation was determined by [³H]Tdr incorporation after 5 d. One of five independent experiments is shown. (C) DCs and CD4⁺CD25⁺ T cells were generated/ isolated from the same donor (donor I). In addition, whole CD4⁺ T cells and CD4⁺ CD25⁺ T cells were isolated from another donor (donor II). 10⁵ whole CD4⁺ T cells per 96 well were cultured with 5 × 10³ DCs per well (i.e., DC/T ratio = 1:20; results were comparable at a DC/T ratio of 1:100, data not shown). CD4⁺CD25⁺ T cells from donor I and donor II were then added, respectively. Proliferation was determined by [³H]Tdr incorporation after 5 d of culture. Results are representative of three independent experiments shown as mean cpm of triplicate cultures. (D) Whole CD4⁺ T cells or CD4⁺CD25⁺ T cells were (10⁵ T cells per 96 well) stimulated with 5 × 10³ allogeneic mature DCs (DC/T ratio = 1:20) (upper two panels). In addition, whole CD4⁺ T cells were cocultured with CD4⁺CD25⁺ T cells at a ratio of 1:1 (10⁵ T cells per 96 well each) and stimulated with allogeneic DCs again at a DC/T ratio of 1:20 in the presence or absence of 10 μg/ml anti–IL-10, 2 μg/ml anti–TGF-β, 500 U/ml IL-2, 50 ng/ml IL-15, or a mixture of 10 U/ml IL-2 and 1 ng/ml IL-15. In a parallel transwell approach the CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T cell ratio of 1:20) in a transwell chamber, and whole CD4⁺ T cell responders were put into the well together with allogeneic DCs again at a DC/T ratio of 1:20. Proliferation after 5 d of culture was determined by [³H]Tdr incorporation. One of four representative experiments is shown.

Figure 3

CD4⁺CD25⁺ T cells if stimulated via the TCR suppress the activation of CD4⁺ and CD8⁺ T cells in a cell contact– and dose-dependent manner. (A and B) MACS^®-sorted whole CD4⁺ (A) and CD8⁺ (B) T cells (10⁵ T cells per 96 well) were added to CD4⁺CD25⁺ T cells at the ratios indicated and stimulated with allogeneic DCs at a DC/CD4⁺ or CD8⁺ T cells ratio of 1:20. Proliferation was determined by [³H]Tdr incorporation after 5 d. One of five independent experiments is shown. (C) DCs and CD4⁺CD25⁺ T cells were generated/ isolated from the same donor (donor I). In addition, whole CD4⁺ T cells and CD4⁺ CD25⁺ T cells were isolated from another donor (donor II). 10⁵ whole CD4⁺ T cells per 96 well were cultured with 5 × 10³ DCs per well (i.e., DC/T ratio = 1:20; results were comparable at a DC/T ratio of 1:100, data not shown). CD4⁺CD25⁺ T cells from donor I and donor II were then added, respectively. Proliferation was determined by [³H]Tdr incorporation after 5 d of culture. Results are representative of three independent experiments shown as mean cpm of triplicate cultures. (D) Whole CD4⁺ T cells or CD4⁺CD25⁺ T cells were (10⁵ T cells per 96 well) stimulated with 5 × 10³ allogeneic mature DCs (DC/T ratio = 1:20) (upper two panels). In addition, whole CD4⁺ T cells were cocultured with CD4⁺CD25⁺ T cells at a ratio of 1:1 (10⁵ T cells per 96 well each) and stimulated with allogeneic DCs again at a DC/T ratio of 1:20 in the presence or absence of 10 μg/ml anti–IL-10, 2 μg/ml anti–TGF-β, 500 U/ml IL-2, 50 ng/ml IL-15, or a mixture of 10 U/ml IL-2 and 1 ng/ml IL-15. In a parallel transwell approach the CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T cell ratio of 1:20) in a transwell chamber, and whole CD4⁺ T cell responders were put into the well together with allogeneic DCs again at a DC/T ratio of 1:20. Proliferation after 5 d of culture was determined by [³H]Tdr incorporation. One of four representative experiments is shown.

Figure 3

CD4⁺CD25⁺ T cells if stimulated via the TCR suppress the activation of CD4⁺ and CD8⁺ T cells in a cell contact– and dose-dependent manner. (A and B) MACS^®-sorted whole CD4⁺ (A) and CD8⁺ (B) T cells (10⁵ T cells per 96 well) were added to CD4⁺CD25⁺ T cells at the ratios indicated and stimulated with allogeneic DCs at a DC/CD4⁺ or CD8⁺ T cells ratio of 1:20. Proliferation was determined by [³H]Tdr incorporation after 5 d. One of five independent experiments is shown. (C) DCs and CD4⁺CD25⁺ T cells were generated/ isolated from the same donor (donor I). In addition, whole CD4⁺ T cells and CD4⁺ CD25⁺ T cells were isolated from another donor (donor II). 10⁵ whole CD4⁺ T cells per 96 well were cultured with 5 × 10³ DCs per well (i.e., DC/T ratio = 1:20; results were comparable at a DC/T ratio of 1:100, data not shown). CD4⁺CD25⁺ T cells from donor I and donor II were then added, respectively. Proliferation was determined by [³H]Tdr incorporation after 5 d of culture. Results are representative of three independent experiments shown as mean cpm of triplicate cultures. (D) Whole CD4⁺ T cells or CD4⁺CD25⁺ T cells were (10⁵ T cells per 96 well) stimulated with 5 × 10³ allogeneic mature DCs (DC/T ratio = 1:20) (upper two panels). In addition, whole CD4⁺ T cells were cocultured with CD4⁺CD25⁺ T cells at a ratio of 1:1 (10⁵ T cells per 96 well each) and stimulated with allogeneic DCs again at a DC/T ratio of 1:20 in the presence or absence of 10 μg/ml anti–IL-10, 2 μg/ml anti–TGF-β, 500 U/ml IL-2, 50 ng/ml IL-15, or a mixture of 10 U/ml IL-2 and 1 ng/ml IL-15. In a parallel transwell approach the CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T cell ratio of 1:20) in a transwell chamber, and whole CD4⁺ T cell responders were put into the well together with allogeneic DCs again at a DC/T ratio of 1:20. Proliferation after 5 d of culture was determined by [³H]Tdr incorporation. One of four representative experiments is shown.

Figure 4

Different cytokine profiles of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells. (A) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 20 ng/ml PMA and 500 μg/ml A23187 Ca²⁺ ionophore for 6 h. 2 μM monensin was added for the last 5 h. Staining of CD3 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for detection of intracellular cytokines using FITC- or PE-conjugated specific Abs. One of nine independent experiments with similar results is shown. Results were identical when T cells were stimulated with platebound anti-CD3 plus soluble anti-CD28 Ab (data not shown). (B) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were activated with platebound anti-CD3 plus soluble anti-CD28. After 48 h of culture analysis of RNA expression was performed by RNase protection assay. (C) After treating cells as described in (B) cytokines in the supernatant were measured by ELISA (one of five independent experiments is shown).

Figure 4

Different cytokine profiles of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells. (A) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 20 ng/ml PMA and 500 μg/ml A23187 Ca²⁺ ionophore for 6 h. 2 μM monensin was added for the last 5 h. Staining of CD3 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for detection of intracellular cytokines using FITC- or PE-conjugated specific Abs. One of nine independent experiments with similar results is shown. Results were identical when T cells were stimulated with platebound anti-CD3 plus soluble anti-CD28 Ab (data not shown). (B) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were activated with platebound anti-CD3 plus soluble anti-CD28. After 48 h of culture analysis of RNA expression was performed by RNase protection assay. (C) After treating cells as described in (B) cytokines in the supernatant were measured by ELISA (one of five independent experiments is shown).

Figure 4

Different cytokine profiles of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells. (A) MACS^®-sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were stimulated with 20 ng/ml PMA and 500 μg/ml A23187 Ca²⁺ ionophore for 6 h. 2 μM monensin was added for the last 5 h. Staining of CD3 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for detection of intracellular cytokines using FITC- or PE-conjugated specific Abs. One of nine independent experiments with similar results is shown. Results were identical when T cells were stimulated with platebound anti-CD3 plus soluble anti-CD28 Ab (data not shown). (B) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were activated with platebound anti-CD3 plus soluble anti-CD28. After 48 h of culture analysis of RNA expression was performed by RNase protection assay. (C) After treating cells as described in (B) cytokines in the supernatant were measured by ELISA (one of five independent experiments is shown).