Human CD4(+)CD25(+) regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells [corrected]

Abstract

It has been recently demonstrated that regulatory CD4(+)CD25(+) CD45RO(+) T cells are present in the peripheral blood of healthy adults and exert regulatory function similar to their rodent counterparts. It remains difficult to understand how the small fraction of these T cells that regulate via direct cell-to-cell contact and not via secretion of immunosuppressive cytokines could mediate strong immune suppression. Here we show that human CD4(+)CD25(+) T cells induce long-lasting anergy and production of interleukin (IL)-10 in CD4(+)CD25(-) T cells. These anergized CD4(+)CD25(-) T cells then suppress proliferation of syngenic CD4(+) T cells via IL-10 but independent of direct cell contact, similar to the so-called type 1 regulatory T (Tr1) cells. This 'catalytic' function of CD4(+)CD25(+) T cells to induce Tr1-like cells helps to explain their central role for the maintenance of immune homeostasis.

Figure 1.

Coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells results in high level IL-10 production. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were MACS^® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or TGF-β could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.

Figure 1.

Coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells results in high level IL-10 production. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were MACS^® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or TGF-β could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.

Figure 1.

Coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells results in high level IL-10 production. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were MACS^® sorted from PBMCs of healthy individuals. These cells were either cultured alone or at a 1:1 ratio and activated with platebound anti-CD3 and soluble anti-CD28 (10 μg/ml, respectively). (A) After various time points supernatants were analyzed for cytokine production by ELISA. IL-10 production peaked 48 h after onset of culture and was markedly higher in the coculture of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells than in the cultures of each of the cell types alone. A representative out of five independent standardized experiments is shown. No elevated levels of INF-α or TGF-β could be measured (data not shown). (B) The different T cell populations were also activated with mature allogeneic DCs (DC/T cell ratio 1:20) compared with anti-CD3 and anti-CD28 (10 μg/ml, respectively). Cytokines were measured 48 h after onset of culture. Results were similar in five independent experiments. (C) For the last 6 h of activation with anti-CD3 and anti-CD28 2 μM monensin was added to the cultures. Staining of CD4 surface expression was performed. Cells were washed, fixed, permeabilized, and stained for intracellular IL-10 using PE-conjugated specific Abs. One of five independent experiments is shown.

Figure 2.

Activated fixed CD4⁺CD25⁺ T cells show similar regulatory potential as viable CD4⁺CD25⁺ T cells and can induce IL-10 production in CD4⁺CD25⁻ T cells. (A) CD4⁺ T cell subpopulations were sorted by MACS^® CD4⁺CD25⁺ T cells were divided into three fractions. One part was activated with platebound anti-CD3 (10 μg/ml) and soluble anti-CD28 (10 μg/ml) overnight and fixed the next day with paraformaldehyde 2% (activated-fixed). The third part was fixed with paraformaldehyde without activation (resting-fixed) and the second part was left untreated (viable). Each fraction was mixed with syngeneic CD4⁺CD25⁻ T cells at a 1:1 ratio (10⁵ T cells per 96 well) and stimulated with platebound anti-CD3 (10 μg/ml) and soluble-anti-CD28 (10 μg/ml). Proliferation was determined by ³[H]Tdr incorporation after 5 d. Results are representative of five independent experiments, shown as mean cpm of triplicate cultures. Similar results were observed when T cells were stimulated with mature allogeneic DCs (DC/T cell ratio of 1:20; data not shown) (B) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were either cultured alone or CD4⁺CD25⁻ T cells were mixed at a 1:1 ratio with activated-fixed, resting-fixed or viable CD4⁺CD25⁺ T cells. T cells were stimulated with mature allogeneic DCs at the same ratio as in A. In a parallel transwell approach CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T ratio 1:20) in a transwell chamber, and CD4⁺CD25⁻ T cells were placed in the well together with allogeneic DCs again at a DC/T ratio of 1:20. IL-10 production was measured by ELISA 48 h after onset of culture. Results were similar in five independent experiments.

Figure 2.

Activated fixed CD4⁺CD25⁺ T cells show similar regulatory potential as viable CD4⁺CD25⁺ T cells and can induce IL-10 production in CD4⁺CD25⁻ T cells. (A) CD4⁺ T cell subpopulations were sorted by MACS^® CD4⁺CD25⁺ T cells were divided into three fractions. One part was activated with platebound anti-CD3 (10 μg/ml) and soluble anti-CD28 (10 μg/ml) overnight and fixed the next day with paraformaldehyde 2% (activated-fixed). The third part was fixed with paraformaldehyde without activation (resting-fixed) and the second part was left untreated (viable). Each fraction was mixed with syngeneic CD4⁺CD25⁻ T cells at a 1:1 ratio (10⁵ T cells per 96 well) and stimulated with platebound anti-CD3 (10 μg/ml) and soluble-anti-CD28 (10 μg/ml). Proliferation was determined by ³[H]Tdr incorporation after 5 d. Results are representative of five independent experiments, shown as mean cpm of triplicate cultures. Similar results were observed when T cells were stimulated with mature allogeneic DCs (DC/T cell ratio of 1:20; data not shown) (B) CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were either cultured alone or CD4⁺CD25⁻ T cells were mixed at a 1:1 ratio with activated-fixed, resting-fixed or viable CD4⁺CD25⁺ T cells. T cells were stimulated with mature allogeneic DCs at the same ratio as in A. In a parallel transwell approach CD4⁺CD25⁺ T cells were stimulated with allogeneic DCs (DC/T ratio 1:20) in a transwell chamber, and CD4⁺CD25⁻ T cells were placed in the well together with allogeneic DCs again at a DC/T ratio of 1:20. IL-10 production was measured by ELISA 48 h after onset of culture. Results were similar in five independent experiments.

Figure 3.

CD4⁺CD25⁻ T cells anergized by CD4⁺CD25⁺ T cells suppress proliferation of CD4⁺ T cells in a IL-10–dependent manner. MACS^® sorted CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were either cultured alone or mixed at a 1:1 ratio (2 × 10⁶ T cells per 24 well) and stimulated with mature allogeneic DCs (DC/T cell ratio 1:20) or immobilized anti-CD3/soluble anti-CD28. After 48 h of culture cells were harvested and one fraction of each population was fixed with paraformaldehyde for 1 h. Viable and fixed cells were cocultured with syngeneic resting CD4⁺CD25⁻ T cells at a 1:1 ratio (10⁵ T cells per 96 well) and stimulated as before with immobilized anti-CD3/soluble anti-CD28 (bottom panel) or mature allogeneic DCs (top panel) in the presence or absence of 10 μg/ml anti–IL-10 Abs. In a parallel transwell approach the three different T cell populations were placed in a transwell chamber and resting CD4⁺CD25⁻ T cells were stimulated with DCs (DC/T cell ratio 1:20; top panel) or platebound anti-CD3/soluble anti-CD28 (bottom panel) in the well. Proliferation after 5 d was determined by ³[HT]Tdr incorporation. One out of four independent experiments is shown.

Figure 4.

(A) Anergized CD4⁺CD25⁻ T cells predominantly secrete IL-10. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were isolated as described and stimulated alone or at a 1:1 ratio with anti-CD3/anti-CD28. 48 h after stimulation supernatant was harvested and analyzed by a cytometric bead array for IL-2, IL-4, IL-5, TNF-α, and INF-γ. Results were similar in five independent experiments. (B) Before mixing CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells at a 1:1 ratio, CD4⁺CD25⁺ T cells were labeled with 0.5 μM CFSE for 15 min. Cells were then mixed and stimulated with immobilized anti-CD3/soluble anti-CD28. After 48 h cells were harvested and sorted on a FACSVantage™. The positive and the negative fraction were then cocultured with syngeneic resting CD4⁺CD25⁻ T cells (10⁵ T cells per 96 well). Proliferation was measured after 5 d by ³[HT]Tdr incorporation. One out of five independent experiments is shown.

Figure 4.

(A) Anergized CD4⁺CD25⁻ T cells predominantly secrete IL-10. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were isolated as described and stimulated alone or at a 1:1 ratio with anti-CD3/anti-CD28. 48 h after stimulation supernatant was harvested and analyzed by a cytometric bead array for IL-2, IL-4, IL-5, TNF-α, and INF-γ. Results were similar in five independent experiments. (B) Before mixing CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells at a 1:1 ratio, CD4⁺CD25⁺ T cells were labeled with 0.5 μM CFSE for 15 min. Cells were then mixed and stimulated with immobilized anti-CD3/soluble anti-CD28. After 48 h cells were harvested and sorted on a FACSVantage™. The positive and the negative fraction were then cocultured with syngeneic resting CD4⁺CD25⁻ T cells (10⁵ T cells per 96 well). Proliferation was measured after 5 d by ³[HT]Tdr incorporation. One out of five independent experiments is shown.