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

Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G - J. Exp. Med. (2001)

Bottom Line: 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.Suppression proved IL-10 independent, yet contact dependent as in the mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Erlangen-Nuremberg, Hartmannstrasse 14, 91052 Erlangen, Germany.

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.

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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).
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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).

Mentions: To analyze and compare the cytokine profiles, freshly sorted CD4+CD25+ and CD4+CD25− T cells were activated with platebound anti-CD3 plus anti-CD28. Supernatants were then analyzed by ELISA, and RNA expression was studied by RNase protection assays. In addition, intracellular cytokine staining was performed to determine the percentage of cells releasing a certain cytokine. As shown in Fig. 4, CD4+CD25− T cells predominantly secreted IFN-γ and IL-2 with little secretion of IL-10 and IL-4, resembling a Th1-like profile. On the other hand, CD4+CD25+ T cells predominantly produced IL-10 and only low levels of IL-2, IL-4, and IFN-γ, resembling Tr1 cells. Comparison of both subpopulations at the RNA level revealed that CD25+ T cells express more IL-10, less IFN-γ, and similar levels of IL-2 mRNA compared with CD25− T cells. IL-1 receptor antagonist mRNA was found predominantly in CD4+CD25+ T cells, whereas significant IL-1β mRNA levels were only present in CD4+CD25− T cells. TGF-β was expressed at similarly low levels in both cell types.


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

Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G - J. Exp. Med. (2001)

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).
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Related In: Results  -  Collection

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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).
Mentions: To analyze and compare the cytokine profiles, freshly sorted CD4+CD25+ and CD4+CD25− T cells were activated with platebound anti-CD3 plus anti-CD28. Supernatants were then analyzed by ELISA, and RNA expression was studied by RNase protection assays. In addition, intracellular cytokine staining was performed to determine the percentage of cells releasing a certain cytokine. As shown in Fig. 4, CD4+CD25− T cells predominantly secreted IFN-γ and IL-2 with little secretion of IL-10 and IL-4, resembling a Th1-like profile. On the other hand, CD4+CD25+ T cells predominantly produced IL-10 and only low levels of IL-2, IL-4, and IFN-γ, resembling Tr1 cells. Comparison of both subpopulations at the RNA level revealed that CD25+ T cells express more IL-10, less IFN-γ, and similar levels of IL-2 mRNA compared with CD25− T cells. IL-1 receptor antagonist mRNA was found predominantly in CD4+CD25+ T cells, whereas significant IL-1β mRNA levels were only present in CD4+CD25− T cells. TGF-β was expressed at similarly low levels in both cell types.

Bottom Line: 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.Suppression proved IL-10 independent, yet contact dependent as in the mouse.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Erlangen-Nuremberg, Hartmannstrasse 14, 91052 Erlangen, Germany.

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.

Show MeSH
Related in: MedlinePlus