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Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes.

Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini V, Romagnani P, Maggi E, Romagnani S - J. Exp. Med. (2002)

Bottom Line: They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells.The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) alpha-chain (CD25) expression in target cells.Such a suppressive activity was partially inhibited by either anti-CTLA-4 or anti-TGF-beta1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R alpha-chain and, therefore, their responsiveness to IL-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University Florence, Italy.

ABSTRACT
Phenotypic markers, localization, functional activities, and mechanisms of action in vitro of CD4(+)CD25(+) T cells, purified from postnatal human thymuses, were investigated. These cells showed poor or no proliferation in mixed lymphocyte culture (MLC), and suppressed in a dose-dependent fashion the proliferative response to allogeneic stimulation of CD4(+)CD25(-) thymocytes. Virtually all CD4(+)CD25(+) thymocytes constitutively expressed cytoplasmic T lymphocyte antigen (CTLA)-4, surface tumor necrosis factor type 2 receptor (TNFR2), and CCR8. They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells. After polyclonal activation, CD4(+)CD25(+) thymocytes did not produce the cytokines interleukin (IL)-2, IL-4, IL-5, IL-13, interferon gamma, and only a very few produced IL-10, but all they expressed on their surface CTLA-4 and the majority of them also transforming growth factor (TGF)-beta1. The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) alpha-chain (CD25) expression in target cells. Such a suppressive activity was partially inhibited by either anti-CTLA-4 or anti-TGF-beta1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R alpha-chain and, therefore, their responsiveness to IL-2. These data demonstrate that CD4(+)CD25(+) human thymocytes represent a population of regulatory cells that migrate in response to the chemokine CCL1/I-309 and exert their suppressive function via the inhibition of IL-2R alpha-chain in target T cells, induced by the combined activity of CTLA-4 and membrane TGF-beta1.

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Cell contact–dependent suppressive activity of CD4+CD25+ human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4+CD25− thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4+CD25+ thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3[H]TdR uptake. (B) CD4+CD25− thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4+CD25+ thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3[H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.
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fig6: Cell contact–dependent suppressive activity of CD4+CD25+ human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4+CD25− thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4+CD25+ thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3[H]TdR uptake. (B) CD4+CD25− thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4+CD25+ thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3[H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.

Mentions: After 6 d of stimulation with insolubilized anti-CD3 plus soluble anti-CD28 mAbs of CFSE-labeled CD4+CD25+ and CD4+CD25− thymocytes, cells were further stimulated with PMA plus ionomycin. In agreement with the results obtained by measuring 3[H]TdR incorporation, the CD4+CD25+ cells showed strongly reduced proliferation, as assessed by CFSE content (Fig. 5 A). Moreover, the same cells did not produce IL-2, IL-4, IL-5, IL-13, or IFN-γ, and only a very few of them produced IL-10, as assessed by flow cytometry at single cell level (Fig. 5 A). However, stimulation with insolubilized anti-CD3 plus soluble anti-CD28 mAbs, induced virtually all CD4+ CD25+ thymocytes to express CTLA-4 on their surface and about a half of them also mTGF-β1 (Fig. 5 B). The latter finding is of interest, since mTGF-β1 has recently been suggested to play a role in the suppressive activity of murine CD4+CD25+ Treg cells (19). To provide direct evidence that mTGF-β1 was also involved in the suppressive activity of CD4+CD25+ human thymocytes, we first assessed the effect of purified CD4+CD25+ thymocytes on the response of CD4+CD25− T cells in MLC by using transwell chambers, in which the proliferative response of CD4+CD25− T cells to irradiated allogeneic non-T cells placed in the lower chamber was evaluated in absence (top chamber) or in presence (bottom chamber) of CD4+ CD25+ T cells. As shown in Fig. 6 A, the proliferative response of CD4+CD25− T cells was strongly inhibited by CD4+CD25+ T cells, but only when they were present in the same chamber, indicating that the suppressive activity of these cells was mediated by cell-to-cell contact and not via the release of soluble factors.


Phenotype, localization, and mechanism of suppression of CD4(+)CD25(+) human thymocytes.

Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini V, Romagnani P, Maggi E, Romagnani S - J. Exp. Med. (2002)

Cell contact–dependent suppressive activity of CD4+CD25+ human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4+CD25− thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4+CD25+ thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3[H]TdR uptake. (B) CD4+CD25− thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4+CD25+ thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3[H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.
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Related In: Results  -  Collection

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fig6: Cell contact–dependent suppressive activity of CD4+CD25+ human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4+CD25− thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4+CD25+ thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3[H]TdR uptake. (B) CD4+CD25− thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4+CD25+ thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3[H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.
Mentions: After 6 d of stimulation with insolubilized anti-CD3 plus soluble anti-CD28 mAbs of CFSE-labeled CD4+CD25+ and CD4+CD25− thymocytes, cells were further stimulated with PMA plus ionomycin. In agreement with the results obtained by measuring 3[H]TdR incorporation, the CD4+CD25+ cells showed strongly reduced proliferation, as assessed by CFSE content (Fig. 5 A). Moreover, the same cells did not produce IL-2, IL-4, IL-5, IL-13, or IFN-γ, and only a very few of them produced IL-10, as assessed by flow cytometry at single cell level (Fig. 5 A). However, stimulation with insolubilized anti-CD3 plus soluble anti-CD28 mAbs, induced virtually all CD4+ CD25+ thymocytes to express CTLA-4 on their surface and about a half of them also mTGF-β1 (Fig. 5 B). The latter finding is of interest, since mTGF-β1 has recently been suggested to play a role in the suppressive activity of murine CD4+CD25+ Treg cells (19). To provide direct evidence that mTGF-β1 was also involved in the suppressive activity of CD4+CD25+ human thymocytes, we first assessed the effect of purified CD4+CD25+ thymocytes on the response of CD4+CD25− T cells in MLC by using transwell chambers, in which the proliferative response of CD4+CD25− T cells to irradiated allogeneic non-T cells placed in the lower chamber was evaluated in absence (top chamber) or in presence (bottom chamber) of CD4+ CD25+ T cells. As shown in Fig. 6 A, the proliferative response of CD4+CD25− T cells was strongly inhibited by CD4+CD25+ T cells, but only when they were present in the same chamber, indicating that the suppressive activity of these cells was mediated by cell-to-cell contact and not via the release of soluble factors.

Bottom Line: They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells.The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) alpha-chain (CD25) expression in target cells.Such a suppressive activity was partially inhibited by either anti-CTLA-4 or anti-TGF-beta1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R alpha-chain and, therefore, their responsiveness to IL-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University Florence, Italy.

ABSTRACT
Phenotypic markers, localization, functional activities, and mechanisms of action in vitro of CD4(+)CD25(+) T cells, purified from postnatal human thymuses, were investigated. These cells showed poor or no proliferation in mixed lymphocyte culture (MLC), and suppressed in a dose-dependent fashion the proliferative response to allogeneic stimulation of CD4(+)CD25(-) thymocytes. Virtually all CD4(+)CD25(+) thymocytes constitutively expressed cytoplasmic T lymphocyte antigen (CTLA)-4, surface tumor necrosis factor type 2 receptor (TNFR2), and CCR8. They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells. After polyclonal activation, CD4(+)CD25(+) thymocytes did not produce the cytokines interleukin (IL)-2, IL-4, IL-5, IL-13, interferon gamma, and only a very few produced IL-10, but all they expressed on their surface CTLA-4 and the majority of them also transforming growth factor (TGF)-beta1. The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) alpha-chain (CD25) expression in target cells. Such a suppressive activity was partially inhibited by either anti-CTLA-4 or anti-TGF-beta1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R alpha-chain and, therefore, their responsiveness to IL-2. These data demonstrate that CD4(+)CD25(+) human thymocytes represent a population of regulatory cells that migrate in response to the chemokine CCL1/I-309 and exert their suppressive function via the inhibition of IL-2R alpha-chain in target T cells, induced by the combined activity of CTLA-4 and membrane TGF-beta1.

Show MeSH
Related in: MedlinePlus