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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.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.

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.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.

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