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Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta.

Nakamura K, Kitani A, Strober W - J. Exp. Med. (2001)

Bottom Line: In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4.In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression.Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
CD4(+)CD25(+) T cells have been identified as a population of immunoregulatory T cells, which mediate suppression of CD4(+)CD25(-) T cells by cell-cell contact and not secretion of suppressor cytokines. In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4. As in prior studies, we found that CD4(+)CD25(+) T cells suppress proliferation of CD4(+)CD25(-) T cells; however, we observed here that such suppression is abolished by the presence of anti-TGF-beta. In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression. Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface. This, plus the fact that we could find no evidence that a soluble factor mediates suppression, strongly suggests that CD4(+)CD25(+) T cells exert immunosuppression by a cell-cell interaction involving cell surface TGF-beta1.

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Comparison of cytokine expression among CD25+, CD25−CD45RBlow and CD25−CD45RBhigh populations of CD4+ T cells. (A) Purified CD4+CD25+, CD4+CD25− CD45RBlow and CD4+CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). 24 h later, cells were incubated with either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken Ig G, washed, and stained with Cy-Chrome™–conjugated streptavidin. Expression of cell surface–bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B–E) 105 CD4+ CD25+, CD4+ CD25−CD45RBlow and CD4+ CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml) and IL-2 (20 U/ml) in 100 μl culture for 72 h. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate. n.d., not detected.
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Figure 10: Comparison of cytokine expression among CD25+, CD25−CD45RBlow and CD25−CD45RBhigh populations of CD4+ T cells. (A) Purified CD4+CD25+, CD4+CD25− CD45RBlow and CD4+CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). 24 h later, cells were incubated with either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken Ig G, washed, and stained with Cy-Chrome™–conjugated streptavidin. Expression of cell surface–bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B–E) 105 CD4+ CD25+, CD4+ CD25−CD45RBlow and CD4+ CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml) and IL-2 (20 U/ml) in 100 μl culture for 72 h. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate. n.d., not detected.

Mentions: In the experiments shown above, we compared CD4+CD25+ T cells and CD4+CD25− T cells and showed that the former population is a high expresser of cell surface–bound and secreted TGF-β1. To exclude the possibility that this is simply because CD4+CD25+ T cells have been subject to prior stimulation through the TCR and that high level expression of TGF-β is simply a feature of previously stimulated T cells, we purified CD25+, CD25−CD45RBlow, and CD25−CD45RBhigh populations of CD4+ T cells. As reported previously 101123, most of the CD4+CD25+ population was CD45RBlow (data not shown). As shown in Fig. 10 A, stimulated CD4+CD25+ T cells expressed abundant cell surface-bound TGF-β, whereas stimulated CD25− CD45RBlow and CD25−CD45RBhigh T cells expressed only small amount of cell surface–bound TGF-β1. In addition, as shown in Fig. 10 B, CD4+CD25+ T cells also secreted significantly higher amount of TGF-β1 into the culture supernatant after stimulation than CD25−CD45RBlow and CD25−CD45RBhigh populations. In contrast, as shown in Fig. 10C–E, CD25−CD45RBlow T cells secreted huge amounts of IL-10, IL-4, and IFN-γ which far exceeded that produced by CD25+ and CD25− CD45RBhigh T cells. Finally, while CD4+CD25+ T cells did not produce high levels of IL-4 or IFN-γ, they did produce quite high amount of IL-10, albeit at levels significantly lower than that of CD4+CD25−CD45RBlow T cells. Taken together, these data show that the expression of high levels of TGF-β in both membrane-bound and soluble forms is a unique feature of CD4+CD25+ regulatory T cells.


Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta.

Nakamura K, Kitani A, Strober W - J. Exp. Med. (2001)

Comparison of cytokine expression among CD25+, CD25−CD45RBlow and CD25−CD45RBhigh populations of CD4+ T cells. (A) Purified CD4+CD25+, CD4+CD25− CD45RBlow and CD4+CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). 24 h later, cells were incubated with either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken Ig G, washed, and stained with Cy-Chrome™–conjugated streptavidin. Expression of cell surface–bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B–E) 105 CD4+ CD25+, CD4+ CD25−CD45RBlow and CD4+ CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml) and IL-2 (20 U/ml) in 100 μl culture for 72 h. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate. n.d., not detected.
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Figure 10: Comparison of cytokine expression among CD25+, CD25−CD45RBlow and CD25−CD45RBhigh populations of CD4+ T cells. (A) Purified CD4+CD25+, CD4+CD25− CD45RBlow and CD4+CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml), and IL-2 (20 U/ml). 24 h later, cells were incubated with either biotin-conjugated chicken anti–TGF-β1 or biotin-conjugated normal chicken Ig G, washed, and stained with Cy-Chrome™–conjugated streptavidin. Expression of cell surface–bound TGF-β1 is shown. Thick lines: anti–TGF-β; thin lines: normal chicken IgG. (B–E) 105 CD4+ CD25+, CD4+ CD25−CD45RBlow and CD4+ CD25−CD45RBhigh T cells were stimulated with plate-bound anti-CD3 (10 μg/ml) and anti–CTLA-4 (10 μg/ml), soluble anti-CD28 (2 μg/ml) and IL-2 (20 U/ml) in 100 μl culture for 72 h. The amount of TGF-β1 (B), IL-10 (C), IL-4 (D), and IFN-γ (E) in culture supernatant was measured by ELISA. In B, 1% Nutridoma/RPMI was used for culture media. The results shown represent the mean ± SEM of triplicate wells with each well measured in duplicate. n.d., not detected.
Mentions: In the experiments shown above, we compared CD4+CD25+ T cells and CD4+CD25− T cells and showed that the former population is a high expresser of cell surface–bound and secreted TGF-β1. To exclude the possibility that this is simply because CD4+CD25+ T cells have been subject to prior stimulation through the TCR and that high level expression of TGF-β is simply a feature of previously stimulated T cells, we purified CD25+, CD25−CD45RBlow, and CD25−CD45RBhigh populations of CD4+ T cells. As reported previously 101123, most of the CD4+CD25+ population was CD45RBlow (data not shown). As shown in Fig. 10 A, stimulated CD4+CD25+ T cells expressed abundant cell surface-bound TGF-β, whereas stimulated CD25− CD45RBlow and CD25−CD45RBhigh T cells expressed only small amount of cell surface–bound TGF-β1. In addition, as shown in Fig. 10 B, CD4+CD25+ T cells also secreted significantly higher amount of TGF-β1 into the culture supernatant after stimulation than CD25−CD45RBlow and CD25−CD45RBhigh populations. In contrast, as shown in Fig. 10C–E, CD25−CD45RBlow T cells secreted huge amounts of IL-10, IL-4, and IFN-γ which far exceeded that produced by CD25+ and CD25− CD45RBhigh T cells. Finally, while CD4+CD25+ T cells did not produce high levels of IL-4 or IFN-γ, they did produce quite high amount of IL-10, albeit at levels significantly lower than that of CD4+CD25−CD45RBlow T cells. Taken together, these data show that the expression of high levels of TGF-β in both membrane-bound and soluble forms is a unique feature of CD4+CD25+ regulatory T cells.

Bottom Line: In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4.In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression.Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
CD4(+)CD25(+) T cells have been identified as a population of immunoregulatory T cells, which mediate suppression of CD4(+)CD25(-) T cells by cell-cell contact and not secretion of suppressor cytokines. In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4. As in prior studies, we found that CD4(+)CD25(+) T cells suppress proliferation of CD4(+)CD25(-) T cells; however, we observed here that such suppression is abolished by the presence of anti-TGF-beta. In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression. Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface. This, plus the fact that we could find no evidence that a soluble factor mediates suppression, strongly suggests that CD4(+)CD25(+) T cells exert immunosuppression by a cell-cell interaction involving cell surface TGF-beta1.

Show MeSH
Related in: MedlinePlus