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Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells.

Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, Roncarolo MG - J. Exp. Med. (2002)

Bottom Line: None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta.Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25.Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan 20132, Italy.

ABSTRACT
T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4(+) T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-beta. The relationship between CD25(+)CD4(+) T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25(+)CD4(+) T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25(-)CD4(+) T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25(+)CD4(+) T cells. Cloned human CD25(+)CD4(+) T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta. Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.

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Tr1 cells can be differentiated from CD25−CD4+ T cells. CD4+ T cells were purified, depleted of CD25+ cells, and activated by anti-CD3 mAbs cross-linked on CD32+ CD80+CD58+ L-cells in the absence (−) or presence of IL-10 plus IFN-α. After two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis (A and B). In B the average percent positive cells from five independent experiments is shown. * indicates a statistically significant difference in the percentage of cytokine producing cells in cultures with IL-10 plus IFN-α, in comparison to those in the absence of cytokines. Error bars represent the SEM. In parallel, polarized T cells were tested for their ability to suppress the proliferation of naive autologous CD4+ T cells to alloantigens. Increasing numbers of cells which had been differentiated in the absence (−) or presence of IL-10 and IFN-α were added to the primary MLR, and after 4 d of culture, [3H]thymidine was added for an additional 16 h. One representative experiment is shown in C. Numbers above the bars in cultures with IL-10 and IFN-α represent the percent inhibition in comparison to the primary MLR alone. D represents the average reduction in proliferation of primary MLRs induced by CD25−CD4+ T cells differentiated in IL-10 and IFN-α in five independent experiments. * indicates a statistically significant (P ≤ 0.05) reduction in proliferation in comparison to primary MLRs in the absence of T cells differentiated in IL-10 and IFN-α.
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fig2: Tr1 cells can be differentiated from CD25−CD4+ T cells. CD4+ T cells were purified, depleted of CD25+ cells, and activated by anti-CD3 mAbs cross-linked on CD32+ CD80+CD58+ L-cells in the absence (−) or presence of IL-10 plus IFN-α. After two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis (A and B). In B the average percent positive cells from five independent experiments is shown. * indicates a statistically significant difference in the percentage of cytokine producing cells in cultures with IL-10 plus IFN-α, in comparison to those in the absence of cytokines. Error bars represent the SEM. In parallel, polarized T cells were tested for their ability to suppress the proliferation of naive autologous CD4+ T cells to alloantigens. Increasing numbers of cells which had been differentiated in the absence (−) or presence of IL-10 and IFN-α were added to the primary MLR, and after 4 d of culture, [3H]thymidine was added for an additional 16 h. One representative experiment is shown in C. Numbers above the bars in cultures with IL-10 and IFN-α represent the percent inhibition in comparison to the primary MLR alone. D represents the average reduction in proliferation of primary MLRs induced by CD25−CD4+ T cells differentiated in IL-10 and IFN-α in five independent experiments. * indicates a statistically significant (P ≤ 0.05) reduction in proliferation in comparison to primary MLRs in the absence of T cells differentiated in IL-10 and IFN-α.

Mentions: Activation of naive CD4+ T cells from cord blood or peripheral blood with anti-CD3 mAbs, cross-linked onto L-cells expressing human CD32, CD58, and CD80, in the presence of IL-10 and IFN-α, resulted in T cell lines containing IL-10–producing cells which suppressed antigen-specific responses in vitro (33). To further investigate whether CD25+CD4+ T cells may represent the precursors of IL-10–producing CD4+ T cells, or be required for the in vitro differentiation of IL-10–producing cells, CD4+ T cells depleted of CD25+ cells were tested in this in vitro differentiation system (33). As shown in Fig. 2 A, a significant increase in IL-10–producing cells was observed in cultures of CD25−CD4+ T cells which had been differentiated in the presence of IL-10 and IFN-α, in comparison to cultures in the absence of polarizing cytokines. Importantly, as Tr1 cells also produce IFN-γ (8, 33), a majority of the IL-10–producing cells also produced IFN-γ. When data from five independent experiments were combined, we observed a significant increase in the percentage of cells expressing IL-10, and expressing both IL-10 and IFN-γ, in cultures of CD25−CD4+ T cells differentiated in the presence of IL-10 and IFN-α, in comparison to control cultures of CD25−CD4+ T cells differentiated in the absence of these cytokines (Fig. 2 B). Cultures of CD25−CD4+ T cells differentiated with IL-10 and IFN-α also displayed a significant reduction in the number of IL-4–producing cells, while the numbers of IL-2 and IFN-γ–producing cells were equivalent to those observed in the absence of IL-10 and IFN-α. Thus, similarly to CD4+CD45RO− T cells (33), CD25−CD4+ T cells from peripheral blood can differentiate into IL-10–producing Tr1 cells in the presence of IL-10 and IFN-α.


Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells.

Levings MK, Sangregorio R, Sartirana C, Moschin AL, Battaglia M, Orban PC, Roncarolo MG - J. Exp. Med. (2002)

Tr1 cells can be differentiated from CD25−CD4+ T cells. CD4+ T cells were purified, depleted of CD25+ cells, and activated by anti-CD3 mAbs cross-linked on CD32+ CD80+CD58+ L-cells in the absence (−) or presence of IL-10 plus IFN-α. After two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis (A and B). In B the average percent positive cells from five independent experiments is shown. * indicates a statistically significant difference in the percentage of cytokine producing cells in cultures with IL-10 plus IFN-α, in comparison to those in the absence of cytokines. Error bars represent the SEM. In parallel, polarized T cells were tested for their ability to suppress the proliferation of naive autologous CD4+ T cells to alloantigens. Increasing numbers of cells which had been differentiated in the absence (−) or presence of IL-10 and IFN-α were added to the primary MLR, and after 4 d of culture, [3H]thymidine was added for an additional 16 h. One representative experiment is shown in C. Numbers above the bars in cultures with IL-10 and IFN-α represent the percent inhibition in comparison to the primary MLR alone. D represents the average reduction in proliferation of primary MLRs induced by CD25−CD4+ T cells differentiated in IL-10 and IFN-α in five independent experiments. * indicates a statistically significant (P ≤ 0.05) reduction in proliferation in comparison to primary MLRs in the absence of T cells differentiated in IL-10 and IFN-α.
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Related In: Results  -  Collection

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fig2: Tr1 cells can be differentiated from CD25−CD4+ T cells. CD4+ T cells were purified, depleted of CD25+ cells, and activated by anti-CD3 mAbs cross-linked on CD32+ CD80+CD58+ L-cells in the absence (−) or presence of IL-10 plus IFN-α. After two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis (A and B). In B the average percent positive cells from five independent experiments is shown. * indicates a statistically significant difference in the percentage of cytokine producing cells in cultures with IL-10 plus IFN-α, in comparison to those in the absence of cytokines. Error bars represent the SEM. In parallel, polarized T cells were tested for their ability to suppress the proliferation of naive autologous CD4+ T cells to alloantigens. Increasing numbers of cells which had been differentiated in the absence (−) or presence of IL-10 and IFN-α were added to the primary MLR, and after 4 d of culture, [3H]thymidine was added for an additional 16 h. One representative experiment is shown in C. Numbers above the bars in cultures with IL-10 and IFN-α represent the percent inhibition in comparison to the primary MLR alone. D represents the average reduction in proliferation of primary MLRs induced by CD25−CD4+ T cells differentiated in IL-10 and IFN-α in five independent experiments. * indicates a statistically significant (P ≤ 0.05) reduction in proliferation in comparison to primary MLRs in the absence of T cells differentiated in IL-10 and IFN-α.
Mentions: Activation of naive CD4+ T cells from cord blood or peripheral blood with anti-CD3 mAbs, cross-linked onto L-cells expressing human CD32, CD58, and CD80, in the presence of IL-10 and IFN-α, resulted in T cell lines containing IL-10–producing cells which suppressed antigen-specific responses in vitro (33). To further investigate whether CD25+CD4+ T cells may represent the precursors of IL-10–producing CD4+ T cells, or be required for the in vitro differentiation of IL-10–producing cells, CD4+ T cells depleted of CD25+ cells were tested in this in vitro differentiation system (33). As shown in Fig. 2 A, a significant increase in IL-10–producing cells was observed in cultures of CD25−CD4+ T cells which had been differentiated in the presence of IL-10 and IFN-α, in comparison to cultures in the absence of polarizing cytokines. Importantly, as Tr1 cells also produce IFN-γ (8, 33), a majority of the IL-10–producing cells also produced IFN-γ. When data from five independent experiments were combined, we observed a significant increase in the percentage of cells expressing IL-10, and expressing both IL-10 and IFN-γ, in cultures of CD25−CD4+ T cells differentiated in the presence of IL-10 and IFN-α, in comparison to control cultures of CD25−CD4+ T cells differentiated in the absence of these cytokines (Fig. 2 B). Cultures of CD25−CD4+ T cells differentiated with IL-10 and IFN-α also displayed a significant reduction in the number of IL-4–producing cells, while the numbers of IL-2 and IFN-γ–producing cells were equivalent to those observed in the absence of IL-10 and IFN-α. Thus, similarly to CD4+CD45RO− T cells (33), CD25−CD4+ T cells from peripheral blood can differentiate into IL-10–producing Tr1 cells in the presence of IL-10 and IFN-α.

Bottom Line: None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta.Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25.Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Via Olgettina 58, Milan 20132, Italy.

ABSTRACT
T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4(+) T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-beta. The relationship between CD25(+)CD4(+) T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25(+)CD4(+) T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25(-)CD4(+) T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25(+)CD4(+) T cells. Cloned human CD25(+)CD4(+) T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta. Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.

Show MeSH
Related in: MedlinePlus