Hepatic stellate cells preferentially expand allogeneic CD4+ CD25+ FoxP3+ regulatory T cells in an IL-2-dependent manner

Abstract

Background: Organ transplantation has been successfully practiced for decades, but the outcome of cell transplantation remains disappointing. This is the case in animal models; liver allografts in mice are spontaneously accepted without requirement of immunosuppression, whereas hepatocyte transplants in the same combination are acutely rejected, apparently resulting from immune attacks because syngeneic hepatocyte transplants survive indefinitely. This suggests that liver nonparenchymal cells play an important role in protecting parenchymal cell from rejection. We have shown that hepatic stellate cells (HpSC), well known to participate in liver repairing and fibrosis, mediate potent immunomodulatory functions through induction of activated T-cell death.

Methods and results: Here, we report that HpSC acquired antigen presenting capacity after activated by interferon-gamma. In contrast to professional antigen-presenting cells dendritic cells that predominantly stimulated CD4+ T cells to generate CD25+ forkhead box P3 (Foxp3)- effector cells, HpSC selectively expanded CD4+ CD25+ Foxp3+ cells in an interleukin-2-dependent manner. These expanded CD4+ CD25+ Foxp3+ cells showed T regulatory cell (Treg) activity in effectively inhibiting T-cell proliferation in responses to anti-CD3 monoclonal antibody or alloantigens in a major histocompatibility complex nonspecific fashion. The Treg cells were expanded from the CD4+ CD25+ population with the help of interleukin-2, independent of B7-H1 and transforming growth factor-beta. Administration of HpSC into allogeneic recipients resulted in expansion of CD4+ CD25+ FoxP3+ cells in vivo.

Conclusion: Liver stromal HpSC acted as nonprofessional antigen-presenting cells, and preferentially expanded CD25+FoxP3+ Treg cells, which may contribute to immune regulation in the liver.

Figure 1. Hepatic stellate cells are capable of presenting antigens to allogeneic T cells

(A) Key molecule expression on hepatic stellate cells (SC), IFN-γ-stimulated hepatic stellate cells (γ-SC) and bone marrow-derived dendritic cells (DC) from B6 (H2^b) mice. Expression of MHC class I, II, CD40, CD80, CD86, B7-H1 and CD54 was displayed as filled histograms analyzed by flow cytometry (empty histograms were isotype controls). (B and C) γ-SC are capable of stimulating proliferative response in allogeneic T cells. (B) In a one way MLR assay, BABL/c (H2^d) spleen T cells (2 × 10⁵) were cultured with graded doses (as indicated) of irradiated SC, γ-SC (both 40 Gy) or DC (20 Gy) from B6 mice for 3 days. T cell proliferation was determined by thymidine uptake. (C) CFSE labeled CD4⁺ T cells (2 × 10⁵) purified from BABL/c spleen cells were cultured with irradiated SC, γ-SC or DC from B6 mice at a ratio of 10:1 for 3, 5 or 7 days. Proliferative response of CD4⁺ T cells was analyzed by CFSE dilution assay. (D) Cytokine profile. The supernatant cytokine levels were assessed following 5 days cultures of BALB/c CD4⁺ T cells with B6 SC or γ-SC (lower panel) or DC (upper panel) using CBA kits. (E) Foxp3 mRNA expression. RNA was isolated from BALB/c CD4⁺ T cells harvested at the end of 5 day-cultures with B6 SC, γ-SC or DC, and examined for Foxp3 mRNA expression by quantitative PCR. The data are representative of three separated experiments.

Figure 1. Hepatic stellate cells are capable of presenting antigens to allogeneic T cells

(A) Key molecule expression on hepatic stellate cells (SC), IFN-γ-stimulated hepatic stellate cells (γ-SC) and bone marrow-derived dendritic cells (DC) from B6 (H2^b) mice. Expression of MHC class I, II, CD40, CD80, CD86, B7-H1 and CD54 was displayed as filled histograms analyzed by flow cytometry (empty histograms were isotype controls). (B and C) γ-SC are capable of stimulating proliferative response in allogeneic T cells. (B) In a one way MLR assay, BABL/c (H2^d) spleen T cells (2 × 10⁵) were cultured with graded doses (as indicated) of irradiated SC, γ-SC (both 40 Gy) or DC (20 Gy) from B6 mice for 3 days. T cell proliferation was determined by thymidine uptake. (C) CFSE labeled CD4⁺ T cells (2 × 10⁵) purified from BABL/c spleen cells were cultured with irradiated SC, γ-SC or DC from B6 mice at a ratio of 10:1 for 3, 5 or 7 days. Proliferative response of CD4⁺ T cells was analyzed by CFSE dilution assay. (D) Cytokine profile. The supernatant cytokine levels were assessed following 5 days cultures of BALB/c CD4⁺ T cells with B6 SC or γ-SC (lower panel) or DC (upper panel) using CBA kits. (E) Foxp3 mRNA expression. RNA was isolated from BALB/c CD4⁺ T cells harvested at the end of 5 day-cultures with B6 SC, γ-SC or DC, and examined for Foxp3 mRNA expression by quantitative PCR. The data are representative of three separated experiments.

Figure 1. Hepatic stellate cells are capable of presenting antigens to allogeneic T cells

(A) Key molecule expression on hepatic stellate cells (SC), IFN-γ-stimulated hepatic stellate cells (γ-SC) and bone marrow-derived dendritic cells (DC) from B6 (H2^b) mice. Expression of MHC class I, II, CD40, CD80, CD86, B7-H1 and CD54 was displayed as filled histograms analyzed by flow cytometry (empty histograms were isotype controls). (B and C) γ-SC are capable of stimulating proliferative response in allogeneic T cells. (B) In a one way MLR assay, BABL/c (H2^d) spleen T cells (2 × 10⁵) were cultured with graded doses (as indicated) of irradiated SC, γ-SC (both 40 Gy) or DC (20 Gy) from B6 mice for 3 days. T cell proliferation was determined by thymidine uptake. (C) CFSE labeled CD4⁺ T cells (2 × 10⁵) purified from BABL/c spleen cells were cultured with irradiated SC, γ-SC or DC from B6 mice at a ratio of 10:1 for 3, 5 or 7 days. Proliferative response of CD4⁺ T cells was analyzed by CFSE dilution assay. (D) Cytokine profile. The supernatant cytokine levels were assessed following 5 days cultures of BALB/c CD4⁺ T cells with B6 SC or γ-SC (lower panel) or DC (upper panel) using CBA kits. (E) Foxp3 mRNA expression. RNA was isolated from BALB/c CD4⁺ T cells harvested at the end of 5 day-cultures with B6 SC, γ-SC or DC, and examined for Foxp3 mRNA expression by quantitative PCR. The data are representative of three separated experiments.

Figure 1. Hepatic stellate cells are capable of presenting antigens to allogeneic T cells

(A) Key molecule expression on hepatic stellate cells (SC), IFN-γ-stimulated hepatic stellate cells (γ-SC) and bone marrow-derived dendritic cells (DC) from B6 (H2^b) mice. Expression of MHC class I, II, CD40, CD80, CD86, B7-H1 and CD54 was displayed as filled histograms analyzed by flow cytometry (empty histograms were isotype controls). (B and C) γ-SC are capable of stimulating proliferative response in allogeneic T cells. (B) In a one way MLR assay, BABL/c (H2^d) spleen T cells (2 × 10⁵) were cultured with graded doses (as indicated) of irradiated SC, γ-SC (both 40 Gy) or DC (20 Gy) from B6 mice for 3 days. T cell proliferation was determined by thymidine uptake. (C) CFSE labeled CD4⁺ T cells (2 × 10⁵) purified from BABL/c spleen cells were cultured with irradiated SC, γ-SC or DC from B6 mice at a ratio of 10:1 for 3, 5 or 7 days. Proliferative response of CD4⁺ T cells was analyzed by CFSE dilution assay. (D) Cytokine profile. The supernatant cytokine levels were assessed following 5 days cultures of BALB/c CD4⁺ T cells with B6 SC or γ-SC (lower panel) or DC (upper panel) using CBA kits. (E) Foxp3 mRNA expression. RNA was isolated from BALB/c CD4⁺ T cells harvested at the end of 5 day-cultures with B6 SC, γ-SC or DC, and examined for Foxp3 mRNA expression by quantitative PCR. The data are representative of three separated experiments.

Figure 1. Hepatic stellate cells are capable of presenting antigens to allogeneic T cells

(A) Key molecule expression on hepatic stellate cells (SC), IFN-γ-stimulated hepatic stellate cells (γ-SC) and bone marrow-derived dendritic cells (DC) from B6 (H2^b) mice. Expression of MHC class I, II, CD40, CD80, CD86, B7-H1 and CD54 was displayed as filled histograms analyzed by flow cytometry (empty histograms were isotype controls). (B and C) γ-SC are capable of stimulating proliferative response in allogeneic T cells. (B) In a one way MLR assay, BABL/c (H2^d) spleen T cells (2 × 10⁵) were cultured with graded doses (as indicated) of irradiated SC, γ-SC (both 40 Gy) or DC (20 Gy) from B6 mice for 3 days. T cell proliferation was determined by thymidine uptake. (C) CFSE labeled CD4⁺ T cells (2 × 10⁵) purified from BABL/c spleen cells were cultured with irradiated SC, γ-SC or DC from B6 mice at a ratio of 10:1 for 3, 5 or 7 days. Proliferative response of CD4⁺ T cells was analyzed by CFSE dilution assay. (D) Cytokine profile. The supernatant cytokine levels were assessed following 5 days cultures of BALB/c CD4⁺ T cells with B6 SC or γ-SC (lower panel) or DC (upper panel) using CBA kits. (E) Foxp3 mRNA expression. RNA was isolated from BALB/c CD4⁺ T cells harvested at the end of 5 day-cultures with B6 SC, γ-SC or DC, and examined for Foxp3 mRNA expression by quantitative PCR. The data are representative of three separated experiments.

Figure 2. Hepatic stellate cells selectively expand CD25⁺FoxP3⁺ Treg cells

(A) Selective expansion of CD25⁺Foxp3⁺ cells by IFN-γ-stimulated hepatic stellate cells (γ-SC). CFSE labels CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured with irradiated (40 Gy) B6 (H2^b) SC, γ-SC at a ratio of 10:1 for 5 days. Fresh BALB/c CD4⁺ T cells, cultured alone or stimulated by irradiated (20 Gy) B6 dendritic cells (DC) at 10: 1 ratio for 5 days, served as controls. At the end of the culture, cells were double stained with mAbs to CD25 and Foxp3 molecules for FACS analysis. Cell expansion was determined by CFSE dilution assay in gated CD25⁺Foxp3⁺ cells (Upper panel). (B) Expanded CD4⁺CD25⁺ cells inhibit T cell proliferative responses. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells purified from a culture (5 days) of BALB/c CD4⁺ T cells with B6 γ-SC were used as the suppressors, and added into a culture of BALB/c CD4⁺ or CD4⁺CD25⁻ T cells (1×10⁵) at 1:1 ratio in the presence or absence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c (syngeneic) spleen cells. The inhibitory activity of suppressor cells was determined by [³H]TdR uptake by reporter cells. The CD4⁺ T cells alone without anti-CD3 mAb and CD4⁺CD25⁺ T cells alone with anti-CD3 were used as controls. (C) Increase in suppressor cell dose resulted in enhanced inhibitory effect. The number of γ-SC expanded CD4⁺CD25⁺ cells was adjusted to a suppressor: responder ratio of 2:1, the proliferation of responder cells was significantly enhanced (p<0.05). (D) CD4⁺CD25⁺ T cells expanded by DC were not suppressive. CD4⁺CD25⁺ T cells purified from a 5-day culture of BALB/c CD4⁺ T cells with B6 DC (DC/T 1:10) were added at 1:1 ratio into BALB/c CD4⁺ T cells in the presence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c spleen cells. The proliferative response was determined by [³H]TdR uptake. (E) The inhibitory activity of CD4⁺CD25⁺ cells is not MHC-specific. The CD4⁺CD25⁺ or CD4⁺CD25⁻ cells purified from a culture of BALB/c CD4⁺ T cells and γirradiated B6 γ-SC at a ratio of 10:1 for 5 days were added into a one way MLR culture in which proliferation of the CFSE labeled responder BALB/c T cells (1x10⁵) was elicited by irradiated B6 or C3H (H2^k, third party) spleen cells (the ratio of suppressor:stimulator:responder = 1:1:1). T cell proliferation was determined by CFSE dilution assay. The data are representative of two separated experiments.

Figure 2. Hepatic stellate cells selectively expand CD25⁺FoxP3⁺ Treg cells

(A) Selective expansion of CD25⁺Foxp3⁺ cells by IFN-γ-stimulated hepatic stellate cells (γ-SC). CFSE labels CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured with irradiated (40 Gy) B6 (H2^b) SC, γ-SC at a ratio of 10:1 for 5 days. Fresh BALB/c CD4⁺ T cells, cultured alone or stimulated by irradiated (20 Gy) B6 dendritic cells (DC) at 10: 1 ratio for 5 days, served as controls. At the end of the culture, cells were double stained with mAbs to CD25 and Foxp3 molecules for FACS analysis. Cell expansion was determined by CFSE dilution assay in gated CD25⁺Foxp3⁺ cells (Upper panel). (B) Expanded CD4⁺CD25⁺ cells inhibit T cell proliferative responses. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells purified from a culture (5 days) of BALB/c CD4⁺ T cells with B6 γ-SC were used as the suppressors, and added into a culture of BALB/c CD4⁺ or CD4⁺CD25⁻ T cells (1×10⁵) at 1:1 ratio in the presence or absence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c (syngeneic) spleen cells. The inhibitory activity of suppressor cells was determined by [³H]TdR uptake by reporter cells. The CD4⁺ T cells alone without anti-CD3 mAb and CD4⁺CD25⁺ T cells alone with anti-CD3 were used as controls. (C) Increase in suppressor cell dose resulted in enhanced inhibitory effect. The number of γ-SC expanded CD4⁺CD25⁺ cells was adjusted to a suppressor: responder ratio of 2:1, the proliferation of responder cells was significantly enhanced (p<0.05). (D) CD4⁺CD25⁺ T cells expanded by DC were not suppressive. CD4⁺CD25⁺ T cells purified from a 5-day culture of BALB/c CD4⁺ T cells with B6 DC (DC/T 1:10) were added at 1:1 ratio into BALB/c CD4⁺ T cells in the presence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c spleen cells. The proliferative response was determined by [³H]TdR uptake. (E) The inhibitory activity of CD4⁺CD25⁺ cells is not MHC-specific. The CD4⁺CD25⁺ or CD4⁺CD25⁻ cells purified from a culture of BALB/c CD4⁺ T cells and γirradiated B6 γ-SC at a ratio of 10:1 for 5 days were added into a one way MLR culture in which proliferation of the CFSE labeled responder BALB/c T cells (1x10⁵) was elicited by irradiated B6 or C3H (H2^k, third party) spleen cells (the ratio of suppressor:stimulator:responder = 1:1:1). T cell proliferation was determined by CFSE dilution assay. The data are representative of two separated experiments.

Figure 2. Hepatic stellate cells selectively expand CD25⁺FoxP3⁺ Treg cells

(A) Selective expansion of CD25⁺Foxp3⁺ cells by IFN-γ-stimulated hepatic stellate cells (γ-SC). CFSE labels CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured with irradiated (40 Gy) B6 (H2^b) SC, γ-SC at a ratio of 10:1 for 5 days. Fresh BALB/c CD4⁺ T cells, cultured alone or stimulated by irradiated (20 Gy) B6 dendritic cells (DC) at 10: 1 ratio for 5 days, served as controls. At the end of the culture, cells were double stained with mAbs to CD25 and Foxp3 molecules for FACS analysis. Cell expansion was determined by CFSE dilution assay in gated CD25⁺Foxp3⁺ cells (Upper panel). (B) Expanded CD4⁺CD25⁺ cells inhibit T cell proliferative responses. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells purified from a culture (5 days) of BALB/c CD4⁺ T cells with B6 γ-SC were used as the suppressors, and added into a culture of BALB/c CD4⁺ or CD4⁺CD25⁻ T cells (1×10⁵) at 1:1 ratio in the presence or absence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c (syngeneic) spleen cells. The inhibitory activity of suppressor cells was determined by [³H]TdR uptake by reporter cells. The CD4⁺ T cells alone without anti-CD3 mAb and CD4⁺CD25⁺ T cells alone with anti-CD3 were used as controls. (C) Increase in suppressor cell dose resulted in enhanced inhibitory effect. The number of γ-SC expanded CD4⁺CD25⁺ cells was adjusted to a suppressor: responder ratio of 2:1, the proliferation of responder cells was significantly enhanced (p<0.05). (D) CD4⁺CD25⁺ T cells expanded by DC were not suppressive. CD4⁺CD25⁺ T cells purified from a 5-day culture of BALB/c CD4⁺ T cells with B6 DC (DC/T 1:10) were added at 1:1 ratio into BALB/c CD4⁺ T cells in the presence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c spleen cells. The proliferative response was determined by [³H]TdR uptake. (E) The inhibitory activity of CD4⁺CD25⁺ cells is not MHC-specific. The CD4⁺CD25⁺ or CD4⁺CD25⁻ cells purified from a culture of BALB/c CD4⁺ T cells and γirradiated B6 γ-SC at a ratio of 10:1 for 5 days were added into a one way MLR culture in which proliferation of the CFSE labeled responder BALB/c T cells (1x10⁵) was elicited by irradiated B6 or C3H (H2^k, third party) spleen cells (the ratio of suppressor:stimulator:responder = 1:1:1). T cell proliferation was determined by CFSE dilution assay. The data are representative of two separated experiments.

Figure 2. Hepatic stellate cells selectively expand CD25⁺FoxP3⁺ Treg cells

(A) Selective expansion of CD25⁺Foxp3⁺ cells by IFN-γ-stimulated hepatic stellate cells (γ-SC). CFSE labels CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured with irradiated (40 Gy) B6 (H2^b) SC, γ-SC at a ratio of 10:1 for 5 days. Fresh BALB/c CD4⁺ T cells, cultured alone or stimulated by irradiated (20 Gy) B6 dendritic cells (DC) at 10: 1 ratio for 5 days, served as controls. At the end of the culture, cells were double stained with mAbs to CD25 and Foxp3 molecules for FACS analysis. Cell expansion was determined by CFSE dilution assay in gated CD25⁺Foxp3⁺ cells (Upper panel). (B) Expanded CD4⁺CD25⁺ cells inhibit T cell proliferative responses. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells purified from a culture (5 days) of BALB/c CD4⁺ T cells with B6 γ-SC were used as the suppressors, and added into a culture of BALB/c CD4⁺ or CD4⁺CD25⁻ T cells (1×10⁵) at 1:1 ratio in the presence or absence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c (syngeneic) spleen cells. The inhibitory activity of suppressor cells was determined by [³H]TdR uptake by reporter cells. The CD4⁺ T cells alone without anti-CD3 mAb and CD4⁺CD25⁺ T cells alone with anti-CD3 were used as controls. (C) Increase in suppressor cell dose resulted in enhanced inhibitory effect. The number of γ-SC expanded CD4⁺CD25⁺ cells was adjusted to a suppressor: responder ratio of 2:1, the proliferation of responder cells was significantly enhanced (p<0.05). (D) CD4⁺CD25⁺ T cells expanded by DC were not suppressive. CD4⁺CD25⁺ T cells purified from a 5-day culture of BALB/c CD4⁺ T cells with B6 DC (DC/T 1:10) were added at 1:1 ratio into BALB/c CD4⁺ T cells in the presence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c spleen cells. The proliferative response was determined by [³H]TdR uptake. (E) The inhibitory activity of CD4⁺CD25⁺ cells is not MHC-specific. The CD4⁺CD25⁺ or CD4⁺CD25⁻ cells purified from a culture of BALB/c CD4⁺ T cells and γirradiated B6 γ-SC at a ratio of 10:1 for 5 days were added into a one way MLR culture in which proliferation of the CFSE labeled responder BALB/c T cells (1x10⁵) was elicited by irradiated B6 or C3H (H2^k, third party) spleen cells (the ratio of suppressor:stimulator:responder = 1:1:1). T cell proliferation was determined by CFSE dilution assay. The data are representative of two separated experiments.

Figure 2. Hepatic stellate cells selectively expand CD25⁺FoxP3⁺ Treg cells

(A) Selective expansion of CD25⁺Foxp3⁺ cells by IFN-γ-stimulated hepatic stellate cells (γ-SC). CFSE labels CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured with irradiated (40 Gy) B6 (H2^b) SC, γ-SC at a ratio of 10:1 for 5 days. Fresh BALB/c CD4⁺ T cells, cultured alone or stimulated by irradiated (20 Gy) B6 dendritic cells (DC) at 10: 1 ratio for 5 days, served as controls. At the end of the culture, cells were double stained with mAbs to CD25 and Foxp3 molecules for FACS analysis. Cell expansion was determined by CFSE dilution assay in gated CD25⁺Foxp3⁺ cells (Upper panel). (B) Expanded CD4⁺CD25⁺ cells inhibit T cell proliferative responses. CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells purified from a culture (5 days) of BALB/c CD4⁺ T cells with B6 γ-SC were used as the suppressors, and added into a culture of BALB/c CD4⁺ or CD4⁺CD25⁻ T cells (1×10⁵) at 1:1 ratio in the presence or absence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c (syngeneic) spleen cells. The inhibitory activity of suppressor cells was determined by [³H]TdR uptake by reporter cells. The CD4⁺ T cells alone without anti-CD3 mAb and CD4⁺CD25⁺ T cells alone with anti-CD3 were used as controls. (C) Increase in suppressor cell dose resulted in enhanced inhibitory effect. The number of γ-SC expanded CD4⁺CD25⁺ cells was adjusted to a suppressor: responder ratio of 2:1, the proliferation of responder cells was significantly enhanced (p<0.05). (D) CD4⁺CD25⁺ T cells expanded by DC were not suppressive. CD4⁺CD25⁺ T cells purified from a 5-day culture of BALB/c CD4⁺ T cells with B6 DC (DC/T 1:10) were added at 1:1 ratio into BALB/c CD4⁺ T cells in the presence of anti-CD3 mAb (2 μg/ml) and irradiated BALB/c spleen cells. The proliferative response was determined by [³H]TdR uptake. (E) The inhibitory activity of CD4⁺CD25⁺ cells is not MHC-specific. The CD4⁺CD25⁺ or CD4⁺CD25⁻ cells purified from a culture of BALB/c CD4⁺ T cells and γirradiated B6 γ-SC at a ratio of 10:1 for 5 days were added into a one way MLR culture in which proliferation of the CFSE labeled responder BALB/c T cells (1x10⁵) was elicited by irradiated B6 or C3H (H2^k, third party) spleen cells (the ratio of suppressor:stimulator:responder = 1:1:1). T cell proliferation was determined by CFSE dilution assay. The data are representative of two separated experiments.

Figure 3. CD25⁺Foxp3⁺ cells are expanded from CD4⁺CD25⁺ population, which requires IL-2

(A) Neutralization of IL-2 eliminates expansion of CD25⁺FoxP3⁺ cells elicited by IFN-γstimulated hepatic stellate cells (γ-SC). CFSE labeled BALB/c (H-2^d) CD4⁺ T cells (2 × 10⁵) were cultured with irradiated (40 Gy) B6 (H-2^b) γ-SC at a ratio of 10:1 for 5 days in the presence of anti-IL-2 (10μg/ml). Rat IgG_2a was used as istotype control. Cells were double stained with anti-CD25 and -Foxp3, and analyzed by flow cytometry. Cell proliferation was assessed by CFSE dilution in CD25⁺Foxp3⁺ populations. (B) γ-SC expand CD25⁺FoxP3⁺ from CD4⁺CD25⁺ population, which requires IL-2. B6 γ-SC were cultured with 2×10⁵ CFSE labeled CD4⁺, CD4⁺CD25⁺ or CD4⁺CD25⁻ cells from BALB/c mice at a ratio of 1:10 for 5 days. Cells were then stained with anti-Foxp3. Proliferation was determined by CFSE dilution in Foxp3⁺ cell populations. To test the effect of IL-2 on Foxp3⁺ cell proliferation, exogenous IL-2 (100 U/ml) was added at the beginning of the cultures in CD4⁺CD25⁺ or CD4⁺CD25⁻ groups. (C) Expansion of CD25⁺Foxp3⁺ cells from CD4⁺CD25⁺ population is demonstrated by using congenic Thy1.1⁺ mice. Flow analysis showed the purity of CD4⁺CD25⁺ cells purified from BALB/c Thy1.1⁺ mice, and CD4⁺CD25⁻ cells purified from normal BALB/c (Thy1.1⁻) mice. Thy1.1⁺CD4⁺CD25⁺ cells were mixed with Thy1.1⁻CD4⁺CD25⁻ cells at a ratio of 1:10 (total 2 × 10⁵, histogram showed containing of 9.7% Thy1.1⁺ cells), and cultured with irradiated B6 γ-SC at 10:1 ratio for 5 days. Cells were further double stained with anti-Thy1.1 and -Foxp3 for flow analyses, showing that CD25⁺FoxP3⁺ cells was increased to 13.7%, among which 95.2% were Thy1.1⁺. The data are representative of three separated experiments.

Figure 3. CD25⁺Foxp3⁺ cells are expanded from CD4⁺CD25⁺ population, which requires IL-2

(A) Neutralization of IL-2 eliminates expansion of CD25⁺FoxP3⁺ cells elicited by IFN-γstimulated hepatic stellate cells (γ-SC). CFSE labeled BALB/c (H-2^d) CD4⁺ T cells (2 × 10⁵) were cultured with irradiated (40 Gy) B6 (H-2^b) γ-SC at a ratio of 10:1 for 5 days in the presence of anti-IL-2 (10μg/ml). Rat IgG_2a was used as istotype control. Cells were double stained with anti-CD25 and -Foxp3, and analyzed by flow cytometry. Cell proliferation was assessed by CFSE dilution in CD25⁺Foxp3⁺ populations. (B) γ-SC expand CD25⁺FoxP3⁺ from CD4⁺CD25⁺ population, which requires IL-2. B6 γ-SC were cultured with 2×10⁵ CFSE labeled CD4⁺, CD4⁺CD25⁺ or CD4⁺CD25⁻ cells from BALB/c mice at a ratio of 1:10 for 5 days. Cells were then stained with anti-Foxp3. Proliferation was determined by CFSE dilution in Foxp3⁺ cell populations. To test the effect of IL-2 on Foxp3⁺ cell proliferation, exogenous IL-2 (100 U/ml) was added at the beginning of the cultures in CD4⁺CD25⁺ or CD4⁺CD25⁻ groups. (C) Expansion of CD25⁺Foxp3⁺ cells from CD4⁺CD25⁺ population is demonstrated by using congenic Thy1.1⁺ mice. Flow analysis showed the purity of CD4⁺CD25⁺ cells purified from BALB/c Thy1.1⁺ mice, and CD4⁺CD25⁻ cells purified from normal BALB/c (Thy1.1⁻) mice. Thy1.1⁺CD4⁺CD25⁺ cells were mixed with Thy1.1⁻CD4⁺CD25⁻ cells at a ratio of 1:10 (total 2 × 10⁵, histogram showed containing of 9.7% Thy1.1⁺ cells), and cultured with irradiated B6 γ-SC at 10:1 ratio for 5 days. Cells were further double stained with anti-Thy1.1 and -Foxp3 for flow analyses, showing that CD25⁺FoxP3⁺ cells was increased to 13.7%, among which 95.2% were Thy1.1⁺. The data are representative of three separated experiments.

Figure 3. CD25⁺Foxp3⁺ cells are expanded from CD4⁺CD25⁺ population, which requires IL-2

(A) Neutralization of IL-2 eliminates expansion of CD25⁺FoxP3⁺ cells elicited by IFN-γstimulated hepatic stellate cells (γ-SC). CFSE labeled BALB/c (H-2^d) CD4⁺ T cells (2 × 10⁵) were cultured with irradiated (40 Gy) B6 (H-2^b) γ-SC at a ratio of 10:1 for 5 days in the presence of anti-IL-2 (10μg/ml). Rat IgG_2a was used as istotype control. Cells were double stained with anti-CD25 and -Foxp3, and analyzed by flow cytometry. Cell proliferation was assessed by CFSE dilution in CD25⁺Foxp3⁺ populations. (B) γ-SC expand CD25⁺FoxP3⁺ from CD4⁺CD25⁺ population, which requires IL-2. B6 γ-SC were cultured with 2×10⁵ CFSE labeled CD4⁺, CD4⁺CD25⁺ or CD4⁺CD25⁻ cells from BALB/c mice at a ratio of 1:10 for 5 days. Cells were then stained with anti-Foxp3. Proliferation was determined by CFSE dilution in Foxp3⁺ cell populations. To test the effect of IL-2 on Foxp3⁺ cell proliferation, exogenous IL-2 (100 U/ml) was added at the beginning of the cultures in CD4⁺CD25⁺ or CD4⁺CD25⁻ groups. (C) Expansion of CD25⁺Foxp3⁺ cells from CD4⁺CD25⁺ population is demonstrated by using congenic Thy1.1⁺ mice. Flow analysis showed the purity of CD4⁺CD25⁺ cells purified from BALB/c Thy1.1⁺ mice, and CD4⁺CD25⁻ cells purified from normal BALB/c (Thy1.1⁻) mice. Thy1.1⁺CD4⁺CD25⁺ cells were mixed with Thy1.1⁻CD4⁺CD25⁻ cells at a ratio of 1:10 (total 2 × 10⁵, histogram showed containing of 9.7% Thy1.1⁺ cells), and cultured with irradiated B6 γ-SC at 10:1 ratio for 5 days. Cells were further double stained with anti-Thy1.1 and -Foxp3 for flow analyses, showing that CD25⁺FoxP3⁺ cells was increased to 13.7%, among which 95.2% were Thy1.1⁺. The data are representative of three separated experiments.

Figure 4. Hepatic stellate cells-induce expansion of CD25⁺Foxp3⁺ CD4⁺ cells is not mediated by B7-H1 or TGFβ

CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured, at a ratio of 10:1 for 5 days, with irradiated (40 Gy) B6 (H2^b) IFN-γ-stimulated hepatic stellate cells (γ-SC). At the end of the culture, cells were double stained with mAbs against CD25 and Foxp3 for FACS analysis. (A) In the presence or absence of blocking antibody against B7-H1 (15 μg/ml) or TGFβ1 (10μg/ml). RatIgG_2a and mouse IgG were used as ispotype controls, respectively. γ-SC from B7-H1 knockout mice (B6 background) instead of that from wild type mice were also tested. (B) Inhibition of TGFβ receptor signaling does not affect HSC-induced Treg cell expansion. SB525334 alone (10 μM) and/or anti-TGFβ1 mAb (10μg/ml) were added at the beginning of culture. A representative Western blot depicting that the presence of SB525334 (10 μM, Sigma-Aldrich) completely inhibited expression of phosphorylated Smed2 in NIH3T3 cells (ATCC) induced by TGF-β (2 ng/ml). The data are representative of two separated experiments.

Figure 4. Hepatic stellate cells-induce expansion of CD25⁺Foxp3⁺ CD4⁺ cells is not mediated by B7-H1 or TGFβ

CD4⁺ T cells (2 × 10⁵) purified from BALB/c (H2^d) spleen cells were cultured, at a ratio of 10:1 for 5 days, with irradiated (40 Gy) B6 (H2^b) IFN-γ-stimulated hepatic stellate cells (γ-SC). At the end of the culture, cells were double stained with mAbs against CD25 and Foxp3 for FACS analysis. (A) In the presence or absence of blocking antibody against B7-H1 (15 μg/ml) or TGFβ1 (10μg/ml). RatIgG_2a and mouse IgG were used as ispotype controls, respectively. γ-SC from B7-H1 knockout mice (B6 background) instead of that from wild type mice were also tested. (B) Inhibition of TGFβ receptor signaling does not affect HSC-induced Treg cell expansion. SB525334 alone (10 μM) and/or anti-TGFβ1 mAb (10μg/ml) were added at the beginning of culture. A representative Western blot depicting that the presence of SB525334 (10 μM, Sigma-Aldrich) completely inhibited expression of phosphorylated Smed2 in NIH3T3 cells (ATCC) induced by TGF-β (2 ng/ml). The data are representative of two separated experiments.

Figure 5. Expansion of CD4⁺CD25⁺FoxP3⁺ cells in vivo by hepatic stellate cells

BALB/c (H2^d) mice were intravenously injected with B6 (H2^b) IFN-γ-stimulated hepatic stellate cells (γ-SC) (10⁶). The mice treated with SC, DC or PBS were used as controls. T cells were isolated from spleen or lymph nodes 5 days later, and were examined for Treg expansion by qPCR and FACS analysis. (A) Enhanced Foxp3 mRNA in lymph nodes, determined by qPCR. The data are expressed as mean ± 1SD of three experiments. (B) Increased CD25⁺FoxP3⁺ cells in the spleen CD4⁺ T cells. T cells purified from spleen cells were multiple-stained with anti-CD4, -CD25 and -FoxP3 mAbs. The incidences of CD25⁺FoxP3⁺ cells in CD4⁺ T cell populations were determined by FACS analysis. (C) Administration of γ-SC results in hyporesponsiveness of T cells to donor Ag. The isolated LN T cells (2 × 10⁵/well) from SC, γ-SC, DC or PBS (none) treated BALB/c mice were restimulated with irradiated (20 Gy) B6 spleen cells at a ratio of 1:1 in triplicate plates for 3 days. The proliferative response of the responder cells was determined by thymidine uptake. The data are representative of two separated experiments.

Figure 5. Expansion of CD4⁺CD25⁺FoxP3⁺ cells in vivo by hepatic stellate cells

BALB/c (H2^d) mice were intravenously injected with B6 (H2^b) IFN-γ-stimulated hepatic stellate cells (γ-SC) (10⁶). The mice treated with SC, DC or PBS were used as controls. T cells were isolated from spleen or lymph nodes 5 days later, and were examined for Treg expansion by qPCR and FACS analysis. (A) Enhanced Foxp3 mRNA in lymph nodes, determined by qPCR. The data are expressed as mean ± 1SD of three experiments. (B) Increased CD25⁺FoxP3⁺ cells in the spleen CD4⁺ T cells. T cells purified from spleen cells were multiple-stained with anti-CD4, -CD25 and -FoxP3 mAbs. The incidences of CD25⁺FoxP3⁺ cells in CD4⁺ T cell populations were determined by FACS analysis. (C) Administration of γ-SC results in hyporesponsiveness of T cells to donor Ag. The isolated LN T cells (2 × 10⁵/well) from SC, γ-SC, DC or PBS (none) treated BALB/c mice were restimulated with irradiated (20 Gy) B6 spleen cells at a ratio of 1:1 in triplicate plates for 3 days. The proliferative response of the responder cells was determined by thymidine uptake. The data are representative of two separated experiments.

Figure 5. Expansion of CD4⁺CD25⁺FoxP3⁺ cells in vivo by hepatic stellate cells

BALB/c (H2^d) mice were intravenously injected with B6 (H2^b) IFN-γ-stimulated hepatic stellate cells (γ-SC) (10⁶). The mice treated with SC, DC or PBS were used as controls. T cells were isolated from spleen or lymph nodes 5 days later, and were examined for Treg expansion by qPCR and FACS analysis. (A) Enhanced Foxp3 mRNA in lymph nodes, determined by qPCR. The data are expressed as mean ± 1SD of three experiments. (B) Increased CD25⁺FoxP3⁺ cells in the spleen CD4⁺ T cells. T cells purified from spleen cells were multiple-stained with anti-CD4, -CD25 and -FoxP3 mAbs. The incidences of CD25⁺FoxP3⁺ cells in CD4⁺ T cell populations were determined by FACS analysis. (C) Administration of γ-SC results in hyporesponsiveness of T cells to donor Ag. The isolated LN T cells (2 × 10⁵/well) from SC, γ-SC, DC or PBS (none) treated BALB/c mice were restimulated with irradiated (20 Gy) B6 spleen cells at a ratio of 1:1 in triplicate plates for 3 days. The proliferative response of the responder cells was determined by thymidine uptake. The data are representative of two separated experiments.