Induction of CD4+CD25+ regulatory T cells by copolymer-I through activation of transcription factor Foxp3 - PubMed (original) (raw)

Comparative Study

. 2005 May 3;102(18):6449-54.

doi: 10.1073/pnas.0502187102. Epub 2005 Apr 25.

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Comparative Study

Induction of CD4+CD25+ regulatory T cells by copolymer-I through activation of transcription factor Foxp3

Jian Hong et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Copolymer-I (COP-I) has unique immune regulatory properties and is a treatment option for multiple sclerosis (MS). This study revealed that COP-I induced the conversion of peripheral CD4+CD25- to CD4+CD25+ regulatory T cells through the activation of transcription factor Foxp3. COP-I treatment led to a significant increase in Foxp3 expression in CD4+ T cells in MS patients whose Foxp3 expression was reduced at baseline. CD4+CD25+ T cell lines generated by COP-I expressed high levels of Foxp3 that correlated with an increased regulatory potential. Furthermore, we demonstrated that the induction of Foxp3 in CD4+ T cells by COP-I was mediated through its ability to produce IFN-gamma and, to a lesser degree, TGF-beta1, as shown by antibody blocking and direct cytokine induction of Foxp3 expression in T cells. It was evident that in vitro treatment and administration with COP-I significantly raised the level of Foxp3 expression in CD4+ T cells and promoted conversion of CD4+CD25+ regulatory T cells in wild-type B6 mice but not in IFN-gamma knockout mice. This study provides evidence for the role and mechanism of action of COP-I in the induction of CD4+CD25+ regulatory T cells in general and its relevance to the treatment of MS.

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Figures

Fig. 1.

Fig. 1.

Induction of Foxp3 mRNA expression by COP-I as a function of time and proliferation rate of PBMC in response to COP-I. (A) PBMC preparations derived from 10 randomly selected healthy individuals were cultured, respectively, in the presence or absence of 40 μg/ml COP-I in complete RPMI medium 1640. Cells were collected at the indicated induction time over a span of 7 days and analyzed for mRNA expression of Foxp3 by real-time PCR. (B) CD4+ T cell preparations purified from the same PBMC specimens described above were cultured in the presence or absence (medium control) of COP-I, respectively, for 4 days. Mixed irrelevant peptides (see Materials and Methods) were used at the same concentration as the control. Cells were harvested for Foxp3 expression by real-time PCR. Data are given as relative mRNA expression. *, Significant statistical differences between COP-I-treated cells and controls (P < 0.01).

Fig. 2.

Fig. 2.

Conversion of CD4+CD25- T cells to CD4+CD25+ regulatory T cells. CD4+CD25- T cell preparations were purified from six healthy individuals and cultured with COP-I or the peptide control, respectively, in the presence of irradiated autologous PBMC as a source of antigen-presenting cells. Cells were collected on day 4 and analyzed for Foxp3 expression in refractionated CD4+CD25- and CD4+CD25+ T cell subsets by real-time PCR (A) and the inhibition on autologous T cell proliferation induced by anti-CD3/CD28 antibodies in inhibition assays (B). *, Significant statistical differences between T cells treated with COP-I and those treated with the peptide control (P < 0.05).

Fig. 3.

Fig. 3.

Immunoblot analysis of Foxp3 expression in CD4+CD25+ T cells after COP-I treatment. PBMC were treated with 40 μg/ml COP-I or control peptides for 4 days. The resulting CD4+CD25+ T cells were subsequently purified and lysed. Lysate was then subjected to 10% SDS/PAGE and followed by immunoblot analysis with an anti-human Foxp3 antibody. An antibody to human β-actin was used as a control.

Fig. 4.

Fig. 4.

Cytokine profile of PBMC in response to COP-I stimulation and antibody blocking experiments. (A) PBMC preparations derived from healthy individuals were cultured in the presence of COP-I or the peptide control at a concentration of 40 μg/ml. Culture supernatants were collected on day 4 and analyzed for the production of the indicated cytokines by ELISA. Data are presented as the mean cytokine concentrations of six individual samples. (B) In parallel experiments, the same PBMC preparations were cultured with 40 μg/ml COP-I in the presence of the indicated purified monoclonal antibodies used at concentrations of 50 ng/ml to 10 μg/ml as instructed, respectively. Cells were collected on day 4 and analyzed for Foxp3 expression by real-time PCR. *, Statistical differences (P < 0.05).

Fig. 5.

Fig. 5.

The effect of IFN-γ on the expression of Foxp3 in human PBMC. PBMC preparations were obtained from 10 healthy individuals and cultured in the presence or absence of the indicated recombinant cytokines at a concentration of 25 ng/ml for 72 h. (A) The resulting cells were analyzed for the expression of Foxp3 by real-time PCR. The data are presented as relative expression of Foxp3. CD4+CD25- T cells were purified from the same PBMC cultured in the presence of recombinant IFN-γ at the indicated concentrations for 72 h. (B) mRNA expression of Foxp3 in the resulting T cells was measured under the same experimental conditions. CD4+CD25- T cells were cultured in the presence of human recombinant IFN-γ (25 ng/ml) and a monoclonal antibody to human IFN-γ (10 μg/ml). An isotype-matched antibody was used at the same concentration as a control. (C) The resulting T cells were analyzed for mRNA expression of Foxp3 by real-time PCR. The results were reproducible in at least three independent experiments. *, Statistical difference between IFN-γ and medium control (P < 0.01); **, statistical difference between anti-IFN-γ and the controls (P < 0.05).

Fig. 6.

Fig. 6.

Induction of Foxp3 expression in T cells by in vitro treatment with COP-I in wild-type and IFN-γ-deficient mice. Splenocytes were derived from wild-type mice (A) and IFN-γ knockout mice (B) of the B6/C57 background. CD4+CD25- T cells were subsequently isolated by magnetic bead separation and cultured in the presence or absence of 40 μg/ml COP-I and irradiated splenocytes predepleted for T cells as a source of antigen-presenting cells. The resulting T cells were collected for Foxp3 mRNA expression on day 4. *, Statistical significance between the COP-I untreated group and the COP-I-induced group (P < 0.01).

Fig. 7.

Fig. 7.

Induction of CD4+CD25+ regulatory T cells in wild-type and IFN-γ knockout mice in response to administration of COP-I. Wild-type and IFN-γ knockout mice were administered COP-I at 5 mg per mouse, which represented an effective dosage demonstrated in previous studies (39). Mice were killed on the indicated days. Splenocytes were obtained and analyzed ex vivo for Foxp3 expression in purified CD4+CD25- and CD4+CD25+ T cell subsets by real-time PCR (A), the proliferative response to anti-CD3/CD28 antibodies (B), and the inhibition on syngeneic T cell proliferation induced by anti-CD3/CD28 antibodies in inhibition assays (C). *, Significant statistical differences between posttreated and pretreated CD4+CD25+ T cells (P < 0.05).

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