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

Jian Hong ^*, Ningli Li , Xuejun Zhang , Biao Zheng , and Jingwu Z. Zhang ^{*, , , , ¶}

Joint Immunology Laboratory of Institute of Health Sciences and Shanghai Institute of Immunology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Second Medical University, Shanghai 200025, China; Departments of ^*Neurology and Immunology, Baylor College of Medicine, Houston, TX 77030; and E-Institute of Shanghai Universities, Shanghai 200025, China

Communicated by Zhu Chen, Shanghai Second Medical University, Shanghai, China, March 27, 2005 (received for review December 3, 2004)

	Abstract

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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- and, to a lesser degree, TGF-1, 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- 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.

IFN- | multiple sclerosis

In this study, we examined the proposed hypothesis that the unique properties of COP-I in the activation of T cells may induce CD4⁺CD25⁺ regulatory T cell responses. The hypothesis was prompted based on our initial discovery that COP-I was able to induce the expression of transcription factor Foxp3 in CD4⁺ T cells, which is associated with CD4⁺CD25⁺ regulatory T cells (19-21). A potential role of COP-I in the induction of CD4⁺CD25⁺ regulatory T cell response is particularly relevant to MS because they have been recognized recently as an important regulatory component that keeps autoreactive T cells in check (19-22). Significant deficiencies in the number or function of these regulatory T cells have been found to correlate with several autoimmune conditions, including MS (23-25). CD4⁺CD25⁺ regulatory T cells can be distinguished from other CD4⁺ activated T cells of nonregulatory functions present in the CD4⁺CD25⁺ T cell pool by the expression of transcription factor Foxp3 (26). Gene transfer of Foxp3 converts naive T cells toward a regulatory T cell phenotype similar to that of naturally occurring CD4⁺ regulatory T cells (19, 27). Experiments were performed here to investigate whether COP-I was able to induce conversion of peripheral CD4⁺CD25^- T cells to CD4⁺CD25⁺ regulatory T cells through the activation of Foxp3 in human and animal systems. Foxp3 expression and regulatory function of T cells were also analyzed ex vivo in MS patients with or without COP-I treatment and in mice administered with COP-I. Human COP-I-specific, short-term T cell lines were generated and characterized. The study described here has provided evidence indicating the role of COP-I in the induction of CD4⁺CD25⁺ regulatory T cells through the activation of Foxp3.

	Materials and Methods

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Cell Stimulation. Fresh peripheral blood mononuclear cells (PBMC) were isolated from blood specimens by Ficoll hypaque separation. PBMC (2 x 10⁵) or purified T cells (1 x 10⁵) were cultured in the presence or absence of 40 µg/ml COP-I alone or 40 µg/ml COP-I with irradiated T cell-depleted PBMC as a source of antigen-presenting cells (2 x 10⁵) in complete RPMI medium 1640 in U-bottom 96-well plates at 37°C in 5% CO₂.On day 4 of the culture, an aliquot of T cells was harvested for Foxp3 gene expression by real-time PCR analysis. Supernatants were collected for cytokine detection, and 1-µCi (1 Ci = 37 GBq) [³H]thymidine (Amersham Pharmacia) was added to the remaining cultures in the last 6 h of culture before cell harvesting.

Foxp3 mRNA Expression by Real-Time PCR. Quantitative real-time RT-PCR was performed on a Prism 7000 sequence detection system (Applied Biosystems). Hypoxanthine phosphoribosyltransferase was used as a reference for sample normalization. Total RNA isolated from PBMC or purified T cells were reverse-transcribed into cDNA by using random hexamer. Human Foxp3 primers (forward, 5'-CAC CTG GCT GGG AAA ATG G-3'; reverse, 5'-GGA GCC CTT GTC GGA TGA T-3') and TaqMan minor groove binder probe (5'-FAM-ACT GAC CAA GGC TTC AT-3') sequences were designed with the PRIMER EXPRESS application program (Applied Biosystems). Mouse Foxp3 primers and probe and all hypoxanthine phosphoribosyltransferase primers and probe were purchased as forms of Assay-on-Demand (Applied Biosystems). The detailed amplification protocol used here is described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. A representative specific amplification of Foxp3 mRNA derived from a human CD4⁺CD25⁺ T cell preparation is shown in Fig. 8, which is published as supporting information on the PNAS web site.

Isolation of Human CD4⁺CD25⁺ Regulatory T Cells. Isolation of human CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25^- T cells was performed by using a human regulatory T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to manufacturer's instructions. Briefly, CD4⁺ T cells were first isolated through negative selection by removing all other cell types. Preisolated CD4⁺ T cells were incubated with 10 µl of magnetic beads conjugated with anti-CD25 antibody (for 10⁷ cells) to separate CD4⁺CD25⁺ and CD4⁺CD25^- T cell populations. The purity of the resulting T cell populations was confirmed to be >97% by flow cytometry.

Generation of Specific T Cell Lines. PBMC were initially seeded out in 96-well round-bottom plates at 20,000 cells per well with irradiated autologous PBMC (100,000 cells per well) as accessory cells in 10% FBS RPMI medium 1640 in the presence of 40 µg/ml COP-I. Cultures were supplemented with IL-2 at 50 units/ml after 48 h. After 7 days, all cultures per wells were assayed for specific reactivity to COP-I in proliferation assays. Briefly, each well was split into four aliquots (10⁴ cells per aliquot) and cultured in duplicate with 10⁵ irradiated autologous PBMC in the presence and absence of COP-I (40 µg/ml). Cultures were kept for 3 days and pulsed with [³H]thymidine at 1 µCi per well during the last 16 h of culture. A positive T cell line was defined as specific for COP-I when cpm were at 1,500 and exceeded the reference cpm (in the absence of COP-I) at least 3-fold. COP-I reactive T cell lines were expanded by restimulation with COP-I under the same conditions described above.

Inhibition Assay. CD4⁺CD25^- T cells (responder) and CD4⁺CD25⁺ T cells (inhibitor) were cocultured at 5 x 10³ per well in the presence or absence of anti-CD3 and anti-CD28 monoclonal antibodies in U-bottom 96-well plates at a responder to inhibitor ratio of 1:1. All cells were cultured in the presence of 10⁵ irradiated accessory cells. At day 7, cell proliferation was measured as described above. The inhibition was calculated as follows: [1 - (experimental cpm per control cpm)] x 100%.

Antibody Blocking Experiments. PBMC were plated out in 96-well plates at 10⁵ cells per well and stimulated with 40 µg/ml COP-I in the presence or absence of the indicated blocking antibodies to various cytokines. The final concentrations of the antibodies as suggested by the manufacturers were as follows: 1 µg/ml for antibodies to IL-8, TNF-, IFN-, and IL-10, 50 ng/ml for IL-1 antibody and 10 µg/ml for TGF-1 antibody. Cells were harvested on day 4 for Foxp3 expression.

Immunization and Mouse T Cell Preparation. Wild-type and IFN- gene knockout C57BL/6 mice were obtained from The Jackson Laboratory. Wild-type and IFN- gene knockout mice were injected s.c. with 1.6 mg of COP-I per injection at day 0, day 2, and day 4 for a total of three injections. Mice were killed at a 4-day interval for the isolation of CD4⁺CD25⁺ T cells. Spleens were gently minced in complete medium containing 10% FBS and CD4⁺ T cells were isolated by using a mouse CD4⁺ T cell negative selection kit (Miltenyi Biotec). T cell-depleted splenocytes of wild-type mice were used as antigen-presenting cells as indicated. To isolate CD4⁺CD25^- T cells, anti-CD25 antibody (10 µl for 10⁷ T cells) conjugated with microbeads was incubated with preselected CD4⁺ T cells before separation, to yield a purity of >95% of CD4⁺CD25^- T cells. For recovery of CD4⁺CD25⁺ T cells, the column bound T cells were flushed off with cold medium. The purity of CD4⁺CD25⁺ T cells was always >95%.

Supporting Information. For further information, see Supporting Materials and Methods and Fig. 8; see also Figs. 9 and 10, which are published as supporting information on the PNAS web site.

	Results

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Induction of Transcription Factor Foxp3 Expression in Human CD4⁺ T Cells by COP-I. After the initial discovery that COP-I induced the expression of transcription factor Foxp3 in human PBMC, the time course of induction of Foxp3 expression in PBMC derived from healthy individuals was evaluated in response to COP-I stimulation by real-time PCR analysis. As shown in Fig. 1A, the peak expression of Foxp3 occurred 4 days after exposed to COP-I. The effect of COP-I on the induction of Foxp3 expression correlated closely with the rate of cell proliferation in PBMC induced by COP-I (data not shown). Furthermore, the observed effect appeared specific for COP-I as the control peptides, although stimulatory to some PBMC preparations, did not alter Foxp3 expression in the same PBMC preparations (Fig. 1 A). Similar results were obtained when purified CD4⁺ T cells were treated with COP-I under the same experimental conditions (Fig. 1B). It was shown that COP-I induced the expression of Foxp3 predominantly in CD4⁺ T cell population of both CD45RA and CD45RO phenotypes (Fig. 9).

View larger version (13K): [in this window] [in a new window]   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).

Conversion of Peripheral CD4⁺CD25^- T Cells to CD4⁺CD25⁺ Regulatory T Cells by COP-I Through the Activation of Foxp3. We then examined whether the induction of Foxp3 expression in CD4⁺ T cells by COP-I represented conversion of peripheral CD4⁺CD25^- T cells to CD4⁺CD25⁺ regulatory T cells. To this end, human CD4⁺CD25-T cells were purified by magnetic bead separation and exposed to COP-I. CD4⁺CD25⁺ T cells rose from background value of 7.99 ± 0.28% to 23.36 ± 0.69% after CD4⁺CD25^- T cell preparations were exposed to COP-I compared with 16.08 ± 0.31% in those treated with the control (P < 0.05, data not shown). In vitro treatment of CD4⁺CD25^- T cells with COP-I led to a significantly elevated level of Foxp3 expression in resulting CD4⁺CD25⁺ T cells (Fig. 2A, P < 0.05). CD4⁺CD25⁺ T cells converted from the CD4⁺CD25^- T cell fractions by COP-I exhibited considerable inhibitory activities on T cell proliferation induced by anti-CD3/CD28 antibodies (Fig. 2B). The increased Foxp3 expression was confirmed in parallel by immunoblot analysis (Fig. 3). The results indicate that COP-I induced conversion of CD4⁺CD25^- to CD4⁺CD25⁺ T cells of regulatory function. Furthermore, human T cell lines were generated from healthy individuals by repeated stimulation with COP-I and characterized for the reactivity, Foxp3 expression and the inhibitory rate. As shown in Table 1, T cell lines reactive to COP-I had high expression of Foxp3 that was roughly 10 times higher than that seen in control T cell lines or T cells stimulated one time with COP-I and displayed considerable inhibitory properties compared to control T cell lines generated by irrelevant peptide control. When compared to control T cell lines, the cytokine profile of COP-I reactive T cell lines was noticeably biased toward high production of IFN- (Table 1).

View larger version (16K): [in this window] [in a new window]   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).

View larger version (34K): [in this window] [in a new window]   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 -actin was used as a control.

Induction of Foxp3 Expression and Regulatory Function in CD4⁺ T Cells by COP-I Is Mediated Through IFN-

View larger version (15K): [in this window] [in a new window]   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).

View larger version (14K): [in this window] [in a new window]   Fig. 5. The effect of IFN- 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- 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- (25 ng/ml) and a monoclonal antibody to human IFN- (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- and medium control (P < 0.01); **, statistical difference between anti-IFN- and the controls (P < 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 6. Induction of Foxp3 expression in T cells by in vitro treatment with COP-I in wild-type and IFN--deficient mice. Splenocytes were derived from wild-type mice (A) and IFN- 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).

View larger version (12K): [in this window] [in a new window]   Fig. 7. Induction of CD4+CD25+ regulatory T cells in wild-type and IFN- knockout mice in response to administration of COP-I. Wild-type and IFN- 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).

	Discussion

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The findings described here are highly significant in the understanding of the mechanism of action of COP-I in relation to its treatment efficacy in MS. In this regard, COP-I may act, through its ability to induce CD4⁺CD25⁺ regulatory T cell response, to compensate a functional deficit in this important regulatory mechanism in MS (23). It is conceivable that this unique property of COP-I in the induction of CD4⁺CD25⁺ regulatory T cells may attribute, at least in part, to the treatment efficacy of COP-I in MS. The observation is also likely to offer a reasonable explanation for, or it may reconcile with, some previously described regulatory functions of COP-I of unknown mechanism, including so-called "bystander" inhibitory effect of COP-I on T cell activation, which is a frequently reported phenomenon (8). The antigen nonspecific inhibitory effect of CD4⁺CD25⁺ regulatory T cell response induced by COP-I is consistent with the spectrum of inhibition induced by COP-I that is often not limited to one antigen (i.e., MBP) and includes various other myelin antigens (16, 32). Furthermore, if the induction of CD4⁺CD25⁺ regulatory T cells is closely associated with the treatment effect of COP-I in MS, one potentially important aspect of the clinical significance of the study may involve the role of Foxp3 as a surrogate biomarker in measurement of treatment efficacy. Currently, the treatment efficacy of COP-I can only be measured 9 months after the treatment by using the standard clinical and magnetic resonance imaging techniques (33). However, it should be cautioned that this study does not completely exclude the role of other regulatory properties of COP-I in the treatment of MS, especially its ability to induce Th2 immunity through repeated administration (5). It is conceivable that these regulatory mechanisms induced by COP-I may work in concert to achieve sufficient immune regulation ultimately beneficial to the clinical course of MS.

Another important aspect of the study is related to defining the mechanism of action potentially responsible for the induction of Foxp3 expression by COP-I in CD4⁺ T cells, which leads to the conversion of CD4⁺CD25^- T cells to CD4⁺CD25⁺ regulatory T cells. It was evident in the study that the observed effect of COP-I is largely mediated through IFN-. It is known from this and other reports that COP-I has the ability to stimulate the production of Th1 and Th2 cytokines, including IFN- (13, 28). The role of IFN- in the induction of Foxp3 expression and CD4⁺CD25⁺ regulatory T cell response is supported by the following experimental evidence: (i) Blocking of IFN- (but not other cytokines also induced by COP-I) by specific antibody resulted in significant inhibition of the effect of COP-I. (ii) Treatment of CD4⁺ T cells with recombinant IFN- led to increased Foxp3 expression. (iii) COP-I failed to induce Foxp3 expression in T cells of IFN- knockout mice in in vitro and in vivo settings. The role of IFN- appears different from that of TGF-1 in the induction of Foxp3 and conversion of CD4⁺CD25^- T cells to CD4⁺CD25⁺ regulatory T cells. TGF-1 requires costimulation of T cells with an anti-CD3 antibody to induce Foxp3 expression (34). As described here and by other investigators (31, 34), in contrast to IFN-, TGF-1 is insufficient when used alone to directly induce Foxp3 expression in T cells. Our observation that the induction of Foxp3 expression by COP-I is partially blocked by antibody to TGF-1 supports the possibility that the involvement of TGF-1 in the induction of Foxp3 expression requires a stimulatory signal provided by COP-I.

The finding that IFN- has the effect of mediating the induction of Foxp3 expression and CD4⁺CD25⁺ regulatory T cells is consistent with a recent report indicating that signal transducer and activator of transcription-1 (STAT1), a signaling molecule closely associated with the IFN- signaling pathway, is critical to the induction of CD4⁺CD25⁺ regulatory T cells (24). The authors demonstrated that STAT1-deficient mice expressing a transgenic T cell receptor against MBP spontaneously developed experimental autoimmune encephalomyelitis, which was attributable to a functional impairment of CD4⁺CD25⁺ regulatory T cells in STAT1-deficient mice (24). Furthermore, the study described here has also raised new questions regarding the role of IFN- in T cell regulation. This is another example adding to the recent debate on the functional role of Th1 and Th2 cytokines in autoimmune conditions, such as MS (35-38). The traditionally held Th1 paradigm is being challenged by mounting evidence that not all Th1 cytokines (e.g., IFN- and TNF-) are necessarily the culprits for MS; in some aspects, they may be beneficial. In conclusion, it is clear from the present study that COP-I acts as an inducer for CD4⁺CD25⁺ regulatory T cell response through the activation of Foxp3 expression. This agent has a potent and selective property for the induction of Foxp3 expression and CD4⁺CD25⁺ regulatory T cells in human and animal experimental systems, making it an excellent tool for the study of CD4⁺CD25⁺ regulatory T cells in future investigations.

	Acknowledgements

 
This work was supported by Chinese Ministry of Science and Technology Grant 863, Projects 04DZ1920, 2002AA216121 and 202CCCD2000; Shanghai Commission of Science and Technology Grants 01JC14036, 20014319207, 04DZ14902, and 03XD14015; National Natural Science Foundation of China Grant 30430650; and National Institutes of Health Grants NS41289 and NS48860 (to J.Z.Z.).

	Footnotes

 
Author contributions: J.Z.Z. designed research; J.H., N.L., X.Z., and B.Z. performed research; J.H., N.L., B.Z., and J.Z.Z. analyzed data; N.L. and B.Z. contributed new reagents/analytic tools; J.Z.Z. wrote the paper; and J.Z.Z. supervised the entire project.

Abbreviations: COP-I, copolymer-I; MBP, myelin basic protein; MS, multiple sclerosis; PBMC, peripheral blood mononuclear cells; Th, T helper.

^¶ To whom correspondence should be addressed at: Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB302, Houston, TX 77030. E-mail: jzang{at}bcm.tmc.edu.

© 2005 by The National Academy of Sciences of the USA

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