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The Journal of Immunology, 2008, 180, 2830-2838
Copyright © 2008 by The American Association of Immunologists, Inc.
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Mitigation of Experimental Allergic Encephalomyelitis by TGF-β Induced Foxp3+ Regulatory T Lymphocytes through the Induction of Anergy and Infectious Tolerance1

Ramesh K. Selvaraj and Terrence L. Geiger2

Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stimulation of naive T lymphocytes in the presence of IL-2 and TGF-β induces the regulatory transcription factor Foxp3, which endows the cells with regulatory functions. To better understand the properties and therapeutic potential of these induced regulatory T cells (iTreg), we examined their immunomodulatory properties in myelin oligodendroglial glycoprotein-induced experimental allergic encephalomyelitis (MOG-EAE). Adoptively transferred iTreg were as potent as natural Foxp3+ Treg in preventing EAE development, and were active both prophylactically and after priming. The iTreg migrated into the CNS in quantity, skewing the ratio of regulatory to effector T lymphocytes. IL-10–/– iTreg failed to suppress disease, demonstrating a critical role for iTreg IL-10 production in their therapeutic activity. MOG-specific T cells from iTreg treated animals were anergic. The cells failed to proliferate in response to Ag except in the presence of exogenous IL-2, and did not secrete or secreted reduced amounts of IL-2, IFN-{gamma}, and IL-17. MOG-specific T cells were not wholly unresponsive though, as they did secrete IL-10 after stimulation. To determine whether iTreg-mediated tolerance was infectious, fostering the development of T lymphocytes that could independently suppress EAE, we purged draining lymph node cells from MOG-immunized, iTreg treated mice of the administered iTreg, and transferred the remaining cells to Ag-inexperienced mice. The transferred cells were able to block EAE development. Thus iTreg are highly potent suppressors of autoimmune encephalomyelitis, and act in an IL-10 dependent manner both through the induction of anergy in effector T cells and through the infectious induction of protective T lymphocytes able to independently suppress disease development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Experimental allergic encephalomyelitis (EAE)3 is an inducible, T cell mediated autoinflammatory disease of the CNS that mimics many of the features of multiple sclerosis (MS) (1, 2). In C57BL/6 mice, EAE is typically induced by immunization with residues 35–55 of myelin oligodendroglial glycoprotein (MOG), a component of the myelin sheath (3). An acute disease follows that may be clinically monitored by the development of paresis/paralysis in affected animals. Early in disease, autoreactive T cells secreting IFN-{gamma} (Th1 cells) and IL-17 (Th17 cells) penetrate the CNS where they damage the myelin sheath. Subsequently, increasing numbers of Foxp3+ regulatory T cells (Treg) enter inflamed sites in the CNS (4, 5, 6). These secrete IL-10 and TGF-β, and limit disease pathology. Mice in which Treg are depleted develop more severe disease than unmanipulated controls, whereas mice supplemented with Treg are protected (7, 8, 9, 10).

Treg develop predominantly in the thymus (11, 12). Seemingly, T cells with an affinity for self-Ag (Ag) between that required for positive and negative selection up-regulate Foxp3 and are diverted into the Treg lineage (13). These cells, also called natural Treg (nTreg), comprise ~3–10% of CD4+ T lymphocytes. They are potent immunosuppressors, and can downmodulate a variety of immune mediated diseases in model systems. Because they are not associated with proinflammatory activities, nTreg have generated considerable interest as an immunotherapeutic agent (14, 15, 16). The low frequency and poor growth in culture of nTreg, however, makes acquiring adequate numbers of cells for adoptive immunotherapy a significant challenge.

Nonregulatory T cells may also up-regulate Foxp3 to levels observed in nTreg. These adaptive or induced Treg (iTreg) can be generated by the stimulation of naive T lymphocytes in the presence of IL-2 and TGF-β (17, 18, 19, 20). We previously showed that iTreg require continuous TGF-β in vitro to maintain Foxp3 expression. After in vivo transfer many iTreg down-regulate Foxp3, though a subset retains Foxp3 for a period of greater than 1 mo (17, 21). iTreg are relatively easy to obtain in large numbers, and expand well in culture. Being derived from conventional {alpha}β T cells, TGF-β-induced iTreg may not be functionally identical with nTreg. Nevertheless they show many properties of nTreg. After priming with TGF-β, T cells produce IL-10 and TGF-β, though limited quantities of effector cytokines, much as nTreg do (17, 19, 20). Further, adoptively transferred iTreg have demonstrated activity in diminishing immunopathology in allotransplant, colitis, gastritis, and diabetes models (22, 23, 24, 25). iTreg therefore are a potentially attractive alternative to nTreg for adoptive immunotherapy.

To better define the functional properties and therapeutic potential of iTreg in a preclinical model of MS, we analyzed their activity in MOG-EAE. We find that iTreg are as potent as nTreg in suppressing disease development, and that they are able to act both prophylactically and therapeutically. The iTreg penetrate the CNS, skewing the Treg: Teff ratio at the site of inflammation. Therapeutic activity is wholly dependent on iTreg production of IL-10 and results in the induction of anergy in MOG-specific effector T cells. Interestingly, iTreg catalyze the development of endogenous regulatory cells that are independently able to suppress EAE, potentially thereby amplifying their effect. Our data therefore demonstrate that iTreg are a capable surrogate for nTreg in the immunotherapy of autoimmune encephalitides, and provide new insights into the mechanism of actions of these cells. Furthermore, these preclinical studies support the continued clinical translation of iTreg as a cellular immunotherapeutic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Mice in which a GFP-FoxP3 fusion has been homologously inserted at the FoxP3 locus have been described (26) and were backcrossed >5 generations onto the C57BL/6 (CD45.2+) background before use. Male mice screened for GFP-FoxP3 were used for experimentation. Some GFP-FoxP3 mice were subsequently bred with CD45.1 congenic mice to obtain CD45.1+ GFP-FoxP3 mice. C57BL/6, congenic CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), and IL10–/– (B6.129P2-Il10tm1Cgn/J) mice were purchased from The Jackson Laboratory. Experimentation was performed in accordance with institutional animal care and use procedures.

Media, reagents, and Abs

Medium for T cell cultures was prepared as described earlier (27). Foxp3-specific Ab (intracellular FoxP3 staining kit, used per manufacturer’s instructions) and recombinant human TGF-β were purchased from eBioscience. All other conjugated and unconjugated Abs were purchased from BD Pharmingen.

Flow cytometry

Cells were stained and analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Sorting was performed using a MoFlo high-speed sorter (DakoCytomation).

Cell purification and cell culture

Lymph node (LN) and spleen cells were collected as described (17). CD4+FoxP3+ (nTreg) and CD4+FoxP3– (non-Treg) cells were isolated by flow cytometric sorting of LN and spleen cells from GFP-Foxp3 KI mice, gating on CD4 and GFP (FoxP3) expression. Sorted cell purity ranged from 97 to 99%. nTregs were grown in medium supplemented with 1 ng/ml PMA, 200 ng/ml ionomycin and 100 IU/ml recombinant human IL-2 (rhIL-2; NCI BRB Repository). CD4+FoxP3– cells were stimulated for seven to nine days with soluble anti-CD3/anti-CD28 (1:750) and supplemented with 100 IU/ml rhIL-2 with or without TGF-β (10 ng/ml) to obtain iTreg or non-Treg cells respectively. IL10–/– iTreg were produced from IL-10–/– mice that lack the GFP-Foxp3 knock-in. CD4+CD25– cells were used to obtain the non-Treg population for expansion and iTreg generation. Cells were split into cytokine-containing medium as needed to prevent overcrowding.

Adoptive transfer of nTreg, iTreg, and non-Treg cells

Aliquots of cell cultures were flow cytometrically analyzed for GFP-Foxp3 expression or by direct staining for Foxp3 to determine the percentage FoxP3+ cells. The total cell number for administration was calculated based on the above percentage so that the dose of iTreg or nTreg indicated in the text was administered. Cells were washed and resuspended in 100 µl of PBS and injected i.v. An identical total number and/or volume of non-Treg cells or PBS was injected for controls.

EAE disease induction

EAE was induced with MOG35–55 (MEVGWYRSPFSRVVHLYRNGK; produced at St. Jude Hartwell Center for Biotechnology) by injecting 50 µg of MOG35–55 emulsified in complete Freund’s adjuvant containing 0.2 mg of H37Ra mycobacterium tuberculosis (Difco Laboratories) in 50 µl s.c. in each hind flank. 200 ng of Bordetella pertussis toxin (Difco Laboratories) was administered i.v. on days 0 and 2. Clinical scoring was as follows: 1, limp tail; 2, hind limb paresis or partial paralysis; 3, total hind limb paralysis; 4, hind limb paralysis and body/front limb paresis/paralysis; 5, moribund. In all experiments that involved EAE disease induction 5 mice per group were used.

Proliferation and cytokine analysis of draining LN and spleen cells

Mice were immunized subcutaneously with 50 µg of MOG35–55 peptide emulsified in complete Freund’s adjuvant in 4 sites, in the left and right hind flank and upper back. 4 x 106 iTreg or non-Treg in 100 µl PBS or PBS was injected i.v. on the same day. At day 18, draining LN and spleen cells were collected. As a negative control, LN and spleen cells were also collected from unimmunized mice. LN or spleen cells were cultured in duplicate in a 96-well plate (5 x 104 CD4+ cells per well) in medium with 2% FCS supplemented with 0, 10 or 100 µg/ml MOG35–55 or 2 µg/ml Con A in the presence or absence of 10 IU/ml rhIL-2. Culture supernatants were assayed at 48 h for cytokines by Bio-Plex (Bio-Rad) according to the manufacturers’ protocols. Alternatively, cultures were pulsed with 1 µCi of [3H]TdR after 72 h and harvested ~16 h later on filtermat for scintillation counting (Wallac-LKB).

Infectious tolerance

Approximately 6 x 106 CD45.1+ iTregs (4 x 106 Foxp3+), 6 x 106 FoxP3– non-Treg, or PBS was administered i.v. into CD45.2+ C57BL/6 mice (5 mice/group) at the time of s.c. immunization with MOG35–55. Draining LN cells were isolated 13 days later and purged of the injected cells by staining for CD45.1 and flow cytometric negative selection. The purged cells were analyzed for residual CD45.1+ cells. A total of 25 x 106 of the purged cells in 200 µl of PBS or saline (control) was directly transferred i.v. into naive C57BL/6 mice (5 mice/group). EAE was induced on the same day and clinical disease was monitored.

CNS cell infiltration

4 x 106 iTreg, an equivalent number of non-Treg, or PBS was administered i.v. into mice, and EAE induced. On day 19, 4 mice/group were sacrificed, perfused with PBS, and T cells were isolated from brain tissue by density centrifugation. Isolated cells were stained with CD4 and CD25-specific Abs and analyzed by flow cytometry.

Statistical analyses

Statistics were calculated with Excel spreadsheet software (Microsoft). Displayed error bars show ± 1 SD. Two-tailed t tests were performed, and a p < 0.05 was considered significant. Representative data from two or more comparable experiments are shown for each figure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of Foxp3 in stimulated CD4+Foxp3– T cells by TGF-β treatment

To examine the induction of Foxp3 by TGF-β, we isolated CD4+Foxp3– T cells by flow cytometric sorting from male hemizygous GFP-Foxp3 KI mice (26), and stimulated them with anti-CD3/anti-CD28, rhIL-2, and TGF-β. Because the GFP-Foxp3 transgene in the KI mice is a chimeric fusion protein that replaces endogenous Foxp3, we could directly assess Foxp3 expression by measuring GFP fluorescence. Foxp3 was up-regulated in >60% of the cells stimulated in the presence of TGF-β (Fig. 1). In the absence of TGF-β no significant up-regulation was seen. The level of Foxp3 and suppressive activity of the TGF-β induced Treg was similar to that of nTreg, and Foxp3 expression was stable in the presence of TGF-β (data not shown and Ref. 17).


Figure 1
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  		FIGURE 1. TGF-β treatment up-regulates FoxP3. CD4+GFP-Foxp3– T cells were flow cytometrically purified from GFP-Foxp3 KI mice, and stimulated with soluble anti-CD3/anti-CD28 Abs and 100 IU/ml rhIL-2 in the presence or absence of 10 ng/ml TGF-β. Representative histogram plots demonstrating up-regulation of GFP-Foxp3 in TGF-β treated cells are shown.

 
Prevention of EAE with iTreg

Adoptively transferred nTreg have been demonstrated to ameliorate EAE symptoms (7, 8). To determine whether the iTreg were similarly active, we adoptively transferred 4 x 106 or 1 x 106 (Fig. 2A, Table I) or larger doses (not shown) of iTreg i.v. into C57BL/6 mice and induced EAE with MOG35–55 peptide. To directly compare the activity of iTreg with nTreg, separate cohorts of mice received the same numbers of CD4+GFP-Foxp3+ nTreg flow cytometrically isolated from the GFP-Foxp3 KI mice and expanded in parallel with the iTreg, though in the absence of TGF-β. Control mice received CD4+GFP-Foxp3– T cells, also expanded in the absence of TGF-β (non-Treg). Mean and peak clinical disease scores were significantly decreased in mice receiving 1 x 106 or larger doses of either iTreg or nTreg when compared with control cells (p < 0.05; Fig. 2A, Table I). In contrast, no significant differences were observed when comparing mice treated with either iTreg or nTreg, or when comparing mice receiving 1 x 106 or 4 x 106 cells of the same type. Therefore either iTregs or nTregs potently and equivalently prevent the development of EAE.


Figure 2
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  		FIGURE 2. EAE blockade with iTregs. EAE was induced by immunizing mice with MOG35–55 peptide followed by pertussis toxin administration. A, Prophylactic treatment is shown. Four x 106 or 1 x 106 GFP-Foxp3+ iTreg, GFP-Foxp3+ nTreg, or GFP-Foxp3– non-Treg cells were i.v. administered into C57BL/6 mice at the time of disease induction. Clinical disease was monitored longitudinally. B, Dose response of iTreg. The indicated number of iTreg or non-Treg, or an equal volume of saline, was administered i.v. on the day of disease induction. Plots show mean clinical score from 5 animals per treatment group and is representative of 2 independent experiments (Table I).

 

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  		Table I. Summary of EAE experimentsa

 
To more precisely define the dose of iTregs capable of suppressing clinical EAE, variable numbers of iTreg, control non-Treg, or saline was administered to mice, and EAE induced. As above, 1 x 106 iTreg efficiently blocked EAE development (Fig. 2B). Lower doses of cells showed graded efficacies. Mice that received 0.75 x 106 cells showed partial protection (p < 0.05 for mean daily score vs PBS and non-Treg) and earlier recovery than the control treated groups. Mice that received 0.5 x 106 iTreg demonstrated more limited protection, which was statistically significant compared with the PBS-treated group (p < 0.05), though not the non-Treg-treated group (p = 0.06). Mice receiving 0.25 x 106 cells developed a similar clinical course as those receiving saline or non-Treg controls. Therefore iTreg demonstrate a steep dose-response relationship, with a 4-fold difference in transferred iTreg distinguishing undetectable from nearly complete disease protection.

Treatment of EAE with iTreg after priming

T cells begin to expand and secrete cytokines within 2–3 days after peptide immunization. After induction of MOG-EAE, autoAg specific T cells migrate to the CNS and a monophasic disease develops with clinical symptoms first detected ~10–15 day after immunization. To determine whether the iTreg could ameliorate disease after initial priming we treated mice with 1 x 106 iTreg at days 0, 4, or 10 after induction (Fig. 3, Table I). Protection was apparent after day 4 treatment, with a mean peak clinical score of 2.4 ± 0.5 in mice receiving iTreg compared with 3.6 ± 0.5 in the non-Treg group. Treatment with the iTreg also led to reduced mean daily score (iTreg, 0.8 ± 0.8; non-Treg 2.2 ± 0.9), and earlier and more complete recovery from disease. iTreg were also effective when administered 10 day after EAE induction, immediately before the first disease symptoms, which developed on day 11. The mean peak disease score of iTreg treated mice was 2.8 ± 0.5 compared with 3.6 ± 0.5 in the non-Treg group. Mean daily scores were significantly diminished (iTreg, 1.4 ± 0.9; non-Treg, 2.3 ± 0.8), and nearly complete recovery was seen. In contrast, ongoing disease symptoms were apparent in the non-Treg control group even 42 day after disease induction. Thus iTreg treatment is effective even after Ag priming.


Figure 3
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  		FIGURE 3. Delayed treatment of EAE with iTreg. EAE disease was induced and monitored as in Fig. 2. One x 106 iTreg or controls were administered on days 0, 4, or 10. Plots show mean clinical score from 5 animals per treatment group and is representative of two independent experiments (Table I).

 
iTreg migration to the CNS

The presence of Treg in the CNS during EAE has been associated with diminished inflammation and resolution of clinical disease (5). CNS Treg that are isolated and adoptively transferred are further able to reduce symptoms of MOG-EAE in immunized recipient mice (6). This implies that Treg migrating to the site of CNS damage are protective, limiting damage mediated by effector T cells. To determine whether the iTreg migrated to the CNS, we adoptively transferred them or control non-Treg into C57BL/6 mice at the time of EAE induction. CNS infiltrating cells were analyzed 19 days later by flow cytometry. iTreg could be distinguished by their expression of the GFP-Foxp3 transgene. Endogenous Treg were also quantified by their coexpression of CD4 and CD25. CD4+CD25+ T cells infiltrating the CNS in MOG-EAE have previously been shown to be virtually exclusively Foxp3+ (6).

After iTreg transfer, 31 ± 5% of CD4+ T cells within the CNS were GFP-Foxp3+ (Fig. 4A). In concordance with the diminished disease severity after iTreg treatment, the absolute numbers of CD4+ T cells infiltrating the CNS after iTreg treatment was lower than after non-Treg or no treatment (Fig. 4B). The fraction of endogenous (GFP–) CD4+CD25+ cells within the CD4+ population, 8–11%, did not substantially differ when comparing mice receiving iTreg, non-Treg, or saline. Thus the transfer of iTreg led to an approximate quadrupling of Treg as a percentage of CD4+ T cells within the CNS. This shows that transferred iTreg migrate into the CNS where they skew the Treg/Teff ratio, potentially limiting local inflammation.


Figure 4
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  		FIGURE 4. Infiltration of Treg and Teff into the CNS. C57BL/6 mice received 4 x 106 iTreg (GFP+), an equivalent number of non-Treg (GFP–), or saline i.v. at the time of EAE induction. On day 19, the mice were sacrificed, perfused with cold saline, and brains were removed. T cells were isolated from brain tissue by density centrifugation and analyzed for CD4, CD25, and GFP-Foxp3 by flow cytometry. Percentage (A) and absolute numbers (B) of CD4+GFP+ (transferred iTreg), CD4+CD25+, and CD4+CD25– infiltrating cells are plotted.

 
iTreg IL-10 production is required for EAE blockade

We and others have shown that IL-10 is critical for the amelioration of EAE symptoms by nTreg (8, 28, 29). To determine whether iTreg had a similar cytokine requirement, we produced iTreg from IL-10–/– mice. As with wild type cells (Fig. 1), FoxP3 was up-regulated in ~60% of purified CD4+CD25– IL-10–/– cells stimulated in the presence of TGF-β (Fig. 5A), demonstrating that endogenous IL-10 is not required for Foxp3 up-regulation. In contrast, fewer than 1% of the IL-10–/– cells similarly stimulated in the absence of TGF-β expressed Foxp3. 1 x 106 iTreg derived either from wild type or IL-10–/– mice were administered to C57BL/6 mice at the time of EAE induction. The wild type iTreg were highly effective in blocking the development of disease (Fig. 5B, Table I). In contrast, IL10–/– iTreg showed no discernible activity, with treated mice experiencing a disease course that was not significantly different from mice receiving saline or IL-10–/– non-Treg in mean and peak disease scores. The failure of iTreg to suppress disease did not appear to be a dose dependent phenomenon, as transfer of 4 x 106 IL-10–/– iTreg similarly failed to protect mice against the development of EAE symptoms (data not shown). Therefore IL-10 production by iTreg is critical for their immunotherapeutic activity in EAE.


Figure 5
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  		FIGURE 5. IL-10 is required for iTreg suppression of EAE. A, IL-10–/– T cells up-regulate Foxp3 in response to TGF-β. CD4+CD25– cells were flow cytometrically isolated from IL10–/– mice and stimulated with anti-CD3/anti-CD28 in the presence of 100 IU/ml rhIL-2 with or without 10 ng/ml TGF-β to obtain iTreg and non-Treg respectively. Cell samples were analyzed before and after culture by intracytoplasmic staining for Foxp3. B, To assess the clinical effect of the IL-10–/– iTreg on EAE, 1 x 106 iTreg (wild type or IL10–/–), non-Treg (IL10–/–), or saline was administered i.v. into C57BL/6 mice at the time of disease induction. Clinical course was monitored. The plot shows mean clinical score from 5 animals per treatment group and is representative of 2 independent experiments (Table I).

 
Infectious tolerance mediated by iTreg

Treg are highly potent suppressors of T cell immunity. This has led to the hypothesis that these cells not only act directly to dampen immune responses, but can catalyze the formation of additional suppressive T cells. We have previously demonstrated that adoptively transferred nTreg can indeed educate the immune system in vivo, converting endogenous lymphocytes into regulatory cells that suppress EAE (28). Considering the high and similar potency of iTreg and nTreg in MOG-EAE, we therefore analyzed whether iTreg could similarly confer infectious tolerance. We bred GFP-Foxp3 KI mice with CD45.1 congenic mice, and generated CD45.1+ iTreg or non-Treg by stimulating CD4+GFP-Foxp3– T cells in the presence or absence of TGF-β. We then adoptively transferred 4 x 106 CD45.1+ iTreg (~ Formula Foxp3+; 6 x 106 total T cells), a similar number of non-Treg, or administered saline i.v. into C57BL/6 mice at the time of immunization with MOG35–55. Draining LN cells were isolated 13 days later. Only ~30% of the CD45.1+ LN cells in the mice receiving iTreg were GFP-Foxp3+. This implies that admixed non-Treg preferentially expanded or that some iTreg down-regulated GFP-Foxp3. As expected, GFP-Foxp3+ cells were not detected in the mice receiving non-Treg (Fig. 6A).


Figure 6
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  		FIGURE 6. Purging of transferred iTreg from draining LN cells and induction of infectious tolerance. Four x 106 GFP-Foxp3+ CD45.1+ iTreg (6 x 106 total CD45.1+ cells), an equivalent number of GFP-Foxp3– non-Treg, or saline was administered i.v. into CD45.2+ C57BL/6 mice at the time of s.c. immunization with MOG35–55. Draining LN cells were isolated 13 days later and purged of the transferred cells by staining for CD45.1 and flow cytometric sorting. A, Flow cytometric analysis of retrieved LN cells before purging is shown. CD45.1 (transferred cells) is plotted vs GFP-Foxp3 expression. B, Flow cytometric analyses postsorting demonstrate few residual CD45.1+ T cells in the purged populations. C, A total of 25 x 106 of the purged draining LN cells from mice treated with iTreg, non-Treg, or saline, or saline control (No cells) was administered i.v. into naive C57BL/6 mice immediately before EAE induction. Clinical disease was monitored. Means of five mice per group are plotted. Results are representative of 2 experiments (Table I).

 
To analyze the influence of the iTreg on endogenous lymphocytes, we then purged the draining LN cells of transferred CD45.1+ cells by flow cytometric sorting. Analysis of the purged cells revealed that only ~0.1% expressed CD45.1 (Fig. 6B). To test the suppressive function of these CD45.1-depleted cells, 25 x 106 were adoptively transferred into C57BL/6 recipients and EAE was induced. Animals receiving cells derived from iTreg treated mice showed substantial protection, with significantly reduced disease symptoms and more rapid disease resolution than control mice not receiving cells (Fig. 6C, Table I). In contrast, transfer of CD45.1– cells from mice treated with non-Treg or saline failed to significantly impact the disease course compared with controls not receiving cells.

These results suggest that cells purged of the transferred iTreg are able to inhibit disease development. However, considering the 0.1% contamination with CD45.1+ cells in the purged populations, the purged cells will also contain ~2.5 x 104 CD45.1+ cells and, in the iTreg cohort, assuming a similar proportion of GFP-Foxp3+ cells pre- and postsorting, ~1 x 104 GFP-Foxp3+ iTreg. This small contaminating population of iTreg is 50-fold less than the minimal 5 x 105 dose required for iTreg therapeutic activity (Fig. 2B) and would not be expected to suppress disease development. We verified this by isolating 2 x 105 flow cytometrically purified CD45.1+ cells (~6 x 104 GFP-Foxp3+) from mice treated with iTreg, transferring them into recipient mice, and inducing EAE as in Fig. 6. The transferred cells failed to influence disease activity in comparison with saline-treated control mice (For control and CD45.1+ cell-treated groups, respective mean maximal disease scores were 3.2 ± 0.4 and 2.8 ± 0.8, p > 0.05, and mean daily scores were 1.6 ± 1.0 and 1.6 ± 0.9, p > 0.05). Therefore iTreg endow endogenous lymphocytes with regulatory properties and these lymphocytes are able to suppress EAE development.

iTreg render host MOG-specific T cells anergic

The development of infectious tolerance implies that the iTreg functionally altered the MOG-specific T cells. To better understand the impact of iTreg on these T cells, we treated mice with iTreg or control cells at the time of immunization with MOG35–55, subsequently isolated draining LN and spleen cells, and evaluated the T cell response to MOG35–55 (Fig. 7, A and C). Draining LN or spleen cells responded essentially identically. Cells from mice receiving either saline or non-Treg showed strong proliferative responses to MOG35–55. As expected, control cells from unimmunized mice failed to respond to MOG35–55. Unexpectedly considering the presence of cells capable of mediating infectious tolerance, cells from iTreg treated mice also showed little or no specific proliferation in response to MOG peptide. Therefore, iTreg treatment suppresses the MOG-specific proliferative response.


Figure 7
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  		FIGURE 7. Anergy in MOG35–55 specific T cells after iTreg treatment. C57BL/6 mice received 4 x 106 iTreg, non-Treg, or saline i.v. at the time of s.c. immunization with MOG35–55. On day 13, draining LN (A and B) or spleen (C and D) cells were isolated. These were cultured for 3 days in the presence of graded doses of MOG35–55 peptide without (A and C) or with (B and D) 10 U/ml rhIL-2. Ag-specific proliferation was measured at 72 h by [3H]thymidine incorporation. E, Cells were cultured without stimulation or stimulated with ConA in the presence or absence of 10 IU/ml rhIL-2. Mean cpm incorporation from two animals per group, each independently assayed in duplicate is plotted. A p < 0.05 comparing iTreg with unimmunized ({ddagger}), saline-treated (°), and non-Treg-treated (*) groups at the 100 µg/ml MOG35–55 stimulation data point is indicated in (A–D). In E, values for iTreg were not significantly different from unimmunized, saline-treated, and non-Treg-treated groups for each experimental condition listed under the abscissa.

 
The minimal proliferative response to MOG by cells from iTreg-treated mice may have resulted from the presence of only small numbers of MOG-specific T cells, for instance due to inefficient priming or deletion, or from the development of anergy, or induced unresponsiveness, among the cells present. Anergic T cells can be induced to respond by stimulation in the presence of exogenous IL-2 in vitro (30). We therefore repeated the proliferation studies, though with the addition of 10 IU/ml rhIL-2 (Fig. 7, B and D). The added IL-2 did not have a significant impact on the proliferation profiles from control mice treated with non-Treg or saline. Cells from unimmunized mice further remained unresponsive to Ag. However, exogenous IL-2 increased the proliferation of MOG-specific T cells from iTreg treated mice to levels seen with control-treated mice. This implies that MOG-specific T cells were adequately primed in the presence of iTreg, however, they were unable to respond ex vivo to Ag in the absence of exogenous IL-2. Anergy among the iTreg treated cells appeared to be specific to the immunogen as the nonspecific mitogen conA induced comparable proliferation in cells from each cohort of mice, the large majority of which will not be Ag-specific, regardless of the added presence of rhIL-2 (Fig. 7E).

To better define the impact of iTreg treatment on the MOG-specific response, we also analyzed cytokine production profiles of draining LN T cells. The LN cells were left unstimulated or stimulated with MOG35–55 in the presence or absence of exogenous rhIL-2. Production of murine IL-2 (mIL-2), IL-4, IFN-{gamma}, IL-17, and IL-10, representative cytokines produced by Th2, Th1, Th17 and regulatory T cells, was measured at 48 h (Fig. 8, A–E). Draining LN cells from immunized mice treated with saline or non-Tg control cells produced a significant amount of mIL-2 in response to MOG35–55 stimulation, either in the presence or absence of exogenous rhIL-2 (Fig. 8A). In contrast, the iTreg treated cells did not specifically produce mIL-2 in response to Ag, and only produced small amounts when exogenous IL-2 was provided. Draining LN cells from control mice also produced significant amounts of the Th1 cytokine IFN-{gamma} with stimulation (Fig. 8B). This was not seen in iTreg treated cells cultured in the absence of IL-2, though exogenous IL-2 did mildly increase IFN-{gamma} production by these cells. In contrast to IFN-{gamma}, IL-4, a Th2 cytokine, was not produced in relevant quantities by any of the cells regardless of stimulation conditions (Fig. 8C).


Figure 8
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  		FIGURE 8. Cytokine production by MOG35–55 specific T cells after iTreg treatment. Cultures were established as in Fig. 8 using draining LN cells from iTreg, non-Treg, or saline (PBS) treated mice. Cells were unstimulated or stimulated with 100 µg/ml MOG35–55 in the presence or absence of 10 IU/ml rhIL-2. At 48 h, culture medium was harvested and analyzed for murine IL-2 (A), IFN-{gamma} (B), IL-4 (C), IL-17 (D), and IL-10 (E). Mean cytokine determinations from two animals per group, each independently assayed in duplicate is plotted. A p < 0.05 for unstimulated vs equivalent 100 µg/ml MOG35–55 stimulated cells (*) and for 100 µg/ml MOG35–55 stimulated cells vs. equivalently stimulated cells from iTreg-treated mice ({ddagger}) are indicated.

 
Th17 cells, which produce IL-17, are essential for the development of MOG-EAE (31), and mice unable to produce Th17 cells do not develop EAE. Interestingly, basal IL-17 production was diminished in cells from the iTreg-treated compared with control-treated mice (Fig. 8D). Stimulation with MOG35–55 further failed to significantly increase production of IL-17 in these cells. In contrast, cells from mice treated with saline or non-Treg produced significant though small amounts of IL-17 in response to Ag stimulation. This low level of Ag-induced IL-17 production is consistent with prior observations demonstrating that only a small percentage of T cells express IL-17 after immunization with MOG (32). These results therefore demonstrate that iTreg treatment inhibits production of effector cytokines, including both IFN-{gamma} and IL-17, as well as the mitogenic cytokine IL-2 in MOG-specific T cells.

IL-10 has been observed to play an important role in the T cell mediated immunoregulation of EAE, and was essential for iTreg activity (Fig. 5). Significant MOG35–55-induced IL-10 production was observed only among the iTreg-treated draining LN cells, and this was seen in the presence or absence of exogenous IL-2 (Fig. 8E). Therefore iTreg induce the development of MOG-reactive T cells that Ag-specifically produce IL-10. Further, these cells are able to block MOG-EAE independently of the inducing iTreg (Fig. 6).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Foxp3+ T cells are critical regulators of adaptive immunity. Deficiency in Foxp3 precipitates early-onset multiorgan autoimmunity (33, 34). The frequency and function of Treg has also been associated with susceptibility to and severity of a number of autoimmune, infectious, and malignant diseases (35, 36, 37, 38, 39). Insight into how manipulation of the Treg compartment influences these diseases is not only biologically but therapeutically significant. Indeed, data demonstrating immunomodulatory activity of adoptively transferred nTreg has been compelling in a number of model systems, supporting their clinical development as a cellular immunotherapeutic. nTreg, however, are present in small numbers in vivo and are anergic in vitro. Isolating and preferentially expanding these cells for immunotherapy has therefore presented considerable challenges (40).

Adaptive Treg, or iTreg, may represent a viable immunotherapeutic alternative to nTreg. iTreg may be induced by stimulating naive, though not memory, T cells in presence of IL-2 and TGF-β (17, 18, 19, 20, 21). Specific interactions with dendritic cells can also promote, though are not essential, for the induction of Foxp3 and expansion of Foxp3+ cells in vitro, and may be essential in vivo (41, 42, 43). We have previously shown that a subpopulation of adoptively transferred iTreg retain Foxp3 for at least a month, residing primarily in the lymph nodes and bone marrow (17). Therefore iTreg are readily generated in vitro and have a sustained lifespan in vivo.

There are documented differences between nTreg and iTreg. Whereas virtually all adoptively transferred nTreg retain Foxp3 long-term, many iTreg downmodulate Foxp3 after transfer and display migration and survival features in vivo similar to non-Treg cells (17). Studies of nTreg have shown that they bear a distinct repertoire with a predilection for self-specificity (13). iTreg, which are derived from the non-regulatory population will lack this self-specificity. Therefore, it remains to be established whether iTreg are functionally and therapeutically equivalent to nTreg.

We present several new findings about the capabilities and mechanism of action of iTreg using an EAE model system. iTreg are highly potent in preventing EAE, acting at low numbers with an activity comparable to that of nTreg. They are able to ameliorate disease even after priming, suggesting that they can act on effector cells and not only on undifferentiated precursors. Transferred iTreg migrate to the CNS, and implicitly can modulate disease at that location.

Interestingly, iTreg, as we and others have previously observed with nTreg (8, 28, 29), operate in an IL-10 dependent manner. IL-10 is a crucial immunomodulatory cytokine in EAE. IL-10 transgenic mice are resistant to EAE, whereas IL-10–/– mice show increased disease severity (44). Th2 T cells ameliorate EAE through IL-10 production (45). CNS IL-10 expression correlates with disease resolution (46). The critical role for IL-10 in the modulation of EAE is not surprising considering the potent activity of this cytokine in subduing activated macrophage and glial cells that play crucial effector roles in the disease (47, 48). Nevertheless, it provides evidence that iTreg and nTreg operate through similar mechanisms.

iTreg appear to alter MOG-specific T cell reactivity in at least two manners. First, they induce anergy in responding T cells, as functionally indicated by the failure of MOG-specific T cells in treated mice to proliferate or produce IL-2 in response to Ag stimulation. The cells do specifically proliferate in response to MOG, however, when exogenous IL-2 is provided. Therefore MOG specific T cells are present and stimulated by Ag, but incapable of producing cytokines required to permit and sustain proliferation in vitro. The induction of anergy by regulatory T cells has been observed in in vitro studies of Treg mediated suppression (49, 50), but to our knowledge, this is the first example of Treg, or more specifically iTreg, inducing anergy in a population in vivo. A similar anergic state has been observed to develop in vivo, however, in other models of immune tolerance (51, 52).

Second, iTreg divert the cytokine profile of MOG-specific T cells. Whereas T cells from control immunized mice produced substantial amounts of IFN-{gamma}, small amounts of IL-17, and no detectable IL-10, mice immunized in the presence of iTreg produced no or smaller amounts of IL-17 and IFN-{gamma}, and significant amounts of IL-10. This suggested that the iTreg converted the MOG-specific T cells into IL-10 secreting cells that may be immunomodulatory in themselves. We tested this by treating MOG-immunized mice with iTreg, purging the transferred iTreg population, and examining the suppressive capacity of the residual cells. We found that these purged cells were potent inhibitors of EAE, indicating that tolerance mediated by the iTreg was transferable, or infectious, to endogenous lymphocytes.

Infectious tolerance by iTreg presents a mechanism by which the cells can amplify their immunomodulatory effects. iTreg are highly potent, and as few as 5 x 105 Ag nonspecific iTreg ameliorated EAE. This potency may arise in part because the cells can catalyze the induction of regulatory activities in additional T cells. Indeed, similar amplification has been observed in vitro (53, 54, 55), and more recently in a transplant model and when using re-directed nTreg to treat EAE in SJL mice (22, 28). Therefore, in a manner analogous to coagulation, in which enzymatic cascades that promote or inhibit fibrin deposition compete, pro- and anti-inflammatory pathways in adaptive immunity may undergo a similarly dynamic competition to regulate inflammatory responses. Further study will be important to dissect out the cell types responsible for infectious tolerance in this and other systems, determine how they are induced by iTreg and nTreg, and define their mechanism of action. In this regards, it is important that studies of nTreg demonstrate not only direct activity on T cells, but also on APCs, particularly dendritic cells. If iTreg indeed act in a manner similar to nTreg, their activities may be indirect, through the modification of dendritic cell function and therefore by altering the priming and activation environment of effector T cells (25, 56).

In summary, we demonstrate that iTreg are highly potent regulators of EAE disease, operate in a cytokine-dependent manner, and produce several changes in effector T cells that can dampen their inflammatory potential. Our results suggest that iTreg may serve as an effective cellular therapeutic for MS or other autoimmune diseases, and support their continued evaluation and therapeutic translation.


   Acknowledgments
 
We thank Richard Cross and Yuxia He for assistance with flow cytometry and cell sorting, Jennifer Smith with bioplex assay, and Sasha Rudensky for providing the GFP-FoxP3 knock-in mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01 AI056153 (to T.L.G.) and by the American Lebanese Syrian Affiliated Charities/St. Jude Children’s Research Hospital (to T.L.G. and R.K.S.). Back

2 Address correspondence and reprint requests to Dr. Terrence L. Geiger, Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, D-4047, Memphis, TN 38105. E-mail address: terrence.geiger{at}stjude.org Back

3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; MS, multiple sclerosis; nTreg, natural regulatory T cell; iTreg, induced regulatory T cell; LN, lymph node; MOG35–55, myelin oligodendroglial glycoprotein 35–55. Back

Received for publication October 3, 2007. Accepted for publication December 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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