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Originally published online as doi:10.1189/jlb.0505271 on October 21, 2005

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(Journal of Leukocyte Biology. 2006;79:140-146.)
© 2006 by Society for Leukocyte Biology

Latent TGF-ß1-transduced CD4+ T cells suppress the progression of allergic encephalomyelitis

Mitsuyuki Murano*,1, Xiaozhong Xiong*,1, Naoko Murano*, James L. Salzer{dagger}, Juan J. Lafaille* and Vincent K. Tsiagbe*,2

* Departments of Pathology and NYU Cancer Institute and
{dagger} Cell Biology, New York University School of Medicine, New York

2 Correspondence: New York University School of Medicine, Department of Pathology, Room 538 MSB, 550 First Avenue, New York, NY 10016. E-mail: tsiagv01{at}med.nyu.edu


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ABSTRACT
 
Systemic injection of small amounts of transforming growth factor-ß (TGF-ß), a cytokine produced by lymphoid and other cells, has a profound effect in protecting mice from the inflammatory demyelinating lesions of experimental allergic encephalomyelitis (EAE; an animal model for multiple sclerosis). However, TGF-ß has side-effects, which might be avoided if the cells producing TGF-ß can be delivered to the affected site in the nervous system to insure its local release in small amounts. Myelin basic protein (MBP)-specific, cloned CD4+ T cells were engineered by retroviral transduction to produce latent TGF-ß. Studies about the spontaneous form of EAE in T cell receptor (TCR)-transgenic recombination-activating gene (RAG)-1–/– mice showed that essentially all of the MBP-specific, TCR-transgenic RAG-1–/– (BALB/cxB10.PL)F1 mice develop spontaneous EAE by the age of 11 weeks. By 12 weeks, 25–50% of the mice have died from disease. A single injection of TGF-ß1-transduced T helper cell type 1 (Th1) cells significantly protected the mice from EAE, and untransduced Th1 cells did not protect. MBP-specific BALB/c Th2 clones, transduced with TGF-ß1-internal ribosome entry site-green fluorescent protein (GFP) significantly reduced EAE induction by untransduced Th1 cells in RAG-1–/– B10.PL mice. Furthermore, the GFP+ TGF-ß1-producing Th2 cells were detectable in the spinal cords of the injected mice.

Key Words: cytokines • gene therapy • multiple sclerosis


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INTRODUCTION
 
Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS), which is a chronic, incapacitating disease characterized by damage to the central nervous system (CNS) myelin [1 ]. It is the most frequent autoimmune disease of the CNS [2 ]. Like many autoimmune diseases, EAE is thought to result from local inflammation by CD4+ T cells with immunoreactivity to myelin antigens [3 ]. The central role of CD4+ T cells in EAE is borne out by their ability to transfer disease to unchallenged hosts [4 ]. These T cells may be induced before experimental autoimmune disorder by systemic exposure to autoantigens [5 ] or may be spontaneously present and expanded during the course of disease. The spontaneous recovery from an initial disease and subsequent resistance to disease have been explained by the expansion of immunoregulatory T cells [6 ]. Spontaneous EAE, which occurs in mice harboring a monoclonal myelin basic protein (MBP)-specific CD4+ {alpha}ß T cell repertoire {achieved by crossing MBP-specific, T cell receptor (TCR)-transgenic mice with recombination-activating gene (RAG)–/– mice or TCR {alpha}–/–/TCR ß–/– double knockout mice [7 , 8 ]}, can be prevented by a single injection of CD4+ splenocytes or thymocytes from wild-type syngeneic mice. The protective regulatory (T-reg) cells need not express the CD25 marker, as CD4 T cell populations depleted of CD25+ T cells were just as effective as the CD25+ ones [9 ]. One of the mechanisms by which such T cells are believed to ameliorate the inflammatory lesions characteristic of autoimmune diseases is by the induction of anti-inflammatory cytokines, most importantly, transforming growth factor- ß (TGF-ß), interleukin (IL)-10, and IL-4. Thus, under certain conditions, neutralization of these cytokines aggravates autoimmune diseases and/or interferes with the effect of immune–T-reg cells [10 11 12 13 14 15 16 17 18 ]. Although treatments with latent TGF-ß, IL-10, or IL-4 only partially protect against autoimmunity, administration of active TGF-ß has been shown to be most effective when given during the late induction phase of EAE [13 ] or at the time of passive induction of EAE with myelin protein-sensitized T cells [19 , 20 ]. Once the disease has developed, TGF-ß cannot cause recovery from EAE. As TGF-ß induces its own production, a few TGF-ß-producing cells retained, as a result of their specificity for myelin protein, may cause neighboring oligodendrocyte and macrophages to produce more TGF-ß. Furthermore, TGF-ß has been reported to enhance the development of immunoregulatory CD8+ T cells in vitro [21 , 22 ].

TGF-ß is produced by most cells, including T cells, in a latent form, attached to a latency-associated protein (LAP), which requires removal to uncover the receptor-binding region of active TGF-ß [23 ]. Removal of LAP in the lymphoid tissue is believed to be accomplished by thrombospondin, plasmin, and/or acidification in macrophages within inflammatory sites [24 , 25 ].

Administration of active TGF-ß into joints of animals has been reported to cause inflammation [26 ], although locally injected TGF-ß counteracted the destructive effect of IL-1 on cartilage [27 ]. Systemic treatment of TGF-ß for a prolonged period may not be practical in patients, as such treatments would induce fibrosis and glomerulonephritis, as has been demonstrated in transgenic mice [28 ]. An alternative method by which TGF-ß might be of clinical use in autoimmune disease would be to introduce latent TGF-ß directly into inflammatory sites, with the hope that it would get activated by inflammatory cells in adequate amounts to thwart the local inflammation. Considering that circulating T cells with specificity for the autoantigen would be the most efficient way for transporting such latent TGF-ß into local lesions, we therefore transduced MBP-specific T cells with cDNA for latent TGF-ß1. Cloned T cells, transduced to constitutively produce latent TGF-ß, were found to be protective for a spontaneous form of EAE in MBP-specific, TCR-transgenic RAG–/– (BALB/cxB10.PL)F1 mice, as observed for the induced form of EAE.


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MATERIALS AND METHODS
 
Animals
MBP-specific, TCR-transgenic mice with disrupted RAG-1 (T/R–) mice on a B10.PL (H-2u) background [7 ] were crossed to RAG1–/– mice on a Balb/c (H-2d) background to obtain H-2d/u mice, which were used for studies. In this model, EAE develops in TCR-transgenic B10.PL mice bearing the {alpha}ß TCR specific for the MBP peptide Ac1–11 [7 ]. When the transgene is present in RAG–/– mice, all the mice develop EAE spontaneously, representing a good MS model insofar as immunization in the absence of adjuvant. For this model, 6- to 8-week-old mice were used. For a passively induced EAE model, 4- to 10-week-old RAG1–/– mice on a B10.PL background were used. All mice were kept in the specific pathogen-free facility at the Skirball Institute (New York University School of Medicine, New York).

Injection of anti-TGF-ß monoclonal antibody (mAb) into MBP-specific, TCR-transgenic RAG1+/+ mice
MBP-specific, TCR-transgenic RAG1+/+ mice on a B10.PL background [7 ] were injected (intraperitoneally) twice a week for 4 weeks, with 1 mg per injection of anti-TGF-ß mAb (2G7), which neutralizes activity of TGF-ß1, TGF-ß2, and TGF-ß3 [29 ], or with isotype-matched control antibody [mouse immunoglobulin G1 (IgG1)] in 0.5 ml phosphate-buffered saline (PBS).

Induction of EAE
For passively induced EAE, MBP-specific T helper cell type 1 (Th1) and Th2 cells were made from TCR-transgenic B10.PL RAG1+ (T/R+) mice [30 ]. MBP-specific Th1 and Th2 cells (5x106) were transferred intravenously (i.v.) into B10.PL RAG1–/– recipients. EAE was graded as follows: level 1, limp tail; level 2, partial hind-leg paralysis; level 3, complete hind-leg paralysis; level 4, front-leg weakness; level 5, moribund, as described [31 ].

Establishment of retroviral construct
The retroviral expression vector, pMIG, containing the green fluorescent probe (GFP), downstream of the internal ribosome entry site (IRES), was provided by Dr. Van Parijs of Center for Cancer Research and Department of Biology Massachusetts Institute of Technology, Cambridge, Massachusetts [32 ]. The cDNA (base pairs 352–1550), encoding latent TGF-ß1, was polymerase chain reaction (PCR)-amplified. The primers used for PCR for TGF-ß1 amplification were: forward, 5'-AATGTTAACATGCCGCCCTCGGGGCT-3'; reverse, 5'-AATGAATTCTCAGCTGCACTTGCAGGAGC-3'. The PCR product was cloned into a plasmid vector pcR2.1 (Invitrogen, Carlsbad, CA). The pCR2.1–TGF-ß1 plasmid was digested with EcoRI and HpaI, and the TGF-ß1 fragment was inserted into the pAcGP67 vector (PharMingen, San Diego, CA). The pAcGP67–TGF-ß1 plasmid was then digested with EcoRI and BglII, and the TGF-ß1 fragment was then inserted into the retroviral vector pMIG and termed pMIG-TGF-ß1-GFP

Establishment of MBP-specific T cell lines producing latent TGF-ß
The ecotropic Phoenix retroviral packaging cell line (Phoenix E) used in the studies was derived from 293 T cells and has been described elsewhere [33 ]. Packaging Phoenix E cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM-C), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. A total of 2 x 106 Phoenix E packaging cells were cultured in 12 ml DMEM-C in 100 mm tissue-culture dishes. Following overnight incubation, cells were transfected with 10 µg retroviral plasmid DNA (Qiagen, Valencia, CA) using a modified version of the calcium phosphate precipitation protocol [33 ]. After 8-12 h of transfection, the calcium phosphate-containing medium was replaced with DMEM-C, and cultures were maintained at 37°C in 5% CO2 for 24-48 h. Viral supernatants from transfected cultures were harvested, filtered through a 0.45-µm filter, and then stored at –80°C. Virus titers were determined using NIH3T3 cells, as described previously [34 ], and only virus stocks with titers >4 x 106 were used for transduction.

MBP-specific T cells (5x106 cells/ml) were cultured in complete RPMI medium (RPMI-C), supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in the presence of 10 µg/ml MBP peptide NAc1–11 (Peptide Synthesis, Keek Biotechnology Resource Center, New Haven, CT) for 24 h. Then, 2–5 x 106 MBP-specific T cells were cultured in six-well plates (0.5 ml/well) and overlaid with 2–3 ml thawed recombinant retroviral supernatant, supplemented with 8 µg/ml protamine sulfate (Sigma Chemical Co., St. Louis, MO). Plates were incubated at 37°C in 5% CO2 for 24 h. Culture medium was exchanged with RPMI-C medium, supplemented with 10 U/ml murine recombinant IL-2, and transferred to incubator at 37°C with 5% CO2 for an additional 24 h [35 ]. GFP-positive cells were sorted by fluorescein-activated cell sorter and maintained in RPMI-C at 37°C. TGF-ß1-GFP-transduced Th2 cells (5x106) were injected i.v. into RAG-1–/– B10.PL mice for studies about a passively induced EAE model.

MBP-specific Th1 cloned cells, kindly provided by Dorf and colleagues [36 ], were derived from BALB/c mice immunized with MBP.

Enzyme-linked immunosorbent assay (ELISA) analysis
For measurements of TGF-ß1 production, T cell clones were cultured in RPMI-1640 serum-free medium containing 1% Nutridoma (Boehringer Mannheim, Germany), and supernatants were harvested at 48 h (for IL-4 and IFN-{gamma}) and 72 h (for TGF-ß). All the T cell clones were stimulated every 2–3 weeks by 10 µg/ml MBP peptide Ac1–11 (MBP 1–11). Samples were assayed after activation of latent TGF-ß by treatment with acid (0.1 M HCl at 4°C for 60 min). The protein content of TGF-ß1 in these supernatants was assayed by ELISA in flat-bottom 96-well Falcon plates (Becton Dickinson and Co., Oxnard, CA), coated with 5 µg/well mAb to TGF-ß1 using natural TGF-ß1 (Genzyme, Cambridge, MA) as a standard and biotinylated mAb to TGF-ß1 (R&D Systems, Minneapolis, MN), streptavidin-peroxidase (Zymed, South San Francisco, CA), and 3,4,5-trimetoxybenzoate substrate solution (Endogen, Woburn, MA) as developing reagents. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). Productions of IL-4 and IFN-{gamma} in culture supernatants were determined by the ELISA kit (BD Biosciences PharMingen, San Diego, CA).

Modulation of EAE by TGF-ß1-transduced cells
For studies using the spontaneous EAE model (BALB/cxB10.PL)F1, mice were given one i.v. injection of 5 x 106 TGF-ß1-transduced Th1 cells beginning at 6 weeks of age and monitored for EAE score, incidence, and mortality, for a period of 7 weeks. For studies using the passively induced EAE model, 4- to 10-week-old B10.PL RAG1–/– mice were given MBP-specific, TCR-transgenic Th1/Th2 cells. Mice were then treated with a single i.v. injection of 5 x 106 TGF-ß1-GFP-transduced Th2 cells and monitored for EAE score and incidence.

Histology
The distribution of TGF-ß1-GFP-transduced Th2 cells in tissue was determined 22 days after adoptive transfer of MBP-specific, TGF-ß1-GFP-transduced Th2 cells. Mice were killed by CO2 narcosis and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cords, spleen, and liver were removed and fixed in 4% PFA at 4°C overnight, followed by cryoprotection in PBS containing 30% sucrose solution at 4°C for 24 h. The samples were embedded into optical cutting temperature compound (Tissue-Tek, Torrence CA) and frozen in liquid nitrogen. Serial tissue sections (4 µm) were prepared and inspected using fluorescence microscopy. Sections were costained with hematoxylin and eosin.

Statistical analysis
Differences in mean EAE severity between groups of mice were evaluated by Student’s t-test.


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RESULTS
 
Effect of anti-TGF-ß on EAE induction in MBP-specific, TCR-transgenic RAG+/+ B10.PL mice
Earlier studies demonstrated a role for endogenously produced TGF-ß in dampening the development of acute as well as relapsing EAE in SJL mice, as borne by the accelerated development of EAE after administration of anti-TGF-ß antibodies [31 ]. It was, therefore, of interest to determine whether neutralization of endogenously produced TGF-ß would accelerate development of spontaneous EAE in MBP-specific, TCR-transgenic RAG1+/+ mice on a B10.PL background [7 ]. MBP-specific, TCR-transgenic RAG-1+/+ B10.PL mice were given repeated injections of anti-TGF-ß mAb (2G7) or control isotype control antibodies for 4 weeks. Although the mice injected with control antibodies did not develop EAE, mice injected with anti-TGF-ß mAb began to develop EAE in 22 days after the initial injection and began to recover 5 days after termination of the treatment (Fig. 1 ).



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  		Figure 1. Induction of EAE in MBP-specific, TCR-transgenic RAG1+/+ B10.PL mice by injection of anti-TGF-ß mAb (2G7). Mice were injected twice a week for 4 weeks with anti-TGF-ß mAb or with isotype control antibodies; days of injection are indicated by vertical arrows. Anti-TGF-ß mAb group ({blacksquare}, n=6); control IgG1 group ({circ}, n=5). This experiment was repeated once with identical results.

TGF-ß production by TGF-ß1-transduced Th2 cells
The MBP-specific Th1 and Th2 cells used in these studies were first tested for their cytokine production in comparison with that of TGF-ß1-transduced Th2 cells. As expected, the Th1 cells produced predominantly IFN-{gamma} but not IL-4, and the Th2 cells produced predominantly IL-4 but not IFN-{gamma} (Fig. 2 ). MBP-specific Th1 cells are known to migrate to the spinal cord and produce lesions resulting in EAE. However, Th2 cells of similar specificity have also been shown to produce lesions in the spinal cord, suggesting they might migrate to these sites. As Th1 and Th2 cells migrate to the CNS, we decided to transduce Th2 cells with TGF-ß1, as the Th2 cells are less aggressive in producing EAE. Although the Th1 and Th2 cells produced endogenous amounts of TGF-ß after stimulation with the MBP peptide (MBP 1–11), the TGF-ß1-transduced Th2 cells produced significantly more (P<0.01) TGF-ß than did the untransduced cells.



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  		Figure 2. IFN-{gamma}, IL-4, and TGF-ß1 production by TGF-ß1-GFP-transduced Th2 and untransduced Th1 and Th2 cells. Cells were stimulated in 24-well tissue-culture plates with 10 µg/ml MBP peptide (Ac1–11) in the presence of {gamma}-irradiated B10.Pl spleen feeder cells (1–2x106 cells in 1 ml) for 2–3 weeks. For IL-4 and IFN-{gamma} determinations, the cells were restimulated with MBP peptide and feeder cells for another 48 h, and for TGF-ß1 determinations, the cultures were restimulated for another 72 h. At the end of the cultures, supernatants were harvested and analyzed by sandwich ELISA. N.D., Not detectable. Results are expressed as mean ± SE (n=5). *, P < 0.05; **, P < 0.01. These determinations were repeated once with identical results.

Kinetics of spontaneous EAE development in MBP TCR-transgenic (BALB/cxB10.PL)F1 mice
The ability of TGF-ß1-transduced Th1 cells to protect (BALB/cxB10.PL)F1 mice from spontaneous EAE development was examined. Spontaneous EAE developed in (BALB/cxB10.PL)F1 mice in 5–6 weeks of age with similar kinetics as mice injected with untransduced Th1 cells (Fig. 3 ). A single injection of as little as 5 x 106 TGF-ß1-transduced Th1 cells significantly reduced the EAE incidence, EAE score, and mortality (Fig. 3A 3B 3C , respectively). The onset of EAE in mice injected with TGF-ß1-transduced cells was delayed by at least 3 weeks, and the first mortality from EAE was delayed by 4 weeks in mice injected with TGF-ß1-transduced Th1 cells.



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  		Figure 3. Time course of spontaneous EAE development in MBP TCR-transgenic (BALB/cxB10.PL)F1 mice, which developed spontaneous EAE 5–6 weeks of age (control mice, {circ}; n=17). Mice were injected i.v. with 5 x 106 untransduced Th1 cells ({square}, n = 9) and TGF-ß-transduced Th1 cells, 5 x 106 ({blacktriangleup}, n=14). Untransduced Th1 cells do not protect from EAE; conversely, TGF-ß1-transduced Th1 cells protect from EAE. (A) EAE incidence; (B) mean EAE score; (C) mortality from EAE. *, P < 0.05; **, P < 0.01. This experiment was repeated once with identical results.

Regulation of passive EAE induction by TGF-ß1-transduced T cells
The role TGF-ß1-transduced Th2 cells in modulation of passively induced EAE was examined in B10.PL RAG–/– mice injected with MBP-specific Th1 and MBP-specific Th2 cells. Although injections of untransduced Th1 MBP-specific or Th2 MBP-specific cells induce EAE (which is absent in unimmunized mice, not shown), the injection of TGF-ß1-transduced Th2 cells alone resulted in a significantly (P<0.05) less EAE score in comparison with injection of untransduced Th1 or Th2 cells (Fig. 4 ). As the Th2 cells used are MBP-specific, it is not surprising that they would be pathogenic in immunodeficient recipients [30 ]. Injection of untransduced Th1 cells together with TGF-ß1-transduced Th2 cells significantly (P<0.05) reduced the EAE score as a result of transduced, MBP-specific Th1 cells (Fig. 4) . Similarly, the injection of untransduced, MBP-specific Th2 cells together with TGF-ß1-transduced Th2 cells significantly (P<0.05) reduced the EAE score in comparison with injection of untransduced, MBP-specific Th2 cells.



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  		Figure 4. Induction of EAE in B10.PL RAG-1–/– recipients of MBP-specific Th1 and Th2 cells. Mice were injected i.v. with 5 x 106 untransduced Th1 cells ({circ}, n = 4); 5 x 106 untransduced Th2 cells ({square}, n=4); 5 x 106 TGF-ß1-transduced Th2 cells ({blacktriangleup}, n=4); 5 x 106 untransduced Th1 cells plus 5 x 106 TGF-ß1-transduced Th2 cells (•, n=4); or 5 x 106 untransduced Th2 cells plus 5 x 106 TGF-ß1-transduced Th2 cells ({blacksquare}, n=3). (A) EAE incidence; (B) mean EAE score. This experiment was repeated once with identical results.

Localizalization of TGF-ß1-transduced T cells in spinal cords
To determine whether the TGF-ß1-transduced Th2 cells traffic to the sites of EAE lesions in the spinal cords, tissue sections of spinal cords from B10.PL RAG–/– mice injected with TGF-ß1-GFP-transduced Th2 cells were examined for the presence of GFP-positive cells. GFP+TGF-ß1-transduced Th2 cells were readily detected in the spinal cords of mice 22 days post-injection, at a time when mice showed evidence of EAE (Fig. 5 ). The GFP+ cells detected in the spinal cord comprised 24% of the DAPI+ nucleated cells. The GFP+ cells are, presumably, producing TGF-ß1, and TGF-ß1-GFP-transduced Th2 cells stimulated with a MBP-specific peptide produce twofold more TGF-ß1 than untransduced Th2 cells under similar tissue-culture conditions (Fig. 2) .



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  		Figure 5. Histological examination of tissue sections of spinal cord from B10.PL RAG–/– mice injected with untransduced, MBP-specific Th2 cells (left) or with TGF-ß1-GFP-transduced Th2 cells (right). The spinal cords of the mice were analyzed after 22 days for the presence of TGF-ß-GFP-transduced Th2 cells. EAE lesions were first noticeable in the spinal cord 22 days following transfer of Th2 cells. Green, GFP-positive T cells; blue, 4',6-diamidino-2-phenylindole (DAPI) nuclear stain. Original magnification = 1000x. Identical results were obtained from examination of tissues from four different mice per treatment group.


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DISCUSSION
 
There is ample evidence that TGF-ß1 protects against relapsing EAE and collagen-induced arthritis [15 , 37 , 38 ]. A fascinating temporal relationship was found in which TGF-ß1 protected mice against acute EAE induction only when given on Days 5–9 but not when given on Days 0–5 or 9–12 after immunization with proteolipid protein (PLP) in complete Freund’s adjuvant [31 ]. No CNS infiltrations of lymphoid cells were detected in protected mice, although MBP- and PLP-responsive T cells could be demonstrated in the peripheral blood and lymphoid tissue of protected mice. These results were interpreted to indicate that TGF-ß1 played a role in preventing sensitized T cells from entering the CNS. The production of endogenous TGF-ß has been demonstrated to be one of the factors involved in protecting against autoimmunity, as shown by the adverse effect of neutralizing anti-TGF-ß on the course of acute [12 , 31 , 39 ] and relapsing EAE [10 ], as well as collagen-induced arthritis [40 ]. As in the case of the above-mentioned models of EAE, administration of anti-TGF-ß mAb accelerated the development of EAE in the spontaneous model of EAE of MBP-specific, TCR-transgenic RAG+/+ B10.PL mice, thus implicating ameliorating role for endogenously produced TGF-ß in EAE.

Many cells, including T cells, produce TGF-ß in latent form, attached to LAP. Removal of LAP is required to uncover the receptor-binding region of active TGF-ß [23 ]. The cleavage, within lymphoid tissue, is believed to occur by thrombospondin, plasmin, and/or acidification in macrophages within sites of inflammation [24 , 25 ]. Studies conducted to determine the role of TGF-ß produced within EAE infiltrates have been inconclusive, as histochemical differentiation between active and latent TGF-ß has not been determined in these studies. Thus, it has not been clear whether the role played by endogenous TGF-ß in autoimmunity is local or systemic. Local administration of active TGF-ß into joints can cause inflammation in animals [26 , 41 ], although a counteracting effect of TGF-ß on the destructive influence of IL-1 on cartilage has been demonstrated as well [27 ]. Until recently, treatments with active TGF-ß, which have been demonstrated to protect against autoimmune disease, have all been systemic. It is well established that TGF-ß treatment over a prolonged period is harmful to patients, as it induces liver fibrosis and glomerulosclerosis, as shown in transgenic mice [28 ].

The gene therapy approach demonstrated in these studies provides a means of delivery of latent TGF-ß to the inflammatory sites, where it can then be activated with ameliorating effects and minimal risk of toxicity. It is of interest that TGF-ß-transduced, MPB-specific Th2 cells produce significantly more TGF-ß1 than untransduced Th2 cells and migrate to the CNS. In the spontaneous EAE model of MBP-TCR-transgenic (BALB/xB10.PL)F1 mice, TGF-ß1-transduced, MBP-specific Th1 cells were effective in reducing the incidence, severity, and mortality from EAE as well as delaying its onset. When EAE was passively induced by injection of MBP-specific Th1 cells in B10.PL RAG–/– mice, TGF-ß1-transduced, MBP-specific Th2 cells were effective in reducing EAE incidence and severity. In this experimental system, it has been shown that untransduced, MBP-specific Th2 cells do not alter the course of Th1 cell-induced EAE [30 ]. Although we have not transduced a T cell line specific to another antigen in our studies, work by colleagues showed that the use of activated cells of unrelated specificity (ovalbumin) did not protect against EAE [42 ]. It is also of interest that administration of TGF-ß1-transduced Th2 cells together with untransduced, MBP-specific Th1 cells protected mice from the adverse effects of untransduced, MBP-specific Th1 cells. As a therapeutic paradigm, the beneficial effect of TGF-ß1-transduced Th2 cells in protecting mice from the pathogenic effects of Th1 cells outweighs the less severe, pathogenic effect of Th2 cells transduced with TGF-ß1.

These studies also demonstrate that the effect of TGF-ß-transduced T cells is most likely mediated by TGF-ß, as the injection of anti-TGF-ß mAb into MBP-specific, TCR-transgenic RAG-1+/+ B10.PL mice removed the protective effect of TGF-ß, and the mice succumbed to EAE. This is consistent with previous studies in which neutralizing anti-TGF-ß antibodies accelerated the development of acute and relapsing EAE in SJL/J mice [31 ]. We also were able to detect GFP+ TGF-ß1-transduced Th2 cells in the spinal cords at a time when the mice exhibited evidence of EAE.

The pathways used by TGF-ß in EAE modulation have not been elucidated completely. Among the immunosuppressive effects of TGF-ß are down-regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and lymphotoxin (LT) production [15 , 43 ], responses to IL-12 [44 ], macrophage, and microglia activation [45 , 46 , 47 ], cytokine-enhanced class II expression [48 ], and migration into the CNS [31 , 49 ]. A number of studies have shown that immunoregulatory effects of T cells in humans and animal are partly a result of TGF-ß production [11 , 15 , 16 , 50 ]. A role for TGF-ß has also been shown in the induction of IL-10 production by macrophages and the inhibition of production of TNF-{alpha} and IL-1 [51 ]. Furthermore, as TGF-ß stimulates its own production [51 ], a few TGF-producing T cells retained in an infiltrate, on the basis of their specificity for myelin protein, may cause glial cells and macrophages in their vicinity to produce TGF-ß.

The temporal and spatial relationship between CNS chemokine expression and disease progression indicates a role for chemokines in MS [52 , 53 ] and suggests that the MBP-specific T cells might travel to the CNS on the basis of chemoattraction. This idea led to studies in which immunoneutralization of chemokines was tested for regulation of EAE. Such studies suggested that CC chemokine ligand 3 (CCL3) and CXC chemokine ligand 10 (CXCL10) specifically regulate the accumulation of T cells, and CCL2 regulates the accumulation of monocytes in the CNS. The main mechanism by which TGF-ß exerts its protective effects on EAE remains to be elucidated. However, examination of the relationship between chemokine and cytokine expression in the CNS during chronic relapsing EAE (ChREAE) suggested a phasic up-regulation of gene expression for chemokine T cell activation gene 3/CCL1, monocyte chemoattractant protein-1/CCL2, macrophage-inflammatory protein-1{alpha} (MIP-1{alpha})/CCL3, MIP-1ß/CCL4, regulated on activation, normal T expressed and secreted/CCL5, and MIP-2/CXCL2–3, as well as cytokines TNF-{alpha}, -ß, LT-ß, IFN-{gamma}, and TGF-ß1 in CNS during exacerbations of ChREAE [54 ]. These studies showed that although the expression of TNF-{alpha}, TNF-ß, LT-ß, and IFN-{gamma} reached peaks during less-advanced stages of the disease and decreased in the more severe phase, only the expression of TGF-ß1 correlated directly with disease severity across the entire disease spectrum. It is interesting that the peak of TGF-ß1 expression occurred 1 day after the peak of chemokine up-regulation.

The data thus far suggest an immunoregulatory role for TGF-ß1, produced by transduced T cells on autoreactive T cells, which in turn, facilitates the control of neighboring inflammatory cells. It is tempting to speculate that one of the beneficial effects of TGF-ß in EAE is its ability to suppress the chemokine system in the CNS. Conversely, an inhibitory role has been demonstrated for TGF-ß1 in blocking nuclear factor-{kappa}B activation and cytokine release, which is stimulated by Toll-like receptor (TLR) 2, 4, and 5 ligand-induced responses involving MyD88 [55 ]. It is quite possible that one of the effects of TGF-ß in blunting EAE could involve inhibition of TLR signaling. Clarification of the effect of TGF-ß on TLR signaling and its impact on innate immunity are pertinent questions that remain to be clarified. Use of TGF-ß1-transduced T cells appears to be a feasible means of treating EAE and possibly MS in humans.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Multiple Sclerosis Society. This work is dedicated to the memory of the late Dr. G. Hochwald and G. Jeanette Thorbecke for their insightful input in these studies. Our thanks go to Drs. G. Dranoff (Dana Farber Institute for Cancer Research, Boston, MA) for the retrovirus engineered packaging cells Dr. Van Parijs (Massachusetts Institute of Technology, Cambridge, Massachusetts) for the retroviral expression vector, and Dr. M. E. Dorf (Harvard Medical School, Boston, MA) for the MBP-specific Th1 cells.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received May 17, 2005; revised August 5, 2005; accepted August 17, 2005.


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