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(Investigative Ophthalmology and Visual Science. 2003;44:4805-4812.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0843
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TGF-ß Inhibits Activation and Uveitogenicity of Primary but Not of Fully Polarized Retinal Antigen-Specific Memory-Effector T Cells

Hui Xu,1,2 Phyllis B. Silver,1 Teresa K. Tarrant,1,3 Chi-Chao Chan,1 and Rachel R. Caspi1

1From the Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland. 2Present address: Department of Immunology, Holland Laboratory of the American Red Cross, Rockville, Maryland; and 3Duke University School of Medicine, Durham, North Carolina.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. TGF-ß exerts suppressive effects on immunity, but its potential applications in therapy of ocular autoimmunity have not been widely explored. In the present study, the effects of TGF-ß on uveitogenic T cells were examined.

METHODS. The effects of TGF-ß on newly primed cells from mice given a uveitogenic regimen of interphotoreceptor retinoid-binding protein (IRBP) were compared with the effects on fully polarized Th1 cells from a long-term uveitogenic T-cell line. The parameters measured were T-cell proliferation, IFN-{gamma} production, induction of IL-12R expression, triggering of pathogenicity, and expression of costimulatory molecules on antigen-presenting cells (APCs) during in vitro exposure to antigen.

RESULTS. TGF-ß suppressed B7.1 expression on APCs in cultures of lymph node cells from immunized mice. It also suppressed T-cell proliferation, IFN-{gamma} production, IL-12 receptor accumulation, and the IL-12-promoted acquisition of uveitogenic function. In contrast, the polarized Th1 cells were either resistant to suppression or were enhanced by TGF-ß.

CONCLUSIONS. The results suggest that TGF-ß suppresses acquisition of effector functions by autopathogenic T cells, in part by interfering with their response to IL-12 through downregulation of IL-12R expression and in part through inhibition of APC function. The data suggest that although TGF-ß may effectively inhibit activation and recruitment of new T cells into the effector pool, it may be less effective in suppressing the reactivation of already polarized memory T cells that are less dependent on IL-12 and costimulation.

TGF-ß is a family of cytokines that includes three structurally and functionally similar isoforms: TGF-ß1, -ß2, and -ß3. The initially identified TGF-ß type is now known as TGF-ß1.1 TGF-ß exerts pleiotropic effects on a wide variety of cell types. With regard to the immune system, TGF-ß has received increasing attention because of its inhibitory effects on immunocompetent cells, including their activation, proliferation, and differentiation.2 Published evidence indicates that TGF-ß inhibits T- and B-cell proliferation, inhibits the secretion of IgG and IgM by B cells, promotes the development of B cells into IgA-secreting cells, suppresses the generation of cytotoxic T cells, and suppresses the growth and function of NK as well as lymphokine-activated killer (LAK) cells.3 4 5 TGF-ß has been shown to inhibit autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), colitis, collagen-induced arthritis, and allograft rejection.6 7 8 9 Treatment with exogenous TGF-ß prevents a relapse of EAE, whereas neutralization of endogenous TGF-ß by administration of anti-TGF-ß antibodies enhances the severity of EAE.10 11

In the eye, TGF-ß is found at high levels and has a central and nonredundant role in the regulation of immune and inflammatory responses.12 13 It has been implicated in the maintenance of the immune privilege of the eye, including direct inhibitory effects on T cells, and in protection from inflammatory damage by FasL-triggered neutrophils. It is a central player in the phenomenon known as anterior chamber-associated immune deviation (ACAID).13 It would therefore be of particular interest to examine the ability of this cytokine to regulate autopathogenic T cells that elicit ocular autoimmune inflammation.

Experimental autoimmune uveoretinitis (EAU) serves as a model for human autoimmune uveitis and can be induced in animals by immunization with retinal antigen(s) or by adoptive transfer of retinal antigen-specific T cells.14 Antigen-specific helper T (Th) cells can exist as functionally polarized Th1 and Th2 subsets that differentiate from a common precursor.15 16 Th1 cells characteristically produce IFN-{gamma} but not IL-4, Th2 cells produce IL-4 but not IFN-{gamma}, and precursor Th0 cells produce both, albeit in lesser amounts.17 Our studies have shown that EAU is mediated by retinal antigen-specific Th1 cells.18 19 IL-12 plays a critical role in the generation of uveitogenic T cells by driving their commitment toward the Th1 pathway. EAU cannot be induced in IL-12-deficient mice or in mice treated with anti-IL-12 antibodies.19 20 21 Thus, EAU shares essential cellular mechanisms with other autoimmune disease models mediated by Th1 cells, including EAE, autoimmune orchitis, arthritis, and others.

TGF-ß can act as a counterregulatory cytokine that inhibits certain IL-12-driven cellular responses, such as IL-12 signaling, including inhibiting the phosphorylation of signal transducers and activators of transcription (STATs).22 23 24 25 We have shown that in T cells specific for myelin basic protein (MBP), an antigen that can induce EAE, there is an accumulation of mRNA for IL-12Rß1 and -ß2 chains upon exposure to MBP. TGF-ß reduces this antigen-driven accumulation of IL-12R mRNA and presumably downregulates expression of the IL-12R by the cells.26 However, in that study we did not examine whether there was a functional impact on the ability of these cells to elicit EAE. In the present study, we explored the effect of TGF-ß on IL-12R expression and the function of uveitogenic T cells, and we examined the possible role of costimulation in these effects. The data indicate a differential effect of TGF-ß on expression of IL-12R and pathogenicity of early and fully polarized uveitogenic effector T cells, and implicate at least in part effects of TGF-ß on antigen-presenting cells (APCs) in this effect.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice
B10.A and B10.RIII mice were purchased from Jackson Laboratories (Bar Harbor, ME). Both males and females between the ages of 6 and 12 weeks were used. The use of laboratory animals conformed to institutional guidelines and to the provisions of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Reagents
Recombinant human (rh) TGF-ß1 was purchased from R&D Systems (Minneapolis, MN) or from PeproTech (Rocky Hill, NJ). Purified recombinant mouse (rm)IL-12 was provided by Maurice K. Gately (Hoffmann-La Roche Inc., Nutley, NJ). Capture antibodies and biotinylated antibodies for IFN-{gamma} ELISA were purchased from BD-PharMingen (San Diego, CA; purified rat anti-mouse IFN-{gamma} monoclonal antibody, 18181D; biotinylated rat anti-mouse IFN-{gamma} monoclonal antibody, 18112D). Horseradish-peroxidase-labeled streptavidin was obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL). Anti-CD11b-FITC (cat. no. 01714A), anti-B7.1-PE (cat. no. 09605B), anti-CD3-FITC (cat. no. 01084D), and anti-CD28-PE (cat. no. 01675B), used for flow cytometry, were purchased from BD-PharMingen. Bovine interphotoreceptor retinoid-binding protein (IRBP) was prepared from bovine retinal tissue by concanavalin A Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) affinity chromatography and fast-performance liquid chromatography as described previously.27 Human IRBP-derived peptide 161-180 (SGIPYIISYLHPGNTILHVD) was synthesized by Fmoc chemistry (model 432A peptide synthesizer; Applied Biosystems, Foster City, CA). Complete Freund’s adjuvant (CFA) was purchased from Sigma-Aldrich Co. (St. Louis, MO) and was supplemented with Mycobacterium tuberculosis strain H37RA to a final concentration of 2.5 mg/mL.

Cells and Cell Culture
Lymph nodes were removed from mice 11 days after immunization and were pooled within each group. Spleen cells from primed mice or naïve mice were dispersed into single-cell suspensions and pooled.

The uveitogenic Th1 cell line specific to a peptide of human IRBP (p16-180) has been described.28 Briefly, the line was derived from draining lymph nodes of B10.RIII mice immunized with human IRBP peptide 161-180, polarized in vitro toward the Th1 phenotype by culture in the presence of antigen, IL-12, and anti-IL-4. Thereafter the cells were maintained by alternating cycles of expansion in IL-2 and restimulation with antigen every 2 to 3 weeks in the presence of syngeneic splenocytes irradiated with 2500 rad as APCs.

For antigen-driven proliferation, triplicate cultures in DMEM supplemented as described29 and containing 1% mouse serum were set up in 96-well U-bottomed culture plates as follows: 5 x 105 primed lymph node cells (LNCs) or 2.5 x 105 T cells plus 2.5 x 105 irradiated APCs, in 100 µL suspension added to 100 µL medium containing various stimulants. Alternatively, anti-CD3 was used as a surrogate for antigen to induce T-cell receptor-driven proliferation.30 31 T cells without APCs (2.5 x 105 per well) or primed LNCs (2.5 x 105 per well) were incubated in anti-CD3-coated tissue culture plates (BD-Falcon Labware, Bedford, MA) in the presence or absence of anti-mouse CD28 (clone 37.51, at 1.25 µg/mL, the optimal concentration determined in preliminary experiments). All proliferation cultures were incubated for 48 hours at 37°C in 10% CO2, and each well was pulsed with 1 µCi of 3H-thymidine overnight (16–18 hours). 3H-TdR uptake was determined by standard liquid scintillation counting. For determination of antigen-driven IFN-{gamma} production, cultures similar to those used for proliferation were set up at 2 x 106 cells/well, and supernatants were harvested at the indicated times after stimulation.

IFN-{gamma} Determination by ELISA
IFN-{gamma} in culture supernatants was analyzed by ELISA. Briefly, 96-well ELISA plates were coated overnight with anti-IFN-{gamma} (100 µL of 2 µg/mL) in 0.1 M NaHCO3 (pH 8.2). The plates were rinsed with washing buffer (PBS pH 7.5, with 0.5% Tween 20 and 0.05% Na azide) and were blocked with blocking buffer (PBS-Tween with 0.1% BSA and 0.05% Na azide) for 2 hours at room temperature. After two washes, duplicates of serially diluted supernatant samples or of recombinant standard were added to the wells, and the plates were incubated overnight at 4°C. After four washes, biotinylated anti-IFN-{gamma} was then applied, followed by six washes. Streptavidin-HRP conjugate (1:5000) was added to the plate for 30 minutes at 37°C, followed by eight washes. Color reaction was performed with o-phenylenediamine dihydrochloride substrate and was terminated by addition of 2 N H2SO4. The plates were read at 490 nm with an ELISA reader (Molecular Devices, Sunnyvale CA).

RT-PCR for IL-12Rß1 and -ß2
Cells stimulated in culture in the presence or absence of TGF-ß were harvested after 18 hours of stimulation. Total RNA was isolated from the cell pellet (Tri Reagent; Molecular Research Center, Cincinnati, OH). Two micrograms RNA was transcribed into cDNA. Samples were normalized by hypoxanthine-guanine phosphoribosyltransferase (HPRT) PCR, as described previously,32 and then were amplified with the primers for IL-12Rs (denaturation: 94°C, 2 minutes 30 seconds; PCR: 94°C, 30 seconds; 57°C, 1 minute; 72°C, 1 minute; 32 cycles; extension: 72°C, 8 minutes). The primers for IL-12Rß1(sense: 5'-CCT GGA GCA GGA GGA ATG TTA CC-3'; anti-sense: 5'-GGG AAA TCT GCA CCT CAA AGC-3') and IL-12Rß2 (sense: 5'-GGG GCT GCA TCC TCC ATT AC-3' and anti-sense: 5'-AAG TGC TGT TTG CTG GAT CTG G-3') were designed (Mac Vector 5.0; Eastman Kodak Co., Rochester, NY), based on cDNA sequences in GenBank for mouse IL-12Rß1 (U2392233 ) and IL-12Rß2 (U6419934 ) (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). PCR products were electrophoresed on 2% agarose gel in the presence of ethidium bromide and photographed. The density of bands was analyzed by computer (Un-scanit software; Silk Scientific, Orem, UT).

Antibody Staining and Flow Cytometric Analysis
LNCs from peptide 161-180-immunized B10.RIII mice were restimulated with the peptide (20 µg/mL) in the presence or absence of TGF-ß (10 ng/mL). The cells were harvested from the culture at 72 hours after immunization, washed, and resuspended in a flow cytometry staining buffer (1 x Hanks’ Balanced Salt Solution [HBS] containing 3% FCS and 0.02% NaN3; FACS buffer; BD Immunocytometry Systems, San Jose, CA), and 1 x 106 cells were costained with anti-CD11b-FITC/anti-B7.1-PE or anti-CD3-FITC/anti-CD28-PE. The cells were incubated with antibody on ice for 30 minutes followed by two washes with flow cytometry buffer. For each sample, 50,000 live cells were collected on a flow cytometer (FACScan; BD Immunocytometry Systems) and were analyzed by computer (FlowJo, ver. 4.0 software; Tree Star, Inc., San Carlos, CA).

Experimental Autoimmune Uveoretinitis
Induction by Active Immunization.
Mice were immunized with 50 µg antigens in a 1:1 (vol/vol) emulsion with CFA in a total volume of 0.2 mL divided among base of the tail and both hind thighs. Bordetella pertussis toxin (PTX; 0.5 µg in a volume of 100 µL) was injected intraperitoneally at the same time. Eyes were collected 21 days after immunization and examined for disease scores by histopathology, as described ahead.

Induction by Adoptive Transfer of Primed LNCs or Long-Term Th1 Cells.
Primed LNCs were collected from p161-180-immunized B10.RIII mice on day 11 after immunization and were stimulated with 10 µg/mL of peptide 161-180 in the presence or absence of IL-12 and 10 ng/mL of TGF-ß for 3 days, essentially as described previously.21 29 The cells were harvested from culture and washed, and 2 x 107 cells were injected into syngeneic naïve mice intraperitoneally. Rested T cells from a peptide 161-180-specific uveitogenic Th1 cell line28 were stimulated in 24-well plates (Corning Costar, Corning, NY) with 2 µg/mL of the antigen in the presence of APCs as for the proliferation assay described earlier, with or without 10 ng/mL TGF-ß. Supernatants collected from the cultured cells were assayed for IFN-{gamma} content as described earlier. Eyes from adoptive transfer recipients were collected 12 days after adoptive transfer (corresponding to approximately 1 week after disease onset), and EAU scores were determined by histopathology.

Determination of EAU Scores
Freshly enucleated eyes were prefixed for 1 hour in 4% buffered glutaraldehyde and postfixed in 10% buffered formaldehyde until they were processed. Fixed and dehydrated tissue was embedded in methacrylate and 4- to 6-µm sections were cut through the pupillary-optic nerve plane. The sections were stained by standard hematoxylin and eosin. Eight to 10 sections cut at different planes were examined for each eye. Severity of EAU was scored on a scale of 0 (no disease) to 4 (maximum disease) in half-point increments, according to a semiquantitative system described previously.35

Statistical Analysis
Experiments were repeated at least twice to confirm the pattern of responses. Statistical analysis was by t-test of replicates. Shown are the mean ± SEM.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Inhibitory Effect of TGF-ß on Antigen-Specific Responses of LNC in Primary Culture, but Not on Cells from an Established T Cell Line
EAU was induced in B10.RIII mice by immunization with a uveitogenic peptide of human IRBP, residues 161-180. EAU in B10.A mice was induced by immunization with native IRBP. To explore the role of TGF-ß in uveitogen-induced cellular responses, we harvested lymph node cells 11 days after immunization and analyzed the ability of the cells to proliferate and produce IFN-{gamma} in response to restimulation in vitro with the immunizing antigen. The results showed that TGF-ß inhibited the antigen-specific proliferation and IFN-{gamma} production of LNCs from both B10.A and B10.RIII mice (Figs. 1a 1b 1c 1d) . TGF-ß also strongly inhibited proliferation of primed and naïve splenocytes stimulated with either plate-bound or soluble anti-CD3 (data not shown).



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  		FIGURE 1. TGF-ß inhibited antigen-driven proliferation and IFN-{gamma} production of recently primed T cells, but not of a polarized uveitogenic T cell line. Primed LNCs were isolated from mice immunized with IRBP (B10.A mice) or peptide 161-180 (B10.RIII mice). Uveitogenic T cells specific to peptide 161-180 (derived from B10.RIII mice) were harvested from cultures. For proliferation, triplicate cultures of 5 x 105 cells per well (2.5 x 105 T cells and 2.5 x 105 APCs for the T-cell line) were restimulated with antigen (50 µg/mL of IRBP or 20 µg/mL of peptide 161-180) in the presence or absence of the indicated amounts of TGF-ß. For IFN-{gamma} production, 2 x 106 cells per well were restimulated with antigen. Supernatants harvested at 18, 36, and 72 hours were assayed by ELISA. (a, c, e) Primary LNCs from B10.A stimulated with IRBP, primary LNCs from B10.RIII stimulated with peptide 161-180, and a polarized T-cell line stimulated with peptide 161-180, respectively. (b, d, f) IFN-{gamma} production from parallel cultures.

 
Similar proliferation and cytokine assays were performed with a peptide 161-180-specific uveitogenic T-cell line derived from B10.RIII mice and carried in vitro for a prolonged period. These cells have a stably polarized Th1 cytokine profile. In contrast to its effect on the responses of primary T cells, TGF-ß actually enhanced proliferation and IFN-{gamma} production of the Th1 line cells in response to the antigen (Figs. 1e 1f) .

Inhibitory Effect of TGF-ß on IL-12-Supported Acquisition of Uveitogenicity by Primed LNC
To induce EAU, antigen-specific lymphocytes must be triggered (activated) with the specific antigen in culture immediately before transfer to the adoptive host. In a previous study, we found that IL-12 is required for EAU to develop, and that addition of IL-12 to the culture step resulted in exacerbated disease by promoting the Th1 phenotype of uveitogen-primed T cells.19 21 In the present study, we examined the potential modulating effect of TGF-ß on adoptively transferred EAU. LNCs from B10.RIII mice immunized with peptide 161-180 collected on day 11 after immunization were cultured in vitro with a combination of peptide 161-180 and IL-12, with or without TGF-ß, for 3 days before they were infused into naïve syngeneic recipients. The results showed, as expected, that IL-12 enhanced the severity of EAU in the recipients. Addition of TGF-ß to the culture abrogated the enhancing effect of IL-12 on EAU (Fig. 2a) . Furthermore, when we assayed the supernatants from these cultures for IFN-{gamma}, we found that cells in which acquisition of uveitogenicity was inhibited by the addition of TGF-ß to the culture step also showed reduced IFN-{gamma} production (Fig. 2b) .



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  		FIGURE 2. TGF-ß counteracted the enhancing effect of IL-12 on (a) uveitogenicity of primed LNCs and (b) their IFN-{gamma} production. LNCs of peptide 161-180-immunized B10R.III mice were stimulated with a combination of antigen (10 µg/mL) and recombinant IL-12 (10 ng/mL) in the presence or absence of TGF-ß (10 ng/mL). The cells were harvested from the cultures at 72 hours, and 20 x 106 cells were infused intraperitoneally into syngeneic recipients. Eyes collected 9 days later were evaluated for EAU on a scale of 0 to 4 (a). IFN-{gamma} was assayed in supernatants of the transferred cells at 72 hours of culture by ELISA (b).

 
Inhibition by TGF-ß of IL-12-Promoted Acquisition of Uveitogenicity and IL-12R mRNA Accumulation in Primed LNC
In a previous study, we reported that TGF-ß inhibits antigen-driven accumulation of IL-12 receptor mRNA in CD4+ encephalitogenic T cells. However, in that study we did not pursue the question of whether this has an in vivo impact on the pathogenic potential of these cells. The purpose of the present experiments was to examine whether TGF-ß-induced reduction in IL-12R mRNA is correlated with inhibition of the acquisition of uveitogenic function by the affected cells. The same primed LNCs that were stimulated with peptide 161-180 in the presence or absence of TGF-ß and were used for the adoptive transfer of EAU in Figure 2 were examined for expression of IL-12R mRNA by RT-PCR. The results showed that antigen stimulation increased the accumulation of both IL-12Rß1 and -ß2 mRNA in LNCs, an increase that was further enhanced in the presence of IL-12. Addition of TGF-ß did not appear to affect IL-12R mRNA levels induced by antigen alone, but counteracted the enhancing effect of IL-12 on the accumulation of mRNA encoding both receptor chains (Fig. 3) . The IL-12-supported acquisition of uveitogenicity and enhancement of IFN-{gamma} production by the LNCs were inhibited in parallel. Thus, IL-12-mediated enhancement of IL-12R expression correlated with the ability of the cells to induce disease.



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  		FIGURE 3. TGF-ß inhibited the antigen-induced accumulation of IL-12Rß1 and -ß2 mRNA in primed LNCs. LNCs from peptide 161-180-immunized B10.RIII mice (1 x 106/mL) were restimulated with the antigen (10 µg/mL) in the presence or absence of TGF-ß (10 ng/mL). The cells were harvested after 18 hours and IL-12Rß1 and -ß2 mRNA was determined by PCR. PCR products were electrophoresed on 2% agarose gel in the presence of ethidium bromide and were photographed. (a) Gel showing actual bands. (b) Corresponding target bands analyzed on computer and shown as the ratio of IL-12R to HPRT. Shown is a representative experiment of two. MW, molecular weight standard.

 
Lack of Effect of TGF-ß on Uveitogenicity and IL-12R Expression of Polarized Effector/Memory Cells
Because the in vitro proliferative response of the long-term Th1-cell line to antigen was insensitive to inhibition by TGF-ß, unlike LNCs in primary culture, we examined the effect of TGF-ß on IL-12R expression and on the uveitogenicity of the line. Line cells that were triggered in vitro in the presence of as much as 10 ng/mL of TGF-ß exhibited undiminished uveitogenicity (Fig 4a) . RT-PCR analysis of IL-12R expression by these cells revealed that TGF-ß did not reduce the accumulation of IL-12R mRNA to a significant extent (Figs. 4b 4c) . These data are compatible with the interpretation that the sensitivity of primary versus polarized T cells of the same antigenic specificity to inhibition by TGF-ß is quite different. The result further supports the notion that uveitogenicity, proliferation, and IL-12R expression by effector T cells are coordinately regulated by TGF-ß, irrespective of their stage of polarization.



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  		FIGURE 4. Polarized uveitogenic Th1 cells were refractory to inhibition of uveitogenicity and IL-12R expression by TGF-ß. Uveitogenic Th1 cells were stimulated with peptide 161-180 in the presence or absence of 10 ng/mL TGF-ß. The cells were harvested after 48 hours of culture. (a) Uveitogenicity in recipients of 2 x 106 cells 12 days after adoptive transfer, according to EAU scores (scale of 0–4). (b) In parallel, the expression of IL-12Rß1 and -ß2 in the transferred cells was analyzed by RT-PCR. MW, molecular weight standard. (c) Density of the bands was analyzed on computer and is shown as the ratio of IL-12R to HPRT.

 
Differential Effect of TGF-ß on Polarized versus Primary T Cells and Their Differential Requirements for Costimulation
TGF-ß is known to inhibit APC expression of costimulatory molecules.36 37 To become activated, T cells in various stages of differentiation require different degrees of accessory molecule participation.38 These effects would be independent of direct TGF-ß effects on the T cells. We therefore examined the effect of TGF-ß on expression of the APC costimulatory molecule B7.1 and its T-cell coreceptor CD28. Draining lymph nodes from peptide 161-180-primed B10.RIII mice were cultured with antigen in the presence or absence of TGF-ß. Expression of B7.1 on APCs (CD11b+ population) and of CD28 on T cells (CD3+ population) was determined by flow cytometry, as described in the Materials and Methods section. B7.1 expression in CD11b+ cells (which function as APCs) was reduced after culture with TGF-ß compared with the control cells (Fig 5) . In contrast, CD28 expression in the CD3+ population (T cells) was undiminished.



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  		FIGURE 5. Effects of TGF-ß on B7.1 and CD28 expression in LNCs. LNCs from B10.RIII mice immunized with peptide 161-180 were cultured with the peptide (20 mg/mL) in the presence or absence of TGF-ß (10 ng/mL). The cells were collected after 72 hours and were costained with anti-CD11b-FITC/anti-B7.1-PE or anti-CD3-FITC/anti-CD28-PE. (a) B7.1 expression in CD11b-positive cells. CD11b positive population was gated and analyzed for B7.1 expression; (b) CD28 expression in T cells. CD3 population was gated and analyzed for CD28 expression. Filled peak: isotype control; green dashed line: unstimulated; blue line: peptide 161-180; red line: peptide+TGF-ß.

 
If ineffective costimulation on the part of the APCs was causally involved in the observed effects, it would be predicted that the effects would be antigen-independent and reversible by excess costimulation. We therefore assayed the proliferative response of peptide 161-180-primed LNCs and of polarized T cells to immobilized anti-CD3, with or without excess costimulation in the form of anti-CD28 antibody, in the presence of graded doses of TGF-ß. Proliferation of the LNCs in response to T-cell receptor (TCR) ligation was progressively inhibited by increasing doses of TGF-ß, an effect that was completely reversed by ligating the CD28 coreceptor (Fig. 6a) . We interpret this finding as showing that the costimulatory signal provided by the agonistic anti-CD28 antibody compensated for the reduced levels of B7.1 on the APCs in the LNC population. In contrast, the proliferative response of the T-cell line to TCR ligation was enhanced rather than inhibited by TGF-ß, recapitulating the response shown in Figure 1 , even at concentrations as high as 40 ng/mL. Costimulation through CD28 did not affect the pattern or the magnitude of the response, consistent with a reduced need for costimulation by more differentiated T cells38 (Fig. 6b) .



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  		FIGURE 6. Inhibition of TCR-driven proliferation in primary LNCs but not in polarized T cells was reversed by excess costimulation. LNCs from peptide 161-180-primed B10.RIII mice (a) or T cells (b), were stimulated with plate-bound anti-CD3, with or without anti-CD28, in the presence of graded doses of TGF-ß. Shown is the percentage of response normalized to control not containing TGF-ß. Stimulated control counts without TGF-ß ranged from 20,000 cpm (T-cell line) to 60,000 cpm (LNCs).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
TGF-ß plays a central role in ocular homeostasis at several levels. The ocular fluids contain microgram quantities of TGF-ß—mostly of the TGF-ß2 isoform, which is integral to the immunologic privilege of the eye.12 13 In addition, TGF-ß is central to induction of the ACAID phenomenon, whereby antigens introduced through (and possibly ones that originate from) the eye elicit a deviant immune response that can be protective against ocular autoimmunity.13 39

Because of the high levels of TGF-ß normally present in the eye, concerns have been raised that effector lymphocytes that are able to overcome the anti-inflammatory threshold and induce EAU may have somehow developed a resistance to TGF-ß-induced regulation. Evidence that might argue against this hypothesis is that extraocularly produced TGF-ß (which would mostly be the TGF-ß1 isoform) can counterregulate pathogenic immunologic responses to retinal antigens and protect from EAU. Thus, protective oral tolerance induced by ingesting IRBP or its immunodominant peptide can involve production of TGF-ß in the Peyer’s patches and spleen.40 41 Furthermore, pregnancy-associated resistance to EAU in mice is accompanied by elevated levels of both TGF-ß1 and -ß2.42 On the basis of this evidence, exogenous TGF-ß could be a promising candidate for the treatment of uveitis. However, it is not known at what level the regulatory effects of TGF-ß on EAU are exerted, whether at the level of the antigen-specific T cell, at the level of the APC, or possibly on the recruited leukocytes that are essential for inducing tissue damage.

In the present study, we set out to examine the effects of TGF-ß on activation and pathogenicity of the uveitogenic lymphocyte at various stages in its maturation toward becoming a polarized Th1 cell. The isoform used in this study was TGF-ß1, representing the most abundant form of TGF-ß; however, other isoforms of TGF-ß were reported to have largely overlapping functions in a spectrum of biological effects relevant to this study.43 44 45 46

We found that TGF-ß markedly suppressed proliferation and IFN-{gamma} production of recently primed cells to IRBP as well as to IRBP peptide 161-180 in two mouse strains. In contrast, TGF-ß did not suppress activation of mature uveitogenic effector-memory T cells, as represented by a uveitogenic T-cell line that had been originally derived under Th1-polarizing conditions. If anything, this cell line was functionally enhanced by TGF-ß.

We propose that the TGF-ß-sensitive, primary cultured T cells represent a phenotype that is not yet fully and stably Th1 polarized. Their incomplete polarization is inferred from the finding that enhanced uveitogenicity and production of IFN-{gamma} are triggered in primary cultured cells if IL-12 is added to the culture.21 29 Further, such primary cultured T cells, when transferred into IL-12-deficient hosts, are able to retain a pathogenic phenotype only if they are polarized before transfer by culture with IL-12.21 The present data show that T-cell populations representing mature (fully polarized) cells versus immature (primary cultured) cells, differ dramatically in their sensitivity to inhibition by TGF-ß. We believe that T-cell populations analogous to the ones represented by the T-cell line and the primary cultured cells may coexist in an autoimmune individual. Long-standing disease and repeated relapses would result in prolonged stimulation and polarization of effector-memory cells. At the same time, newly primed T cells are constantly being recruited into the effector pool as the disease continues to evolve.

The present data indicate that not only proliferation and IFN-{gamma} production, but more important, the enhanced uveitogenicity triggered by IL-12 in the primary cultured cells was abrogated by addition of TGF-ß. This suggests that TGF-ß reduces the uveitogenicity of pathogenic T cells by countering the effects of IL-12. It has been documented in other systems that TGF-ß inhibits the IL-12-induced phosphorylation of JAK2, TYK2, and STAT4.22 However, those data did not exclude the possibility that TGF-ß may also affect IL-12 signaling at the receptor level. In mouse T cells, Gorham et al.47 have shown that TGF-ß inhibits the expression of IL-12Rß2 but not IL-12Rß1 in CD4 T cells. We, in contrast, reported that TGF-ß inhibits expression of both IL12R chains in MBP-specific T cells.26 Similarly, Zhang et al.48 have shown that TGF-ß reduces both IL-12Rß2 and -ß1 expression in peripheral blood mononuclear cells (PBMCs) of patients with tuberculosis. The present data show that addition of TGF-ß to uveitogenic primary T cells cultured with antigen and IL-12 inhibited accumulation of IL-12Rß1and -ß2 mRNA. Conversely, the polarized effector-memory T cells, whose proliferation, IFN-{gamma} production, and pathogenicity are insensitive to TGF-ß-mediated inhibition, were also insensitive to the suppression of IL-12R mRNA expression by TGF-ß (Fig. 4) . Although in our study we are inferring IL-12R expression by examining its mRNA levels (antibodies to mouse IL-12R are not currently available to us), a similar study from another laboratory recently showed that TGF-ß had no effect on the expression of IL-12R on activated human T cells.24 In the aggregate, these data support the notion that, irrespective of their state of polarization, IL-12R expression on effector T cells is regulated coordinately with their ability to activate their uveitogenic potential and undergo clonal expansion.

Although some of these effects could be exerted at the level of the T cell, the reduced expression of B7.1 on APCs in the TGF-ß-treated LNCs (without a parallel decrease of CD28 on the T cells) points also to indirect effects, such as interference with the costimulatory function of APCs. This interpretation is supported by the ability of CD28 ligation to reverse the inhibitory effect of TGF-ß on LNC proliferation in response to TCR engagement. Thus, any deficit in the function of the primary T cells could be overcome by maintaining maximum levels of costimulation. In contrast, the observed resistance of the polarized T cells to the inhibitory effects of TGF-ß would reflect their reduced dependence on costimulation, in line with what has been reported for more differentiated T cells in general.38

Our data may appear to be at variance with findings of Ludviksson et al.,49 who presented evidence that Th2-polarized memory cells become insensitive to TGF-ß, whereas Th1-polarized memory cells remain sensitive. However, in that study the polarized memory cells were generated by stimulating naive ovalbumin TCR transgenic T cells in culture under polarizing conditions for one cycle and resting them for several days before the assay. Thus, these cells might be more comparable to our primary effector-memory cells derived from the explanted lymph nodes of immunized mice than to the fully polarized long-term Th1 cell line.

In summary, our study shows that TGF-ß has an inhibitory effect mainly on naïve or differentiating T cells that are not yet stably committed to the Th1 phenotype and thus can efficiently regulate early stages of the T-cell response. The inhibition affects cells undergoing TCR-driven activation and can combine effects at the level of the T cell as well as at the level of the APCs, including reduced expression of costimulatory molecules on the one hand, and antagonism of the effects of IL-12 on upregulation of IL-12R expression on the other hand. Stably polarized effector T cells appear to have lowered sensitivity to TGF-ß, suggesting that such cells can effectively sustain ocular inflammation in the face of high levels of TGF-ß, such as are present in ocular fluids. It is probable that effector-memory cells at various stages of polarization, from newly primed to mature, coexist in patients with chronic uveitis in whom the disease continues to evolve. Our results suggest that although TGF-ß as a potential immunotherapeutic approach for uveitic disease could limit recruitment of new Th1 cells into the effector pool, adjunct therapies may be needed to target the mature, polarized Th1 effector population.


   Acknowledgements
 
The authors thank Barbara Wiggert, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, for the gift of IRBP.


   Footnotes
 
Submitted for publication August 19, 2002; revised May 22, 2003; accepted May 31, 2003.

Disclosure: H. Xu, None; P.B. Silver, None; T.K. Tarrant, None; C.-C. Chan, None; R.R. Caspi, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Rachael R. Caspi, Laboratory of Immunology, NEI, NIH, Building 10, Room 10N222, Bethesda, MD 20892-1857; rcaspi{at}helix.nih.gov.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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