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Differential Expression of Smad7 Transcripts Identifies the CD4⁺CD45RC^high Regulatory T Cells That Mediate Type V Collagen-Induced Tolerance to Lung Allografts ¹

Teruaki Mizobuchi^2,*,,, Kazuhiro Yasufuku^2,*,,, Yan Zheng, M. Azizul Haque, Kathleen M. Heidler^,, Kena Woods, Gerald N. Smith, Jr., Oscar W. Cummings, Takehiko Fujisawa^*, Janice S. Blum and David S. Wilkes^3,,

^* Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan; and Departments of Medicine, Microbiology and Immunology, and Pathology, Indiana University School of Medicine, Indianapolis, IN 46202

	Abstract

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Regulatory T cells (Tregs) induced by oral tolerance may suppress immunity by production of TGF- that could also enhance Treg activity. However, all cells that are phenotypically Tregs in rats (CD4⁺CD45RC^high-RC^high) may not have regulatory function. Because Smad7 expression in T cells is associated with inflammation and autoimmunity, then lack of Smad7 may identify those cells that function as Tregs. We reported that feeding type V collagen (col(V)) to WKY rats (RT1^l) induces oral tolerance to lung allografts (F344-RT1^lvl) by T cells that produce TGF-. The purpose of the current study was to identify the Tregs that mediate col(V)-induced tolerance, and determine Smad7 expression in these cells. RC^high cells from tolerant rats were unresponsive to allogeneic stimulation and abrogated rejection after adoptive transfer. In contrast, CD4⁺CD45RC^low (RC^low) cells from tolerant rats and RC^high or RC^low cells from normal rats or untreated allograft recipients proliferated vigorously in response to donor Ags, and did not suppress rejection after adoptive transfer. TGF- enhanced proliferation in response to col(V) presented to tolerant RC^high, but not other cells. In contrast to other cells, only RC^high cells from tolerant rats did not express Smad7. Collectively, these data show that the Tregs that mediate col(V)-induced tolerance to lung allografts do not express SMAD7 and, therefore, are permissive to TGF--mediated signaling.

	Introduction

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Peripheral regulatory T cells (Tregs) ⁴ are typically CD4⁺ cells that coexpress CD25 in some murine, human, and rat studies (1, 2, 3, 4, 5, 6), or CD45RC in a rat model of organ transplantation (7). Tregs suppress cell-mediated diseases by production of various cytokines. However, recent studies examining tolerance to organ allografts and autoimmune diabetes show that not all cells that are phenotypically Tregs have regulatory activity (7, 8).

Tregs that result from oral tolerance induction may suppress cellular immunity by membrane-bound or soluble TGF- (reviewed in Ref. 3). Although TGF- is known to be a potent suppressor of T cell differentiation and function (9), recent reports show that priming human T cells in the presence of TGF- in vitro resulted in development of Ag-specific Tregs (10, 11). In addition, TGF- enhanced proliferation in those cells when rechallenged with Ag (10, 11). These potentially conflicting reports showing TGF- suppresses T cell growth in some studies, yet is able to promote expansion/proliferation of Tregs in others, could be explained by subsets of Tregs with variable responses to TGF-.

Differential expression of Smad7, a key intracellular antagonist of TGF--mediated signaling (reviewed in Ref. 9), may prove to be the key for identifying cells that are Tregs as defined by both phenotype and function. In favor of this theory, Nakao et al. (12) showed blockade of TGF--mediated signaling induced by overexpression of Smad7 in T cells abrogated the normal suppressive environment in the lung, which resulted in enhanced Ag-induced inflammation. Additionally, intestinal T cells that mediate inflammatory bowel disease strongly express Smad7, and blocking Smad7 activity in these cells resulted in T cells with regulatory function (13). Similar results were shown in studies examining the T cells that mediate autoimmune kidney disease (14). These data confirm other reports showing that susceptibility to TGF- signaling in T cells has a key role in down-regulating different diseases. Collectively, these studies suggest that T cells that do not express Smad7, which would allow permissiveness to TGF--mediated signaling, may be those cells able to function as Tregs.

We have reported that type V collagen (col(V))-induced oral tolerance abrogates lung allograft rejection by production of TGF- (15, 16, 17). Data showing that neutralizing TGF- recovered cell-mediated immunity to donor Ags suggested that tolerance was mediated by Tregs that produced TGF- (15, 16). However, the specific cells that mediate oral tolerance to any organ allograft, in general, and col(V)-induced tolerance to lung transplants, in particular, have not been reported. The current study determines the cells that mediate col(V)-induced tolerance to lung allografts and determines whether differential expression of Smad7 identifies those cells able to function as Tregs.

	Materials and Methods

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Animals

MHC (RT1)-incompatible male rats were used for the study: Wistar Kyoto (WKY, RT1^l) and Fischer 344 (F344, RT1^lvl) rats (250–300 g at the time of transplantation). All rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in the Laboratory Animal Resource Center at Indiana University School of Medicine (Indianapolis, IN) in accordance with institutional guidelines.

Preparation of collagens

Similar to our prior reports (15, 16, 17, 18), human type V collagen (col(V)), extracted from human placenta and purified by differential NaCl precipitation, was a gift from J. Seyer (VA Hospital, Hampton, VA).

Transplantation model

The orthotopic transplantation of left lung isografts (WKYWKY) or allografts (F344WKY) was performed, as previously reported (15, 16, 17, 19). This rat strain combination is mismatched at MHC class I and matched at MHC class II, as previously reported (15, 16, 17). All transplantation procedures were performed by T. Mizobuchi and K. Yasufuku. The F344WKY transplant model is associated with the development of mild acute rejection by the end of the first week and moderate to severe acute rejection by the end of the second week, which progresses to chronic rejection known as bronchiolitis obliterans (BO) by the tenth week posttransplantation (16, 20). WKYWKY isografts do not develop pathologic lesions at any time point posttransplantation. Survival exceeded 90% in all transplantation groups. No immunosuppressive therapy was given at any time during the experimental period.

Tolerance induction

Oral tolerance was induced, as reported previously (15, 16, 17). Gastric gavage was used to feed WKY rats 10 µg of col(V) dissolved in 0.5 ml of saline every other day for eight feedings. The dose of collagen was chosen because of its effectiveness in our prior studies of oral tolerance to lung allografts (15, 16). Seven days after the last feeding, these rats were used as recipients of lung allografts. In some experiments, WKY rat were fed col(V) without undergoing lung transplantation.

Purification of T lymphocyte subsets

In some experiments, CD4⁺ and CD8⁺ T lymphocytes were purified from splenocytes or lymph node cells from WKY rats by magnetic microbeads (Miltenyi-Biotec, Auburn, CA), per manufacturer’s protocol. The purity of isolated cells was determined by flow cytometry using FITC-labeled mouse anti-rat CD4 or PE-conjugated CD8 Abs (BD PharMingen, San Diego, CA).

In other experiments, purified CD4⁺ and CD8⁺ lymph node T lymphocytes were stained with FITC-labeled mouse anti-rat CD25 Abs and/or PE-labeled mouse anti-rat CTLA4 Abs (both Research Diagnostics, Flanders, NJ). In some experiments, splenic T cells were sorted by flow cytometry into CD4⁺CD45RC^high-RC^high and CD4⁺CD45RC^low-RC^low cells (anti-rat CD4 and CD45RC Abs from BD PharMingen). The purity of sorted cells exceeded 98%.

Adoptive transfer

Spleens were harvested from normal WKY rats, or from untreated WKY rats 2 wk after lung transplantation, or tolerant WKY rats 2 wk postlung transplantation. Individual splenocytes were isolated by mechanical digestion. Unseparated splenocytes or purified CD4⁺ or CD8⁺ splenic T lymphocytes (1 x 10⁷) were injected by tail vein into WKY rats 24 h before transplantation of F344 lung allografts. Other experiments used 3 x 10⁶ RC^high or RC^low cells from different groups of rats for adoptive transfer.

Mixed leukocyte reaction

Splenocytes from F344 or WKY rats (stimulators) were treated with mitomycin C (Sigma-Aldrich, St. Louis, MO) and cocultured in varying ratios with subsets of lymph node T lymphocytes (responders -3 x 10⁵/well) from untreated WKY rats, or transplant recipients (16, 17). In some experiments, cells were cultured in the presence or absence of col(V), with or without rTGF-1 (Roche Diagnostics, Indianapolis, IN). In other studies, transwell chambers were used in which subsets of T cells from WKY rats were placed in the upper wells, and lower wells contained F344 stimulator cells cocultured with subsets of splenic T cells from WKY rats. Cellular proliferation was determined as the mean ± SEM of cpm of [³H]thymidine incorporation in triplicate cultures. Data are reported as a stimulation index, which refers multiples of proliferation in T lymphocytes induced by stimulators compared with proliferation in T cells alone.

Delayed-type hypersensitivity response

Delayed-type hypersensitivity (DTH) responses were performed, as reported previously (15, 16, 17). Two weeks after lung transplantation, lung allograft recipients from different groups received 10⁷ irradiated (3000 rad) donor-derived F344 splenocytes in 30 µl of PBS into the right pinnae by s.c. injection using a 26-gauge needle. The left pinnae received an equal volume of diluent, and served as the control site. Naive WKY rats and WKY rats that received lung isografts were negative controls. The ear thickness was measured with a micrometer caliper (Mitutoyo, Field Tool Supply, Chicago, IL) in a blinded fashion immediately before and 24 h after injection. Ag-specific DTH responses were calculated according to the following formula: specific ear swelling = (right ear thickness at 24 h - right ear thickness at 0 h) - (left ear thickness at 24 h - left ear thickness at 0 h) x 10^-3 mm (19). All data were reported as the mean ± SD of triplicate measurements.

Neutralization of cytokines in DTH and MLR assays

Two or ten weeks posttransplantation, rats received 10⁷ irradiated (3000 rad) donor-derived F344 splenocytes mixed with 5 µg of polyclonal chicken anti-rat TGF- Abs (R&D Systems, Minneapolis, MN) in 30 µl of PBS into the right pinnae by s.c. injection using a 26-gauge needle. The left pinnae received an equal volume of diluent, and served as the control site. For negative controls, a separate group of allograft recipients was given an injection of 10⁷ irradiated (3000 rad) donor-derived F344 splenocytes mixed with 5 µg of control chicken Igs or control goat Igs (R&D Systems) into the right pinnae and diluent into the left. The specific ear swelling was determined, as described above. Control Igs had no effect on the DTH response (15, 16).

Neutralization of TGF- in MLR reactions used anti-TGF- Abs that were kindly provided by P. Heeger (Cleveland Clinic Foundation, Cleveland, OH) (21). Isotype-matched control Abs were used as above.

Stimulation of cytokine production

One hundred and fifty thousand mitomycin C-treated stimulator cells (F344 splenocytes) were cultured alone or with 3.0 x 10⁵ purified CD4⁺, or CD8⁺ WKY lymph node T cells from different experimental groups in 200 µl of serum-free medium in 96-well microtiter plates. After a 48-h incubation, supernatants were harvested and assayed for total TGF-, IL-4, and IL-10 by ELISA, per manufacturer’s protocols (Promega, Madison, WI, and R&D Systems). Specimens were not acidified in performing the assay for TGF-, as reported in our prior studies (15, 16).

RT-PCR

Specific primers for rat Smad7 (5'-CTCAGGCATTCCTCGGAAG-3' and 5'-GCCCTTCACGAAGCTAATC-3') were used to perform RT-PCR in RC^high cells. In brief, the PCR mixture consisted of 5 µl of cDNA, 45 µl of Platinum PCR SuperMix (Invitrogen, San Diego, CA), and 200 nM final concentration of the respective Smad7 primers. To perform amplification, samples were preheated at 94°C (2 min), then denatured at 94°C (1 min), annealed at 55°C (1 min), and extended at 72°C (1 min) over 35 cycles with a prolonged 10-min extension during the last cycle.

All samples were subjected to RT-PCR for the housekeeping gene GAPDH (5'-CAACGGCACAGTCAAGGC-3' and 5'-TGTTGCTGTAGCCATATTC-3'), which served as a positive control and internal standard. RT-PCR products were resolved on 1% agarose gel in 1x Tris-borate-EDTA buffer, visualized by ethidium bromide, and photographed using a ChemiImager 4400 low light image system (Alpha Innotech, San Leandro, CA).

Pathological grading

Two or ten weeks posttransplantation, native and transplanted lungs from each group were harvested, fixed, sectioned, stained, and graded for rejection pathology by a pathologist (O. Cummings) in a blinded fashion without prior knowledge of the transplantation group (15, 16, 17, 19, 22). Similar to these prior reports, the grading system for rejection pathology was the same as used for human lung allograft recipients (23).

Statistics

All data are expressed as the mean ± SEM. Differences between groups were determined by ANOVA. Results were considered statistically different if p values were <0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A hallmark of oral tolerance mediated by activity of Tregs is the ability to adoptively transfer tolerance to naive hosts. Therefore, we first determined whether oral tolerance could be adoptively transferred to naive lung transplant recipients. Tolerance was induced by feeding col(V) to WKY rats before receiving F344 lung allografts, as reported in Materials and Methods. Purified CD4⁺ or CD8⁺ T cells from tolerant rats were adoptively transferred to naive WKY rats before transplantation of F344 lung allografts. At 2 wk posttransplantation, the time to severe acute rejection in untreated allografts (15, 16), the pathology of allograft lungs in recipients that received adoptive transfer of tolerant CD4⁺ T cells revealed minimal rejection, grade 1. In contrast, adoptive transfer of tolerant CD8⁺ T cells resulted in severe acute rejection, grade 4 (data not shown). If untreated, the allograft lung will progress to chronic rejection known as BO by the tenth week posttransplantation (16). Therefore, to determine the duration of the protective effect induced by tolerant cells, these studies were repeated at 10 wk posttransplantation. Fig. 1A shows that isograft lungs have normal histology at 10 wk posttransplantation, and Fig. 1B shows severe rejection with BO in untreated allografts at same time point (see arrow). In contrast, Fig. 1C shows that adoptive transfer of tolerant CD4⁺ T cells down-regulated rejection at 10 wk. However, adoptive transfer of CD8⁺ T cells from tolerant rats did not prevent rejection pathology at any time point and resulted in BO (Fig. 1D, arrowhead). In separate studies examining the kinetics of the rejection response, adoptive transfer of the tolerant CD8⁺ T cells accelerated the onset of rejection (data not shown).

View larger version (61K): [in this window] [in a new window]   FIGURE 1. Pathology in isograft and allograft lungs at 10 wk posttransplantation. WKY (isografts) or F334 (allografts) was transplanted into WKY recipients. Ten weeks posttransplantation, transplanted lungs were harvested and graded for rejection pathology. A, Isograft lung showing normal histology. B, Untreated allograft lung showing extensive mononuclear cells infiltrates, destruction of the allograft, and obliteration of a small airway (arrow), BO. C, Allograft from a rat that received 1 x 107 CD4+ T cells from a tolerant rat 24 h before lung transplantation shows minimal infiltrates with preservation of lung architecture. D, Allograft lung from a WKY rat that received 1 x 107 tolerant CD8+ T cells by adoptive transfer 24 h before lung transplantation. Figure shows extensive infiltration by mononuclear cells and destruction of the allograft and development of BO (arrowhead). All photomicrographs representative of six to eight rats per group (*, p < 0.021 compared with untreated allografts or allograft recipients that received adoptive transfer of CD8+ cells from tolerant rats, x40 original magnification).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. A, Serum TGF- levels in normal WKY rats and WKY rats that received CD4+ or CD8+ T cells from tolerant rats by adoptive transfer 24 h before transplantation of F344 allografts. TGF- assayed by ELISA 2 wk posttransplantation. Data represent the mean ± SEM of TGF- in pg/ml of four to five rats in each group (*, p < 0.008 compared with other groups). B, Reduction of DTH responses to donor alloantigens by adoptive transfer of CD4+ T cells from tolerant WKY rats. A total of 1 x 107 pure CD4+ or CD8+ splenic or lymph node T cells was isolated from tolerant WKY rats 2 wk posttransplantation and adoptively transferred to untreated WKY rats 24 h before transplantation of F344 lung allografts. Two weeks posttransplantation, rats were challenged in the right pinnae with 1 x 107 irradiated (3000 rad) donor-derived F344 splenocytes, and diluent in the left pinnae as a control. To determine the effects of TGF- on suppressed DTH responses, 1 x 107 irradiated donor-derived F344 splenocytes were mixed with anti-TGF- Ab (5 µg/ml) in PBS and injected into the pinnae. The left pinnae received an equal volume of diluent plus splenocytes, and served as the control site. For negative controls, a separate group of tolerant allograft recipients received control Igs with splenocytes into the right pinnae and an equal volume of diluent plus splenocytes into the left pinnae. The ear thickness was measured, as described in Materials and Methods. Data are representative of the mean ± SEM of the DTH in four rats in each group (*, p < 0.005; , p < 0.039).

We next determined whether CD4⁺ T cells from tolerant rats produced TGF- constitutively and/or in response to allogeneic stimulation. To answer this question, CD4⁺ T cells were isolated from tolerant rats and cultured alone or in the presence of F344 splenocytes as a source of donor APCs. The table within Fig. 2 shows that normal CD4⁺ T cells or CD4⁺ T cells from untreated allograft recipients did not produce TGF- constitutively. In contrast, tolerant CD4⁺ T cells produced TGF- constitutively and alloantigen stimulated TGF- production. CD8⁺ T cells from normal rats, untreated allograft recipients, or tolerant rats did not produce TGF- constitutively or in response to alloantigen. IL-4 and IL-10, cytokines produced by Tregs in some studies (reviewed in Ref. 3), were not detected in any of the experimental groups.

Because CD4⁺CD45RC⁺ peripheral T cells in the rat have been shown to be the cells that function as Tregs in some reports (7, 24), we next determined whether these cells were expanded in tolerant allograft recipients and functioned as Tregs. Fig. 3 shows the percentages of CD4⁺CD45RC⁺ cells present in the spleens of each group. CD4⁺CD45RC⁺ cells were increased significantly in tolerant rats (30.7 ± 1.10%, p < 0.05) as compared with normals (24.1 ± 1.26%) or untreated allograft recipients (23.9 ± 0.92%). CD4⁺CD25⁺ T cells or CD4⁺ T cells that express CTLA4, that are Tregs in murine and human studies (3, 25, 26), were increased slightly in tolerant rats. However, coexpression of CD25 or CTLA-4 on CD4⁺ T cells did not identify cells with regulatory function (data not shown).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. CD4 and CD45RC expression in splenic T cells. FACS analysis of splenic T cells for expression of CD4 and CD45RC in normal WKY rats; WKY rats that received F344 allografts, untreated allograft recipients; and col(V)-fed WKY rats that were fed col(V) before transplantation of F344 lung allografts, tolerant allograft recipients. FACS analysis was performed 2 wk posttransplantation. Data are representative of the mean ± SEM of six rats in each group. Boxes represent gates used to sort RChigh and RClow cells in subsequent studies.

View larger version (54K): [in this window] [in a new window]   FIGURE 4. Allograft pathology and DTH responses after adoptive transfer. Untreated WKY rats received 3 x 106 pure RClow cells from untreated allograft recipients (C) or tolerant rats (E), or RChigh from untreated allograft recipients (D) or tolerant rats (F) 24 h before transplantation of F344 lungs. A, Shows normal histology in isograft lungs (A), and severe rejection (extensive mononuclear cell infiltration, grade 4) in untreated allografts (B). Adoptive transfer of RChigh cells from tolerant rats (F) abrogated rejection, resulting in minimal perivascular mononuclear cell infiltrates (grade 1). Adoptive transfer of RChigh or RClow cells from other groups resulted in severe acute rejection, grade 4 (C, D, and E) (x40 magnification). G, DTH responses to donor Ags (F344) in recipient rats 2 wk postadoptive transfer and transplantation. Recipients of isografts do not develop DTH responses to F334 Ags (ear swelling <20 x 10-3 mm), and untreated allograft recipients have brisk DTH to donor Ags, as previously reported. Only adoptive transfer of RChigh cells from tolerant rats suppressed DTH responses to donor Ags (*, p < 0.002 compared with all other allograft recipients). Data representative of three to four rats in each group.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Only RChigh cells from tolerant rats are unresponsive to stimulation (A) and suppress alloantigen-induced stimulation in RClow cells (B). A, RClow cells from tolerant rats (3 x 105; responders, R) were cultured, alone or with 1.5 x 105 F344 stimulator cells, S (stimulator:responder 0:1, 0.1:1, 0.3:1, 0.5:1). B, To determine their ability to suppress proliferation, 1.5 x 105 RChigh cells from tolerant rats were mixed with 1.5 x 105 tolerant RClow cells (50% add back) in coculture with 1.5 x 105 F344 stimulator cells (S:R, 0.5:1). To determine the effect of TGF- on suppression of proliferation, anti-TGF- Abs or isotype-matched control Abs (each 10 µg/ml) were added to MLRs in which responder cells were a mixture of 50% RChigh and 50% RClow cells (total number 3 x 105). In other experiments, RChigh cells were pretreated with anti-TGF-1 Abs (100 µg/ml) before addition to MLR with RClow cells, pretreated. Data reported as a mean ± SEM of stimulation index, as described in Materials and Methods. Proliferation of T cells, alone (0:1), was less than 800 cpm [3H]thymidine incorporation. Data shown are the mean ± SEM of four to six separate experiments in each group.

Studies from other investigators showed that cells primed with Ag in the presence of TGF- in vitro induced the development of Tregs able to proliferate in response to TGF- and Ag (10, 11). Data in the current study showed that col(V)-induced tolerance resulted in increased RC^high cells and these cells function as Tregs in vitro and in vivo. Therefore, expansion of these cells in vivo in the presence of high systemic levels of TGF- could be analogous to that induced by culturing Ag-specific cells in the presence of TGF- in vitro (10, 11). If so, then RC^high T cells from tolerant rats should proliferate in response to col(V) presented by autologous APCs, and TGF- should enhance the proliferative response. Indeed, col(V) induced dose-dependent proliferation in tolerant RC^high cells, and TGF- enhanced this effect (Fig. 6A). Interestingly, RC^high cells from untreated allograft recipients did not proliferate in response to col(V), with or without the addition of exogenous TGF- (Fig. 6B). Similar to a prior report (28), there are few RC^high cells recoverable from untreated rat lung allograft recipients. Therefore, not all culture conditions could be performed with these cells as compared with studies in Fig. 6A using RC^high cells from tolerant rats.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Effect of TGF- on col(V)-induced proliferation in RChigh cells from tolerant rats and untreated allograft recipients. RChigh cells from tolerant rats (A) or untreated allograft recipients (B) were cocultured alone (3 x 105; responders, R) or with irradiated autologous (WKY) splenocytes (1.5 x 105; stimulators, S), in the presence or absence of col(V) and/or TGF-, as shown. [3H]Thymidine incorporation was determined at the completion of a 4-day incubation and reported as a mean ± SEM of a stimulation index, as described in Materials and Methods. All culture conditions were not performed using cells from untreated allograft recipients due to the limited number of these cells, as described in Results. Data shown are representative of three separate experiments.

View larger version (54K): [in this window] [in a new window]   FIGURE 7. Smad7 expression in RChigh T cells from normal rats, untreated allograft recipients, and tolerant rats, as determined by RT-PCR. Smad7 was not expressed in RChigh cells from tolerant rats. Data are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recent studies show that not all cells that are phenotypically Tregs have regulatory function. Depending on the species, Tregs may fall into different categories, e.g., CD4⁺CD25⁺ T cells in mice and humans (2, 3, 4, 5), and RC^high cells in some studies in rats (7). These cells may suppress immunity by production of IL-10 (Tr1 cells), or IL-4, or by membrane-bound or soluble TGF- (Th3 cells) (reviewed in Ref. 3). To the best of our knowledge, the current study is the first to report a Th3-type RC^high cell that results in response to oral tolerance in the rat. It is important to note that Th3 cells have been identified in studies of oral tolerance in rodents, but not all oral tolerance is mediated by these cells (30, 31).

The central role of permissiveness to signaling by TGF- in regulating immune homeostasis by T cells was shown in an early report by Kehrl et al. (32). In that study, and another from Fox et al. (33), T cells that were permissive to TGF-, as evidenced by T cells that produced TGF- in an autocrine manner, were highly suppressive of proliferation in other T cells. It is now clear that permissiveness to TGF- signaling has a key role in determining whether T cells facilitate/induce inflammation or suppress immunity. Indeed, very elegant studies from multiple investigators all showed that interruption of TGF--mediated responses results in exacerbations of inflammatory diseases in lung, bowel, and kidney (12, 13, 14, 34, 35). Moreover, knockout of the TGF- gene results in early lethality in mice due to severe systemic inflammation (36, 37). Conversely, T cells that are permissive to signaling mediated by TGF- appear to have key roles in suppressing immunity or maintaining immune homeostasis in multiple organs (12, 13).

The Smad proteins are a collection of molecules that mediate intracellular signaling in response to TGF- (reviewed in Ref. 38). Although Smads 2, 3, and 4 enhance, Smad7 inhibits signaling by TGF-. Because Smad7 may determine permissiveness to TGF- signaling, then Smad7 may be key in determining whether a cell may contribute to inflammation or suppress immune activation. Indeed, recent reports document that blockade of Smad7 converts a T cell that is autoreactive to cell with regulatory function (12, 13). A common theme emerges from these reports, which is that Smad7 expression or overexpression in T cells results in heightened immunity, autoimmunity, or loss of immune regulation.

In support of this theme, data in the current study showed that only tolerant RC^high cells that did not express Smad7 had regulatory function. Zhai et al. (7) also reported that not all RC^high cells functioned as Tregs. In that study examining the cells mediating tolerance to cardiac allografts induced by partial depletion of CD4⁺ T cells before transplantation, only the RC^high cells from tolerant rats had regulatory function. Tolerance in that study was mediated by IL-4 produced by the Tregs (7). Collectively, these data underscore the fact that phenotypic analysis alone is insufficient to identify the cells that function as Tregs. Moreover, these data indicate the mechanisms of immune suppression induced by Tregs appear to be dependent on the experimental conditions and microenvironment.

Smad7 is expressed constitutively in normal CD4⁺ T cells, and data in the current study showing absence of Smad7 by RT-PCR suggest that col(V)-induced tolerance affects Smad7 expression at the transcriptional level. Although reports suggest that TGF-, IFN-, and IL-7 may induce Smad7 expression, resulting in a negative feedback loop to block TGF- signaling (38, 39), factors that down-regulate Smad7 transcription are not completely understood.

It is also interesting to speculate on the mechanism whereby T cells made tolerant by feeding col(V) suppress alloimmune responses. MHC proteins are the target and stimulus of the rejection response, and prior studies have shown that immunizing transplant recipients with donor-derived MHC peptides before transplantation can induce tolerance to the allograft (40, 41). Because col(V) does not share homology to MHC proteins, then col(V)-induced oral tolerance to lung allografts is not likely to be mediated by MHC-like epitopes within the col(V) molecule. An alternate explanation could be related to linked suppression, in which immune responses to one Ag abrogate immunity to another (42, 43). Linked suppression in the current study is suggested by data showing that col(V)-induced oral tolerance could only be transferred by T cells isolated from lung allograft recipients fed col(V) before transplantation. In contrast, T cells isolated from rats fed col(V) without undergoing lung transplantation could not transfer tolerance (K. Yasufuku and D. S. Wilkes, unpublished observations).

In summary, the current study reports that Tregs that mediate col(V)-induced tolerance to lung allografts are phenotypically CD4⁺CD45RC^high, and suppress alloimmune responses by membrane-bound and soluble TGF-. However, it is important to note that not all cells of this phenotype function as Tregs, as data showed that these cells in normals and untreated allograft recipients did not have regulatory function. The characteristic that distinguished the Tregs in tolerant rats from the other groups was permissiveness to TGF--mediated signaling, as shown by lack of Smad7 in these cells. Collectively, these data provide mechanistic reasons to explain why all cells that are phenotypically Tregs and function in a TGF--dependent manner do not have regulatory function. Moreover, the current study identifies Smad7 as a potential target for therapeutic intervention in the immune responses that mediate lung allograft rejection.

	Footnotes

 
¹ This work was supported by National Institutes of Health AI33418 to J.S.B.; a grant from the Indiana Chapter of the Arthritis Foundation to M.A.H.; and grants from the National Institutes of Health, HL69727, HL60797, and HL/AI67177, to D.S.W.

² T.M. and K.Y. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. David S. Wilkes, Division of Pulmonary and Critical Care Medicine, Indiana University School of Medicine, 1001 West Tenth Street, OPW 425, Indianapolis, IN 46202. E-mail address: dwilkes{at}iupui.edu

⁴ Abbreviations used in this paper: Treg, regulatory T cell; BO, bronchiolitis obliterans; col(V), type V collagen; DTH, delayed-type hypersensitivity.

Received for publication January 24, 2003. Accepted for publication May 23, 2003.

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