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Protection against Autoimmunity in Nonlymphopenic Hosts by CD4⁺CD25⁺ Regulatory T Cells Is Antigen-Specific and Requires IL-10 and TGF-¹

Departments of Medicine and Immunology, Duke University Medical Center, Durham, NC 27710

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25⁺ regulatory T cells (T_Reg) play a critical role in the control of autoimmunity. However, little is known about how T_Reg suppress self-reactive T cells in vivo, thus limiting the development of T_Reg-based therapy for treating autoimmune diseases. This is in large part due to the dependency on a state of lymphopenia to demonstrate T_Reg-mediated suppression in vivo and the unknown Ag specificity of T_Reg in most experimental models. Using a nonlymphopenic model of autoimmune pneumonitis and T_Reg with known Ag specificity, in this study we demonstrated that these T_Reg can actively suppress activation of self-reactive T cells and protect mice from fatal autoimmune pneumonitis. The protection required T_Reg with the same Ag specificity as the self-reactive T cells and depended on IL-10 and TGF-. These results suggest that suppression of autoimmunity by T_Reg in vivo consists of multiple layers of regulation and advocate for a strategy involving Ag-specific T_Reg for treating organ-specific autoimmunity, because they do not cause generalized immune suppression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Representing 5–10 and 2–4% of the peripheral CD4⁺ T cells in healthy adult mice and humans, respectively, CD4⁺CD25⁺regulatory T cells (T_Reg)³ play important roles in the maintenance of self-tolerance and the control of autoimmunity (1, 2, 3). These T_Reg have been characterized by the expression of IL-2R -chain (CD25), CTLA-4 (4, 5), glucocorticoid-induced TNFR (GITR) (6, 7), and more recently identified T_Reg-specific transcription factor, forkhead/winged helix transcription factor (Foxp3) (8, 9, 10). It has been shown that the majority of CD4⁺CD25⁺ T_Reg are of thymic origin (11, 12, 13), although in some cases T_Reg may be generated in the periphery as well (13, 14, 15, 16). Despite these progresses, how T_Reg suppress self-reactive T cells in vivo remains uncertain, thus impeding the development of therapeutic strategies involving T_Reg for treating autoimmune diseases.

It has been shown that in vitro, CD4⁺CD25⁺ T_Reg-mediated suppression on T cell activation requires cell-cell contact, but is independent of cytokines such as IL-10 or TGF- (17, 18). In contrast to in vitro models, IL-10 and TGF- have been implicated in T_Reg-mediated suppression in vivo; however, their precise roles remain controversial, because they are model and context dependent (19, 20, 21, 22, 23). This uncertainty could be due in part to the dependency on a state of lymphopenia to demonstrate T_Reg-mediated suppression in most in vivo models. The presence of homeostatic signals in the setting of lymphopenia could complicate the interpretation of T_Reg-mediated immune regulation in vivo. Consistent with this idea, recent studies indicated that dysregulated expansion of potentially pathogenic T cells in a lymphopenic environment can be prevented even by naive T cells regardless of their specificity as a side effect of their response to homeostatic stimulation (24). Therefore, a nonlymphopenic model is required to address the mechanisms of T_Reg-mediated suppression in vivo.

Although CD4⁺CD25⁺ T_Reg-mediated suppression in vitro is non-Ag specific (18, 25), the role of Ag specificity in T_Reg-mediated suppression in vivo remains unknown. In fact, some early experiments suggested a role of Ag specificity in T_Reg cell-mediated regulation in vivo. It has been shown in the day 3 thymectomy models that splenocytes from female donors or orchidectomized male donors were less effective in preventing autoimmune orchitis than those from normal male donors (26). Furthermore, CD4⁺ T cells from athyroid rats effectively suppressed autoimmune diabetes, but not autoimmune thyroiditis (27). Thus, to determine whether CD4⁺CD25⁺ T_Reg regulate autoimmunity toward pathogenic T cells with the same Ag specificity or different epitopes in a bystander manner, it is necessary to develop a model using CD4⁺CD25⁺ T_Reg with known Ag specificity for regulation of autoimmunity.

To address these questions directly, we used a nonlymphopenic model of autoimmune pneumonitis in influenza hemagglutinin (HA)-transgenic mice (C3-HA) that express HA as a self-Ag in a variety of organs, including lung (28, 29). Previous studies have shown that adoptive transfer of naive HA-specific T cells from TCR-HA transgenic mice (clone 6.5) into C3-HA^high mice that express high levels of the HA transgene led to activation of HA-specific T cells and development of fatal autoimmune pneumonitis within 4–7 days after transfer (30). We have also observed that the naive HA-specific T cells failed to elicit mortality when transferred into C3-HA^high and TCR-HA double-transgenic mice. In this study we showed that C3-HA^high and TCR-HA double-transgenic mice had abundant HA-specific CD4⁺CD25⁺ T cells with regulatory function. Adoptive transfer of purified HA-specific CD4⁺CD25⁺ T_Reg into C3-HA^high mice suppressed activation of naive HA-specific T cells and protected mice against fatal autoimmune pneumonitis. The suppression in vivo was dependent on IL-10 and TGF- and required T_Reg with the same Ag specificity as the self-reactive T cells. Using this approach, we demonstrate that CD4⁺CD25⁺ T_Reg can suppress self-Ag-driven activation of autoreactive T cells in vivo and prevent autoimmunity in a nonlymphopenic environment through both IL-10 and TGF- in an Ag-specific manner.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

B10.D2 and BALB/c mice (H-2^d) were purchased from The Jackson Laboratory. The C3-HA transgenic mice were provided by Dr. D. Pardoll (Johns Hopkins University, Baltimore, MD) and have been described previously (28, 29, 31). The 6.5 TCR-HA transgenic mice on the BALB/c background that express a TCR recognizing an I-E^d-restricted HA epitope (¹¹⁰SFERFEIFPKE) (12) were provided by Dr. H. von Boehmer (Harvard University, Boston, MA) (32). Some of these mice were backcrossed for more than nine generations onto the Thy1.1⁺ or Thy1.2⁺ B10.D2 genetic background. The DO11.10 TCR-OVA transgenic mice in BALB/c background that express a TCR recognizing an I-A^d-restricted OVA epitope (³²³ISQAVHAAHAEINEAGR) (33) were purchased from The Jackson Laboratory (33). RAG-2^–/– mice were purchased from Taconic Farms. TCR-OVA, TCR-HA, and TCR-HA x C3-HA^high on a RAG-2^–/– background were bred in our animal facility. All mice used in these studies were between 8 and 12 wk of age. Experimental procedures were performed in accordance with protocols approved by the Animal care and use committee of Duke University Medical Center.

Adoptive transfer

Naive clonotypic HA- and OVA-specific T cells were prepared from TCR-HA and TCR-OVA transgenic mice on a RAG-2^–/– background as described previously (34) and transferred into recipients in 0.2 ml of HBSS. For transfer of clonotypic CD4⁺CD25⁺ T cells, single-cell suspensions were prepared from TCR-HA x C3-HA double-transgenic mice. CD4⁺ T cells were enriched through depleting non-CD4 cells by a mixture of biotinylated mAbs, followed by anti-biotin MicroBeads (Miltenyi Biotec). Enriched CD4⁺ T cells were then subjected to cell sorting gated on 6.5⁺CD4⁺CD25⁺ vs 6.5⁺CD4⁺CD25^– cells with a high speed cell sorter FACSVantage (BD Biosciences). The purity of FACS sorted populations of cells was >98%.

In vivo Ab blocking

For in vivo cytokine blocking experiments, mice were injected with 0.5 mg of anti-TGF- (1D11) Ab (R&D Systems), anti-IL-10R (1B1.3a) Ab (BD Pharmingen), or the control rat IgG (Jackson ImmunoResearch Laboratories) i.v. 6 h before adoptive transfer of naive transgenic T cells. Mice were monitored for survival or were harvested for analyses.

Flow cytometry

The following mAbs were purchased from BD Pharmingen: anti-CD4 (RM4-5), anti-Thy1.1 (OX-7), anti-CD44 (1M7), anti-IL-10 (JES5), anti-CD25 (7D4), anti-CTLA4 (UC10), anti-TGF- (A75-3.1), anti-TCR-OVA (KJ1-26), and anti-IFN- (XMG1.2). Anti-GITR mAb (108619) was purchased from R&D Systems. Anti-TCR-HA Abs (6.5) were purified and conjugated in our laboratory. Cells were stained with the desired mAbs and subjected to FACS analysis using FACSCalibur (BD Biosciences). To assess the production of IFN- or IL-10 intracellularly, cells were incubated in the presence of 10 µg/ml I-E^d-HA epitope peptide and 5 µg/ml brefeldin A containing Golgi-Plug (BD Pharmingen) for 6 h at 37°C. After washing, cells were stained with CD4 and Thy1.1 and permeabilized to detect intracellular IFN- or IL-10 using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s instructions.

Real-time RT-PCR

Total RNA was extracted from 5 x 10⁵ sorted cells using TRIzol (Invitrogen Life Technologies). Oligo(dT)₁₅-primed cDNA was generated with ImProm-II reverse transcriptase (Promega). The expression of Foxp3 and hypoxanthine phosphoribosyltransferase (HPRT) was quantified by real-time PCR using the TaqMan Universal PCR MasterMix (Applied Biosystems) as well as the following primers and internal fluorescent probes: Foxp3, 5'-GGC CCT TCT CCA GGA CAG A-3', 5'-GCT GAT CAT GGC TGG GTT GT-3', and 5'-FAM ACT TCA TGC ATC AGC TCT CCA CTG TGG AT-TAMRA-3'; or HPRT, 5'-TGA AGA GCT ACT GTA ATG ATC AGT CAA C-3', 5'-AGC AAG CTT GCA ACC TTA ACC A-3', and 5'-FAM-TGC TTT CCC TGG TTA AGC AGT ACA GCC C-TAMRA-3'. The normalized value for Foxp3 mRNA expression in each sample was calculated as the relative quantity of Foxp3 derided by the relative quantity of HPRT (x100).

Proliferation assay

Naive 6.5⁺CD4⁺ cells (2.5 x 10⁴) from TCR-HA mice were cultured in the presence of irradiated (3000 rad) B10.D2 splenocytes (2.5 x 10⁵) and 10 µg/ml of the I-E^d-HA peptide. For in vitro suppression experiments, sorted 6.5⁺CD4⁺CD25⁺ (2.5 x 10⁴) or 6.5⁺CD4⁺CD25^– (2.5 x 10⁴) cells were added to the wells. Incorporation of [³H]thymidine (1 µCi/well) during the last 18 h of a 66-h culture was measured by scintillation counting.

Dendritic cell (DC) preparation

Bone marrow-derived DCs were generated as previously described (34). Briefly, bone marrow cells were cultured in the presence of murine GM-CSF (1000 U/ml) and IL-4 (500 U/ml) for 5 days. On day 5, cells were harvested and transferred onto a new plate in the above-described DC medium with addition of TNF (500 U/ml). On day 7, DCs were harvested and loaded with HA and OVA peptides for immunization.

Histopathology and immunohistochemistry

Paraffin sections (5 µm) were stained with H&E according to standard procedures. Random sections were examined for histopathology in a blinded fashion. Detection of donor-derived Thy1.1⁺ cells was performed by immunohistochemistry as previously described (31).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
A model of fatal autoimmune pneumonitis

To determine whether CD4⁺CD25⁺ T_Reg can inhibit self-Ag driven activation of autoreactive T cells in vivo in the absence of homeostatic signals, we first further characterized a previously described mouse model of autoimmune pneumonitis using C3-HA transgenic mice (30). Adoptive transfer of 1.2 x 10⁶ naive HA-specific T cells (Thy1.1⁺) into C3-HA^high mice that express high levels of HA in a variety of organs, including lung, led to 100% mortality in recipients within 5–6 days after transfer (Fig. 1A). Pulmonary pathology revealed that these animals developed severe pneumonitis characterized by perivascular and alveolar infiltration (Fig. 1B). Immunohistochemistry with anti-Thy1.1 Ab indicated that the pulmonary infiltrate was mainly composed of donor-derived, self-reactive T cells (Fig. 1B). Four days after transfer of CFSE-labeled naive HA-specific T cells, vigorous proliferation was detected in spleen and hilar lymph nodes (LNs) including at least six divisions in C3-HA^high mice (Fig. 1C). Extensive proliferation resulted in activation of self-reactive T cells and development of effector function, indicated by their ability to produce IFN- (Fig. 1C). Neither mortality nor T cell activation was observed when naive HA-specific T cells were transferred into nontransgenic B10.D2 mice or C3-HA^low mice that express low levels of HA in the lung (500- to 1000-fold lower than that of C3-HA^high mice) (29), suggesting that induction of fatal pneumonitis was Ag specific and was dependent on the levels of self Ag expression in the target organ (Fig. 1, A and B). The mortality of C3-HA^high mice was also related to the number of clonotypic T cells transferred in a dose-dependent manner (Fig. 1D).

View larger version (61K): [in this window] [in a new window]   FIGURE 1. A model of fatal autoimmune pneumonitis. A, Nontransgenic B10.D2, C3-HAhigh, or C3-HAlow mice (n = 10 for each group) were adoptively transferred with naive HA-specific T cells (Thy1.1+) and monitored for survival. Data represent the Kaplan-Meier survival curve, indicating the percent survival over time after T cell transfer. B, In some experiments mice were harvested 4 days after transfer of T cells and examined for pulmonary pathology by H & E staining and for T cell infiltration by immunohistochemistry using Thy1.1 mAb. C, CFSE-labeled naive HA-specific T cells (Thy1.1+) were used for transfer. Four days later, lymphocytes from hilar LNs were analyzed for in vivo divisions (by CFSE dilution) and function (by intracellular IFN- staining). For CFSE dilution, events were gated on CD4+Thy1.1+ T cells; for intracellular IFN- staining, events were gated on CD4+ T cells. D, C3-HAhigh mice (n = 10 for each group) were adoptively transferred with 5 x 106, 2.5 x 106, 1.2 x 106, 6 x 105, 3 x 105, or 1 x 105 of naive HA-specific T cells and monitored for survival. Representative data of three independent experiments are shown.

We next looked at whether the observed mortality can be induced by intercrossing TCR-HA transgenic mice with C3-HA^high mice. Although there was a reduction in the number of TCR-HA x C3-HA^high double-transgenic litters compared with that of TCR-HA or C3-HA^high single-transgenic ones, the double-transgenic mice survive well after birth. Furthermore, these double-transgenic mice became refractory to naive HA-specific T cell-induced pulmonary lethality (Fig. 2A). No mortality was observed even with a dose of 5 x 10⁶ HA-specific T cells (Fig. 2A). When CFSE-labeled naive HA-specific T cells were transferred into these double-transgenic mice, very little activation of self-reactive T cells, characterized by poor proliferation and no effector function, was detected in hilar LNs (Fig. 2B). These results suggested that protection against lethal pneumonitis in C3-HA^high x TCR-HA double-transgenic mice was probably due to lack of self-reactive T cell activation.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Protection from fatal autoimmune pneumonitis in C3-HAhigh x TCR-HA double-transgenic mice. A, C3-HAhigh or C3-HAhigh x TCR-HA mice (n = 10 for each group) were adoptively transferred with 1.2 x 106 or 5 x 106 of naive HA-specific T cells and monitored for survival. B, CFSE-labeled naive HA-specific T cells (Thy1.1+) were transferred into C3-HAhigh or C3-HAhigh x TCR-HA mice, and cells were analyzed for in vivo divisions and function of TCR-HA T cells. For CFSE dilution, events were gated on CD4+Thy1.1+ T cells; for intracellular IFN- staining, events were gated on CD4+ T cells. Representative data of two independent experiments are shown.

We next observed that in double-transgenic mice, there was a large population of clonotypic HA-specific CD4⁺ T cells (6.5⁺CD4⁺) that expressed CD25 (Fig. 3). These 6.5⁺CD4⁺CD25⁺ T cells (referred to as 6.5⁺CD25⁺) were abundant in secondary lymphoid tissues, such as spleen and peripheral LNs (Fig. 3A). They expressed high levels of CTLA-4, GITR, IL-10, and TGF- compared with 6.5⁺CD25^– T cells from the same mouse (Fig. 4A). Thus, the phenotype of 6.5⁺CD25⁺ T cells suggested that they were T_Reg. Indeed, when the expression of the T_Reg-specific marker, Foxp3 (8, 9, 10), was analyzed by real-time PCR, high levels of Foxp3 mRNA were detected in sorted 6.5⁺CD25⁺ T cells (Fig. 4B). The level of Foxp3 expression in 6.5⁺CD25⁺ T cells was comparable to that in naturally occurring polyclonal CD4⁺CD25⁺ T_Reg cells from B10.D2 mice (Fig. 4B).

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Generation of clonotypic CD4+CD25+ T cells in C3-HAhigh x TCR-HA double-transgenic mice. A, Thymus, spleen, and peripheral LNs were harvested from C3-HAhigh or C3-HAhigh x TCR-HA mice at 8 wk of age. Single-cell suspensions were stained with anti-CD4, anti-TCR-HA (6.5), and anti-CD25. For thymus, events were gated on 6.5+CD4+CD8– cells; for spleen and LNs, events were gated on 6.5+CD4+ cells. B, Cells were harvested from 8-wk-old C3-HAhigh RAG-2–/– or C3-HAhigh x TCR-HA RAG-2–/– mice and analyzed as described.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Characteristics of clonotypic CD4+CD25+ T cells. A, Production of CTLA-4, GITR, IL-10, and TGF-. Cells from spleens of C3-HAhigh x TCR-HA double-transgenic mice were stained with anti-CD4, anti-TCR-HA (6.5), and anti-CD25 in combination with anti-CTLA-4, anti-GITR, anti-IL-10, or TGF-. Events were gated on 6.5+CD4+CD25– vs 6.5+CD4+CD25+ cells. B, Expression of Foxp3. Total RNA samples were extracted from 6.5+CD25+, 6.5+CD25–, or 6.5–CD25+ cell populations isolated from C3-HAhigh x TCR-HA double-transgenic mice as well as polyclonal CD4+CD25+ T cells from B10.D2 mice as a control. Normalized Foxp3 mRNA abundance is derived from the ratio of Foxp3 to HPRT expression (x100), and data are presented as the mean ± SD. C, In vitro function of clonotypic 6.5+CD25+ T cells. Naive HA-specific T cells were cultured with APC and 10 µg/ml of the I-Ed-HA peptide alone (Control) or with sorted 6.5+CD25– or 6.5+CD25+. In some experiments, naive T cells and 6.5+CD25+ cells were separated by a Transwell devise (6.5+CD25+ Transwell); in others, 10 µg/ml anti-IL-10 (6.5+CD25+ and IL-10 Ab) or anti-TGF- Ab (6.5+CD25+ and TGF- Ab) was added to the cultures. Cultures were labeled with [3H]thymidine and harvested for scintillation counting. Results are expressed as the mean ± SD.

Clonotypic CD4⁺CD25⁺ T cells suppress naive TCR-HA T cell response in vitro

To confirm that 6.5⁺CD25⁺ T_Reg possess regulatory function, we analyzed their suppressive activity in vitro. Purified 6.5⁺CD25⁺ cells potently suppressed the proliferation of naive HA-specific T cells, whereas 6.5⁺CD25^– cells failed to do so (Fig. 4C). Separation of 6.5⁺CD25⁺ T_Reg from naive HA-specific T cells by a Transwell abrogated suppression of these cells (Fig. 4C). In addition, the suppressive activity of 6.5⁺CD25⁺ T_Reg did not require IL-10 or TGF-, because the addition of anti-IL-10 or anti-TGF- Ab did not ablate suppression (Fig. 4C). Thus, similar to the property of naturally occurring polyclonal CD4⁺CD25⁺ T_Reg (17, 18), these results indicate that the suppressive activity of 6.5⁺CD25⁺ T_Reg is cell contact dependent and cytokine independent in vitro.

Clonotypic CD4⁺CD25⁺ T_Reg protect against lethal autoimmune pneumonitis in vivo

We next examined the capacity of 6.5⁺CD25⁺ T_Reg cells to inhibit fatal autoimmune pneumonitis in C3-HA^high mice. Purified 6.5⁺CD25⁺ T_Reg cells were then transferred into C3-HA^high recipients, which were subsequently challenged i.v. with naive HA-specific T cells (Thy1.1⁺) to induce lethal autoimmune pneumonitis. Adoptive transfer of 6.5⁺CD25⁺ T_Reg completely protected C3-HA^high mice from pulmonary lethality (Fig. 5A) compared with 100% mortality in the C3-HA^high mice that received only naive HA-specific T cells (Fig. 1A). However, transfer of 6.5⁺CD25^– T cells generated from the same group of mice failed to prevent mortality (Fig. 5A). The protection from fatal pneumonitis by 6.5⁺CD25⁺ T_Reg cells was accompanied by a notable reduction in perivascular and alveolar infiltration compared with no reduction of infiltration by transfer of 6.5⁺CD25^– T cells (Fig. 5B). Immunohistochemistry with anti-Thy1.1 Ab indicated that transfer of 6.5⁺CD25⁺ T_Reg cells inhibited the infiltration of donor-derived self-reactive T cells (Thy1.1⁺). When CFSE-labeled naive HA-specific T cells were used for the challenge experiment, transfer of 6.5⁺CD25⁺ T_Reg cells, but not 6.5⁺CD25^– T cells, suppressed proliferation as well as the development of effector function by HA-specific T cells (Fig. 5C). Taken together, these results showed that the clonotypic CD4⁺CD25⁺ T_Reg possessed the capacity to protect mice from lethal autoimmune pneumonitis through suppressing the activation of self-reactive T cells in nonlymphopenic mice.

View larger version (59K): [in this window] [in a new window]   FIGURE 5. Protection against fatal autoimmune pneumonitis by clonotypic CD4+CD25+ TReg cells. C3-HAhigh mice were adoptively transferred with 6 x 105 of sorted 6.5+CD25+, 6.5+CD25– or 6.5–CD25+ T cells from C3-HAhigh x TCR-HA double-transgenic mice or polyclonal CD4+CD25+ T cells from B10.D2 mice. These mice were then challenged with 1.2 x 106 of naive HA-specific T cells (Thy1.1+) and monitored for survival and pulmonary pathology. A, Kaplan-Meier survival curve. Data represent the percent survival over time after transfer of naive HA-specific T cells. B, Pulmonary pathology. Four days after transfer of naive HA-specific T cells, pulmonary pathology was examined by H & E staining or immunohistochemistry for infiltration by donor-derived T cells using Thy1.1 mAb. C, Four days after transfer, cells from hilar LNs were analyzed for in vivo divisions and function of HA-specific T cells. For CFSE dilution, events were gated on CD4+Thy1.1+ T cells; for intracellular IFN- staining, events were gated on CD4+ T cells. D, Different doses (1.2 x 106, 2.5 x 106, or 5 x 106) of polyclonal CD4+CD25+ T cells from B10.D2 mice were transferred into C3-HAhigh mice, which were then challenged with 1.2 x 106 of naive HA-specific T cells (Thy1.1+) and monitored for survival. Representative data of three independent experiments are shown.

To test whether polyclonal T_Reg can also suppress naive HA-specific T cell-induced pneumonitis, we purified 6.5^–CD25⁺ T_Reg cells from C3-HA^high x TCR-HA double-transgenic mice or polyclonal naturally occurring CD4⁺CD25⁺ T_Reg from nontransgenic B10.D2 mice. The levels of Foxp3 expressed by these T_Reg were comparable to those expressed by 6.5⁺CD25⁺ T_Reg cells (Fig. 4B). There Foxp3⁺ T_Reg were then transferred into C3-HA^high recipients, which were subsequently challenged i.v. with naive HA-specific T cells (Thy1.1⁺). Transfer of 6.5^–CD25⁺ T_Reg or polyclonal CD4⁺CD25⁺ T_Reg cells did not protect mice from fatal pneumonitis (Fig. 5A). Neither reduction of pulmonary infiltrate (Fig. 5B) nor suppression of naive HA-specific T cell activation (Fig. 5C) was noted in mice transferred with 6.5^–CD25⁺ T_Reg or polyclonal CD4⁺CD25⁺ T_Reg. These results suggested that suppression of self-reactive T cell activation and prevention of autoimmunity by T_Reg in vivo required T_Reg cells with the same Ag specificity.

To test whether differential expansion of clonotypic 6.5⁺CD25⁺ T_Reg vs polyclonal CD4⁺CD25⁺ T_Reg in C3-HA^high mice contributes to the observed Ag specificity in T_Reg-mediated suppression in vivo, we first transferred purified 6.5⁺CD25⁺ T_Reg or polyclonal CD4⁺CD25⁺ T_Reg cells and followed their expansion over time in vivo (Table I). Transfer of 6.5⁺CD25⁺ T_Reg and CD4⁺CD25⁺ T_Reg cells led to 3- to 4-fold and 2- to 2.5-fold expansion, respectively, by day 15 in both spleen and hilar LNs in C3-HA^high mice, consistent with previous observations that T_Reg are not anergic in vivo and can undergo limited expansion in response to Ag stimulation (35, 36, 37, 38). Because we observed that expansion of 6.5⁺CD25⁺ T_Reg was slightly more efficient than that of polyclonal CD4⁺CD25⁺ T_Reg, we examined whether transfer of escalated doses of polyclonal CD4⁺CD25⁺ T_Reg protected C3-HA^high mice from HA-specific, T cell-induced pneumonitis. As shown in Fig. 5D, no protection was observed even with a dose of 5 x 10⁶ of CD4⁺CD25⁺ T_Reg (8-fold higher than 6.5⁺CD25⁺ T_Reg), suggesting that the slight difference in expansion did not contribute to the observed Ag specificity in T_Reg-mediated suppression in vivo.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Clonotypic TReg cell-mediated suppression in vivo is Ag specific. BALB/c mice were transferred with a mixture of 2 x 105 of naive HA-specific T cells (HA) and 2 x 105 of naive OVA-specific T cells (OVA) with or without coinjection of 2 x 105 sorted HA-specific TReg cells (6.5+CD25+) i.v. These mice were then immunized with 1 x 106 mature DCs (DC) or DCs loaded with 10 µg/ml I-Ed-HA peptide and 10 µg/ml I-Ad-OVA peptide (DC/Pep). Four days later, splenocytes were harvested and stained for FACS analysis. A, In vivo expansion of HA-specific (CD4+Thy1.1+) or OVA-specific (CD4+KJ1–26+) T cells. B, Quantitative analysis of Ag-specific T cells. Representative data of two independent experiments are shown.

Clonotypic T_Reg-mediated protection in vivo is mediated by IL-10 and TGF-

Although IL-10 and TGF- did not seem to play a role in 6.5⁺CD25⁺ T_Reg-mediated suppression in vitro (Fig. 4C), 6.5⁺CD25⁺ T_Reg cells from C3-HA^high x TCR-HA double-transgenic mice secreted high levels of IL-10 and TGF- (Fig. 4A). Thus, it was of interest to determine whether IL-10 and TGF- played a role in the protection of mice from lethal autoimmune pneumonitis. 6.5⁺CD25⁺ T_Reg cells were transferred into C3-HA^high recipients, and mice were challenged with naive HA-specific T cells. Before transfer, mice were treated with 0.5 mg of anti-IL-10, anti-TGF-, or control Ab. Surprisingly, mice treated with anti-IL-10 or anti-TGF- Ab succumbed with 100% mortality within 8 days after transfer of naive HA-specific T cells (Fig. 7A). In vivo blocking of IL-10 or TGF- was associated with increased proliferation and gain of effector function by the self-reactive T cells in the presence of 6.5⁺CD25⁺ T_Reg cells (Fig. 7B). These results suggest that in addition to Ag-specific regulation by T_Reg, both IL-10 and TGF- were required for T_Reg-mediated suppression in vivo and protection from the lethal autoimmune pneumonitis.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. IL-10 and TGF- are required for the protection by clonotypic CD4+CD25+ TReg cells. A, Naive HA-specific T cells (Thy1.1+) were transferred into C3-HAhigh mice pretreated with sorted 6.5+CD25+ cells. Six hours before the transfer of naive HA-specific T cells, these mice were treated with 0.5 mg of anti-IL-10 Ab, anti-TGF- Ab, or irrelevant rat IgG (Control Ab) i.v. and monitored for survival. B, CFSE-labeled naive HA-specific T cells were used for transfer. Four days later, lymphocytes from hilar LNs were analyzed for in vivo divisions and function of HA-specific T cells. For CFSE dilution, events were gated on CD4+Thy1.1+ T cells; for intracellular IFN- staining, events were gated on CD4+ T cells. Representative data of two independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Despite accumulating evidence that CD4⁺CD25⁺ T_Reg play a crucial role in the control of autoimmunity (1, 2), very little is known about how they function in vivo. This is largely due to the limitations in current models for T_Reg-mediated suppression in vivo. One of them is that most models heavily rely on a state of lymphopenia to demonstrate T_Reg-mediated suppression (39, 40, 41, 42, 43). The presence of homeostatic signals in the lymphopenic environment has made it difficult to discern whether T_Reg-mediated suppression on naive or effector T cells in vivo is due to competition for space or cytokines (24) or to active suppression of self-Ag-driven activation of autoreactive T cells. Using a nonlymphopenic model of autoimmune pneumonitis, in this study we provide direct evidence that CD4⁺CD25⁺ T_Reg can inhibit self-Ag-driven activation of autoreactive T cells in vivo in the absence of homeostatic signals. Furthermore, our findings that only 6.5⁺CD25⁺ T_Reg, but not 6.5⁺CD25^– T, cells inhibit activation of naive HA-specific T cells suggest that this is a process of active suppression, rather than intraclonal T cell competition for available space or cytokines.

Another limitation is that in most in vivo models, the Ag specificity of the CD4⁺CD25⁺ T_Reg cells is unknown. Thus, whether CD4⁺CD25⁺ T_Reg cells suppress the activation of self-reactive T cells with the same Ag specificity or different ones in a bystander fashion remains to be defined despite the idea that T_Reg-mediated suppression in vitro is non-Ag specific (18, 25). Some early studies suggest that Ag specificity may play a role in T_Reg-mediated regulation in vivo (26, 27). Using a model of HA-specific T_Reg, we have provided evidence that suppression of self-reactive T cells and prevention of autoimmune pneumonitis by T_Reg in vivo is Ag specific. Similarly, HA-specific T_Reg have been shown to specifically inhibit HA-specific allograft rejection (44, 45) or tumor rejection (46). Whether this reflects the mode of action for naturally occurring T_Reg in a nonlymphopenic setting remains to be confirmed, because T_Reg development in these transgenic TCR mice might be different from that of naturally occurring T_Reg cells in nontransgenic mice, which might influence the function of these transgenic T_Reg cells. Thus, it will be important to develop a model that allows us to study how naturally occurring T_Reg suppress autoimmunity in a nonlymphopenic host. Furthermore, the delineation of how T_Reg are generated in the thymus will also help us to better understand their function.

How T_Reg cells suppress activation of naive T cells with the same Ag specificity remains to be defined. We could envision three potential mechanisms for the observed Ag specificity in T_Reg-mediated suppression in vivo: 1) T_Reg could compete with self-reactive T cells for the available MHC/peptide ligands on DCs; 2) if cell-cell contact is also necessary for suppression in vivo, then the binding to DCs that present the same MHC/peptide ligand could serve as a mechanism to bring T_Reg and self-reactive T cells together; and 3) T_Reg could down-regulate the function of DCs that present the same MHC/peptide ligand to self-reactive T cells. Thus, future work should delineate these potential mechanisms responsible for T_Reg-mediated suppression in vivo.

In addition to the requirement for Ag specificity, we found that T_Reg-mediated suppression in vivo was critically dependent on immunosuppressive cytokines, IL-10 and TGF-. These data suggest that the mechanisms by which T_Reg-mediated suppression in vivo are far more complex and require multiple layers of regulation. How, then, do IL-10 and TGF- contribute to Ag-specific immune regulation by T_Reg in vivo? Our data indicate that both IL-10 and TGF- may contribute to T_Reg-mediated suppression in vivo by inhibiting the expansion and effector functions of self-reactive T cells. These suppressive cytokines could exert an effect on self-reactive T cells directly or through DCs indirectly. These observations are consistent with the findings of a previous study that IL-10 may play a role in the control effector T cell expansion by CD4⁺CD25⁺ T_Reg (47). Furthermore, it has been shown that TGF- signaling is required for in vivo expansion of CD4⁺CD25⁺ T_Reg (48, 49), suggesting that TGF- may also contribute to T_Reg-mediated suppression in vivo by controlling the number of available Ag-specific T_Reg. Consistent with previous observations on naturally occurring T_Reg cells (22, 23, 35, 47), we have shown that clonotypic 6.5⁺CD25⁺ T_Reg cells produce high levels of TGF- and IL-10. However, many other cell types may also produce soluble IL-10 and/or TGF-, including endogenous polyclonal T_Reg in our system (50, 51, 52, 53). Thus, it is not clear what type of cells produce IL-10 and/or TGF-, which are required for the observed immune suppression by Ag-specific T_Reg cells. Thus, future studies will focus on identifying the cellular source of IL-10 and TGF- as well as defining the precise roles of IL-10 and TGF- in Ag-specific T_Reg cell-mediated suppression in vivo.

In conclusion, we have demonstrated in a model of autoimmune pneumonitis that CD4⁺CD25⁺ T_Reg cells can suppress self-Ag-driven activation of self-reactive T cells and protect mice from fatal pneumonitis in vivo. In this nonlymphopenic model, the suppression and protection by T_Reg are Ag specific and dependent on both IL-10 and TGF-. These results indicate that T_Reg can actively suppress autoimmunity in an Ag-specific manner and suggest an appealing therapeutic strategy using Ag-specific T_Reg for treating autoimmune diseases, because they would not cause generalized immune suppression.

	Acknowledgments

 
We thank Dr. Drew Pardoll for the C3-HA transgenic mice, and Dr. Harald von Boehmer for the 6.5 TCR-HA transgenic mice.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was in part supported by National Institutes of Health Grant CA093659 (to Y.Y.).

² Address correspondence and reprint requests to Dr. Yiping Yang, Departments of Medicine and Immunology, Duke University Medical Center, Box 3502, Durham, NC 27710. E-mail address: yang0029{at}mc.duke.edu

³ Abbreviations used in this paper: T_Reg, regulatory T cell; DC, dendritic cell; Foxp3, forkhead/winged helix transcription factor; GITR, glucocorticoid-induced TNFR; HA, hemagglutinin; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph node.

Received for publication April 12, 2005. Accepted for publication July 21, 2005.

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