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Induction of IL-10 Suppressors in Lung Transplant Patients by CD4⁺25⁺ Regulatory T Cells through CTLA-4 Signaling¹

Ankit Bharat^*, Ryan C. Fields^*, Elbert P. Trulock, G. Alexander Patterson^*, and Thalachallour Mohanakumar^2,*,

^* Department of Surgery, Department of Pathology, Division of Cardiothoracic Surgery, and Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
T cell-mediated autoimmunity to collagen V (col-V), a sequestered yet immunogenic self-protein, can induce chronic lung allograft rejection in rodent models. In this study we characterized the role of CD4⁺CD25⁺ regulatory T cells (Tregs) in regulating col-V autoimmunity in human lung transplant (LT) recipients. LT recipients revealed a high frequency of col-V-reactive, IL-10-producing CD4⁺ T cells (T_IL-10 cells) with low IL-2-, IFN--, IL-5-, and no IL-4-producing T cells. These T_IL-10 cells were distinct from Tregs because they lacked constitutive expression of both CD25 and Foxp3. Expansion of T_IL-10 cells during col-V stimulation in vitro involved CTLA-4 on Tregs, because both depleting and blocking Tregs with anti-CTLA4 F(ab')₂ mAbs resulted in loss of T_IL-10 cells with a concomitant increase in IFN- producing Th1 cells (TIFN- cells). A Transwell culture of col-V-specific T_IL-10 cells with Th1 cells (those generated in absence of Tregs) from the same patient resulted in marked inhibition of IFN- and proliferation of T_IFN- cells, which was reversed by neutralizing IL-10. Furthermore, the T_IL-10 cells were HLA class II restricted because blocking HLA class II on APCs resulted in the loss of IL-10 production. Chronic lung allograft rejection was associated with the loss of Tregs with a concomitant decrease in T_IL-10 cells and an increase in T_IFN- cells. We conclude that LT patients have col-V-specific T cells that can be detected in the peripheral blood. The predominant col-V-specific T cells produce IL-10 that suppresses autoreactive Th1 cells independently of direct cellular contact. Tregs are pivotal for the induction of these "suppressor" T_IL-10 cells.

	Introduction

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Intrathymic clonal deletion of T cells with a high-affinity self-reactive TCR eliminates the majority of lymphocytes reactive to self-peptides (1, 2, 3). However, the intrathymic deletion is an incomplete process, and a significant proportion of self-reactive T cells that escape into the periphery can potentially induce autoimmunity (4, 5, 6, 7). Furthermore, the "central" tolerogenic mechanisms do not eliminate T cells reactive to sequestered Ags that are not expressed within the thymus (8). Following solid organ transplantation, peripheral mechanisms may also significantly contribute to the development of autoimmunity. For example, cellular immunity against an alloantigen can spread to additional epitopes within the parent or other self-proteins, so-called intramolecular and intermolecular epitope spreading, respectively (6, 9). Furthermore, inflammation within the allograft can lead to the release of previously "cryptic" determinants and neoantigens that may trigger autoimmunity. During inflammation, up-regulation of MHC and associated costimulatory molecules along with resultant infiltration by new APCs can lead to the lowering of the T cell activation threshold, thereby priming autoreactive T cells with "low-affinity" TCRs that were previously below their activation potential.

Lung allografts sustain multiple injuries due to ischemia-reperfusion, alloimmunity, external pathogens, and gastroesophageal reflux (10, 11, 12, 13). Such an inflammatory milieu is conducive for the development of autoimmunity. Recently, collagen V (col-V)³ has been demonstrated to represent a sequestered self-protein localized in the lung tissue that can induce autoimmunity and contribute to lung allograft and isograft rejection in both animal models and human subjects (14, 15, 16). However, the pathogenesis of autoimmunity to col-V following lung transplantation remains unclear. CD4⁺25⁺ T cells have emerged as potent modulators of both alloimmunity and autoimmunity (17). Nevertheless, the mechanisms of CD4⁺25⁺ T cell-mediated suppression of conventional T cells are unclear. In addition, the role of CD4⁺25⁺ T cells in maintaining peripheral tolerance to self-proteins after human allotransplantation has not been investigated. In this study, we demonstrate that CD4⁺25⁺ T cells prevent autoimmunity to col-V following human lung transplantation by inducing the development of IL-10-producing T cells.

	Materials and Methods

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Human subjects

Ten patients that underwent lung transplantation at the Washington University Medical Center/Barnes-Jewish Hospital were randomly selected for the study after obtaining informed consent. The PBMCs were isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation (Pharmacia) and stored at –135°C until evaluation. The cell yield ranged between 0.1 and 4.0 x 10⁶ PBMCs per milliliter of whole blood. The samples selected for analysis were obtained at least 1 year posttransplantation and when the patients were free of any acute or chronic rejection. The mean age of transplantation was 52.0 ± 8.1, and the male to female ratio was 4:6. The end stage pulmonary pathologies were chronic obstructive pulmonary disease (n = 7), -1-antitrypsin deficiency (n = 1), and cystic fibrosis (n = 2). Most of the transplants were bilateral (n = 9). Chronic lung allograft rejection (bronchiolitis obliterans syndrome (BOS)) was diagnosed according to standard International Society for Heart and Lung Transplantation guidelines (18). The standard immunotherapy protocol for all patients consisted of cyclosporine A, azathioprine, and prednisone.

Antibodies

The unconjugated and fluorochrome-conjugated mouse anti-human CD4 (clone RPA-T4), CD25 (clone M-A251), and isotype control Abs were purchased from BD Biosciences. Anti-human APC-CD49d (integrin ₄ chain; clone 9F10), PE/Cy5-integrin ₇ (clone FIB504), PE/Cy5-CD29 (integrin ₁; clone MAR4), and isotype controls were also purchased from BD Biosciences. Anti-human IL-10 mAb (50 µg/ml; clone JES3-19F1; BD Pharmingen) and pan-specific anti-soluble TGF- Abs (20 µg/ml; AB-100-NA; R&D Systems) were used for neutralization experiments. Anti-human CTLA-4 F(ab')₂ mAbs (10 µg/ml) were purchased from Ancell. Hybridomas for anti-HLA class II mAb KuIA2 (framework anti-HLA-DR) were produced in our laboratory (19).

Development of cell lines

PBMCs from lung transplant (LT) patients were stimulated with irradiated (3000 rad) autologous PBMCs (1:1 ratio) with 20 µg/ml human col-V (BD Biosciences) in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 15% heat-inactivated, endotoxin-free FBS (HyClone), L-glutamine (2 mmol/liter), nonessential amino acids (100 µmol/L), HEPES buffer (25 mmol/L), sodium pyruvate (1 mmol/L), penicillin (100 U/ml), streptomycin (100 µg/ml), rIL-2 (20 U/ml; Chiron), and T-stim medium without PHA (10%; BD Biosciences). The cultures were supplemented with fresh IL-2 at a concentration of 20U/ml every 4 days. The cells were re-stimulated six times every 8–10 days. After the sixth stimulation the cells were rested for 10 days, after which CD4⁺ T cells were purified from the cell lines using negative immunomagnetic separation (Miltenyi Biotec). The depletional mixture in the immunomagnetic separation contains anti-CD8, -CD14, -CD16, -CD19, -CD36, -CD56, -CD123, -TCR-, and CD235a (glycophorin A). Flow cytometric analysis confirmed the depletion of non-CD4⁺ T cells, i.e., CD8⁺ T cells, T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and erythroid cells.

ELISPOT assay

ELISPOT assays were performed as described previously (20). Briefly, MultiScreen 96-well filtration plates (Millipore) were coated overnight at 4°C with 5.0 µg/ml capture human cytokine-specific mAb (BD Biosciences) in 0.05 M carbonate-bicarbonate buffer (pH 9.6). The plates were blocked with 1% BSA for 2 h and washed three times with PBS. Subsequently, 3 x 10⁵ cells were cultured in triplicate in the presence of col-V (40 µg/ml) and irradiated feeder autologous PBMCs (APCs) (1:1 ratio). After 48 h, the plates were washed three times with PBS and three times with 0.05% PBS/Tween 20. Then, 2.0 µg/ml biotinylated human cytokine-specific mAb (BD Biosciences) in PBS/BSA/Tween 20 was added to the wells. After an overnight incubation at 4°C, the plates were washed three times and HRP-labeled streptavidin (BD Biosciences) diluted 1/2000 in PBS/BSA/Tween 20 was added to the wells. After 2 h, the assay was developed by 3-amino-9-ethylcarbazole substrate reagent (BD Biosciences) for 5–10 min. The plates were washed with tap water to stop the reaction and air dried. The spots were analyzed in an ImmunoSpot Series I analyzer (Cellular Technology), and the results were expressed as spots per million cells (spm). Any spots obtained by culturing T cell lines with APCs alone were subtracted from the number of spots in the experimental cultures.

Proliferation assay

Cells from col-V-specific cell lines were seeded in triplicate cultures in flat 96-well plates (Falcon; BD Labware) at a concentration of 2 x 10⁵ cells/well in the presence of col-V (40 µg/ml) and APCs (1:1). After 4 days, the cultures were pulsed with [³H]thymidine (1 µCi/well) for 18 h, after which [³H]thymidine incorporation into DNA was determined by means of liquid scintillation counting. The results were expressed as counts per minute after subtracting the counts obtained from the culturing cells with autologous APCs.

Coculture

Coculture experiments were performed in 24-well Transwell plates (Costar) with 0.4-µm membrane supports. For all experiments the ratio of cells on either side of membrane was 1:1. At the end of coculture, CD4⁺ T cells were negatively selected and col-V-specific response was measured using ELISPOT assays.

Activation-fixation of regulatory T cells (Tregs)

Activation-fixation of natural Tregs was performed as described earlier (21). Briefly, CD4⁺CD25⁺ T cells were separated from the PBMCs using immunomagnetic separation (Miltenyi Biotec). The yield of Tregs varied between 0.3 and 8%. First, CD4⁺ T cells were negatively purified using the non-CD4 depletional Ab mixture. Then, CD25⁺ Tregs were positively selected from the enriched CD4⁺ T cells. The purity of CD4⁺CD25⁺ T cells obtained by this protocol was >98% (not shown). The CD4⁺CD25⁺ T cells obtained were then treated with anti-CD3/CD28 Abs (Dynal Biotech). Following this treatment, the CD4⁺CD25⁺ T cells were washed thoroughly and fixed in 10% paraformaldehyde for 15 min. In parallel, PBMCs were depleted of the CD25⁺ T cells using anti-CD25 microbeads. The feeder PBMCs depleted of CD25⁺ T cells, which had <1% CD25⁺ T cells, were kept in overnight culture and then irradiated (3000 rad). The irradiated PBMCs and the isolated CD4⁺CD25⁺ T cells were then added with col-V to the developing cell lines (1:1 ratio). In separate experiments, nonstimulating anti-CTLA4 F(ab')₂ Abs were used to block CTLA-4 on CD4⁺CD25⁺ T cells. CD4⁺CD25⁺ T cells were activated overnight using anti-CD3/CD28 treatment. Following this step, the activated CD4⁺CD25⁺ T cells were incubated with the anti-CTLA4 F(ab')₂ (10 µg/ml) Abs for 30 min at room temperature and then washed three times.

Statistical Analysis

Statistical analysis was performed using Microsoft Excel 2002 and GraphPad Prism, version 4.02 (GraphPad Software). Paired or unpaired two-tailed Student and Fisher exact t tests were used as appropriate to compare the col-V response. Statistical significance was defined at p < 0.05.

	Results

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Tregs induce IL-10 predominant CD4 T cell immunity to col-V in LT patients

The col-V-specific response was first analyzed in either the presence or absence of Tregs. The PBMCs from LT patients were stimulated with col-V in the presence of irradiated autologous PBMCs acting as APCs (n = 10) to develop cell lines. The clinical and demographic profile of these patients is presented in Table I. In parallel, the PBMCs from five of these patients were stimulated in the absence of CD25⁺ T cells. CD25⁺ T cells were depleted from the original PBMC sample used to generate the cell line and from the APCs used during stimulations. Following the last stimulation, the CD4⁺ T cells were negatively selected. The cells obtained were >90% CD4⁺ (data not shown). Col-V-specific response was measured by performing ELISPOT assays in the presence of irradiated autologous APCs and col-V. For cell lines generated in the absence of Tregs, the autologous APCs used for the ELISPOT assay were also depleted of CD25⁺ T cells. Of the 10 LT patients tested, cell lines could be established in eight. These cell lines generated in the presence of CD4⁺CD25⁺ T cells revealed a high frequency of IL-10-producing CD4+ T cells (T_IL-10 cells) reactive to col-V (291 ± 24.17 spm) and a low frequency of IFN--producing T cells (T_IFN- cells, 57.33 ± 24.84 spm). The T_IL-10 cells did not constitutively produce any IL-10 in the absence of APCs and col-V: (0 spm). In addition, there was low cross-reactivity with an unrelated protein OVA (12.4 ± 9.5 spm) and collagen type II (32.2 ± 10.5 spm). However, depletion of CD25⁺ T cells led to IFN- predominance (250.67 ± 52.67 spm) with low IL-10 (39.84 ± 13.84 spm; p = 0.001) in all of the five patients tested (Fig. 1). This result indicated that the amplification of col-V-specific T_IL-10 cells was dependent on Tregs. Depletion of Tregs led to the preferential expansion of T_IFN- cells that represent Th1 cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Tregs induced IL-10-predominant immunity to col-V. Col-V-specific cell lines were developed from the PBMCs of LT patients (n = 8/10) by repeated stimulation with col-V in either the presence of absence of CD25+ T cells. In parallel, the isolated CD4+CD25+ Tregs were activated using CD3/CD28 beads overnight and subsequently fixed with paraformaldehyde. These activated/fixed (act-fixed) Tregs were then reconstituted back into the cell cultures. The col-V-specific response was measured using IL-10 (filled bars) and IFN- (open bars) ELISPOT assays. The data are representative of all experiments done in triplicate, and the SE bars (1–4; p = 0.001) are illustrated. The results are expressed as spots/million cells (spm).

The possibility of differentiation of Tregs into T_IL-10 cells was investigated next. The PBMCs were stimulated with col-V in the presence of activated but fixed Tregs. The original PBMCs were depleted of the CD25⁺ T cells as described in Materials and Methods. The autologous APCs were also depleted of CD25⁺ T cells. CD4⁺CD25⁺ Tregs were isolated using immunomagnetic separation and cultured in the presence of anti-CD3/CD28 beads overnight, after which they were fixed in paraformaldehyde and washed thoroughly. Then, the CD25⁺ T cell-depleted APCs were irradiated and mixed with the activated-fixed Tregs and used as feeders for the CD25⁺ T cell-depleted PBMCs at a 1:1 ratio in the presence of col-V. This procedure prevents any cytokine production from CD4⁺CD25⁺ Tregs yet maintains their cell contact-dependent suppressive functions (21, 22). The cell lines generated in this fashion had high IL-10 (221 ± 25.10 spm) and low IFN- (85 ± 25.12 spm) in response to col-V (Fig. 1), suggesting that the predominant T_IL-10 cells were distinct but still required the presence of CD4⁺CD25⁺ Tregs for development.

T_IL-10 did not express CD25 and Foxp3 constitutively

To further confirm that the T_IL-10 cells were distinct from Tregs, we analyzed the expression of CD25 and Foxp3. Following in vitro stimulation, the cells were rested for 10 days and the CD4 T cells were purified by negative selection as described in Materials and Methods. Following this step, CD25⁺ T cells were positively depleted from the purified CD4 T cells. The remaining CD4⁺25^– T cells were used to perform a col-V-specific IL-10 ELISPOT assay in the presence of CD25⁺ T cell-depleted autologous APCs and in a real-time RT PCR to quantify Foxp3 expression. There was no significant decrease in the IL-10 response after depleting the CD25⁺ T cells from the cell cultures (291.0 ± 24.17 spm vs 212.0 ± 24.25 spm; p = 0.08), indicating that the predominant T_IL-10 cells did not express CD25 constitutively (Fig. 2A). Further, the real-time PCR assay indicated that these cells did not express Foxp3 (Fig. 2B). These results indicated that the T_IL-10 cells were CD25 and Foxp3 negative and, therefore, distinct from Tregs.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. TIL-10 cells were distinct from Tregs. Col-V-specific T cell lines were developed from the PBMCs of LT patients after stimulation with col-V. Following the last stimulation, the cell lines were rested and then CD4+ T cells were purified. The CD25+ T cells were then positively depleted from the purified CD4+ T cells. Then, the remaining CD4+25– T cells were used to perform IL-10 ELISPOT in presence of autologous irradiated APCs depleted of CD25+ T cells (A) and real-time RT-PCR for Foxp3 (B). As control, the Foxp3 expression of CD25– conventional T cells from same patients and CD4+25+ Tregs is shown. The data are representative of four separate experiments done in triplicate, and the SE bars are illustrated for the ELISPOT assay. The results are expressed as spots per million cells.

T_IL-10 suppressed autologous T_IFN- cells (Th1 cells) without direct contact

Previous studies have clearly demonstrated that in vitro suppression mediated by CD4⁺CD25⁺ Tregs is cytokine independent and cell contact-dependent (23, 24, 25, 26, 27). We first confirmed these findings. Neutralization with high concentrations of anti-IL-10 or anti-TGF- did not reverse the suppression of conventional CD25^– T cells during coculture with CD4⁺CD25⁺ Tregs (Fig. 3A). Moreover, Transwell separation of CD4⁺CD25⁺ Tregs from the CD4⁺CD25^– T cells completely abrogated the suppression of CD4⁺CD25^– T cells (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. CD4+CD25+ T cell-mediated suppression of conventional CD25– T cells is contact dependent. A, Freshly isolated CD4+CD25+ or CD4+CD25– T cells from LT patients at a 1:1 ratio were cocultured and stimulated with CD3/CD28 beads for 6 days. In parallel, neutralizing IL-10 or TGF- mAbs were added to the cultures. Total cellular proliferation was measured using a [3H]thymidine incorporation assay. B, CD4+CD25– T cells (3 x 105 cells/well) were cocultured in the presence of increasing numbers of CD4+CD25+ Tregs separated by a Transwell membrane and stimulated with CD3/CD28 beads for 6 days. Total cellular proliferation was measured using a [3H]thymidine incorporation assay and has been illustrated as counts per minute.

IFN-

IFN-

IFN-

IFN-

IFN-

IFN-

View larger version (18K): [in this window] [in a new window]   FIGURE 4. TIL-10 cells suppressed col-V reactive TIFN- cells. PBMCs from the same patients were stimulated with col-V either in the presence (IL-10 cell line) or absence (IFN- cell line) of CD25+ T cells three times. Following this step, the IL-10 and IFN- cell lines were transferred to a Transwell plate separating the IL-10 (above) and IFN- cells (below) at 1:1 ratio. Under these conditions, the IL-10 and IFN- cell lines were cocultured for another three rounds of col-V stimulation either in the presence or absence of anti-IL-10 mAbs or isotype (rat IgG2a) control Abs. After six stimulations, the col-V-specific response was analyzed by IFN- (open bars) and IL-10 (filled bars) ELISPOT assays (A) and [3H]thymidine incorporation (B). For the IFN- cell lines all APCs used were depleted of CD25+ T cells. The data represent the effects of coculture in IFN- cell lines from three separate experiments, and the SE bars are represented (1–3; p < 0.01). The results for ELISPOT assays are expressed as spots/million cells (spm) and for [3H]thymidine incorporation as counts per minute.

We investigated whether T_IL-10 cells were self-HLA class II restricted. Following stimulation, T_IL-10 cells were activated with autologous APCs and col-V in the presence of increasing doses of anti-HLA class II (KuIA2) or isotype control (mouse IgG1) Abs. KuIA2 mAbs demonstrated a dose-dependent suppression of IL-10 production (Fig. 5). In contrast, there was no decrease in IL-10 production with the isotype control Abs, indicating that the T_IL-10 cells were HLA class II restricted.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. TIL-10 cells were HLA class II restricted. Col-V cell lines were developed from the PBMCs of LT patients after stimulation with col-V and autologous APCs. Following stimulation, the HLA class II on the APCs was first blocked by incubating with increasing concentrations of KuIA2 mAbs, and then the IL-10 response was analyzed using ELISPOT assays (solid line). Isotype control (mouse IgG1) Abs were used as control (broken line). The data are representative of three separate experiments done in triplicate, and the SE bars are illustrated. The results are expressed as spots per million cells.

Induction of T_IL-10 by CTLA-4 on Tregs

The induction of T_IL-10 cells by Tregs was next investigated. As previously described (26), CTLA-4 was found to be up-regulated on Tregs following activation (Fig. 6A). We speculated that CTLA-4 may have a role in the induction of T_IL-10 cells by Tregs. To investigate this possibility, the CD25⁺ T cells were positively depleted from the original PBMC sample and the APCs were used to generate the cell line. The isolated CD4⁺CD25⁺ Tregs were activated using the CD3/CD28 ligation. Then, CTLA-4 on CD4⁺CD25⁺ T cells was blocked by nonstimulating anti-CTLA-4 F(ab')₂ Abs. These activated but CTLA-4-blocked CD4⁺CD25⁺ Tregs were reconstituted back into the cell cultures along with irradiated (CD25⁺ T cell-depleted) autologous APCs and col-V. The cells were treated similarly at each round of stimulation. We found a significant decline in the frequency of T_IL-10 cells by blocking the CTLA-4 molecule of Tregs (136.2 ± 45.1 vs 280.0 ± 30.23 spm; p = 0.039). However, the IL-10 response was preserved by using isotype control Abs (251.0 ± 45.2; p = 0.67) (Fig. 6B). These results indicate that CTLA-4 was in part responsible for the induction of T_IL-10 cells by Tregs.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Expansion of TIL-10 cells involves CTLA-4 on Tregs. A, CD4+CD25+ Tregs up-regulate CTLA-4 within 24 h of activation. B, Col-V specific cell lines were developed from the PBMCs of LT patients after stimulation with col-V. In parallel cultures Tregs were depleted from the cell cultures. The isolated Tregs were activated using CD3/CD28 costimulation and then incubated with anti-CTLA-4 F(ab')2 Abs. Then, the activated and CTLA-4-blocked Tregs were reconstituted back into the cell cultures. Isotype mouse IgG1 Abs were used as control. The frequency of TIL-10 cells was analyzed using IL-10 ELISPOT assay. The data are representative of four separate experiments done in triplicate, and the SE bars are illustrated. The results are expressed as spots per million cells.

Loss of Tregs and expansion of T_IFN- cells during BOS development

As illustrated in Table I, four of the patients included in the study developed BOS. Three of these BOS patients, and none of the BOS-negative patients, revealed a significant decline in the frequency of CD4⁺CD25⁺ Tregs at the time of BOS development (p = 0.04, Fig. 7A). Concomitantly, there was a loss of T_IL-10 cells (Fig. 7B) and an expansion of T_IFN- cells (Fig. 7C) in these same patients.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Loss of Tregs during BOS development. Three (, , and ) of the four patients (, , •, and ) that developed BOS demonstrated a decline in the frequency of CD4+CD25+ Tregs in the peripheral blood (A). The loss of Tregs correlated with a decline in TIL-10 cells (B) and a concomitant increase in TIFN- cells (C). The frequency of Tregs is expressed as percentage of CD4+CD25+ T cells present in the PBMCs. TIL-10 cells and TIFN- cells are expressed as spots per million cells (spm). Post-Tx, posttransplantation.

View larger version (42K): [in this window] [in a new window]   FIGURE 8. Loss of 47 Tregs during BOS development. PBMCs were stained using CD4-FITC, CD25-PE, 4-allophycocyanin (Alpha-4 APC), and 7-PE/Cy5 (Beta-7 PE-Cy5). 4+7+ Tregs were analyzed after gating first on CD4+CD25+ T cells. The three patients tested were WU01 (Pt1), WU07 (Pt2), and WU08 (Pt3).

	Discussion

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Naturally occurring Tregs have emerged as potent mediators of peripheral tolerance. Depletion of Tregs has been shown to result in the development of autoimmunity (17). Tregs have also been shown to modulate transplantation tolerance (43, 44). Several previous reports have established that the suppressive properties of Tregs in vitro are dependent on direct cellular interactions (24, 45, 46). However, if the suppressive properties of Tregs are dependent on direct cellular contact, it is difficult to explain how a subset of T cells that comprises only 5–6% of total PBMCs can effectively mediate suppressive functions. Using in vitro model of mitogenic stimulation, Dieckmann et al. (21) have recently shown that Tregs might induce anergy and IL-10 production in naive T cells, converting them into suppressor cells, a phenomenon termed "infectious tolerance". This may serve to complement the peripheral suppression of Tregs. We demonstrated that the Tregs were crucial for the development of T_IL-10 cells recognizing a self-protein (Fig. 1). These T_IL-10 cells were distinct from Tregs because they did not express CD25 and Foxp3 constitutively (Fig. 2). Moreover, the T_IL-10 cells were capable of suppressing the autoreactive T_IFN- cells through IL-10 production (Fig. 4). The suppression of T_IFN- cells during coculture with T_IL-10 cells in the Transwell plates was not mediated by Tregs (present in IL-10 cell lines), because activated Tregs alone in a similar setup did not inhibit T_IFN- cells (data not shown). Furthermore, depletion of Tregs following development of col-V cell lines did not prevent IL-10 production (Fig. 2B). Also, it has been previously established that Tregs cannot mediate suppression of conventional T cells if separated by a physical barrier (22, 24, 45). Although T_IL-10 cells suppressed T_IFN- cells, no increase in IL-10 induction was observed in Th1 cell lines after coculture (Fig. 4). This finding further suggests that direct cellular interactions with Tregs alone could induce suppressive (IL-10) activity in conventional T cells.

The mechanisms through which the Tregs induce suppressive activity in the CD25^– T cells are yet unclear. In these experiments, reconstitution of activated/fixed Tregs induced T_IL-10 cell expansion (Fig. 1). Moreover, blocking CTLA-4 on Tregs significantly decreased the IL-10 response (Fig. 6B). Hence, these experiments demonstrate that induction of T_IL-10 cells involved direct contact-dependent interactions with Tregs and was in part mediated by CTLA-4 (Fig. 6). However, it is unclear whether the Tregs interacted via CTLA-4 directly with CD25^– T cells to induce IL-10 or indirectly through APCs. Tregs have recently been shown to trigger the induction of IDO in dendritic cells (DCs) through CTLA-4 signaling (47, 48, 49). IDO then catalyzes the conversion of tryptophan to kynurenine and other metabolites that have potent immunosuppressive effects in the local environment of the DCs. Furthermore, Tregs can also down-regulate expression of both CD80 and CD86 on DCs making Ag presentation inefficient (50), which might in turn anergize the concerned T cells. However, the Tregs can also function in the absence of APCs (24). Tr1-activity can also be generated by DCs in the presence of high IL-10 (51). Hence, Tregs may interact with dendritic cells through CTLA-4 to induce IL-10 production, which can induce an IL-10 predominant phenotype in conventional T cells.

Two subsets of natural Tregs have recently been described, characterized by either ₄₇ or ₄₁ integrins (28). Tregs expressing ₇ induce IL-10-production, whereas those with ₁ integrin preferentially induce TGF- in conventional CD4⁺ T cells upon polyclonal stimulation. In the present study, there was a loss of ₄⁺₇⁺ Tregs during BOS development with a concomitant decrease in T_IL-10 cells and an increase in T_IFN- cells. Interestingly, these patients also revealed a loss of ₄⁺₁⁺ Tregs (data not shown), indicating the possibility of deranged TGF--mediated regulation during BOS. Taken together, our results indicate that propagation of infectious tolerance by Tregs may form an integral regulatory circuit that maintains peripheral tolerance to self-Ags in human allograft recipients.

	Acknowledgments

 
We express our gratitude to Billie Glasscock for her secretarial assistance.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grant HL56643 (to T.M.). R.F. is recipient of the International Society for Heart and Lung Transplantation Research Fellowship Award.

² Address correspondence and reprint requests to Dr. Thalachallour Mohanakumar, Washington University School of Medicine, Department of Surgery, Box 8109-3328 Clinical Sciences Research Building, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: kumart{at}wustl.edu

³ Abbreviations used in this paper col-V, collagen V; BOS, bronchiolitis obliterans syndrome; DC, dendritic cell; LT, lung transplant; MMP, metalloproteinase; spm, spots per million cells; Treg, regulator T cell; T_IFN- cell, IFN--producing Th1 cell; T_IL-10 cell, IL-10-producing CD4⁺ T cell.

Received for publication December 23, 2005. Accepted for publication June 29, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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