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Gliadin-Specific Type 1 Regulatory T Cells from the Intestinal Mucosa of Treated Celiac Patients Inhibit Pathogenic T Cells

Carmen Gianfrani^1,*, Megan K. Levings^2,,, Claudia Sartirana, Giuseppe Mazzarella^*, Gianvincenzo Barba^*, Delia Zanzi, Alessandra Camarca^*,, Gaetano Iaquinto, Nicola Giardullo, Salvatore Auricchio, Riccardo Troncone^*, and Maria-Grazia Roncarolo^,¶

^* Institute of Food Science-Consiglio Nazionale delle Ricerche, Avellino, Italy; San Raffaele Telethon Institute for Gene Therapy (Hospital San Raffaele-Telethon Institute for Gene Therapy). Milan, Italy; Department of Pediatrics and European Laboratory for the Investigation of Food-Induced Diseases, University Federico II, Naples, Italy; Gastroenterology Unit, Moscati Hospital, Avellino, Italy; and ^¶ Vita-Salute San Raffaele University, Milan, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Celiac disease (CD)³ is a common intestinal disorder caused by altered immune responses to dietary gliadin present in wheat gluten and to proteins from the related cereals barley and rye (1). The hallmark of CD is enteropathy of the small intestine, characterized by flattening of the villous architecture and massive infiltration of T cells which release proinflammatory cytokines, such as IL-2 and IFN-, in response to gliadin (1, 2). Currently, the only available treatment for CD is withdrawal of gliadin from the diet, and in patients following a strict gluten-free diet (treated CD) the intestinal architecture is completely restored. Nevertheless, even after many years of a gluten-free diet, CD patients never acquire tolerance to gliadin, and re-exposure to this Ag results in recurrence of acute disease. These findings suggest that pathogenic T cells remain in the intestine throughout the life of treated CD patients.

The mechanisms responsible for normal intestinal homeostasis toward harmless intestinal Ags such as gliadin are not well understood (3). Recently, several studies have suggested that the immunosuppressive cytokines such as IL-10 and TGF- have an important role in maintaining intestinal tolerance (4, 5, 6, 7, 8, 9, 10, 11). For example, mice which are genetically deficient for IL-10 develop a severe form of enterocolitis, similar to human inflammatory bowel disease (IBD) (12), and have increased susceptibility to autoimmune diseases such as rheumatoid arthritis (13) and experimental autoimmune encephalomyelitis (14). In contrast, colitis, which develops in SCID mice after transfer of CD4⁺CD45RB^high T cells, can be prevented by IL-10 (15).

The immunomodulatory properties of IL-10 are at least partially due to its role in the differentiation of a subset of CD4⁺ T regulatory cells, known as type 1 regulatory T cells (Tr1) (16, 17). Tr1 cells possess a unique profile of cytokine production characterized by high levels of IL-10 and TGF-, normal levels of IFN-, very low amounts of IL-2, and little or no IL-4 (18). Importantly, Tr1 cells down-regulate naive and memory T cell responses upon local secretion of IL-10 and TGF- (18). Thus, via the induction of Tr1 cells, IL-10 has a central role in maintaining intestinal tolerance. It is possible that a dysfunction or deficiency in Tr1 cells is involved in the breakdown of tolerance in intestinal disorders such as CD and IBD (19, 20, 21).

Interestingly, we and others have recently shown that, in addition to proinflammatory cytokines, the inflamed CD mucosa also contains high levels of T cell-derived IL-10 when compared with treated CD or normal donors (22, 23). It is possible that in acute CD, although IL-10 is produced, the levels are insufficient to down-regulate the massive Th1/Th0 immune responses induced by gliadin. Indeed, addition of exogenous IL-10 to mucosal cultures from treated CD patients can suppress gliadin-induced T cell activation and cytokine production ex vivo. (22) Therefore, similar to what has been described with alloantigens, (24), IL-10 can induce hyporesponsiveness to food Ags, and under noninflammatory conditions may also induce the differentiation of Tr1 cells in the gut.

Although it is widely assumed that IL-10 and Tr1 cells have a central role in regulating responses to intestinal Ags, including those from commensal bacteria and dietary proteins, (6, 7, 20), to date the derivation of dietary Ag-specific Tr1 cells from human intestinal mucosa has not been described. We therefore investigated whether gliadin-specific Tr1 cells are present in the mucosa of CD patients and whether they can mediate suppression of T cell responses to dietary gliadin.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Organ culture and generation of gliadin-specific T cell lines (TCL)

Jejunal explants were obtained from 12 CD patients (7 treated, mean age, 31.1 years; range, 18–49 years; 5 untreated, mean age, 23.6 years; range, 10–34 years) and 5 healthy subjects (mean age, 25.8 years; range, 10–50 years). Both treated CD and control subjects were serum negative for anti-endomysium Abs and had a noninflamed mucosa. All subjects gave informed consent to the proposed study. Mucosal explants were either immediately digested or cultured for 24 h in vitro with a peptic-tryptic digest of gliadin in the absence (control (ctl) biopsy) or presence of recombinant human IL-10 (IL-10 biopsy) (50 ng/ml; R&D Systems). Biopsies were digested with 1 mg/ml collagenase A for 1 h at 37°C. Cells obtained from fresh biopsies and ctl biopsies were stimulated with 1 x 10⁶ irradiated (3500 rad) autologous PBMC and 50 µg/ml transglutaminase-treated peptic-tryptic-gliadin (indicated as gliadin) for an additional 7 days. Cells from the IL-10 biopsy were stimulated with gliadin and PBMC in the presence of IL-10 (200 ng/ml; IL-10-TCL). To generate short-term TCLs, cells were restimulated with irradiated autologous PBMC and gliadin (50 µg/ml). Every 3–4 days, medium (X-Vivo15 medium supplemented with 5% AB⁺ pooled human serum and antibiotics all provided from BioWhittaker) was replenished and IL-15 (10 ng/ml) was added. After 18–21 days of culture, TCLs were assayed for gliadin specificity and either cryopreserved or kept in culture as long-term TCLs by restimulating with gliadin followed by cyclic (14 days) restimulation with a feeder-cell mixture containing PHA (1 µg/ml) and IL-2 (100 IU/ml) as previously described (25).

IFN- ELISPOT assay

TCLs were tested for gliadin recognition by an IFN- ELISPOT assay as previously described (22). Briefly, 0.3–0.5 x 10⁵ T cells were plated in the presence of 1 x 10⁵ autologous PBMC that had been pulsed overnight with medium or gliadin (50 µg/ml) in 96-well plates (Millipore) coated with anti- IFN- mAbs. In the experiments with neutralizing mAbs, T cells were preincubated with anti-IL-10R (10 µg/ml, clone 3F9; BD Pharmingen) or anti-TGF- (10 µg/ml, clone 1D11; R&D Systems) mAbs before addition of gliadin-pulsed APC. All experiments were performed in duplicate. After 36–40 h of incubation, spot-forming cells (SFC) were counted by an immunospot analyzer (A.EL.VIS).

Cloning of intestinal TCLs

Ctl-TCLs and IL-10-TCLs from two treated CD patients (CD041001 male, 28 years old, DR3/5, DQ2; CD090401 female, 27 years old, DR3/14, DQ2) and the normal donor ND090102 (male, 31 years old, DR5/DR7, DQ2) were plated at 1 cell/well in the presence of 5 x 10⁴ allogeneic irradiated (6,000 rad) PBMC, 5 x 10³ irradiated (10,000 rad) JY (an EBV-lymphoblastoid cell line (LCL)), and 0.05 µg/ml PHA (Roche). At days 3, 7, and 10, cells were fed with fresh medium containing IL-2 (40 IU/ml). T cell clones (TCCs) were screened after 14 days for IL-10 and IL-4 production following activation of one-half of each well with immobilized anti-CD3 (10 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs in a final volume of 200 µl of complete medium. IL-10-producing TCC were expanded by cyclic restimulation with feeder cells as described above.

Proliferation and cytokine production

Ten to 14 days after restimulation, TCLs and TCCs were analyzed for proliferation and/or cytokine production in response to gliadin and/or polyclonal activation. Cells (0.3–0.5 x 10⁵) were plated in 96-well round-bottom plates in the presence of 0.3–0.5 x 10⁵ (ratio 1:1) irradiated autologous EBV-LCLs, which had been pulsed overnight with gliadin (50 µg/ml) or medium alone, or stimulated with immobilized anti-CD3 (10 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs in 200 µl of complete medium. After 48 h of incubation, T cells were pulsed for 16 h with 1µCi/well [³H]thymidine (Amersham Pharmacia).

In parallel, supernatants from TCCs were collected after 24 h (for IL-2) or 48 h (for IL-10, IL-4, IFN-, and TGF-). For detection of IL-2, IL-10, IL-4, or IFN-, specific capture and detection mAbs were used (BD Pharmingen) and ELISAs were performed as described previously (25). Amounts of TGF- were determined by a commercial ELISA kit (R&D Systems). Sensitivity of assays was as follows: IL-10, 9 pg/ml; IL-4, 9 pg/ml; IL-2, 15 pg/ml; IFN-, 62 pg/ml; and TGF-, 62 pg/ml.

TCLs and TCCs were considered responsive to gliadin when proliferation and/or IFN- (ELISPOT/ELISA) production were 2-fold greater than those of cells cultured in medium alone.

For suppression experiments, CD3-depleted PBMC from DQ2⁺ healthy donors were used as APC. Increasing numbers of effector Th0 or Tr1 cell clones (up to 1 x 10⁵) were added to 0.5 x 10⁵ effector Th0 cell clones and cocultured in the presence of 0.5 x 10⁵ APC, which had been pulsed overnight with gliadin. Proliferation was evaluated after 48 h as described above.

Statistical analysis

Data are expressed as mean ± SD, mean (range) or median (upper and lower quartile) as indicated. Statistical analysis was performed by a nonparametric Wilcoxon test for paired data or Mann-Whitney U test for independent samples, as appropriate (26). Two-sided p values <0.05 were considered statistically significant. Statistical analysis was performed by SPSS 11.0 (SPSS).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IL-10-induced unresponsiveness in TCLs from treated CD patients results in low proliferation and cytokine production following gliadin stimulation

We previously reported that incubation of treated CD mucosa with exogenous IL-10 for 24 h inhibits gliadin-induced T cell activation and IFN- production (22). Herein, we further characterized the gliadin-specific T cells generated in the presence of IL-10. Short-term TCLs were generated from mucosal explants of three treated CD patients (CD041001, CD090401, CD140102) and three normal donors (ND090102, ND140102, ND200203) upon organ culture with gliadin in the absence (ctl-TCLs) or presence of IL-10 (IL-10-TCLs) (see Materials and Methods). Upon restimulation with gliadin (in the absence of IL-10), the ctl-TCLs from only two CD patients (CD041001, CD090401) proliferated (mean stimulation index (SI) ± SD: 3.6 ± 1.8), whereas the corresponding IL-10-TCLs displayed low Ag-dependent proliferation (mean SI ± SD: 1.5 ± 0.7) (Fig. 1A). As expected, TCLs obtained from normal mucosa failed to proliferate in response to gliadin in any condition (mean SI ± SD: 1.1 ± 0.4 and 1.3 ± 0.3 in ctl-TCLs and IL-10-TCLs, respectively) (Fig. 1A).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Gliadin-dependent T cell responses are present in the intestinal mucosa of treated CD patients and are susceptible to IL-10-induced hyporesponsiveness. Short-term TCLs were obtained from small intestinal mucosa of three celiac patients (CD) and three normal donors (ND). Mucosal explants were cultured for 24 h with gliadin alone (ctl-TCLs) or in presence of IL-10 (IL-10-TCLs). TCLs obtained from IL-10-cultured biopsies underwent an additional 7 days of in vitro treatment with IL-10. A, Proliferative responses of TCLs from patient CD041001 and normal donor ND090102 are illustrated. T cells (3 x 104) were plated in the presence of 5 x 104 autologous EBV-LCLs, which were pulsed with medium or gliadin (50 µg/ml). Proliferative responses, evaluated after 48 h of incubation, were considered positive when the SI was >2. Numbers above the columns indicate the corresponding SI values. One representative experiments of three performed for each TCL is shown. B, Cellular phenotypes of short-term ctl-TCLs and IL-10-TCLs from one representative CD patient (CD041001) of three analyzed are shown. Numbers indicate percentage of double-positive cells present in the R1 (alive cells) region.

IL-10-induced unresponsiveness to gliadin in intestinal T cells is mediated by IL-10 and TGF-

Ag-driven activation of T cells in the presence of IL-10 results in a long-lasting Ag-specific unresponsiveness mediated by Tr1 cells which secrete both IL-10 and TGF- (18, 24). We therefore investigated whether the unresponsiveness of the IL-10-TCLs was mediated by endogenous IL-10 and/or TGF- production. Similar to Fig. 1, gliadin-dependent IFN-- secreting T cells were present at high frequencies in short-term ctl-TCLs from the three CD patients analyzed (CD090401, CD041001, CD140102), as determined by ELISPOT: median (range) of IFN- secreting cells (IFN--SFC)/10⁶ cells: 200 (170–1380) (Fig. 2A). By contrast, a reduction of the IFN- response to gliadin stimulation was observed in IL-10-TCLs: median IFN--SFC: 40 (40–220) (p < 0.05, Wilcoxon test) (Fig. 2A). Addition of neutralizing anti-IL-10 receptor or anti-TGF- mAbs in the IL-10-TCLs reversed the anergic state of the gliadin-specific cells and led to a positive response to gliadin stimulation (>2-fold response to medium alone). More specifically, the addition of anti-IL-10R rescued IFN- responses in two of three CD patients (CD090401, CD041001); the addition of anti-TGF- rescued IFN- responses in all three CD patients tested (CD090401, CD041001, CD140102): median IFN--SFC/10⁶ cells: 120 (50–1990) and 150 (140–2520) in the presence of anti-IL-10R and anti-TGF- mAbs, respectively, in comparison to 50 (40–410) in the presence of medium alone (Fig. 2A). Interestingly, we also observed a consistent trend to increased production of IFN- from the ctl-TCLs upon addition of either anti-IL-10R or anti-TGF- mAbs: median IFN--SFC: 510 (300–2210) and 400 (200–2050) in the presence of anti-IL-10R and anti-TGF- mAbs, respectively. Isotype Abs were used as control in stable intestinal TCL and no effect was observed (data not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. IL-10 and TGF- mediate the down-regulation of gliadin-specific IFN- production. A, Short-term TCLs, obtained from the intestinal mucosa of three treated CD patients (CD041001, CD090401, CD140102) that were cultured for 24 h in the presence of gliadin alone (ctl-TCLs) or gliadin plus IL-10 (IL-10-TCLs), were analyzed for their capacity to recognize gliadin. Autologous PBMC, which had been cultured overnight with medium or gliadin (50 µg/ml), were used as APC. In some cases, TCLs were incubated with anti-IL-10R (10 µg/ml) or anti-TGF- (10 µg/ml) mAbs for 10 min before adding the APC. The number of gliadin-reactive cells was determined by an IFN- ELISPOT assay. Data are expressed as mean ± SD of duplicate experiments performed for each patient. The TCL response to gliadin was considered positive when IFN- production was >2-fold response to medium alone. Statistical significance was evaluated by the nonparametric Wilcoxon test for paired data, comparing responses to gliadin alone in ctl-TCLs with response in IL-10-TCLs from all three patients (n = 3, p < 0.05). B–E, Similar experiments were performed with long-term TCLs, which were generated by polyclonal restimulation of the short-term TCLs obtained from treated (B and C) and untreated (D and E) CD mucosa. Proliferative responses (B and D) and IFN- production (C and E) by the TCLs in response to gliadin were evaluated in the absence or presence of neutralizing anti-IL-10R (10 µg/ml) and/or anti-TGF- mAbs (10 µg/ml). Results of TCLs from one treated (CD041001) and one untreated patient (CD171204) are illustrated (mean ± SD of duplicate experiments) and were representative of TCLs from 10 different (5 treated and 5 untreated) CD patients. Results from these 10 patients are reported in the text; statistical significance was evaluated by the nonparametric Wilcoxon test for paired data, comparing responses to gliadin alone with gliadin plus both neutralizing Abs in n = 5 treated (B and C) (p < 0.05) and n = 5 untreated (D and E) (p < 0.05).

To further investigate whether IL-10- and/or TGF--producing T cells may be present in the TCLs derived from ctl-biopsies (i.e., without addition of exogenous IL-10), we generated long-term TCLs from a larger panel of both treated (n = 5, CD230204, CD041001, CD310504, CD041005, CD041005.2) and untreated (n = 5, CD171204, CD151004, CD061204, CD011204, CD200503) patients by restimulating short-term TCLs with gliadin and thereafter with PHA (see Materials and Methods). Thereafter, long-term TCLs were tested for gliadin recognition in the absence or presence of either anti-IL-10R or anti-TGF- or both mAbs. As shown in Fig. 2, B–E, both proliferation (B and D) and production of IFN-, assessed by ELISA (C and E), were significantly enhanced in the presence of both neutralizing mAbs in treated (B and C) as well as in untreated (D and E) TCLs. Statistical significance was evaluated with the Wilcoxon test comparing proliferative responses (SI) and IFN- production (level of IFN- detected in cell supernatants) in response to gliadin in the presence of anti-IL-10R and anti-TGF- mAbs with responses in the presence of gliadin alone in both groups of patients. In treated CD, SI (median (upper and lower quartile)) was: 17.6 (1.7–95.3) in cultures with anti-IL-10R and anti-TGF- mAbs vs 8.9 (1.1–62.8) in cultures with gliadin alone (n = 5, p < 0.05); IFN- (net IFN-/10⁶ cells, median (upper and lower quartile)) was: 7.5 ng/ml, (6.8–8.7) vs 2.8 ng/ml (2.1–3.6), (n = 5, p < 0.05). Similarly, in untreated CD, SI was: 1.6 (1.3–3.5) vs 1.2 (1.1–2.5), (n = 5, p < 0.05) and IFN- was: 7.7 ng/ml (0.6–13.5) vs 3.4 ng/ml (0.09–7.7), (n = 5, p < 0.05). Although a large variability in the magnitude of proliferative responses and IFN- production to gliadin was observed among CD patients, a trend toward higher proliferative responses in treated compared with untreated CD was detected. However, the differences between the two cohorts of patients were not statistically significant in any experimental condition (p > 0.5, Mann-Whitney U test).

These data suggest that although gliadin-specific T effector cells may predominate in the intestinal mucosa of CD patients, Tr1 cells are also present in vivo both in treated (noninflamed) and untreated (inflamed) CD mucosa.

Gliadin-specific T cells are restricted by the CD-associated HLA-DQ2 molecule

Several studies have found that T cells isolated from CD mucosa recognize gliadin peptides in the context of HLA-DQ2 or DQ8 (27, 28, 29, 30, 31, 32). To assess the MHC restriction of the TCLs isolated in this study, DQ2⁺ APC from three normal donors were used individually (ND.1 DR4/17, DQ2/3; ND.2 DR3/11, DQ2; ND.3 DR1/DR7, DQ1/2), or as a pool (ND.1 + ND.2), to present gliadin to ctl-TCLs. When gliadin was presented by APC from any of the three DQ2⁺ normal donors, the CD ctl-TCLs proliferated vigorously at levels equivalent to those obtained when gliadin was presented by autologous EBV-LCLs (Fig. 3A). Similar results were obtained upon analysis of cytokine production (Fig. 3B). Importantly, no responses were observed when DQ2^neg APC from two different normal donors (ND.4 DR7/8, DQ3; ND.5 DR11, DQ7) were used. Because DQ2 is the only MHC II allele shared by these three normal donors, these data indicate that the response in these CD patient-derived TCLs is restricted to this allele.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. T cells from CD patients recognize gliadin in the context of the HLA-DQ2 heterodimer. To assess the MHC restriction of the gliadin-specific immune response, ctl-TCLs from patient CD041001 (DR3/5) were stimulated with APC (PBMC depleted of CD3+ cells) from three different DQ2+ normal donors (ND.1 DR4/17, DQ2/3; ND.2 DR3/11, DQ2; ND.3 DR1/DR7, DQ1/2). PBMC were used either individually or as a pool of two donors (ND.1 and ND.2). CD3-depleted PBMC from two different DQ2– normal donors (ND.4 DR7/8, DQ3; ND.5 DR11, DQ7) were used as negative control. APC were pulsed overnight with medium or gliadin (50 µg/ml). As a positive control, TCL was stimulated with autologous EBV-LCLs that had been pulsed with medium or gliadin (50 µg/ml). After 48 h, proliferation (A) or production of IFN- (B) was determined. Numbers above the columns in A indicate the SI. Representative results of one (mean ± SD of duplicates) of three experiments performed are illustrated.

Based on our findings that anti-IL-10R and/or anti-TGF- mAbs could enhance gliadin-induced proliferation and cytokine production in both ctl- and IL-10-TCLs, derived from CD patients, we next investigated whether gliadin-specific Tr1 cells are present in ctl-TCLs and/or IL-10-TCLs. TCCs were isolated by limiting dilution from ctl-TCLs and IL-10-TCLs from two treated CD patients (CD041001 and CD090401) and a DQ2⁺ normal donor (ND090102). To evaluate the frequency of Tr1 cells, TCCs were screened after 14 days for their capacity to produce IL-10 following stimulation with anti-CD3/CD28 mAbs. Cell clones found to be positive for IL-10 were subsequently screened for IL-4 production, and Tr1 cell clones were defined based on the presence of IL-10 and the absence of IL-4 (Table I). Interestingly, in patient CD041001 we observed a similar frequency of Tr1 cell clones in both ctl-TCLs and IL-10-TCLs (10.7% in ctl-TCLs vs 10% in IL-10-TCLs). In patient CD090401, a slightly increased frequency of Tr1 cell clones was observed in IL-10-TCLs (10.7%) compared with ctl-TCLs (7.8%). In this patient the frequency of IL-10⁺ cells was significantly higher compared with that of Tr1 cells, suggesting the presence of T cells with a Th2 phenotype (IL-10⁺IL-4⁺). The frequency of Tr1 cells in TCLs from the normal mucosa was very low in both ctl-TCLs and IL-10-TCLs, although a higher percentage of Tr1 clones was detectable in IL-10-TCLs (4.8% in IL-10-TCLs vs 1.1% in ctl-TCLs).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Both gliadin-specific Tr1 and Th0 T cells are present in intestinal mucosa of treated CD patients. A, IL-10+ TCCs derived from intestinal TCLs generated in the presence of gliadin alone (ctl-TCLs) or gliadin plus IL-10 (IL-10-TCLs) were tested for their capacity to proliferate in response to gliadin presented by autologous EBV-LCLs after 48 h of culture. TCCs 1–13 from ctl-TCLs and TCCs 10/1, 10/2, 10/3 obtained from IL-10 TCLs derived from patient CD041001 are illustrated. B and C, The MHC restriction of representative Th0 (TCC.6) and a representative Tr1 (TCC.9) TCC was tested. TCCs were stimulated with a pool of APC from two different DQ2+ normal donors that had been pulsed with medium or gliadin (50 µg/ml). After 48 h, cultures were tested for proliferation (B) or production (C) of IFN-. Numbers above the columns in B indicate the SI. Representative results of one (mean ± SD of duplicates) of three experiments performed for each TCC are reported.

Intestinal mucosa Tr1 cell clones suppress gliadin-reactive Th0 cell clones

We next investigated whether the gliadin-specific Tr1 cell clones could suppress Ag-specific proliferative responses of pathogenic T cells. Suppression experiments with two representative Tr1 cell clones (TCC.9 and TCC.7) are shown in Fig. 5. In the case of Tr1 clone TCC.9, 57% suppression of the proliferative response of the Th0 cell clone TCC.3 was observed at a 1:1 ratio (Fig. 5A). A similar magnitude of suppression was observed with Tr1 clone TCC.7 on the gliadin-induced proliferative response of Th0 cell clone TCC.6. Importantly, the suppressive effects of the Tr1 cells were found to be dose dependent (Fig. 5B). The Tr1 cell clones were capable of suppressing the gliadin-induced proliferation of T effector cells at very low cell numbers (11% suppression at 1:0.1 (Th0:Tr1) cell ratio). As expected, none of the Th0 cell clones tested displayed suppressive activity.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Gliadin-specific Tr1 cell clones suppress proliferative responses to gliadin of intestinal DQ2-restricted Th0 cell clones. The responder Th cell clones, TCC.3 (A) and TCC.6 (B) (5 x 104 cell/well), were stimulated with gliadin-pulsed, CD3-depleted PBMC (5 x 104 cell/well) from a DQ2+ normal donor either alone, or in the presence of autologous, gliadin-specific, suppressor Tr1 cell clones TCC.9 (A) and TCC.7 (B). A, TCC.3 and TCC.9 were plated at a ratio 1:1 (5 x 104:5 x 104). B, Different ratio of responder (TCC.6) and suppressor (TCC.7) TCCs were used: 5 x 104 responder cells were added to a decreasing number of suppressor cells (Tr1) or control cells (Th0), starting from 10 x 104 cells. Proliferative responses were evaluated by assessing [3H]thymidine incorporation after 48 h. Representative results of the SI (expressed as mean ± SD of duplicates) of one of two experiments performed for each Tr1 clones are illustrated. Numbers on the top of the bars represented the percentage of suppression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Interestingly, no evidence of gliadin-specific T cells, either in terms of proliferative or cytokine responses, was detected in normal donors. Upon cloning of cells from the normal intestinal mucosa, we observed a small increase in the frequency of IL-10⁺ TCC derived from the IL-10-TCLs in comparison to ctl-TCLs. However, we were unable to expand and characterize any IL-10-producing TCC from normal mucosa. Therefore, although we cannot formally exclude that Tr1 cells are present in the mucosa of normal donors, we can conclude that gliadin-specific T cells are not detectable, which is consistent with previous observations (Refs. 28, 29, 30 and C. Gianfrani, unpublished observations). This may be due to the fact that gliadin-specific T cells are only generated in CD patients and that the normal immune system simply ignores this food Ag. Alternatively, the frequency of gliadin-specific T cells in the normal mucosa may be extremely low and/or they may have an intrinsically low proliferative capacity which hampers the possibility to clone them in vitro (34, 35). By contrast, Tr1 cells specific for desmoglein 3, the autoantigen of pemphigus vulgaris, are found at high frequency in the peripheral blood of healthy donors and only in a minority of patients (36, 37). Our results are consistent with the hypothesis that gliadin-specific Tr1 cells are recruited to/differentiated in the inflamed intestinal mucosa during the acute disease, possibly as consequence of local production of high levels of IL-10 (22). However, in the acute stage of disease, Tr1 cells are clearly unable to efficiently down-regulate the massive inflammatory response driven by gliadin. Nevertheless, they must remain in the treated mucosa as long-lived memory T cells. In accordance with this hypothesis, we observed that TCLs generated from the intestinal mucosa of treated as well as untreated CD patients contained both gliadin-specific T effector and Tr1 cells. Indeed, proliferation and production of IFN- in response to gliadin increased in the presence of anti-IL-10R and/or anti-TGF--neutralizing mAbs, and Tr1 cell clones were isolated from CD patients. Interestingly, challenge of treated mucosa with gliadin in the presence of exogenous IL-10 resulted in further down-regulation of gliadin-specific responses, (22), indicating that, at least in treated patients, the pathogenic T cells are still susceptible to some form of immune modulation.

Although exogenous IL-10 was able to modulate gliadin-specific responses in vitro, an increase in the percentage of Tr1 cell clones was observed in cultures from only one of the two treated CD patients tested. We have previously shown that although IL-10 is necessary for the differentiation of Tr1 cells, it is not sufficient (17). Therefore, these in vitro cultures may have lacked an essential cofactor(s) which led to inefficient differentiation of Tr1 cells. To clarify the effect of IL-10 on differentiation/expansion of intestinal mucosa Tr1 cells, cloning from a larger cohort of CD patients is required.

The role of Ag-specific Tr1 cells in controlling immune responses in murine intestinal mucosa is well established. For example, IL-10-secreting cells from Peyer’s patches are responsible for active suppression in low-dose oral tolerance to OVA Ag, (10), and cecal bacterial Ag-specific Tr1 inhibit the immune response to enteric flora (38). Moreover, mucosal administration of IL-10 along with repetitive feeding of low doses of myelin basic protein or insulin results in prevention of experimental autoimmune encephalomyelitis or diabetes in NOD mice, respectively, and in both cases is associated with enhanced production of IL-10 by T cells (39). Reports on the role of IL-10 and Tr1 cells in the human intestinal mucosa are more limited. Khoo et al. (35) showed that lymphocytes from human intestinal mucosa, but not from mesenteric lymph nodes, inhibited peripheral responses to Escherichia coli via an IL-10- and TGF--dependent mechanism. Our data are consistent with these findings and demonstrate that Tr1 cells arise in response to dietary Ags in human intestine. Moreover, these Ag-specific Tr1 cells suppressed the responses of Ag-specific effector T cells, a phenomenon that has not been previously reported.

In vivo administration of IL-10 has already been used in an attempt to decrease inflammation in a variety of intestinal diseases. Clinical trials in which IL-10 was delivered s.c. in CD patients who were refractory to gluten-free diet regimen proved ineffective (40). Furthermore, i.v. or s.c. administration of IL-10 to patients with active CD or ulcerative colitis gave unclear results on the therapeutic efficacy of IL-10 treatment (41, 42, 43, 44, 45, 46). Several reasons can account for the overall failure of IL-10 therapy to treat inflammatory mucosal diseases, including an inappropriate route of administration, the short serum half-life of IL-10, and its pleiotropic activities (47). The current study has important implications for an IL-10-based therapy for CD. A future therapeutic approach to silence gliadin-induced intestinal inflammation could include administration of IL-10 through the intestinal lumen, to recruit and/or differentiate gliadin-specific Tr1 cells in treated (i.e., noninflamed) CD patients. In addition, in vitro expanded gliadin-specific Tr1 cells that could be reintroduced into CD patients. In this context, it is noteworthy to mention a recent study from Van Montfrans et al. (48) who described the generation of IL-10-producing CD4⁺ Tr cells with a specific gut-homing capacity as a novel tool to deliver IL-10 to the intestinal mucosa of IBD patients.

In addition to Tr1 cells, many other regulatory T cell subsets have been described to control inflammation and maintain immune tolerance to harmless Ags in intestinal tissues. Among these, the natural occurring CD4⁺CD25⁺ cells have been the most extensively characterized (6, 7, 8, 9, 10, 11). Although the role of this CD4⁺CD25⁺ cell population in controlling auto- and alloimmunity has been largely demonstrated, only recent studies reported their involvement in orally induced tolerance (49, 50, 51, 52). The proliferative response of CD4⁺CD25^neg cells to enteroantigens was markedly suppressed by CD4⁺CD25⁺ Tr cells in mesenteric lymph nodes (50). Interestingly, Brandtzaeg and coworkers (52) found an increased percentage of circulating CD4⁺CD25⁺ cells in children who outgrew milk allergy (tolerant), compared with children who retained a clinically active milk allergy. The gliadin-specific Tr1 cell clones isolated in this study did not express high levels of CD25, a marker that we have previously found to be a hallmark for naturally occurring CD4⁺CD25⁺ Tr cells (25). However, we cannot exclude that CD4⁺CD25⁺ Tr cells may also have a role in controlling the immune response to gliadin. Further studies are underway to address this question.

In conclusion, our data demonstrate that gliadin-reactive Tr1 cells are present in the intestinal mucosa of CD patients and suggest that methods to enhance their numbers and/or function can be a novel tool to suppress immune responses toward gliadin or other Ags that are introduced daily into the gastrointestinal system.

	Acknowledgments

 
We thank Dr. Beatrice De Giulio for assistance in gliadin preparation and Drs. Caterina Anania and Giuseppe Falanga at the Malzoni Clinic in Avellino for their help in cell irradiation. We gratefully thank Drs. Massimo Conese and Katherina Fleischhauer for helpful discussions.

	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 Address correspondence and reprint requests to Dr. Carmen Gianfrani, Istituto di Scienze dell’Alimentazione-Consiglio Nazionale delle Ricerche, Via Roma 52A/C, 83100, Avellino, Italy. E-mail address: cgianfrani{at}isa.cnr.it

² Current address: Department of Surgery, University of British Columbia, Vancouver, B.C.

³ Abbreviations used in this paper: CD, celiac disease; Tr1, type 1 regulatory T cell; IBD, inflammatory bowel disease; TCL, T cell line; TCC, T cell clone; SI, stimulation index; LCL, lymphoblastoid cell line; ctl, control.

Received for publication July 14, 2005. Accepted for publication June 16, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Sollid, L. M.. 2000. Molecular basis of coeliac disease. Annu. Rev. Immunol. 18: 53-81. [Medline]
2. Lahat, N., S. Shapiro, R. Karban, R. Gerstein, A. Kinarty, A. Lerner. 1999. Cytokine profile in coeliac disease. Scand. J. Immunol. 49: 441-446. [Medline]
3. Faria, A. M. C., H. L. Weiner. 1999. Oral tolerance: mechanism and therapeutic application. Adv. Immunol. 73: 153-264. [Medline]
4. Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376: 177-180. [Medline]
5. Friedman, A., H. L. Weiner. 1994. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl. Acad. Sci. USA 91: 6688-6692. [Abstract/Free Full Text]
6. Chen, Y., V. K. Kuchroo, J. I. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell-clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265: 1237-1240. [Abstract/Free Full Text]
7. Perez-Machado, M. A., P. Ashwood, M. A. Thomson, F. Latcham, R. Sim, J. A. Walker-Smith, S. H. Murch. 2003. Reduced transforming growth factor-1-producing T cells in the duodenal mucosa of children with food allergy. Eur. J. Immunol. 33: 2307-2315. [Medline]
8. Ke, Y., K. Pearce, J. P. Lake, H. K. Ziegler, J. A. Kapp. 1997. T lymphocytes regulate the induction and maintenance of oral tolerance. J. Immunol. 158: 3610-3618. [Abstract]
9. Thorstenson, K. M., A. Khoruts. 2001. Generation of anergic and potentially immunoregulatory CD25⁺CD4⁺ T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen. J. Immunol. 167: 188-195. [Abstract/Free Full Text]
10. Tsuji, N. M., K. Mizumachi, J. Kurisaki. 2003. Antigen-specific, CD4⁺CD25⁺ regulatory T cell clones induced in Peyer’s patches. Int. Immunol. 15: 525-534. [Abstract/Free Full Text]
11. Dubois, B., L. Chapat, A. Goubier, M. Papiernik, J. F. Nicolas, D. Kaiserlian. 2003. Innate CD4⁺CD25⁺ regulatory T cells are required for oral tolerance and control CD8⁺ T cells mediating skin inflammation. Blood 102: 3295-3301. [Abstract/Free Full Text]
12. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, W. Muller. 1993. Interleukin-10 deficient mice develop chronic enterocolitis. Cell 75: 263-274. [Medline]
13. Samoilova, E. B., J. L. Horton, Y. Chen. 1998. Acceleration of experimental autoimmune encephalomyelitis in interleukin-10-deficient mice: roles of interleukin-10 in disease progression and recovery. Cell. Immunol. 188: 118-124. [Medline]
14. Bettelli, E., M. P. Das, E. D. Howard, H.L. Weiner, R. A. Sobel, V. K. Kuchroo. 1998. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J. Immunol. 161: 3299-3306. [Abstract/Free Full Text]
15. Powrie, F., M. W. Leach, S. Mauze, S. Menon, L. B. Caddle, R. L. Coffman. 1994. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RB^hiCD4⁺ T cells. Immunity 1: 553-562. [Medline]
16. Groux, H., A. O’Garra, M. Bigler, J. de Vries, M. G. Roncarolo. 1997. A CD4⁺ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737-742. [Medline]
17. Levings, M., R. Sangregorio, F. Galbiati, S. Squadrone, R. de Waal Malefyt, M. G. Roncarolo. 2001. IFN- and IL-10 induce the differentiation of human type 1 regulatory cells. J. Immunol. 166: 5530-5539. [Abstract/Free Full Text]
18. Roncarolo, M. G., R. Bacchetta, C. Bordignon, S. Narula, M. K. Levings. 2001. Type 1 T regulatory cells. Immunol. Rev. 182: 68-79. [Medline]
19. Mowat, A.. 2003. Anatomical basis of tolerance and immunity to intestinal antigens. Nat. Rev. Immunol. 3: 331-341. [Medline]
20. Groux, H., F. Powrie. 1999. Regulatory T cells and inflammatory bowel disease. Immunol. Today 20: 442-446. [Medline]
21. Fiocchi, C.. 1998. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115: 182-205. [Medline]
22. Salvati, V., G. Mazzarella, C. Gianfrani, M. Levings, R. Stefanile, B. De Giulio, G. Iaquinto, N. Giardullo, S. Auricchio, M. G. Roncarolo, R. Troncone. 2005. Recombinant human IL-10 suppresses gliadin-dependent T-cell activation in ex vivo cultured celiac intestinal mucosa. Gut 54: 46-53. [Abstract/Free Full Text]
23. Forsberg, G., O. Hernell, S. Melgar, A. Israelsoon, S. Hammarstrom, M. L. Hammarstrom. 2002. Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease. Gastroenterology 123: 667-678. [Medline]
24. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4⁺T cells. J. Exp. Med. 184: 19-29. [Abstract/Free Full Text]
25. Levings, M. K., R. Sangregorio, C. Sartirana, A. L. Moschin, M. Battaglia, P. Orban, M. G. Roncarolo. 2002. Human CD25⁺CD4⁺ T suppressor cell clones produce transforming growth factor-, but not interleukin-10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196: 1335-1346. [Abstract/Free Full Text]
26. Olsen, C. H.. 2003. Guest commentary: review of the use of statistic in infection and immunity. Infect. Immun. 71: 6686-6692. [Free Full Text]
27. Lundin, K. E. A., H. Scott, T. Hansen, G. Paulsen, T. S. Halstensen, O. Fausa, E. Thorsby, L. M. Sollid. 1993. Gliadin-specific, HLA-DQ(1*0501,_1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J. Exp. Med. 188: 187-196.
28. Molberg, O., K. Kett, H. Scott, E. Thorsby, L. M. Sollid, K. E. A. Lundin. 1997. Gliadin-specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand. J. Immunol. 46: 103-108. [Medline]
29. Nilsen, E. M., K. E. A. Lundin, P. Krajci, H. Scott, L. M. Sollid, P. Brandtzaeg. 1995. Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon-. Gut 37: 766-776. [Abstract/Free Full Text]
30. Troncone, R., C. Gianfrani, G. Mazzarella, L. Greco, J. Guardiola, S. Auricchio, P. De Bernardinis. 1998. The majority of gliadin-specific T cell clones from the coeliac small intestinal mucosa produce both g-interferon and IL4. Dig. Dis. Sci. 43: 156-161. [Medline]
31. Vader, W., D. Stepniak, T. Kooy, L. Mearin, A. Thompson, J. J. van Rood, L. Spaenij, F. Koning. 2003. The HLA-DQ2 gene dose effect in coeliac disease is directly related to the magnitude and breadth of gluten-specific T-cell responses. Proc. Natl. Acad. Sci. USA 100: 12390-12395. [Abstract/Free Full Text]
32. Kim, C. Y., H. Quarsten, E. Bergseng, C. Khosla, L. M. Sollid. 2004. Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in coeliac disease. Proc. Natl. Acad. Sci. USA 101: 4175-4179. [Abstract/Free Full Text]
33. Bacchetta, R., C. Sartirana, M.K. Levings, C. Bordignon, S. Narula, M. G. Roncarolo. 2002. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines. Eur. J. Immunol. 32: 2237-2245. [Medline]
34. Ebert, E. C.. 1989. Proliferative responses of human intraepithelial lymphocytes to various T-cell stimuli. Gastroenterology 97: 1372-1381. [Medline]
35. Khoo, U. Y., I. E. Proctor, A. J. S. Macpherson. 1997. CD4⁺ T cell down-regulation in human intestinal mucosa: evidence for intestinal tolerance to luminal bacterial antigens. J. Immunol. 158: 3626-3634. [Abstract]
36. Veldman, C., A. Hohne, D. Dieckmann, G. Schuler, M. Hertl. 2004. Type I regulatory T cells specific for desmoglein 3 are more frequently detected in healthy individuals than in patients with pemphigu vulgaris. J. Immunol. 172: 6468-6475. [Abstract/Free Full Text]
37. Yudoh, K., H. Matsuno, F. Nakazawa, T. Yonezawa, T. Kimura. 2000. Reduced expression of the regulatory CD4⁺ T cell subset is related to Th1/Th2 balance and disease severity in rheumatoid arthritis. Arthritis Rheum. 43: 617-627. [Medline]
38. Cong, Y., C. Weaver, A. Lazenby, C. O. Elson. 2002. Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora. J. Immunol. 169: 6112-6119. [Abstract/Free Full Text]
39. Slavin, A. J., R. Maron, H. Weiner. 2001. Mucosal administration of IL-10 enhances oral tolerance in autoimmune encephalomyelitis and diabetes. Int. Immunol. 13: 825-833. [Abstract/Free Full Text]
40. Mulder, C. J. J., P. J. Wahab, J. W. R. Meijer, E. Metselaar. 2001. A pilot study of recombinant human interleukin-10 in adults with refractory coeliac disease. Eur. J. Gastroenterol. Hepatol. 13: 1183-1188. [Medline]
41. Schreiber, S., T. Heinig, H. G. Thiele, A. Raedler. 1995. Immunoregulatory role of Interleukin-10 in patients with inflammatory bowel disease (IBD). Gastroenterology 108: 1434-1444. [Medline]
42. Fedorak, R. F., A. Gangl, C. O. Elson, P. Rutgeerts, S. Schreiber, G. Wild, S. Hanauer, A. Kilian, M. Cohard, A. LeBeaut, B. Feagan. 2000. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn’s disease. Gastroenterology 119: 1473-1482. [Medline]
43. Annacker, O., C. Asseman, S. Read, F. Powrie. 2003. Interleukin-10 in the regulation of T cell-induced colitis. J. Autoimm. 20: 277-279.
44. Van Deventer, S., C. O. Elson, R. N. Fedorak. 1997. Multiple doses of intravenous interleukin-10 in steroid-refractory Crohn’s disease: Crohn’s disease study group. Gastroenterology 113: 383-389. [Medline]
45. Schreiber, S., R. Fedorak, O. Nielsen, G. Wild, N. Williams, S. Nikolaus, M. Jacyna, B. Lashner, A. Gangl, P. Rutgeerts, et al 2000. Safety and efficacy of recombinant human interleukin-10 in chronic active Crohn’s disease. Gastroenterology 119: 1461-1472. [Medline]
46. Madsen, K.. 2002. Combining T cells and IL-10: a new therapy for Crohn’s disease. Gastroenterology 123: 2140-2144.
47. Colpaert, S., K. Vanstraelen, Z. Liu, F. Penninckx, K. Geboes, P. Rutgeerts, J. Ceuppens. 2002. Decreased lamina propria effector cell responsiveness to interleukin-10 in ileal Crohn’s disease. Clin. Immunol. 102: 68-72. [Medline]
48. Van Montfrans, C., E. Hooijberg, M. Rodriguez Pena, E. De Jong, H. Spits, A. Velde, S. Van Deventer. 2002. Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer. Gastroenterology 123: 1877-1888. [Medline]
49. Hauet-Broere, F., W. Unger, J. Garssen, M. Hoijer, G. Kraal, J. Samsom. 2003. Functional CD25^– and CD25⁺ mucosal regulatory T cells are induced in gut-draining lymphoid tissue within 48 h after oral antigen application. Eur. J. Immunol. 33: 2801-2810. [Medline]
50. Gad, M., A. Pedersen, N. Kristensen, H. Claesson. 2004. Demonstration of strong enterobacterial reactivity of CD4⁺CD25^– T cells from conventional and germ-free mice which is counter-regulated by CD4⁺CD25⁺T cells. Eur. J. Immunol. 34: 695-704. [Medline]
51. Nakamura, K., A. Kitani, I. Fuss, A. Pedersen, N. Harada, H. Nawata, W. Strober. 2004. TGF-1 plays an important role in the mechanism of CD4⁺CD25⁺ regulatory T cell activity in both humans and mice. J. Immunol. 172: 834-842. [Abstract/Free Full Text]
52. Karlsson, M. R., J. Rugtveit, P. Brandtzaeg. 2004. Allergen-responsive CD4⁺CD25⁺ regulatory T cells in children who have outgrown cow’s milk allergy. J. Exp. Med. 199: 1679-1688. [Abstract/Free Full Text]

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