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Servicios de Endocrinología (M.M., M.A.G.-L.) e Inmunología (H.d.l.F., F.S.-M.), Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain; and Departamento de Inmunología (N.F.-V., B.A.-S., A.M.-U., R.G.-A.), Facultad de Medicina, Universidad Autónoma de San Luis Potosí, 78210 San Luis Potosí S.L.P., México
Address all correspondence and requests for reprints to: Roberto González-Amaro, M.D., Ph.D., Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de San Luis Potosi, Ave. V. Carranza 2405, 78210 San Luis Potosí, S.L.P. México. E-mail: rgonzale{at}uaslp.mx.
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Abstract |
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Objective: The objective of the study was to analyze different regulatory T cell subsets in patients with AITD.
Design: We studied by flow cytometry and immunohistochemistry different T regulatory cell subsets in peripheral blood mononuclear cells (PBMCs) and thyroid cell infiltrates from 20 patients with AITD. In addition, the function of TREG lymphocytes was assessed by cell proliferation assays. Finally, TGF-ß mRNA in thyroid tissue and its in vitro synthesis by thyroid mononuclear cells (TMCs) was determined by RNase protection assay and quantitative PCR.
Results: PBMCs from AITD patients showed an increased percent of CD4+ lymphocytes expressing glucocorticoid-induced TNF receptor (GITR), Foxp3, IL-10, TGF-ß, and CD69 as well as CD69+CD25bright, CD69+TGF-ß, and CD69+IL-10+ cells, compared with controls. TMCs from these patients showed an increased proportion of CD4+GITR+, CD4+CD69+, and CD69+ cells expressing CD25bright, GITR, and Foxp3, compared with autologous PBMCs. Furthermore, a prominent infiltration of thyroid tissue by CD69+, CD25+, and GITR+ cells, with moderate levels of Foxp3+ lymphocytes, was observed. The suppressive function of peripheral blood TREG cells was defective in AITD patients. Finally, increased levels of TGF-ß mRNA were found in thyroid tissue, and thyroid cell infiltrates synthesized in vitro significant levels of TGF-ß upon stimulation through CD69.
Conclusions: Although T regulatory cells are abundant in inflamed thyroid tissue, they are apparently unable, in most cases, to downmodulate the autoimmune response and the tissue damage seen in AITD.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Regulatory T cells have an important role in immune tolerance to self-antigens and exert a key homeostatic effect in the immune system (6). There are two main subsets of CD4+ regulatory/suppressor T lymphocytes, the natural T regulatory cells (TREG cells) and an apparently heterogeneous cell population of T lymphocytes (type 1 regulatory T cells or Tr1, Th3 lymphocytes, Tr1-like cells), which are generated in the periphery (7, 8, 9). TREG cells are generated in the thymus, are able to recognize foreign and self-antigens, and are characterized by a constitutive high expression of CD25 (7). These cells are apparently anergic, dependent of IL-2, and express CTLA-4, and the glucocorticoid-induced TNF receptor (GITR) (6, 7). In addition, it has been demonstrated that the development of TREG cells in thymus is regulated by the forkhead-winged-helix family transcription factor Foxp3 (10). The key role of Foxp3 in the generation of TREG cells has been demonstrated in Foxp3–/– mice, which exhibit a scurfy phenotype with a fatal lymphoproliferative disorder (11).
Other CD4+ lymphocytes with immunoregulatory function have been described, mainly Tr1 cells (8). These regulatory cells are also antigen specific and characterized by the synthesis of IL-10 and TGF-ß and are generated in the periphery from CD4+CD25– lymphocytes (6, 8). Because CTLA-4, CD25, and GITR behave as lymphocyte activation markers, Tr1 cells could express (or not) these molecules. However, it is thought that these regulatory cells do not express Foxp3 (6, 7, 8, 9, 10). On the other hand, it has been reported that CD69+ T lymphocytes also exert a regulatory function, mainly through the synthesis of TGF-ß (12, 13). However, the relationship between CD69+ regulatory T cells and Tr1 lymphocytes has not been defined.
The role of regulatory T cells in the pathogenesis of thyroid autoimmune conditions has been mainly explored in animal models of endocrine organ-specific diseases (14). In this regard, an early report of Asano et al. (15) showed that neonatal thymectomy in mice eliminates CD25+ cells in the periphery and induces various organ-specific autoimmune diseases, including thyroiditis, when these animals are transplanted with neonatal thymuses. In addition, it has been shown that the tolerating effect of oral administration of thyroglobulin in mice is mediated by the induction of regulatory T cells that synthesize TGF-ß (16). Works that are more recent indicate that chemokines, specifically macrophage chemoattractant protein-1, are important in the attraction of TREG cells to the inflamed thyroid tissue (17) and that the engagement of CTLA-4 facilitates the generation of CD4+CD25+ lymphocytes and the synthesis of TGF-ß in thyroid tissue (18). The role of tolerogenic dendritic cells in the generation of CD4+CD25+ regulatory T cells that are able to synthesize IL-10 and suppress or prevent experimental autoimmune thyroiditis in mice has also been reported (19, 20). However, there are only a few studies on regulatory T cells in humans with autoimmune thyroiditis, including those on CD8+ suppressor cells (21, 22) and the different reports on the immune dysregulation, polyendocrinopathy X-linked syndrome, a condition characterized by Foxp3 mutations and TREG cell deficiency (23). The aim of this work was to carry out a quantitative and functional study of CD4+ regulatory T cells in patients with AITD. We found that different T regulatory cell subsets are detected in the peripheral blood and thyroid tissue from these patients. Some of these cell subsets are apparently abundant and able to synthesize TGF-ß. However, the suppressive function of these cells seems to be defective, and its presence in the thyroid gland does not seem to modify the clinical course and the inflammatory phenomenon seen in AITD.
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Patients and Methods |
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Surgical thyroid tissue and peripheral blood were obtained from 12 patients with GD and eight with HT. Diagnosis was established on commonly accepted clinical, laboratory, and histological criteria (1). All GD patients had relapsed after antithyroid drug treatment and were euthyroid under carbimazole therapy at the time of surgery. All had received iodine preoperatively. Normal thyroid tissues were obtained from unaffected glands of patients undergoing parathyroidectomy. All GD thyroid specimens studied showed high lymphocytic infiltration. Patients with HT who were operated had long-standing, very large compressive goiters. All were under treatment with thyroid hormone replacement, and thyroid hormone levels were normal. Peripheral blood was also obtained from 13 healthy individuals and 12 additional patients (eight with GD and four with HT). In all cases, an informed consent was obtained, and this study was approved by the local hospital ethical committee.
Cells
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) cushions. To isolate thyroid mononuclear cells (TMCs), thyroid specimens were minced and passed through a steel mesh. Then mononuclear cells (MCs) were isolated by Ficoll-Hypaque cushions, washed two times, and resuspended in RPMI 1640 tissue culture medium. Cell viability, assessed by trypan blue dye exclusion, was always higher than 95%.
CD4+ lymphocytes were purified using a MACS LS separation column (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, PBMCs were incubated with a biotinylated antibody cocktail for negative selection of CD4+ cells (Miltenyi) for 15 min at 4 C, washed, incubated with antibiotin microbeads (Miltenyi) for 20 min at 4 C, and washed. Then CD4+ lymphocytes were negatively selected using a MACS LS separation column (Miltenyi). For isolation of CD4+CD25+ cells, CD4+ lymphocytes were incubated with anti-CD25 microbeads (Miltenyi) for 15 min at 4 C, followed by positive selection using an additional separation column. CD4+CD25– lymphocytes were also recovered. Cell purity was always greater than 90%, as assessed by flow cytometry analysis.
Flow cytometry analysis
Cells were washed and double stained with the indicated monoclonal antibodies (mAbs), e.g. an anti-CD4-PerCP, and an anti-CD25-fluorescein isothiocyanate (FITC) (both from Becton Dickinson, San Jose, CA). Finally, cells were washed, fixed with p-formaldehyde, and analyzed by flow cytometry using the Cell Quest software and a FACSCalibur cytometer (Becton Dickinson). For these analyses, lymphocytes were electronically gated based on their forward and side scatter characteristics, and then double-positive cells were detected by analyzing at least 5000 lymphocytes. In all cases, negative controls of cell staining using isotype-matched irrelevant mAbs (Becton Dickinson) were run. For the detection of intracellular antigens, specific mAbs for CTLA-4 (BD PharMingen, San Jose, CA), Foxp3 (PCH101 clone, eBioscience, San Diego, CA), or IL-10 (PharMingen) were used. A double-labeling procedure was performed, first with an anti-CD4-FITC or an anti-CD69-FITC mAb (both from BD PharMingen) and then followed by fixation with 4% p-formaldehyde (10 min, room temperature), and permeabilization with 0.01% of saponine and 10% of fetal bovine serum in PBS for 5 min on ice. Finally, cells were additionally stained with the indicated mAb (i.e. anti-CTLA-4) and analyzed by flow cytometry. In some assays, three-color flow cytometry analysis was performed, using anti-CD4-PerCP, anti-CD25-FITC (Becton Dickinson), and anti-Foxp3-PE (eBioscience) mAbs. In these assays, careful color compensation was performed, and their intraassay reproducibility was corroborated. In the case of TGF-ß detection, a biotinylated specific mAb plus streptavidin-FITC (both from BD PharMingen) were used.
Immunohistochemical analysis
Cryostat sections (4 µm thick) were obtained from snap-frozen thyroid tissue embedded in Tissue-Tek OCT medium (Ames; Miles Laboratories, Elkhart, IN) stored at –80 C. Tissue sections were stained by an indirect immunoperoxidase method. Briefly, acetone-fixed tissue sections were sequentially incubated with the indicated mAb and a peroxidase-conjugated rabbit antimouse Ig (Dako, Glostrup, Denmark). Each incubation was followed by three washes with Tris-buffered saline solution (pH 7.4). Finally, sections were developed with the Graham-Karnovsky reagent containing 0.5 mg/ml 3,3'-diaminobenzidine (Sigma) and hydrogen peroxide and then counterstained with Carazzi’s hematoxylin.
Functional analysis of TREG cells
Nonregulatory CD4+CD25– T cells (1 x 105) were mixed or not with CD4+CD25+ regulatory T cells (1 x 104 or 1 x 105) in the presence of anti-CD3 and anti-CD28 mAb plus a cross-linker goat antimouse Ig (Sigma). Then cells were cultured for 72 h in complete culture medium in 96-well plates. 3H-methyl-thymidine (3H-TdR, 1.0 µCi/well, specific activity 5.0 Ci/mM, NEN Life Science Products, Boston, MA) was added for the last 12 h of culture, and at the end of incubation cells were harvested and proliferation was determined using a liquid scintillation counter. All these experiments were run in triplicate, and the results were expressed as the percent of cell proliferation, according to the following formula: percent cell proliferation = (counts per minute of CD4+CD25– plus CD4+CD25+ cells/cpm of CD4+CD25– cells alone) x 100.
In separate experiments, the suppressive function of CD4+CD25+ lymphocytes on cell proliferation was assessed via fluorescent label partition and flow cytometry analysis. In this assay, cells are labeled with a fluorescent probe that irreversibly couples to cellular proteins, and when cells divide, their fluorescence shows a 2-fold decrement. Briefly, CD4+CD25+ and CD4+CD25– cells were mixed at 1:10 ratio, labeled with carboxyfluorescein diacetate (CFDA) succinimidyl ester (Molecular Probes, Eugene, OR), and stimulated through CD3 and CD28 for 72 h. Then the percent of divided cells was detected by flow cytometry analysis. Finally, in some assays the suppressive activity of CD25bright cells was analyzed by comparing the cell proliferation of PBMCs depleted or not of these cells (24). In brief, PBMCs were depleted or not of CD25bright cells with anti-CD25-coated microbeads (1:8 ratio) and a MACS LB separation column (Miltenyi). Then cells were stimulated with an anti-CD3 and anti-CD28 mAb plus a cross-linker goat antimouse Ig for 72 h. In these assays cell proliferation was assessed by either 3H-TdR incorporation or CFDA succinimidyl ester labeling, as stated above.
In vitro stimulation of TGF-ß mRNA synthesis
TMCs and PBMCs were suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated for 2 h in the presence of purified anti-CD69 TP1/8 mAbs (10 µg/ml) plus a rabbit antimouse IgG (Sigma). Control cells were incubated with medium alone or the secondary antimouse cross-linker antibody. In separate experiments, TMCs and PBMCs were incubated with 1.0 µg/ml recombinant human GITR ligand (GITRL) (TNFSF18, Sigma) for 2 h. At the end of incubation, cells were centrifuged and then used for TGF-ß mRNA analysis.
RNase protection assay and quantitative real-time RT-PCR analyses
Total RNA was isolated from TMCs using the Ultraspec RNA reagent (Biotecx, Houston, TX). RNase protection assays were performed on 2.5–5.0 µg of RNA using Riboquant protection assay templates and reagents from PharMingen (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. For RT-PCR, 2.0 µg of DNase I-treated RNA were reverse transcribed with Moloney leukemia virus reverse transcriptase (Roche Diagnostics Ltd., Lewes, UK). Then real-time PCR was performed in a Lightcycler rapid thermal cycler system (Roche, Mannheim, Germany) using the following primers: TGF-ß forward, GGA CAC CAA CTA TTG CTT CAG, reverse, TCC AGG CTC CAA ATG TAG G; and glyceraldehyde-3-phosphate dehydrogenase forward, TGG GTG TGA ACC ACG AA, reverse, ACA GCT TTC CAG AGG G. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase expression and measured in parallel in each sample.
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Results |
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, A and C). Nevertheless, a significantly higher proportion of CD4+GITR+, CD4+Foxp3+, and CD4+ cells synthesizing IL-10 or TGF-ß was detected in patients with AITD (see Figs. 1A and 5A; also data not shown). Likewise, higher levels of CD4+CD69+, CD69+CD25bright, and CD69+ lymphocytes producing IL-10 or TGF-ß were observed in patients with this condition (Fig. 1, B and D). In contrast, no significant differences were found in the levels of CD69+ cells coexpressing GITR or the transcription factor Foxp3, and no significant correlation was detected between the expression of these antigens and thyroid hormone levels. No apparent differences in the levels of cell subsets studied were observed in HT, compared with GD (data not shown).
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, similar levels of several CD4+ lymphocyte subpopulations were detected, including CD4+CD25bright, CD4+Foxp3+, and CD4+CTLA-4+ cells as well as CD4+ lymphocytes synthesizing IL-10 or TGF-ß. However, a significantly higher proportion of CD4+GITR+ cells was detected in thyroid tissue, compared with peripheral blood (Fig. 2A). In addition, different CD69+ cell subsets were also significantly enriched in thyroid tissue, including those coexpressing GITR, Foxp3, and high levels of CD25 (Fig. 2B). In contrast, no significant differences were detected in the case of CD69+ cells synthesizing the immunoregulatory cytokines IL-10 or TGF-ß (Fig. 2B). As in peripheral blood, no apparent differences of the cell subsets studied were observed in tissues from HT, compared with GD (data not shown).
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, A and B). Likewise, a significant proportion of the inflammatory cell infiltrate showed intracellular expression of the transcription factor Foxp3 and CTLA-4 (Fig. 3C and data not shown). Finally, there was a significant expression of GITR and a striking infiltration by GITRL+ cells (data not shown and Fig. 3D).
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, when the inhibition of cell proliferation by peripheral blood CD4+CD25+ cells was tested on autologous CD4+CD25– lymphocytes by a 3H-TdR incorporation assay, we found a significantly diminished suppressive effect in cells from AITD patients, compared with controls (P < 0.05, Mann-Whitney U test). These results were confirmed using a cell proliferation assay based in the detection of partition of the cell fluorescent tracker CFDA by flow cytometry (Fig. 4B and data not shown). Furthermore, using a different suppression assay, based in the depletion of CD25bright lymphocytes and CFDA cell labeling, we found a similar diminished regulatory activity of TREG cells in PBMCs from three additional patients with AITD, compared with healthy controls (mean of the difference in the percent of divided cells in nondepleted and depleted cultures: 5.3% in patients and 16.6% in controls; Fig. 4B and data not shown).
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, lower panel, patient 2, with HT and heavy cell infiltration of the gland), a very poor suppressive function was exerted. This defective regulatory function was also detected in their PBMCs. In contrast, in the other case (patient 1, with HT and moderate cell infiltration of the gland), a strong suppressive activity was detected in both TMCs and PBMCs (Fig. 4A, lower panel).
As stated above, flow cytometry data showed the presence of CD4+ synthesizing TGF-ß in the inflammatory cell infiltrate of autoimmune thyroiditis (Fig. 5A). Immunohistochemical analysis confirmed the presence of a significant proportion of TGF-ß+ cells in the inflamed gland (Fig. 5B). Further analysis with an RNase protection assay showed heterogeneous but high levels of the different isoforms of TGF-ß mRNA in glands from autoimmune thyroiditis (Fig. 5, C and D).
Because it has been shown in mice that the cross-linking of CD69 induces the synthesis and release of TGF-ß by T cells (12), we explored this phenomenon in the infiltrating MCs from autoimmune thyroiditis. As shown in Fig. 5, CD69 engagement induced the synthesis of TGF-ß mRNA by thyroid MCs from patients with autoimmune thyroiditis. In contrast, this phenomenon was not observed in the PBMCs from the same patients (Fig. 5E).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Although experimental models of autoimmune thyroiditis have provided important information on the pathogenesis of this condition, it is evident that there are important differences between these animal models and AITD in humans (27, 28). Therefore, we assessed the number and functional status of regulatory T cells in human AITD. In this work, we found that the PBMCs from patients with AITD exhibit high to moderate levels of different CD4+ T cell subsets with regulatory phenotype, including CD4+GITR+ and CD4+ Foxp3+ cells as well as CD4+IL-10+ lymphocytes, which likely correspond to TREG and Tr1 cells, respectively (6, 8). In addition, these patients show enhanced levels of cells that very likely exert a regulatory function, including CD69+CD25bright and CD69+ cells synthesizing IL-10 and TGF-ß (12, 13). Accordingly, an important proportion of the thyroid inflammatory cell infiltrate corresponded to lymphocytes with a regulatory phenotype, including CD4+GITR+, CD69+CD25bright, and CD69+Foxp3+ cells. Therefore, it is feasible that at least some of these cell subsets migrate from thyroid tissue to peripheral blood and vice versa, in a homing phenomenon similar to that observed for memory T cells in chronically inflamed tissues (3). In this regard, a similar enrichment in TREG lymphocytes has been described in the inflammatory cell infiltrate of the rheumatoid synovium (29). Likewise, it has been previously found that CD69+ lymphocytes are able to down-regulate the inflammatory phenomenon seen in collagen-induced arthritis in mice and that this effect is mainly mediated by TGF-ß (12). Accordingly, our data indicate that the inflammatory cell infiltrates of autoimmune thyroiditis contain a significant number of CD69+ and TGF-ß+ lymphocytes. In addition, our results confirm that CD69 engagement induces the synthesis of this immunoregulatory cytokine and further support the importance of CD69+ lymphocytes as regulatory cells.
As in the case of rheumatoid arthritis (29), it is of interest that the autoimmune/inflammatory process of human thyroiditis does not seem to be significantly affected by the noticeable presence of T regulatory cells, including Foxp3+ lymphocytes. In rheumatoid arthritis, it has been found that TREG cells have a defective function and that anti-TNF
therapy enhances their suppressive activity (30, 31). Our functional assays of suppression of cell proliferation performed with peripheral blood CD4+CD25+ lymphocytes indicate that most patients with AITD also have a defective regulatory function. Therefore, it is very likely that in most patients with autoimmune thyroiditis, as in rheumatoid arthritis, TREG cells are unable to down-regulate the inflammatory phenomenon.
In this regard, we found a striking expression of GITRL in the inflamed glands studied, and it has been described that its receptor, GITR, may act as a costimulatory molecule in TREG cells, favoring their proliferation and inhibiting their regulatory activity (32). Thus, it is very likely that in thyroid autoimmune diseases, the activity of regulatory cells is being inhibited by the proinflammatory milieu of the diseased gland, including the abundant presence of GITRL+ cells. We consider that our suppression assays performed with CD4+CD25+ cells from thyroid tissue support this possibility. In this regard, we found one case with high suppressive activity and another one with a very defective function. In the latter patient, a heavy inflammatory cell infiltrate was seen, with a prominent presence of GITRL+ cells. In contrast, in the former patient, we observed a moderate cell infiltrate with a low proportion of GITRL+ cells. However, it is evident that it is necessary to study additional patients to further support this hypothesis.
Another interesting possibility to explain our apparent paradoxical results is that once immune tolerance is broken and the inflammatory/destructive phenomenon is ongoing, the activity of effector T cells would overcome the suppressive effect of TREG cells (33). In this regard, it has been described that CD4+CD25– T cells from rheumatoid arthritis synovial fluid are more difficult to suppress than cells from the peripheral blood (34). In addition, it has been recently reported that CD4+CD25– cells from MRL/Mp autoimmune mice show a reduced sensitivity to suppression by autologous TREG lymphocytes (35), a phenomenon that could be related to defects in intracellular signaling molecules such as Cbl-b (36). Our data on the enhanced number of IL-10+ lymphocytes and TGF-ß synthesis as well as the induction of synthesis of this cytokine through CD69 in TMCs support this interesting possibility. Therefore, our results suggest that in most patients with AITD, some regulatory cell subsets (i.e. TREG cells) seem to exert a defective suppressive function, whereas other regulatory lymphocytes (i.e. Tr1 or Th3 cells) seem to be functionally intact, but their target cells are refractory to them.
In summary, this study shows that different regulatory T cell subsets are present in the peripheral blood and thyroid tissue from patients with AITD. Some of these cell subsets are apparently abundant and able to synthesize the immunoregulatory cytokines IL-10 and TGF-ß. However, in most cases the presence of these regulatory cells does not seem to modify the clinical course and the inflammatory phenomenon seen in these conditions. We think that these data are very important for the potential therapeutic use in AITD of immunoregulatory cytokines (37); in vitro-generated TREG cells; and even tolerogenic dendritic cells, which mainly exert their effect through the induction of regulatory T cells (6, 13, 16, 20). Finally, we consider that it is necessary to perform additional studies to fully elucidate the complex role of regulatory cells in AITD.
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Acknowledgments |
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Footnotes |
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First Published Online June 27, 2006
Abbreviations: AITD, Autoimmune thyroid disease; CFDA, carboxyfluorescein diacetate; FITC, fluorescein isothiocyanate; GD, Graves’ disease; GITR, glucocorticoid-induced TNF receptor; GITRL, GITR ligand; HT, Hashimoto’s thyroiditis; 3H-TdR, 3H-methyl-thymidine; mAb, monoclonal antibody; MC, mononuclear cell; PBMC, peripheral blood MC; TMC, thyroid MC.
Received October 25, 2005.
Accepted June 19, 2006.
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