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Impairs Both T Regulatory Cell Function and Protection against Autoimmune Diabetes1
Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212
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Abstract |
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(mTGF-) and sustained protection against diabetes, whereas Tregs expanded during insulitis had minimal mTGF- and could not protect against diabetes. The Tregs probably operate suppressive function through mTGF-, because Ab blockade of mTGF- nullifies protection against diabetes. Surprisingly, young Tregs that modulated pathogenic T cells maintained stable frequency over time in the protected animals, but decreased their mTGF- at the age of 8 wk. More strikingly, these 8-wk-old mTGF--negative Tregs, which were previously protective, became unable to confer resistance against diabetes. Thus, a developmental decline in active mTGF- nullifies Treg function, leading to a break in tolerance and the onset of diabetes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(mTGF-) on Tregs has recently been shown to mediate cell contact inhibition of pathogenic T cells (10) and play a critical role in Treg suppressive function (11, 12, 13). In fact, anti-islet CD8+ T cells expressing a dominant negative TGF- receptor transgene could not be targeted by Tregs in vivo (14). In this study an approach for peptide delivery on Ig was developed, and a treatment regimen was defined that expanded mTGF--positive Tregs and protected animals against diabetes. Moreover, we found that an abrupt decline in mTGF- expression on Tregs accompanied by a loss of suppressive functions transpire during the transition to destructive insulitis and progression to diabetes. Indeed, when the glutamic acid decarboxylase (GAD65) 524–543 peptide (designated GAD1) (15, 16) was genetically engineered into an Ig molecule, the resulting Ig-GAD1 expanded Tregs expressing active mTGF- and protected young mice against diabetes. However, Ig-GAD1 given to 8-wk-old mice with progressive insulitis induced Tregs lacking mTGF- and did not protect against diabetes. Interestingly, 6-wk-old Tregs, whether from Ig-GAD1 treated or naive NOD mice, expressed mTGF- and delayed diabetes when cotransferred with diabetogenic splenocytes into NOD.scid mice. However, 8- or 26-wk-old Tregs, whether from naive or Ig-GAD1-treated nondiabetic animals, had minimal mTGF- and could not protect NOD.scid mice against passive diabetes. Furthermore, blockade of mTGF- with Abs before transfer into NOD.scid mice nullifies the protective function of the otherwise suppressive 6-wk-old Tregs. Together, these results indicate that a decline in cell surface expression of active TGF- during transition to insulitis is responsible for the loss of suppressive function of Tregs and the resulting onset of diabetes. |
Materials and Methods |
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NOD (H-2g7) and NOD.scid mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IL-4-deficient (IL-4–/–) and IL-10-deficient (IL-10–/–) NOD mice were previously described (17, 18). All mice were maintained in the animal facility for the duration of experiments, and the experimental procedures performed on these animals were conducted according to the guidelines of the institutional animal care and use committee.
Assessment of diabetes
Mice were bled from the tail vein weekly, and the blood samples were used to assess both glucose content and anti-insulin Abs. For measurement of glucose, a drop of blood was directly placed on a test strip, and the glucose content was read using a FreeStyle blood glucose-monitoring system (TheraSense, Alameda, CA). For detection of anti-insulin Abs, the blood was allowed to coagulate for 1 h at room temperature, and the serum was separated and used for ELISA. A mouse was considered diabetic when the blood glucose was >300 mg/dl for 2 consecutive weeks.
Antigens
Peptides.
All peptides used in this study were purchased from Metabion (Munich, Germany) and were purified by HPLC to >90% purity. Insulin -chain (INS) peptide (SHLVEALYLVCGERG) encompasses a diabetogenic epitope corresponding to aa residues 9–23 of INS (19). GAD1 peptide (SRLSKVAPVIKARMMEYGTT) corresponds to aa residues 524–543 of GAD65 (20, 21). GAD2 peptide (TYEIAPVFVLLEYVT) corresponds to aa residues 206–220 of GAD65 (22). Hen egg lysozyme (HEL) peptide (AMKRHGLDNYRGYSL) encompasses a nondiabetogenic epitope corresponding to aa residues 11–25 of HEL (23). INS, GAD1, GAD2, and HEL peptides are presented to T cells in association with I-Ag7 MHC class II molecules.
Ig chimeras.
Ig-GAD1 and Ig-HEL are chimeras expressing GAD1 and HEL peptides, respectively. Insertion of GAD1 and HEL nucleotide sequences into the CDR3 of the H chain variable region of 91A3 IgG2b, 
Ig, was conducted as previously described (24). Large-scale cultures of transfectoma cells were used in DMEM containing 10% iron-enriched calf serum (BioWhittaker, Walkersville, MD). Purification of Ig-GAD1 and Ig-HEL was conducted on separate columns of rat anti-mouse -chain mAb coupled to cyanogen bromide-activated 4B Sepharose (Amersham Biosciences, Piscataway, NJ). Aggregation of the Ig chimeras was conducted by precipitation with 50% saturated (NH4)2SO4 as previously described (24).
Generation of T cell clones
A T cell clone specific for GAD1 peptide was generated by immunizing NOD mice with 50 µg of GAD1 peptide in 200 µl of PBS/CFA (v/v) s.c. in the footpads and at the base of each limb. After 10 days, the draining lymph nodes were removed, and T cells were stimulated in vitro for two rounds in the presence of irradiated (3000 rad), syngeneic splenocytes, 5% T-Stim supplement (Collaborative Biomedical Products, Bedford, MA), and GAD1 peptide (15 µg/ml). Cloning of a T cell line specific for GAD1 was accomplished by limiting dilution. The culture medium used to carry out these stimulations and other T cell activation assays in this study was DMEM supplemented with 10% FCS (HyClone, Logan, UT), 0.05 mM 2-ME, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µg/ml gentamicin sulfate.
Isolation of T cells
CD4+ T lymphocytes were isolated from the spleen by positive selection on microbeads (Miltenyi Biotech, Auburn, CA). For CD4+CD25+ T cells, splenic cells were depleted of RBC, and CD4+ lymphocytes were separated by negative selection using the Miltenyi CD4 T cell isolation kit. The CD4+CD25+ T cells were isolated by positive selection using anti-CD25-coupled Miltenyi microbeads. The CD25-negative fraction (CD4+CD25–) was used as a control for CD4+CD25+ T cells. All procedures were conducted according to Miltenyi’s instructions.
Isolation of BSA-APCs
Partial purification of splenic APC was accomplished by floating fresh NOD spleen cells on a dense BSA gradient, and the cells were then washed in plain culture medium and used in T cell activation assays.
Flow cytometric analyses
For staining of CD4, CD25, and CD62L, purified splenic CD4+ T cells (1.5 x 106) were incubated with anti-CD4-PE, anti-CD25-allophycocyanin (or isotype control rat IgG1-allophycocyanin), and anti-CD62L-FITC (or isotype control rat IgG2a-FITC) for 30 min at 4°C and washed with buffer. The cells were fixed with 2% formaldehyde for 20 min at room temperature and then analyzed. Events (30–50 x 103) were collected on a FACSVantage flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest software 3.3 (BD Biosciences). Staining for CTLA-4 was conducted as follows: purified islet and splenic CD4+ T cells (2 x 106 cells) were incubated with anti-CTLA-4-PE (4F10) or isotype control hamster IgG1-PE for 2 h at 37°C, followed by anti-CD4-FITC and anti-CD25-allophycocyanin or isotype control rat IgG1-allophycocyanin for 30 min at 4°C. The cells were then washed, fixed with 2% formaldehyde, and analyzed as described above. Anti-CD4-FITC or -PE, anti-CD25-allophycocyanin, anti-CD62L-FITC, anti-CTLA-4-PE, rat IgG1-allophycocyanin, rat IgG2a-FITC, and hamster IgG1-PE were purchased from BD Pharmingen (San Diego, CA). Staining for surface expression of active TGF- was conducted as previously described (10). Briefly, purified CD4+ T cells (2 x 106 cells) were incubated with anti-CD4-FITC, anti-CD25-allophycocyanin (or isotype control rat IgG-allophycocyanin), and biotin-conjugated anti-TGF-1 (BAF240) or with isotype control chicken IgY-biotin for 30 min at 4°C and washed with buffer. Subsequently, the cells were stained with PE-conjugated streptavidin for 30 min at 4°C. The cells were then washed, fixed with 2% formaldehyde, and analyzed as described above. Biotin-conjugated anti-TGF-1 and chicken IgY were purchased from R&D Systems (Minneapolis, MN).
Proliferation assays
For presentation of Ig-GAD1 to a specific T cell clone, irradiated (3000 rad) NOD splenocytes (5 x 105 cells/50 µl/well) were incubated with 100 µl of Ag, and 1 h later, GAD1-specific T cells, TCC-GAD1-1F6 (5 x 104 cells/well/50 µl), were added. After 3-day incubation, 1 µCi of [3H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells were then harvested on a Mach III harvester (Tomtec, Hamden, CT), and incorporated [3H]thymidine was counted on a Trilux 1450 Microbeta counter (Wallac, Gaithersburg, MD) using Microbeta 270.004 software (Wallac). For activation of splenic T cells after Ig chimera treatment, purified CD4+ T cells (2.5 x 105 cells/well) were incubated with irradiated (3000 rad) BSA-APCs (5 x 105) and 20 µg/ml peptide for 72 h. After the incubation, 1 µCi of [3H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells were then harvested and counted as described above. For alloantigen-induced expansion, isolated CD4+CD25– T cells (2 x 105 cells/well) were incubated for 5 days with T cell-depleted C57BL/6 splenic cells (1 x 105 cells/well) and increasing numbers of CD4+CD25+ T cells. The CD4+CD25+ T cells were incubated for 2 h with or without anti-TGF- Ab (1D11) or mouse IgG isotype control and washed before addition to the alloantigen reaction mix. The culture was pulsed 8 h before harvesting with 1 µCi of [3H]thymidine and then counted.
Cytokine production by Tregs
Splenic CD4+CD25+ or CD4+CD25– T cells (2.5 x 105 cells/well) were stimulated with 30 µg/ml peptide for 48 h in the presence of irradiated (3000 rad) BSA-APCs (5 x 105 cells/well). Subsequently, cytokine production was assessed by ELISA from 100 µl of culture supernatant.
Detection of cytokines in cell cultures
Detection of IL-10 was conducted by ELISA according to BD Pharmingen’s standard protocol. The capture Ab was rat anti-mouse IL-10, JES5-2A5, and the biotinylated anti-cytokine Ab was rat anti-mouse IL-10, JES5-16E3. Both Abs were purchased from BD Pharmingen. Detection of TGF- was performed according to the procedure outlined by R&D Systems. To activate latent TGF- to the immunoreactive form, samples were acidified by the addition of HCl (20 mM) for 10 min at room temperature, then neutralized by NaOH/HEPES solution. The capture Ab was mouse anti-TGF-1, -2, and -3 1D11 mAb, and the biotinylated anti-cytokine Ab was chicken anti-TGF-1 (BAF240). Both Abs were purchased from R&D Systems. All assays were read on a SpectraMAX 190 counter (Molecular Devices, Sunnyvale, CA) and analyzed using SOFTmax PRO 3.1.1 software. Graded amounts of recombinant mouse IL-10 (BD Pharmingen) and TGF- (R&D Systems) were included in all experiments for construction of standard curves. The cytokine concentration in culture supernatants was extrapolated from the linear portion of the standard curve.
Depletion of Tregs
For depletion of CD4+CD25+ T cells in vivo, mice were injected with 1 mg of anti-CD25 Ab (PC61)/mouse alone or in conjunction with aggregated (agg) Ig-GAD1 treatment. Rat IgG (1 mg/mouse) was used as a control.
Suppression of passive diabetes by Tregs
Splenic cells were harvested from untreated (nil) and Ig chimera-treated mice at the ages indicated. Subsequently, splenic CD4+CD25+ and CD4+CD25– T cells were purified and resuspended in PBS. Additionally, spleens from recently diabetic NOD female mice (
2 wk diagnosed) were harvested, and the isolated diabetogenic splenocytes (used to induce diabetes in NOD.scid) were resuspended in PBS. Then, 5 x 105 cells CD4+CD25+ or CD4+CD25– T cells were coinjected i.v. with 1 x 107 diabetogenic splenocytes into NOD.scid mice (4–8 wk of age). In some experiments the CD4+CD25+ T cells were incubated for 2 h with 100 µg/ml anti-TGF- (1D11) or isotype control mouse IgG before cotransfer with diabetogenic splenocytes into NOD.scid mice.
Real-time PCR for Foxp3 expression
Total RNA was extracted from cells using TRIzol reagent. RT and DNA amplification were performed according to the one-step protocol using 300 ng of total RNA and a QuantiTect SYBR Green real-time PCR kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Primer pairs were as follows: Foxp3, 5'-GGCCTTCTCCAGGACAGA-3' and 5'-GCTGATCATGGCTGGGTTGT-3'; and -actin, 5'-AGAGGGAAATCGTGCGTGAC-3' and 5'-CAATAGTGATGACCTGGCCGT-3'. Real-time PCR was performed on a Cepheid Smart Cycler (Sunnyvale, CA), and the results were analyzed by the comparative threshold cycle (CT) method using Smart Cycler software. The comparative CT method allows relative quantitation of gene expression to be performed where CT represents the cycle where detection of an increased signal associated with exponential growth of PCR product begins. Furthermore, 
CT values represent the difference between a sample CT and a normalizer CT such as -actin. For comparisons of gene expression, the CT values are used and represent the difference between the sample CT and a reference CT. Finally, quantitation using the formula 2–CT provides a comparative expression level for comparisons of differing conditions, such as treatments or ages. This comparative expression level, therefore, represents a fold difference from that of the reference level.
Insulin autoantibody assay
Detection of insulin autoantibodies (IAA) in the serum of NOD mice was conducted by ELISA as follows. Microtiter plates (no. 3369; Corning Glass, Corning, NY) were coated with 50 µl of sodium bicarbonate solution (pH 9.6) containing 10 µg/ml porcine insulin (Sigma-Aldrich, St. Louis, MO) for 16 h at 4°C. The plates were then washed three times with PBS-0.05% Tween 20, and free plastic sites were saturated by incubation with 2.5% casein (in 0.3 M NaCl, pH 7) for 2 h at room temperature. Subsequently, serum samples (1/200 dilutions) were added, and the plates were incubated for 16 h at 4°C. Biotin-conjugated, rat anti-mouse mAb (100 µl at 1 µg/ml) was added, and the plates were incubated for 1 h at room temperature. Bound anti-mouse mAb was revealed by incubation with a casein solution containing 2.5 mg/ml avidin peroxidase for 30 min at room temperature, followed by addition of ABTS substrate. The samples were read at 405 nm on a Spectramax 190. A sample is considered IAA positive when the OD405 is >0.2. This cutoff line of 0.2 was chosen because serum samples from 10 SJL mice, which are not prone to diabetes development and presumably do not produce insulin-specific autoantibodies, never exceeded 0.05 OD405 (4-fold less than cutoff).
Statistical analysis
The 
2 test was used for data analysis among experimental and control groups. Cytokine levels were compared using Student’s t test for unpaired samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells made against GAD1 peptide proliferated upon stimulation with GAD1, but not the negative control HEL peptide (Fig. 1a). Ig-GAD1, but not the negative control Ig-HEL, was presented to these specific cells and induces their proliferation (Fig. 1b). These data indicated that Ig-GAD1 is internalized by APCs, and the GAD1 peptide is released and presented to the T cells in a specific manner. It was recently shown that aggregation of Ig-myelin chimeras cross-linked Fc
R on APCs and increased the myelin peptide’s tolerogenic functions (24, 25, 26). Whether such regimens operate through expansion of Tregs is unknown. Administration of agg Ig-GAD1 into young NOD mice induced the expansion of cells with a regulatory phenotype. Indeed, the number of CD25+ T cells among all CD4+ T lymphocytes rose from 5.0% in the agg Ig-HEL-treated mice to 10.5% in the animals given agg Ig-GAD1 (Fig. 1, c and d). Nil or soluble (sol) Ig-GAD1-treated mice had 4–6% CD4+CD25+ T cells (data not shown). These CD4+CD25+ T cells had an increased mRNA expression of the Forkhead/winged helix transcription factor (Foxp3) gene relative to their CD4+CD25– counterparts (Fig. 1e), concurring with a Treg phenotype (27, 28). These Tregs did not secrete IL-10 or TGF- (Fig. 1, f and g), but displayed significant suppressive functions against their CD4+CD25– counterparts (Fig. 1h). Without a doubt, the CD4+CD25– T cells mounted significant MLR proliferation against T cell-depleted allogeneic C57BL/6 splenocytes, but a marked decrease in the proliferation was observed when CD4+CD25+ T cells were added to the culture (Fig. 1h). Thus, treatment with agg Ig-GAD1 resulted in expansion of T cells with both phenotypic and functional marks of Tregs. Additional in vivo analyses seem to associate these Tregs with a significant delay of diabetes. In fact, mouse recipients of agg Ig-GAD1 treatment reduced their spontaneous proliferative responses to diabetogenic peptides such as INS, GAD1, and GAD2 in comparison with animals recipient of the control molecule agg Ig-HEL (Fig. 2a). It should be noted that HEL peptide, although restricted to I-Ag7-like GAD1 peptide, is not a self-determinant, and NOD mice do not develop spontaneous responses against it. Thus, the lack of proliferation against HEL peptide in Ig-GAD1- and Ig-HEL-treated mice is due to the absence of a spontaneous response, rather than to down-regulatory functions by the chimeras. Furthermore, a significant level of protection against diabetes was observed in these animals. Indeed, only 40% of mice treated with agg Ig-GAD1 developed diabetes compared with 70% of control agg Ig-HEL treated and 80% of nil animals (Fig. 2b). It should be noted that some protection was seen with the control agg Ig-HEL in the early stage of disease, which is probably due to bystander suppression by IL-10 produced by the APCs upon cross-linking of FcRs (24). The sol form of Ig-GAD1, which did not expand CD4+CD25+ Tregs, supported a delay in disease onset through 20 wk of age (10% incidence of diabetes vs 50% for both nil and agg Ig-HEL). However, the incidence of disease rose to 80%, which is similar to that in the control sol Ig-HEL-treated group (data not shown). A prolonged treatment regimen, consisting of a weekly injection of 300 µg of agg chimeras from wk 4–12 and biweekly injections thereafter until wk 30 of age, produced only a slight enhancement of disease prevention; 30% of the mice became diabetic by wk 30 (Fig. 2c) vs 40% in the short treatment group (Fig. 2b). The nil as well as agg Ig-HEL groups displayed similar incidences of disease as the short treatment regimen (Fig. 2, b and c). A prolonged regimen with sol Ig-GAD1 remains less effective, because only transient protection was observed at wk 20, and most of the mice became diabetic by wk 26 of age (Fig. 2d). Hence, the results indicate that a short treatment at the preinsulitis stage is sufficient to induce optimal protection by agg Ig-GAD1. The delay of disease onset is most likely controlled by Tregs. This statement stems from the observation that the percentage of CD4+CD25+ T cells was maintained at expanded (10% of total CD4+ cells) levels through 26 wk of age relative to the 5% obtained with the untreated mice (Fig. 2e). Moreover, depletion of these Tregs at the preinsulitis stage nullified the suppressive effects of agg Ig-GAD1. Indeed, 90% of the mice given anti-CD25 Ab during treatment with agg Ig-GAD1 became diabetic by wk 26 of age, whereas only 30% of the animals displayed hyperglycemia when rat IgG replaced anti-CD25 Ab (Fig. 2f). Interestingly, anti-CD25 Ab alone did not affect the pattern of disease, indicating that interference with activated pathogenic T cells was minimal. Overall, these results indicate that agg Ig-GAD1 expands cells with a phenotypic pattern characteristic of T regulatory cells and operates protection against diabetes through the suppressive function of these Tregs.
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Natural Tregs arise in the normal T cell repertoire to contribute to the maintenance of self-tolerance (1, 2). Gradual loss of function by Tregs is viewed as one of the lead mechanisms for development of autoimmunity in maturing NOD mice (7, 9). To address the issue of ineffectiveness of maturing Tregs, we began by examining whether treatment with agg Ig-GAD1 expanded Tregs in older mice positive for IAA, a sign indicative of insulitis (31, 32) and an ongoing disease process. Accordingly, mice were given agg Ig-GAD1 during the week of IAA seroconversion (which occurs at 8–11 wk of age) as well as 7 and 14 days later and then tested for expansion of Tregs. The results indicate that the number of CD4+CD25+ T cells had increased from 6.1% in the untreated mice to 10.1% in the age-matched, agg Ig-GAD1-treated animals (Fig. 4, a and b). However, when these animals were monitored for blood glucose levels, hyperglycemia was as prevalent as in the control untreated or agg Ig-HEL-treated groups, indicating a lack of protection against diabetes (Fig. 4c). In fact, the agg form of Ig-GAD1 had a similar result as the sol form, which is not effective in expanding Tregs (Fig. 4c). Overall, mice with progressive insulitis are able to expand Tregs, but fail to protect themselves against diabetes. Subsequently, the splenic CD4+CD25+ T cells from these mice were isolated and tested for suppression of passive diabetes mediated by pathogenic splenocytes of recently diabetic mice. These Tregs, however, were unable to protect the NOD.scid mice from diabetes; the survival pattern of the recipient mice was similar to that of animals given only the diabetogenic splenocytes (Fig. 4d). However, CD4+CD25+ T cells from the young NOD mice treated at 4, 5, and 6 wk of age were protective; 80% of the recipient animals were free of diabetes. It is thus logical to suspect that a decline in the suppressive function of Tregs is responsible for the lack of protection against the disease. To further address this matter, maturing natural and agg Ig-GAD1-expanded Tregs were isolated at different time points and tested for suppression of passive diabetes by cotransfer with diabetogenic splenocytes into NOD.scid mice. Fig. 5 shows that 70–80% of NOD.scid mice given young (6-wk-old) agg Ig-GAD1-expanded or natural (from untreated animals) Tregs remain free of diabetes (Fig. 5, a and b). The CD4+CD25– counterparts had no significant effect on diabetes, and by wk 5 posttransfer, all animals became diabetic, as in the NOD.scid mice recipient of diabetogenic splenocytes without any Treg cotransfer. However, neither expanded nor natural Tregs taken at 8 wk of age (intermediate cells) could confer protection against the disease, and the incidence of diabetes was similar to that in animals that received the CD4+CD25– counterparts (Fig. 5, c and d). Similarly, Tregs taken from protected 26-wk-old mice did not confer significant delay of diabetes relative to their CD4+CD25– counterparts or the mice recipient of diabetogenic splenocytes without cotransfer (Fig. 5, e and f). Overall, these results indicate that Tregs abruptly lose their suppressive function at 8 wk of age and do not regain effectiveness by 26 wk of age.
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expression on Tregs is responsible for loss of suppressive functionTo investigate the mechanism underlying the loss of suppressive function, we began by ascertaining that the Tregs have not lost their phenotypic characteristics. Therefore, the intermediate (8-wk-old) Tregs were analyzed for CTLA-4 (33, 34), CD62L (35), and Foxp3 (27, 28) expression and compared with their young counterparts. It is shown that CTLA-4 expression on intermediate Tregs, whether expanded or natural, was similar to levels in young counterparts (Fig. 6, a–c). Similarly, CD62L expression was as significant on the intermediates as on the young Tregs (Fig. 6, d–f). Real-time PCR analysis revealed that Foxp3 mRNA expression was comparable in the intermediate Tregs vs their young counterparts (Fig. 6, g and h). Thus, the phenotypic characteristics of the Tregs were not altered over the transition from 6–8 wk of age.
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, it is likely that they conduct their suppressive function through physical contact (36). In recent years, mTGF- has been suspected to be a major player in cell contact-mediated suppression by Tregs (10, 11, 12, 13). Furthermore, dominant negative expression of TGF- receptor type II on target effector CD8+ Treg led to exacerbation of diabetes (14). The study then focused on analysis of surface expression of TGF- on Tregs and its involvement in the functional discrepancy among young and intermediate Tregs. Accordingly, both young (6-wk-old) and intermediate (8-wk-old) expanded Tregs were assessed for cell mTGF-. Fig. 7 shows that 39.0% of young expanded Tregs had mTGF-. In contrast, only 12.1% of the intermediate population displayed mTGF- (Fig. 7, a and b). Similar finding was observed with natural Tregs, as mTGF- expression was reduced from 30.0% on the young Tregs to just 11.8% on their older counterparts (Fig. 7, c and d). Interestingly, Tregs from male NOD mice, which usually have lower incidence and delayed disease (37), did not decrease mTGF- (Fig. 7, e and f). Indeed, 35.1% of the intermediate Tregs had significant mTGF-, and this does not seem to reflect diminished expression, as 36.2% of the young Tregs also displayed mTGF-. A decline in mTGF- probably diminishes the suppressive function of Tregs. This statement emanates from the observation that blockade of mTGF- abrogates the suppressive function of young Tregs both in vitro and in vivo (Fig. 7, g and h). In fact, young Tregs reduced the proliferation of their CD4+CD25– counterparts to allogeneic cells. However, the alloreactivity of these effector cells was restored when the young Tregs were coated with anti-TGF- Ab, but not isotype control mIgG (Fig. 7g). In vivo, when the young Tregs were coated with anti-TGF- Ab before cotransfer with diabetogenic splenocytes, the recipient NOD.scid mice developed diabetes (Fig. 7h). However, coating with mIgG instead of anti-TGF- sustained resistance against diabetes. Therefore, young NOD Tregs require the activity of mTGF- to effectively suppress the function of effector cells.
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on Tregs lessens immune regulation of effector cells, leading to the onset of destructive insulitis and progression to diabetes. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Treatment with agg Ig-GAD1 at the age of 4 wk reduced the spontaneous proliferative T cell responses that usually develop in NOD mice and delayed diabetes (Fig. 2). Depletion of Tregs during administration of agg Ig-GAD1 resulted in a lack of protection against diabetes. Moreover, titration of Tregs in the protected animals indicated maintenance of the elevated frequency for the 30-wk period of clinical monitoring. Therefore, it appears that agg Ig-GAD1-mediated delay of diabetes is dependent on Treg function. In fact, DC-expanded specific Tregs proved potent against diabetes (38). In addition, it seems likely that suppression of diabetogenic T cells by the Tregs is mediated by cell contact, rather than cytokines. This statement stems from the observations that Tregs were unable to secrete TGF- or IL-10, and treatment with Ig-GAD1 was effective against diabetes in both IL-10–/– and IL-4–/– mice (Fig. 3). Surprisingly, however, treatment with agg Ig-GAD1 was not effective against diabetes in 8-wk-old, IAA-positive mice despite expansion of Tregs (Fig. 4). Functional analysis of these mature cells indicated an inability to suppress passive diabetes, whereas counterparts expanded before IAA seroconversion protected against the disease. These findings, although puzzling, suggested that young Tregs, which protect against the disease and maintain a significant frequency thereafter, lose their suppressive functions over time and become unable to oppose the disease. In an attempt to explore this postulate, maturing Tregs were isolated at different ages and tested for protection against diabetes. The findings indicated that 6-wk-old Tregs, which we refer to as young Tregs, are endowed with suppressive functions and protect against passive diabetes (Fig. 5). These results agree with reports showing that young Tregs protect against aggressive diabetes mediated by islet diabetogenic T cells for even a longer period of time (35). In contrast, 8- and 26-wk-old Tregs, which we refer to as intermediate and aged Tregs, respectively, were unable to confer resistance against the disease (Fig. 5). The fact that natural Tregs do not decline in number over time in naive untreated mice (Fig. 2) and protect against diabetes when tested as young, rather than intermediate or aged Tregs (Fig. 5), again indicates a time-sensitive loss of function.
Analysis of the expression of Treg markers on these nonprotective cells showed an unaffected phenotype, because CTLA-4, CD62L, and Foxp3 expressions were similar on young protective and older nonprotective Tregs, whether natural or expanded by agg Ig-GAD1 (Fig. 6). In the face of this dilemma, we resorted to exploring any involvement of mTGF- in this loss of function by Tregs. Surprisingly, the young Tregs expressed significant levels of active mTGF-, but over a transition period of 2–3 wk, during which IAA seroconversion took place and an abrupt decline in mTGF- expression transpired, persisting up to wk 26 of age (Fig. 7). This decline was not observed with Tregs of male NOD mice and thus concurs with the protection observed with aged male Tregs (6). Interestingly, blockade of mTGF- by anti-TGF- Ab abolished the suppressive function of young Tregs, leading to a lack of protection against diabetes. These findings indicate that a decline in mTGF- during the transition to IAA seroconversion nullifies the suppressive function of Tregs. Thus, although the cells remain expandable by agg Ig-GAD1 and maintain a significant frequency, an abrupt loss of mTGF- during maturation drives a loss of function and a lack of protection against diabetes. These results suggest that Tregs are able to suppress pathogenic T cells up to wk 8 of age, then a loss of mTGF- occurs, which nullifies their suppressive function, leading to a lack of protection at later stages of the disease. It should be noted, however, that this Treg functional impairment would not affect protected animals, because their pathogenic T cells have already been down-regulated. Given that mTGF- has been implicated in Treg function (10, 11, 12, 13), the age-dependent decline in its expression bodes well with the report describing a loss of function by Tregs at 16 wk of age (9). Also, this would provide a mechanism for circumstances under which disease eruption occurs despite the presence of an unaltered frequency of Tregs (6, 7). The abrupt transition for loss of function at 8 wk of age may be critical for massive release of diabetogenic cells from suppression to ensure perpetuation of the 6- to 8-wk-long destructive insulitis and resultant onset of diabetes (39). Although, this observation sheds light on the loss of function by Tregs operating suppression through mTGF-, other mechanisms may be in place for cells operating through cell surface expression of GITR (40, 41), production of IL-10 (42, 43), or secreted TGF- (44, 45). In fact, we found that Ig-INS, a chimera expressing INS9–23 peptide expands Tregs that produce IL-10 and protects young animals against diabetes (43). However, at later stages of the disease when the diabetogenic T cells reach the islets and become activated, IL-10 down-regulates their CTLA-4, thus hindering the CTLA-4 inhibitory pathway, to sustain T cell activation and nullify the protective function of Tregs (43). Compensatory mechanisms seem to be available, however, because stimulation with anti-CD3 Ab at later stages of the disease mobilizes Tregs that secrete TGF- and protects against the disease (44). Finally, these findings shed light on the efficacy of Ag- and cytokine-based approaches against diabetes at early, but not later, stages of the disease.
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Acknowledgments |
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1 This work was supported by start-up funds from the University of Missouri School of Medicine. S.J.S. was supported by a fellowship from the University of Missouri Arts and Sciences Undergraduate Research Mentor Program and a scholarship from the University of Missouri Life Sciences Undergraduate Research Opportunity Program. J.J.B. was supported by Predoctoral Training Grant T32GM08396-13 from National Institute of General Medical Sciences. 
2 Current address: University of Virginia, Beirne B. Carter Center for Immunology Research, MR4 Building, Charlottesville, VA 22908-1386.
3 Address correspondence and reprint requests to Dr. Habib Zaghouani, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, M616 Medical Sciences Building, Columbia, MO 65212. E-mail address: zaghouanih{at}health.missouri.edu
4 Abbreviations used in this paper: Treg, T regulatory cell; agg, aggregated; CT, threshold cycle; Foxp3, Forkhead/winged helix transcription factor gene; GAD65, glutamic acid decarboxylase-65; HEL, hen egg lysozyme; IAA, insulin autoantibody; INS, insulin -chain; mTGF-, membrane-bound TGF-; nil, untreated; NOD, nonobese diabetic; sol, soluble; T1D, type I diabetes.
Received for publication August 12, 2004. Accepted for publication October 8, 2004.
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) were used as controls. h, Proliferation of the CD4+CD25– fraction (2 x 105 cells/well) was assessed by [3H]thymidine incorporation after 5-day incubation in the presence of allogeneic C57BL/6 splenocytes (1 x 105 cells/well) alone or together with increasing numbers of CD4+CD25+ counterparts. Each bar represents the mean ± SD of triplicate wells.
) or Ig-HEL (
) were included for control purposes. a, p < 0.05; b, p<0.05; c, p<0.01 (compared with nil groups).
) T cells from either untreated (Natural) or agg Ig-GAD1-treated (Expanded) mice were isolated at wk 6 (a and b; young), wk 8 (c and d; intermediate), or wk 26 (e and f; aged) of age. The cells were then coinjected i.v. with splenic cells derived from recently diabetic NOD females into recipient NOD.scid mice, and blood glucose levels were monitored every 7 days for a period of 56 days post-transfer. A group injected with diabetic splenocytes only (No transfer) was included for control purpose. Shown is the percentage of mice free of diabetes. These results are representative of two independent experiments.
