Insulin Treatment in Patients With Type 1 Diabetes Induces

Original Article Insulin Treatment in Patients With Type 1 Diabetes Induces Upregulation of Regulatory T-Cell Markers in Peripheral Blood Mononuclear Cells Stimulated With Insulin In Vitro Minna Tiittanen,1 Johanna T. Huupponen,1 Mikael Knip,2,3 and Outi Vaarala1,4

Patients with type 1 diabetes are treated with daily injections of human insulin, an autoantigen expressed in thymus. Natural CD4ⴙCD25high regulatory T-cells are derived from thymus, and accordingly human insulin–specific regulatory T-cells should exist. We had a chance to study peripheral blood mononuclear cells (PBMCs) from children with type 1 diabetes both before and after starting insulin treatment, and thus we could analyze the effects of insulin treatment on regulatory T-cells in children with type 1 diabetes. PBMCs were stimulated for 72 h with bovine/ human insulin. The mRNA expression of regulatory T-cell markers (transforming growth factor-␤, Foxp3, cytotoxic T-lymphocyte antigen-4 [CTLA-4], and inducible co-stimulator [ICOS]) or cytokines (␥-interferon [IFN-␥], interleukin [IL]-5, IL-4) was measured by quantitative RT-PCR. The secretion of IFN-␥, IL-2, IL-4, IL-5, and IL-10 was also studied. The expression of Foxp3, CTLA-4, and ICOS mRNAs in PBMCs stimulated with bovine or human insulin was higher in patients on insulin treatment than in patients studied before starting insulin treatment. The insulininduced Foxp3 protein expression in CD4ⴙCD25high cells was detectable in flow cytometry. No differences were seen in cytokine activation between the patient groups. Insulin stimulation in vitro induced increased expression of regulatory T-cell markers, Foxp3, CTLA-4, and ICOS only in patients treated with insulin, suggesting that treatment with human insulin activates insulin-specific regulatory T-cells in children with newly diagnosed type 1 diabetes. This effect of the exogenous autoantigen could explain the difficulties to detect in vitro T-cell proliferation responses to insulin in newly diagnosed patients. Furthermore, autoantigen treatment–induced activation of regulatory Tcells may contribute to the clinical remission of the disease. Diabetes 55:3446 –3454, 2006

From the 1Department of Viral Diseases and Immunology, Laboratory for Immunology, National Public Health Institute, Helsinki, Finland; the 2Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland; the 3 Department of Pediatrics, Tampere University Hospital, Tampere, Finland; and the 4Department of Molecular and Clinical Medicine, Linko¨ping University, Linko¨ping, Sweden. Address correspondence and reprint requests to Minna Tiittanen, National Public Health Institute, Department of Viral Diseases and Immunology, Laboratory for Immunology, Mannerheimintie 166, 00300 Helsinki, Finland. E-mail: [email protected]. Received for publication 31 January 2006 and accepted in revised form 6 September 2006. CTLA-4, cytotoxic T-lymphocyte antigen-4; ICOS, inducible co-stimulator; IAA, insulin autoantibody; IFN-␥, ␥-interferon; IL, interleukin; PBMC, peripheral blood mononuclear cell; TGF, transforming growth factor; Th, T-helper. DOI: 10.2337/db06-0132 © 2006 by the American Diabetes Association. 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.

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ype 1 diabetes is a disease that results from the immune-mediated destruction of insulin-producing ␤-cells in the pancreatic islets (1). Autoimmunity in type 1 diabetes is directed against several ␤-cell autoantigens, including insulin, which is used for the treatment of the disease. A humoral immune response to endogenous insulin is common during the preclinical phase of the disease (2– 4), and insulin autoantibodies (IAAs) are used for the prediction of type 1 diabetes (5,6). After starting exogenous insulin therapy, most of the children with newly diagnosed type 1 diabetes enter into a remission phase, during which they require less exogenous insulin. Insulin treatment is also known to modulate autoimmunity reflected by increasing levels of circulating insulin antibodies (7). Immunological factors have been suggested to be responsible, at least partly, for the decrease in the insulin demands following the start of insulin therapy. Treatment of type 1 diabetes with an autoantigen itself offers an interesting model to study the changes in autoantigen-specific immune responses during continuous exposure to the exogenously administrated autoantigen, namely insulin. The autoimmune attack on the ␤-cells is considered to be T-helper (Th)1 mediated (8 –10). CD4⫹ T-cells are divided into subsets, such as Th1 and Th2 effector T-cells and regulatory T-cells according to their functional profile. Regulatory T-cells can be separated into naturally occurring regulatory T-cells, which originate from the thymus, and adaptive regulatory T-cells, which are induced in the periphery (11,12). Natural regulatory T-cells characteristically express the CD4, CD25, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and Foxp3 transcription regulator, which is considered to be a specific marker for CD4⫹CD25high regulatory T-cells (13–15). In humans, it has been shown that Foxp3-expressing CD4⫹CD25high regulatory T-cells can be induced from peripheral CD4⫹CD25⫺ T-cells upon stimulation (16,17). Our aim was to study the possible effect of exogenous insulin treatment on the insulin-specific immune response with a special emphasis on regulatory T-cell–associated markers. For this study, we collected blood samples from children with type 1 diabetes at diagnosis before starting exogenous insulin treatment, from recently diagnosed children on insulin therapy and from unaffected children. We stimulated peripheral blood mononuclear cells (PBDIABETES, VOL. 55, DECEMBER 2006

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TABLE 1 Distribution of sex, age, blood glucose concentration at the time of sampling, and A1C values 5– 6 months after the diagnosis in children with newly diagnosed type 1 diabetes Patient group Sex (male/female) Age (years) Blood glucose (mmol/l) A1C (%)

0

1

7/5 8.5 (1.9⫺12.5) 19.8 (15.2⫺32.6) 7.4 (6.5–8.6)

11/10 7.8 (1.8⫺14.4) 7.6 (3.1–14.6) 7.4 (6.0–9.7)

Data are median (range). Group 0 refers to patients before starting insulin treatment and group 1 to patients on insulin treatment.

MCs) with insulin in vitro and measured the expression of regulatory T-cell–related factors (transforming growth factor (TGF)-␤, Foxp3, CTLA-4, and inducible co-stimulator [ICOS]) and cytokines (␥-interferon [IFN]-␥, interleukin [IL]-5, and IL-4) with quantitative RT-PCR and studied the secretion of IFN-␥, IL-5, IL-4, IL-2, and IL-10 by cytometric bead array. RESEARCH DESIGN AND METHODS Our study included 33 patients with newly diagnosed type 1 diabetes. In 12 of 33, the blood sample was obtained at diagnosis before starting exogenous insulin therapy (group 0), whereas 21 patients had been treated with insulin for 1–21 days (median 8 days, group 1). Table 1 shows sex distribution, age, blood glucose concentration at the time of sampling, and HbA1c (A1C) values 5– 6 months after the diagnosis in the patients in both groups. During the same period, we also studied 10 (7 males) nondiabetic children without acute infections or autoimmune diseases as control subjects. The median age in the control group was 6.7 years (range 1.2–15.8). In addition, 12 recently diagnosed type 1 diabetic patients (4 male) and 4 healthy children (3 male) were included in the analyses of CD4⫹CD25⫹ cells and Foxp3-expressing cells by means of flow cytometry. In this series, the median age of the patients was 4.6 years (range 1.1–12.8) in group 0 (n ⫽ 7) and 6.6 years (3.6 –13.2) in group 1 (n ⫽ 5). The median age of the control children included in the flow cytometric analyses was 7.5 years (3.5–10.1). The study protocol was approved by the ethical committees of the participating hospitals (Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland, and Jorvi Hospital, Espoo, Finland). Cell culture for RT-PCR and cytometric bead array. PBMCs were isolated from heparinized blood by Ficoll-Paque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) and were suspended in RPMI1640 containing 5% human AB⫹ serum (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) and 25 ␮g/ml gentamicin (Life Technologies, Paisley, Scotland). PBMCs were cultured in U-bottomed 96-well cell culture plates, 2 ⫻ 105 cells (200 ␮l) per well in triplicate wells with or without antigens. Twenty micrograms per milliliter tetanus toxoid (National Public Health Institute, Helsinki, Finland), 300 ␮g/ml bovine insulin (purified from bovine pancreas, cell culture tested; Sigma, St. Louis, MO), and 300 ␮g/ml human insulin (yeast recombinant protein; Boehringer Mannheim, Mannheim, Germany) were used as antigens. After incubation for 72 h, the supernatants and the cells were collected and frozen. The cell pellets were frozen in the lysis buffer of the RNA kit (see methods in QUANTITATIVE RT-PCR). Quantitative RT-PCR. RNA was extracted from frozen cell pellets with the GenElute Mammalian Total RNA Miniprep kit (Sigma). Reverse transcription was performed in a total volume of 20 ␮l by using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Before adding multiscribe reverse transcriptase enzyme, the reaction mixture was treated with 0.01 units/␮l DNase (Boehringer Mannheim). Real-time quantitative PCR was performed using TaqMan PDAR (predeveloped assay reagents) or assayson-demand primers/probes and the ABI Prism 7700 Sequence Detection System (Applied Biosystems) in triplicate wells. PDAR primers/probes for IFN-␥ (cat. no. 4327052), IL-5 (4327039), IL-4 (4327038), and TGF-␤ (4327054) and assays-on-demand for Foxp3 (Hs00203958_m1), CTLA-4 (Hs00175480_m), and ICOS (Hs00359999_m1) were used. Ribosomal 18S RNA was used as an endogenous control (PDAR 4310893E and assays-on-demand Hs99999901_s1). On every plate, the expression of the same marker mRNA was measured in a calibrator sample that was prepared by stimulating PBMCs from a healthy volunteer with phytohemagglutinin (Sigma) for 48 h. The reaction mixture contained TaqMan Universal PCR DIABETES, VOL. 55, DECEMBER 2006

Master Mix, 1 ⫻ primers/probes (both from Applied Biosystems), and 1.8 ␮l of template cDNA in 25 ␮l total volume. Thermal cycling conditions were 2 min at 50°C, 10 min at 95°C, and 50 cycles of 15 s at 95°C and 1 min at 60°C. The quantities of the cytokines were analyzed by a comparative threshold cycle (Ct) method (as recommended by Applied Biosystems). ⌬Ct stands for the difference between Ct of the marker gene and Ct of the 18S gene, whereas ⌬⌬Ct is the difference between the ⌬Ct of the analyzed sample and ⌬Ct of the calibrator. Calculation of 2⫺⌬⌬Ct then gives a relative amount of the analyzed sample compared with the calibrator, both normalized to an endogenous control (18S). The relative amount (2⫺⌬⌬Ct) of IFN-␥ and CTLA-4 was multiplied by 1,000, the relative amount of Foxp3 and ICOS by 100, and the relative amounts of IL-5, IL-4, and TGF-␤ by 10 to get whole numbers for plots. The relative amount of each marker detected in the wells cultured without antigen was subtracted from the relative amount of respective marker detected in the antigen-stimulated wells. Cytometric bead array for IFN-␥, IL-5, IL-4, IL-2, and IL-10. Secreted IFN-␥, IL-5, IL-4, IL-2, and IL-10 were measured with a flow cytometry– based cytometric bead array kit (cat. no. 550749; BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. The amount of each secreted cytokine in the wells cultured without antigen was subtracted from the amount of respective cytokine expressed in the antigen-stimulated wells. Analysis of Foxp3 by flow cytometry. PBMCs were cultured with or without the antigens for 5– 6 days in 24-well cell culture plate, 2 ⫻ 106 cells (2 ml) per well, in order to detect antigen-induced Foxp3 expression in flow cytometric analysis. The staining protocol was adapted from the article by Roncador et al. (18). Briefly, the cells were fixed with 1% paraformaldehyde and 0.05% Tween-20 PBS overnight at 4°C. Cells were treated twice for 30 min with 100 units/ml DNase (Roche Diagnostics, Mannheim, Germany). Before staining, the cells were blocked with 1% goat IgG (Caltag, Burlingame, CA) in staining buffer that contained 3% FCS, 0.5% Tween 20, and 0.05% Na-azide in PBS. Mouse anti-human Foxp3 antibody (clone 150D/E4 cell culture supernatant was a kind gift from Dr. Alison H. Banham, University of Oxford, Oxford, U.K.) was diluted (1:50) in blocking buffer and incubated for 1 h at room temperature. Cell culture supernatant with irrelevant mouse monoclonal antibody was used as a control antibody. Alexa fluor 488 –labeled goat anti-mouse IgG (Molecular Probes, Leiden, the Netherlands) was used as a secondary antibody in 1:500 dilution. Before cell surface staining, the cells were blocked with 1% mouse IgG (Caltag) for 20 min. CD4-PerCP (Becton Dickinson, San Jose, CA) and CD25-PE (Miltenyi Biotech, Bergisch Gladbach, Germany) were used for surface staining at room temperature for 20 min. Cells were analyzed by FACSCalibur and CellQuest Pro software (Becton Dickinson). T-cell proliferation test. PBMCs isolated by density gradient centrifugation were cultured in U-bottomed 96-well cell culture plate, 1 ⫻ 105 cells (200 ␮l) per well, in quadruplicates with the same antigen concentrations as described in CELL CULTURE FOR RT-PCR AND CYTOMETRIC BEAD ARRAY. After incubation for 5 days, 1 ␮Ci of tritiated thymidine (Amersham, Buckinghamshire, U.K.) was added to each well and the cells were harvested 16 –18 h later to measure the incorporation of radioactivity. The proliferative response has been presented as a stimulation index calculated by dividing the median counts per minute measured from the antigen-stimulated wells by median counts per minute detected in the wells cultured without antigen. Insulin autoantibody assay. IAAs were analyzed with a specific radioligand assay as previously described (19). The assay had a disease sensitivity of 58% and a disease specificity of 98% in the 2005 Diabetes Autoantibody Standardization Program workshop. Statistical analysis. The nonparametric Mann-Whitney U test was used for comparisons between the groups. The Spearman rank correlation test was applied to analyze correlations between different parameters (SPSS 10.0 for Windows; SPSS, Chicago, IL). P values ⬍0.05 were considered significant.

RESULTS

No differences were seen between the study groups in any immunological parameters of the unstimulated PBMCs (data not shown). Insulin-induced Foxp3, CTLA-4, and ICOS mRNA expression. The insulin-induced expression of Foxp3-, CTLA-4 –, and ICOS-specific mRNAs in PBMCs was higher in newly diagnosed children who had received insulin treatment than in children with type 1 diabetes studied before starting insulin therapy. Both bovine and human insulin induced higher expression of Foxp3 (P ⫽ 0.040 and P ⫽ 0.055, respectively, and median [means ⫾ SD] relative amounts were 10.6 [14 ⫾ 15] and 2.8 [6.0 ⫾ 7.8] for bovine 3447

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FIG. 1. Foxp3-specific mRNA detected by RT-PCR in PBMCs stimulated with bovine (A) or human (B) insulin. Samples from patients in group 0 were taken at diagnosis of type 1 diabetes before starting exogenous insulin therapy. Patients in group 1 were treated with insulin for 1–21 days (median 8 days). Results are shown as relative amount (100 ⴛ 2ⴚ⌬⌬Ct) after a 72-h stimulation of PBMCs in the analyzed sample compared with the calibrator, both normalized to an endogenous control. Horizontal solid lines represent median values and dashed lines mean values of the groups.

insulin and 4.6 [8.1 ⫾ 11] and 0.1 [2.0 ⫾ 3.6] for human insulin), CTLA-4 (P ⫽ 0.035; 8.1 [10 ⫾ 10] and 0.4 [3.6 ⫾ 5.2] for bovine insulin and P ⫽ 0.009; 2.7 [6.1 ⫾ 10] and 0 [0.6 ⫾ 1.1] for human insulin), and ICOS (P ⫽ 0.025; 9.1 [16 ⫾ 25] and 6.9 [6.3 ⫾ 3.8] for bovine insulin and P ⫽ 0.018; 6.6 [6.7 ⫾ 2.4] and 3.6 [4.0 ⫾ 2.0] for human insulin) in diabetic children receiving insulin therapy compared with the diabetic children before starting insulin therapy (Figs. 1, 2, and 3). When compared with healthy children, the children who had received exogenous insulin therapy showed higher expression of Foxp3 mRNA (P ⫽ 0.002; median [mean ⫾ SD] relative amount 4.0 [3.2 ⫾ 2.2] for control subjects), CTLA-4 mRNA (P ⫽ 0.002; median relative amount 0.2 [1.2 ⫾ 1.6] for control subjects), and ICOS mRNA (P ⫽ 0.005; median relative amount was 5.0 [4.9 ⫾ 2.1] for control subjects) than the unaffected children after bovine insulin stimulation of PBMCs. The bovine insulin–induced expression of Foxp3 mRNA 3448

FIG. 2. CTLA-4 –specific mRNA expression in response to bovine (A) or human (B) insulin after stimulation for 72 h in patients with newly diagnosed type 1 diabetes. In group 0, patients were tested before insulin therapy and in group 1 1–21 days (median 8 days) after starting insulin treatment. Results are shown as relative amounts (1,000 ⴛ 2ⴚ⌬⌬Ct). Horizontal solid lines show medians and dashed lines means for each group.

correlated with the expression of bovine insulin–induced CTLA-4 and ICOS mRNAs (r ⫽ 0.78, P ⬍ 0.001 for CTLA-4; r ⫽ 0.69, P ⬍ 0.001 for ICOS). The human insulin–induced expression of Foxp3 mRNA correlated with the human insulin–induced expression of CTLA-4 and ICOS mRNAs (r ⫽ 0.71, P ⬍ 0.001 for CTLA-4; r ⫽ 0.55, P ⫽ 0.003 for ICOS). The bovine insulin– and human insulin–induced expression levels of Foxp3, CTLA-4, and ICOS mRNAs in PBMCs correlated as well (r ⫽ 0.44, P ⫽ 0.011 for Foxp3; r ⫽ 0.42, P ⫽ 0.016 for CTLA-4; and r ⫽ 0.45, P ⫽ 0.017 for ICOS). The human insulin–induced Foxp3 mRNA expression in insulin-treated patients correlated inversely with A1C values measured 5– 6 months after diagnosis (r ⫽ ⫺0.66, P ⫽ 0.038). There was a direct correlation between bovine insulin–induced Foxp3 mRNA expression studied after treatment and A1C values of the patients determined 5– 6 months after diagnosis (r ⫽ 0.74, P ⫽ 0.010). No correlation was observed between Foxp3 mRNA and the blood glucose concentrations at the time of sampling or the age of all subjects. The duration of insulin treatment in paDIABETES, VOL. 55, DECEMBER 2006

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FIG. 3. ICOS-specific mRNA measured by means of RT-PCR after stimulation of PBMC for 72 h with bovine insulin (A) or with human insulin (B). Group 0 represents patients with type 1 diabetes at diagnosis before starting exogenous insulin therapy and group 1 recent-onset patients after treatment with insulin for 1–21 days (median 8 days). The expression level of mRNA is shown as relative units (100 ⴛ 2ⴚ⌬⌬Ct). Median expressions are marked with horizontal solid lines and mean expressions with dashed lines.

tients in group 1 did not correlate with the mRNA expression of any regulatory T-cell markers (data not shown). Insulin-induced Foxp3 in CD4ⴙCD25high cells. Insulin stimulation of PBMCs in vitro induced an increase in the number of CD4⫹CD25high cells detected with flow cytometric analysis. The proportion of bovine insulin–induced CD4⫹CD25high cells among CD4⫹ cells varied between 0.1 and 4.7% and that of human insulin–induced cells between 0 and 0.4% in children with newly diagnosed type 1 diabetes before starting insulin treatment. Respectively, the proportion of bovine insulin–induced CD4⫹CD25high cells among CD4⫹ cells varied between 1.2 and 9.0% and that of human insulin–induced cells between 0.2 and 1.7% in insulin-treated children. The proportion of bovine insulin–induced CD4⫹CD25high cells among CD4⫹ cells varied between 0.8 and 2.6% and that of human insulin–induced cells between 0 and 1.7% in the control children. The cells cultured without antigen showed very few CD4⫹CD25high cells among CD4⫹ cells in all children (median 0.1%, mean 0.3%). The expression of Foxp3 in CD4⫹CD25high cells was DIABETES, VOL. 55, DECEMBER 2006

FIG. 4. Flow cytometric analysis of a representative patient at diagnosis of type 1 diabetes. A: The CD4ⴙCD25high cells are gated for B and C. The bovine insulin–induced expression of Foxp3 is presented in B and the human insulin–induced expression of Foxp3 in C. The gray histograms represent the staining with anti-Foxp3 antibody and the uncolored histograms are stained with a control antibody.

studied by flow cytometry in three patients with newly diagnosed type 1 diabetes. Foxp3 expressing cells among CD4⫹CD25high cells varied between 42.7 and 57.9% after in vitro bovine insulin stimulation (Fig. 4A and B). Also, human insulin induced CD4⫹CD25high cells and approxi3449

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FIG. 5. IL-4 –specific mRNA expression detected by RT-PCR in PBMCs stimulated with bovine (A) or human (B) insulin for 72 h. Samples from patients in group 0 were taken at diagnosis of type 1 diabetes before starting insulin therapy, and in group 1 the patients were treated with insulin for 1–21 days (median 8 days). Results are shown as relative amounts (10 ⴛ 2ⴚ⌬⌬Ct). Horizontal solid lines represent median values and dashed lines mean values of the groups.

mately half of these cells (52.0 – 62.5%) expressed Foxp3 as well (Fig. 4C), although the absolute number of CD4⫹CD25high cells induced by human insulin was lower than that induced by bovine insulin. Insulin-induced cytokine expression. No differences in cytokine activation were observed between the patients studied before or after starting insulin treatment. Both groups of children with newly diagnosed type 1 diabetes, tested either before or after starting exogenous insulin therapy, had higher expression of IL-4 mRNA than unaffected children in response to stimulation with bovine insulin (P ⫽ 0.007 for group 0 and P ⫽ 0.002 for group 1; median [mean ⫾ SD] relative amounts were 5.9 [5.0 ⫾ 3.1] for group 0, 6.1 [21 ⫾ 38] for group 1, and 0 [0.9 ⫾ 1.8] for control subjects) and with human insulin (P ⫽ 0.033 for group 0 and P ⫽ 0.057 for group 1; medians were 1.3 [2.2 ⫾ 1.7], 2.3 [4.5 ⫾ 5.6], and 0 [0.6 ⫾ 0.9]) (Fig. 5). Also, the secretion of IL-4 by insulin-stimulated PBMCs was higher in insulin-treated patients than in healthy children. The recent-onset patients who had received exogenous insulin had higher levels of IL-4 than the unaffected children in response to bovine insulin (P ⫽ 0.014; median [mean ⫾ SD] levels were 0.8 [1.0 ⫾ 1.0] and 0 [0.1 ⫾ 0.2] pg/ml). The levels of IFN-␥–, IL-5–, or TGF-␤–specific mRNAs or secreted IFN-␥, IL-5, IL-2, or IL-10 did not differ between the groups after insulin stimulations. Tetanus toxoid–induced response. The groups did not differ in the quantities of tetanus toxoid–induced cytokine or regulatory T-cell marker–specific mRNAs (Table 2). The children with newly diagnosed type 1 diabetes tested before insulin therapy had lower levels of secreted IFN-␥ in response to tetanus toxoid than the nondiabetic children (P ⫽ 0.033; medians [means ⫾ SD] were 53.4 [135 ⫾ 160] and 642 [1,012 ⫾ 1,069] pg/ml). No differences could be observed between the groups in response to tetanus toxoid in IL-4, IL-5, IL-2, and IL-10 secretion. Proliferative response and antigen-induced cytokine or regulatory T-cell marker expression. The two groups of patients with type 1 diabetes and the unaffected children did not differ in their T-cell proliferation response after stimulation with bovine or human insulin or with tetanus toxoid. The proliferation response to bovine insulin correlated with the quantities of bovine insulin–induced IFN-␥ or IL-5

TABLE 2 The expression of Foxp3-, CTLA-4 –, and ICOS-specific mRNAs in response to tetanus toxoid, bovine, or human insulin after 72 h cell culture 0

Patient group 1

Control

⫺⌬⌬Ct

Foxp3 mRNA (100 ⫻ 2 ) Tetanus toxoid Bovine insulin Human insulin CTLA-4 mRNA (1,000 ⫻ 2⫺⌬⌬Ct) Tetanus toxoid Bovine insulin Human insulin ICOS mRNA (100 ⫻ 2⫺⌬⌬Ct) Tetanus toxoid Bovine insulin Human insulin

17.4 (0.3–38.8) 2.8 (0–23.9) 0.1 (0–9.7)

16.8 (0–298.5) 10.6 (0.3–57.5) 4.6 (0–40.0)

10.1 (4.6–270.8) 4.0 (0–5.7) 0.9 (0–9.0)

5.0 (0⫺41.6) 0.4 (0⫺15.7) 0 (0⫺2.3)

15.5 (0.9⫺57.7) 8.1 (0⫺36.0) 2.7 (0⫺41.8)

14.7 (0⫺350.3) 0.2 (0⫺3.6) 0.2 (0⫺5.6)

3.7 (0⫺10.9) 1.4 (0⫺5.4) 0

5.4 (0⫺55.6) 5.9 (0⫺98.3) 0.8 (0⫺6.2)

1.3 (0⫺111.9) 0 (0⫺4.8) 0.4 (0⫺2.1)

Data are median (range). The type 1 diabetic patients in group 0 were studied at diagnosis before starting insulin treatment and those in group 1 after treatment with insulin for 1⫺21 days (median 8 days). 3450

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n ⫽ 24 for proliferation and r ⫽ ⫺0.45, P ⫽ 0.008, n ⫽ 34 for age). DISCUSSION

FIG. 6. Correlation between the stimulation index in T-cell proliferation and the expression of Foxp3 mRNA in response to bovine (A) and human insulin (B) in all subjects (patients with type 1 diabetes and unaffected control subjects) (A: r ⴝ 0.51; P ⴝ 0.003 and B: r ⴝ 0.22; P ⴝ 0.273 in Spearman’s rank correlation).

at mRNA (r ⫽ 0.54, P ⫽ 0.002 for IFN-␥ and r ⫽ 0.62, P ⬍ 0.001 for IL-5) and at protein level (r ⫽ 0.61, P ⫽ 0.001 for IFN-␥ and r ⫽ 0.65, P ⬍ 0.001 for IL-5) in the total series. The bovine insulin–induced stimulation indexes correlated with the bovine insulin–induced expression of Foxp3 and CTLA-4 mRNAs (r ⫽ 0.51, P ⫽ 0.003; r ⫽ 0.43, P ⫽ 0.016) (Fig. 6A shows the correlation between stimulation index and Foxp3 mRNA expression.) In contrast, the human insulin–induced expression of IFN-␥, IL-5, Foxp3, or CTLA-4 did not correlate with the proliferation response induced by human insulin (Fig. 6B shows the correlation between stimulation index and Foxp3 mRNA expression), but there was an inverse correlation between the quantity of human insulin–induced TGF-␤ mRNA and the proliferation response induced by human insulin (r ⫽ ⫺0.41, P ⫽ 0.029). The proliferative response to bovine and human insulin correlated with each other (r ⫽ 0.52, P ⫽ 0.002). IAAs and insulin-induced cellular immune response. The levels of IAAs correlated with the quantities of IL-4 and TGF-␤ mRNAs in response to human insulin in patients with newly diagnosed type 1 diabetes (r ⫽ 0.46, P ⫽ 0.027, n ⫽ 23 for IL-4 and r ⫽ 0.60, P ⫽ 0.003, n ⫽ 23 for TGF-␤). The proliferative response induced by human insulin, as well as the age of the patients, inversely correlated with the levels of IAAs (r ⫽ ⫺0.42, P ⫽ 0.044, DIABETES, VOL. 55, DECEMBER 2006

The exogenous insulin treatment in children with newly diagnosed type 1 diabetes resulted in increased expression of regulatory T-cell–related markers Foxp3, CTLA-4, and ICOS after in vitro stimulation of PBMCs with insulin. No effect of insulin treatment was seen on the characteristics of immune responses to an unrelated antigen, tetanus toxoid. We had a unique possibility to study children with newly diagnosed type 1 diabetes before the start of insulin treatment. The expression of regulatory T-cell markers Foxp3, CTLA-4, and ICOS mRNAs in response to in vitro insulin stimulation was lower in patients with type 1 diabetes who had not received insulin therapy. This suggests that exogenous insulin expands or activates the population of insulin-specific regulatory T-cells in the periphery. The phenomenon of autoantigen-induced activation of regulatory T-cells in the periphery has been reported in experimental animal studies (20,21), but to our knowledge this is the first report indicating that it may occur also in humans. Naturally arising Foxp3-expressing CD4⫹CD25high regulatory T-cells are produced in the thymus (11,12). These endogenous regulatory T-cells specifically express Foxp3, which encodes a transcription factor responsible for the development and function of regulatory T-cells (13–15). Transduction of Foxp3 suppresses IL-2 production but upregulates the expression of regulatory T-cell–associated molecules, such as CD25 and CTLA-4. The X-linked immunodeficiency syndrome IPEX is caused by mutations in Foxp3 and is associated with autoimmune disease in multiple endocrine organs (type 1 diabetes and thyroiditis), inflammatory bowel disease, and allergies (22). Thus, natural regulatory T-cells play a key role in the control of immune responses to self and nonself in humans. The T-cell receptor repertoire of natural regulatory T-cells is skewed toward recognizing complexes of self-peptide and MHC expressed in thymus and periphery (23). Insulin is expressed as a self-peptide in thymus (24), and thus insulin-specific regulatory T-cells should be present in humans. It has been shown that regulatory T-cells undergo antigen-specific proliferation in vivo after antigenic stimulation (25). Based on these observations, we hypothesized that treatment of diabetic children with a disease-specific autoantigen, human insulin, induces the expansion and/or activation of autoantigen-specific regulatory T-cells in vivo. To detect this, we measured the mRNA levels of regulatory T-cell–associated molecules after in vitro stimulation of PBMCs with insulin. The blood volume received from the children, especially at diagnosis of type 1 diabetes before the start of insulin therapy, limited the number of PBMCs available for our studies. For this reason, analyses at single-cell level were not possible. The upregulation of the CD4⫹CD25high cell population expressing Foxp3 at protein level was confirmed by flow cytometry in a subgroup of the children. As a control antigen, we used tetanus toxoid, an antigen unrelated to type 1 diabetes. The increase in the expression of regulatory T-cell–related markers was seen after insulin treatment only when PBMCs were stimulated with human or bovine insulin and not after tetanus toxoid stimulation, which supports the interpretation that in vivo insulin stimulation specifically results in an expansion of 3451

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insulin-reactive Foxp3-expressing regulatory T-cells. An increased expression of CTLA-4 and ICOS in in vitro insulin-stimulated PBMCs was also seen in the patients on insulin therapy. CTLA-4 is constitutively expressed by regulatory T-cells but is also expressed after activation in other T-cells. CTLA-4 blockade prevents regulatory T-cell activation (26), and CTLA-4 may be an important molecule to signal activation in regulatory T-cells together with the T-cell receptor. CTLA-4 may also directly mediate suppression by interacting with CD80/86 on responder T-cells. Similarly to CTLA-4, ICOS is a member of the family of CD28 proteins (27). It is expressed in both CD4 and CD8 T-cells after their activation. ICOS regulates T-cell differentiation and cytokine production and provides critical signals for immunoglobulin production and class switching. In a mouse model of autoimmune diabetes, it has been shown that regulatory T-cells are present in the insulitis during the pre-diabetic stage and that their regulatory activity is dependent on the presence of ICOS (28). The expression of these different regulatory T-cell markers, Foxp3, CTLA-4, and ICOS, strongly correlated in our assay. Since human insulin is expressed in thymus as a selfantigen, naturally arising regulatory T-cells in humans are primarily specific to human insulin. We also studied the response to bovine insulin for several reasons. Bovine insulin is encountered in the diet, and the regulatory mechanisms specific to bovine insulin may thus be induced in the intestinal immune system (i.e., peripherally [29]). Bovine insulin differs from human insulin by three amino acids, which is sufficient to make it more immunogenic in humans as observed when patients with type 1 diabetes were treated with bovine insulin (30). The increase in Foxp3 and IL-4 expression was more pronounced after bovine insulin stimulation than with human insulin. Due to the limited difference of only three amino acids between these two insulin molecules, the immune response cross-reacts (31,32). In our study, a strong correlation was seen between human insulin– and bovine insulin–induced regulatory T-cell markers. However, we found differences in the immune response to human and bovine insulin. Insulin from different species may induce functionally different immune responses at the cellular level, as suggested by studies in animal models (33,34). In vitro stimulation of PBMCs with insulin activates both effector T-cells and regulatory T-cells present in the culture wells. The Foxp3 activation induced by bovine insulin did not suppress, but rather correlated with, the proliferation of the effector cells in vitro and showed a direct correlation with A1C after diagnosis. Bovine insulin seemed to induce regulatory T-cells de novo as a consequence of immune activation. These kind of regulatory T-cells may not be as suppressive as the thymus-derived regulatory T-cells (11). Instead, in vitro human insulin–induced Foxp3 response showed an inverse correlation with subsequent A1C levels, suggesting a link to the recovery of ␤-cell function after the diagnosis. However, we could not find any correlation between the duration of insulin treatment and the expression of Foxp3 mRNA or other regulatory T-cell markers after in vitro insulin stimulation (r ⫽ 0.07, P ⫽ 0.763 for Foxp3 mRNA after bovine insulin stimulation), which may indicate that the activation of regulatory T-cells occurs already soon after the start of insulin therapy. On the other hand, the number of individuals studied here was relatively small, and thus the results should be interpreted cautiously. In nonobese diabetic mice, it has been shown 3452

that an expansion of CD4⫹CD25high regulatory T-cells in the pancreatic islets (35) and in vitro (21) inhibits the presentation of overt diabetes. As exogenous insulin treatment does induce a regulatory T-cell response to insulin, it might contribute to the remission period. A recently published work (36) reported that in vivo challenge with food allergens induced CD4⫹CD25high regulatory T-cells in children who had become tolerant to the allergen. The insulin-induced activation of a regulatory T-cell population together with insulin-specific IL-4 increment may explain, at least partly, the limited capacity of T-cell assays to detect insulin-specific responses in patients with type 1 diabetes (37– 40). We could not see any difference in T-cell proliferation in response to insulin between the patients and the unaffected children, and similar findings have been reported by other study groups (41– 43). An inverse correlation has been reported between IAAs and insulin-induced T-cell proliferation (4,42) and, indeed, was also seen in our study. Notably, the IAA levels of the patients correlated with insulin-induced IL-4 and TGF-␤ mRNAs, cytokines that inhibit cell proliferation. Our finding of IL-4 upregulation in response to insulin in children with type 1 diabetes, although seemingly in conflict with the concept of type 1 diabetes as a Th1mediated disease, is in agreement with the observation of IL-13–secreting insulin-specific T-cell lines derived from the pancreatic lymph nodes of patients with type 1 diabetes (44). Although the highest increase in insulin-induced IL-4 expression was seen in recent-onset patients receiving insulin therapy, the increased insulin-specific IL-4 expression at the level of mRNA was also observed in children with type 1 diabetes before starting insulin treatment. This suggests that an insulin-specific immune response of Th2 type is also associated with the autoimmune response to endogenous insulin in patients with type 1 diabetes and that exogenous insulin further enhances such reactivity. This observation is in accordance with previous publications reporting IL-4 and IL-10 secretion in response to preproinsulin epitopes in individuals with type 1 diabetes– associated autoantibodies (45) and increased mitogeninduced secretion of IL-4 by insulin from PBMCs (46). Also, the occurrence of IgG4-subclass IAAs in nondiabetic autoantibody-positive children (47) is in agreement with our finding of insulin-induced IL-4 activation, a cytokine inducing IgG4 production. In addition, Achenbach et al. (48) observed that autoantibodies of IgG1 subclass represent low predictive value for type 1 diabetes, and the risk increases when the response to ␤-cell autoantigens spreads to include IgG2 and IgG4 subclasses. It is also noteworthy that IL-4 activates the regulatory T-cell population (49). The present observations suggest that insulin treatment induces activation of insulin-specific regulatory T-cells in patients with recent-onset type 1 diabetes. These results in humans, indicating that the administration of a thymusexpressed self-antigen activates regulatory T-cells, support the concept of the induction and expansion of regulatory T-cells by their antigen in the periphery. This effect of insulin may interfere with the insulin-specific T-cell assays performed in patients with type 1 diabetes. Furthermore, the enhancement of regulatory T-cell responses to insulin induced by insulin treatment may play a role in the induction of the remission phase. The transient nature of the remission suggests defects in the mechanisms of T-cell regulation as reported in patients with type 1 diabetes (50). DIABETES, VOL. 55, DECEMBER 2006

M. TIITTANEN AND ASSOCIATES

ACKNOWLEDGMENTS

This work was supported by the Academy of Finland, the Juvenile Diabetes Research Foundation International, the Sigrid Juselius Foundation, and the Finnish Cultural Foundation. We thank Dr. Alison H. Banham (University of Oxford, U.K.) for providing us the Foxp3 antibody. We are also grateful to Kristiina Luopaja¨rvi, MD, for compiling the data on blood glucose concentrations and A1C values in the patients. We thank Harry Lybeck, Annika Cederlo¨f, Sinikka Tsupari, and Riitta Pa¨kkila¨ for technical assistance. REFERENCES 1. Kaufman FR: Type 1 diabetes mellitus. Pediatr Rev 24:291–300, 2003 2. Palmer JP, Asplin CM, Clemons P, Lyen K, Tatpati O, Raghu PK, Paquette TL: Insulin autoantibodies in insulin-dependent diabetes before insulin treatment. Science 222:1337–1339, 1983 3. Keller RJ: Cellular immunity to human insulin in individuals at high risk for the development of type I diabetes mellitus. J Autoimmun 3:321–327, 1990 4. Dubois-LaForgue D, Carel J-C, Bougne`res P-F, Guillet J-G, Boitard C: T-cell response to proinsulin and insulin in type 1 and pretype 1 diabetes. J Clin Immunol 19:127–134, 1999 5. Verge CF, Gianani R, Kawasaki E, Yu L, Pietropaolo M, Jackson RA, Chase HP, Eisenbarth GS: Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes 45:926 –933, 1996 6. Kulmala P, Savola K, Petersen JS, Va¨ha¨salo P, Karjalainen J, Lo¨ppo¨nen T, Dyrberg T, Åkerblom HK, Knip M, the Childhood Diabetes in Finland Study Group: Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes. J Clin Invest 101:327–336, 1998 7. Deckert T: The immunogenicity of new insulins. Diabetes 34 (Suppl. 2):94 –96, 1985 8. Liblau RS, Singer SM, McDevitt HO: Th1 and Th2 CD4⫹ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 16:34 –38, 1995 9. Berman MA, Sandborg CI, Wang Z, Imfeld KL, Faldivar FJ, Dadufalza V, Buckingham BA: Decreased IL-4 production in new onset type I insulindependent diabetes mellitus. J Immunol 157:4690 – 4696, 1996 10. Kallman BA, Huther M, Tubes M, Feldkamp J, Bertrams J, Gries FA, Lampeter EF, Kolb H: Systemic bias of cytokine production toward cell-mediated immune regulation in IDDM and toward humoral immunity in Graves’disease. Diabetes 46:237–243, 1997 11. Fontenot JD, Rudensky AY: A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol 6:331–337, 2005 12. Sakaguchi S: Naturally arising Foxp3-expressing CD25⫹CD4⫹ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6:345–352, 2005 13. Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4⫹CD25⫹ regulatory T cells. Nat Immunol 4:330 –336, 2003 14. Khattri R, Cox T, Yasayko SA, Ramsdell F: An essential role for Scurfin in CD4⫹CD25⫹ T regulatory cells. Nat Immunol 4:337–342, 2003 15. Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, Sakaguchi S: Crucial role of FOXP3 in the development and function of human CD25⫹CD4⫹ regulatory T cells. Int Immunol 16:1643–1656, 2004 16. Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF: Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4⫹CD25- T cells. J Clin Invest 112:1437– 1443, 2003 17. Rao PE, Petrone AL, Ponath PD: Differentiation and expansion of T cells with regulatory function from human peripheral lymphocytes by stimulation in the presence of TGF-{beta}. J Immunol 174:1446 –1455, 2005 18. Roncador G, Brown PJ, Maestre L, Hue S, Martinez-Torrecuadrada JL, Ling KL, Pratap S, Toms C, Fox BC, Cerundolo V, Powrie F, Banham AH: Analysis of FOXP3 protein expression in human CD4⫹CD25⫹ regulatory T cells at the single-cell level. Eur J Immunol 35:1681–1691, 2005 19. Ronkainen MS, Ha¨ma¨la¨inen A-M, Koskela P, Åkerblom HK, Knip M, the Finnish TRIGR Study Group: Pregnancy induces nonimmunoglobulin insulin-binding activity in both maternal and cord blood serum. Clin Exp Immunol 124:190 –196, 2001 20. Seddon B, Mason D: Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J Exp Med 189:877– 882, 1999 DIABETES, VOL. 55, DECEMBER 2006

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