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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patients with type 1 diabetes are suffering from defects in immune regulatory cells. Their siblings may be at increased risk of type 1 diabetes especially if they are carriers of certain human leucocyte antigen (HLA) alleles. In a prospective non-randomized study, we intended to evaluate 31 healthy siblings of paediatric patients with type 1 diabetes and explore immune regulatory populations of CD4+CD25+ T cells and natural killer (NK) T cells. Tested siblings of type 1 diabetes patients were stratified according to the HLA-associated risk of possible diabetes development. Immune regulatory function of CD4+CD25+ T cells was tested in vitro. Significant differences in CD4+CD25+ but not in NK T cells have been identified. Siblings of type 1 diabetes patients carrying high risk HLA alleles (DQA1*05, DQB1*0201, DQB1*0302) had significantly lower number of immune regulatory CD4+CD25+ T cells than the age-matched healthy controls or siblings carrying low-risk HLA alleles (DQB1*0301, DQB1*0603, DQB1*0602). Regulatory function of CD4+CD25+ T cells demonstrated a dose-escalation effect. In siblings of type 1 diabetes patients, the defect in immune regulatory CD4+CD25+ T cells exists in association with genetic HLA-linked risk for type 1 diabetes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Type 1 diabetes (T1D) arises from a breakdown of tolerance to islet antigens resulting in uncontrolled T-cell-mediated autoimmune destruction of insulin-producing β-cells in the pancreas. Autoreactive subsets of CD4+ T-helper (Th) lymphocytes recognize self-antigens and after activation preferentially produce Th1 cytokine spectrum that initiate the autoimmune process. For further development, the presence of autoreactive CD8+ cytotoxic T (Tc) lymphocytes is necessary as well [1]. To prevent reactivity against self-tissues, autoreactive T cells can be controlled through active suppression by different types of regulatory T cells [2–4]. Sakaguchi has demonstrated an immune regulatory role of CD4+CD25+ T cells (Tregs) naturally occurring in peripheral blood [5]. Elimination of these cells early in life results in development of various T-cell-mediated autoimmune diseases while reconstitution of CD4+CD25+ T-cell population prevents such pathological effects [2–6]. Recent data describe immune regulatory subpopulation of human CD4+ T cells as cells with high expression of CD25 (CD4+CD25high) and same functional characteristics as CD4+CD25+ regulatory cells in mice [7]. Other putative markers include cell-surface expression of CD62L+, CD45RBlow, glucocorticoid-induced tumour necrosis factor receptor (GITR), overexpression of CTLA-4 and intracellular expression of transcriptional repressor FoxP3 (forkhead box P3) [2, 4, 8]. The suppression mechanisms of regulatory cells are indirect through secretion of anti-inflammatory cytokines such as interleukin 4 (IL-4), IL-10, transforming growth factor β (TGF-β) or direct through cell–cell contact, for example via the perforin pathway [2–4, 8]. Evidence that natural Tregs are antigen specific is still limited [4].

Recent studies demonstrate multiple defects in T-cell regulation of T1D individuals [1, 7, 9, 10]. In contrary, limited information exists about siblings of T1D patients that may have an increased risk of T1D. In this work, we focused on immune regulatory populations of CD4+CD25+ T cells and natural killer T cells (NKT) in siblings of children and adolescents with T1D. We were particularly interested in revealing potential defects in these regulatory T-cell populations in healthy individuals, especially siblings of T1D patients who may be at an increased genetic risk of developing T1D. Early identification of at risk individuals may lead to an early therapeutic intervention prior to complete destruction of insulin-producing cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Study subjects.  Heparinized blood samples were obtained from 31 healthy siblings (10 females, 21 males of age 1–20, median age 13 years) of children with T1D followed at the 1st Department of Pediatrics, University Hospital Brno. Healthy controls (16 females, 20 males of age 1–17, median age 10 years) were consecutively recruited from healthy children and adolescents undergoing minor surgery with no family or personal history of T1D or any other autoimmune disease. Blood samples of all study subjects were taken after signing the informed consent approved by the Ethical Committee of the University Hospital Brno, Brno, Czech Republic. A complete human leucocyte antigen (HLA)-DQA1 and HLA-DQB1 genotyping was carried out by polymerase chain reaction (PCR) with sequence-specific primers in all T1D siblings. A stratification of HLA-linked genetic risk was performed according to the T1D prediction programme of the Czech Republic [11] and divided into three groups with high, standard or low risk (Table 1). Sera of all T1D siblings were examined by radioimmunoassay (RIA) (Solupharm, Brno, Czech Republic) for the presence of autoantibodies against islet antigens glutamic acid decarboxylase 65 (GADA) and tyrosinephosphatase (IA-2A). Levels above 1 IU/ml for GADA as well as for IA-2A (more than 2 standard deviations) were considered positive. GAD65 and IA-2 autoantibodies were screened every 12 months in a high-risk group and every 24 months in a standard-risk group. T1D siblings with positive autoantibodies had a standard intravenous glucose tolerance test (ivGTT) and the first phase of insulin response (FPIR) was assessed as described previously [12, 13]. FPIR levels above the fifth percentile were considered normal [12].

Table 1.   Sibling characteristics and an estimated human leucocyte antigen (HLA)-linked genetic risk of type 1 diabetes
HLA characteristicsNo. of subjectsHLAAutoantibodies positive casesImpaired ivGTT
  1. ivGTT, intravenous glucose tolerance test.

High risk 8DQA1*05 DQB1*0201 DQB1*030210
Standard risk11Other than high or low risk21
Low risk12DQB1*0301 DQB1*0603 DQB1*060210
Age1–20, median age 13 years
Sex9 females, 18 males

Flow cytometry.  Flow cytometric determination of the lymphocyte populations from whole blood was performed by the following cell markers: anti-CD4, anti-CD8, anti-CD3, anti-CD25, anti-TCRα24 and anti-TCRβ11 labelled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-cyanin 5 (PC5) or phycoerythrin-cyanin 7 (PC7) (Immunotech, Marseille, France). Samples were analysed by a four-colour flow cytometry on a CytomicsTM FC 500 cytometer (Beckman Coulter, Miami, FL, USA). Data were analysed using the CXP Software (Beckman Coulter).

Immunomagnetic cell sorting.  Immunomagnetic sorting of CD4+CD25+ T cells was performed from peripheral blood mononuclear cells (PBMC) obtained by Histopaque (Sigma-Aldrich, Prague, Czech Republic) gradient centrifugation of the whole blood by the CD4+CD25+ Human Regulatory T Cell Isolation Kit in two steps according to the manufacturer's instructions on a VarioMACS (Miltenyi Biotec, Bergish Gladbach, Germany). Briefly, the first step immunomagnetically eliminated major cell populations except CD4+ T cells that were labelled in the second step with anti-CD25 and positively selected by magnetic beads. The purity of CD4+CD25+ Tregs was more than 97%.

CD4+CD25+ Tregs testing in vitro.  CD4+CD25+ Tregs were used alone or were mixed in different ratios (1:1–1:4) with autologous CD4+CD25 T cells to test their ability to suppress reactivity of lymphocytes to different antigenic stimuli such as irradiated allogeneic PBMC (1:1 ratio to tested CD4+CD25 T cells) or phytohaemagglutinin (PHA) 10 μg/ml (Sigma-Aldrich, Prague, Czech Republic). Cells were cultured at 37 °C and 5% CO2 atmosphere in a complete media (CM) containing X-VIVO 10, 50 mg/l gentamycin, 2 mml-glutamine, 25 mg/ml HEPES (BioWhittaker, Walkersville, MD, USA) and 10% heat-inactivated human AB-serum (Sigma-Aldrich). Cell concentration was 1.0 × 106 cells/ml CM, usually 0.5 × 106 of CD4+CD25 cells were tested. Cell activation to the antigen was measured by the production of interferon gamma (IFN-γ) on the surface of activated CD3+ T cells using the Secretion Assay Cell Detection Kit (Miltenyi Biotec) according to the manufacturer's instructions by flow cytometry. To exclude dead irradiated PBMC, labelling with propidium iodide was used. As a positive control, PBMC with no CD4+CD25+ Tregs were used. As a negative control, unstimulated PBMC were tested.

Statistical analysis.  The four age-matched groups (control, low , standard and high risk) were compared using Kruskal–Wallis test due to asymmetric data distribution. Any results with P-value of less than 0.05 were considered significant. For in vitro testing of CD4+CD25+ Tregs, a descriptive statistic of means and standard deviation was used. All analyses were done using Statistica for Windows 7.1 and Microsoft Office Excel 2003.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

HLA-linked risk, autoantibodies and ivGTT in siblings of T1D patients

HLA-linked risk was evaluated in 31 siblings of T1D patients. There were eight, 11, 12 subjects in high-, standard- and low-risk groups respectively. In five subjects the presence of GAD65 and/or IA-2 autoantibodies was detected. One subject from the standard-risk group was repeatedly positive for GAD65 autoantibodies and had FPIR lesser than first percentile in the ivGTT. The remaining four autoantibody-positive subjects had FPIR values above fifth percentile which was considered normal.

CD4+CD25+ T cells

CD4+CD25+ cells as well as CD4+CD25hi cells were evaluated by flow cytometry in siblings of T1D patients. Gating strategy is demonstrated in Fig. 1. Study subjects were divided into three groups based on a predicted HLA-linked risk of T1D and the evaluation of CD4+CD25+ and CD4+CD25hi T-cell populations were performed for each group. A statistically significant decrease of both CD4+CD25hi T cells and CD4+CD25+ was noticed in the high-risk group in comparison to healthy age-matched controls (P < 0.0001 and <0.0001 respectively) as well as to the low-risk group (P = 0.049 and 0.0014 respectively) (see Fig. 2). Standard-risk group had a significant decrease of CD4+CD25hi T cells (P = 0.011) but not CD4+CD25+ T cells (P = 0.36) in comparison to healthy controls. No significant difference was detected between standard- and high-risk groups or standard- and low-risk groups.

image

Figure 1.  Gating strategy for identification of CD4+CD25+ and CD4+CD25hi cells. Cells were gated based on CD3 and CD4 positivity, expression of CD25, and divided into CD4+CD25 or CD4+CD25+ cells. Cells with expression of CD25 exceeding that of CD4CD25+ cells within PBMC (predominantly activated B cells) were considered CD4+CD25hi cells. Due to a smaller size in the forward scatter channel (data not shown) CD4+CD25hi cells showed a slightly lower CD4 expression then the rest of CD4+ cells.

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image

Figure 2.  Percentage of (A) CD4+CD25+ T cells and (B) CD4+CD25hi cells in siblings with high (HI), standard (STD) and low (LO) human leucocyte antigen (HLA)-linked risk in comparison to healthy controls (CTR).

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NKT cells

NKT cells defined as CD3-positive, TCR-α 24-positive and TCR-β 11-positive cells were examined in all siblings of T1D patients and compared with age-matched healthy controls. No difference was detected among the risk groups of siblings or between siblings of T1D patients and healthy controls (Fig. 3).

image

Figure 3.  Percentage of natural killer T (NKT) cells in siblings with high (HI), standard (STD) and low (LO) human leucocyte antigen (HLA)-linked risk in comparison to healthy controls (CTR).

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Regulatory effect of CD4+CD25+ T cells

Immune regulatory CD4+CD25+ T cells were isolated by magnetic separation from PBMC of healthy donors. Sorted population of CD4+CD25+ T cells contained approximately 0.5–1% of the original number of PBMC with more then 97% purity of CD4+CD25+ T cells. Autologous PBMC were activated either by PHA or by irradiated allogeneic PBMC and Tregs were added in different ratios to demonstrate their regulatory effect based on IFN-γ production. Tregs were able to suppress activation of PBMC in a dose-dependent fashion (Fig. 4).

image

Figure 4.  Dose-dependent suppression with CD4+CD25+ T cells. CD4+CD25+ Tregs were added to activated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner in a ratio 1:1, 2:1 and 4:1 (PBMC:Tregs), non-activated PBMC were used as a negative control (NC) and activated PBMC without addition of Tregs were used as a positive control (PC). Interferon gamma (IFN-γ) production was measured after 48 h. Data represent at least three independent experiments: (A) activation of PBMC by phytohaemagglutinin and (B) activation of PBMC by irradiated allogeneic PBMC.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

A deficiency in the number or function of Tregs can contribute to the onset of T1D as previously documented by several studies in the NOD mice as well as in humans [1–3, 6]. In children with T1D and their healthy siblings the data are rather limited. Kukreja et al. [14] were able to demonstrate defects affecting both the CD4+CD25+ T cells and NKT cells in patients with T1D as well as defects in NKT cells in 12 siblings of those patients. Unfortunately, CD4+CD25+ T cells in siblings of patients with diabetes were not studied in that study. In our previous study, we were able to demonstrate defects in T1D children only in CD4+CD25+ T cells but not in NKT cells [15]. Here, we focused on siblings of T1D patients for whom, to our knowledge, no data are available.

Siblings of T1D patients can be also stratified based on predicted genetic HLA-linked risk for the development of T1D in the future. That stratification has been previously described for the Czech population [11]. For the purpose of our study, we divided the subject population into three groups with high, standard and low risk of T1D. We were able to demonstrate a close association between HLA-linked risks and the CD4+CD25+ T cell population, i.e. the higher the genetic risk the lower the CD4+CD25+ Tregs population. As expected, the differences were more pronounced in CD4+CD25hi cells that mainly contain Tregs cells rather then the general population of CD4+CD25+ T cells that contains regulatory as well as recently activated T cells. The standard-risk group revealed a significant decrease of CD4+CD25hi T cells but not CD4+CD25+ in comparison to healthy controls. This can be explained by rather insufficient classification of the standard-risk group that is defined by exclusion of HLA alleles for high- or low-risk groups. In general, regardless HLA type, siblings of patients with T1D have an increased risk of T1D [11, 14] and one of the mechanisms can involve diminished numbers of Tregs. We were able to demonstrate decreased number of Tregs in this group of healthy siblings of T1D patients.

We completed our study with a set of experiments that document the regulatory effect of immune magnetically isolated CD4+CD25+ T cells from peripheral blood. Despite a strong activation by mitogen or allogeneic PBMC, the suppressive effect of CD4+CD25+ T cells was obvious in a dose-dependent manner. These data are in agreement with previously published results describing regulatory effect of CD4+CD25+ T cells [3, 7, 10].

In conclusion, the defect in immune regulatory CD4+CD25+ T cells has been described in siblings of paediatric patients with T1D. This defect is associated with an increased HLA-associated risk of T1D and thus confirms the hypothesis that an autoimmune mechanism that, in general, leads to target organ destruction, is initiated well before the clinical manifestation of the disease appears. Further research and a careful long-term immunological monitoring of regulatory T cells in individuals at risk of T1D based on HLA-linked risk can be helpful in considering an early possible intervention to prevent a complete destruction of target organ or tissue.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We would like to acknowledge Drs Ondrej Cinek and Marta Pechova for the HLA typing and antibody screening of study subjects.

This work was supported in part by Ministry of Health, Czech Republic, project no. 00064203.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References