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. 2007 Jun;121(2):189–196. doi: 10.1111/j.1365-2567.2007.02557.x

Reduced CCR4, interleukin-13 and GATA-3 up-regulation in response to type 2 cytokines of cord blood T lymphocytes in infants at genetic risk of type 1 diabetes

Kristiina Luopajärvi 1, Susanne Skarsvik 2, Jorma Ilonen 3,4, Hans K Åkerblom 1, Outi Vaarala 2,5
PMCID: PMC2265934  PMID: 17244154

Abstract

Aberrancies in T-cell polarization including expression of chemokine receptors have been reported in human leucocyte antigen (HLA) class II associated autoimmune diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis. We asked whether these aberrancies are present at birth in newborn infants carrying the HLA risk haplotypes for T1D. Sixty-seven cord blood (CB) samples from infants were screened for T1D-associated HLA risk genotypes (HLA-DR4-DQ8 and/or DR3-DQ2 without protective alleles). CB lymphocytes were stimulated with phytohaemagglutinin in type 1 (interleukin (IL)-12, anti-IL4) or type 2 (IL-4, anti-IL12) cytokine environment for 6 days. The expression of chemokine and cytokine receptors on T cells was determined by flow cytometry, secretion of cytokines was analysed with enzyme-linked immunosorbent assay, and transcription factors were analysed using real-time reverse transcriptase–polymerase chain reaction. After culture of CB lymphocytes in type 2 cytokine environment newborn infants carrying DR4-DQ8 haplotype (n = 18) showed reduced percentage of CD4 T cells expressing CCR4 (P = 0·009) and the level of CCR4 mRNA was decreased (P = 0·008). In addition, lower secretion of IL-13 and expression of GATA-3 in CB lymphocytes cultured in type 2 cytokine environment were found in the infants with DR4-DQ8 haplotype (P = 0·020 and P = 0·004, respectively) in comparison to newborn infants without DR4-DQ8 and DR3-DQ2 haplotypes (n = 37). Poor in vitro induction of type 2 immune responses in newborn infants with DR4-DQ8 haplotype suggests that the HLA genotype associated with risk of autoimmunity may affect the T cell polarization already at birth, which in turn may contribute to the risk for autoimmunity later in life.

Keywords: CCR4, T lymphocytes, type 1 diabetes

Introduction

The immunological mechanisms underlying the association of human leucocyte antigen (HLA) class II alleles with autoimmune diseases are not fully understood. Allelic variation in the binding of autoantigen derived peptides and thereby differences in the presentation of the pathogenic epitope to immune system are the first plausible explanation for the generation of the antigen-specific autoimmune response attacking the target tissue. It is, however, of interest that the same HLA class II haplotypes are associated with several autoimmune diseases, in which the target autoantigens are different. HLA DR3-DQ2 haplotype is associated with several autoimmune diseases including type 1 diabetes (T1D), coeliac disease, Addison's disease, and systemic lupus erythematosus and HLA DR4 haplotypes are also associated with both T1D and rheumatoid arthritis. Accordingly, the mechanisms of HLA risk genotypes leading to autoimmunity may be mediated by non-antigen-specific ways and certain HLA genotypes may support the development of autoimmunity by other mechanisms.1,2 Few studies suggest that HLA risk genotypes of T1D are associated with aberrancies in the cytokine secretion profile.3

Aberrancies in T-cell polarization including expression of chemokine receptors have been reported in HLA class II haplotype associated autoimmune diseases, such as T1D and rheumatoid arthritis. The migration of T cells into target tissue is achieved by inflammatory mediators (cytokines and chemokines) and different receptors for these mediators are associated with certain functional subsets of T cells. Naïve T cells have been reported to express CXCR4 and CCR7.4 So called type 1 T helper (Th1) cells, which contribute to the development of cytotoxic immune responses, express interleukin (IL)-12 receptor β2 chain,5 IL18R,6 CCR5, CXCR3, and CXCR6 and mainly produce interferon-γ (IFN-γ), whereas type 2 T helper (Th2) cells, which support the humoral immunoglobulin E (IgE)-associated immune responses, express CCR4 and CCR3 and secrete IL-4, IL-5 and IL-13.7–9 In addition, Th1 and Th2 cell polarization have been shown to involve the transcription factors T-bet10 and GATA-3,11,12 respectively.

The aim of this study was to characterize the in vitro regulation of T-cell polarization in the newborn infants at genetic risk for T1D. Thus, the expression of cytokine and chemokine receptors on T cells cultured in type 1 or 2 cytokine environment was investigated by flow cytometry, the secretion of cytokines from CB lymphocytes was analysed by enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of transcription factors and the chemokine receptor CCR4 in CB lymphocytes were measured by real-time reverse transcriptase–polymerase chain reaction (RT–PCR).

Subjects and methods

Subjects

CB ethylenediaminetetra-acetic acid (EDTA) samples were randomly received from newborn infants taking part in the Trial to Reduce IDDM in Genetically at Risk (TRIGR) project with a first-degree relative (mother, father, or full sibling) with type 1 diabetes (n = 27) at the Department of Obstetrics, University of Helsinki, Helsinki City Maternity Hospital, and Jorvi Hospital or in the Finnish Dietary Intervention Trial for Prevention of Type 1 Diabetes (FINDIA) project with no affected family member (n = 40) at Jyväskylä Central Hospital and Kuopio University Hospital. The inclusion criterion was gestation age of ≥35 weeks. According to the protocols of the TRIGR and FINDIA studies, infants who carry HLA-DQB1 genotypes *0302/*02, or *0302/x (x stands for alleles other than *0301, or *0602), HLA DQA1*05-DQB1*02/y or HLA DQA1*03-DQB1*02/y (y stands for alleles other than DQA1*0201-DQB1*02, DQB1*0301, DQB1*0302, DQB1*0602, or DQB1*0603) were defined as susceptible to type 1 diabetes. In this study we included newborn infants carrying DR4-DQ8 (DQB1*0302) haplotype (n = 18), and infants with DR3-DQ2 (HLA DQA1*0501-DQB1*02) haplotype (n = 12). Newborn infants (n = 37) carrying neither DR4-DQ8 nor DR3-DQ2 haplotype were also included. The study protocol has been approved by local Ethics Committee for Paediatrics, Adolescent Medicine and Psychiatry (Helsinki, Finland), Coordinating Ethics Committee at the Hospital District of Helsinki and Uusimaa (Helsinki, Finland), and Ethics Committee in Kuopio University Hospital (Kuopio, Finland).

HLA genotyping

Genotyping was performed on CB EDTA samples using a method previously described.13,14

Cell culture

CB mononuclear cells were isolated from cord blood by Ficoll-Paque density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and were suspended at 106 cells/ml in culture medium, which was RPMI-1640 containing 5% inactivated human AB+ serum (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland), l-glutamine (2 mmol/l; Gibco, Grand Island, NY) and gentamicin (25 µg/ml; Sigma-Aldrich Inc., St Louis, MO). The monocytes were depleted from the cell suspension by plastic adherence (1 hr at 37° in 5% CO2). The non-adherent lymphocytes were stimulated (2 × 106 cells/well) in the presence of either phytohaemagglutinin (PHA)-P (5 µg/ml; Sigma-Aldrich Inc.), recombinant human IL-12 (200 pg/ml; BD PharMingen, San Diego, CA), and anti-human IL-4 (50 ng/ml; BD PharMingen) for generating a Th1/Tc1-line, or with PHA-P (5 µg/ml), recombinant human IL-4 (0·4 ng/ml; Sigma-Aldrich Inc.), and anti-human IL-12 (1 µg/ml; BD PharMingen) for generating a Th2/Tc2-line. On day 2, the T-lymphocyte lines were expanded by addition of recombinant human IL-2 (32 pg/ml; BD PharMingen). Supernatants from the cultures were collected on day 4 for further analysis of cytokine profile. Also, half of the medium was replaced and cytokines (IL-12 or IL-4), antibodies (anti-IL-4 or anti-IL-12), and IL-2 were added again. After 6 days of cultures the cells were characterized for expression of chemokine and cytokine receptors by flow cytometry. In addition, lymphocytes were collected and stored at −70° in lysis buffer (GenElute Mammalian Total RNA Miniprep kit; Sigma) until mRNA analysis by real-time RT–PCR.

Flow cytometry for chemokine receptor analysis

The cultured cells were resuspended in phosphate-buffered saline (PBS) with 0·5% bovine serum albumin (staining buffer). The following antibodies were added with 5 µl/300 000 cells in 200 µl staining buffer according to the recommendations of the manufacturer: (1) fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) to CXCR4, CXCR3, CCR3, CCR7 (clones 12G5, 49801, 61828·111, 150503, respectively; R & D Systems, Minneapolis, MN), or CCR5 (clone SD7; BD PharMingen); (2) phycoerythrin (PE)-conjugated mAb to IL-18R, CCR2, CXCR6 (clones 70625, 48607·211, 56811, respectively; R & D Systems), IL-12Rβ2 (clone 2B6/12β2; BD PharMingen), or CCR4 (clone 1G1; BD PharMingen; (3) PerCP conjugated anti-human CD8 (clone SK1; BD PharMingen); (4) allophyco-cyanin (APC)-conjugated anti-human CD4 (clone SK3; BD PharMingen) for 30 min. After staining, cells were incubated with 2 ml of fluorescence-activated cell sorting (FACS) Lysing Solution (Becton Dickinson, San Jose, CA) for 10 min at room temperature to lyse erythrocytes, and analysed with four-colour flow cytometry using a FACSCalibur and CellQuest software (Becton Dickinson). The lymphocytes were gated on forward and side scatter.

Real-time RT–PCR quantification

Total RNA and cDNA was prepared and the mRNA expression of GATA-3, T-bet, and chemokine receptor CCR4 was detected by real time RT–PCR as previously described.15 TaqMan Gene Expression assays were used to measure transcription levels of the selected genes (T-bet, Hs00203436_m1; GATA-3, Hs00231122_m1; CCR4, Hs00747615_s1; Applied Biosystems, Foster City, CA). Real time RT-PCR was performed by using TaqMan Universal PCR master mix (Applied Biosystems) and ABI Prism 7700 Sequence Detection System (Applied Biosystems Sequence detector) in triplicate wells. Ribosomal RNA 18S was applied as an endogenous control (cat no Hs99999901_s1; Applied Biosystems).

The quantities of the markers were analysed by a comparative threshold cycle (CT) method. ΔCT stands for the difference between CT of target gene and CT of the 18S gene, whereas ΔΔCT is the difference between ΔCT of the analysed sample and ΔCT of the calibrator. Calculation of 2–ΔΔCT then gives a relative amount of the analysed sample compared to the calibrator, both normalized to an endogenous control (18S). The relative amounts (2–ΔΔCT) of CCR4, T-bet, and GATA-3 were multiplied by 100 to get whole numbers for plots.

ELISA for IFN-γ and IL-13

ELISA was used for detection of IFN-γ and IL-13 as previously described.16

Statistical analysis

The Kruskal–Wallis test was used as pretest for comparison of three unrelated groups and the Mann–Whitney U-test was applied for comparison of two unrelated groups. For comparison of two related parameters the Wilcoxon Signed Ranks Test was used and for correlation analysis of variables Spearman's rank order correlation coefficient was applied (SPSS 11.0 for Windows). P-values below 0·05 were considered significant.

Results

The generation of type 1 and type 2 T cells

The expression of T-bet mRNA in CB lymphocytes cultured in type 1 cytokine environment compared to type 2 cytokine environment was higher (Wilcoxon Signed Rank Test P < 0·001, median levels 22·0 and 2·0, respectively) and GATA-3 mRNA was lower (Wilcoxon Signed Rank Test, median levels 20·8 and 45·4) in newborn infants without either HLA DR4-DQ8 or DR3-DQ2 haplotypes (Fig. 1). In addition, expression of IL-12Rβ2-chain (Wilcoxon Signed Rank Test P < 0·001, median levels 23·5 and 1·9) and the secretion of IFN-γ (Wilcoxon Signed Rank Test P < 0·001, median levels 163 291 pg/ml and 77 pg/ml) were higher, and the secretion of IL-13 was lower (Wilcoxon Signed Rank Test P < 0·001, median levels 146·0 pg/ml and 599·6 pg/ml) when CB lymphocytes were cultured in a type 1 cytokine environment compared to a type 2 cytokine environment. No significant differences were found in CXCR4 or CCR4 expression between CD4+ T cells cultured in type 1 and 2 cytokine environments, although a trend for increased numbers of CCR4 positive cells were found in CD4+ T cells cultured in type 2 cytokine environment when compared to type 1 cytokine environment (P = 0·08). The secretion of IL-13 correlated positively with expression of CCR4 after culture in type 2 cytokine environment (Spearman correlation r = 0·528, P = 0·001).

Figure 1.

Figure 1

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Relative T-bet and GATA-3 mRNA levels. T-bet and GATA-3 mRNA levels in CB lymphocytes from infants carrying no DR4-DQ8 and DR3-DQ2 haplotypes after culture in type 1 and type 2 cytokine environment. Horizontal lines indicate median values and P-values of Wilcoxon Signed Rank Test are shown in the figure.

Chemokine receptor CXCR4 expression on cord blood T lymphocytes

The three groups of the infants divided according to HLA haplotype differed in the percentage of CD4+ T cells expressing CXCR4 in type 1 cytokine environment (Kruskal–Wallis P = 0·004, median levels 46·9%, 51·8%, and 25·7%(Fig. 2a), and in type 2 cytokine environment (Kruskal–Wallis P = 0·026, median levels 54·1%, 48·4%, and 23·8%) (Fig. 2b). Also the expression of CXCR4 on CD8+ T cells cultured in type 1 cytokine environment differed between the three groups (Kruskal–Wallis P = 0·011, median levels 40·3%, 53·4%, and 29·2%, respectively). The expression of CXCR4 was higher on CD4+ T cells in infants carrying either DR4-DQ8 or DR3-DQ2 haplotype. Expression of CXCR4 on CD8+ T cells cultured in type 1 cytokine environment was higher in infants carrying DR3-DQ2 haplotype compared to infants without either DR4-DQ8 or DR3-DQ2 haplotypes (Mann– Whitney P = 0·006).

Figure 2.

Figure 2

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Percentage of T lymphocytes expressing CXCR4. Percentage of CD4+ T cells expressing CXCR4 from infants carrying DR4-DQ8, DR3-DQ2, or no DR4-DQ8 and DR3-DQ2 haplotypes after culture of CB lymphocytes in type 1 (a) or type 2 (b) cytokine environment. Horizontal lines indicate median values and P-values of Mann–Whitney test are shown in the figure. NS, not significant

Chemokine receptor CCR4 expression on cord blood T lymphocyte

The three groups of the infants divided according to HLA haplotype differed in percentage of CD4+ T cells expressing CCR4 in type 1 cytokine environment (Kruskal–Wallis P = 0·033, median levels 80·0%, 78·7%, and 87·3%, respectively). The percentage of CD4+ T cells expressing CCR4 was reduced in infants carrying DR4-DQ8 haplotype (Fig. 3a). Also, the groups differed in the median fluorescence intensity of CCR4 on CD4+ T cells cultured in type 1 cytokine environment (Kruskal–Wallis P = 0·014, median levels 11·5, 9·7, and 17·8). The median fluorescence intensity of CCR4 on CD4+ T cells was reduced on CD4+ T cells in infants carrying DR4-DQ8 (Mann–Whitney P = 0·024) or DR3-DQ2 (Mann–Whitney P = 0·016) haplotype compared to infants carrying no DR4-DQ8 and DR3-DQ2 haplotypes. The percentage of CD8+ T cells expressing CCR4 cultured in type 1 cytokine environment did not differ between the three groups (Kruskal–Wallis P = 0·248, median levels 38,1%, 43,0%, and 48,3%, respectively).

Figure 3.

Figure 3

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Percentage of T lymphocytes expressing CCR4. Percentage of CD4+ T cells expressing CCR4 from infants carrying DR4-DQ8, DR3-DQ2, or no DR4-DQ8 and DR3-DQ2 haplotypes when CB lymphocytes were cultured in a type 1 (a) or type 2 (b) cytokine environment. Horizontal lines indicate median values and P-values of Mann–Whitney test are shown in the figure. NS, not significant.

The three groups of the infants divided according to HLA haplotype differed in percentage of CD4+ T cells expressing CCR4 in type 2 cytokine environment (Kruskal–Wallis P = 0·009, median levels 70·3%, 73·3%, and 94·1%, respectively). CCR4 was reduced in infants carrying DR4-DQ8 or DR3-DQ2 haplotype (Fig. 3b). Also, the median fluorescence intensity of CCR4 on CD4+ T cells cultured in type 2 cytokine environment differed (Kruskal–Wallis P = 0·001, median levels 10·1, 8·2, 25·7). The median fluorescence intensity of CCR4 on CD4+ T cells was reduced in infants carrying DR4-DQ8 (Mann–Whitney P = 0·003) or DR3-DQ2 (Mann–Whitney P = 0·006) haplotype compared to infants carrying no DR4-DQ8 and DR3-DQ2 haplotypes. The percentage of CD8+ T cells expressing CCR4 cultured in type 2 cytokine environment differed between the three groups (Kruskal–Wallis P = 0·027, median levels 32·7%, 42·6%, and 64·0%). The expression of CCR4 was reduced on CD8+ T cells in infants carrying DR4-DQ8 haplotype compared to neonates without DR4-DQ8 or DR3-DQ2 haplotypes (Mann–Whitney P = 0·008).

There were no differences in expression of CCR2, CCR3, CCR5, CCR7, CXCR3, CXCR6, IL-18R, and IL-12Rβ2-chain on CD4+ or CD8+ T cells in infants carrying DR4-DQ8 or DR3-DQ2 haplotype compared to infants without DR4-DQ8 or DR3-DQ2 haplotypes after culture in type 1 or type 2 cytokine environment.

Levels of CCR4, T-bet and GATA-3 mRNA

The expression of CCR4 mRNA in CB lymphocytes after culture in a type 2 environment differed between the study groups (Kruskal–Wallis P = 0·008, median levels 38·5, 65·1, and 106·8, respectively). In infants carrying DR4-DQ8 haplotype the relative level of CCR4 mRNA in CB lymphocytes was decreased. The expression levels of GATA-3 mRNA in CB lymphocytes cultured in a type 2 environment showed differences between the three groups of the infants (Kruskal–Wallis P = 0·004, median levels 23·6, 31·5, and 45·4, respectively). The relative level of GATA-3 mRNA in CB lymphocytes was reduced in infants carrying DR4-DQ8 haplotype (Fig. 4). There was no difference in expression of T-bet between the groups.

Figure 4.

Figure 4

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Relative levels of CCR4 and GATA-3 mRNA. CCR4 and GATA-3 mRNA level in CB lymphocytes from infants carrying DR4-DQ8, DR3-DQ2, or no DR4-DQ8 and DR3-DQ2 haplotypes after culture in type 2 cytokine environment. Horizontal lines indicate median values and P-values of Mann–Whitney test are shown in the figure. NS, not significant.

Cytokine production of cord blood lymphocytes

IL-13 response of CB lymphocytes cultured in a type 2 cytokine environment differed between groups (Kruskal–Wallis P = 0·02, median levels 194·8 pg/ml, 214·9 pg/ml, and 599·6 pg/ml, respectively). The secretion of IL-13 by CB lymphocytes in infants carrying DR4-DQ8 haplotype was reduced (Fig. 5). There was no difference in expression of IFN-γ between groups.

Figure 5.

Figure 5

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Secretion of IL-13. Expression level of IL-13 on CB lymphocytes from infants carrying DR4-DQ8, DR3-DQ2, or no DR4-DQ8 and DR3-DQ2 haplotypes after culture in type 2 cytokine environment. Horizontal lines indicate median values and P-values of Mann–Whitney test are shown in the figures. NS, not significant.

Discussion

CB T cells were polarized to Th1 and Th2 type T cells in the cytokine environment as demonstrated by differences in the levels of transcription factors T-bet and GATA-3, expression of IL-12R, and secretion of cytokines (IFN-γ, IL-13) in Th1 and Th2 lines. IL-13 was used as marker for Th2 cytokines because IL-4 was present in cultures for induction of deviation.

Our results indicated that the in vitro T-cell response to type 2 cytokine stimulation is less pronounced at birth in infants carrying DR4-DQ8 haplotype. This was demonstrated as poor induction of GATA-3, IL-13 and CCR4 by type 2 cytokine environment. In the differentiation of Th2 cells, IL-4 signalling and signal transducer and activator of transcription-6 activation induces expression of the transcription factor GATA-3,17 which in turn binds mainly to promotors of IL-512 and IL-13.18 It has also been reported that GATA-3 may be the main factor determining the expression level of IL-13.19 Our results indicated decreased expression of GATA-3 and lower secretion of IL-13 after culture of CB lymphocytes in type 2 inducing conditions in infants with DR4-DQ8 haplotype. Th2 cells have been shown to express high levels of CCR4,4 which we detected to be reduced on T cells cultured in both the type 1 and type 2 cytokine environment in infants carrying DR4-DQ8 or DR3-DQ2 haplotype. However, we did not find such a pronounced difference in the expression of CCR4 between T cells cultured in type 1 or 2 cytokine environments suggesting that CCR4 up-regulation is not closely linked to T cell polarization. However, our results suggest that infants with DR4-DQ8 haplotype have an impaired in vitro up-regulation of Th2 cell responses or a general defect in the generation of Th2 cells. Th2 cells are important in the suppression of Th1 immune responses and impairments in Th2 cell differentiation may contribute to an imbalance of the immune response, which favours the development of Th1 responses.

Impairments in T-cell responses to the type 2 cytokine environment associated with certain HLA risk haplotypes for T1D may not imply a specific risk of beta-cell autoimmunity but rather be a marker of more general immune aberrancy, which contributes to the development of immune-mediated diseases. HLA-DR4 allele is associated with increased risk of developing T1D, but also with increased risk of rheumatoid arthritis.20 The development of coeliac disease is also associated with partly the same HLA risk alleles as for T1D.21 It is possible that HLA associated aberrancies in the T-cell development underlie immune-mediated diseases by allowing autoimmunity to develop, but other disease-specific factors are involved in triggering the disease.

Fetal immunity can be influenced by the immunological features of mothers with T1D,22 but in our study there were no difference in immune responses in neonates born to mothers with T1D or to healthy mothers (data not shown). In addition, the way of delivery has been reported to influence the immune system in infants23 but no differences were found between infants born by caesarean section or normal delivery (data not shown). The number of infants born by caesarean section was low, however.

Although we studied newborn infants with a genetic risk of T1D, our findings show similarities with observations in patients with T1D. In patients with newly diagnosed and longstanding T1D, Lohmann et al. have reported reduced expression of CCR4 on freshly isolated T cells and this correlated to low PHA-induced production of IL-4.24 In our study we observed decreased production of IL-13 in infants carrying DR4-DQ8 haplotype and this correlated with low number of CD4 T cells expressing CCR4. Furthermore, in patients with T1D, reduced mRNA levels of IL-4 in unstimulated peripheral blood mononuclear cells have been reported giving further support for a defect in Th2 cell differentiation in patient with T1D.25 Also in subjects at high risk of T1D, the secretion of IL-13 in whole blood cultures stimulated with PHA was lower.26 The studies including patients with T1D are discrepant, however, and also decreased type 1 responsiveness in peripheral blood T cells has been reported.15,27 When we studied the effect of HLA haplotype on T-cell polarization at birth the findings are not modified by metabolic or immunological changes related to the disease.

After culture of CB lymphocytes in type 1 and type 2 cytokine environment, infants carrying DR4-DQ8 or DR3-DQ2 haplotype had increased numbers of CD4 T cells expressing CXCR4 compared to infants with no DR4-DQ8 or DR3-DQ2 haplotypes. CXCR4 has been reported to be expressed on naïve T cells8 and our results may reflect general impairments in differentiation and maturation of T cells in these infants with DR4-DQ8 or DR3-DQ2 haplotype.

In conclusion, individuals with HLA DR4-DQ8 haplotype appear to have poor in vitro maturation of T cells as response to type 2 cytokines already at birth. This may, at least, partly explain why these infants are prone to develop autoimmunity later in their life when they encounter the disease-specific triggers that drive autoimmunity, for example to beta-cells, and lead to the development of T1D.

Acknowledgments

The authors thank the TRIGR Study Group and FINDIA Study Group. This work was supported by NICHD and NIDDK, NIH, Canadian Institutes of Health Research, the Juvenile Diabetes Research Foundation International, the European Union (Fifth Framework), the European Foundation for the Study of Diabetes, the Academy of Finland, and Finska Läkaresällskapet.

We are indebted to Hilkka Puttonen, Anne Björk and Minna Matikainen for their commitment to the studies. We thank Anneli Suomela and Harry Lybeck for skilful technical assistance.

Abbreviations

APC

allophycocyanin

CB

cord blood

CCR

chemokine receptor, CC

CXCR

chemokine receptor, CXC

ELISA

enzyme-linked immunosorbent assay

FACS

fluorescence-activated cell sorter

FITC

fluorescein isothiocyanate

HLA

human leucocyte antigen

IFN-γ

interferon-γ

IL

interleukin

PBS

phosphate-buffered saline

PE

phycoerythrin

RT–PCR

Reverse transcriptase–polymerase chain reaction

T1D

type 1 diabetes

Tc

T cytotoxic lymphocyte

Th

T helper lymphocyte

TRIGR

Trial to Reduce IDDM in Genetically at Risk

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