Abstract
Aims/hypothesis
The pancreatic beta cell response to cytokines is crucial for the development of type 1 diabetes in the NOD mouse. For example, beta cell production of suppressor of cytokine signalling-1 (SOCS-1) protects against diabetes. This finding and other recent studies indicated that cytokine-stressed beta cells might contribute to disease progression by affecting the pancreatic lymphocyte infiltrate. The aim of this study was to provide insight into how the beta cell influences the pancreas-infiltrating T cell repertoire.
Methods
Lymphocytes isolated from Socs1-transgenic (tg) and non-tg NOD mice were analysed by flow cytometry. mRNA and protein levels in pancreatic islets were measured by real-time PCR and immunofluorescence analysis, respectively.
Results
The percentages of regulatory T cells, total counts and ratios between infiltrating CD8+ and CD4+ T cells, and the expression of killer cell lectin-like receptor subfamily K, member 1 (NKG2D) on CD8+ T cells did not differ in pancreases from prediabetic Socs1-tg and non-tg NOD mice. However, a striking difference in the percentages of CD8+ T cells specific for glucose 6-phosphatase catalytic subunit-related protein 206–214 was found, showing that SOCS-1 prevents the accumulation of high percentages of self-reactive CD8+ T cells in the pancreas. It was also found that protection from diabetes in Socs1-tg NOD mice correlated with a reduced expression of Cxcl10 mRNA in IFN-γ treated islets.
Conclusions/interpretation
This study highlights an important role for the beta cell in the local regulation of the diabetogenic process. By responding to the pro-inflammatory pancreas milieu it strongly influences the islet-reactive T cell repertoire in the pancreas.
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Introduction
Parenchymal cells may regulate local inflammatory processes. Recent studies have indicated that the target tissue, including the insulin-producing pancreatic beta cell, is an active participant in autoimmunity [1]. The NOD mouse spontaneously develops diabetes as a result of a T cell-dependent destruction of beta cells [2]. How the beta cell might affect the diabetogenic process besides providing autoantigens remains largely to be established.
We recently showed that overexpression of Socs1 specifically in beta cells protects NOD mice from developing diabetes [3]. Suppressor of cytokine signalling-1 (SOCS-1) is a negative regulator of Janus kinase/signal transducer and activator of transcription signalling that inhibits cellular responses to cytokines, including IFN-γ [4]. Disease protection in Socs1 transgenic (tg) NOD mice was not associated with central T cell tolerance to beta cell antigens or altered insulitis scores in prediabetic animals aged ≤14 weeks. In contrast, adoptive transfer experiments using pancreatic-infiltrating lymphocytes revealed a remarkably low ability of cells isolated from Socs1-tg NOD mice to cause diabetes in NOD.Scid recipients (Scid is also known as Prkdc) [3]. These observations raised the question of whether cytokine-exposed beta cells (e.g. following IFN-γ release by infiltrating immune cells) might affect the accumulating pancreatic lymphocyte repertoire.
Diabetes modelled in the NOD mouse is in part regulated by the frequencies of regulatory T cells (Tregs) and specific pathogenic T cell clones infiltrating the pancreas [2, 5, 6]. Here we took advantage of the Socs1-tg NOD mouse model to study whether cytokine-stressed beta cells influence the T cell repertoire on a local level.
Methods
Animals and diabetes monitoring
The Socs1-tg and 8.3 T cell receptor (TCR)-tg NOD mouse models have been described previously [3, 7, 8]. The mice were kept in a specific pathogen-free environment at the Karolinska Institutet. All experiments were conducted in accordance with institutional guidelines and approved by the local ethics committees. Diabetes development was followed by measuring venous blood glucose concentrations under non-fasting conditions using Glucometer Elite strips (Bayer, Stockholm, Sweden). Mice were considered diabetic after two consecutive measurements of blood glucose >13.0 mmol/l, and onset of diabetes was dated from the first of the sequential measurements.
Adoptive transfers
Splenocytes from non-diabetic 5- to 7-week-old 8.3 TCR-tg NOD mice were harvested and adherent cells were removed by a 1 h incubation in Petri dishes. The cells were stained with 5 μmol/l 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (Sigma, Stockholm, Sweden) and injected i.v.
Islet and lymphocyte isolation
Islets were isolated from 6- to 7-week-old mice in order to retrieve islets that did not have significant numbers of mononuclear infiltrates or significant beta cell destruction. The islets were cultured for 6–7 days before experiments were initiated, as mononuclear cells surrounding the islets are released during this preculture period [7]. The islets were then treated with IFN-γ (1,000 U/ml; Calbiochem, distributed by VWR International, Stockholm, Sweden) [9] for 6 h (real time [RT] PCR) or 24 h (confocal microscopy). Pancreatic-infiltrating lymphocytes were isolated from 16- to 18-week-old mice. In order to remove circulating blood, animals were perfused (with PBS) prior to pancreas retrieval. Total lymphocytes were isolated from pancreases digested with collagenase P (Roche, Stockholm, Sweden) using Lymphoprep (Fresenius Kabi, Uppsala, Sweden) or total T cells were isolated using magnetic cell sorting (MACS) with positive selection of CD90 (Miltenyi Biotec, distributed by Fisher Scientific, Västra Frölunda, Sweden), according to the manufacturers’ instructions.
Flow cytometry
Following incubation with antibodies to CD16/CD32 cells were stained with anti-NKG2D or IgG isotype control (FITC-labelled) (BioLegend, distributed by Nordic Biosite, Täby, Sweden), anti-CD8a (allophycocyanin [APC]-labelled), anti-CD4 (APC-labelled) (BD Pharmingen, Stockholm, Sweden) and/or tetramers NRPV7 and tumour-derived H-2Kd-binding peptide (TUM), as a negative control (phycoerythin [PE]-labelled) [6]. Intracellular staining with antibodies against forkhead box P3 (FOXP3) and IgG2a isotype control (PE-labelled) were performed using a FOXP3 Buffer Set (eBioscience, San Diego, CA, USA). Samples were analysed using a FACSCalibur Instrument and CellQuest (BD Pharmingen, Stockholm, Sweden) or FlowJo software (Tree Star, Olten, Switzerland).
RNA isolation and RT-PCR
Total RNA was isolated and cDNA synthesised as described [10]. RT-PCR analyses were run with TaqMan Gene Expression assays (Applied Biosystems, Stockholm, Sweden). The mRNA expression level of Cxcl10 was normalised against the mRNA expression level of the housekeeping gene Gapdh.
Fluorescent staining and confocal microscopy
Islets were fixed in formalin, embedded in paraffin and cut in 4 μm thick sections. After rehydration the sections were boiled for 5 min in citrate buffer (0.01 mmol/l, pH6), followed by incubation in avidin and biotin block (Vector Laboratories, distributed by Immunkemi F&D AB, Järfälla, Sweden) for 15 min each. The sections were then incubated in PBS-saponin containing 0.1% (wt/vol.) BSA-c (an acetylated and partly linearised form of BSA with an increased net negative charge; Aurion, distributed by SMS gruppen, Hørsholm, Denmark) for 30 min. Sections were incubated with a primary antibody against chemokine (C-X-C motif) ligand 10 (CXCL10) (R&D Systems, Abingdon, UK) overnight followed by the secondary biotinylated antibody (donkey anti-goat; Jackson ImmunoResearch, Suffolk, UK) and the streptavidin-conjugated fluorophore Alexa Fluor 549 (Molecular Probes, Stockholm, Sweden) in the dark. Thereafter the sections were incubated in the primary antibody to insulin (polyclonal anti-swine insulin; Dako Cytomation, Stockholm, Sweden) for 2 h 30 min, followed by biotinylated anti-guinea pig IgG (Vector Laboratories) and streptavidin-conjugated Alexa Fluor 488 (Molecular Probes) as above. The nuclei were visualised using mounting medium containing DAPI (Vectashield, Vector Laboratories). For evaluation, a Leica confocal scanner TCS SP II, coupled to a Leica DMR microscope was used.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 4 software. Differences in diabetes incidence were determined using Kaplan–Meier life-table analysis. RT-PCR data were analysed using a Kruskal–Wallis test (non-parametric ANOVA). Comparisons of T cell populations in the pancreas were performed using a Mann–Whitney test and the frequencies of chemokine (C-X-C motif) receptor 3 (CXCR3) with a paired t test. Bar graphs represent means ± SD.
Results
SOCS1 production protects from diabetes and changes the islet cell response to IFN-γ
As the incidence of diabetes in the NOD mouse may vary between different mouse colonies and animal facilities, we first confirmed the protection from diabetes in Socs1-tg NOD mice from the colony bred at the Karolinska Institutet (Fig. 1a [3]). To determine the magnitude of protection that islet production of SOCS-1 provides against IFN-γ we next studied the expression of Cxcl10, a gene encoding an IFN-inducible chemokine produced in the pancreas during diabetes development ([11, 12], M. Hultcrantz and M. Flodström-Tullberg, unpublished observation). The expression of Cxcl10 was significantly upregulated in IFN-γ-treated islets from non-tg NOD mice, while the induced expression was lower and not significant in islets from Socs1-tg NOD mice (Fig. 1b). In agreement with a perturbed, yet notable expression of Cxcl10 in islets from SOCS-1-tg mice, confocal microscopy showed that the production of CXCL10 was not completely blocked in Socs1-tg beta cells. This analysis also revealed that only few non-beta cells produce CXCL10 following IFN-γ treatment (Fig. 1c–f, and data not shown).
No difference in major T cell populations among lymphocytes infiltrating the Socs1-tg NOD pancreas
Insulitis scores in Socs1-tg NOD mice are comparable to those of non-tg NOD mice up to the age of 14 weeks [3]. Adoptive transfer studies using lymphocytes isolated from the pancreases of prediabetic (16- to 18-week-old) mice suggested that the composition of infiltrating lymphocytes might differ between Socs1-tg and non-tg NOD mice [3]. Based upon these observations, we set out to characterise the infiltrating T cell pool in non-diabetic 16- to 18-week-old Socs1-tg and non-tg mice. No difference in the total counts of CD8+ and CD4+ T cells (Electronic supplementary material [ESM] Fig. 1a,b) or in the ratio of CD4+ to CD8+ T cells (ESM Fig. 1c) was found.
Cell-surface expression of NKG2D on CD8+ T cells is important for diabetes development in the NOD mouse [13]. Islets from NOD mice produce IL-15 [14], a cytokine that induces cell-surface expression of NKG2D on CD8+ T cells [15]. As SOCS-1 reduces Il15 mRNA expression by cytokine-stimulated islets in vitro [16], we postulated that the environment in the Socs1-tg NOD pancreas could be less prone to induce cell-surface expression of NKG2D on infiltrating CD8+ T cells. However, the frequency of NKG2D+ cells within the CD8+ T cell population was the same in Socs1-tg and non-tg NOD mice (ESM Fig. 1d).
Tregs play a critical role in delaying autoimmunity in the NOD mouse [5] and target–T cell interactions may promote local generation of Tregs [17]. To investigate whether beta cells might affect the numbers of infiltrating Tregs, and if the lower incidence of diabetes in Socs1-tg NOD mice correlated with an increased pool of Tregs, we next studied FOXP3-producing CD4+ T cells with a known regulatory phenotype [18]. First, we looked at the absolute numbers of FOXP3+ cells infiltrating the Socs1-tg (n = 10) and non-tg (n = 9) NOD pancreas. Similar counts of cells were obtained from the two groups of mice (not shown). We next assessed the percentage of CD4+ T cells producing FOXP3, but found no differences (ESM Fig. 1e).
Lower frequency of self-reactive T cells in pancreas, pancreatic lymph nodes (PLNs) and peripheral blood of Socs1-tg NOD mice
The risk of diabetes development correlates with the frequency of islet-specific T cells in the peripheral blood and pancreas of NOD mice [6, 19]. Islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) 206–214-specific CD8+ T cells, identified using H-2Kd tetramers complexed with the high-affinity NRPV7 mimetic peptide, become particularly prominent during the weeks just before diabetes onset [6, 20, 21]. Here we observed a striking difference in the percentage of IGRP206–214-specific cells within the CD8+ T cell population infiltrating the pancreas of non-diabetic Socs1-tg and non-tg NOD mice aged 16–18 weeks (Fig. 2). The percentage of IGRP-specific CD8+ T cells was also lower in PLNs and peripheral blood of Socs1-tg NOD mice (Fig. 2). The relevance of SOCS-1 in preventing beta cell damage caused by T cells with this specificity was shown by protection from diabetes in Socs1-tg mice crossed with 8.3 TCR-tg NOD mice harbouring T cells recognising the 206–214 epitope within the IGRP protein (Fig. 3a, [16]).
We next evaluated whether a lower percentage of IGRP206–214-specific T cells could be a result of a decreased priming of T cells in the PLNs. However, transfers of splenocytes from young 8.3 TCR-tg NOD mice suggested that T cell priming was not altered in the PLNs of Socs1-tg NOD mice (Fig. 3b).
Following activation in the lymph node, recruitment of activated T cells to inflamed tissue is dependent on the production of chemokines in the tissue and chemokine receptors on the activated T cell. As the recruitment of islet-specific T cells may be influenced by pancreatic CXCL10 production and CXCR3 cell-surface expression on T cells [11, 12] (see also Fig. 1b) we next studied cell-surface expression of CXCR3 on total CD8+ T cells and NRPV7+CD8+ T cells in the pancreas, PLNs and peripheral blood of non-tg and Socs1-tg NOD mice. Although the vast majority of T cells present in pancreases are CXCR3+ (Fig. 3e), there was a striking difference in the percentage of CXCR3+ cells between total CD8+ T cells and the NRPV7+CD8+ T cells in PLN and blood (Fig. 3c,d). While about 40–80% of the NRPV7+CD8+ cells were CXCR3+ only 10–20% of the total CD8+ T cells were CXCR3+, indicating that the NRPV7+CD8+ T cells are more likely to be recruited by an increase in the islet production of CXCL10.
Discussion
Immune reactions to self are regulated by central and peripheral tolerance mechanisms [22, 23]. Recent studies have indicated that the target organ itself may provide an additional level of control [1]. The present study suggests that the beta cell in the NOD mouse actively contributes to the pathogenic process by modulating the autoreactive T cell repertoire in the pancreas.
In this study the Socs1-tg NOD mouse model was used to investigate how cytokine-stressed beta cells contribute to the diabetogenic process. Production of SOCS-1 by beta cells protects NOD mice [3] and TCR-tg 8.3 NOD mice ([16], and present study) from diabetes. SOCS-1 efficiently blocks the biological effects of IFN-γ [3, 24], but also other cytokines such as IFN-α, IL-4 and IL-6 [4, 7], which may also influence diabetes development. IFN-γ together with the cytokine IL-1 causes beta cell damage in vitro [25]. SOCS-3, another member of the SOCS family that has some functional overlap with SOCS-1, has been shown to block the response to IL-1 [26]. This indicates that SOCS-1 may also provide protection from the actions of IL-1.
Our adoptive transfer studies using splenocytes from 8.3 TCR-tg NOD (Fig. 3b) and BDC2.5-tg NOD (M. Flodström-Tullberg, unpublished observation) showed that T cell priming in the PLN occurs normally in the Socs1-tg NOD mouse. In addition, our studies suggest that beta cells exposed to pro-inflammatory cytokines like IFN-γ do not have a major influence on the overall recruitment of lymphocytes to the pancreas in the NOD mouse ([3], and present study). This is further supported by previous studies in double transgenic 8.3 TCR-tg×Socs1-tg NOD mice [16], and supports a notion that autoreactive T cell clones are influenced locally by indirect or direct interactions with target cells.
It was recently shown that effector CD4+ T cells convert into FOXP3+ Tregs upon encounter with neurons, thereby suppressing experimental autoimmune encephalitis [17]. Although CD4+ T cells are present in high numbers in the Socs1-tg NOD pancreas no signs of increased numbers of CD4+FOXP3+ cells were observed in the pancreas (ESM Fig. 1e) or PLNs (not shown). A local regulation of NKG2D cell-surface expression on infiltrating CD8+ T cells was also not supported by our findings, suggesting that islet cell production of IL-15 [13, 14, 16] may not have a potent effect on the recruited CD8+ T cell population.
The present study shows that the percentage of IGRP206–214-specific CD8+ T cells is dramatically lower in the Socs1-tg NOD pancreas compared with non-tg NOD pancreas. We also observed a lower frequency of these cells in PLNs and peripheral blood of Socs1-tg mice. This is in agreement with previous studies showing that the frequency of IGRP206–214-specific CD8+ T cells in the blood reflects the frequency in the pancreas and can predict disease development in NOD mice [6, 19].
The observation that the frequency of IGRP206–214-specific CD8+ T cells is reduced in the pancreas of Socs1-tg mice, together with the finding that the total CD8+ T cell counts did not differ, clearly suggests that beta cells have a local effect on islet-specific T cell clones. Several, non-mutually exclusive, mechanisms may contribute to this. Cytokines may directly cause islet cell damage and thereby the release of antigens, supporting an efficient restimulation of islet-specific T cell clones upon their arrival in the pancreas. Importantly, SOCS-1 prevents the cytokine-induced production of molecules such as MHC class I [16] and CD40 (M. Hultcrantz and M. Flodström-Tullberg, unpublished observation), suggesting that the pro-inflammatory milieu in the NOD pancreas normally enhances CD8+ T cell recognition of beta cells and increases beta cell susceptibility to damage induced via CD40 ligand [27]. The former mechanism is supported by a study demonstrating that a block in the cell-surface expression of MHC class I specifically in beta cells impairs the accumulation of autoreactive CD8+ T cells in islets without affecting priming in the PLNs [28]. Furthermore, exogenously added IGRP peptide has been shown to increase the recognition and specific killing of Socs1-expressing islets [29].
Although no difference was seen in the overall numbers of pancreas-infiltrating CD4+ and CD8+ T cells it cannot be excluded that beta cell production of chemokines locally affects the specific recruitment and/or stimulates the retention of islet-specific T cells after their initial priming in the PLN. Indeed, we observed a lower expression of Cxcl10 by IFN-exposed Socs1-tg islet cells (Fig. 1b), a gene encoding a chemokine shown to be of importance in other models of type 1 diabetes [11, 12, 30]. We also found that the receptor CXCR3 is present more frequently on NRPV7+CD8+ T cells than on the bulk CD8+ T cells in PLNs and peripheral blood (Fig. 3c,d), suggesting a preferential recruitment of NRPV7+CD8+ T cells to the inflamed pancreas. Finally, our preliminary transfer experiments have demonstrated a reduced retention of CFSE-labelled CD8+ T cells from 8.3 TCR-tg mice in pancreases of Socs1-tg NOD mice compared non-tg NOD mice (M. Hultcrantz and M. Flodström-Tullberg, unpublished observation).
A long prediabetic period with an innocuous insulitis such as that observed in the NOD mouse may not be a typical hallmark of type 1 diabetes in humans. However, insulitis has been observed in non-diabetic organ donors positive for three or four autoantibodies [31]. Moreover, lymphocytes are often found in or around islets of newly diagnosed patients (e.g. [32–35]), indicating that their recruitment is crucial for disease development. CXCL10 is an interesting candidate chemokine since newly diagnosed type 1 diabetes patients have elevated serum levels of CXCL10 [36], and human T cells expressing CXCR3 have been shown to home to the islets in NOD.Scid mice [37]. More recent data from a patient with newly onset type 1 diabetes showed CXCL10 production in islets and CXCR3 expression on pancreas-infiltrating lymphocytes [38]. That the percentage of IGRP-specific T cells was lower in the Socs1-tg NOD mouse pancreas is also of potential interest for human type 1 diabetes, as IGRP-specific T cell clones have been identified in type 1 diabetes patients [39, 40]. Prevention of the migration of these cells and other potentially autoreactive T cells to the islets by a block in CXCL10 production may be a therapeutic measure worth trying in individuals with a high risk of developing type 1 diabetes [41].
In summary, the present study suggests that the beta cell response to cytokines is essential for the accumulation of pathogenic T cells and the progression from insulitis to diabetes in the NOD mouse. This highlights an additional level of regulation that is present in non-lymphoid tissue and dependent on the target cell itself.
Abbreviations
- CFSE:
-
5-(and 6-) carboxyfluorescein diacetate succinimidyl ester
- CXCL10:
-
chemokine (C-X-C motif) ligand 10
- CXCR3:
-
chemokine (C-X-C motif) receptor 3
- FOXP3:
-
forkhead box P3
- IGRP:
-
islet-specific glucose 6-phosphatase catalytic subunit-related protein
- NKG2D:
-
killer cell lectin-like receptor subfamily K, member 1
- PLN:
-
pancreatic lymph node
- RT:
-
real time
- SOCS-1:
-
suppressor of cytokine signalling-1
- TCR:
-
T cell receptor
- tg:
-
transgenic
- Tregs:
-
regulatory T cells
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Acknowledgements
The authors thank N. Sarvetnick, The Scripps Research Institute, La Jolla, CA, USA, for fruitful discussions. We also thank M. Hühn, Karolinska Institutet, Stockholm, Sweden, for critical reading of the manuscript and A. Hoffman, Karolinska Institutet, for excellent technical assistance with the confocal microscopy. This work was supported by a Marie Curie International Reintegration Grant (M. Flodström-Tullberg), a transitional award from the Juvenile Diabetes Research Foundation (M. Flodström-Tullberg), the European Foundation for the Study of Diabetes (M. Flodström-Tullberg), the Swedish Foundation for Strategic Research (M. Flodström-Tullberg), and the Swedish Research Council (M. Flodström-Tullberg). P. Santamaria is supported by the Canadian Institutes of Health Research, and is a Scientist of the Alberta Heritage Foundation for Medical Research. Some parts of this work were presented as an abstract at the 9th International Congress of the Immunology of Diabetes Society and American Diabetes Association Research Symposium (Miami, 2007) and in a thesis by M. Hultcrantz, Department of Medicine, Karolinska Institutet, Stockholm (Interferons in antiviral defense and autoimmunity: focus on type 1 diabetes) defended in 2008 at the Karolinska Institutet, Sweden.
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ESM Fig. 1
Major T cell populations among lymphocytes infiltrating the Socs1-tg NOD pancreas. The absolute counts of CD8+ (a) and CD4+ (b) T cells, the ratio of CD8+/CD4+ (c) and the frequencies of NKG2D+/CD8+ (d) and FOXP3+/CD4+ (e) T cells isolated from Socs1-tg and non-tg NOD pancreases were measured by flow cytometry, as described in the Methods. Horizontal bars indicate the mean values (PDF 2.51 MB)
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Hultcrantz, M., Jacobson, S., Hill, N.J. et al. The target cell response to cytokines governs the autoreactive T cell repertoire in the pancreas of NOD mice. Diabetologia 52, 299–305 (2009). https://doi.org/10.1007/s00125-008-1193-7
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DOI: https://doi.org/10.1007/s00125-008-1193-7