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Abstract

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

Insulin is a critical autoantigen for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. About 80% of NOD females and 30–40% of NOD males develop diabetes. However, Insulin2 (Ins2) knockout NOD mice develop autoimmune diabetes with complete penetrance in both sexes, at an earlier age, and have stronger autoimmune responses to insulin. The severe diabetes phenotype observed in NOD-Ins2−/− mice suggests that lack of Ins2 expression in the thymus may compromise immunological tolerance to insulin. Insulin is a prototypical tissue specific antigen (TSA) for which tolerance is dependent on expression in thymus and peripheral lymphoid tissues. TSA are naturally expressed by medullary thymic epithelial cells (mTEC), stromal cells in peripheral lymphoid tissues and bone marrow (BM)-derived cells, mainly CD11c+ dendritic cells. The natural expression of TSA by mTEC and stromal cells has been shown to contribute to self-tolerance. However, it is unclear whether this also applies to BM-derived cells naturally expressing TSA. To address this question, we created BM chimeras and investigated whether reintroducing Ins2 expression solely by NOD BM-derived cells delays diabetes development in NOD-Ins2−/− mice. On follow-up, NOD-Ins2−/− mice receiving Ins2-expressing NOD BM cells developed diabetes at similar rates of those receiving NOD-Ins2−/− BM cells. Diabetes developed in 64% of NOD recipients transplanted with NOD BM and in 47% of NOD mice transplanted with NOD-Ins2−/− BM (P = ns). Thus, NOD-Ins2−/− BM did not worsen diabetes in NOD recipients and Ins2 expression by NOD BM-derived cells did not delay diabetes development in NOD-Ins2−/− mice.


Introduction

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

Immunological self-tolerance is established and maintained through mechanisms taking place in both thymus (central tolerance) and peripheral lymphoid tissues (peripheral tolerance) [1]. Recent studies show that a critical component of self-tolerance is mediated by the expression of self-molecules, including those that are expressed by specific tissues or cells (tissue specific antigens (TSA)) in both the thymus and peripheral lymphoid tissues (reviewed in [2, 3]). The ability of lymphatic tissues to directly produce TSA ensures the availability of these self-molecules for presentation to lymphocytes and the induction of deletion or other regulatory mechanisms that promote self-tolerance. Paradoxically, many TSA are also autoantigens in autoimmune diseases. Variation in their expression, determined by genetic polymorphisms, alternative splicing and possibly epigenetic regulation of gene expression, can influence the efficiency of thymic selection and other tolerogenic processes, in turn modifying susceptibility to autoimmune disease [2, 4–11].

Insulin is a prototypical TSA expressed in the thymus and an important autoantigen in type 1 diabetes, an autoimmune disease resulting in the loss of pancreatic β cells and insulin-dependency [12]. In man, the thymic expression of insulin is regulated by genetic polymorphism in the insulin gene region [6, 7]. The non-obese diabetic (NOD) mouse is a well characterized model of type 1 diabetes [13] in which insulin is a critical autoantigen [14]. Mice have two insulin genes (Ins1 and Ins2) [15]. While both mouse insulin genes are expressed in pancreas, Insulin2 (Ins2) is the predominant insulin gene expressed in the mouse thymus. Ins1 mRNA has been rarely reported in thymus [16, 17] and other studies have found it absent [18] or extremely low [19]. The expression levels of Ins2 in the NOD mouse thymus are similar to those of non-diabetes prone strains at 2 weeks of age [20] but become lower at 3 weeks of age [21]. Insulin and other TSA are also expressed in peripheral lymphoid tissues both in man and mouse [4, 5, 22–26]. Importantly, the expression of Ins2 decreases sharply in the pancreatic lymph node of NOD mice but not in a control strain starting at about 4 weeks of age [27]. Thus, reduced levels of Ins2 expression in both thymus and peripheral lymphoid tissues may favour loss of tolerance to insulin in NOD mice. Further support for this hypothesis derives from the observation that NOD-Ins2 knockout (NOD-Ins2−/−) mice develop diabetes at much higher rates than NOD mice. Indeed, up to100% of NOD-Ins2−/− mice (both sexes) develop diabetes compared to about 80% in NOD females and 30% in NOD males. Moreover, diabetes develops at much faster rates since most NOD-Ins2−/− mice have diabetes by 15–20 weeks of age compared to NOD mice, many of which develop diabetes by 30–32 weeks of age [28, 29]. NOD-Ins2−/− mice also have stronger autoimmunity against insulin [28, 30]. Thus, disease penetrance, disease severity and insulin autoimmunity seem to be worsened by the lack of Ins2 expression in lymphoid tissues.

TSA expression in the thymus has been primarily assigned to a subset of medullary thymic epithelial cells (mTEC) (reviewed in [2]), in which expression of many but not all TSA is controlled by the autoimmune regulator transcription factor (AIRE) [17] and it is believed to promote negative selection [31]. However, there is evidence that bone marrow (BM)-derived cells, mainly CD11+ dendritic cells, also express TSA in the thymus. Insulin gene expression and insulin protein has been detected in CD11c+ cells, both in man [22, 23, 32] and rodents [16, 23, 33]. While some studies have not detected TSA expression in CD11c+ cells [34, 35], discordant results could be explained by differences in methodology [36] and by the fact that TSA expression levels appear lower in DC compared to mTEC [16, 26]. CD11c+ cells expressing insulin and other TSA were also detected in the circulation [23, 32]. More recently, TSA transcripts have been described in AIRE-expressing stromal cells in mouse peripheral lymphoid tissues [25, 26]. Stromal cell populations expressing TSA were shown to mediate deletion of autoreactive CD8 T cells using transgenic models [25, 26]. However, insulin gene transcripts were not detected in these AIRE-expressing stromal cells (Mark Anderson, personal communication) and were not regulated by AIRE [26]. Thus, insulin gene transcription in peripheral lymphoid tissues has only been observed in CD11c+ cells [16, 23]. Moreover, recent evidence shows that Ins2-expressing BM-derived cells do traffic to the thymus in transplantation experiments [37].

Studies have explored the contribution of insulin expression by mTEC and BM-derived CD11c+ cells. Thymus and BM chimeras in Ins2−/− mice (not on the NOD background) suggest that Ins2 expression by thymic stromal cells but not BM-derived cells is important for regulating immune responsiveness to insulin following immunization [30, 38]. However, the effect on the development of spontaneous autoimmune responses has not been assessed. Using the very stringent NOD-Ins2−/− model, we investigated the potential contribution to self-tolerance of NOD BM-derived cells, which naturally express Ins2. We created BM chimeras and studied whether Ins2 expression by NOD BM-derived cells influenced diabetes development in NOD-Ins2−/− chimeric mice.

Materials and methods

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

Our studies were performed at the Diabetes Research Institute, University of Miami School of Medicine and at the Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center. Studies were approved by the Animal Care and Use Committees at each institution, respectively.

Experimental mice.  NOD/ShiLtJ and NOD.129S2(B6)-Ins2tm1Jja/GseJ homozygous mice (in short NOD-Ins2−/−) were established in the laboratory in Denver [39] and later donated to the The Jackson Laboratory (Bar Harbour, ME, USA; Jax stock number 005036). As noted, NOD-Ins2−/− mice feature increased disease severity, penetrance and earlier age of onset compared to NOD mice. For whole BM chimera experiments performed in Miami we elected to use male mice. This choice was made because the disease is more aggressive in female mice and the difference in diabetes incidence between NOD and NOD-Ins2−/− mice is greater in male mice (30% versus up to 100%) than in female mice (80% versus 100%) [28, 29]. Moreover, male NOD-Ins2−/− mice tend to develop diabetes at a slightly slower rate and older age than females, although disease still develops much earlier than NOD males. Because experiments in Miami were performed with mice shipped from the Jackson Laboratory when 5–6 weeks old, using male mice helped reducing the chance the mice may be already diabetic at the time of BM transplantation. Female mice were used for the BM transplantation experiments performed at the Barbara Davis Center. They were bred and housed under specific pathogen-free conditions at the University of Colorado Center for Comparative Medicine (CCM) in Aurora, Colorado. To assess reconstitution, the NOD/ShiLtJ used as BM donors were congenic mice that transgenically expressed green fluorescent protein (GFP), generated by backcrossing (more than 11 times) with GFP transgenic mice kindly provided by the Kappler and Marrack’s laboratory at the National Jewish Medical and Research Center, Denver, CO, USA [40].

Bone marrow transplantation.  In experiments performed in Miami, male donor mice (5–8 weeks old) were euthanized by cervical dislocation under general anaesthesia. To harvest BM cells, femurs and tibiae were collected into cold RPMI Medium 1640 complemented by 0.5 mg/ml gentamicin and 25 mm HEPES. BM was flushed with cold medium to filter through a sterile nylon mesh and collected by centrifugation. Male recipient mice (5–9 weeks old) received a single total body irradiation (950 rads) and donor BM (10 × 106 cells) without further manipulation infused i.v. in the tail vein within 1–3 h of irradiation. All mice were provided with 6 mm HCl supplemented water post-bone marrow transplantation (BMT). Any mice showing signs of physical distress in the immediate post-BMT period were euthanized and excluded from analysis. BM reconstitution was monitored by assessing blood cell counts, haematocrit and haemoglobin levels on samples taken 2–3 weeks after BMT with standard laboratory methods. We verified that reconstitution with NOD marrow restored the transcription of Ins2 mRNA in the NOD-Ins2−/− background, using quantitative realtime RT-PCR. Total RNA from thymus and pancreas was extracted with Trizol (Invitrogen; Carlsbad, CA, USA). Reverse transcription and PCR was performed using standard reagents and methods (Applied Biosystems; Foster City, CA, USA), including primers-probes oligonucleotide reagents which were Hs99999901_S1 for S18 ribosomal RNA (control) and Mm00731595_gH for Ins2. We have previously reported that Ins2 mRNA is not detectable in NOD-Ins2−/− mice [18]. In additional experiments performed in Denver, female mice received T and B cell depleted BM cells to exclude any potential influence from mature T and B cells in the BM inoculum. To deplete mature T and B cells, harvested BM cells were cultured with anti-mouse CD4, CD8, and CD45R Magnetic Beads followed by autoMACSTM cell separation (130–049–201, 130–049–401, and 130–049–501, respectively, Miltenyi Biotec; Auburn, CA, USA). CD4, CD8, and CD45R-negative BM cell populations were then used for BMT in 4 week-old recipient mice which received irradiation (360 rads twice 4 h apart) and T and B cell depleted BM (10 × 106 cells) intravenous infusion. To assess the efficacy of cell depletion, pre- and post-cell depleted BM cells were stained with anti-CD4 (APC conjugated, clone RM4-5), anti-CD8 (FITC conjugated, clone 53–6.7), and anti-CD19 (PE conjugated, clone 1D3) antibodies (BD Biosciences; San Jose, CA, USA) and analysed using a BD FACSCalibur™ (Becton-Dickinson; Franklin Lakes, NJ, USA). To assess the reconstitution of transferred BM cells, recipient mice (NOD/ShiLtJ mice that received BM cells from NOD mice expressing GFP) were bled at 3 and 6 weeks after BM transplant. After lysis of erythrocytes, peripheral blood cells were stained with anti-CD4 (PE conjugated, clone RM4-5), anti-CD8 (PE conjugated, clone 53–6.7), and anti-CD19 (PE conjugated, clone 1D3) antibodies (BD Biosciences) and then analysed for GFP positivity in each cell population using a FACSCalibur™. Overall, 170 mice were included in this study as BMT recipients. BMT failed in 6 mice (3.6%) and they were sacrificed after 2 days. One mouse developed a thymoma 24 weeks after the transplant, before developing diabetes. An additional four mice were sacrificed for other reasons (infections, abscesses) before completing 30 weeks of follow-up. These mice were not included in the analysis, leaving a total of 159 mice, divided in eight groups of mice with the different donor–recipient combinations, and control mice (Table 1).

Table 1.   Donor–recipient combinations and control groups.
GroupBM donor miceRecipient miceUnmanipulated mice
  1. BM, bone marrow; NOD, non-obese diabetic.

  2. Groups A and B included NOD and NOD-Ins2−/− unmanipulated mice which were followed for comparison but did not receive BMT. Groups C–F included recipients of whole BM cells. Groups C and D include NOD-Ins2−/− recipient mice of BM cells from NOD-Ins2−/− and NOD donor mice, respectively. Groups E and F included NOD recipient mice of BM cells from NOD-Ins2−/− and NOD donor mice, respectively. Groups G and H included mice that received BM cells depleted of CD4+, CD8+ and CD45R+ cells. Recipient mice in groups C–F were males, while recipient mice in groups G–H were females.

A  NOD (n = 39)
B  NOD-Ins2−/− (n = 26)
CNOD-Ins2−/−, whole marrowNOD-Ins2−/− (n = 19) 
DNOD, whole marrowNOD-Ins2−/− (n = 23) 
ENOD-Ins2−/−, whole marrowNOD (n = 17) 
FNOD, whole marrowNOD (n = 25) 
GNOD-Ins2−/−, lymphocyte depletedNOD-Ins2−/− (n = 4) 
HNOD, lymphocyte depletedNOD-Ins2−/− (n = 4) 

Monitoring of diabetes development.  Mice were tested weekly for urine glucose levels with Diastix test strips (Bayer Corporation; Elkhart, IN, USA). In glycosuric mice, blood glucose levels were measured with a metre (One Touch Ultra 2; LifeScan, Milpitas, CA, USA). Mice were considered diabetic and euthanized when three consecutive blood glucose readings were greater than 250 mg/dl. The onset of diabetes was dated from the first of the sequential glycemia measurements above 250 mg/dl.

Statistical analysis.  Diabetes incidence was compared using survival curves with the log-rank test (GraphPad PrismTM; GraphPad Software Inc., San Diego, CA, USA).

Results

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

Ins2 expression or lack of Ins2 expression by BM-derived cells does not influence diabetes development in whole BM chimera experiments

We investigated whether the natural expression of Ins2 by NOD BM-derived cells can influence diabetes development in NOD-Ins2−/− mice. Thus, we transplanted BM cells from NOD into 5–9 week-old NOD-Ins2−/− males after total body irradiation (950 rads). As a control we transplanted BM cells from NOD-Ins2−/− donors into NOD-Ins2−/− recipients. Virtually all mice were properly reconstituted by 2–3 weeks after BMT, as assessed by blood cell counts, haemoglobin and haematocrit levels (not shown). We further confirmed reconstitution and the presence of donor BM-derived cells by demonstrating Ins2 mRNA in the thymus by quantitative realtime RT-PCR (Fig. 1). However, we did not observe differences in the onset of diabetes in NOD-Ins2−/− recipients of NOD BM, compared with those transplanted with NOD-Ins2−/− BM. The overall incidence of diabetes was similar in both groups, reaching 100% at 30 weeks post-BMT (Fig. 2A). The onset of diabetes in both transplanted groups was delayed slightly compared to the spontaneous disease onset development in non-manipulated NOD and NOD-Ins2−/− mice, but not significantly (Fig. 2C). We also investigated whether the lack of Ins2 expression by BM-derived cells could accelerate diabetes in NOD mice. To this end, we transplanted whole BM from NOD-Ins2−/− and NOD donors (control) into 5–9 week-old male NOD mice after total body irradiation (950 rads). After 30 weeks, diabetes developed in 47% of NOD recipients transplanted with NOD-Ins2−/− BM and in 64% of those transplanted with NOD BM (P = ns, Fig. 2B). As expected, the incidence of diabetes was higher in the NOD-Ins2−/− control group (88%) compared with the unmanipulated NOD control group (56%) (P = 0.02, Fig. 2C).

image

Figure 1.  NOD BM-derived cells restore Ins2 transcription in the NOD-Ins2−/− thymus after BMT. Realtime RT-PCR amplification plot shows cycle number versus normalized reporter (Rn) fluorescence. The data demonstrate Ins2 mRNA transcripts in the thymus of a NOD-Ins2−/− recipient, 19 days after BMT with NOD BM cells. The same sample yielded no product when omitting the reverse transcription enzyme (RT-control). We used the pancreas from a non-diabetic Balb/c mouse as positive control for Ins2 transcription. 18S ribosomal RNA was used as a control of RNA quality.

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image

Figure 2.  NOD whole BM does not delay or accelerate diabetes development in NOD-Ins2−/− male mice. Panel A: survival curves showing the incidence of diabetes (%) in NOD-Ins2−/− mice after BMT with NOD or NOD-Ins2−/− whole bone marrow. Panel B: survival curves showing the incidence of diabetes (%) in NOD mice after BMT with NOD-Ins2−/− or NOD whole bone marrow. Panel C: survival curves showing the incidence of diabetes in unmanipulated NOD and NOD-Ins2−/− mice.

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Transplantation of lymphocyte-depleted NOD BM into NOD-Ins2−/− mice does not delay diabetes development

Freshly isolated BM cell population might contain some mature autoreactive T cells and B cells that may target pancreatic β cells. These autoreactive lymphocytes may potentially contribute to diabetes development in BM chimeric mice. To test this hypothesis, we performed BMT in additional mice using BM which was depleted of mature T and B cells. Fig. 3A shows the percentages of CD4+, CD8+ and CD19+ lymphocytes before and after their removal using antibody-conjugated magnetic bead depletion. The procedure effectively depleted these lymphocyte populations from the inoculum to <2%. As shown in Fig. 3B, NOD-Ins2−/− mice receiving BM cells from either NOD mice or NOD-Ins2−/− mice developed diabetes within 15 weeks following BMT. Both recipients and donors used in this particular experiment were female mice, and thus mice developed diabetes relatively faster than male recipients receiving whole BMT. To test whether transplanted BM cells differentiated in recipient mice, we analysed the reconstitution of donor cells using NOD BM cells transgenically expressing GFP. We tested two NOD mice that received NOD GFP+ BM cells after the same amount of irradiation. Greater than 90% of CD8 T cells and B cells (CD19+ cells) were replaced by GFP+ cells at 3 weeks after transplant, but only 60% of CD4 T cells were GFP+ at this time point. However, 90–95% of CD4 and CD8 T cells and almost 100% of B cells in peripheral blood were GFP+ 6 weeks after BM transplantation (Fig. 3C), suggesting that radiation eliminated most host lymphocytes and that the donor–recipient cells reconstituted the host and differentiated. Thus, it is likely that in the mice treated with BMT diabetes development was mediated by the lymphocytes derived from the donor BM cells.

image

Figure 3.  NOD, lymphocyte-depleted BM does not delay diabetes development in NOD-Ins2−/− female mice. Panel A: frequency of CD4+, CD8+, and CD19+ in freshly isolated bone marrow before and after depletion with antibody-conjugated magnetic micro-beads. Representative flow cytometry plots are shown. Panel B: survival curves showing the incidence of diabetes (%) in NOD-Ins2−/− female mice after BMT with NOD or NOD-Ins2−/− lymphocyte-deleted bone marrow at 4 weeks of age. All mice in both groups developed diabetes by 15 weeks after transplantation. Panel C: NOD mice were irradiated with 360 rads, twice, followed by BMT with CD4, CD8, and CD19-depleted bone marrow cells isolated from NOD mice transgenic for green fluorescent protein (GFP) to assess reconstitution. CD4, CD8, and CD19 cells from NOD-GFP mice were assessed in peripheral blood by flow cytometry. Percentage of donor-derived GFP-positive cells (mean value of two mice) is shown at 3 and 6 weeks after BMT.

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Discussion

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

Insulin is a critical autoantigen for diabetes development in NOD mice [14]. There is growing evidence suggesting that reduced and lack of Ins2 expression in the thymus and lymphoid tissues may play a role in favouring loss of tolerance to insulin in NOD mice. This effect appears even more prominent in NOD-Ins2−/− mice, most of which develop diabetes with increased severity and at younger age. Using this very stringent model, we investigated the potential contribution to self-tolerance of NOD BM-derived cells, as CD11c+ cells are the only cells so far shown to naturally express Ins2 in both thymus and peripheral lymphoid tissues. Moreover, experimental data show that BM-derived cells contribute to Ins2 expression in the thymus in the absence of Ins2 expression in the mTEC [37]. Studies in transgenic mice have also shown that TSA expression by BM-derived cells can be tolerogenic and results in activation induced cell death in the periphery [24]. However, there are no studies that specifically address the role of the natural Ins2 expression by BM-derived cells.

Thus, we created BM chimeras to determine whether Ins2 expression by NOD BM-derived cells influenced diabetes development in NOD-Ins2−/− chimeric mice. Our data show that Ins2-expressing NOD BM cells did not alter the rapid and severe course of diabetes development in NOD-Ins2−/− recipient mice (both male and female mice). BMT with either whole BM or lymphocyte-depleted BM yielded similar results. NOD-Ins2−/− BM did not accelerate diabetes development in NOD recipients. Mice were properly reconstituted, as collectively shown by conventional monitoring of BM-derived hematopoietic cells and the presence of donor-derived GFP-expressing cells. Both female and male mice developed diabetes several weeks after BMT, further suggesting that diabetes development was likely mediated by donor-derived cells. Reconstitution was further confirmed by the detection of Ins2 mRNA in the NOD-Ins2−/− thymus after BMT with NOD BM cells. This finding also confirms that Ins2-expressing BM-derived cells can migrate to the thymus. This is consistent with an earlier study that showed restoration of Ins2 mRNA expression in the thymus when this was transplanted under the kidney capsule. [37].

At first glance, our results appear to refute our hypothesis that Ins2-expression by BM-derived CD11+ cells may contribute to self-tolerance. However, there are limitations to our study and several alternative explanations are worth discussing. First, we used a very stringent model in which disease course may be very difficult to alter. Because insulin autoimmunity is a major driving force for autoimmunity in NOD and even more so in NOD-Ins2−/− mice [28, 30, 41], it is possible that anti-insulin responses might have already been triggered by the time we could perform BMT in our mice. In fact, some of our male recipients were already diabetic before BMT could be performed, typically by 5–6 weeks of age. In these mice we could demonstrate very severe insulitis and β cell destruction (not shown). It is also possible that early insulin autoimmunity could have caused significant β cell damage before BMT, and that autoimmune responses to other autoantigens could have driven progression to overt diabetes after BMT. Thus, a more specific readout for this study would have been an assessment of insulin-specific T cells responses.

Another consideration is that NOD mice may not be the ideal source of Ins2-expressing BM cells to restore tolerance to insulin. In fact, the expression levels of Ins2 in the NOD mouse thymus are lower than in non-diabetes prone strains [21] and Ins2 mRNA levels decrease significantly in thymus, spleen and the pancreatic lymph node starting at about 4 weeks of age [27]. Therefore, Ins2 expression by NOD BM-derived cells may be too low or decline over time in critical tissues, which may prevent the mediation of effective tolerogenic signals. In agreement with this hypothesis, transgenic Ins2 overexpression in MHC class II-positive cells (which include both cells in the thymus and BM) completely prevents insulitis and diabetes development in NOD mice [42]. Moreover, Steptoe et al. showed that BMT with BM cells or hemopoietic stem cells from these transgenic mice protected NOD mice from diabetes [43]. Hemopoietic stem cells induced to express Ins2 by retroviral transduction also protected NOD mice from insulitis [44]. In addition to levels of autoantigen, the protective effects observed using these transgenic or genetically manipulated cells may be ascribed to differences in Ins2 processing and presentation compared to the natural expression of Ins2, since the genetically manipulated cells expressed Ins2 under the control of the MHC class II promoter.

Finally, there is mounting evidence that CD11c+ DC can induce regulatory T cells in vitro, which have been used therapeutically in NOD mice [45–48]. There is also evidence that AIRE and TSA expression in the thymus are important for the selection of regulatory T cells in the thymus [49, 50]. Thus, it is possible that Ins2 expression by CD11c+ cells in peripheral lymphoid tissues may help maintaining or activating the pool of insulin-specific regulatory T cells originally selected in the thymus [51]. Given the recent evidence that the regulatory T cell compartment in the NOD strain is also compromised [52], reconstitution with Ins2-expressing NOD BM may not restore tolerance to insulin if this depends on a functionally intact regulatory T cell compartment or if this is unresponsive due to the inefficient selection of insulin-specific regulatory T cells that could be stimulated and maintained by Ins2 presentation by CD11c+ cells.

Based on the above considerations, we propose that further studies are required to fully dissect the potential role of Ins2 expression by CD11c+ cells in self-tolerance. Perhaps BMT in NOD-Ins2−/− recipient mice using NOD marrow together with infusion of non-obese resistant regulatory T cells (a related but non-diabetes prone strain), or even better regulatory T cells from insulin B9-23 TCR transgenic mice [53] could assist in addressing issues of levels of antigen expression as well as antigen specificity.

Author contributions

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

Martin-Pagola, Zahr, Molano, Vendrame, and Snowhite performed the experimental work. Martin-Pagola, Pileggi, Ricordi, Eisenbarth, Nakayama, and Pugliese conceived the study design. Pileggi, Molano, Martin-Pagola, Nakayama, and Pugliese performed data analysis and interpretation. Martin-Pagola, Pileggi, Eisenbarth, Nakayama and Pugliese co-wrote the manuscript.

Acknowledgment

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

This work was supported by the Diabetes Research Institute Foundation (Hollywood, FL), the Juvenile Diabetes Research Foundation (JDRF Center Grants 4-2004-361 and 4-2007-1056) and by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, grant DK-55969). F.V. is the recipient of JDRF Post-doctoral Research Fellowship Award (JDRF 3-2008-32). M.N. is supported by NIDDK award DK-80885.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Author contributions
  8. Acknowledgment
  9. References
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