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Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin-conditioned donor DCs could effectively induce CD4+CD25+Foxp3+ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro-differentiated CD4+CD25+Foxp3+ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow-derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4+CD25+Foxp3+ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro-induced CD4+CD25+Foxp3+ iTregs exerted donor-specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG−/− hosts upon coadoptive transfer with T-effector cells. The CD4+CD25+Foxp3+ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4+CD25+Foxp3+ iTregs were further able to induce endogenous naïve T cells to convert to CD4+CD25+Foxp3+ T cells. We conclude that rapamycin-conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen-specific CD4+CD25+Foxp3+ iTregs. Such in vitro-generated donor-specific CD4+CD25+Foxp3+ iTregs are able to effectively control allogeneic islet graft rejection.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) have been increasingly documented to play a critical role in suppressing allogeneic graft rejection and promoting allograft tolerance (1). In several tolerance regimens where Tregs have been implicated in induction and/or maintenance of tolerance (2,3), graft protection is transferable to naïve hosts via adoptive transfer of Tregs isolated from the tolerized hosts. These observations have generated significant interests in testing the possibility of utilizing ex vivo-generated CD4+CD25+Foxp3+ Tregs for therapeutic prevention of allograft rejection and donor-specific tolerance induction for transplantation.
Two types of Tregs exist in the periphery. Natural Tregs (nTregs) are thymic-derived CD4+CD25+Foxp3+ T cells, whereas induced Tregs (iTregs) are CD4+CD25+Foxp3+ T cells differentiated from CD4+CD25−Foxp3− T cells in the periphery (4,5). Consequently, two different strategies have been employed for ex vivo generation of antigen-specific CD4+CD25+Foxp3+ T cells, both with the ultimate goals of: (1) obtaining sufficient numbers of Tregs for adoptive transfer in vivo; and (2) enhancing antigen specificity, which could potentially enhance suppression potency and diminish unwanted nonspecific suppression (6,7). The first strategy expands nTregs under culture conditions that provide activation of T-cell receptor (TCR), costimulation signals and IL-2 (8). Anti-CD3/CD28 antibodies significantly expand the numbers of polyclonal nTregs in cell cultures, while further selection for direct and indirect alloantigen specificities significantly enhance the ability of such ex vivo-expanded nTregs to suppress allograft rejection (9,10). The second approach converts naïve CD4+CD25−Foxp3− T cells to CD4+CD25+Foxp3+ Treg cells under culture conditions that provide TCR and costimulation signals of appropriate strengths, but more importantly provide transforming growth factor-β1 (TGF-β1), the single best-documented potent inducer of Foxp3 expression (11). While adoptive transfer of ex vivo-induced Tregs has been shown to suppress autoimmunity in several disease models (12–14), their role in suppressing allogeneic transplant rejection is less clear. The characteristics of antigen-presenting cells, including but not limited to, Dendritic cells (DCs), that are conducive to ex vivo iTreg conversion are also not well defined (15,16).
Recent work has determined that DCs alternatively differentiated in the presence of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (referred to as R-DCs hereafter) selectively expand nTregs (17,18). Adoptive transfer of alloantigen-pulsed, recipient origin R-DCs prolonged allogeneic heart and skin graft survival (17,19). This acquired ability of R-DCs to inhibit graft rejection is likely a consequence of the effect of rapamycin on DC differentiation and maturation (20), although its effects on DC antigen uptake, presentation and cytokine production have also been described (21). Here, we show that DCs preconditioned with rapamycin are also potent inducers of iTreg differentiation in allogeneic cell cultures, and such ex vivo-differentiated iTregs effectively suppress islet allograft rejection in vivo.
BALB/c, C57BL/6, SJL, Thy1.1 congenic C57BL/6, C57BL/6.Rag−/− and C57BL/6.GFP-Foxp3 knock-in mice were purchased from The Jackson Laboratory. Mice were used according to protocols approved by the Animal Care and Use Committee at Northwestern University.
Conditions for BMDC generation, DC-T cell cocultures, purification of the resulting iTregs, Thy1.1 and Thy1.2 cells for adoptive transfers are detailed in Supporting Information.
DC viability was determined by annexin V and propidium iodide staining using the apoptosis detection kit from BD Biosciences (Franklin Lakes, NJ). Details for assessing DC antigen uptake/processing, RNA preparation, primer and probe information are provided in Supporting Information.
Details are provided in Supporting Information.
Statistical analysis was performed using Student’s unpaired t-test for mixed lymphocyte reactions, cytokine analysis and cell quantifications. Analysis of variance was used to analyze islet graft survival. P-values of <0.05 were considered to be statistically significant.
Bone marrow-derived donor (BALB/c) DCs were initially utilized in cocultures with recipient (B6) CD4+CD25−Foxp3−T cells in the presence or absence of TGF-β1 for 7–10 days. In addition, IL-2 was added to support Treg growth and retinoic acid (RA) (22,23) was added to synergize with TGF-β1 for Foxp3 induction. As shown in Figure 1A, in the absence of TGF-β1, activated T cells were induced to express cell surface CD25 without expression of Foxp3. In contrast, in the presence of TGF-β1, a distinct population of activated T cells also concurrently expressed high levels of Foxp3, albeit only a small percentage (2.7%). Recent reports suggest that DCs alternatively differentiated in pharmacological agents are more conducive for stimulating and expanding nTregs (17,24). We next asked whether preconditioning donor DCs with rapamycin could enhance their ability to induce Foxp3 upon coculture with naïve T cells. Surprisingly, a short preconditioning with rapamycin for 18 h after a 6-day standard BMDC differentiation culture significantly enhanced the donor DCs’ capacity for inducing Foxp3 expression in T cells in the presence of TGF-β1, such that up to 32.4% of CD4+ T cells became Foxp3+ (Figure 1A). We also studied the effect of TGF-β1 preconditioning on the DCs’ capacity for inducing Foxp3+ cells given previous work demonstrating tolerogenicity of TGF-β DCs (25). As shown in Figure 1A, although preconditioning of DCs with TGF-β1 alone only modestly enhanced the DCs’ ability to induce Foxp3+ T cells, it could further potentiate the effect of rapamycin such that pretreatment of DCs with a combination of rapamycin and TGF-β1 allowed induction of up to 45.7% of Foxp3+ T cells. Therefore, for subsequent experiments we used combined rapamycin + TGF-β1 18-h preconditioning of DCs (referred to as (R+T)-DCs hereafter). To evaluate the individual contribution by TGF-β1, IL-2 and RA in the generation of iTregs, we compared the above DC-T cultures with TGF-β1 alone, TGF-β1 + IL-2 or TGF-β1 + IL-2 + RA using (R+T)-DCs. As shown in Supporting Figure S1, TGF-β1 alone induced ~60% conversion to Foxp3+ cells, but with a poor cell yield. Addition of IL-2 increased the cell yield by 5-fold, but also enhanced single positive CD25+Foxp3− T cells (activation without conversion). Further addition of RA to TGF-β1 and IL-2 preserved both an increased cell yield and a high Foxp3 conversion rate (63%). The overall yield of the desired CD4+CD25+ T cells under this condition was on average 30–40% of input cells (Supporting Figure S1).
As shown in Figure 1B, compared to DCs without preconditioning, (R+T)-DCs expressed fairly comparable levels of cell surface class II (MFI: 536 ± 173 for (R+T)-DCs vs. 594 ± 186 for control DCs), CD40 (MFI 135 ± 49 vs. 187 ± 51), CD86 (MFI 321 ± 170 vs. 380 ± 180) and CD80 (245 ± 99 vs. 247 ± 79). The viability, antigen uptake/processing ability of the DCs were not altered by the preconditioning (Supporting Figures S2, S3).
We next measured cytokine production by (R+T)-DCs compared to control DCs by RT-PCR (Figure 1C). (R+T)-DCs expressed ~50% of TNF-α compared to control DCs whereas TGF-β1 expression was enhanced by ~3-fold (p = 0.0173 and 0.0397 for TNF-α and TGF-β, respectively, from five individual experiments). No differences were observed in the expression of IL-10, indoleamine 2,3-dioxygenase (IDO), or IL-12p40.
We examined the effect of DC preconditioning on naïve T-cell activation, proliferation and IFN-γ production in a 5-day mixed lymphocyte reaction (MLR). As shown in Figure 1D, R-DCs and (R+T)-DCs exhibited progressively decreased ability to stimulate CD4+ T-cell activation measured by up-regulation of CD25. Among the activated T cells, there was a significant delay in cell proliferation measured by CFSE dilution. The calculated percentage of precursors of the activated CD25+ population that underwent division was 65%, 43% and 33% for cultures with control DCs, R-DCs and (R+T)-DCs, respectively. Furthermore, there was a profound reduction in IFN-γ production by T cells cocultured with preconditioned DCs, most markedly with (R+T)-DCs.
Phenotypically, the CD4+CD25+ T cells induced by (R+T)-DCs in the presence of TGF-β1 exhibited upregulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) (Figure 1E) (12) compared to naïve T cells. In addition, expression of the homing receptor L-selectin (CD62L) was downregulated compared to that of naïve T cells, although a significant percentage (~26%) remained CD62L+ (Figure 1E). Interestingly, CD4+CD25+ T cells resulting from cultures with control DCs also showed upregulation of GITR and CTLA-4, although their expression of CD62L was further downregulated (~9%) compared to that seen in the CD4+CD25+ T cells induced by (R+T)-DCs (Figure 1E).
We enriched the resulting CD4+CD25+Foxp3+ T cells from the DC-T cultures by first depleting CD11C+ DCs followed by positive selection of CD25+ cells by MACS purification. On average, among the resulting CD4+CD25+ T cells, 60–70% were CD4+CD25+Foxp3+ with the remaining 30–40% being CD4+CD25+Foxp3− T cells. In vitro and in vivo functional assays were performed with such MACS-purified CD4+CD25+ T cells unless otherwise specified. We first asked whether the in vitro-differentiated CD4+CD25+ T cells exhibit regulatory function. We tested the ability of these cells to suppress MLRs. As shown in Figure 2A, the induced CD4+CD25+ T cells were anergic themselves to donor DC stimulation, and when added in graded amounts to responder cells, that is, naïve recipient T cells, they exerted a dose-dependent suppression of proliferation. This suppression is donor-specific, as suppression was not seen with third-party SJL stimulator cells. IFN-γ production by the recipient T cells was similarly inhibited by the induced CD4+CD25+ T cells in a dose-dependent, donor-specific manner (Figure 2B).
To test the in vivo suppressive activity of the in vitro-differentiated CD4+CD25+Foxp3+ T cells, we used an adoptive transfer model in RAG−/− mice. Diabetic (streptozotocin-treated) B6.RAG−/− mice transplanted with BALB/c islet grafts were adoptively transferred with 1 × 10 7 naïve B6 Thy1.2 T cells alone or together with 5 × 10 6 B6 CD4+CD25+ iTreg obtained as described above. As shown in Figure 3A, B6 Thy1.2 cells alone led to rejection of all islet grafts between day 14 and day 20 after adoptive transfer. In contrast, cotransfer of 5 × 106 iTregs significantly delayed islet graft rejection. The earliest rejection did not occur until day 25 after adoptive transfer, and 30% of the grafts were permanently protected. This protection is donor-specific, as transplanted third-party SJL islet grafts were not protected by the iTregs induced with BALB/c DCs.
Migration of iTreg to the islet graft was examined by histology. To do so, we took advantage of the Foxp3-GFP knock-in mice and generated Foxp3gfp+ donor-specific iTregs, FACS sorted for CD4+CD25+Foxp3gfp+ T cells, coinjected them with naïve B6 Thy1.2 T cells and tracked the migration of the Foxp3gfp+ cells into the islet graft. As shown in Figure 3B, at day 10 posttransplant, some Foxp3gfp+ iTreg cells have accumulated in the islet graft (top left panel, shown as yellow cells indicating double staining with GFP (green) and Foxp3 (red)). This accumulation persisted at day 17 posttransplantation (top middle panel) as the islet graft maintained robust insulin secretion (top right panel). Interestingly, there was also a significant number of Foxp3+GFP− cells (shown as red cells indicating single staining with Foxp3) in the islet graft, suggesting that a significant number of Treg cells from the coinjected B6 Thy1.2 population also infiltrated the islet graft. In contrast, significantly fewer numbers of Foxp3+GFP− cells were seen in islet grafts from mice injected with B6 Thy1.2 cells alone without the iTreg (Figure 3B lower panels). Consistent with histological findings, FACS analysis of islet grafts at day 14 posttransplantation showed that in (+)iTreg grafts, 24.4% of total CD4+ cells were Foxp3+, whereas in (−)iTreg grafts, only 5.9% of total CD4+ cells were Foxp3+ (data not shown).
We next investigated the in vivo behavior of the induced CD4+CD25+Foxp3+ T cells after adoptive transfer. To do so, we coinjected Thy1.2 iTreg cells with congenic Thy1.1 naïve B6 cells at a 1:2 ratio as above in RAG−/− mice bearing allogeneic islet grafts, and tracked the two populations in the draining lymph nodes (dLNs), nondraining LNs (non-dLNs) and the spleen. As shown in Figure 4A, 14 days postadoptive transfer, the ratio of Thy1.2 iTregs to Thy1.1 cells remained relatively close to 1:2 in most compartments with the exception of the blood where the ratio dropped to ~1:10 (data not shown). Furthermore, these Thy1.2+ iTregs were able to maintain high levels of expressions of Foxp3 and CD25 in the dLNs (74% Foxp3+CD25+) and the non-dLNs (57% Foxp3+CD25+), although there was a significant loss of the phenotype of this population in the spleen (9% Foxp3+CD25+) (Figure 4A). The injected iTregs underwent multiple cycles of cell division within the first 7 days of adoptive transfer in the dLNs in the RAG−/− hosts, as shown by CFSE dilution (Figure 4B). However, similar degrees of cell division were seen in the spleen and non-dLNs (data not shown). In addition, when we examined as early as 3 days postadoptive transfer, again similar degrees of cell divisions were seen among the dLNs, non-dLNs and the spleen (data not shown). These findings suggested that this proliferation likely represented homeostatic proliferation in the lymphopenic RAG−/− hosts (26,27), rather than antigen-driven proliferation. Significantly, despite rigorous cell proliferation in this setting, the iTreg phenotype was well maintained in the graft dLNs as shown in Figure 4B.
We next examined whether the coadoptively transferred iTregs would suppress priming of effector cells in vivo. To do so, we adoptively transferred Thy1.2 iTregs and Thy1.1 naïve B6 T cells, and examined the Thy1.1+ cells 2–3 weeks posttransfer. As shown in Figure 5A, in the dLN of the graft, Thy1.1+ cells showed downregulated CD4+IFN-γ hi population in the (+) iTreg mice compared to the (−) iTreg mice (from 14.1% to 9.1%, ~35% decrease). A similar diminishment in CD8+IFN-γ hi population was also seen (from 10.9% to 8.1%, ~26% decrease). Interestingly, decrease of the IFN-γ hi populations was not seen in the spleen or non-dLNs (Figure 5A). We then further subjected these T cells to in vitro restimulation with donor APCs. As shown in Figure 5B, T cells from the (+) iTreg recipient mice showed significantly fewer IFN-γ producing CD4+ and CD8+ cells compared to T cells from the (−) iTreg mice (Figure 5B) upon in vitro restimulation. These findings suggest that the cotransferred iTregs significantly inhibited priming of both donor-specific CD4+ and CD8+ effector cells in vivo.
Interestingly, when gated on Thy1.1+ cells, a significantly higher percentage of Thy1.1+Foxp3+CD25+ T cells were seen in the dLNs of the (+) iTreg mice (35.2%) compared to the (−) iTreg mice (23.4%) (p = 0.03 from 2 independent experiments) 2–3 week posttransfer (Figure 6A). This difference was not observed in non-dLNs or the spleen. This suggested that the presence of the Thy1.2+ iTregs in the dLNs enhanced the recruitment/expansion and/or induction of Tregs from the comigrated Thy1.1 T cells. To differentiate between expansion of existing Tregs versus induction of new Tregs from the Thy1.1+ cells, we further sorted for the CD25− fraction from Thy1.1+ cells prior to injection. As shown in Figure 6B, despite the removal of the CD25+ Treg population prior to adoptive transfer, there was a small but distinct population (~5%) among Thy1.1+ cells that became Foxp3+ in the dLNs of the (+) iTreg mice, which was not seen in the (−) iTreg mice. This finding suggests that together with recruitment/expansion of existing Tregs, de novo induction of new Tregs by the injected iTregs contributed to the enhanced number of endogenous Tregs seen in the graft dLNs.
In this article, we show that donor DCs preconditioned with rapamycin could effectively differentiate CD4+CD25+Foxp3+ Tregs from naïve CD4+CD25−Foxp3−T cells of recipient origin. TGF-β further synergizes with rapamycin in increasing the efficiency of this differentiation. The mechanisms by which combined rapamycin and TGF-β signaling increases the ability of DCs to induce de novo Foxp3 expression in T cells are not clear. We have observed some, but not dramatic, differences in expression of class II and costimulatory molecules between preconditioned and control DCs (Figure 1B). In addition, the short overnight preconditioning led to decreased TNF-α but enhanced TGF-β production by the DCs (Figure 1C). These findings are consistent with published data demonstrating the involvement of AKT-mTOR in the activation of NF-κB, which can subsequently activate TNF-α expression via several NF-κB binding sites in its promoter (28,29). Others have shown that mTOR inhibition also leads to induction of IL-1β production and subsequent upregulation of IL-1R-like 1 (20). TGF-β1 has also been shown to positively regulate its own expression in a number of cell types (30,31). It is not clear if these effects are independently or synergistically exerted by rapamycin and TGF-β1 in our system. It is also not clear if the preconditioned DCs are resistant to maturation signals such as CD40 stimulation or if their phenotype will further alter upon interaction with alloreactive T cells. Regardless, the combined effects of mTOR inhibition and TGF-β on DCs ultimately compromise the DCs’ ability to prime effector responses (Figure 1D), while favoring differentiation of iTregs. Indeed, Turnquist et al. have showed that rapamycin-conditioned DCs preferentially allowed enrichment of Foxp3+ Tregs, although there the enriched Tregs were a consequence of expansion of nTregs, rather than de novo induction of iTregs (18). To our knowledge, ours is the first report to describe the efficacy of rapamycin-conditioned DCs for de novo induction of CD4+CD25+Foxp3+ iTregs in vitro. In addition to providing a potential avenue for in vitro induction of antigen-specific Tregs for therapeutic uses, this model will likely allow further understanding of signaling requirements of DCs in the process of peripheral induction of Tregs through careful cellular and molecular analysis.
CD4+CD25+Foxp3+ Tregs have been shown to mediate transplant tolerance in a variety of experimental systems. In human organ and bone marrow transplantations, favorable graft outcome has also been frequently associated with a robust Treg population (32–34). While the use of expanded nTregs has been more widely studied (8,35–38), generation of iTregs carries the advantage of having abundantly available precursors and not having to sort for the small population of nTregs through cumbersome purification. However, upon adoptive transfer, just as nTregs, iTregs encounter similar issues with respect to stability of Foxp3 expression, engraftment, trafficking and homing and maintenance of suppressive capacity (39,40). We have shown that iTregs induced by allogeneic (R+T)-DCs readily traffic to the allograft and graft dLNs. They maintain fairly stable expression of Foxp3 in the dLNs, but quickly lose Foxp3 expression in the spleen. This pattern of stability of Foxp3 expression suggests that continuous presence of cognate antigens contributes in part to the maintenance of iTreg phenotype. Importantly, we have not observed conversion of iTregs to IFN-γ or IL-17 secreting cells (data not shown) despite the reported plasticity of iTregs in other models (40).
Significant and reproducible prolongation of islet graft survival was observed here by adoptive transfer of 5 × 106 MACS-purified iTregs with 1 × 107 naïve T cells. This ratio of Treg: naïve T cells is lower or comparable to that used in several other published reports using ex vivo expanded Tregs (9,10,41). FACS sorting for Foxp3GFP+ cells might further enrich cells with true regulatory function, therefore further enhance in vivo suppression, although this approach would be clinically less feasible. Additionally, several means for improving the iTreg efficacy can now be explored, particularly in the context of islet cell transplantation. First, it has been shown that initial trafficking of Tregs to the inflamed islet allograft is critical for suppression of alloimmunity (42). Consequently, direct deposition of Tregs to the islet allograft might be more efficacious in preventing graft rejection. Second, as acute rejection progresses, alloimmunity of indirect antigen specificities becomes predominant. Consequently, Tregs with indirect antigen specificities might be more potent in suppressing later-stage alloimmunity, therefore should be generated and tested in our system. This concept has been recently demonstrated in models of chronic rejection (10,43). Third, it is likely that in order to establish and maintain a favorable Treg: Teff ratio, repetitive injections of iTregs and perhaps at higher doses would be necessary. Last, the rigorousness of antigen specificity of the iTregs generated in our system warrants further investigation. In this regard, further experiments comparing our iTregs to anti-CD3/CD28-induced polyclonal iTregs or nTregs as well as using crisscross strain combinations will provide definitive evidence for donor-antigen specificity.
Another characteristic of the iTregs induced by (R+T)-DCs in our system is their ability to recruit endogeneous Tregs to the dLNs (Figure 6A) and islet grafts (Figure 3B). While Treg lymphoid homing has been associated with CD62L, CCR7 and possibly CXCR5, Treg trafficking to transplant organs/tissues requires different signals including CCR4, CCR5 and CD103 (44). Local elaboration of TGF-β and IL-2 may further allow expansion of migrated Tregs. It is unclear in our model what signals are utilized by the iTregs to recruit/expand endogenous Tregs to the dLNs or islet grafts. In addition, they also seem to have the ability to convert endogenous naïve T cells to become additional induced Tregs (Figure 6B), a process that has been previously described as infectious tolerance (45,46). This process is thought to mediate through antigen-driven interactions with the same APCs by Tregs and naïve T cells (45,46). Therefore, it is not surprising that this was only observed in the dLNs where such interactions are likely to be most robust due to the relative high density of alloantigen-loaded APCs. This process can potentially prompt a cascade of self-amplification that helps maintain a robust state of allotolerance. iTregs induced by various protocols may have different ability to mediate infectious tolerance depending on the multitude of mechanisms involved, including expression of cell surface TGF-β (47), catabolism of essential amino acids (48,49) or generation of extracellular adenosine (50). Additional optimization of culture protocols may further enhance this ability and contribute to enhanced suppression of the generated iTregs through infectious tolerance.
In clinical transplantation, it is likely that induction therapies that debulk host lymphocytes will remain necessary to establish an initial favorable Treg: Teff ratio (51). Therefore, the RAG−/− model employed here is highly relevant in that it allows examination of iTreg behavior in a lymphopenic environment, similar to that created by depletion therapies. Indeed, therapies utilizing ex vivo-generated Tregs have invariably been used in combination with transient lymphocyte depletion for durable efficacy (9,41,52). Ongoing experiments are testing the efficacy of our iTregs in immune competent mice in combination with transient immunosuppression.
In summary, we have demonstrated that donor BMDCs pharmacologically conditioned with rapamycin and TGF-β are potent inducers of recipient CD4+CD25+Foxp3+ Tregs in in vitro cultures in the continued presence of TGF-β. Such in vitro-differentiated Tregs suppress acute rejection of islet allografts, maintain relatively stable phenotype and possibly mediate infectious tolerance in vivo. This protocol further provides the basis for future exploration of in vitro-generated human iTregs for a variety of clinical applications.
This work was supported in part by the National Institute of Health Career Award 1K08DK070029-01 (XL), Type 1 Diabetes Pathfinder Award DP2DK083099-01 (XL) and the Juvenile Diabetes Research Foundation Regular Research Grant 1-2007-1005 (SDM and XL).
Conflict of Interest Statement
The authors declare no conflict of interest.
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