A Defect in Lineage Fate Decision during Fetal Thymic Invariant NKT Cell Development May Regulate Susceptibility to Type 1 Diabetes1
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免疫学杂志 2005年第11期
Abstract
A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4+CD8+ double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4–CD8– double-negative T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.
Introduction
The NOD mouse spontaneously develops a form of type 1 diabetes (T1D)3 characterized by the progression from insulitis (infiltration of dendritic cells, macrophages, T cells, and B cells into pancreatic islets) at 3–4 wk of age to islet cell destruction and the onset of disease during 4–6 mo of age. Self-reactive T cells can be isolated from mice and humans free of autoimmune disease (1, 2). Because protection against an autoimmune disease in healthy mice and individuals may be mediated by different types of regulatory T cells, it follows that a deficiency in the number and function of a given subset(s) of regulatory T cells can mediate the onset of such a disease, including T1D (1, 2, 3).
Invariant NK T (iNKT) cells constitute a subset of regulatory T cells that express NK cell markers and a highly invariant TCR, V14J18, V8.2/7/2 in mice that can recognize lipid Ags in a CD1-restricted manner (3). A numerical and functional deficiency in iNKT cells is detectable both in PBLs of diabetic patients (2) and in the thymus and spleen of prediabetic NOD mice as early as 3 wk of age (3, 4, 5). An increase in the number of iNKT cells that leads to a correction of this deficiency in NOD mice is sufficient to decrease insulitis and prevent T1D in several experimental systems, including intrathymic transfer, enhanced development through transgenic expression of the iNKT TCR chain (V14J18), and proliferation induced by activation through the CD1d-restricted sphingoglycolipid Ag, -galactosylceramide (-GalCer) (6, 7, 8). Nonetheless, our understanding of the mechanism(s) of protection from T1D in NOD mice by iNKT cells requires further investigation.
Fully developed CD4+ and CD8+ T cells can first be detected in an embryonic murine thymus ex vivo on the day of birth (19 days postconception, embryonic day 19 (E19)) (9), and embryonic thymus-derived T cells from NOD mice are capable of infiltrating a syngeneic pancreas in vitro (10). It is conceivable, therefore, that T cells capable of infiltrating pancreatic islets might already be developed in these mice by day 1 after birth. Yet, despite this early generation of potentially autoreactive T cells, it is curious that a deficiency in iNKT cells in the thymus of NOD mice was reported to first manifest at the time of peri-insulitis, i.e., at 3 wk of age (11). To resolve this apparent discrepancy and given that defective T cell activation occurs concomitantly with the onset of insulitis in NOD mice (12), we investigated whether a deficiency in iNKT cells arises earlier than 3 wk of age, and in particular during embryonic thymic T cell development. We reasoned that if this were the case, it might mediate the early pathogenesis of T1D and render NOD mice more susceptible to T1D at birth.
Similar to conventional T cells, iNKT cells pass through the CD4+CD8+ double-positive (DP) stage of T cell development and undergo positive and negative selection (13, 14). Moreover, several stages of embryonic T cell development are conserved with respect to specificity and order during adult T cell development. Embryonic T cells pass through the four CD4–CD8– double-negative (DN) stages of development between E12 and E14, a DP stage that appears at E17-E18 and a mature T cell stage at E19-day 1 after birth (9, 15). CD4+ or CD8+ single-positive (SP) thymocytes that appear at E15-E17 are phenotypically (CD4+ or CD8+, TCR–) and functionally consistent with that of a precursor to DP thymocytes, initially described in the adult thymus as an immature SP (ISP) T cell (11, 16). Despite reports that common precursors mediate embryonic and adult T cell development and accumulating evidence for a conventional pathway of development for mainstream T cells and iNKT cells, an alternate T cell pathway of development has been proposed for both types of T cells (17, 18, 19, 20, 21).
In this study, we further investigated the kinetics and pathway of development of iNKT cells in NOD mice by analyzing whether a deficiency in iNKT cells first occurs during embryonic intrathymic T cell development, and if so, at what stage of intrathymic T cell development. We found that an intrathymic iNKT cell deficiency in both embryonic and adult NOD mice is not present at the DP thymocyte stage of development, but rather manifests subsequent to positive selection of DP thymocytes from a lineage-specific depletion of DN T cells shortly before birth during E18-day 1. The latter DN T cells are enriched in DN iNKT cells. Our findings suggest that during embryonic thymic T cell development in NOD mice, conventional T cells and iNKT cells develop along a common pathway until the DP stage, after which iNKT cells and DN T cells are depleted and result in an iNKT cell deficiency. We also present data that suggest that the latter intrathymic iNKT cell deficiency may be corrected by stimulation of development with the Notch-1 ligand, Delta-1.
Materials and Methods
Fetal thymic lobes were collected at E13 and cultured in complete RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-ME, 100 IU/ml penicillin, 1 mM sodium pyruvate, and 15 mM HEPES buffer, pH 7.3. RPMI 1640 and all supplements were obtained from Life Technologies. Culture medium was also supplemented with soluble Delta-1-Fc or Jagged-1-Fc (a kind gift from M. Bhatia, Robarts Research Institute, London, Ontario, Canada), where indicated. Lobes (10–15 per well) were cultured in a six-well plate (Costar) at 37°C and 7% CO2 for 3–21 days with a partial medium replacement every 3 days.
Flow cytometry
Thymocytes (105-106) were stained at 4°C for 1.5 h and washed three times with PBS/FBS. Fetal thymic culture samples were preincubated in propidium iodide (Sigma-Aldrich) for 15 min before analysis. A FACSCalibur with CellQuest software (BD Biosciences) and FlowJo software (Treestar) was used for flow cytometry. Dead cells were excluded through gating based on forward and side scatter. For fetal thymic organ cultures, dead cells were further excluded by gating on cells negative for propidium iodide staining. Adult thymocytes were stained with -GalCer-loaded CD1d tetramers conjugated to PE, anti-TCR FITC, anti-CD8 CyChrome (PE Cy5), and anti-CD4 allophycocyanin. Fetal thymocytes were stained with -GalCer-loaded CD1d tetramers allophycocyanin, anti-TCR FITC, anti-heat-stable Ag FITC, anti-CD8 PE, or anti-CD4 allophycocyanin. To evaluate any nonspecific binding of the latter test Abs to NOD and B6 adult and embryonic thymocytes, the following isotype controls were used: hamster IgG2 FITC (for anti-TCR FITC), rat IgG2a PE Cy5 (for anti-CD8 PE Cy5), rat IgG2a allophycocyanin (for anti-CD4 allophycocyanin), rat IgG2b FITC (for anti-heat-stable Ag FITC), and rat IgG2a PE (for anti-CD8 PE). All mAbs used for staining were obtained from BD Biosciences. -GalCer-loaded PE or allophycocyanin-conjugated CD1d tetramers were prepared in our laboratory, as described (22).
Results
iNKT cells are depleted after the DP stage in an adult NOD thymus
The pathway of iNKT cell development is similar to that of conventional T cell development, and includes a precursor at the DP thymocyte stage of development (13). To determine whether iNKT cell development in a NOD thymus is blocked at or before the DP stage of thymocyte development, we examined female NOD and C57BL/6 (B6) thymi at 8 wk of age for their numbers of iNKT and conventional TCR T cells in the DP, mature CD4+ or CD8+ SP, and DN thymocyte subsets (Fig. 1). iNKT cells were detected by double staining with an anti-TCR mAb and -GalCer-loaded CD1d tetramers (Tet+), as described (8). FACS analyses demonstrated that NOD mice are deficient in DN thymocytes at this age (Fig. 1A). This DN thymocyte deficiency consists of an iNKT cell (Tet+TCR+) deficiency in a NOD thymus relative to a B6 thymus (Fig. 1B). Note that the number of iNKT cells is also reduced in CD8+, CD4+ SP, and DP thymocytes, but to a much lesser extent.
iNKT cell deficiency results from a lineage-specific depletion of DN T cells
CD4 and CD8 coreceptors augment physical interactions between TCR and MHC in the periphery and define a Th cell or cytolytic T cell function. Due to their influence on T cell function, CD4 and CD8 lineage fate decisions in the thymus have been studied extensively, whereas DN T cell lineage decision has often been overlooked (23). To determine whether the depletion of DN iNKT cells may be due to a lineage decision event, we examined the numbers of conventional T cells (Tet–TCR+) in the thymus of 8-wk-old NOD and B6 mice. We found that the number of conventional DN T cells was significantly decreased in a NOD thymus relative to a B6 thymus (Fig. 1B). In contrast, a significantly greater number of CD4+ T cells and CD8+ T cells were detected in a NOD thymus than a B6 thymus. These results indicate that a lineage-specific depletion of DN T cells after the DP stage of development may mediate an iNKT cell deficiency detectable in the thymus of an 8-wk-old NOD mouse.
iNKT cell deficiency arises during NOD fetal thymic development
At E13 in a fetal mouse, the first wave of common lymphoid precursors enters into the thymic lobes. These precursors are predicted to give rise to T cells between days 1 and 14 after birth (24). To determine whether this first wave of thymic precursors in a NOD thymus results in an iNKT cell deficiency, NOD and B6 thymic lobes were removed from embryos at E13 and introduced into fetal thymic organ culture. This prevents further input of thymic precursors or other developmental stimuli. Interestingly, E13 NOD thymic precursors developed 10-fold fewer iNKT cells than age-matched B6 thymic precursors (Fig. 2). This iNKT cell deficiency was apparent in an E13 NOD thymus despite the fact that 4- to 5-fold more conventional T cells developed in a NOD thymus than in an age-matched B6 thymus (Fig. 2). Thus, an iNKT cell deficiency in NOD mice arises in an E13 thymus in the absence of in vivo developmental stimuli or further common lymphoid precursor input.
NOD and B6 fetal thymocytes develop at comparable rates
Thymocyte development in a fetal B6 and BALB/c thymus parallels that of an adult thymus and progresses through three main precursor stages of mature T cells: DN TCR– thymocytes, ISP CD4+ or CD8+ TCR– thymocytes, and DP TCR–/+ thymocytes (9, 15). To ensure that fetal NOD thymocytes develop at a rate comparable to that in B6 and BALB/c mice and with the same main precursor stages during positive and negative selection, we examined the main precursor stages and phenotype of mature T cells by FACS analyses beginning at E17, the initial time of appearance of DP thymocytes in fetal B6 mice. From E17 to day 14 after birth, NOD and B6 thymi consist of similar populations of DN, DP, and CD4+ or CD8+ SP T cells (Fig. 3A). Gating on DN, DP, and CD4+CD8– or CD4–CD8+ populations, according to Fig. 3A, all NOD and B6 thymocytes were found to be TCR– at E17 (Fig. 3B). These findings indicate that CD4+ and CD8+ SP thymocytes are ISP T cells at this stage, and that development has progressed in both strains to an early DP stage before TCR-mediated positive or negative selection. At E18, DP thymocytes begin to express TCR, allowing for positive and negative selection, and mature T cells expressing TCR emerge among the CD4+, CD8+, and DN subsets from E18 to day 1 after birth. This finding suggests that positive and negative selection may occur for the first time at E18 in both strains. The distribution of thymocyte subsets from day 1 to 14 in NOD and B6 mice resembles that of adult mice as more DN, CD4+, and CD8+ SP T cells are evident. During this period of development, the latter subsets further mature, as indicated by the loss of CD24 expression (Fig. 3B). Thus, phenotypic analyses of fetal B6 and NOD thymocytes indicate that DP TCR+ thymocytes develop and allow for the positive and negative selection of mature T cells (TCR+, CD24+/–) at E18.
Deficiency in DN T cells and iNKT cells develops before birth
An iNKT cell deficiency in NOD mice was initially demonstrated as a DN T cell deficiency, which after correction by intrathymic transfer of syngeneic DN TCR+ thymocytes prevents insulitis and T1D (6). Our results indicate that reduced numbers of both DN iNKT cells and conventional T cells are present in an adult NOD thymus compared with a B6 thymus (Fig. 1). Furthermore, an iNKT cell deficiency is most prominent in DN TCR+ T cells after the DP stage of thymocyte development in an adult NOD thymus (Fig. 1B). To determine whether a similar depletion of DN TCR+ T cells begins with the emergence of TCR+ DP thymocytes at E18, we examined the distribution of thymocyte populations during late embryogenesis. At E18, a deficiency of mature DN T cells (TCR+CD24–) was found to develop in a NOD thymus relative to a B6 thymus (Fig. 3B). This deficiency arises concomitantly with the emergence of DP TCR+ thymocytes, which time it is presumably followed by TCR-mediated positive and negative selection. A role for TCR-mediated positive and negative selection in DN T cell depletion is strengthened by the finding of consistently lower numbers of CD24+ DN T cells in a NOD thymus. The CD24+ DN T cell is a precursor to mature DN T cells (TCR+CD24–), and it arises immediately after the DP thymocyte stage. This CD24+ DN T cell subset was consistently low in number in a NOD thymus beginning at E18, and was detectable in statistically significant numbers only by day 1 after birth. At the latter time, significantly more mature CD4+ and CD8+ SP T cells develop in a NOD thymus than B6 thymus (Fig. 3B). These results are similar to that found in an adult NOD thymus, indicating that in both embryonic and adult NOD there is a lineage-specific deficiency of DN T cells.
Previously, the earliest detection of iNKT cells by flow cytometry was reported to be 0.01% of thymocytes on day 5 after birth in a B6 thymus (25). iNKT cells comprise only 0.3–0.4% of adult NOD thymocytes, and are therefore difficult to detect by flow cytometry until adequate numbers of mature T cells have accumulated. To circumvent this technical difficulty and estimate the relative number of iNKT cells during thymic development, we used real-time PCR to quantify the expression of invariant TCR V14J18 chain mRNA specific for iNKT cells relative to that of -actin mRNA. This approach was used to determine whether selection of NOD TCR+ DP thymocytes at E18 depletes iNKT cells and results in an iNKT cell deficiency at birth. Fig. 3 shows that both NOD and B6 thymocytes progress to an early DP stage at E17 without detectable TCR surface expression. As TCR rearrangement is reported to occur at E17 (26), we were unable to detect V14J18 mRNA expression at E13 or E15 (our unpublished observations). As TCR mRNA is readily detected at E17, it is reasonable to assume that the majority of TCR mRNA detected at this stage is expressed by the large group of TCR– DP thymocytes, which first develop at this time with a rather low level of TCR mRNA expression in DN4 and ISP thymocytes. Thus, V14J18 mRNA levels at E17 should be indicative of the relative number of iNKT cells at an early DP or less mature precursor stage.
At E17, the ratio of V14J18 mRNA:-actin mRNA was found to be greater in NOD thymocytes than in B6 thymocytes (Fig. 4). This finding suggests that a larger number of iNKT cell precursors and/or a higher level of TCR mRNA expression is present in a NOD than B6 thymus at and before the TCR– DP stage of thymocyte development. At E18, DP thymocytes express TCR on their surface, which enables TCR-mediated positive and negative selection. This results in the emergence of mature DN T cells and a decrease in the level of V14J18 mRNA in NOD thymocytes, which most likely accounts for the similar ratio of V14J18 mRNA:-actin mRNA observed in NOD and B6 thymocytes at E18 (Fig. 4). At days 1 and 7 after birth, the estimated iNKT cell deficiency becomes more pronounced in a NOD thymus relative to a B6 thymus. Similar to the findings in adult NOD thymocytes, this kinetic analysis of iNKT cell depletion in fetal NOD thymocytes suggests that depletion occurs during the positive and negative selection of DP thymocytes between E18 and day 1 after birth.
Delta-1 stimulates the development of thymic iNKT and conventional T cells
The Notch family of molecules is linked to many aspects of T cell development, including the CD4/CD8 lineage decision (27, 28). The Notch ligand, Delta-1, was recently shown to promote conventional T cell development during the first three stages of DN thymocyte development (29, 30). Delta-1, but not Jagged-1, enhances the development of cells with a phenotype of conventional T cell and NKT precursors in cultures of human cord blood cells (31). To determine the effect of Delta-1 and Jagged-1 on iNKT cell development, soluble Fc-conjugated forms of these ligands were added to cultures of E13 B6 DN thymocytes, and the development of iNKT cells was estimated by semiquantitative RT-PCR analyses of V14J18 mRNA. Delta-1-Fc, but not Jagged-1-Fc, stimulated V14J18 mRNA expression in a dose-dependent manner (Fig. 5A). To determine whether Delta-1 enhances iNKT cell development by a pathway independent of conventional T cells, as previously suggested (17, 18, 19), fetal thymic organ cultures established in the presence or absence of Delta-1-Fc and Jagged-1-Fc were examined for V14J18 and control V3J15 expression by RT-PCR. TCR V3J15 is a gene that was characterized in the B6 T cell repertoire, and primers were designed to study the rearrangement of this gene based on analyses of its expression in a conventional CD4+ T cell reactive to proinsulin-derived C peptide (32) (National Center for Biotechnology Information accession: Z86026). Delta-1-Fc and Jagged-1-Fc did not yield detectable changes in the number of thymocytes in these cultures (our unpublished observations). However, the levels of TCR V14J18 (iNKT cell-specific) and TCR V3J15 (conventional T cell-specific) mRNA were significantly increased in thymocytes cultured in the presence of Delta-1-Fc, but not Jagged-1-Fc (Fig. 5, B and C). These observations suggest that Delta-1 can stimulate the development of iNKT cells in addition to conventional T cells.
Next, we determined whether the relative level of Delta-1 expression is decreased in a NOD fetal thymus, and if so, whether this decreased expression mediates the development of an iNKT cell deficiency in NOD mice. Comparison of the ratios of Delta-1:-actin mRNA levels expressed in NOD and B6 E13 thymocytes by real-time PCR revealed that the relative level of Delta-1 mRNA expression is reduced 2-fold in NOD vs B6 thymocytes (Fig. 5D). These findings suggest that a decrease in Delta-1 expression during T cell development may mediate the generation of the intrathymic iNKT cell deficiency noted in NOD embryos.
Discussion
Our results demonstrate for the first time that an iNKT cell deficiency, which is known to be associated with susceptibility to T1D, develops in NOD mice before birth. Previous studies of this deficiency failed to detect iNKT cells either in a NOD fetal thymus or in a newborn NOD thymus before 3 wk of age (26). An earlier attempt to detect V14J18 mRNA in a B6 fetal thymus by PCR analysis was unsuccessful possibly due to the fact that E9 embryos were used and DP thymocytes with V14J18 gene rearrangement appear in the thymic primordium 8–9 days later. Indeed, we were also unable to detect V14J18 mRNA in E13 or E15 thymic lobes (our unpublished observations). Moreover, the inability to detect iNKT cells in a NOD thymus by flow cytometry before 3 wk of age is not surprising, because this population comprises only 0.3% of thymocytes in a B6 thymus that contains a higher percentage of iNKT cells at 3 wk after birth (25). iNKT cells comprise a relatively small subset in the thymus, and require several rounds of T cell development to accumulate and become detectable by tetramer staining and flow cytometry.
Application of a real-time PCR approach enabled us to show that an iNKT cell deficiency begins with a depletion of these cells at E18, and that this deficiency is evident in NOD mice on day 1 after birth. Although adult and embryonic T cell development are often assumed to differ (21), we contend that many of these differences may be minor and ascribable to the different stages of development that occur in the thymus at a given day during embryonic life. Our results indicate that in NOD mice the development of an autoimmune T cell repertoire develops before birth and is then conserved during adult T cell development. This raises the issue that further investigation of the similarities between embryo and adult T cell development is required. Moreover, the fact that a day 7 neonatal mouse is considered to be the immunological equivalent of a newborn human (33) coupled with the finding that an iNKT cell deficiency is established by day 1 after birth in NOD mice further emphasizes the need for additional research to be directed toward the early detection and treatment of T1D in humans.
A defect in central tolerance (negative selection) has been reported to be causal to susceptibility to T1D in NOD mice (34, 35). In addition, NOD thymic epithelium, which is enriched in self Ag for positive and negative selection, was shown to induce insulitis upon transfer into diabetes-resistant B6 mice (36). Furthermore, our demonstration in this study that an intrathymic deficiency in iNKT cells appears after the DP thymocyte stage in embryonic NOD mice points toward a defect in positive selection of iNKT cells in these mice. Collectively, these findings suggest that central tolerance, including defects in the negative selection of autoreactive T cells and positive selection of regulatory T cells, such as iNKT cells, is an important factor that determines susceptibility to T1D in NOD mice.
We have obtained evidence that the depletion of iNKT cells from the thymus that occurs after the DP stage of development may be lineage specific. This conclusion is strengthened by our detection of a numerical deficiency in DN conventional T cells and iNKT cells both in a NOD adult thymus at 8 wk of age and in a NOD fetal thymus at E18. Recent reports indicate that iNKT cells and conventional T cells follow a similar developmental pathway, including progression through the DP stage of T cell development and positive and negative selection (13, 14). Based on our results in this study, we propose that DN conventional T cells and DN iNKT cells make a common lineage decision, and that this lineage decision is defective in a developing NOD thymus according to a model shown in Fig. 6. Pre-iNKT DP thymocytes and earlier precursors are not deficient in a fetal NOD thymus; in fact, these pre-iNKT thymocytes appear to be more numerous in a NOD than B6 thymus at E17. TCR protein expression in DP thymocytes at E18 allows for a selection event that preferentially depletes DN T cells. This lineage-specific depletion of DN T cells results in an iNKT cell deficiency at day 1 after birth, and this deficiency is conserved after birth in the adult NOD thymus. Hence, our studies not only highlight the importance of analysis of the relationship of DN T cell fates and lineage decisions as they relate to the pathogenesis of autoimmune disease, but also provide the first indication that lineage decision in early intrathymic T cell development may govern susceptibility to autoimmune T1D.
In addition to thymic epithelial stimulation, other factors may also affect the positive or negative selection of iNKT cells in a NOD thymus. Intrathymic cotransfer of thymic precursors (1:1) from diabetes-susceptible NOD and diabetes-resistant ACK mice can normalize the frequency of TCR+DX5+ iNKT cells in a NOD thymus (37). The Notch ligand, Delta-1, may be the source of such a survival signal during the positive and negative selection of DP thymocytes. Previously, Delta-1 was shown to provide survival signals to all of three subsets of DN TCR– T cell precursors (29, 30), and to enhance T and possibly NKT cell development from human cord blood cells (31). Nonetheless, loss of function experiments in Delta-1 knockout mice demonstrated that T cell development was not altered in these mice possibly due to a compensation of function by Delta-4 in these mice (38). Despite functional overlap between Delta-1 and Delta-4, our finding that Delta-1, but not Jagged-1, can enhance conventional T and iNKT cell development supports the notion that Notch ligands differentially promote T cell development. Furthermore, our detection of a dose-dependent effect of Delta-1 on iNKT cell development demonstrates that despite overlap in Delta-1/Delta-4 function, the local concentration of Delta-1 in the thymus may affect T cell development. Thus, our observation that the amount of Delta-1 mRNA is significantly decreased in E13 NOD thymocytes compared with an age-matched B6 thymus supports the notion that a reduced level of Delta-1 protein in a NOD thymus may elicit in part the deficiency in DN T cells and iNKT cells in NOD mice. Future experimentation is required to investigate the effects of Delta-1 on the development of DN T cells, iNKT cells, and other T cell populations in NOD mice and the effects of altered T cell development on T1D development in NOD mice. Such analyses may also reveal whether a reduced intrathymic level of Delta-1 gives rise to a general thymic lymphopenia in NOD mice, which is purported to be a susceptibility factor for T1D in these mice (8, 29, 30, 39).
In conclusion, this study further underscores the important role that central tolerance may play in controlling the susceptibility to autoimmune disease, and T1D in particular. Our observations demonstrate that lineage fate decisions in positive selection during the intrathymic development of conventional DN and iNKT cells before birth may be an important feature of central tolerance that, if defective, can predispose to the outcome of autoimmune disease.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Juvenile Diabetes Research Foundation Grant 1-2001-351 (to T.L.D.), Canadian Institutes of Health Research Grant MOP 64886 (to T.L.D.), National Institutes of Health Grant RR 18789 (to J.M.V.), and Cobre grant in stem cell biology and regenerative medicine (to J.M.V.). T.L.D. is the Sheldon H. Weinstein Scientist at the Robarts Research Institute and University of Western Ontario.
2 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Robarts Research Institute, 100 Perth Drive, POB 5015, London, Ontario N6A 5K8, Canada. E-mail address: del{at}robarts.ca
3 Abbreviations used in this paper: T1D, type 1 diabetes; -GalCer, -galactosylceramide; E, embryonic day; DN, double negative; DP, double positive; iNKT, invariant NK T cell; SP, single positive; ISP, immature SP.
Received for publication December 9, 2004. Accepted for publication March 21, 2005.
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Laloux, V., L. Beaudoin, D. Jeske, C. Carnaud(Melany J. D. Wagner*, Sha)
A numerical and functional deficiency in invariant NKT (iNKT) cells detectable by 3 wk of age in the thymus and spleen mediates the pathogenesis of type 1 diabetes in NOD mice, but the stage of T cell development at which this deficiency first occurs is unknown. We report in this study that this deficiency develops after the CD4+CD8+ double-positive stage of thymic T cell development and is due to a lineage-specific depletion of CD4–CD8– double-negative T cells and iNKT cells from the thymus between embryonic day 18 and day 1 after birth. Thus, an inheritable defect in a lineage fate decision that elicits a deficiency in fetal thymic iNKT cell development may predispose to susceptibility to type 1 diabetes.
Introduction
The NOD mouse spontaneously develops a form of type 1 diabetes (T1D)3 characterized by the progression from insulitis (infiltration of dendritic cells, macrophages, T cells, and B cells into pancreatic islets) at 3–4 wk of age to islet cell destruction and the onset of disease during 4–6 mo of age. Self-reactive T cells can be isolated from mice and humans free of autoimmune disease (1, 2). Because protection against an autoimmune disease in healthy mice and individuals may be mediated by different types of regulatory T cells, it follows that a deficiency in the number and function of a given subset(s) of regulatory T cells can mediate the onset of such a disease, including T1D (1, 2, 3).
Invariant NK T (iNKT) cells constitute a subset of regulatory T cells that express NK cell markers and a highly invariant TCR, V14J18, V8.2/7/2 in mice that can recognize lipid Ags in a CD1-restricted manner (3). A numerical and functional deficiency in iNKT cells is detectable both in PBLs of diabetic patients (2) and in the thymus and spleen of prediabetic NOD mice as early as 3 wk of age (3, 4, 5). An increase in the number of iNKT cells that leads to a correction of this deficiency in NOD mice is sufficient to decrease insulitis and prevent T1D in several experimental systems, including intrathymic transfer, enhanced development through transgenic expression of the iNKT TCR chain (V14J18), and proliferation induced by activation through the CD1d-restricted sphingoglycolipid Ag, -galactosylceramide (-GalCer) (6, 7, 8). Nonetheless, our understanding of the mechanism(s) of protection from T1D in NOD mice by iNKT cells requires further investigation.
Fully developed CD4+ and CD8+ T cells can first be detected in an embryonic murine thymus ex vivo on the day of birth (19 days postconception, embryonic day 19 (E19)) (9), and embryonic thymus-derived T cells from NOD mice are capable of infiltrating a syngeneic pancreas in vitro (10). It is conceivable, therefore, that T cells capable of infiltrating pancreatic islets might already be developed in these mice by day 1 after birth. Yet, despite this early generation of potentially autoreactive T cells, it is curious that a deficiency in iNKT cells in the thymus of NOD mice was reported to first manifest at the time of peri-insulitis, i.e., at 3 wk of age (11). To resolve this apparent discrepancy and given that defective T cell activation occurs concomitantly with the onset of insulitis in NOD mice (12), we investigated whether a deficiency in iNKT cells arises earlier than 3 wk of age, and in particular during embryonic thymic T cell development. We reasoned that if this were the case, it might mediate the early pathogenesis of T1D and render NOD mice more susceptible to T1D at birth.
Similar to conventional T cells, iNKT cells pass through the CD4+CD8+ double-positive (DP) stage of T cell development and undergo positive and negative selection (13, 14). Moreover, several stages of embryonic T cell development are conserved with respect to specificity and order during adult T cell development. Embryonic T cells pass through the four CD4–CD8– double-negative (DN) stages of development between E12 and E14, a DP stage that appears at E17-E18 and a mature T cell stage at E19-day 1 after birth (9, 15). CD4+ or CD8+ single-positive (SP) thymocytes that appear at E15-E17 are phenotypically (CD4+ or CD8+, TCR–) and functionally consistent with that of a precursor to DP thymocytes, initially described in the adult thymus as an immature SP (ISP) T cell (11, 16). Despite reports that common precursors mediate embryonic and adult T cell development and accumulating evidence for a conventional pathway of development for mainstream T cells and iNKT cells, an alternate T cell pathway of development has been proposed for both types of T cells (17, 18, 19, 20, 21).
In this study, we further investigated the kinetics and pathway of development of iNKT cells in NOD mice by analyzing whether a deficiency in iNKT cells first occurs during embryonic intrathymic T cell development, and if so, at what stage of intrathymic T cell development. We found that an intrathymic iNKT cell deficiency in both embryonic and adult NOD mice is not present at the DP thymocyte stage of development, but rather manifests subsequent to positive selection of DP thymocytes from a lineage-specific depletion of DN T cells shortly before birth during E18-day 1. The latter DN T cells are enriched in DN iNKT cells. Our findings suggest that during embryonic thymic T cell development in NOD mice, conventional T cells and iNKT cells develop along a common pathway until the DP stage, after which iNKT cells and DN T cells are depleted and result in an iNKT cell deficiency. We also present data that suggest that the latter intrathymic iNKT cell deficiency may be corrected by stimulation of development with the Notch-1 ligand, Delta-1.
Materials and Methods
Fetal thymic lobes were collected at E13 and cultured in complete RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-ME, 100 IU/ml penicillin, 1 mM sodium pyruvate, and 15 mM HEPES buffer, pH 7.3. RPMI 1640 and all supplements were obtained from Life Technologies. Culture medium was also supplemented with soluble Delta-1-Fc or Jagged-1-Fc (a kind gift from M. Bhatia, Robarts Research Institute, London, Ontario, Canada), where indicated. Lobes (10–15 per well) were cultured in a six-well plate (Costar) at 37°C and 7% CO2 for 3–21 days with a partial medium replacement every 3 days.
Flow cytometry
Thymocytes (105-106) were stained at 4°C for 1.5 h and washed three times with PBS/FBS. Fetal thymic culture samples were preincubated in propidium iodide (Sigma-Aldrich) for 15 min before analysis. A FACSCalibur with CellQuest software (BD Biosciences) and FlowJo software (Treestar) was used for flow cytometry. Dead cells were excluded through gating based on forward and side scatter. For fetal thymic organ cultures, dead cells were further excluded by gating on cells negative for propidium iodide staining. Adult thymocytes were stained with -GalCer-loaded CD1d tetramers conjugated to PE, anti-TCR FITC, anti-CD8 CyChrome (PE Cy5), and anti-CD4 allophycocyanin. Fetal thymocytes were stained with -GalCer-loaded CD1d tetramers allophycocyanin, anti-TCR FITC, anti-heat-stable Ag FITC, anti-CD8 PE, or anti-CD4 allophycocyanin. To evaluate any nonspecific binding of the latter test Abs to NOD and B6 adult and embryonic thymocytes, the following isotype controls were used: hamster IgG2 FITC (for anti-TCR FITC), rat IgG2a PE Cy5 (for anti-CD8 PE Cy5), rat IgG2a allophycocyanin (for anti-CD4 allophycocyanin), rat IgG2b FITC (for anti-heat-stable Ag FITC), and rat IgG2a PE (for anti-CD8 PE). All mAbs used for staining were obtained from BD Biosciences. -GalCer-loaded PE or allophycocyanin-conjugated CD1d tetramers were prepared in our laboratory, as described (22).
Results
iNKT cells are depleted after the DP stage in an adult NOD thymus
The pathway of iNKT cell development is similar to that of conventional T cell development, and includes a precursor at the DP thymocyte stage of development (13). To determine whether iNKT cell development in a NOD thymus is blocked at or before the DP stage of thymocyte development, we examined female NOD and C57BL/6 (B6) thymi at 8 wk of age for their numbers of iNKT and conventional TCR T cells in the DP, mature CD4+ or CD8+ SP, and DN thymocyte subsets (Fig. 1). iNKT cells were detected by double staining with an anti-TCR mAb and -GalCer-loaded CD1d tetramers (Tet+), as described (8). FACS analyses demonstrated that NOD mice are deficient in DN thymocytes at this age (Fig. 1A). This DN thymocyte deficiency consists of an iNKT cell (Tet+TCR+) deficiency in a NOD thymus relative to a B6 thymus (Fig. 1B). Note that the number of iNKT cells is also reduced in CD8+, CD4+ SP, and DP thymocytes, but to a much lesser extent.
iNKT cell deficiency results from a lineage-specific depletion of DN T cells
CD4 and CD8 coreceptors augment physical interactions between TCR and MHC in the periphery and define a Th cell or cytolytic T cell function. Due to their influence on T cell function, CD4 and CD8 lineage fate decisions in the thymus have been studied extensively, whereas DN T cell lineage decision has often been overlooked (23). To determine whether the depletion of DN iNKT cells may be due to a lineage decision event, we examined the numbers of conventional T cells (Tet–TCR+) in the thymus of 8-wk-old NOD and B6 mice. We found that the number of conventional DN T cells was significantly decreased in a NOD thymus relative to a B6 thymus (Fig. 1B). In contrast, a significantly greater number of CD4+ T cells and CD8+ T cells were detected in a NOD thymus than a B6 thymus. These results indicate that a lineage-specific depletion of DN T cells after the DP stage of development may mediate an iNKT cell deficiency detectable in the thymus of an 8-wk-old NOD mouse.
iNKT cell deficiency arises during NOD fetal thymic development
At E13 in a fetal mouse, the first wave of common lymphoid precursors enters into the thymic lobes. These precursors are predicted to give rise to T cells between days 1 and 14 after birth (24). To determine whether this first wave of thymic precursors in a NOD thymus results in an iNKT cell deficiency, NOD and B6 thymic lobes were removed from embryos at E13 and introduced into fetal thymic organ culture. This prevents further input of thymic precursors or other developmental stimuli. Interestingly, E13 NOD thymic precursors developed 10-fold fewer iNKT cells than age-matched B6 thymic precursors (Fig. 2). This iNKT cell deficiency was apparent in an E13 NOD thymus despite the fact that 4- to 5-fold more conventional T cells developed in a NOD thymus than in an age-matched B6 thymus (Fig. 2). Thus, an iNKT cell deficiency in NOD mice arises in an E13 thymus in the absence of in vivo developmental stimuli or further common lymphoid precursor input.
NOD and B6 fetal thymocytes develop at comparable rates
Thymocyte development in a fetal B6 and BALB/c thymus parallels that of an adult thymus and progresses through three main precursor stages of mature T cells: DN TCR– thymocytes, ISP CD4+ or CD8+ TCR– thymocytes, and DP TCR–/+ thymocytes (9, 15). To ensure that fetal NOD thymocytes develop at a rate comparable to that in B6 and BALB/c mice and with the same main precursor stages during positive and negative selection, we examined the main precursor stages and phenotype of mature T cells by FACS analyses beginning at E17, the initial time of appearance of DP thymocytes in fetal B6 mice. From E17 to day 14 after birth, NOD and B6 thymi consist of similar populations of DN, DP, and CD4+ or CD8+ SP T cells (Fig. 3A). Gating on DN, DP, and CD4+CD8– or CD4–CD8+ populations, according to Fig. 3A, all NOD and B6 thymocytes were found to be TCR– at E17 (Fig. 3B). These findings indicate that CD4+ and CD8+ SP thymocytes are ISP T cells at this stage, and that development has progressed in both strains to an early DP stage before TCR-mediated positive or negative selection. At E18, DP thymocytes begin to express TCR, allowing for positive and negative selection, and mature T cells expressing TCR emerge among the CD4+, CD8+, and DN subsets from E18 to day 1 after birth. This finding suggests that positive and negative selection may occur for the first time at E18 in both strains. The distribution of thymocyte subsets from day 1 to 14 in NOD and B6 mice resembles that of adult mice as more DN, CD4+, and CD8+ SP T cells are evident. During this period of development, the latter subsets further mature, as indicated by the loss of CD24 expression (Fig. 3B). Thus, phenotypic analyses of fetal B6 and NOD thymocytes indicate that DP TCR+ thymocytes develop and allow for the positive and negative selection of mature T cells (TCR+, CD24+/–) at E18.
Deficiency in DN T cells and iNKT cells develops before birth
An iNKT cell deficiency in NOD mice was initially demonstrated as a DN T cell deficiency, which after correction by intrathymic transfer of syngeneic DN TCR+ thymocytes prevents insulitis and T1D (6). Our results indicate that reduced numbers of both DN iNKT cells and conventional T cells are present in an adult NOD thymus compared with a B6 thymus (Fig. 1). Furthermore, an iNKT cell deficiency is most prominent in DN TCR+ T cells after the DP stage of thymocyte development in an adult NOD thymus (Fig. 1B). To determine whether a similar depletion of DN TCR+ T cells begins with the emergence of TCR+ DP thymocytes at E18, we examined the distribution of thymocyte populations during late embryogenesis. At E18, a deficiency of mature DN T cells (TCR+CD24–) was found to develop in a NOD thymus relative to a B6 thymus (Fig. 3B). This deficiency arises concomitantly with the emergence of DP TCR+ thymocytes, which time it is presumably followed by TCR-mediated positive and negative selection. A role for TCR-mediated positive and negative selection in DN T cell depletion is strengthened by the finding of consistently lower numbers of CD24+ DN T cells in a NOD thymus. The CD24+ DN T cell is a precursor to mature DN T cells (TCR+CD24–), and it arises immediately after the DP thymocyte stage. This CD24+ DN T cell subset was consistently low in number in a NOD thymus beginning at E18, and was detectable in statistically significant numbers only by day 1 after birth. At the latter time, significantly more mature CD4+ and CD8+ SP T cells develop in a NOD thymus than B6 thymus (Fig. 3B). These results are similar to that found in an adult NOD thymus, indicating that in both embryonic and adult NOD there is a lineage-specific deficiency of DN T cells.
Previously, the earliest detection of iNKT cells by flow cytometry was reported to be 0.01% of thymocytes on day 5 after birth in a B6 thymus (25). iNKT cells comprise only 0.3–0.4% of adult NOD thymocytes, and are therefore difficult to detect by flow cytometry until adequate numbers of mature T cells have accumulated. To circumvent this technical difficulty and estimate the relative number of iNKT cells during thymic development, we used real-time PCR to quantify the expression of invariant TCR V14J18 chain mRNA specific for iNKT cells relative to that of -actin mRNA. This approach was used to determine whether selection of NOD TCR+ DP thymocytes at E18 depletes iNKT cells and results in an iNKT cell deficiency at birth. Fig. 3 shows that both NOD and B6 thymocytes progress to an early DP stage at E17 without detectable TCR surface expression. As TCR rearrangement is reported to occur at E17 (26), we were unable to detect V14J18 mRNA expression at E13 or E15 (our unpublished observations). As TCR mRNA is readily detected at E17, it is reasonable to assume that the majority of TCR mRNA detected at this stage is expressed by the large group of TCR– DP thymocytes, which first develop at this time with a rather low level of TCR mRNA expression in DN4 and ISP thymocytes. Thus, V14J18 mRNA levels at E17 should be indicative of the relative number of iNKT cells at an early DP or less mature precursor stage.
At E17, the ratio of V14J18 mRNA:-actin mRNA was found to be greater in NOD thymocytes than in B6 thymocytes (Fig. 4). This finding suggests that a larger number of iNKT cell precursors and/or a higher level of TCR mRNA expression is present in a NOD than B6 thymus at and before the TCR– DP stage of thymocyte development. At E18, DP thymocytes express TCR on their surface, which enables TCR-mediated positive and negative selection. This results in the emergence of mature DN T cells and a decrease in the level of V14J18 mRNA in NOD thymocytes, which most likely accounts for the similar ratio of V14J18 mRNA:-actin mRNA observed in NOD and B6 thymocytes at E18 (Fig. 4). At days 1 and 7 after birth, the estimated iNKT cell deficiency becomes more pronounced in a NOD thymus relative to a B6 thymus. Similar to the findings in adult NOD thymocytes, this kinetic analysis of iNKT cell depletion in fetal NOD thymocytes suggests that depletion occurs during the positive and negative selection of DP thymocytes between E18 and day 1 after birth.
Delta-1 stimulates the development of thymic iNKT and conventional T cells
The Notch family of molecules is linked to many aspects of T cell development, including the CD4/CD8 lineage decision (27, 28). The Notch ligand, Delta-1, was recently shown to promote conventional T cell development during the first three stages of DN thymocyte development (29, 30). Delta-1, but not Jagged-1, enhances the development of cells with a phenotype of conventional T cell and NKT precursors in cultures of human cord blood cells (31). To determine the effect of Delta-1 and Jagged-1 on iNKT cell development, soluble Fc-conjugated forms of these ligands were added to cultures of E13 B6 DN thymocytes, and the development of iNKT cells was estimated by semiquantitative RT-PCR analyses of V14J18 mRNA. Delta-1-Fc, but not Jagged-1-Fc, stimulated V14J18 mRNA expression in a dose-dependent manner (Fig. 5A). To determine whether Delta-1 enhances iNKT cell development by a pathway independent of conventional T cells, as previously suggested (17, 18, 19), fetal thymic organ cultures established in the presence or absence of Delta-1-Fc and Jagged-1-Fc were examined for V14J18 and control V3J15 expression by RT-PCR. TCR V3J15 is a gene that was characterized in the B6 T cell repertoire, and primers were designed to study the rearrangement of this gene based on analyses of its expression in a conventional CD4+ T cell reactive to proinsulin-derived C peptide (32) (National Center for Biotechnology Information accession: Z86026). Delta-1-Fc and Jagged-1-Fc did not yield detectable changes in the number of thymocytes in these cultures (our unpublished observations). However, the levels of TCR V14J18 (iNKT cell-specific) and TCR V3J15 (conventional T cell-specific) mRNA were significantly increased in thymocytes cultured in the presence of Delta-1-Fc, but not Jagged-1-Fc (Fig. 5, B and C). These observations suggest that Delta-1 can stimulate the development of iNKT cells in addition to conventional T cells.
Next, we determined whether the relative level of Delta-1 expression is decreased in a NOD fetal thymus, and if so, whether this decreased expression mediates the development of an iNKT cell deficiency in NOD mice. Comparison of the ratios of Delta-1:-actin mRNA levels expressed in NOD and B6 E13 thymocytes by real-time PCR revealed that the relative level of Delta-1 mRNA expression is reduced 2-fold in NOD vs B6 thymocytes (Fig. 5D). These findings suggest that a decrease in Delta-1 expression during T cell development may mediate the generation of the intrathymic iNKT cell deficiency noted in NOD embryos.
Discussion
Our results demonstrate for the first time that an iNKT cell deficiency, which is known to be associated with susceptibility to T1D, develops in NOD mice before birth. Previous studies of this deficiency failed to detect iNKT cells either in a NOD fetal thymus or in a newborn NOD thymus before 3 wk of age (26). An earlier attempt to detect V14J18 mRNA in a B6 fetal thymus by PCR analysis was unsuccessful possibly due to the fact that E9 embryos were used and DP thymocytes with V14J18 gene rearrangement appear in the thymic primordium 8–9 days later. Indeed, we were also unable to detect V14J18 mRNA in E13 or E15 thymic lobes (our unpublished observations). Moreover, the inability to detect iNKT cells in a NOD thymus by flow cytometry before 3 wk of age is not surprising, because this population comprises only 0.3% of thymocytes in a B6 thymus that contains a higher percentage of iNKT cells at 3 wk after birth (25). iNKT cells comprise a relatively small subset in the thymus, and require several rounds of T cell development to accumulate and become detectable by tetramer staining and flow cytometry.
Application of a real-time PCR approach enabled us to show that an iNKT cell deficiency begins with a depletion of these cells at E18, and that this deficiency is evident in NOD mice on day 1 after birth. Although adult and embryonic T cell development are often assumed to differ (21), we contend that many of these differences may be minor and ascribable to the different stages of development that occur in the thymus at a given day during embryonic life. Our results indicate that in NOD mice the development of an autoimmune T cell repertoire develops before birth and is then conserved during adult T cell development. This raises the issue that further investigation of the similarities between embryo and adult T cell development is required. Moreover, the fact that a day 7 neonatal mouse is considered to be the immunological equivalent of a newborn human (33) coupled with the finding that an iNKT cell deficiency is established by day 1 after birth in NOD mice further emphasizes the need for additional research to be directed toward the early detection and treatment of T1D in humans.
A defect in central tolerance (negative selection) has been reported to be causal to susceptibility to T1D in NOD mice (34, 35). In addition, NOD thymic epithelium, which is enriched in self Ag for positive and negative selection, was shown to induce insulitis upon transfer into diabetes-resistant B6 mice (36). Furthermore, our demonstration in this study that an intrathymic deficiency in iNKT cells appears after the DP thymocyte stage in embryonic NOD mice points toward a defect in positive selection of iNKT cells in these mice. Collectively, these findings suggest that central tolerance, including defects in the negative selection of autoreactive T cells and positive selection of regulatory T cells, such as iNKT cells, is an important factor that determines susceptibility to T1D in NOD mice.
We have obtained evidence that the depletion of iNKT cells from the thymus that occurs after the DP stage of development may be lineage specific. This conclusion is strengthened by our detection of a numerical deficiency in DN conventional T cells and iNKT cells both in a NOD adult thymus at 8 wk of age and in a NOD fetal thymus at E18. Recent reports indicate that iNKT cells and conventional T cells follow a similar developmental pathway, including progression through the DP stage of T cell development and positive and negative selection (13, 14). Based on our results in this study, we propose that DN conventional T cells and DN iNKT cells make a common lineage decision, and that this lineage decision is defective in a developing NOD thymus according to a model shown in Fig. 6. Pre-iNKT DP thymocytes and earlier precursors are not deficient in a fetal NOD thymus; in fact, these pre-iNKT thymocytes appear to be more numerous in a NOD than B6 thymus at E17. TCR protein expression in DP thymocytes at E18 allows for a selection event that preferentially depletes DN T cells. This lineage-specific depletion of DN T cells results in an iNKT cell deficiency at day 1 after birth, and this deficiency is conserved after birth in the adult NOD thymus. Hence, our studies not only highlight the importance of analysis of the relationship of DN T cell fates and lineage decisions as they relate to the pathogenesis of autoimmune disease, but also provide the first indication that lineage decision in early intrathymic T cell development may govern susceptibility to autoimmune T1D.
In addition to thymic epithelial stimulation, other factors may also affect the positive or negative selection of iNKT cells in a NOD thymus. Intrathymic cotransfer of thymic precursors (1:1) from diabetes-susceptible NOD and diabetes-resistant ACK mice can normalize the frequency of TCR+DX5+ iNKT cells in a NOD thymus (37). The Notch ligand, Delta-1, may be the source of such a survival signal during the positive and negative selection of DP thymocytes. Previously, Delta-1 was shown to provide survival signals to all of three subsets of DN TCR– T cell precursors (29, 30), and to enhance T and possibly NKT cell development from human cord blood cells (31). Nonetheless, loss of function experiments in Delta-1 knockout mice demonstrated that T cell development was not altered in these mice possibly due to a compensation of function by Delta-4 in these mice (38). Despite functional overlap between Delta-1 and Delta-4, our finding that Delta-1, but not Jagged-1, can enhance conventional T and iNKT cell development supports the notion that Notch ligands differentially promote T cell development. Furthermore, our detection of a dose-dependent effect of Delta-1 on iNKT cell development demonstrates that despite overlap in Delta-1/Delta-4 function, the local concentration of Delta-1 in the thymus may affect T cell development. Thus, our observation that the amount of Delta-1 mRNA is significantly decreased in E13 NOD thymocytes compared with an age-matched B6 thymus supports the notion that a reduced level of Delta-1 protein in a NOD thymus may elicit in part the deficiency in DN T cells and iNKT cells in NOD mice. Future experimentation is required to investigate the effects of Delta-1 on the development of DN T cells, iNKT cells, and other T cell populations in NOD mice and the effects of altered T cell development on T1D development in NOD mice. Such analyses may also reveal whether a reduced intrathymic level of Delta-1 gives rise to a general thymic lymphopenia in NOD mice, which is purported to be a susceptibility factor for T1D in these mice (8, 29, 30, 39).
In conclusion, this study further underscores the important role that central tolerance may play in controlling the susceptibility to autoimmune disease, and T1D in particular. Our observations demonstrate that lineage fate decisions in positive selection during the intrathymic development of conventional DN and iNKT cells before birth may be an important feature of central tolerance that, if defective, can predispose to the outcome of autoimmune disease.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Juvenile Diabetes Research Foundation Grant 1-2001-351 (to T.L.D.), Canadian Institutes of Health Research Grant MOP 64886 (to T.L.D.), National Institutes of Health Grant RR 18789 (to J.M.V.), and Cobre grant in stem cell biology and regenerative medicine (to J.M.V.). T.L.D. is the Sheldon H. Weinstein Scientist at the Robarts Research Institute and University of Western Ontario.
2 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Robarts Research Institute, 100 Perth Drive, POB 5015, London, Ontario N6A 5K8, Canada. E-mail address: del{at}robarts.ca
3 Abbreviations used in this paper: T1D, type 1 diabetes; -GalCer, -galactosylceramide; E, embryonic day; DN, double negative; DP, double positive; iNKT, invariant NK T cell; SP, single positive; ISP, immature SP.
Received for publication December 9, 2004. Accepted for publication March 21, 2005.
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