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 CD4CD8 double-negative αβ 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.

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 β 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, Vα14Jα18, Vβ8.2/7/2 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 TCRα chain (Vα14Jα18), and proliferation induced by activation through the CD1d-restricted sphingoglycolipid Ag, α-galactosylceramide (α-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 CD4CD8 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+, TCRαβ) 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 αβ T cells shortly before birth during E18-day 1. The latter DN αβ T cells are enriched in DN iNKT cells. Our findings suggest that during embryonic thymic T cell development in NOD mice, conventional αβ T cells and iNKT cells develop along a common pathway until the DP stage, after which iNKT cells and DN αβ 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.

NOD/Del mice were bred in a specific pathogen-free environment at the Robarts Research Institute. C57BL/6 mice were obtained from Charles River Laboratories or The Jackson Laboratory and then bred at the Robarts Research Institute. The day of the vaginal plug was observed and counted as day 0 of gestation (E0).

Mice were euthanized by asphyxiation in a CO2 chamber. Thymi were removed and kept on ice in Dulbecco’s PBS (Invitrogen Life Technologies) supplemented with 10% FBS (Sigma-Aldrich) until further use. Embryonic and neonatal thymic lobes were isolated using a dissecting microscope (Niko type 102) and were kept on ice in PBS/FBS until used. Thymi were dissociated into single cells by passage through a 40-μm nylon mesh (BD Biosciences). Erythrocytes were removed from thymocyte preparations by lysis in potassium-ammonium chloride buffer (0.15 M NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.3). Thymocyte viability was determined by trypan blue staining (Sigma-Aldrich). CD4CD8 (DN) thymocytes were isolated by using MiniMacs columns (Miltenyi Biotec), according to manufacturer’s protocol, with the exception that PBS/FBS was used instead of PBS.

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.

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 α-GalCer-loaded CD1d tetramers conjugated to PE, anti-TCRαβ FITC, anti-CD8α CyChrome (PE Cy5), and anti-CD4 allophycocyanin. Fetal thymocytes were stained with α-GalCer-loaded CD1d tetramers allophycocyanin, anti-TCRαβ FITC, anti-heat-stable Ag FITC, anti-CD8α 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-TCRαβ FITC), rat IgG2a PE Cy5 (for anti-CD8α PE Cy5), rat IgG2a allophycocyanin (for anti-CD4 allophycocyanin), rat IgG2b FITC (for anti-heat-stable Ag FITC), and rat IgG2a PE (for anti-CD8α PE). All mAbs used for staining were obtained from BD Biosciences. α-GalCer-loaded PE or allophycocyanin-conjugated CD1d tetramers were prepared in our laboratory, as described (22).

Thymocyte RNA was isolated using RNeasy spin columns (Qiagen), according to the manufacturer’s instructions, with the addition of a poly(C) (Sigma-Aldrich) RNA carrier. RNA preparations were pretreated with DNase (Invitrogen Life Technologies), according to the manufacturer’s instructions. Reverse transcription was performed with Superscript II (Invitrogen Life Technologies) and oligo(dT) (Invitrogen Life Technologies), according to manufacturer’s instructions, and with the addition of an RNase inhibitor (Invitrogen Life Technologies). cDNA was then treated with RNase (Invitrogen Life Technologies), according to the manufacturer’s instructions. Semiquantitative PCR analyses were performed by analysis of PCR samples at 15, 20, 25, 30, and 35 cycles. Real-time PCR analysis was performed by determining the fluorescence of a Syber I master mix (Sigma-Aldrich) measured by Stratagene MX 4000. The annealing temperature was 63°C for all PCR, and a brief 82°C step was added to the end of each cycle in real-time PCR cycles to melt primer dimers. The primers were designed using Primer3 software (〈http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi〉), as follows: Vα3Jα15, 5′-AAGGAGTGAATGGCTTCGAG-3′ and 5′-TGAGTCCCAGCTCCAAAATG-3′; Vα14Jα18, 5′-GACAAAACGTCAAATGGGAGA-3′ and 5′-CTGGGTCTGGATGTCAGGT-3′; Delta-1, 5′-TTAGCATCATTGGGGCTACC-3′ and 5′-TAAGTGTTGGGGCGATCTTC-3′; and β-actin, 5′-TGTTACCAACTGGGACGACA and 5′-CTCTCAGCTGTGGTGGTGAA-3′.

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 TCRαβ 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-TCRαβ mAb and α-GalCer-loaded CD1d tetramers (Tet+), as described (8). FACS analyses demonstrated that NOD mice are deficient in DN thymocytes at this age (Fig. 1,A). This DN thymocyte deficiency consists of an iNKT cell (Tet+TCRαβ+) deficiency in a NOD thymus relative to a B6 thymus (Fig. 1 B). Note that the number of iNKT cells is also reduced in CD8+, CD4+ SP, and DP thymocytes, but to a much lesser extent.

FIGURE 1.

An iNKT cell deficiency arises after the DP thymocyte stage of development in a NOD thymus and is restricted mainly to the DN lineage of iNKT and conventional αβ T cells. Mononuclear thymocytes isolated from 8-wk-old NOD (n = 5) and C57BL/6 (B6) (n = 5) mice were stained, as described in Materials and Methods, with either α-GalCer CD1d PE tetramers (Tet), anti-TCRαβ FITC, anti-CD4 allophycocyanin, anti-CD8α CyChrome, or IgG isotype controls. A, DP, DN, CD4+ SP, or CD8+ SP subsets were gated. B, iNKT cells (Tet+TCRαβ+) and conventional αβ T cells (TetTCRαβ+) were detected within DP, DN, CD4+ SP, or CD8+ SP subsets. Mean values of absolute cell number ± SD of three flow cytometric samples are shown. ∗, p < 0.05.

FIGURE 1.

An iNKT cell deficiency arises after the DP thymocyte stage of development in a NOD thymus and is restricted mainly to the DN lineage of iNKT and conventional αβ T cells. Mononuclear thymocytes isolated from 8-wk-old NOD (n = 5) and C57BL/6 (B6) (n = 5) mice were stained, as described in Materials and Methods, with either α-GalCer CD1d PE tetramers (Tet), anti-TCRαβ FITC, anti-CD4 allophycocyanin, anti-CD8α CyChrome, or IgG isotype controls. A, DP, DN, CD4+ SP, or CD8+ SP subsets were gated. B, iNKT cells (Tet+TCRαβ+) and conventional αβ T cells (TetTCRαβ+) were detected within DP, DN, CD4+ SP, or CD8+ SP subsets. Mean values of absolute cell number ± SD of three flow cytometric samples are shown. ∗, p < 0.05.

Close modal

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 αβ T cells (TetTCRαβ+) in the thymus of 8-wk-old NOD and B6 mice. We found that the number of conventional DN αβ T cells was significantly decreased in a NOD thymus relative to a B6 thymus (Fig. 1 B). In contrast, a significantly greater number of CD4+ αβ T cells and CD8+ αβ T cells were detected in a NOD thymus than a B6 thymus. These results indicate that a lineage-specific depletion of DN αβ 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.

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 αβ 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.

FIGURE 2.

An iNKT cell deficiency is detectable in NOD fetal thymic organ cultures. Fetal thymic lobes from E13 NOD (n = 46) and C57BL/6 (n = 49) embryos were cultured for 21 days. Mononuclear thymocytes were then isolated, stained with vehicle (A) and α-GalCer CD1d allophycocyanin tetramers (B), and anti-TCRαβ FITC or IgG isotype control and iNKT cells (Tet+TCRαβ+), and conventional αβ T cells (TetTCRαβ+) were detected by flow cytometry. One of two reproducible flow cytometric analyses is shown.

FIGURE 2.

An iNKT cell deficiency is detectable in NOD fetal thymic organ cultures. Fetal thymic lobes from E13 NOD (n = 46) and C57BL/6 (n = 49) embryos were cultured for 21 days. Mononuclear thymocytes were then isolated, stained with vehicle (A) and α-GalCer CD1d allophycocyanin tetramers (B), and anti-TCRαβ FITC or IgG isotype control and iNKT cells (Tet+TCRαβ+), and conventional αβ T cells (TetTCRαβ+) were detected by flow cytometry. One of two reproducible flow cytometric analyses is shown.

Close modal

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 TCRαβ thymocytes, ISP CD4+ or CD8+ TCRαβ thymocytes, and DP TCRαβ−/+ 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. 3,A). Gating on DN, DP, and CD4+CD8 or CD4CD8+ populations, according to Fig. 3,A, all NOD and B6 thymocytes were found to be TCRαβ at E17 (Fig. 3,B). 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 TCRαβ-mediated positive or negative selection. At E18, DP thymocytes begin to express TCRαβ, allowing for positive and negative selection, and mature T cells expressing TCRαβ 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. 3 B). Thus, phenotypic analyses of fetal B6 and NOD thymocytes indicate that DP TCRαβ+ thymocytes develop and allow for the positive and negative selection of mature T cells (TCRαβ+, CD24+/−) at E18.

FIGURE 3.

A deficiency in DN αβ T cells develops in NOD mice before the day of birth (E19). Mononuclear thymocytes were isolated from NOD and B6 embryos at E17 (B6, n = 15; NOD, n = 15), E18 (B6, n = 20; NOD n = 20), day 1 (B6, n = 18; NOD, n = 20), and day 14 (B6, n = 4; NOD, n = 4) after birth. A, The percentages of DP, DN, CD4+ SP, and CD8+ SP thymocytes present at E17, E18, day 1, and day 14 were analyzed by flow cytometry as in Fig. 2. B, The CD4+ SP αβ thymocytes (CD4+CD8), CD8+ SP αβ thymocytes (CD4CD8+), DP αβ thymocytes (CD4+CD8+), and DN αβ thymocytes (CD4CD8) detected in A were gated and then analyzed for their respective levels of CD24 and TCRαβ surface expression by flow cytometry as in Fig. 2. The percentages of cells with varying levels of CD24 and TCRαβ expression for each of the gated SP, DP, and DN subsets are presented as the mean values ± SD of three flow cytometry samples. CD4+ SP αβ thymocytes (CD4+CD8TCRαβ+CD24), CD8+ SP αβ thymocytes (CD4+CD8TCRαβ+CD24), DP αβ thymocytes (CD4+CD8+TCRαβ+CD24), and DN αβ thymocytes (CD4CD8TCRαβ+CD24) were detected. SDs ranged from 5 to 15% of the mean values and were generally <10%. ∗, p < 0.05.

FIGURE 3.

A deficiency in DN αβ T cells develops in NOD mice before the day of birth (E19). Mononuclear thymocytes were isolated from NOD and B6 embryos at E17 (B6, n = 15; NOD, n = 15), E18 (B6, n = 20; NOD n = 20), day 1 (B6, n = 18; NOD, n = 20), and day 14 (B6, n = 4; NOD, n = 4) after birth. A, The percentages of DP, DN, CD4+ SP, and CD8+ SP thymocytes present at E17, E18, day 1, and day 14 were analyzed by flow cytometry as in Fig. 2. B, The CD4+ SP αβ thymocytes (CD4+CD8), CD8+ SP αβ thymocytes (CD4CD8+), DP αβ thymocytes (CD4+CD8+), and DN αβ thymocytes (CD4CD8) detected in A were gated and then analyzed for their respective levels of CD24 and TCRαβ surface expression by flow cytometry as in Fig. 2. The percentages of cells with varying levels of CD24 and TCRαβ expression for each of the gated SP, DP, and DN subsets are presented as the mean values ± SD of three flow cytometry samples. CD4+ SP αβ thymocytes (CD4+CD8TCRαβ+CD24), CD8+ SP αβ thymocytes (CD4+CD8TCRαβ+CD24), DP αβ thymocytes (CD4+CD8+TCRαβ+CD24), and DN αβ thymocytes (CD4CD8TCRαβ+CD24) were detected. SDs ranged from 5 to 15% of the mean values and were generally <10%. ∗, p < 0.05.

Close modal

An iNKT cell deficiency in NOD mice was initially demonstrated as a DN αβ T cell deficiency, which after correction by intrathymic transfer of syngeneic DN TCRαβ+ 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 TCRαβ+ T cells after the DP stage of thymocyte development in an adult NOD thymus (Fig. 1,B). To determine whether a similar depletion of DN TCRαβ+ T cells begins with the emergence of TCRαβ+ DP thymocytes at E18, we examined the distribution of thymocyte populations during late embryogenesis. At E18, a deficiency of mature DN αβ T cells (TCRαβ+CD24) was found to develop in a NOD thymus relative to a B6 thymus (Fig. 3,B). This deficiency arises concomitantly with the emergence of DP TCRαβ+ 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 αβ T cell depletion is strengthened by the finding of consistently lower numbers of CD24+ DN αβ T cells in a NOD thymus. The CD24+ DN αβ T cell is a precursor to mature DN αβ T cells (TCRαβ+CD24), and it arises immediately after the DP thymocyte stage. This CD24+ DN αβ 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. 3 B). 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 Vα14Jα18 chain mRNA specific for iNKT cells relative to that of β-actin mRNA. This approach was used to determine whether selection of NOD TCRαβ+ 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 TCRαβ surface expression. As TCRα rearrangement is reported to occur at E17 (26), we were unable to detect Vα14Jα18 mRNA expression at E13 or E15 (our unpublished observations). As TCRα mRNA is readily detected at E17, it is reasonable to assume that the majority of TCRα mRNA detected at this stage is expressed by the large group of TCRαβ DP thymocytes, which first develop at this time with a rather low level of TCRα mRNA expression in DN4 and ISP thymocytes. Thus, Vα14Jα18 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 Vα14Jα18 mRNA:β-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 TCRαβ mRNA expression is present in a NOD than B6 thymus at and before the TCRαβ DP stage of thymocyte development. At E18, DP thymocytes express TCRαβ 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 Vα14Jα18 mRNA in NOD thymocytes, which most likely accounts for the similar ratio of Vα14Jα18 mRNA:β-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.

FIGURE 4.

A deficiency in iNKT cells develops in a NOD thymus on the day of birth. Mononuclear thymocytes from NOD and B6 embryos were isolated at E17 (B6, n = 16; NOD, n = 13), E18 (B6, n = 20; NOD, n = 20), day 1 (B6, n = 8; NOD, n = 7), and day 7 (B6, n = 5; NOD, n = 5) after birth. The number of iNKT cells was estimated by a standard curve method of analysis of real-time PCR quantitation of Vα14Jα18 mRNA expression normalized to β-actin mRNA expression. Mean values ± SD of three quantitative PCR are shown. ∗, p < 0.05.

FIGURE 4.

A deficiency in iNKT cells develops in a NOD thymus on the day of birth. Mononuclear thymocytes from NOD and B6 embryos were isolated at E17 (B6, n = 16; NOD, n = 13), E18 (B6, n = 20; NOD, n = 20), day 1 (B6, n = 8; NOD, n = 7), and day 7 (B6, n = 5; NOD, n = 5) after birth. The number of iNKT cells was estimated by a standard curve method of analysis of real-time PCR quantitation of Vα14Jα18 mRNA expression normalized to β-actin mRNA expression. Mean values ± SD of three quantitative PCR are shown. ∗, p < 0.05.

Close modal

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 αβ 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 αβ 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 Vα14Jα18 mRNA. Delta-1-Fc, but not Jagged-1-Fc, stimulated Vα14Jα18 mRNA expression in a dose-dependent manner (Fig. 5,A). To determine whether Delta-1 enhances iNKT cell development by a pathway independent of conventional αβ 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 Vα14Jα18 and control Vα3Jα15 expression by RT-PCR. TCR Vα3Jα15 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 αβCD4+ 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 Vα14Jα18 (iNKT cell-specific) and TCR Vα3Jα15 (conventional αβ 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 αβ T cells.

FIGURE 5.

Delta-1 enhances the development of iNKT and conventional T cells, and its expression is reduced in a NOD fetal thymus. A, Notch-mediated TCR Vα14Jα18 mRNA induction is Notch ligand specific and dose dependent. DN thymocytes from 25 E13 B6 embryos were cultured in triplicate in the presence of various concentrations of Delta-1 or Jagged-1 for 24 h. Semiquantitative RT-PCR was performed for each replicate to obtain relative levels of TCR Vα14Jα18 mRNA expression. Mean values of TCR Vα14Jα18 mRNA levels are shown. B and C, Delta-1 induces TCR Vα mRNA expression characteristic of iNKT cells and DN TCRαβ cells. Thymic lobes from E13 B6 embryos (n = 28) were cultured in complete RPMI 1640 supplemented with 5 nM soluble Delta-1. Typical bands and mean values ± SD from three separate and reproducible semiquantitative PCR are shown. D, Delta-1 mRNA expression is reduced in E13 NOD (n = 22) thymocytes compared with age-matched B6 (n = 20) thymocytes. Data shown represent mean values ± SD of three separate semiquantitative PCR experiments. ∗, p < 0.05.

FIGURE 5.

Delta-1 enhances the development of iNKT and conventional T cells, and its expression is reduced in a NOD fetal thymus. A, Notch-mediated TCR Vα14Jα18 mRNA induction is Notch ligand specific and dose dependent. DN thymocytes from 25 E13 B6 embryos were cultured in triplicate in the presence of various concentrations of Delta-1 or Jagged-1 for 24 h. Semiquantitative RT-PCR was performed for each replicate to obtain relative levels of TCR Vα14Jα18 mRNA expression. Mean values of TCR Vα14Jα18 mRNA levels are shown. B and C, Delta-1 induces TCR Vα mRNA expression characteristic of iNKT cells and DN TCRαβ cells. Thymic lobes from E13 B6 embryos (n = 28) were cultured in complete RPMI 1640 supplemented with 5 nM soluble Delta-1. Typical bands and mean values ± SD from three separate and reproducible semiquantitative PCR are shown. D, Delta-1 mRNA expression is reduced in E13 NOD (n = 22) thymocytes compared with age-matched B6 (n = 20) thymocytes. Data shown represent mean values ± SD of three separate semiquantitative PCR experiments. ∗, p < 0.05.

Close modal

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:β-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. 5 D). 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.

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 Vα14Jα18 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 Vα14Jα18 gene rearrangement appear in the thymic primordium 8–9 days later. Indeed, we were also unable to detect Vα14Jα18 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 αβ 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 αβ 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 αβ 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 αβ T cells. This lineage-specific depletion of DN αβ 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 αβ 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.

FIGURE 6.

Proposed role for Delta-1 in NOD T cell development. Delta-1 may play a role in the selection of DP thymocytes by enhancing the positive selection of DN αβ T cells, including iNKT cells. In contrast, a deficient amount of Delta-1 in a NOD thymus may decrease the number of DN αβ T cells positively selected in a lineage-specific manner, as shown (X). Thus, relative to that in a B6 thymus, a Delta-1 deficiency in a NOD thymus may be responsible for the selection of a decreased number of DN αβ T cells, including iNKT cells, and an increased number of conventional CD4+ and CD8+ αβ T cells.

FIGURE 6.

Proposed role for Delta-1 in NOD T cell development. Delta-1 may play a role in the selection of DP thymocytes by enhancing the positive selection of DN αβ T cells, including iNKT cells. In contrast, a deficient amount of Delta-1 in a NOD thymus may decrease the number of DN αβ T cells positively selected in a lineage-specific manner, as shown (X). Thus, relative to that in a B6 thymus, a Delta-1 deficiency in a NOD thymus may be responsible for the selection of a decreased number of DN αβ T cells, including iNKT cells, and an increased number of conventional CD4+ and CD8+ αβ T cells.

Close modal

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 TCRαβ+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 TCRαβ 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 αβ 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 αβ T cells and iNKT cells in NOD mice. Future experimentation is required to investigate the effects of Delta-1 on the development of DN αβ 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 αβ and iNKT cells before birth may be an important feature of central tolerance that, if defective, can predispose to the outcome of autoimmune disease.

We thank Dr. Mitch Kronenberg for providing the protocol for the α-GalCer-loaded CD1d tetramers, Dalam Ly for preparing the tetramers, Dr. Mickie Bhatia for providing soluble Jagged-1, and Melissa Bellyou and her staff for providing animal husbandry assistance.

The authors have no financial conflict of interest.

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.

3

Abbreviations used in this paper: T1D, type 1 diabetes; α-GalCer, α-galactosylceramide; E, embryonic day; DN, double negative; DP, double positive; iNKT, invariant NK T cell; SP, single positive; ISP, immature SP.

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