Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.

Type 1 diabetes mellitus results from the autoimmune destruction of insulin-producing pancreatic islet β cells leading to insulin deficiency and increased blood glucose levels. The β cell destruction is due to the breakdown of the mechanisms maintaining immune tolerance to self-tissues and is caused by islet-infiltrating cytotoxic CD4+ and CD8+ T cells (14), as well as self-reactive autoantibodies specific for β cell Ags (5). The molecular mechanisms underlying the loss of immune tolerance and leading to islet β cell killing in type 1 diabetes are largely unknown.

Invariant NKT (iNKT) cells are a highly conserved, innate-like, CD1d-restricted T cell population, which contributes to the induction and maintenance of immune tolerance (6). They exert their tolerogenic functions via both cellular contact and an ability to rapidly produce copious amounts of cytokines, thereby instructing tolerogenic dendritic cells (79), enhancing the function of regulatory T cells (10), and inhibiting autoreactive B cells (11, 12) and pathogenic T cells (13).

In the NOD mouse model, several iNKT-targeting experimental approaches prevent the onset of type 1 diabetes, including adoptive transfer of iNKT cells (13), overexpression of CD1d within the pancreatic islets (14), transgenic overexpression of the iNKT-TCR (15), or in vivo stimulation of iNKT cells with lipid Ags (1618). The first evidence for a potential protective role of iNKT cells in human type 1 diabetes came from studies in monozygotic twins who were discordant for the disease (19, 20). These studies demonstrated both a significant reduction of circulating iNKT cells and a loss of IL-4 gene expression and cytokine production in the iNKT clones from the twin who developed diabetes (1921). Consistent with these results, another study demonstrated a loss of IL-4 production in iNKT clones derived from the pancreatic lymph nodes of people with type 1 diabetes but not from control donors without type 1 diabetes (22). In contrast, other studies failed to demonstrate significant changes in the frequency (23) or function (24, 25) of circulating iNKT cells in larger cohorts of people with type 1 diabetes. Whereas the results from these studies challenge the role of iNKT cells in human type 1 diabetes, they may also suggest that more subtle changes in the iNKT clonal repertoire underlie the loss of immune tolerance leading to autoimmunity.

Human NKT cells are activated via their CD1d-restricted semi-iNKT-TCR, which consists of an invariant TCR Vα24-Jα18 chain and a semivariant TCR Vβ11 chain. Our studies have previously revealed that the human iNKT repertoire is composed of clones with widely varying iNKT-TCR affinities for CD1d (26). This clonal heterogeneity of the iNKT repertoire is rooted in the only V region of the human iNKT-TCR, the CDR3β loop of Vβ11 (26). Depending on the composition of its short amino acid sequence, the CDR3β loop can enable auxiliary Ag-independent protein–protein contacts with CD1d, thereby providing additional binding energy to the interaction (26). The clonally distributed variations within the CDR3β loop can drive potent functional differences between iNKT clones expressing high- and low-affinity TCRs for CD1d. Indeed, high-affinity iNKT clones, that is, expressing high-affinity TCR, but not low-affinity iNKTs proliferate and secrete various cytokines following stimulation with endogenous CD1d Ags or synthetic partial agonist Ags such as OCH.

In this study, we reasoned that an imbalance of the iNKT repertoire with regard to high- and low-affinity iNKT clones may lead to aberrant iNKT-mediated immune regulation in type 1 diabetes, and that such an imbalance would not necessarily need to be associated with a detectable decrease of circulating iNKT cells in autoimmune patients. To address this hypothesis, we carried out a cross-sectional case control study to compare the clonal iNKT repertoire with regard to iNKT-TCR affinity and function in people with recent onset type 1 diabetes and matched healthy controls.

Fresh heparinized blood was obtained from 19 people between 18 and 35 y of age with diagnosis of type 1 diabetes and disease duration of ≤4 y and from 28 healthy controls (Table I) following the approval by the National Research Ethics Service Committee South Central (REC reference 09/H0502/107). There were no differences in age and sex distribution between the two groups. Written informed consent was obtained from all participants and their identity was encrypted immediately after inclusion into the study.

Table I.
Demographic characteristics of people with type 1 diabetes and healthy subjects
nFemale (%)Age (y), Mean ± SDDuration of Disease (mo), Mean ± SD
Type 1 diabetes 19 42.1 25.2 ± 3.17 24.2 ± 15 
Healthy 28 46.4 25 ± 3.82 N/A 
p Value N/A 1.0 0.82 N/A 
nFemale (%)Age (y), Mean ± SDDuration of Disease (mo), Mean ± SD
Type 1 diabetes 19 42.1 25.2 ± 3.17 24.2 ± 15 
Healthy 28 46.4 25 ± 3.82 N/A 
p Value N/A 1.0 0.82 N/A 

The age between the two groups was compared using a two-tailed Student t test, assuming equal variances. The sex distribution between the two groups was compared with the Fisher exact test. N/A, not applicable.

For all assays, KRN7000 (INstruchemie, Delfzijl, the Netherlands) and OCH (Enzo Life Sciences) were solubilized to 200 μg/ml by sonication at 80°C in vehicle (water containing 150 mM NaCl, 0.5% Tween 20). Lipid-loaded CD1d/β2-microglobulin complexes were refolded using oxidative refolding chromatography (27) and tetramerized as previously described (28). For the generation of CD1d/MACSiBead microparticles, 10 μg of biotinylated CD1d-KRN7000 or CD1d-OCH monomers and 5 μg of each biotinylated anti-CD2 and anti-CD28 Abs were conjugated to 5 × 107 anti-biotin MACSiBead microparticles (all Miltenyi Biotec, Surrey, U.K.) and stored at 4°C in PBS containing 2 mM EDTA, 0.5% BSA, and 0.4% sodium azide.

The following reagents were used for iNKT analysis: PE–anti-Vα24 (clone C15), FITC–anti-Vβ11 (clone C21) (Beckman Coulter, High Wycombe, U.K.), allophycocyanin–anti-CD3 (clone UCHT1; ImmunoTools, Friesoythe, Germany), allophycocyanin–anti-CD4 (clone OKT-4), and PE-conjugated CD1d tetramers loaded with either KRN7000 or OCH. Propidium iodide (Sigma-Aldrich, Dorset, U.K.) was used to exclude dead cells. Cells (1 × 105) from each iNKT clone or 0.5 × 106 cells from the iNKT lines were stained with either KRN7000 or OCH tetramers for 45 min on ice, washed twice with cold PBS containing 1% FCS, and acquired on a FACSAria II (Beckton Dickinson). The data were analyzed on FACSDiva software (Beckton Dickinson) and FlowJo. To stain for surface CD1d expression, we used PE–anti-CD1d Ab (clone 51.1) and mouse IgG2bκ-PE isotype control Ab (both from Miltenyi Biotec). Then, 1 × 105 monocytes or B cells were stained with each Ab for 30 min at 4°C and washed twice with cold PBS/1% FCS. The cells were acquired on a FACSAria II and the data were analyzed using BD FACSDiva software.

PBMCs were isolated from venous blood by density gradient centrifugation. For the generation of iNKT clones, single live CD3+Vα24+Vβ11+ T cells were sorted ex vivo in 96-well plates and cultured in the presence of 1 μg/ml PHA (Sigma-Aldrich) and gamma-irradiated (35 Gy) autologous feeder PBMCs in complete culture media (RPMI 1640 [Lonza] containing 10% FCS, 2% human AB serum, 1 mM sodium pyruvate, 1% nonessential amino acids, 1% l-GlutaMAX, 100 IU/ml of penicillin, 100 μg/ml streptomycin [all from Sigma-Aldrich], 50 μM 2-ME, 400 IU/ml of IL-2 [Proleukin; Chiron], 10 ng/ml IL-7, and 10 ng/ml IL-15 [both from ImmunoTools]). Following initial expansion, the media were replenished with complete media containing 200 IU/ml of IL-2, 2 ng/ml IL-7, and 2 ng/ml IL-15.

In vitro iNKT expansion using autologous CD14+ monocytes, CD19+ B cells, or MACSiBead microparticles.

One million PBMCs depleted from CD14+ monocytes and CD19+ B cells were cultured with either 1) 1 × 105 autologous CD14+ monocytes or 1 × 105 CD19+ B cells pulsed with vehicle alone or with 200 ng/ml of each lipid (OCH or KRN7000); or 2) with 5 × 105 CD1d-OCH/MACSiBead microparticles or 5 × 105 CD1d-KRN7000/MACSiBead microparticles in complete medium at 37°C, 5% (v/v) CO2. One week later, the culture media were replenished with fresh complete RPMI supplemented with 200 IU of IL-2, 2 ng/ml IL-7, and 2 ng/ml IL-15, and the cultures were analyzed for iNKT expansion after 14 d.

Cytokine assays.

For plate-bound cytokine analyses, frozen iNKT clones were thawed and restimulated with allogeneic gamma-irradiated (35 Gy) feeder PBMCs in the presence of 1 μg/ml PHA, 400 IU of IL-2, 10 ng/ml IL-7, and 10 ng/ml IL-15 in complete RPMI. Following in vitro iNKT clone expansion, the cells were restained with CD1d-KRN7000 and CD1d-OCH tetramers to confirm iNKT identity and iNKT-TCR affinity, respectively. Next, 96-well Nunc MaxiSorp plates were coated overnight at 4°C with 10 μg/ml streptavidin. The plates were washed with cold PBS, and 2 μg/ml human biotinylated CD1d-KRN7000 or CD1d-OCH monomers were added for 1 h at 37°C. After two more washes, 1 × 105 live cells from each iNKT clone were added to the wells and incubated at 37°C, 5% (v/v) CO2 in complete RPMI without cytokines. Prior to addition to the wells, the cells from each iNKT clone were washed twice in warm sterile PBS. The cell culture supernatants were collected after 24 h and analyzed for cytokine production using the Ready-SET-Go! ELISA sets from eBioscience according to the manufacturer’s recommendations.

GraphPad Prism software was used for statistical analysis, and a two-tailed p value ≤0.05 was considered statistically significant. High- and low-affinity iNKT clones were identified following staining with CD1d-OCH tetramers (26). iNKT-TCR affinity values for each clone were expressed as the ratio of the median fluorescent intensity (MFI) of CD1d-OCH tetramer staining normalized to the MFI of the anti-Va24 Ab staining, to account for differences in TCR expression. The distributions of the data were tested with a D’Agostino and Pearson omnibus normality test. Data that did not pass the normality test were analyzed with the nonparametric Mann–Whitney U test, and normally distributed data were analyzed with the unpaired Student t test.

Nineteen people with type 1 diabetes aged 18–35 y and diabetes duration of <4 y and 28 healthy people of similar age and sex were included in this study. The demographic characteristics of our cohort are presented in Table I.

We first sought to compare the ex vivo iNKT frequency in peripheral blood between people with type 1 diabetes and healthy people of similar age and sex. We used two validated protocols for human iNKT quantitation in these samples by employing an Ab-based and a tetramer-based flow cytometry approach (Fig. 1). The results obtained with these protocols showed a high correlation in both healthy control participants (Pearson r = 0.99) and people with type 1 diabetes (Pearson r = 0.67) (Supplemental Fig. 1A, 1B). Consistent with recent reports (23, 25, 29), the frequency of circulating iNKT cells was similar (p = 0.38) between the two groups (Fig. 1D, 1F), and no correlation was found with either the duration of diabetes (Pearson r = 0.15, p = 0.55) (Supplemental Fig. 1E) or the age of the participants (controls, Pearson r = −0.14, p = 0.45; people with type 1 diabetes, Pearson r = −0.18, p = 0.47) (Supplemental Fig. 1C, 1D). We also assessed the ex vivo proportion of iNKT cells expressing high-affinity TCRs (Fig. 1G, 1H). For this, we used CD1d-OCH tetramers that selectively stain iNKT cells expressing high-affinity TCRs, but fail to detectably bind to iNKT cells expressing low-affinity TCR. The results of these experiments failed to demonstrate significant differences in the ex vivo proportion of iNKT cells expressing high-affinity TCR (p = 0.356). However, owing to the very low ex vivo frequencies of high-affinity iNKT cells in peripheral blood, these analyses could not be adequately powered.

FIGURE 1.

Ex vivo frequency of total human iNKT cells in peripheral blood. Live PBMCs from people with type 1 diabetes and matched healthy controls were stained with anti-CD3/anti-Vβ11 Abs (A and B) and with either anti-Vα24 Abs (C and D) or recombinant human CD1d-KRN7000 tetramers (E and F), or recombinant human CD1d-OCH tetramers (G and H). To determine the proportion of iNKT cells expressing high-affinity TCR (H) in healthy donors (black circles) and people with type 1 diabetes (gray circles), the percentage of CD3+/Vβ11+/CD1d-OCH tetramer+ cells was divided by the percentage of CD3+/Vβ11+/CD1d-KRN7000 tetramer+ cells. Horizontal lines show medians.

FIGURE 1.

Ex vivo frequency of total human iNKT cells in peripheral blood. Live PBMCs from people with type 1 diabetes and matched healthy controls were stained with anti-CD3/anti-Vβ11 Abs (A and B) and with either anti-Vα24 Abs (C and D) or recombinant human CD1d-KRN7000 tetramers (E and F), or recombinant human CD1d-OCH tetramers (G and H). To determine the proportion of iNKT cells expressing high-affinity TCR (H) in healthy donors (black circles) and people with type 1 diabetes (gray circles), the percentage of CD3+/Vβ11+/CD1d-OCH tetramer+ cells was divided by the percentage of CD3+/Vβ11+/CD1d-KRN7000 tetramer+ cells. Horizontal lines show medians.

Close modal

Having established a similar frequency of circulating iNKT cells in people with type 1 diabetes and controls, we next examined the functionality of the iNKT repertoire in the two study cohorts. First, we compared the in vitro expansion of total iNKT cells between people with type 1 diabetes and healthy controls in response to autologous monocytes and B cells pulsed with either the partial iNKT agonist Ag OCH or the strong agonist Ag KRN7000 (Fig. 2). To account for interindividual differences in the precursor frequency of CD1d-expressing APCs, we cocultured a fixed number of monocyte- and B cell–depleted PBMCs (CD14CD19 PMBCs) with equal numbers of either lipid- or vehicle-pulsed autologous CD14+ monocytes or B cells. As expected, no iNKT expansion was observed in vehicle-pulsed cultures (data not shown), and total iNKT proliferation was greater in response to KRN7000 compared with OCH stimulation in all subjects, independent of the study group (Fig. 2A, 2B, top panels). Furthermore, the fold expansion of total iNKT cells in the cultures was similar between healthy controls and people with type 1 diabetes in response to either KRN7000 or OCH stimulation (Supplemental Fig. 2A–D).

FIGURE 2.

In vitro expansion of high-affinity iNKT cells in response to full (KRN7000) and partial (OCH) iNKT agonists. Representative flow cytometry plots of total iNKT (A and B, top panels) or high-affinity iNKT expansion (A and B, bottom panels) in response to vehicle (untreated), KRN7000-pulsed, or OCH-pulsed autologous monocytes. The proportion of high-affinity iNKTs that expanded in the cultures in response to either monocytes (C and D) or B cells (E and F) pulsed with KRN7000 or OCH, and in response to CD1d-lipid–conjugated MACSiBeads (G and H), was determined by dividing the proportion of CD1d-OCH tetramer+ iNKTs by the proportion of total iNKTs that expanded in the cultures. Box plots indicate the first and third quartiles and median. (I and J) CD1d surface expression normalized to isotype control Ab in CD14+ monocytes (I) or CD19+ B cells (J). *p < 0.05, **p < 0.01.

FIGURE 2.

In vitro expansion of high-affinity iNKT cells in response to full (KRN7000) and partial (OCH) iNKT agonists. Representative flow cytometry plots of total iNKT (A and B, top panels) or high-affinity iNKT expansion (A and B, bottom panels) in response to vehicle (untreated), KRN7000-pulsed, or OCH-pulsed autologous monocytes. The proportion of high-affinity iNKTs that expanded in the cultures in response to either monocytes (C and D) or B cells (E and F) pulsed with KRN7000 or OCH, and in response to CD1d-lipid–conjugated MACSiBeads (G and H), was determined by dividing the proportion of CD1d-OCH tetramer+ iNKTs by the proportion of total iNKTs that expanded in the cultures. Box plots indicate the first and third quartiles and median. (I and J) CD1d surface expression normalized to isotype control Ab in CD14+ monocytes (I) or CD19+ B cells (J). *p < 0.05, **p < 0.01.

Close modal

Next, we assessed the expansion of iNKT cells expressing high-affinity TCRs in the same in vitro cultures (Fig. 2A, 2B, bottom panels). For this, we used CD1d-OCH tetramers to selectively stain iNKT cells expressing high-affinity TCRs. The results from these experiments showed similar fold expansion of the high-affinity iNKT subset (CD3+/Vβ11+/CD1d-OCH tetramer+ cells) between the two cohorts in response to both lipids (Supplemental Fig. 2G–J). However, compared with healthy controls the proportion of expanded high-affinity iNKT cells among total iNKTs in the monocyte cultures from people with type 1 diabetes remained significantly lower in response to both KRN7000 and OCH stimulation (KRN7000 pulsed, p = 0.035; OCH-pulsed, p < 0.001) (Fig. 2C, 2D). Furthermore, the proportion of expanded high-affinity iNKT cells was significantly lower in people with type 1 diabetes compared with healthy controls in response to OCH-pulsed B cells (p = 0.049) (Fig. 2F). These results suggested that the fold expansion of total iNKT cells was similar between healthy controls and people with type 1 diabetes because the loss of high-affinity iNKT cells in the in vitro cultures from people with type 1 diabetes was masked by the expansion of the numerically predominant low-affinity iNKT subset.

Although in the previous experiments we carefully controlled for the number of APCs and responder cells, we next tested whether the observed lower proportion of high-affinity iNKTs in type 1 diabetes was due to monocyte- or B cell–related differences in Ag presentation or CD1d surface expression. To assess iNKT expansion in the absence of APCs, we cocultured CD14CD19 PBMCs with MACSiBead microparticles conjugated to recombinant human CD1d/KRN7000 or CD1d/OCH monomers, anti-CD2 and anti-CD28. Similar to the results obtained with monocytes and B cells, no significant difference in the fold expansion of total and high-affinity iNKT cells was observed between the two study groups in response to CD1d/KRN7000 or CD1d/OCH MACSiBeads (Supplemental Fig. 2E, 2F, 2K, and 2L). However, compared with healthy controls, the proportion of expanded high-affinity iNKT cells from people with type 1 diabetes was significantly lower in response to CD1d-OCH–conjugated MACSiBead microparticles (p = 0.013) (Fig. 2H), whereas no difference was seen in response to KRN7000 stimulation (Fig. 2G). Finally, CD1d surface expression on CD14+ monocytes and CD19+ B cells was similar between people with type 1 diabetes and healthy donors (Fig. 2I, 2J).

Following from the above results, we sought to determine whether the significantly decreased proportion of high-affinity iNKT cells after in vitro expansion in people with type 1 diabetes may be due to a lower proportion of high-affinity iNKTs in the ex vivo clonal repertoire. In preparation for these experiments, we first characterized the clonal distribution of the iNKT repertoire in healthy donors. For this, single Vα24+Vβ11+ T cell clones from five healthy volunteers were sorted ex vivo and expanded using the nonspecific T cell mitogen PHA as previously described in 2Materials and Methods (Fig. 3). The identity of all 24 expanded clones was confirmed by using CD1d-KRN7000 tetramers (Fig. 3A), whereas CD1d-OCH tetramers were used to determine their iNKT-TCR affinity (Fig. 3B). Finally, we quantified the IL-2 production from these clones in response to KRN7000 and OCH (Fig. 3C). IL-2 production increased in relationship to both the potency of the Ag and the increasing iNKT-TCR affinity. These results were consistent with our previous findings (26) that the healthy clonal iNKT repertoire comprises clones of widely different TCR affinities for CD1d, driving the potency of the iNKT functional response (Fig. 3C).

FIGURE 3.

Clonal composition of the healthy iNKT repertoire. iNKT clones from five healthy donors were expanded in vitro as described in 2Materials and Methods, and stained with either CD1d-KRN7000 tetramers (A) to confirm iNKT identity or CD1d-OCH tetramers (B) to determine iNKT-TCR affinity. To account for differences in TCR expression, the MFI of the CD1d-OCH tetramer staining for each clone was divided by the MFI of the anti-Vα24 Ab stain for the same clone representing the true affinity of the iNKT-TCR (C). iNKT clones with TCR affinity within the first quartile (iNKT-TCR affinity of <0.3) are considered low affinity and clones above the third quartile (iNKT-TCR affinity of >0.8) are considered high affinity. Inset in (C) shows IL-2 production from the same clones segregated into low, medium, and high-affinity according to iNKT-TCR affinity.

FIGURE 3.

Clonal composition of the healthy iNKT repertoire. iNKT clones from five healthy donors were expanded in vitro as described in 2Materials and Methods, and stained with either CD1d-KRN7000 tetramers (A) to confirm iNKT identity or CD1d-OCH tetramers (B) to determine iNKT-TCR affinity. To account for differences in TCR expression, the MFI of the CD1d-OCH tetramer staining for each clone was divided by the MFI of the anti-Vα24 Ab stain for the same clone representing the true affinity of the iNKT-TCR (C). iNKT clones with TCR affinity within the first quartile (iNKT-TCR affinity of <0.3) are considered low affinity and clones above the third quartile (iNKT-TCR affinity of >0.8) are considered high affinity. Inset in (C) shows IL-2 production from the same clones segregated into low, medium, and high-affinity according to iNKT-TCR affinity.

Close modal

Following these results, we generated 115 iNKT clones from 16 people with type 1 diabetes and 138 iNKT clones from 19 healthy people and compared the distribution of the iNKT clonal repertoire between the two groups with regard to the level of iNKT-TCR expression (Fig. 4A, 4B) and iNKT-TCR affinity (Fig. 4C–F). Staining with anti-Vα24 Abs (Fig. 4A) or CD1d-KRN7000 tetramers (Fig. 4B) demonstrated that the surface TCR expression of the iNKT clones from people with type 1 diabetes was significantly decreased (CD1d-KRN7000 tetramer, p < 0.0001; anti-Vα24, p = 0.0044). Next, by using CD1d-OCH tetramers (Fig. 4C) we ranked all iNKT clones from both study groups by their TCR affinity. The results of this analysis showed that compared with healthy people, the clonal iNKT repertoire in people with type 1 diabetes is skewed toward clones expressing lower affinity iNKT-TCRs (p < 0.0001) (Fig. 4C, 4E). To account for differences in the level of surface iNKT-TCR expression between people with type 1 diabetes and healthy controls, the CD1d-OCH tetramer stains were normalized to TCR expression (Fig. 4D, 4F). These results confirmed that people with type 1 diabetes have a significantly lower frequency of iNKT cells expressing high-affinity TCRs, independently of the level of TCR expression (p = 0.0007) (Fig. 4D, 4F). Furthermore, type 1 diabetes was negatively associated with the proportion of in vitro–expanded high-affinity iNKT clones (Pearson r = −0.40, p = 0.021). This resulted in an overall lower empirical cumulative distribution frequency of iNKT-TCR affinity in people with type 1 diabetes (Fig. 4E, 4F, gray lines).

FIGURE 4.

Clonal composition of the iNKT repertoire in people with type 1 diabetes. (A and B) The iNKT identity of in vitro–expanded single CD3+Vα24+Vβ11+ T cell clones was determined by staining with anti-Vα24/anti-Vβ11 Abs and CD1d-KRN7000 tetramers, respectively. The level of surface iNKT-TCR expression was determined by the MFI of the Vα24 stain (A) or the MFI of the CD1d-KRN7000 tetramer stain (B). To determine iNKT-TCR affinity, clones were stained with CD1d-OCH tetramers (C) and the MFI of the CD1d-OCH tetramer staining was normalized to the MFI of the Vα24 stain (D), which reflects the true iNKT-TCR affinity regardless of the level of surface TCR expression. (E and F) Empirical cumulative distribution frequency (ECDF) of iNKT-TCR affinity for healthy people (black line) or people with type 1 diabetes (gray line). CD4 expression was determined for all iNKT clones (A–D; orange circles represent CD4 iNKT clones, black circles show CD4+ iNKT clones). The proportion of CD4+ and CD4 iNKT clones (G) and their iNKT-TCR affinity (H) were determined for healthy people and people with type 1 diabetes. In (H), values show medians, and in parentheses the 25th and 75th percentiles. The p values were determined with the Mann–Whitney U test, and horizontal lines show medians. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Clonal composition of the iNKT repertoire in people with type 1 diabetes. (A and B) The iNKT identity of in vitro–expanded single CD3+Vα24+Vβ11+ T cell clones was determined by staining with anti-Vα24/anti-Vβ11 Abs and CD1d-KRN7000 tetramers, respectively. The level of surface iNKT-TCR expression was determined by the MFI of the Vα24 stain (A) or the MFI of the CD1d-KRN7000 tetramer stain (B). To determine iNKT-TCR affinity, clones were stained with CD1d-OCH tetramers (C) and the MFI of the CD1d-OCH tetramer staining was normalized to the MFI of the Vα24 stain (D), which reflects the true iNKT-TCR affinity regardless of the level of surface TCR expression. (E and F) Empirical cumulative distribution frequency (ECDF) of iNKT-TCR affinity for healthy people (black line) or people with type 1 diabetes (gray line). CD4 expression was determined for all iNKT clones (A–D; orange circles represent CD4 iNKT clones, black circles show CD4+ iNKT clones). The proportion of CD4+ and CD4 iNKT clones (G) and their iNKT-TCR affinity (H) were determined for healthy people and people with type 1 diabetes. In (H), values show medians, and in parentheses the 25th and 75th percentiles. The p values were determined with the Mann–Whitney U test, and horizontal lines show medians. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Finally, we assessed the expression of the CD4 coreceptor in these iNKT clones with relationship to iNKT-TCR affinity. Neither the frequency (Fig. 4G) nor the TCR affinity of the iNKT clones was associated with their CD4 phenotype (Fig. 4H).

To separately assess the function of high- and low-affinity iNKT clones in type 1 diabetes compared with healthy controls, we measured the clonal production of IFN-γ, IL-4, IL-13, and GM-CSF following stimulation with plate-bound CD1d-KRN7000 or CD1d-OCH monomers (Fig. 5). For these experiments, we thawed frozen iNKT clones and simultaneously restimulated 32 clones from nine people with type 1 diabetes and 29 clones from nine healthy people as described previously in 2Materials and Methods. Prior to the plate-bound stimulation, we reconfirmed their cell viability, iNKT identity, and TCR affinity (Supplemental Fig. 3A, 3B). Compared to healthy people, iNKT cells from people with type 1 diabetes produced significantly more GM-CSF (p = 0.0001), IL-13 (p = 0.007), IL-4 (p = 0.0002), and IFN-γ (p = 0.0023) in response to plate-bound CD1d-KRN7000 monomers (Fig. 5A–D, left panels). This increased production of GM-CSF, IL-13, IL-4, and IFN-γ in type 1 diabetes was confined to the low-affinity iNKT subset (GM-CSF, p < 0.0001; IL-13, p = 0.0024; IL-4, p = 0.0095; IFN-γ, p = 0.011) (Fig. 5A–D, right panels). A significant increase in GM-CSF and IL-13 production in people with type 1 diabetes was also observed in response to plate-bound CD1d-OCH monomers (GM-CSF, p = 0.0067; IL-13, p = 0.02) (Fig. 5E, 5F, left panels), and it was similarly confined to the iNKT subset expressing low-affinity TCRs (GM-CSF, p = 0.0004; IL-13, p = 0.0008) (Fig. 5E, 5F, right panels). Furthermore, low-affinity iNKT clones from people with type 1 diabetes also produced significantly more IL-4 (p = 0.0043) (Fig. 5G), but not IFN-γ (Fig. 5H), in response to plate-bound CD1d-OCH monomers.

FIGURE 5.

Cytokine production from iNKT clones of people with type 1 diabetes and healthy controls. iNKT clones from nine people with type 1 diabetes (gray circles, n = 32) and nine healthy controls (black circles, n = 29) were thawed and restimulated in vitro with either plate-bound CD1d-KRN7000 monomers (AD) or plate-bound CD1d-OCH monomers (EH) as described in 2Materials and Methods. The cell culture supernatants were collected after 24 h for cytokine analysis. The figures show cytokine production (picograms per milliliter) from all iNKT clones (left panels) and from the same clones segregated by iNKT-TCR affinity (right panels). Only clones of iNKT-TCR affinity within the upper quartile (high affinity) and the lower quartile (low affinity) were chosen for this analysis. Horizontal lines show medians. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

Cytokine production from iNKT clones of people with type 1 diabetes and healthy controls. iNKT clones from nine people with type 1 diabetes (gray circles, n = 32) and nine healthy controls (black circles, n = 29) were thawed and restimulated in vitro with either plate-bound CD1d-KRN7000 monomers (AD) or plate-bound CD1d-OCH monomers (EH) as described in 2Materials and Methods. The cell culture supernatants were collected after 24 h for cytokine analysis. The figures show cytokine production (picograms per milliliter) from all iNKT clones (left panels) and from the same clones segregated by iNKT-TCR affinity (right panels). Only clones of iNKT-TCR affinity within the upper quartile (high affinity) and the lower quartile (low affinity) were chosen for this analysis. Horizontal lines show medians. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Due to conflicting reports regarding the function of CD4+ versus CD4 iNKT clones, we also segregated the iNKT clones that were used for cytokine analysis by surface CD4 expression (Fig. 6, Supplemental Fig. 3C). In agreement with previously published reports, CD4 iNKT clones from healthy individuals produced significantly more GM-CSF (p < 0.0001) and IFN-γ (p = 0.0012) in response to plate-bound CD1d-KRN7000 stimulation (Fig. 6A, 6D). However, in people with type 1 diabetes, CD4+ and CD4 iNKT clones produced equal amounts of these cytokines in response to plate-bound KRN7000 stimulation (Fig. 6A, 6D). As a result, the CD4+ iNKT clones from people with type 1 diabetes produced significantly more GM-CSF (p = 0.0021) and IFN-γ (p = 0.0003) compared with CD4+ iNKT clones from healthy controls. In response to weaker stimulation with plate-bound CD1d-OCH (Fig. 6E–H), the CD4+ iNKT clones from people with type 1 diabetes produced significantly more GM-CSF (p < 0.0001) (Fig. 6E) and similar amounts of IL-13, IL-4, and IFN-γ (Fig. 6F–H) compared with healthy CD4+ iNKT clones.

FIGURE 6.

Cytokine production from CD4+ and CD4 iNKT clones of people with type 1 diabetes and healthy controls. The same iNKT clones from Fig. 5 were also segregated by their CD4 surface expression and the cytokine production in response to plate-bound CD1d-KRN7000 (AD) and CD1d-OCH (EH) stimulation was determined in CD4 versus CD4+ iNKT clones. Black circles indicate iNKT clones from healthy donors; open gray circles indicate iNKT clones from people with type 1 diabetes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6.

Cytokine production from CD4+ and CD4 iNKT clones of people with type 1 diabetes and healthy controls. The same iNKT clones from Fig. 5 were also segregated by their CD4 surface expression and the cytokine production in response to plate-bound CD1d-KRN7000 (AD) and CD1d-OCH (EH) stimulation was determined in CD4 versus CD4+ iNKT clones. Black circles indicate iNKT clones from healthy donors; open gray circles indicate iNKT clones from people with type 1 diabetes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Conflicting evidence exists regarding the role of iNKT cells in human type 1 diabetes (29). Whereas earlier reports found a significant reduction of circulating iNKT cells in type 1 diabetes (19, 21), subsequent studies using more accurate methods for ex vivo iNKT identification could not replicate those findings (23, 25).

The present study assessed quantitative as well as qualitative changes of the iNKT repertoire in people with recent onset of type 1 diabetes compared with matched control subjects. Similarly to previous studies from Lee et al. (24) and Roman-Gonzales et al. (25), we found no differences in the ex vivo frequency of circulating total CD1d-restricted iNKT cells between the two groups. We then carried out more detailed analyses of the iNKT repertoire, including assessments of the clonal composition with regard to iNKT-TCR affinity and function. These studies revealed a selective loss of iNKT clones expressing high-affinity TCRs (high-affinity iNKT) in people with type 1 diabetes, resulting in a shift of the repertoire toward iNKT clones bearing lower affinity iNKT-TCRs (low-affinity iNKT).

First, we attempted to measure the ex vivo proportion of circulating iNKT cells bearing high-affinity TCR in both study groups (Fig. 1G, 1H) using our previously validated CD1d tetramer-based method (26). Due to the low numbers of circulating iNKT cells in the study subjects, these experiments failed to reveal statistically significant differences in the ex vivo proportion of high-affinity iNKT cells between healthy people and people with type 1 diabetes. To overcome the challenge of low ex vivo iNKT frequency, we then carried out a series of in vitro tests to determine the composition and function of the clonal iNKT repertoire in people with type 1 diabetes and healthy donors.

Our in vitro analysis of polyclonal iNKT expansion in response to lipid-pulsed autologous APCs strongly indicated a lower precursor frequency of high-affinity iNKT cells in type 1 diabetes. Similar results were obtained when we used synthetic CD1d/OCH-conjugated MACSiBeads for iNKT expansion instead of autologous APCs. Furthermore, surface CD1d expression levels on both monocytes and B cells were similar in both study groups. Taken together, these data indicated that the lower proportion of high-affinity iNKT cells was not caused by aberrant CD1d Ag presentation in type 1 diabetes, but rather by iNKT-intrinsic factors.

To address this further, we analyzed a large panel of ex vivo–sorted iNKT clones from people with type 1 diabetes and healthy donors for their binding to CD1d tetramers loaded with either the partial agonist Ag OCH (CD1d/OCH tetramers) or the full agonist KRN7000 (CD1d/KRN tetramers). We have previously shown that this CD1d tetramer–based method reliably ranks iNKT clones according to their true TCR affinity for CD1d (26), and it also correlates with iNKT function (Fig. 3). By using a panel of healthy donors’ iNKT clones, we confirmed a strong positive correlation between iNKT-TCR affinity and IL-2 cytokine production (Fig. 3). Subsequent comparative analyses of the clonal repertoire between people with type 1 diabetes and healthy controls confirmed the qualitative shift of the iNKT repertoire in type 1 diabetes toward low-affinity iNKTs. Additionally, people with type 1 diabetes had a significantly decreased iNKT-TCR expression consistent with previous Ag encounter. Functional analyses also revealed a significantly increased secretion of GM-CSF, IL-13, and IL-4 from the low-affinity iNKT clones, both in response to KRN7000 and OCH. Importantly, the results from both our clonal and functional studies were not confounded by CD4 expression on the iNKT clones. The iNKT-TCR affinity was significantly lower in people with type 1 diabetes regardless of CD4 expression (Fig. 4), and similarly to previous reports, our results confirm an increased Th1 cytokine production from CD4-expressing iNKT clones in people with type 1 diabetes (Fig. 6) (23, 30, 31).

A potential caveat of our study is that we cannot rule out differences between high- and low-affinity iNKT clones with regard to their propensity for in vitro proliferation and survival. However, this would be expected to equally affect iNKT cells from healthy people and people with type 1 diabetes. Our data revealed significant differences in the clonal repertoire between people with type 1 diabetes and healthy controls, with a shift toward low-affinity iNKT in people with type 1 diabetes. Taken together, these results provide the first evidence for an asymmetrical activation of high- and low-affinity iNKT clones in type 1 diabetes. Furthermore, they support the notion that high- and low-affinity iNKT clones exert differential regulatory roles in autoimmune inflammation such as human type 1 diabetes.

In conclusion, our results from a small cohort of people with type 1 diabetes of short duration and healthy controls of similar age and sex show, to our knowledge for the first time, that in human type 1 diabetes the clonal iNKT repertoire is skewed toward iNKT clones expressing lower affinity iNKT-TCRs. Recently, we have reported similar results in a cohort of early rheumatoid arthritis patients (32) suggesting that our observations may extend to other autoimmune diseases. Several preclinical studies in autoimmune mouse models have pointed to a key regulatory role for iNKT cells in autoimmune inflammation, including type 1 diabetes. Although the results of our study support the notion that iNKT cells may be a therapeutic target in people with type 1 diabetes, deeper insights into the early changes of the iNKT repertoire in type 1 diabetes, even before onset of overt disease, are required to advance this idea. Unraveling the reasons behind the in vivo shifts of high- and low-affinity iNKT cells in type 1 diabetes that we observed in this study, as well as understanding the differential roles of these populations for induction and maintenance of immune tolerance in humans, may inspire new powerful therapies for autoimmunity in the future.

We thank the study volunteers for their time and Dr. Geraint Dingley and Dr. Hitasha Rupani from the Department of Clinical and Experimental Sciences at the Faculty of Medicine, University of Southampton, for sample collection. We thank Dr. Michael Breen for revising the manuscript and for statistical support. We also thank Dr. Regina Teo for technical support and Dr. Richard Jewell and Dr. Carolann McGuire at the Faculty of Medicine for endless flow cytometry support.

This work was supported by the Parnell Fund–Southampton Hospital Charity (registered charity no. 1051543) and by Diabetes UK Project Grant 13/0004749.

The online version of this article contains supplemental material.

Abbreviations used in this article:

iNKT

invariant NKT

MFI

median fluorescence intensity.

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The authors have no financial conflicts of interest.

Supplementary data