OBJECTIVE—Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing β-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo.

RESEARCH DESIGN AND METHODS—We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2β/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared.

RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2β/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42–56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice.

CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and β-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

Autoimmune type 1 diabetes is a chronic, complex autoimmune disease mediated by the infiltration of autoreactive lymphocytes into the pancreas, resulting in the destruction of the insulin-producing β-cells in the islets of Langerhans (1). The nonobese diabetic (NOD) mouse is a spontaneous murine model of type 1 diabetes and involves many of the same autoantigens targeted by human T-cells (15). CD4+ T-cells appear to be essential in both the early and late stages of the disease, as evidenced by the ability of multiple CD4+ T-cell clones to transfer disease (68). Additionally, anti-CD4 therapy can prevent the onset of disease in NOD mice (9).

Over two dozen islet specific autoantigens have been implicated in the initiation and/or pathogenesis of diabetes (6). While T-cell responses to many of these antigens have been studied (e.g., GAD [1013], insulin [14,15], the tyrosine phosphatase-like protein insulinoma-associated protein 2 (IA2) [islet cell antigen 512] [1618], and IA2β [phogrin] [19,20]), their relative contribution to disease initiation or pathogenesis remains unclear. Furthermore, the specificities of the majority of diabetogenic T-cell clones isolated from NOD mice are still unknown (8). A key question that remains unresolved is the extent to which autoantigenicity and pathogenicity are linked.

The hallmark of preclinical type 1 diabetes is the infiltration of CD4+ T-cells into the pancreatic islets. While many cell types participate in the disease process, CD4+ T-cells command a central role in the initiation, regulation, and progression of the disease. Clearly autoantigen specificity plays a critical role in governing CD4+ T-cell entry into islets. However, the ability to study these processes using unmanipulated T-cell populations that have a broad range of insulitic and diabetogenic potential has been greatly limited. T-cell receptor (TCR) transgenic (Tg) mice are powerful tools for the analysis of autoimmune diseases such as type 1 diabetes. However, only five major histocompatibility complex class II–restricted, autoantigen-specific Tg NOD lines have been described (BDC-2.5, BDC-6.9, NY4.112-4.1, and a GAD286–300 Tg), providing a limited phenotypic range (2125). We developed a new approach for the rapid generation of TCR Tg mice, refered to as retrogenic (Rg) mice, using retroviral-mediated stem cell gene transfer and novel self-cleaving 2A peptide–linked multicistronic retroviral vectors that express both TCR chains plus a fluorescent protein marker in a single vector (2630). This approach allows us to generate and analyze multiple TCR Rg mice on a variety of defined genetic backgrounds in less than 2 months. This is particularly important in studying diabetes, for which background genes have a significant effect on disease outcome (31). Furthermore, the phenotype of Tg mice can be affected by additional variables such as the level and timing of TCR expression and the possibility of insertional mutagenesis by the integrated transgene. These issues are eliminated in Rg mice because the same expression system is used for all TCRs; multiple, independently generated mice are analyzed; and retroviral integration is widespread. This facilitates the direct comparison between multiple TCRs.

The goal of this study was to assess the link between autoreactivity versus pathogenicity of multiple autoantigen-specific TCRs using the Rg system. We chose to clone and generate a panel of Rg mice expressing twelve different TCRs specific for four known autoantigens (various epitopes of GAD65, IA2, phogrin, and insulin) and four different TCRs specific for unknown islet antigens. All four known autoantigens have been implicated in the disease onset, but their relative contribution is still controversial. With this panel of Rg mice, we addressed two important questions. First, are all autoreactive TCRs pathogenic and capable of initiating insulitis and diabetes in the absence of other T-cells and B-cells? Second, is there a defined relationship between the extent of insulitis, the time of diabetes onset, and the incidence of diabetes?

NOD.scid mice were obtained from The Jackson Laboratory and bred in-house. B6.H2g7 mice were provided by Christophe Benoist and Diane Mathis (Joslin Diabetes Center, Boston, MA) (31) and crossed onto an RAG-1−/− background. Diabetes incidence was monitored on a biweekly, weekly, or more frequent basis by testing for the presence of glucose in the urine by Clinistix (Bayer, Elkhart, IN). Mice testing positive by Clinistix were then tested with a One Touch Ultra glucometer (Lifescan, Milpitas, CA) for blood glucose levels and were considered diabetic if their blood glucose was >200 mg/dl for retroviral-mediated stem cell gene transfers or 400 mg/dl for adoptive transfers. All mice were bred and housed at the St. Jude Animal Resources Center (Memphis, TN) in a Helicobacter-free specific pathogen free facility following state, national, and institutional mandates. The St. Jude Animal Resources Center is accredited by the American Association for the Accreditation of Laboratory Animal Care. All animal experiments followed animal protocols approved by the St. Jude institutional animal care and use committee.

TCR retroviral constructs.

All TCRs were generated as 2A-linked single open reading frame inserts using RT-PCR and cloned into a murine stem cell virus–based retroviral vector with a green fluorescent protein (GFP) marker as previously described (26,2830). Details of cloning strategies and primer sequences are available on request (amanda.burton@stjude.org). TCRs were cloned by PCR using plasmid or cDNA obtained from Phogrin13 and Phogrin18 T-cell clones; 10.23 hybridoma from John Hutton (Barbara Davis Center for Diabetes, Aurora, CO); BDC-6.9 and BDC-10.1 T-cell clones from Kathryn Haskins (University of Colorado Health Sciences Center, Denver, CO); BDC-2.5 TCR plasmids from Luc Teyton (Scripps Research Institute, La Jolla, CA); NY4.1 TCR plasmids from Pere Santamaria (University of Calgary, Alberta, Canada); 1A4 TCR plasmids from Roland Tisch (University of North Carolina, Chapel Hill, NC); 530.45.19 hybrid from Eli Sercarz (Torrey Pines Institute for Molecular Studies, San Diego, CA); and PA15.14B12, PA19.5E11, PA18.10F10, PA17.9G7, PA18.9H7 and PA21.14H4 hybridomas generated in our lab by Paula Arnold. The 12-4.4v1 and 12-4.1 TCR constructs were generated de novo from the published sequence data (12-4.1 accession numbers: DQ172905 for α-chain and DQ180320 for β-chain) and information generously provided by George Eisenbarth (Barbara Davis Center for Childhood Diabetes). For TCR with unknown Vα and Vβ usage, Vβ was determined by flow cytometry using Vβ-biotinylated antibodies and streptavidin phycoerythrin (PE). Vα usage was determined by RT-PCR with Vα primers, cloning into the PCR Blunt II-TOPO vector (Invitrogen, Carlsbad, CA), and sequencing as previously described (32). All TCR 2A-linked vectors utilize the PTV1.2A sequence except for PA21.14H4, NY4.1, and PA18.10F10, which utilize the TaV.2A sequence (27,28). The BDC-10.1 and BDC-6.9 vectors contain a gly-ser-gly linker between the Cα region and the PTV1.2A sequences. The BDC-2.5 vector lacks this linker, while the remaining vectors contain a gly between the Cα region and the 2A sequences.

Retroviral-mediated stem cell gene transfer, flow cytometric analysis, and cell sorting.

Retroviral-mediated stem cell gene transfer was performed as previously described (2630). At 35, 70, and 140 days posttransplant, spleens, inguinal lymph nodes (ILNs), and pancreatic lymph nodes (PLNs) were harvested, processed, counted, and stained with TCRβ(H57)-PE/and CD4-APC (BD Biosciences Pharmingen, San Diego, CA). For functional assays, splenocytes were stained with CD4-PE (BD Biosciences Pharmingen, San Diego, CA) and sorted with a fluorescence-activated cell sorter (FACS) for GFP+/CD4+ on a MoFlow (Dako, Fort Collins, CO).

Adoptive transfers.

At 28 days posttransplant, GFP+CD4+TCR+ splenocytes from NOD.scid TCR Rg mice were purified by FACS and injected intravenously into NOD.scid recipient mice.

Islet isolation.

Pancreata were perfused by injecting 3 ml collagenase P (1.5 μg/ml in Hanks’ Balanced Salt Solution [HBSS]) (Roche, Indianapolis, IN), harvested, and placed in 3–5 ml collagenase P on ice. The pancreata were then incubated at 37°C for 16–20 min, after which 7 ml Hanks’ balanced salt solution (HBSS) with 5% fetal bovine serum (FBS) was added and the tissues pelleted at 1,000 rpm for 3 min. The pellet was washed twice with 7 ml 5% FBS/HBSS and resuspended in 7 ml 5% FBS/HBSS. Islets were handpicked and dissociated with 1 ml cell dissociation buffer (Invitrogen, Carlsbad, CA) and incubated at 37°C for 5 min. After vortexing, the dissociation process was repeated twice. Cells were placed in 10 ml 5% FBS/HBSS, centrifuged for 10 min at 1,300 rpm, resuspended in 5% FBS/HBSS, and irradiated at 3,000 rads.

Functional assays.

To determine the activation index of TCRs with known specificities, 5 × 105 splenocytes of mice at 70 or 140 days posttransplant were cultured with or without peptide in either supplemented Eagle's minimum essential medium or RPMI with 10% FBS for 48 h in 96-well flat-bottom plates. In some instances, splenocytes were FACS sorted as described above, and 2.5 × 104 purified T-cells were cultured with irradiated (3,000 rads) NOD/LtJ splenocytes with or without peptide for 48 h. The wells were then pulsed with 1μCi/well of [3H]-thymidine for the final 8 or 24 h and then harvested. The peptides used were: IA2678–688, GAD217–236, GAD284–300, GAD206–220, GAD510–524, GAD524–538, GAD530–543, Hen egg white lysozyme (HEL)11–25, insulin B9–23, IA2β640–659, and IA2β755–777. For TCRs of unknown specificity, 2.5 × 104 purified GFP+/CD4+ splenic T-cells were cultured with 5.0 × 104 irradiated purified islets from 11- to 19-week-old NOD/LtJ mice in supplemented Eagle's minimum essential medium with 10% FBS in 96-well round-bottom plates for 72 h, pulsed with 1μCi/well of [3H]-thymidine for 18 h, and then harvested.

Insulitis scores.

Pancreata of NOD.scid TCR retrogenic mice were harvested at 28, 70, and 140 days posttransplant; placed into 10% buffered formalin; and embedded in paraffin; 4 μm–thick sections were cut at 150-μm step-sections and stained with hematoxylin and eosin at the St. Jude Histology Core Facility. An average of 90–100 islets per mouse were scored in a blinded manner using the following metric: no insulitis (normal islet and no infiltration), peri-insulitis (infiltration on edges of islet or 0–20% of islet infiltrated) or insulitis (infiltration of 30–100% of islet).

Statistics.

The time to diabetes (survivor function) for each group of mice was estimated using the Kaplan-Meier log-rank test both overall and pairwise within each experiment. The overall Type I error rate for each experiment was controlled at the 0.05 level using the Bonferroni adjustment for multiple comparisons.

Cloning and expression of β-cell–specific TCRs.

GAD65 is one of the principal autoantigens in human type 1 diabetes and was one of the first autoantigens implicated in diabetes in NOD mice (10,11). Nevertheless, its role as a target autoantigen remains controversial (24,26,33,34). We cloned and expressed seven different TCRs specific for the major immunogenic GAD epitopes and others that have been proposed to modulate disease progression (Table 1) (35,36). All of the GAD-specific TCRs, with the exception of 1A4, were derived by immunizing NOD mice with peptide. 1A4 was isolated from the islets of a nonimmunized, nondiabetic NOD mouse. Four different TCRs based on relevant epitope immunogenicity were chosen: PA15.14B12 and PA19.5E11 (specific for GAD206–220, the primary immunogenic epitope of GAD65) and 1A4 and PA17.9G7 (which covered the second and third most immunogenic epitopes GAD221–235 and GAD284–300, respectively) (35,36). We also cloned and expressed three different TCRs specific for the COOH-terminus portion of GAD65 thought to be important in the initiation of islet infiltration and β-cell destruction, two of which—PA18.9H7 and 530.45.19—cover the GAD524–538 and GAD530–543 epitope, respectively, and PA18.10F10, specific for GAD510–524, just upstream of epitope GAD524–543 (Table 1). 530.45.19 and PA18.9H7 are particularly intriguing because it has been reported that spontaneously arising, GAD-reactive T-cells in NOD mice use Vβ4+ (530.45.19) and are specific for the 530–543 epitope, while TCRs that recognize GAD524–538 are primarily generated by immunization, use Vβ12 (PA18.9H7) and may have regulatory activity (10,13,34).

Insulin has also been implicated in the diabetogenic response. A considerable number of T-cells infiltrating the islets react to insulin, with >90% recognizing the insulin B9–23 epitope (15,37). Recently it has been shown that insulin I/II null NOD mice expressing an insulin transgene with a mutation in the critical insulin B9–23 epitope did not develop disease, suggesting that it is a primary autoantigenic epitope (38). T-cell clones generated against insulin B9–23 both accelerate diabetes onset in NOD mice and confer disease following adoptive transfer into NOD.scid mice (15). The 12-4.1 and 12-4.4v1 TCRs were originally isolated from the islets of prediabetic NOD mice and were reconstructed by PCR from published sequences (see research design and methods and Table 1 for details) and expressed in NOD.scid mice.

Insulinoma-associated tyrosine phosphatase-like protein (IA2) or islet cell antigen 512 is found in the secretory granules of β-cells (17,39) and is a potential target of CD4+ T-cells (16,18,40). We cloned the TCR 10.23, which is specific for IA2 and was derived through immunizations with whole protein. We later identified the epitope as IA2676–688 (data not shown). In addition, given the implication of IA2β (phogrin)-reactive CD4+ T-cells in disease pathogenesis (19,20,41), two phogrin-specific TCRs, phogrin13 and phogrin18, were cloned from T-cell lines established by immunization with whole protein (41).

Included in this study were clonotypic BDC-2.5, BDC-6.9, BDC-10.1, and NY4.1 TCRs specific for undefined β-cell epitopes. BDC-2.5, BDC-6.9, and BDC-10.1 were the first diabetogenic CD4+ T-cell clones described and were originally isolated from the spleen and lymph nodes of diabetic NOD mice by Haskins and colleagues (8,42,43), while NY4.1 was one of six CD4+ T-cell clones that were isolated from the islets of acutely diabetic NOD mice and were reactive to islet cells (44). These clones are highly diabetogenic upon transfer into NOD.scid mice or when expressed as a transgene in NOD mice (BDC-2.5, BDC-6.9, and NY4.1) (2123,45,46). We have previously generated BDC-2.5 and NY4.1 Rg mice (26), which were included in this study as positive controls for the other TCR Rg mice. Likewise, Rg mice expressing PA21.14H4, an H-2Ag7–restricted, HEL11–25-specific TCR, were included as negative controls.

TCR Rg mice were established using NOD.scid mice as both bone marrow donors and recipients. This enabled us to examine the ability of Rg T-cells to infiltrate the islets and mediate diabetes in the absence of other T-cells or B-cells. For each TCR, we examined expression and reconstitution levels, T-cell proliferative capacity, and the incidence of insulitis and diabetes in the Rg mice.

Expression and function of β-cell–specific TCRs.

All of the TCRs were expressed in mice and had comparable levels of GFP (Fig. 1 and data not shown). However, there was variability in TCR expression levels compared with the NOD control and in the number of GFP+/TCR+/CD4+ Rg T-cells in the spleen, ILN, and PLN (Figs. 1 and 2). This is likely due to the influence of thymic selection and the availability of selecting ligands, as well as homeostatic and antigen-driven proliferation in the periphery. Importantly, TCR expression level and T-cell numbers were consistent within each group, suggesting that this variability was due to intrinsic characteristics of each TCR rather then experimental variance. Also, there was a correlation between the number of T-cells in the spleen compared with the ILN and PLN within each TCR Rg group (Fig. 2).

The GAD65-specific TCRs were reactive to and specific for their respective peptides, albeit with differing efficiencies (Table 1 and Fig. 3A and B). While 12-4.1 and 12-4.4v1 recognize the same epitope (insulin B9–23), the significant difference in stimulation index observed is likely due to differences in cognate ligand affinity caused by differences in complimentarity-determining region/Vβ usage (Table 1). Furthermore, differences in IA2- and phogrin-specific TCR expression in 10.23, phogrin13, and phogrin18 Rg mice may explain the differing reactivity observed (Table 1 and Figs. 1 and 3A). However, Rg T-cells were antigen specific and T-cells isolated from the spleen and PLN reacted equivalently, as exemplified with phogrin18 T-cells (Fig. 3C and data not shown). Finally, all of the unknown islet antigen–specific TCRs—BDC-2.5, BDC-6.9, BDC-10.1, and NY4.1—were expressed at detectable levels (Fig. 1). The level of TCR expression was more variable among some of the Rg mice. This was particularly evident with BDC-2.5 T-cells and may, in part, be due to the relatively low level of TCR expression seen in these mice compared with other Rg T-cells (Figs. 1 and 2). The functionality of these Rg T-cells was determined by measuring proliferation in response to purified, irradiated islets from 11- to 19-week-old NOD mice. BDC-10.1 Rg T-cells were strongly reactive to irradiated islets, while BDC-2.5 T-cells responded weakly compared with the control HEL-specific PA21.14H4 Rg T-cells, Rg T-cells alone, and islets alone (Fig. 3D and data not shown). In contrast, minimal reactivity was seen with BDC-6.9 and NY4.1 Rg T-cells, suggesting that either these islets express insufficient levels of the antigenic epitope to elicit proliferation in vitro or, more likely, that T-cells derived from these Rg mice are less sensitive than the parent T-cell clones.

Insulitis and incidence of diabetes of Rg mice.

Next, we evaluated the extent of insulitis 28, 70, and/or 140 days post–bone marrow transplant and the incidence of diabetes for 140 days in the 17 TCR Rg mouse lines. Essentially, no peri-insulitis or insulitis was observed in any of the GAD-specific Rg mice (Table 1 and Fig. 4). Likewise, minimal infiltration of IA2676–688–specific 10.23 T-cells or IA2β/phogrin640–659–specific phogrin13 T-cells was seen in the respective Rg mice. This was surprising because the phogrin-specific T-cell clones phogrin15 and phogrin12 infiltrate rat islets transplanted under the kidney capsule and destroy β-cells (41). However, demonstrable peri-insulitis (∼11%) and insulitis (∼6%) were observed in IA2β/phogrin755–777–specific phogrin18 Rg mice, although this was not manifested until 140 days posttransfer (Table 1 and Fig. 4). The differential ability of these phogrin-specific T-cells to infiltrate islets may be due to differences in epitope recognition or TCR affinity/avidity (Table 1). Not surprisingly, none of these Rg mice developed diabetes (Table 1).

Analysis of the two insulin B9–23 Rg mice was particularly intriguing. While significant peri-insulitis and insulitis were observed, insulitis was greater in the 12-4.1 versus the 12-4.4v1 Rg mice (Table 1). Furthermore, diabetes developed in 33% of the 12-4.1 Rg mice (ID50 –60 days), while none of the 12-4.4v1 Rg mice became diabetic. The difference in diabetes incidence may be due to differences in the relative avidity of the corresponding T-cells to antigen (Table 1). The peri-insulitis/insulitis score appeared to decline by 140 days posttransfer. Given that a proportion of the 12-4.1 Rg mice becomes diabetic, it is possible that this reduced insulitis score is due to the reduced disease incidence in the remaining mice. However, this cannot account for the decline in the 12-4.4v1 Rg mice and suggests that the insulitis resolves with time in these mice.

As previously reported, BDC-2.5 and NY4.1 Rg mice developed significant insulitis ∼28 days posttransfer, which is remarkable given that it takes ∼50 days for complete bone marrow reconstitution and maximal T-cell population of the periphery. There was also substantial insulitis in the BDC-10.1 Rg mice (87% for BDC-10.1 vs. 93% for BDC-2.5 at 28 days posttransfer [Table 1 and Fig. 4]). Indeed, there was a correlation between the percentage of infiltrating CD4+ Rg T-cells and the severity of insulitis and incidence of diabetes (BDC-10.1, 5.9% CD4+ T-cells in the islets; BDC-2.5, 5.1%; 12-4.1, 3.6%; phogrin18, 0.6%; and PA21.14H4, 0.2%).

Diabetes incidence in the BDC-2.5 Rg mice was 100% with an ID50 of 52 days. Curiously, the rate of diabetes development in the NY4.1 Rg mice was comparable (ID50 –46 days) but the incidence was only 71% (Table 1 and Fig. 5A). Insulitis development in the BDC-6.9 Rg mice was delayed, with no insulitis at day 28 but substantial insulitis at days 70 and 140 posttransfer. Consistent with this, diabetes onset in the BDC-6.9 Rg mice was delayed (ID50 –81 days; incidence –56%), in accordance with observations made with their Tg counterparts (22). Diabetes onset in the BDC-10.1 Rg mice was rapid and highly penetrant with all the mice hyperglycemic by day 33 (ID50 –27 days) (Fig. 5A). Given the diabetogenic potential of the BDC-10.1 TCR and the flexibility of the Rg system, BDC-10.1 Rg T-cells were generated on a C57BL/6 background using the B6g7.RAG-1−/− mice. Interestingly, BDC-10.1 Rg T-cells on a nonautoimmune background mediated diabetes, albeit at a slower rate (ID50 − 76 days) and reduced frequency (80%) (Fig. 5A).

To further evaluate the diabetogenic potency of the BDC-10.1 Rg T-cells, GFP+CD4+ T-cells were sorted from BDC-10.1, NY4.1, and the control TCR PA21.14H4 Rg mice and adoptively transferred into NOD.scid mice (Fig. 5B). As expected, the HEL-specific T-cell recipients failed to develop diabetes, while mice receiving NY4.1 Rg T-cells initially developed diabetes by day 55 posttransfer, and by day 138 all recipients were diabetic. Strikingly, all of the BDC-10.1 T-cell recipients developed diabetes by day 13 posttransfer. Finally, we adoptively transferred titrated numbers of purified BDC-10.1 Rg T-cells into NOD.scid mice to define the minimal number required to mediate disease (Fig. 5C). Remarkably, only 50,000 BDC-10.1 Rg T-cells were required to cause rapid diabetes onset in 100% of recipients by day 13, while as few as 1,000 BDC-10.1 Rg T-cells caused diabetes in 80% of recipients. Taken together, these data highlight the increased diabetogenic potential of BDC-10.1 T-cells.

Type 1 diabetes is a complex disease mediated by T-cells of multiple specificities, many of which have yet to be determined. Important questions remain concerning what governs T-cell islet entry, what makes a T-cell diabetogenic, and what the relationship is between autoreactivity and pathogenicity. While the generation of TCR Tg mice has provided important insight into the disease process, these studies represent a limited repertoire of potential autoantigen epitope-specific TCRs that might mediate type 1 diabetes. Our study demonstrates the utility of the Rg approach for the generation and analysis of mice expressing a large panel of TCRs with varied specificity. These data show that for the panel of TCRs studied, relatively few promote islet entry and β-cell destruction in the absence of other T-cells and B-cells. Indeed, it is noteworthy that four of the five TCRs that promoted diabetes when expressed in Rg mice recognize unknown antigens.

It has recently been suggested that GAD-reactive T-cells may not be as critical to the initiation and progression of type 1 diabetes as originally thought (10,11,33). Our data support this view. Despite the high immunogenicity of GAD epitopes (206–220, 221–235 and 284–300) and the large number of Rg T-cells in the spleens and lymph nodes, no β-cell destruction was seen in the seven GAD-reactive TCR Rg mouse lines analyzed. While some peri-insulitis was observed in some mice, particularly those expressing TCR specific for distal GAD epitopes (e.g., 530.45.19 specific for GAD530–543), this was not significantly greater compared with HEL-specific Rg mice (PA21.14H4). While we cannot rule out a role for GAD-reactive T-cells in the later stages of pathogenesis or in concert with other T-cells (34), our data suggest that T-cells expressing the current panel of clonotypic TCRs specific for GAD are not capable of initiating insulitis and diabetes on their own. Furthermore, several studies suggest that certain GAD-reactive TCRs are expressed by regulatory T-cells, an issue that was not addressed in this study. While the IA2-specific Rg T-cells did not infiltrate the islets, suggesting that IA2 may not be an important autoantigenic epitope in the early stages of type 1 diabetes, both phogrin-specific T-cell populations infiltrated islets to varying degrees. However, although phogrin18 mediated significant peri-insulitis/insulitis, likely due to increased TCR expression and T-cell number, this required 140 days to develop. Taken together, these data suggest that some of the most commonly studied type 1 diabetes autoantigens—GAD, IA2 and IA2β/phogrin—may have a reduced role in initiating type 1 diabetes.

On the other hand, the importance of insulin as an autoantigen (38,47) was reaffirmed in this study. Both insulin-specific TCRs conferred significant insulitis, while only 12-4.1 caused diabetes. Given that these TCRs recognize the same epitope, the phenotypic distinctions observed are likely due to differences in TCR Vβ (Vβ2 vs. Vβ12) and complimentarity-determining region usage, and/or affinity/avidity as the 12-4.1 Rg T-cells have a considerably higher stimulation index against the insulin B9–23 epitope. This suggests that while autoantigen and epitope availability are important factors in mediating the disease process, TCR affinity is likely an important factor.

Differences in the diabetogenicity of TCRs from BDC-2.5, BDC-6.9, and BDC-10.1 in Rg mice would not have been predicted from their pathogenicity as T-cell clones, which is very similar in adoptive transfers into young NOD or NOD.scid recipients (8,43). The hierarchy of insulitic and diabetogenic potential exhibited by the three TCRs that had previously been expressed as transgenes was largely as expected (BDC-2.5 > NY4.1 > BDC-6.9) (2123). The most surprising observation was the diabetogenic potential of Rg T-cells expressing the BDC-10.1 TCR. These data suggest that this TCR may recognize a critical epitope in disease etiology and/or may have an optimal affinity/avidity for maximal islet infiltration and β-cell destruction. It is important to note that despite the autoreactivity of BDC-2.5, BDC-10.1, and NY4.1, all three TCRs were expressed, and Rg T-cells escaped thymic selection (albeit to a lesser extent in the spleen compared with other Rg T-cells). This suggests that diabetogenicity was attributed to the specificity of the TCR and not to differences in the levels of TCR expression or the number of Rg T-cell present in mice.

Taken together, these data suggest that autoantigen-specific TCRs can be segregated into three phenotypic groups: 1) TCRs that fail to mediate T-cell islet entry but may play a role in the later stages of the disease (e.g., TCRs specific for GAD and IA2), 2) TCRs that mediate islet infiltration but not β-cell destruction and thus may only contribute significantly to type 1 diabetes if potentiated by diabetogenic T-cells (e.g., certain phogrin- and insulin-specific TCRs), and 3) TCRs that mediate (to varying degrees) insulitis and β-cell destruction and thus may be key initiators/propagators of type 1 diabetes (e.g., certain TCRs specific for insulin and unknown islet antigens). Understanding the molecular distinctions between these three populations will be critically important. In addition, gaining insight into the levels and timing of autoantigen expression/availability as well as the location/phenotype of the APCs presenting autoantigens will be important and may explain the differences we observed between the autoreactive Rg T-cells. In conclusion, our data suggest that autoreactivity does not imply pathogenicity, as most autoreactive TCRs, on their own, appear to be nonpathogenic.

FIG. 1.

Expression of Rg T-cells in NOD.scid mice. Flow cytometric analysis of splenocytes from female NOD.scid TCR Rg mice 32–39 days posttransplant was performed. Splenocytes were gated on live (based on forward/side scatter) GFP+ cells and dot plots of CD4 vs. TCR (anti–TCRβ-H57) expression depicted. The numbers in the top right quadrant refer to the percentage of TCR+CD4+ T-cells. Note that the level of TCR expression and the number of T-cells was more variable among BDC-2.5 Rg mice, with the example depicted representative of those on the low end (See Fig. 2 for a mean of all mice examined).

FIG. 1.

Expression of Rg T-cells in NOD.scid mice. Flow cytometric analysis of splenocytes from female NOD.scid TCR Rg mice 32–39 days posttransplant was performed. Splenocytes were gated on live (based on forward/side scatter) GFP+ cells and dot plots of CD4 vs. TCR (anti–TCRβ-H57) expression depicted. The numbers in the top right quadrant refer to the percentage of TCR+CD4+ T-cells. Note that the level of TCR expression and the number of T-cells was more variable among BDC-2.5 Rg mice, with the example depicted representative of those on the low end (See Fig. 2 for a mean of all mice examined).

FIG. 2.

Reconstitution levels of TCR retrogenic mice. The spleens, ILNs, and PLNs from the NOD.scid TCR Rg mice were removed and single cell suspensions prepared, counted, and stained for TCR and CD4 expression. The number of Rg T-cells was determined from the percentage of GFP+/TCR+/CD4+ cells and the total number of cells in the spleen and lymph nodes. The data represents the means ± SE of 3–6 mice per group. *Not determined.

FIG. 2.

Reconstitution levels of TCR retrogenic mice. The spleens, ILNs, and PLNs from the NOD.scid TCR Rg mice were removed and single cell suspensions prepared, counted, and stained for TCR and CD4 expression. The number of Rg T-cells was determined from the percentage of GFP+/TCR+/CD4+ cells and the total number of cells in the spleen and lymph nodes. The data represents the means ± SE of 3–6 mice per group. *Not determined.

FIG. 3.

T-cells from TCR retrogenic NOD.scid mice are functional and specific for their cognate antigen. A: 5 × 105 splenocytes from TCR NOD.scid Rg mice were incubated with varying concentrations of peptide for 48 h, pulsed with 1 μCi, and harvested 24 h later. Background counts from T-cells alone were subtracted at each antigen concentration. Data are representative of two experiments. B: GFP+/CD4+ T-cells from PA17.9G7 and PA21.14H4 Rg mice were purified by FACS and incubated with or without 5 μmol/l GAD284–300 or HEL11–25 peptide presented by irradiated NOD splenocytes for 48 h, pulsed with 1 μCi, and harvested 18 h later. Data represent means ± SE of three independent experiments. C: Phogrin18 Rg T-cells were purified by FACS based on GFP and CD4 expression and incubated with a titration of various peptides presented by irradiated NOD splenocytes for 48 h, pulsed with 1 μCi, and harvested 18 h later. Data represent means ± SE of three independent experiments. D: BDC2.5, BDC10.1, and PA21.14H4 Rg T-cells were sorted and incubated with or without irradiated 11- to 19-week-old NOD/LtJ islets for 72 h, pulsed with 1μCi, and harvested 18 h later. The data represent the means ± SE of 2–4 independent experiments.

FIG. 3.

T-cells from TCR retrogenic NOD.scid mice are functional and specific for their cognate antigen. A: 5 × 105 splenocytes from TCR NOD.scid Rg mice were incubated with varying concentrations of peptide for 48 h, pulsed with 1 μCi, and harvested 24 h later. Background counts from T-cells alone were subtracted at each antigen concentration. Data are representative of two experiments. B: GFP+/CD4+ T-cells from PA17.9G7 and PA21.14H4 Rg mice were purified by FACS and incubated with or without 5 μmol/l GAD284–300 or HEL11–25 peptide presented by irradiated NOD splenocytes for 48 h, pulsed with 1 μCi, and harvested 18 h later. Data represent means ± SE of three independent experiments. C: Phogrin18 Rg T-cells were purified by FACS based on GFP and CD4 expression and incubated with a titration of various peptides presented by irradiated NOD splenocytes for 48 h, pulsed with 1 μCi, and harvested 18 h later. Data represent means ± SE of three independent experiments. D: BDC2.5, BDC10.1, and PA21.14H4 Rg T-cells were sorted and incubated with or without irradiated 11- to 19-week-old NOD/LtJ islets for 72 h, pulsed with 1μCi, and harvested 18 h later. The data represent the means ± SE of 2–4 independent experiments.

FIG. 4.

Insulitis incidence in TCR Rg NOD.scid mice. Hematoxylin and eosin–stained pancreata of NOD.scid retrogenic mice were scored for lymphocytic infiltration of the islets at 28, 70, and 140 days posttransplant. Insulitis was scored (∼100 islets per mouse) using the following metric: no insulitis (normal islet; no infiltration), peri-insulitis (infiltration on edges of islet or 0–20% of islet infiltrated), or insulitis (infiltration of 30–100% of islet). The average score of 4–17 mice (Table 1) is presented with SEs (∼800 slides in total). An alternative scoring system was also used for comparison, which gave comparable results (BDC10.1–0.85 ± 0.04, BDC2.5–0.89 ± 0.02, NY4.1–0.59 ± 0.08, BDC6.9, and 14H4–0) (48).

FIG. 4.

Insulitis incidence in TCR Rg NOD.scid mice. Hematoxylin and eosin–stained pancreata of NOD.scid retrogenic mice were scored for lymphocytic infiltration of the islets at 28, 70, and 140 days posttransplant. Insulitis was scored (∼100 islets per mouse) using the following metric: no insulitis (normal islet; no infiltration), peri-insulitis (infiltration on edges of islet or 0–20% of islet infiltrated), or insulitis (infiltration of 30–100% of islet). The average score of 4–17 mice (Table 1) is presented with SEs (∼800 slides in total). An alternative scoring system was also used for comparison, which gave comparable results (BDC10.1–0.85 ± 0.04, BDC2.5–0.89 ± 0.02, NY4.1–0.59 ± 0.08, BDC6.9, and 14H4–0) (48).

FIG. 5.

Diabetes incidence in TCR Rg NOD.scid mice. A: NOD.scid and B6.g7.RAG-1−/− TCR retrogenic mice were monitored for diabetes onset for 140 days. B: GFP+CD4+TCRβ+ cells were FACS purified from BDC-10.1, NY4.1, and PA21.14H4 NOD.scid Rg mice 28 days posttransplant, adoptively transferred into NOD.scid recipient mice, and monitored for diabetes. C: A titration of purified BDC-10.1 NOD.scid Rg T-cells were transferred into NOD.scid mice and monitored for diabetes. The tables of P values on the right were generated using the log-rank test both overall and pairwise within each experiment. The P value was based at the 0.05 significance level using the Bonferroni adjustment for multiple comparisons.

FIG. 5.

Diabetes incidence in TCR Rg NOD.scid mice. A: NOD.scid and B6.g7.RAG-1−/− TCR retrogenic mice were monitored for diabetes onset for 140 days. B: GFP+CD4+TCRβ+ cells were FACS purified from BDC-10.1, NY4.1, and PA21.14H4 NOD.scid Rg mice 28 days posttransplant, adoptively transferred into NOD.scid recipient mice, and monitored for diabetes. C: A titration of purified BDC-10.1 NOD.scid Rg T-cells were transferred into NOD.scid mice and monitored for diabetes. The tables of P values on the right were generated using the log-rank test both overall and pairwise within each experiment. The P value was based at the 0.05 significance level using the Bonferroni adjustment for multiple comparisons.

TABLE 1

The expression and insulitogenic and diabetogenic potential of the T-cell receptors used

TCRAntigen specificityTCR usage
Function
Peri-insulitis/Insulitis
Diabetes
SI ± SD*n28 days70 days140 daysnIncidence§ (%)ID-5n
PA15.14B12 GAD206–220 4.7 8.2 ND — ND 0/0 0/0 — 
PA19.5E11 GAD206–220 4.7 8.2 29.2 ± 18.3 ND 0.6/0 0.3/0 — 10 
1A4 GAD217–236 4.4 21.4 ± 7.9 ND 0.4/0 1.1/0 10 — 13 
PA17.9G7 GAD284–300 4.5 10 ND — ND 0/0 ND — 
PA18.10F10 GAD510–524 1.4 10 18.9 ± 3.0 ND 1.0/0 0/1.5 10 — 
PA18.9H7 GAD524–538 6.2 12 5.9 ± 3.1 ND 1.6/1.6 0/0 — 
530.45.19 GAD530–543 13.3 2.0 ± 0.5 ND 0.8/1.2 2.8/1.0 12 — 
12–4.1 Insulin B9–23 13.3 15.0 ± 2.0 ND 41.2/46.0 33.3/20.0 33 60 
12–4.4v1 Insulin B9–23 13.3 12 2.5 ± 0.5 ND 14.5/13.8 6.0/3.2 10 — 
10.23 IA2676–688 10.7 10 34.2 ± 12.7 ND 0.9/0 0/0 12 — 
Phogrin13 IA2β640–659 10.1 16 1.0 ± 0.04 ND 2.5/0.6 0.3/0 11 — 10 
Phogrin18 IA2β755–777 15.1 11 22.8 ± 10.2 10 ND 2.0/1.0 11.4/5.6 11 — 10 
BDC-2.5 Islet Ag 1.5 NA** — 6.8/93.0 27.1/58.8 NA†† 100 52 19 
BDC-6.9 Islet Ag 13.1 NA** — 0.3/0 20.9/22.7 24.4/24.8 56 81 
BDC-10.1 Islet Ag 9.2 15 NA** — 9.5/87.0 NA†† NA†† 100 27 15 
NY4.1 Islet Ag 13.1 11 NA** — 29.7/56.7 37.1/50.7 24.9/12.3 10 71 46 21 
PA21.14H4 HEL11–25 13.1 7.8 ± 5.0 1.3/0 2.1/0 6.1/1.2 17 — 16 
TCRAntigen specificityTCR usage
Function
Peri-insulitis/Insulitis
Diabetes
SI ± SD*n28 days70 days140 daysnIncidence§ (%)ID-5n
PA15.14B12 GAD206–220 4.7 8.2 ND — ND 0/0 0/0 — 
PA19.5E11 GAD206–220 4.7 8.2 29.2 ± 18.3 ND 0.6/0 0.3/0 — 10 
1A4 GAD217–236 4.4 21.4 ± 7.9 ND 0.4/0 1.1/0 10 — 13 
PA17.9G7 GAD284–300 4.5 10 ND — ND 0/0 ND — 
PA18.10F10 GAD510–524 1.4 10 18.9 ± 3.0 ND 1.0/0 0/1.5 10 — 
PA18.9H7 GAD524–538 6.2 12 5.9 ± 3.1 ND 1.6/1.6 0/0 — 
530.45.19 GAD530–543 13.3 2.0 ± 0.5 ND 0.8/1.2 2.8/1.0 12 — 
12–4.1 Insulin B9–23 13.3 15.0 ± 2.0 ND 41.2/46.0 33.3/20.0 33 60 
12–4.4v1 Insulin B9–23 13.3 12 2.5 ± 0.5 ND 14.5/13.8 6.0/3.2 10 — 
10.23 IA2676–688 10.7 10 34.2 ± 12.7 ND 0.9/0 0/0 12 — 
Phogrin13 IA2β640–659 10.1 16 1.0 ± 0.04 ND 2.5/0.6 0.3/0 11 — 10 
Phogrin18 IA2β755–777 15.1 11 22.8 ± 10.2 10 ND 2.0/1.0 11.4/5.6 11 — 10 
BDC-2.5 Islet Ag 1.5 NA** — 6.8/93.0 27.1/58.8 NA†† 100 52 19 
BDC-6.9 Islet Ag 13.1 NA** — 0.3/0 20.9/22.7 24.4/24.8 56 81 
BDC-10.1 Islet Ag 9.2 15 NA** — 9.5/87.0 NA†† NA†† 100 27 15 
NY4.1 Islet Ag 13.1 11 NA** — 29.7/56.7 37.1/50.7 24.9/12.3 10 71 46 21 
PA21.14H4 HEL11–25 13.1 7.8 ± 5.0 1.3/0 2.1/0 6.1/1.2 17 — 16 
*

Stimulation index (SI) of splenocytes activated with peptide.

Mice analyzed.

Percentage of islets showing either peri-insulitis (defined as infiltration around islet or 0–20% of islet infiltrated) or insulitis. The data are represented as peri-insulitis/insulitis at 28, 70, or 140 days posttransplant.

§

Incidence of diabetes.

Number of days posttransplant that 50% of the mice that became diabetic had a blood glucose level >400mg/dl.

The 12–4.4 sequence used in this study was reconstructed from information obtained from the study by Wegmann et al. (15). However, as a result of changes in Jα nomenclature since publication of their article, an alternate Jα was used in its construction (a modified Jα42 [SGGSNYKLTFGKGTKLSVKS] rather than Jα53 [SGGSNYKLTFGKGTLLTVTP]). It is important to note that 12–4.4v1 retains insulin reactivity and mediates significant islet infiltration and thus represents a very useful research reagent. As this is now a variant of the original 12–4.4, we refer to this TCR as 12–4.4v1.

**

Not applicable (NA) because the peptide specificity of the TCR is not known. The inapplicable TCRs were activated with whole islets to determine functionality.

††

Not applicable because the mice were all diabetic by 70 and 140 days posttransplant. ND, not determined.

Published ahead of print at http://diabetes.diabetesjournals.org on 21 February 2008. DOI: 10.2337/db07-1129.

C.J.W. and D.A.A.V. share senior authorship of this article.

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.

J.H was supported by National Institutes of Health grants R01 DK052068 and P30 DK57516. P.S. was supported by the Canadian Institutes of Health Research and is a Scientist of the Alberta Heritage Foundation for Medical Research. D.A.A.V. was supported by funds from the Juvenile Diabetes Research Foundation International (1-2004-141 [The Robert and Janice Compton Research Grant in honor of Elizabeth S. Compton] and 1-2006-847), a pilot project from the Cooperative Study Group for Autoimmune Disease Prevention (U19 AI050864-05 [George Eisenbarth, principal investigator]), the St. Jude Cancer Center Support CORE grant (CA-21765), and the American Lebanese Syrian Associated Charities.

We are very grateful to Kate Vignali, Yao Wang, and Smaroula Dilioglou for technical assistance; to the Vignali lab for assistance with harvesting bone marrow; to Richard Cross, Jennifer Rogers, and Yuxia He for flow cytometry analysis; to the St. Jude Hartwell Center staff for oligo synthesis and DNA sequencing, the staff of the St. Jude ARC Histology Laboratory, and the Animal Husbandry Unit and Flow Cytometry and Cell Sorting Shared Resource facility staff for AutoMACS. We thank Luc Teyton and Christophe Benoist for the BDC-2.5 TCR plasmids and Christophe Benoist and Diane Mathis for the B6.H2g7 mice.

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Supplementary data