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*Diabetes Type 1
The Journal of Immunology, 2006, 177: 7588-7598.
Copyright © 2006 by The American Association of Immunologists, Inc.

Loss of Invariant Chain Protects Nonobese Diabetic Mice against Type 1 Diabetes1

Richard J. Mellanby*, Chad H. Koonce{dagger}, Anthony Monti{dagger}, Jenny M. Phillips*, Anne Cooke2,* and Elizabeth K. Bikoff2,3,{dagger}

* Department of Pathology, University of Cambridge, Cambridge, United Kingdom; and {dagger} Wellcome Trust Center for Human Genetics, University of Oxford, Oxford, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The invariant (Ii) chain acts as an essential chaperone to promote MHC class II surface expression, Ag presentation, and selection of CD4+ T cells. We have examined its role in the development of type 1 diabetes in NOD mice and show that Ii chain-deficient NOD mice fail to develop type 1 diabetes. Surprisingly, Ii chain functional loss fails to disrupt in vitro presentation of islet Ags, in the context of NOD I-Ag7 molecules. Moreover, pathogenic effector cells could be shown to be present in Ii chain-deficient NOD mice because they were able to transfer diabetes to NOD.scid recipients. The ability of these cells to transfer diabetes was markedly enhanced by depletion of CD25 cells coupled with in vivo anti-CD25 treatment of recipient mice. The numbers of CD4+CD25+Foxp3+ T cells in thymus and periphery of Ii chain-deficient NOD mice were similar to those found in normal NOD mice, in contrast to conventional CD4+ T cells whose numbers were reduced. This suggests that regulatory T cells are unaffected in their selection and survival by the absence of Ii chain and that an alteration in the balance of effector to regulatory T cells contributes to diabetes prevention.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The loss of tolerance underlying T cell-mediated destruction of insulin-producing pancreatic beta cells in type 1 diabetes has been investigated intensely. The spontaneous autoimmune disease that develops in NOD mice closely resembles human type 1 diabetes and provides an excellent animal model for studying differentiation of pathogenic Th1 effectors (1). At least 20 regulatory loci with the ability to influence disease have been identified, but genetic control of diabetes susceptibility is most strongly linked to the MHC class II gene products responsible for guiding CD4+ T cell responses (2). Recent x-ray crystal structural studies provide considerable insight into common features shared by diabetogenic class II alleles (3, 4, 5). I-Ag7 expressed in NOD mice and human HLA-DQ8 molecules, strongly associated with diabetes, both lack the conserved aspartic acid residue at beta57 that pairs with the conserved arginine at {alpha}76. The loss of this salt bridge widens the P-9 pocket of the groove and may consequently introduce a bias in the selection of the self-peptide repertoire, but it remains unknown how this shift affects presentation of islet Ags. The ability of protective MHC alleles to influence disease outcome has been attributed to several possible mechanisms such as more effective central tolerance, competition for self-peptides, and/or production of regulatory T cells (Tregs)4 (6, 7, 8, 9, 10, 11, 12, 13, 14).

MHC class II Ag presentation depends on invariant chain (Ii) actions as a chaperone (15). The Ii chain associates with class II at early stages of assembly to prevent irreversible misfolding of the nascent groove and promote release from resident chaperones such as GRP94/BiP and Erp72/calnexin responsible for endoplasmic reticulum (ER) quality control (16, 17, 18). By the dileucine-targeting signal in its cytoplasmic domain, Ii chain also transports class II to specialized endosomal compartment(s) where exposure to proteolytic enzymes and the nonconventional MHC class II molecule DM promotes Ag capture (19, 20, 21). Ii chain-mutant strains described to date display defective class II surface expression, peptide occupancy, and Ag presentation activities, but allele-specific differences have been shown to influence Ii chain requirements during class II subunit assembly and CD4+ T cell development (22, 23, 24, 25). Whereas Ii chain loss in b haplotype mice causes severe disturbances, d and k haplotype Ii chain mutants display relatively relaxed phenotypes. Indeed, secondary proliferative responses upon peptide challenge were hardly affected in BALB/c mutants (24). Similarly, Ii chain-independent class II Ag presentation pathways have been described in k haplotype dendritic cells (26). Production of IgG Abs and protective CD4+ T cell responses in the absence of Ii chain expression have also been documented (25, 27, 28, 29). On the other hand, the Ii chain plays an essential role in CNS autoimmunity and allergen-induced lung inflammatory responses (30, 31). These experiments analyzing b haplotype mutants suggest that Ii chain-dependent pathways control autoimmune disease susceptibility.

We previously found that Ii chain loss in NOD mice results in reduced levels of surface class II and ER retention of high m.w. class II aggregates (32). Thus, as for other strains, NOD Ii chain mutants display defective class II export, but we also observe in this exceptional background that Ii chain loss fails to disrupt selection of mature B cell subsets. As B cells have been shown to be important in the development of type 1 diabetes in NOD mice, this permitted us to analyze the effects of Ii chain loss on diabetes development in the absence of any B cell abnormalities. Ii chain-independent class II Ag presentation has been described extensively in dendritic cells, the key class II-expressing cell type responsible for stimulating pathogenic CD4 T cells during diabetes development. As stated above, there are haplotype differences in the effects of Ii chain deficiency on Ag presentation and T cell development, and we were therefore interested to examine the consequences of Ii chain deficiency on the presentation of islet Ags in NOD mice. The present experiments were designed to test whether Ii chain deficiency influences the presentation of islet Ag to autoreactive T cells and the development of type 1 diabetes in NOD mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Wild-type NODLt/J, BDC2.5NOD, and NOD.scid mice were bred and maintained under barrier conditions in the Biological Services facility of the Department of Pathology, University of Cambridge. They received standard laboratory food and water ad libitum. NOD.scid and irradiated mice were maintained in microisolator cages with filtered air and handled under sterile conditions in a laminar flow hood. The experimental protocols described in this report have been approved by the Ethical Review Committee of the University of Cambridge. Ii chain-mutant mice generated by backcrossing the targeted allele onto the B10.BR/SgSn (H-2k) background, B.C-9, a strain congenic with C57BL/6 but expressing the Igha allotype of BALB/c (22), or the NOD/LtJ background have been described previously (32). For Ii chain NOD-mutant stocks established at the 10th backcross generation, linkage markers associated with Idd-recessive loci were confirmed to be NOD-derived by microsatellite analysis by Charles River Laboratories Genetic Testing Services. Blood glucose levels were measured using One Touch test strips (Lifescan; Johnson & Johnson), and values > 250 mg/dl were considered positive for diabetes.

Abs and reagents

FITC, PE, or biotin-conjugated anti-CD3 (145-2C11), CD4 (RM4-5), CD11b (M1/70), CD11c (HL3), CD19 (ID3), Vbeta4 (KT4), MHC class II (OX6), and CD25 (PC61) were purchased from BD Biosciences or Serotec. Anti-Foxp3 (clone FJK-16s) was from eBioscience. Flow cytometry was performed using standard cell staining protocols as recommended by the vendors. The biotin-conjugated peptides OVA323–339 (ISQAVHAAHAEINEAAGR) and Ii chain CLIP85–101 (KPVSQMRMATPLLMRPM) were from Quality Control Biochemicals. The BDC2.5 T cell-specific peptide mimetope 1040-55 described by Judkowski et al. (33) (RTRPLWVRME) was from Southampton Polypeptides.

Histology

Pancreases were processed for wax histology. Five-micrometer sections were taken at eight levels (200 µm apart) and stained with H&E. Total islets per section were counted, and the degree of cellular infiltration was scored as follows: noninfiltrated; peri-islet infiltrate where infiltration is restricted and occupies up to 20% of the islet area; and intraislet infiltrate where 21–100% of the islet is infiltrated and architecture is disrupted. Between 400 and 600 islets were scored. Photographs were taken using a Zeiss Axiophot microscope.

Dendritic cell isolation

Spleens were cut into small fragments and incubated for 30 min in HBSS containing 0.5 mg/ml Liberase CI (Roche Biochemicals). EDTA was added to give a final concentration of 0.01 M. Fragments were disrupted by passing through a plastic cell strainer (Falcon; BD Biosciences), washed with HBSS, and the cells were resuspended in 1% BSA/PBS/0.01 M EDTA. Dendritic cells were then positively selected using anti-CD11c-coated microbeads, according to the manufacturer’s instructions (Miltenyi Biotec).

Alternatively, bone marrow-derived dendritic cells were prepared by flushing precursor cells from mouse femurs and tibias using a narrow gauge syringe needle, and erthyrocytes were lysed. The cells were resuspended in complete medium (IMDM (Invitrogen Life Technologies)) supplemented with 10% FCS, 100 µg/ml streptomycin (Sigma-Aldrich), and 60 µg/ml penicillin (Sigma-Aldrich), supplemented with 10 ng/ml rIL-4 and 20 ng/ml rGM-CSF (PeproTech), and cultured in flat-bottom 6-well plates at a density of 1.5 x106/ml. Nonadherent granulocytes were removed on day 3, and immature bone marrow-derived dendritic cells were harvested at day 7.

T cell isolation

For in vitro proliferation assays, spleen cells harvested from 8-wk-old BDC2.5NOD male mice were depleted of non-CD4+ T cell populations using microbeads, according to the manufacturer’s instructions (Miltenyi Biotec).

Alternatively, for in vivo proliferation assays, spleen cell suspensions from BDC2.5NOD mice were incubated with an Ab depletion mixture, followed by goat anti-rat IgG-coated beads (Polysciences), according to the manufacturer’s instructions, and then the nonadherent T cells were collected.

For transfer to NOD.scid recipients, spleen cell suspensions depleted of RBCs by lysis in ammonium chloride buffer were incubated with a mixture of FITC-conjugated CD11b, CD11c, and OX6 (anti-MHC class II), passed through a MoFlo cell sorter (DakoCytomation), and nonstained cells were collected. This T cell sort routinely yields 90–95% CD3+ T cells, and the percentage of NOD CD4+ T cells is higher (~55%) than NOD Ii– (~30%). The percentages of CD25+ T cells within the CD4+ T cell pool is ~10% in NOD and ~25% in NOD Ii– mice. CD25-depleted T cells were selected by adding FITC anti-CD25 to the Ab depletion mixture. Any remaining contaminating CD25+ cells were inactivated in vivo by giving two injections of 2 mg of anti-CD25.

In vitro proliferation assays

BDC2.5NOD CD4+ T cells at 5 x 107/ml in PBS were incubated with 5 µM CFSE at 37°C for 30 min, washed in PBS, and resuspended in complete medium. Splenic dendritic cells purified as described above were resuspended at 5 x 106/ml, pulsed with the BDC2.5 peptide mimetope (1 µg/ml) at 37°C for 1 h, then washed and resuspended in complete medium. CFSE-labeled BDC2.5NOD T cells (1 x 105) were incubated in a 96-well round-bottom plate (Falcon; BD Biosciences) at 37°C for 72 h with 104, 5 x 103, or 103 splenic dendritic cells in a total volume of 200 µl/well. Cells were harvested after 72 h, stained for Vbeta4, the Vbeta-chain expressed by BDC2.5NOD T cells, and analyzed by flow cytometry.

Intact pancreatic islets were isolated from male NOD mice by infusing the common bile duct in situ with collagenase. The pancreas was then digested at 37°C, and the islets were enriched on a Eurocollins-Ficoll gradient, washed in HBSS containing BSA, and handpicked using a siliconized micropipette. For Ag presentation assays, 1 x 104 bone marrow or splenic dendritic cells were incubated in a round-bottom 96-well plate together with macerated islets (~50 islets/well) in 200 µl of complete medium at 37°C for 3 h. The dendritic cells were then gently washed three times with fresh complete medium, and 1 x 105 CFSE-labeled BDC2.5NOD CD4+ T cells were added to each well. Seventy-two hours later, proliferation was assessed by analyzing CFSE dilution gating on Vbeta4-positive cells.

In vivo proliferative responses of BDC2.5NOD T cells

CFSE-labeled BDC2.5NOD T cells (1 x 107) were injected into the lateral tail vein of individual wild-type or Ii chain-deficient NOD mice. After 72 h, spleens, pancreatic, and mesenteric lymph nodes were harvested. Single-cell suspensions were stained for Vbeta4 and analyzed by flow cytometry.

Adoptive transfers into NOD.scid recipients

T cells (5 x 106) from 8-wk-old, female, nondiabetic NOD, or NOD Ii– mice were injected i.v. into the lateral tail vein of 6-wk-old NOD.scid females. Recipients of CD25-depleted T cells also received 1 mg of anti-CD25 (PC61) injected i.p. at the time of transfer, followed by a second injection of 1 mg of anti-CD25 (PC61) 48 h later. Mice were sublethally irradiated (650 rad) and then injected with 2 x 107 splenocytes from diabetic NOD donors. Urinary glucose levels were tested using Diastix every 7 days (Bayer). Recipients scored as diabetic had urinary glucose concentrations of ≥55 mM, on two occasions separated by at least 7 days.

In vitro CD4+CD25+ T cell suppressor assays

To evaluate functional activities of CD4+CD25+ T cells, we tested proliferative responses and cytokine production by CD4+CD25– T cells stimulated in the presence of anti-CD3 and APCs. These CD4+CD25– effectors and CD4+CD25+ Tregs were purified using microbeads, according to the manufacturer’s instructions (Miltenyi Biotec). CFSE-labeled CD4+CD25– T cells were resuspended in complete medium. Cell cultures were set up as follows, and the exact cell numbers are stated in the figure legends. CFSE-labeled CD4+CD25– T cells were cultured for 72 h in a round-bottom 96-well plate (Falcon; BD Biosciences) with APCs consisting of irradiated, CD4+ T-depleted, red cell-lysed splenocytes, 1 µg/ml anti-CD3, and a variable number of CD4+CD25+ T cells. Cells from each well were harvested after 72 h, stained for CD4, and analyzed by flow cytometry. Alternatively, proliferation was assayed by addition of 1 µCi of [3H]thymidine (Amersham Biosciences) for the final 16 h, and incorporation was measured in a liquid scintillation counter. Results are expressed as the mean counts per minute ± SE of triplicate wells.

Culture supernatants harvested after 72 h were also assessed for the presence of IFN-{gamma} using a sandwich ELISA (R&D Systems). The concentration was calculated by comparison with IFN-{gamma} standard preparations, according to the manufacturer’s instructions.

Statistics

Data were analyzed using the GraphPad Prism computer package. The Mann-Whitney U test was used to assess differences between nonparametric groups. Log-rank analysis was used in Fig. 7 for comparison of survival curves. Values of p are indicated in the figures. Results were considered to be significant if p was <0.05.


Figure 7
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  		FIGURE 7. Depletion of CD25+ T cells reveals the presence of pathogenic CD4+ T effectors in NOD Ii chain mutants. NOD.scid females were reconstituted with 5 x 106 total or CD25-depleted Ii chain mutant T cells or 5 x 106 NOD T cells. The CD25+ Treg subset selected in NOD Ii chain mutants markedly decreases the incidence and delays the onset of diabetes compared with NOD Ii nondepleted T cells (p < 0.0005). NOD T cells (5 x 106) transferred diabetes faster than NOD Ii T cells (p < 0.005) but at a slower rate than CD25-depleted NOD Ii– T cells (p < 0.01).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ii chain-deficient NOD mice express functionally empty I-Ag7 molecules with enhanced peptide-binding capabilities and reduced percentages of mature CD4+ T cells

To test whether Ii chain-dependent class II pathways are essential for development of type 1 diabetes, we generated NOD mutants. We crossed the null allele onto the NOD background and set up homozygous matings at the 10th backcross generation. The presence of linkage markers associated with NOD-derived recessive Idd loci, necessary for the onset of disease, was confirmed by microsatellite analysis. We previously reported that NOD Ii chain mutants display decreased levels of surface I-Ag7 and that splenic B cells express high m.w. class II aggregates unable to escape ER quality control (32). To further characterize class II maturation defects, the present experiments examine peptide-binding activities. Wild-type and Ii chain-deficient NOD spleen cells were incubated with biotinylated peptides, followed by FITC avidin, and surface staining was assessed by flow cytometry. As shown in Fig. 1A, mutant spleen cells consistently display markedly enhanced reactivity toward exogenously added peptides, in comparison with wild-type NOD splenocytes. Superior binding capabilities were observed in the presence of either OVA or CLIP peptides (Fig. 1A). Thus, we conclude that Ii chain loss in NOD mice creates a substantial pool of empty or loosely occupied I-Ag7 molecules.


Figure 1
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  		FIGURE 1. Ii chain-deficient NOD mice display enhanced peptide binding capabilities and reduced numbers of mature CD4+ T cells in the thymus and periphery. A, NOD wild-type (1) or Ii chain mutant (2) splenocytes were cultured for 5 h at 37°C with biotin-conjugated peptides or medium alone as indicated and subsequently stained with FITC-labeled avidin and analyzed by FACS. B, Thymus, spleen, and pooled lymph node (brachial, mesenteric, inguinal, and axillary) cell suspensions were stained for CD4 and CD8 expression and analyzed by flow cytometry. The numbers refer to the percentages of total cells within the indicated gates. Representative data from one of seven identical experiments with similar results are shown.

 
Allele-specific differences govern Ii chain contributions to CD4+ T cell maturation. Thus, Ii chain loss in b haplotype mice results in dramatically reduced numbers of mature CD4+ T cells, but in contrast, d and k haplotype mutants display relatively subtle CD4+ T cell defects (23, 24, 25). As shown in Table I and Fig. 1B, we also found here that Ii chain-deficient NOD mice produce fewer mature CD4+ T cells. Although total cell numbers in thymus, spleen, and pooled lymph nodes of NOD and Ii-deficient NOD mice were not significantly different, there was a significant reduction in the numbers and percentages of CD4+ T cells (Fig. 1 and Table I). The reduced percentage of CD4+ T cells seen in pooled lymph nodes of Ii-deficient NOD mice (Fig. 1B) was also seen in pancreatic lymph nodes (29% in NOD Ii chain mutants in contrast to 60% in NOD pancreatic lymph nodes).


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  		Table I. Absolute numbers of cells in the spleen, thymus, pooled lymph nodes (brachial, mesenteric, inguinal, and axillary) and pancreatic lymph nodes in NOD and Ii chain-deficient NOD mice

 
NOD Ii chain mutants fail to develop type 1 diabetes

Recent evidence suggests that Ii chain-independent pathways favor selection of the Th1 subset (34). Thus, the few mature CD4+ T cells present in Ii chain mutants display the ability to stimulate delayed-type hypersensitivity and inflammatory responses in vivo and produce IFN-{gamma} but not the Th2 cytokine IL-4 upon challenge in vitro. This suggests that type 1 diabetes in NOD Ii chain mutants might develop normally due to substantial activation of pathogenic Th1 effectors. To examine this possibility, we compared blood glucose levels in wild-type and Ii chain-deficient NOD mice for 30 wk. As shown in Fig. 2A, the onset of disease in wild-type NOD females begins at ~15 wk of age, and ~60% were diabetic by 30 wk. In striking contrast over the same time interval, NOD Ii chain mice remained diabetes-free. No symptoms of disease have ever been observed over the past 4 years in specific pathogen-free facilities located in Boston or Oxford. Our NOD Ii chain mutants display a dramatic protection against type 1 diabetes.


Figure 2
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  		FIGURE 2. Ii chain-deficient NOD mice fail to develop autoimmune diabetes. A, The onset of diabetes was evaluated by measurement of blood glucose levels in Ii chain mutants alongside age-matched control wild-type females. The incidence of diabetes has been absolutely zero over the past 4 years, with mutants routinely observed for over 6 mo of age. B and C, The percentage of islets with a given degree of infiltration at 5 mo of age. D, A typical example of normal pancreatic islets seen in the majority of Ii chain mutant females. E, A typical example of substantial mononuclear infiltration occasionally seen in the pancreatic islets of Ii chain mutants. Magnification, x20. 

 
To investigate whether Ii chain loss disrupts early recruitment of inflammatory Th1 effectors to pancreatic islets and/or progression to overt disease, we examined histological sections for evidence of insulitis. As shown in Fig. 2, B–E, the vast majority of Ii chain-mutant islets are normal. Occasionally, we observe peri-islet infiltrates, but Ii chain-deficient NOD mice fail to display evidence of aggressive insulitis. Nevertheless, the presence of islet infiltration suggests that islet reactive T cells might be present in Ii-deficient NOD mice.

Presentation of islet Ags by the Ii chain-independent class II pathway stimulates autoreactive CD4+ T cells

The simplest explanation is that Ii chain mutants are protected because they lack the ability to present islet Ags. To examine this possibility, wild-type or Ii chain-deficient splenic dendritic cells were pulsed in vitro with peptide or macerated NOD islets, and the ability to stimulate proliferative responses in the presence of CFSE-labeled TCR-transgenic BDC2.5NOD T cells was assessed by dilution of the CFSE label. Consistent with the results in Fig. 1, we also found here that Ii chain mutants display efficient peptide-loading capabilities (Fig. 3A). Similarly, intact islet Ags were presented equally well by either wild-type or Ii chain-deficient splenic dendritic cells (Fig. 3B). As a control, no response was detectable with BDC2.5NOD CD4+ T cells cultured in the absence of Ag-pulsed APCs (Fig. 3B). These results demonstrate that dendritic cells from Ii chain-deficient NOD mice can stimulate BDC2.5NOD T cells in vitro. The same conclusions were reached analyzing islet Ag presentation by bone marrow-derived dendritic cells (data not shown).


Figure 3
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  		FIGURE 3. Presentation of islet Ags by Ii chain-deficient dendritic cells (DCs). A, CD11c+ splenic DCs from age-matched wild-type or Ii chain NOD mutants were incubated for 1 h with cognate peptide ligand, washed, and then cultured for 72 h with CFSE-labeled BDC2.5NOD T cells at various ratios as indicated. Representative profiles gated on Vbeta4-positive cells from triplicate wells are shown. B, CD11c+ splenic DCs (1 x 104) were incubated for 3 h with macerated islets, subsequently washed, and then cultured for 72 h with 1 x 105 CFSE-labeled BDC2.5 NOD T cells. Wild-type and Ii chain mutant DCs display equivalent Ag presentation capabilities in the presence of peptide or intact islets.

 
It is of course possible that NOD Ii chain mutants fail to develop diabetes due to defective presentation of diabetogenic epitopes locally in the draining pancreatic lymph node. To test this possibility, we transferred CFSE-labeled BDC2.5NOD T cells into wild-type and Ii chain-mutant NOD mice and measured proliferation in the pancreatic lymph nodes 72 h later. As shown in Fig. 4, there was no significant difference in the proportion of BDC2.5NOD CD4+ T cells that have proliferated in the pancreatic lymph nodes of NOD or Ii-deficient NOD mice. As a control, proliferation of BDC2.5NOD T cells was undetectable in recipient spleens or mesenteric lymph nodes (Fig. 4B). Thus, NOD Ii chain mutants have the ability to present islet Ags and efficiently activate BDC2.5NOD T cells under physiological conditions.


Figure 4
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  		FIGURE 4. Ii chain-deficient NOD mice can present islet Ag. CFSE-labeled BDC2.5NOD T cells (1 x 107) were transferred into 9- to 12-wk-old female wild-type or Ii chain mutant NOD mice. Pancreatic lymph nodes were harvested at 72 h, and proliferation was measured by dilution of CFSE label in Vbeta4-positive T cells. A, Representative histograms. B, Summary of experiments. There is no significant difference between the percentages of dividing BDC2.5NOD T cells recovered in the pancreatic lymph nodes upon transfer into either wild-type (n = 9) or Ii chain-deficient (n = 7) recipients (p = 0.17). There is limited proliferation of BDC2.5NOD T cells in the spleen and MLN of both NOD and Ii-deficient NOD mice. C, Spleen cells (2 x 107) from diabetic NOD donors were transferred into sublethally irradiated NOD and Ii-deficient NOD recipients. Almost all wild-type NOD recipients were diabetic within 4 wk of transfer (15 of 16), and all Ii chain mutant recipients became diabetic within 15 wk of transfer.

 
To determine whether Ii-deficient APCs can support a pathological T cell response, we examined whether disease can be transferred to NOD Ii chain-mutant recipients. Sublethally irradiated wild-type and age-matched Ii chain-mutant NOD mice were injected i.v. with splenocytes from diabetic NOD donors and closely monitored to evaluate the onset and incidence of disease. From Fig. 4C, it can be seen that, as expected, virtually all the wild-type NOD recipients were diabetic within 4 wk of transfer. Although the Ii chain-mutant recipients showed a delay in the onset of diabetes, all became diabetic by 15 wk. These results demonstrate that NOD Ii chain mutants can present diabetogenic epitopes and activate pathogenic CD4+ T cells directed against islet Ags in vivo, albeit less effectively than wild-type NOD mice. As we have never seen spontaneous diabetes in Ii chain-deficient NOD mice, this suggests that the protection afforded by loss of Ii chain cannot simply be explained by class II Ag presentation defects.

Increased proportions of CD4+CD25+Foxp3+ Tregs in Ii chain-deficient mice expressing diverse MHC haplotypes

Considerable evidence suggests that development of type 1 diabetes in NOD mice depends on the gradual age-related loss of CD4+CD25+Foxp3+ T cells with regulatory activities (35). Perhaps Ii chain mutants fail to develop disease due to the presence of increased numbers of Tregs. We first compared the percentages of CD4+CD25+ T cells in age-matched wild-type and Ii chain-deficient NOD mice. As shown in Fig. 5A, Ii chain mutants consistently display increased proportional representation of CD4+CD25+ T cells in the spleen and pancreatic lymph nodes. Recent experiments demonstrate that the forkhead-winged helix transcription factor Foxp3 plays an essential role in specification of the regulatory CD4+ T cell subset (36, 37, 38, 39). Unlike CD25, Foxp3 is not up-regulated in recently activated CD4+ T cells, so its expression is considered a definitive functional marker for Tregs. Consistent with the results above, NOD Ii chain mutants contain increased percentages of CD4+Foxp3+ T cells, and interestingly, this was already evident at early stages of CD4+ T cell differentiation in the thymus (Fig. 5B). The absolute numbers of CD4+Foxp3+ T cells, however, were comparable in NOD and Ii chain-deficient NOD thymus, spleen, and lymph node, indicating that absence of Ii does not compromise Treg selection and survival (Fig. 5C and data not shown).


Figure 5
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  		FIGURE 5. Ii chain loss favors development of CD4+CD25+Foxp3+ Tregs in the context of diverse MHC haplotypes. CD25 or Foxp3 and CD4 coexpression were analyzed by FACS on single-cell suspensions as indicated. A, The percentage of CD4+ T cells coexpressing CD25 is significantly increased in Ii chain-deficient NOD mutant spleens and pancreatic lymph nodes. B, The percentage of CD4+ T cells coexpressing Foxp3 is significantly increased in the thymus and periphery of Ii chain mutant NOD mice. C, The absolute numbers of CD4+Foxp3+ cells in the spleen of NOD and Ii chain-deficient NOD mice are not significantly different. In contrast, the number of CD4+Foxp3– cells are significantly lower in Ii chain-deficient NOD compared with NOD mice. D and E, The developmental shift toward CD4+CD25+Foxp3+ Tregs was also observed in Ii chain-deficient B10.BR (H-2k) mice and was especially noticeable in B.C-9 (H-2b) Ii chain mutants. 

 
The exceptional susceptibility of NOD mice to autoimmune diabetes has been associated with defective central tolerance mechanism(s) (40, 41, 42). We speculated whether the bias toward CD4+CD25+Foxp3+ Tregs is simply due to loss of Ii chain function and/or dependent on the activities of additional NOD-derived background genes. Therefore, it was of interest to examine the proportional representation of CD4+CD25+ T cells in Ii chain-deficient strains carrying diverse genetic backgrounds. As shown in Fig. 5, D and E, increased percentages of CD4+CD25+ T and CD4+Foxp3+ T cells were detected readily in B10.BR (H-2k) and B.C-9 (H-2b) Ii chain mutants. As previously shown for NOD, these increased percentages of Tregs did not reflect any change in the absolute number of CD4+CD25+Foxp3+ T cells but rather a reduction in the conventional CD4+ T cell number (data not shown). Thus, we conclude that Ii chain-independent class II pathways permit the development and survival of the CD4+ CD25+Foxp3+ regulatory subset.

CD4+CD25+ Tregs in NOD Ii chain mutants suppress CD4+ T cell proliferation and IFN-{gamma} production in a dose-dependent fashion

The suppressive activities of CD4+CD25+ T cells in NOD Ii chain mutants were assessed in vitro initially by establishing their ability to inhibit the proliferative response of CD4+CD25– T cells from NOD Ii-deficient mice induced by coculture in the presence of NOD Ii APCs and anti-CD3. Spleen-derived CD4+CD25+ T cells from NOD Ii-deficient mice were able to inhibit proliferation of their own CD4+CD25– T cells in a dose-dependent manner as assessed by both dilution of CFSE (Fig. 6A) and inhibition of thymidine incorporation (Fig. 6Bi). This inhibition of proliferation was comparable to that seen when NOD CD4+CD25+ spleen T cells were cocultured with NOD CD4+CD25– T cells, NOD APCs, and anti-CD3 (Fig. 6Bii). This inhibitory activity of NOD Ii–CD4+CD25+ T cells was also demonstrated by their dose-dependent inhibition of IFN-{gamma} production (Fig. 6C). CD4+CD25+ T cells from both NOD and Ii-deficient NOD mice were able to suppress NOD CD4+CD25– T cell proliferation. The inhibition by NOD Ii-deficient CD4+CD25+ T cells appeared to be more potent, particularly at lower Treg:T effector ratios (Fig. 6D). This was confirmed by thymidine incorporation (data not shown). No response was seen in any of the experimental systems when APCs or CD4+CD25+ T cells were cultured on their own (data not shown). As Tregs from Ii-deficient NOD mice manifest comparable Treg activity in vitro, the relative changes in the proportions of conventional CD4+ T cells to Tregs seen in mutant mice might result in increased operational Treg activity in vivo.


Figure 6
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  		FIGURE 6. CD4+CD25+ Tregs from Ii chain-deficient NOD mutants display dose dependent suppressor activities. A, Varying numbers of NOD Ii chain-deficient CD4+CD25+ Tregs were cultured with their own CFSE-labeled CD4+CD25– T cells and their own APC and anti-CD3 for 72 h. Proliferation was evaluated by dilution of CFSE. Representative histograms of triplicate wells are shown. B, Varying numbers of NOD Ii chain-deficient (i) or NOD (ii) CD4+CD25+ Tregs were cultured with their own CD4+CD25– T cells and their own APC and anti-CD3. Proliferation was measured by thymidine incorporation. C, IFN-{gamma} was measured by a sandwich ELISA in the supernatants (72 h) of the experiment described in A. Control cultures lacking APC or CD4+CD25– effector T cells gave barely detectable responses. D, Varying numbers of NOD Ii chain-deficient (i) or NOD (ii) CD4+CD25+ Tregs were cultured with CFSE-labeled NOD CD4+CD25– T cells, NOD APC, and anti-CD3 for 72 h. Proliferation was evaluated by dilution of CFSE. Representative histograms of triplicate wells are shown. In the experiments described in A–C, the number per well of CD4+CD25– effectors was 1 x 105, and the number of APC was 5 x 105. In the experiment described in D, the number per well of CD4+CD25– T cells was 5 x 104, and the number of APC was 2.5 x 105. 

 
CD4+CD25+ Tregs selected in NOD Ii chain mutants regulate autoreactive T cell function in vivo

It is conceivable that NOD Ii chain mutants are diabetes-free due to the unimpaired development of CD4+CD25+Foxp3+ Tregs. An alternative but not mutually exclusive view is that Ii chain mutants fail to develop disease due to the absence of pathogenic Th1 cells. To determine whether such cells were present in Ii-deficient NOD mice, we transferred T cells from NOD Ii chain-mutant mice to NOD.scid recipients. As a positive control, we also transferred T cells from age-matched NOD mice. It can be seen from Fig. 7 that NOD.scid recipients of mutant T cells did develop diabetes but showed a delayed onset and reduced incidence of diabetes compared with wild-type NOD T cells. This could be because of fewer effector cells (1.1 x 106 vs 2.5 x 106) or more regulatory cells (0.4 x 106 vs 0.27 x 106). To determine whether Tregs played any role in the reduced ability of T cells from Ii chain-deficient NOD mice to transfer diabetes, we also examined the ability of CD25-depleted Ii chain-mutant T cells to transfer diabetes to NOD.scid recipients. Recipients of these cells additionally received anti-CD25 (PC61) at the time of transfer and also 48 h later. The incidence of diabetes onset in recipients of CD25-depleted T cells was increased dramatically compared with T cells from Ii mutant mice, and disease onset had a faster kinetic than that observed with either mutant or wild-type T cells. These results strongly argue that conventional autoreactive CD4+ T cells with the ability to mediate destruction of pancreatic islets are present in NOD Ii chain mutants, but CD25+ Tregs prevent them from causing disease.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The key structural feature shared by diabetogenic NOD I-Ag7 and HLA-DQ8 class II molecules is the presence of a non-Asp residue at beta57. This substitution disrupts the salt bridge with a conserved arginine residue at {alpha}76 and opens the P-9 side pocket of the groove (2, 3, 4, 5). Exactly how this structural change contributes to the loss of tolerance to pancreatic islet self-Ags is unknown. Its exceptional SDS instability implied that I-Ag7 might be only loosely occupied by low-affinity peptides, but direct-binding assays have failed to support this idea (43, 44, 45). Recent evidence suggests that I-Ag7 and DQ8 molecules preferentially bind to peptides with an acidic residue at the P-9 anchor position (46), but whether these class II peptide sequences isolated from B cell lymphomas and the diabetogenic epitopes presented by dendritic cells in pancreatic lymph nodes share a common binding motif has yet to be determined.

Ii chain and DM contributions to selection of diverse class II peptides in professional APCs have been documented extensively (23, 25, 47, 48, 49). In the present study, we demonstrate for the first time that Ii chain actions as a class II chaperone are essential for development of type 1 diabetes in NOD mice. Class II maturation defects in NOD Ii chain mutants closely resemble those previously described in the context of different MHC haplotypes. As expected, Ii chain-deficient NOD mice display markedly reduced levels of surface class II and a substantial pool of functionally empty I-Ag7 with enhanced peptide-binding capabilities. As for Ii chain-independent class II export and peptide capture previously described in k haplotype dendritic cells (26) and mutant thymi (32), we also observe here that Ii chain loss in NOD mice hardly perturbs Ag presentation. Thus, Ii chain-deficient NOD dendritic cells efficiently present islet Ag and stimulate autoreactive CD4+ T cells. As for BALB/c Ii chain mutants (24), we also observe here slightly decreased numbers of mature CD4+ T cells with 2-fold fewer mature CD4+ T cells being selected in the thymus and present in the periphery.

MHC class II expression in the thymus is necessary for selection of CD4+CD25+Foxp3+ Tregs (37, 50). Recent evidence suggests that CD4+ T cells with increased affinities for class II self-peptide(s) are fated to become Tregs (51, 52, 53, 54, 55, 56). On the other hand, direct measurements of TCR activation thresholds by this subset have yet to be reported, and selective expression of a distinct TCR repertoire specific for different self-peptide ligand(s) could equally well account for previous observations. In the present study, we demonstrate a development bias that arises as a result of selection of CD4+ T cells in the absence of Ii chain. Increased percentages of CD4+CD25+Foxp3+ T cells were observed universally in Ii chain-deficient mice carrying different genetic backgrounds. However, this increased percentage was due to a loss of mature conventional CD4+ T cells, whereas the numbers of CD4+CD25+Foxp3+ T cells were unaffected by Ii chain deficiency. This increased proportional representation of CD4+CD25+Foxp3+ T cells in both thymic and peripheral mature CD4+ T cell populations of Ii-deficient NOD mice most likely reflects reduced positive selection of conventional CD4+ T cells in the Ii chain-deficient mouse or selective survival of the Treg population. It is interesting to note that previous work has shown that apparent increased proportions of CD4+CD25+ T cells in transgenic mice expressing an agonist ligand were caused by substantial clonal deletion of conventional CD4+ T cells, whereas CD4+CD25+ T cells showed resistance to apoptosis (57).

Numerous reports demonstrate that mature CD4+ T cells selected in Ii chain mutants display a broad TCR repertoire (25, 58, 59, 60, 61). Ii chain mutants display a phenotype characteristic of recently activated CD4+ T cells and a bias toward Th1 subset development (34, 61, 62, 63). In NOD mice, we find that while the numbers of Foxp3-expressing CD4+ T cells is unaffected by Ii chain deficiency, the development of conventional CD4+ T cells is reduced. This results in an alteration in the proportion of conventional CD4+ T cells to Tregs in the periphery of these Ii chain-mutant mice. Under physiological conditions described to date, both CD4+ subpopulations of T cells coexist and dominant suppression probably reflects a competition for rate-limiting interactions with dendritic cells (64). The alteration in the numbers of Tregs and effector T cells is very likely to be an important protective mechanism in Ii-deficient NOD mice since previous adoptive transfer studies have clearly established that alterations of Treg cell numbers can markedly influence the ability of effector cells to transfer disease (Ref. 35 and our unpublished observations).

Ii chain-deficient NOD mice fail to develop type 1 diabetes; not a single mutant mouse has shown symptoms of disease over the past 4 years. Although development of the conventional mature CD4+ T cells is impaired in these mice, we observed that pathogenic CD4+ T cells are still generated in Ii-deficient NOD mice as they are able to efficiently transfer diabetes to immunocompromised recipients if CD25+ T cells are removed. This suggests that the increased relative proportions of Tregs play a role in diabetes prevention in NOD Ii chain-deficient mice. However, enhanced Treg numbers are unlikely to be the only mechanism involved in the protection of Ii-deficient NOD mice since spleen cells from diabetic NOD mice transfer disease faster in sublethally irradiated NOD mice than Ii-deficient NOD mice. This observation suggests that reduced Ag presentation may also be involved in the protection from diabetes in Ii-deficient NOD mice. Impaired Ag presentation cannot itself be the only mechanism involved in diabetes prevention in these mutant mice since transfer of spleen cells from diabetic NOD mice is able to initiate diabetes in NOD Ii recipients. Both mechanisms may contribute to the profound protection from diabetes in NOD Ii chain-mutant mice.

The increased proportional representation of Tregs in the Ii-deficient NOD mice cannot simply be due to a reduction in the numbers of conventional CD4+ T cells because in DM mutant mice the relative proportion of Tregs to conventional CD4+ T cells is unaffected despite their markedly reduced total number of CD4+ T cells (Ref. 50 and our unpublished observations). Perhaps due to decreased levels of surface class II, CD4+ T cell selection simply favors TCR with increased affinities. On the other hand, NOD mice lacking cathepsin L (cat L) activity similarly display a striking protection against type 1 diabetes due to increased percentages of CD4+CD25+Foxp3+ T cells (65). Cat L loss in NOD mice fails to perturb Ii chain processing or class II peptide occupancy. Indeed, mature I-Ag7 produced in the absence of cat L is indistinguishable from wild type. In this case, the increased percentages of CD4+CD25+Foxp3+ T cells cannot be explained by class II maturation defects. As for Ii chain-deficient NOD mice, cat L mutants display decreased numbers of mature CD4+ T cells, strengthening earlier suggestions that cat L enzymatic activities directly influence self-peptide repertoire selection (66). It would seem that the repertoire of peptides available for T cell selection in Ii-deficient mice predominantly influences the conventional CD4+ T cell repertoire and leaves the Treg population unaffected. It will be interesting to learn whether the CD4+CD25+Foxp3+ Tregs present in NOD Ii chain and cat L-mutant strains share a common TCR repertoire.

Striking cell type-specific differences governing quality control of class II assembly and export have been documented previously. In contrast to splenic B cells, Ii chain activities are nonessential for class II maturation in k haplotype thymi and dendritic cells (26, 32). Besides its ability to promote peptide capture by the exogenous pathway, Ii chain also extinguishes presentation of endogenous self-Ags such as beta2-microglobulin (59, 67, 68, 69). Thymic medullary and cortical epithelial cell lines display distinct class II pathways and peptide profiles, but exactly how these cell types regulate CD4+ T cell development is unknown (70, 71, 72, 73, 74). A widely held view is that negative selection in the thymus depends on expression of cognate self-peptide ligands such as insulin by medullary epithelial cells (75, 76, 77). Our data show that Ii chain loss has a profound effect on conventional CD4+ T cell development while leaving the development of Tregs untouched, thus indicating that the presentation pathways governing selection and/or survival differ for these two CD4+ T cell populations. These results strongly argue that the ligands seen by CD4+CD25+Foxp3+CD4+ Tregs are presented via the alternative Ii chain-independent class II pathway.

Ii chain requirements during CD4+ T cell maturation were described over a decade ago (23, 25, 78). The present experiments demonstrate that Ii chain functions as a class II chaperone also control the dynamic balance between Th1 effectors and Tregs. Despite severely reduced numbers of mature CD4+ T cells, selection of the CD4+CD25+Foxp3+ regulatory subset proceeds efficiently in the absence of Ii chain. Ii chain-deficient NOD mice described in the present report should prove useful for studying dominant tolerance mechanism(s) and CD4+CD25+Foxp3+ Treg repertoire selection.


   Acknowledgments
 
We are grateful to Drs. Elizabeth Robertson, Brigitta Stockinger, and Zoltan Fehervari for their helpful discussions and comments regarding our manuscript. We also thank Dr. Nicky Parish, Dr. Gerald Chu, and Barry Potter for their histological input and Nigel Miller for his assistance with the cell sorting. We would also like to acknowledge the input of George Kenty and Shanda Porter in the generation of the Ii chain-deficient NOD mice.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Wellcome Trust. Back

2 A.C. and E.K.B. are joint senior authors. Back

3 Address correspondence and reprint requests to Dr. Elizabeth K. Bikoff, Wellcome Trust Center for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom. E-mail address: elizabeth.bikoff{at}well.ox.ac.uk Back

4 Abbreviations used in this paper: Treg, regulatory T cell; ER, endoplasmic reticulum; cat, cathepsin; Ii, invariant; PLN, pancreatic draining lymph node; MLN, mesenteric lymph nodes. Back

Received for publication March 20, 2006. Accepted for publication August 31, 2006.


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 Introduction
 Materials and Methods
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