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Diabetes 55:2098-2105, 2006
DOI: 10.2337/db05-0810
© 2006 by the American Diabetes Association
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Deficiency in NOD Antigen-Presenting Cell Function May Be Responsible for Suboptimal CD4+CD25+ T-Cell–Mediated Regulation and Type 1 Diabetes Development in NOD Mice

Pascale Alard, Jean N. Manirarora, Sarah A. Parnell, Jason L. Hudkins, Sherry L. Clark, and Michele M. Kosiewicz

From the Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky

Address correspondence and reprint requests to Pascale Alard, Department of Microbiology and Immunology, University of Louisville, 319 Abraham Flexner Way, Bldg. 55A, Rm. 405, Louisville, KY 40202. E-mail: p0alar01{at}gwise.louisville.edu

Abbreviations: APC, antigen-presenting cell; CTLA-4, cytotoxic T-lymphocyte–associated antigen-4; Foxp3, Forkhead box P3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GITR, glucocorticoid-induced tumor necrosis factor receptor; PE, phycoethrin; PerCP, peridinin chlorophyll protein


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various defects in antigen-presenting cells (APCs) and T-cells, including regulatory cells, have been associated with type 1 diabetes development in NOD mice. CD4+CD25+ regulatory cells play a crucial role in controlling various autoimmune diseases, and a deficiency in their number or function could be involved in disease development. The current study shows that NOD mice had fewer CD4+CD25+ regulatory cells, which expressed normal levels of glucocorticoid-induced tumor necrosis factor receptor and cytotoxic T-lymphocyte–associated antigen-4. We have also found that NOD CD4+CD25+ cells regulate poorly in vitro after stimulation with anti-CD3 and NOD APCs in comparison with B6 CD4+CD25+ cells stimulated with B6 APCs. Surprisingly, stimulation of NOD CD4+CD25+ cells with B6 APCs restored regulation, whereas with the reciprocal combination, NOD APCs failed to activate B6 CD4+CD25+ cells properly. Interestingly, APCs from disease-free (>30 weeks of age), but not diabetic, NOD mice were able to activate CD4+CD25+ regulatory function in vitro and apparently in vivo because only spleens of disease-free NOD mice contained potent CD4+CD25+ regulatory cells that prevented disease development when transferred into young NOD recipients. These data suggest that the failure of NOD APCs to activate CD4+CD25+ regulatory cells may play an important role in controlling type 1 diabetes development in NOD mice.

The disease process in the spontaneous animal model for human type 1 diabetes, the nonobese diabetic (NOD) mouse, closely resembles the human disease because it is characterized by autoimmune destruction of insulin-producing ß-cells in pancreatic islets. Moreover, CD4 and CD8 T-cells appear to play a primary role in the specific destruction of ß-cells (1,2). A growing body of evidence suggests that dysregulation of the immune response in NOD mice plays a major role in the development of type 1 diabetes (3). NOD mice have a defect in central tolerance (4) and exhibit a variety of abnormalities in immune function, including defects in T-cell function (5). More importantly, both NOD mice and patients with type 1 diabetes exhibit a deficiency in two regulatory T-cell populations, natural killer T-cells and CD4+CD25+ T-cells (6–9). In addition to defects in regulation, defects have been reported in the antigen-presenting cells (APCs) of both NOD mice and type 1 diabetic patients (10–18).

A growing body of evidence has shown that the naturally occurring CD4+CD25+ regulatory T-cells control the development of a variety of autoimmune diseases (19–21). The CD4+CD25+ T-cell population comprises 5–10% of the CD4+ T-cells both in normal naive mice (i.e., in the peripheral lymphoid organs and thymus) (22,23) and in the circulation of normal humans (24–26). The CD4+CD25+ regulatory T-cells appear to control effector T-cell proliferation through a direct cell-cell interaction (26,27). Moreover, activation of CD4+CD25+ T-cells is absolutely required for the regulation to take place in vitro (26,27). The constitutive expression of various markers by this regulatory cell population distinguishes it from unactivated CD4+CD25– T-cells. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is expressed on the majority of CD4+CD25+ regulatory T-cells (80–90%) (28). Cytotoxic T-lymphocyte–associated antigen-4 (CTLA-4) is also expressed by CD4+CD25+ regulatory T-cells and may be required both to stimulate their function as well as for their development (29,30). Finally, the transcription factor Forkhead box P3 (Foxp3) has recently been shown to play an essential role in CD4+CD25+ regulatory T-cell development, as indicated by the reduction of CD4+CD25+ regulatory T-cells in the thymus and periphery of Foxp3-deficient mice (30,31).

CD4+CD25+ regulatory T-cells are reduced in NOD mice (9) and have been shown to prevent diabetes very effectively when expanded in vitro and transferred into NOD mice (32). In humans, these cells appear to play an equally important role in controlling the immune response (22,25). CD4+CD25+ regulatory T-cells have been shown to be impaired in type 1 diabetic patients (7) and exhibit impaired function in multiple sclerosis patients (33). Moreover, CD28–/– and B7–/– NOD mice develop a higher incidence and earlier onset of diabetes than (non-knockout) NOD mice, which appears to be attributable to a reduced number of CD4+CD25+ T-cells in the periphery (8). Taken together, these data strongly suggest that a deficiency or dysfunction of the CD4+CD25+ regulatory T-cell population could be an important factor in the development of diabetes in NOD mice. CD4+CD25+ regulatory T-cells from NOD mice may not regulate potentially pathogenic T-cells, thereby leading to the development of diabetes.

In the current study, we investigated the nature of the immunoregulatory defect present in NOD mice relative to CD4+CD25+ regulatory T-cells, and we determined whether the small percentage of NOD mice that never develop diabetes possess potent CD4+CD25+ regulatory T-cells. We have found that CD4+CD25+ regulatory T-cells from NOD mice are not intrinsically deficient. However, the inability of NOD APCs to activate NOD CD4+CD25+ regulatory T-cells appears to be involved in the failure of NOD CD4+CD25+ cells to regulate in vitro. In addition, we show here that NOD mice that remain diabetes free possess APCs capable of activating NOD CD4+CD25+ regulatory T-cells in vitro, and they also possess potent CD4+CD25+ regulatory T-cells capable of controlling pathogenic cells in vivo.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
C57BL/6 and NOD female mice from 3 to 35 weeks of age were in our barrier facility under SPF conditions and obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained according to institutional animal care and use committee guidelines.

Labeling for fluorescence-activated cell sorting.
We labeled 1 x106 lymph node cells with APC–anti-CD25, peridinin chlorophyll protein (PerCP)-anti-CD4, fluorescein isothiocyanate–anti-CD62L, phycoethrin (PE)–anti-CTLA-4 (Pharmingen, San Diego, CA), or PE–anti-GITR (R&D, Minneapolis, MN) antibodies for 20 min in Dulbecco’s PBS, 1% FCS, and 0.1% NaNO3 and washed them twice. For CTLA-4 labeling, cells were fixed and permeabilized according to the manufacturer’s instructions.

mRNA extraction and real-time PCR.
mRNA was extracted, using a Picopure RNA isolation kit (Arcturus, Mountain View, CA), and reverse transcribed, using a Taq Man reverse transcriptase kit (Applied Biosystems, Foster City, CA). cDNA was amplified in duplicate by real-time PCR, using a SYBR Green PCR kit (Applied Biosystems) with primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Foxp3. Relative Foxp3 mRNA amounts were normalized with respect to GAPDH mRNA amounts. The fold increase by comparison to CD4+CD25– T-cells is represented (fold change = 1 for CD4+CD25– T-cells). Primer sequences are: GAPDH: 5'-GGA-GCG-AGA-CCC-CAC-TAA-CA-3' and 5'-ACA-TAC-TCA-GCA-CCG-GCC-TC-3'; and Foxp3: 5'-CCC-ACC-TAC-AGG-CCC-TTC-TC-3' and 5'-GGC-ATG-GGC-ATC-CAC-AGT-3'.

Isolation of CD4+CD25+ T-cells.
Lymphocytes were obtained from lymph nodes and spleens and processed in Hank’s balanced salt solution containing 2% heat-inactivated FCS (Hyclone, Logan, UT). CD4 cells were enriched on a CD4 cell-enrichment column (R&D) labeled with PE–anti-CD25 antibody, incubated with anti-PE beads (Miltenyi Biotech, Auburn, CA), and then passed through a magnetic column according to the manufacturer’s instructions. CD4+CD25+ T-cell purity was consistently >90%.

Cell culture and T-cell proliferation assay.
CD4+CD25– T-cells (2.5 x 104 per well in 96-well round-bottom plates) were cultured for 3 days at 37°C in 5% CO2 with irradiated spleen cells as APCs (2 x 105 per well) and anti-CD3 antibody at 10 µg/ml, with or without CD4+CD25+ T-cells (≤2.5 x 104 cells per well) at an optimal 1:1 regulatory-to-responder cell ratio, as shown in previous publications (26,27,31,34). The cell cultures were pulsed on day 3 with 0.5 µCi [3H]thymidine for the last 18 h.

Adoptive transfer assay and assessment of diabetes.
Spleen cells or pancreatic lymph node cells were collected from sick or nonsick NOD mice and pooled. Splenic CD90+ and CD90– were purified, using CD90 beads and MS columns (Miltenyi Biotech). In some experiments, spleen cells were depleted of CD25+ cells by incubation with 7D4 cell (American Type Culture Collection) supernatant and rabbit complement (Cedarlane, Hornby, Canada). We intravenously injected 40 x106 CD90– cells or 10 x 106 CD90+ splenic cells into 4-week-old female NOD mice. Blood glucose was monitored weekly until 30 weeks of age, and diabetes incidence was determined when the glucose measurement was >33 mmol/l for 2 consecutive weeks.

Statistical analysis.
Data were analyzed using either Student’s t test, ANOVA and the Tukey-Kramer multiple comparisons test, or the Mann-Whitney U test. Experiments were performed at least twice with reproducible results. One representative experiment is shown in each figure.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pre-diabetic NOD mice have lower percentages of CD4+CD25+ T-cells in their lymphoid tissue.
Controversial data have been reported concerning the proportion of CD4+CD25+ regulatory T-cells in the lymphoid organs of NOD mice. One study has shown that the percentage of CD4+CD25+ regulatory T-cells is normal in the periphery of NOD mice (35). In contrast, lower levels of CD4+CD25+ regulatory T-cells have been found in autoimmune disease–prone mice, including NOD mice, and could be associated with autoimmune disease development (8,9). To evaluate CD4+CD25+ T-cells in our colony, lymph nodes and spleens from NOD or B6 mice were analyzed for the presence of CD4+CD25+ T-cells. Because timing could have been a reason for the conflicting findings reported above, female NOD and B6 mice of various ages were used for this study. Lymph nodes and spleens were collected from individual mice, and cells were labeled with PerCP–anti-CD4 and PE–anti-CD25 antibodies. We have also found that NOD mice exhibit a significant decrease in the percentage of CD4+CD25+ T-cells in lymph node and spleen by 9 weeks of age in comparison to B6 mice (Fig. 1B). However, in 3-week-old mice, only a slight difference could be found in the lymph node but not in the spleen (Fig. 1A).


Figure 1
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  		FIG. 1. Impairment of CD4+CD25+ T-cell percentage in NOD mice. Lymph node (LN) and spleen (SPL) cells from 10 individual 3-week-old (A) and 9-week-old (B) NOD and B6 mice were labeled for CD4 and CD25 and analyzed by fluorescence-activated cell sorting. Each point represents an individual animal.

 
CD4+CD25+ regulatory T-cells from NOD mice express normal levels of GITR and CTLA-4.
Various molecules, including CTLA-4 and GITR, have been shown to be critical for the development and/or regulation of CD4+CD25+ regulatory T-cells (30). In addition, CD62L, a homing receptor for naive T-cell migration into the lymph node, is expressed by a good portion of CD4+CD25+ regulatory T-cells and can help distinguish between regulatory and activated cells because its downregulation generally indicates an activated or memory cell phenotype. We examined whether CD4+CD25+ regulatory T-cells from adult NOD mice at different stages of the disease (i.e., pre-diabetic or sick) expressed different levels of these molecules compared with CD4+CD25+ regulatory T-cells from age-matched B6 mice. Lymph node and spleens were collected from individual NOD or B6 mice and labeled with anti-CD4 and anti-CD25 antibodies and either anti-GITR, anti–CTLA-4, or anti-CD62L antibodies. Normal levels of CTLA-4, GITR, and CD62L were expressed by CD4+CD25+ regulatory T-cells harvested from either lymph nodes (Fig. 2) or spleens (online appendix Fig. 1A [available at http://diabetes.diabetesjournals.org]) of pre-diabetic NOD mice. The percentage of CD4+CD25+ cells expressing CD62L, however, was reduced in sick mice (>26 weeks) (Fig. 3 and online appendix Fig. 1B). These data indicate that although CD4+CD25+ regulatory T-cells from NOD mice express normal levels of GITR and CTLA-4, they express decreased levels of CD62L as they become sick.


Figure 2
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  		FIG. 2. Phenotype of CD4+CD25+ cells in pre-diabetic NOD mice. Lymph node cells from prediabetic NOD mice were labeled for CD4, CD25, and either GITR, CTLA-4, or CD62L and analyzed by fluorescence-activated cell sorting by gating on CD4+CD25+ or CD4+CD25– cells.

 

Figure 3
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  		FIG. 3. Phenotype of CD4+CD25+ cells in sick NOD mice. Lymph node cells from sick NOD mice were labeled for CD4, CD25, and either GITR, CTLA-4, or CD62L and analyzed by fluorescence-activated cell sorting by gating on CD4+CD25+ or CD4+CD25– cells.

 
NOD CD4+CD25+ regulatory T-cells are functional in vitro when properly stimulated.
A recent study has suggested that CD4+CD25+ regulatory T-cells from NOD mice may be not effective in in vitro assays that test for regulatory function (36). Because these experiments were performed in the presence of NOD APCs, and APCs from adult NOD mice have been shown to be abnormal at various levels (10,13–15), including aberrant cytokine production, defective antigen presentation, and differentiation, we examined whether defective regulatory cell function in vitro could be caused by a deficiency in the ability of NOD APCs to activate CD4+CD25+ regulatory T-cells. To test this possibility, we compared the ability of B6 APCs and NOD APCs to stimulate NOD CD4+CD25+ regulatory function in vitro. NOD CD4+CD25– responder and CD4+CD25+ regulatory T-cells isolated and pooled from adult B6 or NOD mice were cultured with pooled B6 or NOD APCs and soluble anti-CD3 antibody. Figure 4A shows that CD4+CD25+ regulatory T-cells from NOD mice that were stimulated with NOD APCs (right section, fourth column) were much less efficient at suppressing CD4+CD25– cells than B6 CD4+CD25+ regulatory T-cells stimulated with B6 APCs (left section, second column). Interestingly, when NOD CD4+CD25+ cells were stimulated with B6 APCs in vitro in the same experiment, they became as efficient as B6 CD4+CD25+ cells at suppressing responder cell proliferation (Fig. 4A, left section, fourth column). For comparison, we determined whether B6 CD4+CD25+ regulatory cells could be activated by NOD APCs in vitro. We found hat NOD APCs were also not able to effectively activate B6 CD4+CD25+ regulatory T-cells (Fig. 4A, right section, second column). Percent inhibition of proliferation was represented in Fig. 4B to aid in the interpretation of the data. Altogether, these data suggest that CD4+CD25+ regulatory T-cells from NOD mice are not intrinsically deficient. However, NOD APCs appear to deliver a nonoptimal activation signal to CD4+CD25+ regulatory T-cells, thereby preventing optimal regulation. Although NOD APCs have been shown previously to be impaired in their ability to stimulate responder T-cells and a cell population referred to as suppressor T-cells (37), the current report shows for the first time that NOD APCs lack the ability to stimulate natural CD4+CD25+ regulatory cell function.


Figure 4
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  		FIG. 4. NOD CD4+CD25+ regulatory cells are not functionally defective. B6 or NOD CD4+CD25– responder cells were cultured alone with either B6 or NOD irradiated spleen cells (APC) and anti-CD3 or cocultured at a 1:1 (regulatory-to-responder) ratio with B6 or NOD CD4+CD25+ cells. *P < 0.001, **P < 0.01, and ***P < 0.05 significant difference from the group without CD4+CD25+ cells. Counts per minute (CPM) (A) and inhibition (B) are shown.

 
NOD APCs are poor stimulators of CD4+CD25+ regulatory T-cell function in vitro.
Numerous reports have shown that APCs from adult NOD mice are deficient at various levels (10,13–15), including aberrant cytokine production, defective antigen presentation, and differentiation. APCs from NOD mice could become deficient as the disease progresses, or they could be deficient from birth. APCs from individual 3-week-old NOD mice (i.e., before development of insulitis) and from individual 9-week-old NOD mice (i.e., after insulitis but before diabetes onset) were tested for their ability to induce 1) B6 responder cell proliferation and 2) CD4+CD25+ regulatory T-cell function. Responder cells stimulated with 3-week-old NOD APCs proliferated much less than when stimulated with 3-week-old B6 APCs (Fig. 5A), suggesting that NOD APCs have an impaired ability to activate T-cells in general. Because of this dramatic difference in proliferation, the regulation experiments performed with APCs from individual mice (10 per group) are presented in Fig. 5B as a percent inhibition of proliferation. As shown in Fig. 5B, the regulatory cell function (i.e., percent inhibition of proliferation) induced by 3-week-old NOD APCs was significantly reduced in comparison to that induced by APCs from age-matched B6 mice, and this was observed using either B6 or NOD CD4+CD25+ regulatory T-cells. These results suggest that APCs from NOD mice are deficient early in life at stimulating both responder and regulatory cells and that CD4+CD25+ regulatory T-cells may not be optimally activated even before insulitis develops. Similarly, APCs from 9-week-old NOD mice (pre-diabetic) (Fig. 5C) as well as APCs from 30-week-old sick NOD mice (Fig. 6B) induced reduced regulation in comparison to APCs from age-matched B6 mice.


Figure 5
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  		FIG. 5. NOD APCs are poor activators of regulatory cell function. B6 CD4+CD25– responder cells were cultured alone with either B6 or NOD irradiated spleen cells (APC) and anti-CD3 or cocultured at a 1:1 (regulatory-to-responder) ratio with B6 or NOD CD4+CD25+ cells, respectively. Counts per minute (CPM) for CD4+CD25– cell proliferation (A) and percent inhibition (B) are shown. A: Proliferation in 3-week-old mice. B and C: Regulatory cell function in 3-week-old mice (B) and 9-week-old mice (C). *P < 0.05, **P < 0.01, and ***P < 0.001 significant difference from the group without CD4+CD25+ cells, respectively.

 

Figure 6
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  		FIG. 6. APCs from disease-free NOD mice effectively induce regulatory cell function. CD4+CD25– responder cells were cultured alone with irradiated spleen cells (APC) from either individual B6, sick NOD, or disease-free NOD mice and anti-CD3 (A) or at a 1:1 (regulatory-to-responder) ratio with CD4+CD25+ cells from NOD mice (B). Counts per minute (CPM) for CD4+CD25– cell proliferation (A) and percent inhibition (B) are shown. B: Regulatory cell function. *P < 0.03 significant difference from the B6 APC control. Ab, antibody.

 
APCs from 30-week-old disease-free NOD mice effectively stimulate CD4+CD25+ regulatory T-cell function in vitro.
The reason for which a small percentage of NOD mice (<10% in our facility) never develop diabetes is still not clear. We hypothesized that APCs from 30-week-old disease-free NOD mice may not be deficient in their ability to activate CD4+CD25+ regulatory T-cells and may thereby induce optimal regulation. To test this hypothesis, we collected spleens from individual NOD mice that were still disease free after 30 weeks, and we tested their ability to activate responder cell proliferation (Fig. 6A) and, more importantly, CD4+CD25+ regulatory T-cells (Fig. 6B). APCs from B6 or disease-free NOD mice could induce similar levels of CD4+CD25– responder cell proliferation (Fig. 6A). However, proliferation was significantly decreased when responder cells were stimulated with APCs from sick NOD mice (Fig. 6A). Moreover, the inhibition of proliferation of responder cells by CD4+CD25+ regulatory T-cells was similar when cells were stimulated with APCs from B6 or disease-free NOD mice, but it was significantly less when cells were stimulated with APCs from diabetic NOD mice (Fig. 6B). Therefore, the APCs from disease-free NOD mice, in contrast to APCs from NOD mice that develop disease, appear to have the ability to optimally activate CD4+CD25+ regulatory T-cells and responder CD4+CD25– cells.

Potent CD4+CD25+ regulatory cells reside in the spleens of disease-free, but not diabetic, 30-week-old NOD mice.
Normal APC function in disease-free NOD mice could impact regulatory cells at several different levels, including the proper activation of CD4+CD25+ regulatory T-cells that can control pathogenic T-cells in vivo, as well as increasing the percentage of CD4+CD25+ regulatory T-cells present in the lymphoid organs. We first examined whether cells harvested from lymphoid organs of disease-free NOD mice (>30 weeks of age) had the ability to prevent diabetes development directly in NOD mice and whether these cells were CD4+CD25+ regulatory T-cells. As shown in Fig. 7A (line labeled NS CD90+), CD90+ cells purified from the spleens of disease-free NOD mice (nonsick) could prevent type 1 diabetes when transferred into 4-week-old NOD mice. However, when CD90+ cells were depleted of CD25 cells, they were incapable of preventing diabetes (Fig. 7A, line labeled NS CD90+CD25–) and in fact dramatically accelerated disease development. We found that 95% of CD90+ cells injected were CD3+, and 99% of CD90+ cells were CD25– (online appendix Fig. 2A and B). In addition, splenic CD90– cells from neither sick nor nonsick NOD mice could prevent disease when transferred into 4-week-old NOD mice (Fig. 7B).


Figure 7
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  		FIG. 7. Spleen cells from disease-free NOD mice prevent diabetes development. CD90– and CD90+ spleen cells from 30-week-old sick (S) or nonsick (NS) NOD mice were purified or depleted of CD25+ cells. Then, 10 x 106 CD90+ CD25+ (n = 6) or 10 x 106 CD90+ CD25– spleen cells (n = 7) (A) or 40 x 106 CD90– spleen cells from nonsick (n = 10) or sick mice (n = 10) (B) were injected into 4-week-old NOD mice. Blood glucose was monitored weekly and diabetes incidence determined. *Incidence is significantly different from the untreated controls at P < 0.005. wk, week.

 
Next, we examined whether disease-free NOD mice exhibit an increase in the percentage of CD4+CD25+ T-cells. Lymph nodes and spleens from individual disease-free and sick NOD mice (>30 weeks of age) were collected and cells were labeled with PerCP–anti-CD4 and PE–anti-CD25 antibodies as well as fluorescein isothiocyanate–anti-CD62L antibody because expression of CD62L by CD4+CD25+ cells was reduced in the spleens of sick NOD mice (Fig. 3). Flow cytometric analysis revealed that the percentage of CD4+CD25+ T-cells expressing CD62Lhigh was also significantly decreased in the spleens (Fig. 8A) of nonsick NOD mice. The percentage of CD4+CD25+CD62Lhigh cells was still significantly decreased in the lymph nodes (data not shown) and in the spleens (Fig. 8B) of nonsick NOD mice. Moreover, Foxp3 mRNA expression in unpurified spleen cells from age-matched nonsick and sick NOD mice showed no significant difference (Fig. 8C).


Figure 8
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  		FIG. 8. Percentages of CD4+CD25+ T-cells in spleens of nonsick NOD mice. Spleen cells from 30-week-old sick (S) and nonsick (NS) NOD mice were labeled for CD4, CD25, and CD62L and analyzed by fluorescence-activated cell sorting (A and B), and Foxp3 mRNA levels were quantified by real-time PCR (C). Normalized Foxp3 mRNA expression levels in the samples relative to normalized Foxp3 mRNA expression levels in CD4+CD25– cells (fold change = 1) is shown (C). Each point represents an individual animal.

 
These data suggest that spleens of disease-free NOD mice possess pathogenic cells that are kept under control by functional CD4+CD25+ regulatory T-cells. Although disease-free NOD mice still exhibit a decrease in the percentage of CD+CD25+ cells, these cells have regained an optimal regulatory activity.


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NOD mice have major defects in immunoregulation (3,4,6,8,9) and APCs (10,13–15), which could have a direct impact on the development of diabetes. In the current study, we show that the CD4+CD25+ regulatory T-cells from NOD mice do not have an intrinsic defect but that the NOD APCs are defective in their ability to stimulate or activate the CD4+CD25+ regulatory T-cells.

CD4+CD25+ regulatory T-cells were found in lower percentages in the peripheral lymphoid organs of NOD mice, as has been reported previously in adult mice (8,9). CD4+CD25+ regulatory T-cells from NOD mice expressed normal levels of some critical molecules associated with CD4+CD25+ regulatory T-cell development and regulation, including CTLA-4 and GITR. Moreover, NOD CD4+CD25+ regulatory T-cells downregulated the proliferation of responder CD4+CD25– T-cells in vitro as efficiently as control CD4+CD25+ regulatory T-cells when stimulated with B6 APCs. These data suggest that the immunoregulatory defect may not be within the CD4+CD25+ regulatory T-cells themselves. Recent studies have reported that NOD CD4+CD25+ regulatory T-cells that have been activated in vitro or in vivo are capable of preventing diabetes development much more efficiently (32,36), suggesting that stimulation of CD4+CD25+ regulatory T-cells in NOD mice is not optimal. In contrast, transfer of CD4+CD25+ regulatory T-cells controls gastritis and oophoritis development in BALB/c and (A/JxB6)F1 mice, respectively, without requiring preactivation (21,38). Taken together, these data suggest that CD4+CD25+ regulatory T-cells require extra stimulation to control diabetes in NOD mice.

NOD mice have abnormalities that affect antigen presentation, including defects in the protein kinase C activation pathway (15) as well as low expression levels of CD80 and CD86 (39), which affect costimulation. Moreover, the decreased expression of CD86 on NOD APCs has been shown to impair T-cell activation (11). Our data show that the proliferation of CD4+CD25– responder T-cells is dramatically impaired when stimulated with NOD versus B6 APCs, confirming that NOD APCs are impaired in their ability to stimulate the T-cell response. Because activation of CD4+CD25+ T-cells is absolutely required for regulation to take place in vitro (26,27), we hypothesized that NOD APCs may not be capable of activating CD4+CD25+ T-cells properly. The data presented in this study show that from as early as 3 weeks of age, APCs from NOD mice are impaired in their ability to activate CD4+CD25+ T-cell–mediated regulation. A recent study reported that the ability of diabetogenic effector cells to be counterregulated decreases with age in NOD mice (40). We did not find a decrease in the ability of CD25– cells from 30-week-old NOD mice to be suppressed, as shown in Fig. 6. This discrepancy is likely caused by the fact that the 30-week-old mice used in the current study were disease free. Moreover, the percent inhibition shown in that recent report (40) is low (~40–60%), corroborating our findings that regulation in vitro is not optimal when NOD APCs are used. The identification of the deficient APC type, as well as the deficient phenotype, is currently under evaluation. Our data indicate that NOD APCs do not optimally stimulate either CD4+CD25– responder T-cells or CD4+CD25+ T-cells. This result could perhaps be explained by the decreased expression of costimulatory molecules (e.g., B7) on the surface of NOD APCs (11,39). When compared with intact NOD mice, CD28–/– and B7–/– NOD mice develop a higher incidence and earlier onset of diabetes that appears to be caused by a reduced number of CD4+CD25+ T-cells in the periphery (8). Moreover, B7 costimulation plays an important role in the maintenance of self-tolerance by sustaining CD4+CD25+ regulatory T-cells (41,42). Finally, conversion of CD4+CD25– cells into CD4+CD25+ regulatory T-cells requires B7 costimulation (34). Altogether, these results suggest that deficiency in B7 expression by NOD APCs could be involved in abnormal induction of this cell population, and this may explain the impaired level of peripheral CD4+CD25+ T-cells in NOD mice and the development of disease.

A small percentage of NOD mice in our colony do not develop diabetes (5–10%). In contrast to NOD mice that develop disease, our data show that these disease-free NOD mice possess not only potent CD4+CD25+ T-cells but also functional APCs. Splenic APCs from disease-free NOD mice stimulated CD4+CD25– responder T-cells and CD4+CD25+ mediated regulation as effectively as B6 APCs. Furthermore, splenic T-cells from disease-free NOD mice, after being transferred into 4-week-old NOD mice, were able to prevent disease in a significant percentage of mice. These data corroborate previous studies showing that CD4+ T-cells that reside in the spleens of young pre-diabetic NOD mice can prevent diabetes in a transfer model (43–45). We show in the current study that the cells responsible for disease protection appear to be CD4+CD25+ T-cells because depletion of CD25+ cells from splenic T-cells abolishes the ability of these T-cells to prevent diabetes development. Thus, our results demonstrate the presence, in the spleens of 30-week-old disease-free NOD mice, of potent APCs and CD4+CD25+ regulatory T-cells that are capable of controlling diabetogenic cells. There is a possibility that the impaired functional capacity of APCs in NOD mice with overt disease is a consequence (i.e., the result of exposure to high levels of glucose), not a cause, of their resistance to diabetes development. However, two of our observations suggest that this APC defect is unlikely to be a consequence of the disease: 1) APCs from 3-week-old NOD mice without disease symptoms (i.e., destruction of islet and high blood glucose) already exhibit impaired function (Fig. 5), and 2) APCs from 8-week-old transgenic (OVE26 on an FVB background) mice that express calmodulin under the rat insulin II promoter and consequently exhibit hyperglycemia (>600 mg/dl) for 8 consecutive weeks (46) exhibit normal function (data not shown). In a recent study, anti-CD3 antibody treatment has been shown to induce a potent population of CD4+CD25+ regulatory T-cells that could inhibit transfer of disease and gain the ability to optimally suppress the proliferation of CD4+CD25– responder T-cells in vitro in the presence of NOD APCs (36). In the light of our findings, these data suggest that strong preactivation of CD4+CD25+ regulatory T-cells bypasses the need for optimal APC stimulation. We have not yet identified the APCs responsible for the increased CD4+CD25+ T-cell regulatory activity. Many studies have shown that NOD mice do not produce normal myeloid dendritic cells in vivo (13,38) and that mature dendritic cells transferred into NOD mice can prevent diabetes development (47–49). Moreover, mature dendritic cells are the only APCs that can trigger in vitro proliferation of CD4+CD25+ T-cells (50). For these reasons, there is a possibility that disease-free NOD mice possess a particular population of dendritic cells capable of activating CD4+CD25+ T-cells.

Together, the data presented in this study suggest that NOD mice that develop diabetes do not exhibit an inherent defect in CD4+CD25+ regulatory T-cells, but they possess a deficient APC compartment that does not allow for efficient activation of these regulatory T-cells. In contrast, APCs from NOD mice that do not develop diabetes are capable of activating CD4+CD25+ regulatory T-cells and induce potent CD4+CD25+ regulatory T-cells that keep pathogenic cells under control. Therefore, targeting the APC population may provide a therapeutic approach for inducing and maintaining peripheral tolerance in autoimmune disease–prone individuals.


   ACKNOWLEDGMENTS
 
This research was supported by a junior faculty award from the American Diabetes Association and a basic grant from the University of Louisville School of Medicine.

The authors thank Mike Myers for technical assistance, Chris Worth for cell sorting, and the staff of the animal facility for animal care.


   FOOTNOTES
 
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.

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

Received for publication June 24, 2005 and accepted in revised form March 28, 2006


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shizuru JA, Taylor-Edwards C, Banks BA, Gregory AK, Fathman CG: Immunotherapy of the nonobese diabetic mouse: treatment with an antibody to T-helper lymphocytes. Science 240:659–662, 1988[Abstract/Free Full Text]
  2. Bendelac A, Carnaud C, Boitard C, Bach JF: Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates: requirement for both L3T4+ and Lyt-2+ T cells. J Exp Med 166:823–832, 1987[Abstract/Free Full Text]
  3. Delovitch TL, Singh B: The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727–738, 1997[Medline]
  4. Kishimoto H, Sprent J: A defect in central tolerance in NOD mice. Nat Immunol 2:1025–1031, 2001[Medline]
  5. Rapoport MJ, Jaramillo A, Zipris D, Lazarus AH, Serreze DV, Leiter EH, Cyopick P, Danska JS, Delovitch TL: Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice. J Exp Med 178:87–99, 1993[Abstract/Free Full Text]
  6. Gombert JM, Herbelin A, Tancrede-Bohin E, Dy M, Carnaud C, Bach JF: Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse. Eur J Immunol 26:2989–2998, 1996[Medline]
  7. Kukreja A, Cost G, Marker J, Zhang C, Sun Z, Lin-Su K, Ten S, Sanz M, Exley M, Wilson B, Porcelli S, Maclaren N: Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 109:131–140, 2002[Medline]
  8. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA: B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431–440, 2000[Medline]
  9. Wu AJ, Hua H, Munson SH, McDevitt HO: Tumor necrosis factor-{alpha} regulation of CD4+CD25+ T cell levels in NOD mice. Proc Natl Acad Sci U S A 99:12287–12292, 2002[Abstract/Free Full Text]
  10. Alleva DG, Pavlovich RP, Grant C, Kaser SB, Beller DI: Aberrant macrophages cytokine production is a conserved feature among autoimmune-prone mouse strains: elevated interleukin (IL)-12 and an imbalance in tumor necrosis factor-{alpha} and IL-10 define a unique cytokine profile in macrophages from young nonobese diabetic mice. Diabetes 49:1106–1115, 2000[Abstract]
  11. Dahlen E, Hedlund G, Dawe K: Low CD86 expression in the nonobese diabetic mouse results in the impairment of both T cell activation and CTLA-4 up-regulation. J Immunol 164:2444–2456, 2000[Abstract/Free Full Text]
  12. Jansen A, van Hagen M, Drexhage HA: Defective maturation and function of antigen-presenting cells in type 1 diabetes. Lancet 345:491–492, 1995[Medline]
  13. Lee M, Kim AY, Kang Y: Defects in the differentiation and function of bone marrow-derived dendritic cells in non-obese diabetic mice. J Korean Med Sci 15: 217, 2000
  14. Piganelli JD, Martin T, Haskins K: Splenic macrophages from the NOD mouse are defective in the ability to present antigen. Diabetes 47:1212–1218, 1998[Abstract]
  15. Serreze DV, Gaskins HR, Leiter EH: Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J Immunol 150:2534–2543, 1993[Abstract]
  16. Takahashi K, Honeyman MC, Harrison LC: Impaired yield, phenotype, and function of monocyte-derived dendritic cells in humans at risk for insulin-dependent diabetes. J Immunol 161:2629–2635, 1998[Abstract/Free Full Text]
  17. Weaver DJJ, Poligone B, Bui T, Abdel-Motal UM, Baldwin AS, Tisch R: Dendritic cells from nonobese diabetic mice exhibit a defect in NF-{kappa}B regulation due to a hyperreactive I{kappa}B kinase. J Immunol 167:1461–1468, 2001[Abstract/Free Full Text]
  18. Yan G, Shi L, Penfornis A, Faustman DL: Impaired processing and presentation by MHC class II proteins in human diabetic cells. J Immunol 170:620–627, 2003[Abstract/Free Full Text]
  19. Asano M, Toda M, Sakaguchi N, Sakaguchi S: Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med 184:387–396, 1996[Abstract/Free Full Text]
  20. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164, 1995[Abstract]
  21. Suri-Payer E, Amar AZ, Thornton AM, Shevach EM: CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol 160:1212–1218, 1998[Abstract/Free Full Text]
  22. Dieckermann D, Plottner H, Berchtold S, Berger T, Shuler G: Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J Exp Med 193:1303–1310, 2001[Abstract/Free Full Text]
  23. Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi S: Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol 162:5317–5326, 1999[Abstract/Free Full Text]
  24. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH: Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J Exp Med 193:1285–1294, 2001[Abstract/Free Full Text]
  25. Stephens LA, Mottet C, Mason D, Powrie F: Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro. Eur J Immunol 31:1247–1254, 2001[Medline]
  26. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, Shimizu J, Sakaguchi S: Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 10:1969–1980, 1998[Abstract/Free Full Text]
  27. Thornton AM, Shevach EM: CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 188:287–296, 1998[Abstract/Free Full Text]
  28. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S: Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3:135–142, 2002[Medline]
  29. Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S: Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 192:303–310, 2000[Abstract/Free Full Text]
  30. Sakaguchi S: Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22:531–562, 2004[Medline]
  31. Khattri R, Yasayko SA, Ramsdell F: An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 4:337–342, 2003[Medline]
  32. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller E, McDevitt HO, Bonyhadi M, Bluestone JA: In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199:1455–1465, 2004[Abstract/Free Full Text]
  33. Viglietta V, Allan C, Weiner H, Hafler DA: Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 199:971–979, 2004[Abstract/Free Full Text]
  34. Liang S, Alard P, Zhao Y, Parnell S, Clark SL, Kosiewicz MM: Conversion of CD4+CD25- cells into CD4+CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. J Exp Med 201:127–137, 2005[Abstract/Free Full Text]
  35. Berzins SP, Venanzi ES, Benoist C, Mathis D: T-cell compartments of pre-diabetic NOD mice. Diabetes 52:327–334, 2003[Abstract/Free Full Text]
  36. Belghith M, Bluestone JA, Barriot S, Megret J, Bach JF, Chatenoud L: TGF-ß-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 9:1202–1208, 2003[Medline]
  37. Serreze DV, Leiter EH: Defective activation of T suppressor cell function in nonobese diabetic mice: potential relation to cytokine deficiencies. J Immunol 140:3801–3807, 1988[Abstract]
  38. Alard P, Thompson C, Agersborg SS, Thatte J, Setiady Y, Samy E, Tung KS: Endogenous oocyte antigens are required for rapid induction and progression of autoimmune ovarian disease following day-3 thymectomy. J Immunol 166:4363–4369, 2001[Abstract/Free Full Text]
  39. Strid J, Lopes L, Marcinkiewicz J, Petrovska L, Nowak B, Chain BM, Lund T: A defect in bone marrow derived dendritic cell maturation in the nonobese diabetic mouse. Clin Exp Immunol 123:375–381, 2001[Medline]
  40. You S, Belghith M, Cobbold S, Alyanakian MA, Gouarin C, Barriot S, Garcia C, Waldmann H, Bach JF, Chatenoud L: Autoimmune diabetes onset results from qualitative rather than quantitative age-dependent changes in pathogenic T-cells. Diabetes 54:1415–1422, 2005[Abstract/Free Full Text]
  41. Lohr J, Knoechel B, Jiang S, Sharpe AH, Abbas AK: The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat Immunol 4:664–669, 2003[Medline]
  42. Tang Q, Henriksen K, Boden E, Tooley A, Ye J, Subudhi S, Zheng X, Strom T, Bluestone JA: Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol 171:3348–3352, 2003[Abstract/Free Full Text]
  43. Boitard C, Yasunami R, Dardenne M, Bach JF: T cell-mediated inhibition of the transfer of autoimmune diabetes in NOD mice. J Exp Med 169:1669–1680, 1989[Abstract/Free Full Text]
  44. Lepault F, Gagnerault MC: Characterization of peripheral regulatory CD4+ T cells that prevent diabetes onset in nonobese diabetic mice. J Immunol 164:240–247, 2000[Abstract/Free Full Text]
  45. Hutchings PR, Cooke A: The transfer of autoimmune diabetes in NOD mice can be inhibited or accelerated by distinct cell populations present in normal splenocytes taken from young males. J Autoimmun 3:175–185, 1990[Medline]
  46. Epstein PN, Overbeek PA, Means AR: Calmodulin-induced early-onset diabetes in transgenic mice. Cell 58:1067–1073, 1989[Medline]
  47. Feili-Hariri M, Dong X, Alber SM, Watkins SC, Salter RD, Morel PA: Immunotherapy of NOD mice with bone marrow–derived dendritic cells. Diabetes 48:2300–2308, 1999[Abstract]
  48. Naumov YN, Bahjat KS, Gausling R, Abraham R, Exley MA, Koezuka Y, Balk SB, Strominger JL, Clare-Salzer M, Wilson SB: Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets. Proc Natl Acad Sci U S A 98:13838–13843, 2001[Abstract/Free Full Text]
  49. Shinomiya M, Fazle Akbar SM, Shinomiya H, Onji M: Transfer of dendritic cells (DC) ex vivo stimulated with interferon-gamma (IFN-gamma) down-modulates autoimmune diabetes in non-obese diabetic (NOD) mice. Clin Exp Immunol 117:38–43, 1999[Medline]
  50. Yamazaki S, Iyoda T, Tarbell K, Olson K, Velinzon K, Inaba K, Steinman RM: Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells. J Exp Med 198:235–247, 2003[Abstract/Free Full Text]

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