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The Journal of Immunology, 2007, 179, 764 -774
Copyright © 2007 by The American Association of Immunologists, Inc.
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Activating Fc{gamma} Receptors Participate in the Development of Autoimmune Diabetes in NOD Mice1

Yoshihiro Inoue*, Tomonori Kaifu*, Akiko Sugahara-Tobinai*, Akira Nakamura*, Jun-Ichi Miyazaki{dagger} and Toshiyuki Takai2,*

* Department of Experimental Immunology, and the Core Research for Evolutional, Science and Technology Program of the Japan Science and Technology Agency, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; and {dagger} Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 diabetes mellitus (T1D) in humans is an organ-specific autoimmune disease in which pancreatic islet beta cells are ruptured by autoreactive T cells. NOD mice, the most commonly used animal model of T1D, show early infiltration of leukocytes in the islets (insulitis), resulting in islet destruction and diabetes later. NOD mice produce various islet beta cell-specific autoantibodies, although it remains a subject of debate regarding whether these autoantibodies contribute to the development of T1D. Fc{gamma}Rs are multipotent molecules that play important roles in Ab-mediated regulatory as well as effector functions in autoimmune diseases. To investigate the possible role of Fc{gamma}Rs in NOD mice, we generated several Fc{gamma}R-less NOD lines, namely FcR common {gamma}-chain (FcR{gamma})-deficient (NOD.{gamma}–/–), Fc{gamma}RIII-deficient (NOD.III–/–), Fc{gamma}RIIB-deficient (NOD.IIB–/–), and both FcR{gamma} and Fc{gamma}RIIB-deficient NOD (NOD.null) mice. In this study, we show significant protection from diabetes in NOD.{gamma}–/–, NOD.III–/–, and NOD.null, but not in NOD.IIB–/– mice even with grossly comparable production of autoantibodies among them. Insulitis in NOD.{gamma}–/– mice was also alleviated. Adoptive transfer of bone marrow-derived dendritic cells or NK cells from NOD mice rendered NOD.{gamma}–/– animals more susceptible to diabetes, suggesting a possible scenario in which activating Fc{gamma}Rs on dendritic cells enhance autoantigen presentation leading to the activation of autoreactive T cells, and Fc{gamma}RIII on NK cells trigger Ab-dependent effector functions and inflammation. These findings highlight the critical roles of activating Fc{gamma}Rs in the development of T1D, and indicate that Fc{gamma}Rs are novel targets for therapies for T1D.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Insulin-dependent diabetes mellitus or type 1 diabetes (T1D)3 is a pancreatic islet-specific autoimmune disease affecting ~0.4% of the population (reviewed in Refs. 1, 2, 3, 4, 5). Similar to other organ-specific as well as systemic autoimmune diseases in humans, susceptibility to T1D is determined by genetic elements as well as by environmental factors such as infectious agents and diet. In human T1D, multiple genetic loci (defined as IDDM1–19) are responsible for development of the disease, MHC class II alleles being the strongest genetic determinant. The NOD mouse spontaneously exhibits infiltration of inflammatory cells in pancreatic islets (insulitis) even at an early age, which induces autoimmune diabetes later, and has become the most extensively studied model of T1D in humans. Crossing of NOD mice with nondiabetic strains of mice revealed multiple loci (Idd1–24) determining susceptibility to T1D throughout the entire murine chromosomes (1). The most important locus is, as in humans, the MHC class II genes, candidates being IAg7 and IE (Idd1). Some candidate genes in non-MHC Idd loci have also been suggested (1, 6), such as Il-2 (Idd3) and Cd152 (Idd5.1). However, our knowledge of the interplay between molecular as well as cellular components participating at the onset of the disease and exacerbation remains far from complete.

Fc receptors for IgG (Fc{gamma}Rs) are modulators of IgG autoantibodies generated against different tissues in autoimmunity (7, 8). In the mouse system, the Fc{gamma}R family includes high-affinity Fc{gamma}RI, and low-affinity Fc{gamma}RIIB and Fc{gamma}RIII (reviewed in Refs. 7, 8, 9, 10, 11), and recently described, intermediate-affinity Fc{gamma}RIV (12, 13, 14). A unique inhibitory receptor, Fc{gamma}RIIB, occurs as a monomeric form with the ITIM, whereas Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV associate with the ITAM-harboring FcR common {gamma}-chain (FcR{gamma}) homodimer, which mediates trafficking of the receptors to the cell surface and transduction of the activating signal. Mice deficient in FcR{gamma}, which do not express any activating Fc{gamma}Rs (Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV), are resistant to various autoimmune disorders such as collagen-induced arthritis and glomerulonephritis, whereas those deficient in inhibitory Fc{gamma}RIIB are prone to developing autoimmune diseases such as systemic lupus erythematosus (7, 13, 15). These findings suggest the possible participation of activating as well as inhibitory Fc{gamma}Rs also in T1D, irrespective of whether their effect is as a pivot or a modifier. Supporting this notion, inhibitory Fc{gamma}RIIB expression in NOD mice was reduced (16, 17) due to multiple mutations in the promoter of the gene, as was the case for other autoimmune-prone mouse strains (17, 18), whereas that of Fc{gamma}RIII was not altered (16). In addition, Fc{gamma}RI in NOD mice exhibited altered IgG binding specificity and surface expression (19). However, the involvement of these activating or inhibitory Fc{gamma}Rs has not been demonstrated directly in the context of T1D in NOD mice to date.

We sought to determine whether activating and inhibitory Fc{gamma}Rs participate in the development of T1D. To this end, we generated various Fc{gamma}R-deficient NOD lines, including those deficient in FcR{gamma} (NOD.{gamma}–/–), Fc{gamma}RIII (NOD.III–/–), Fc{gamma}RIIB (NOD.IIB–/–), and both FcR{gamma} and Fc{gamma}RIIB (NOD.null). In this study, we show significant protection from the spontaneous onset of T1D in the NOD lines lacking expression of activating Fc{gamma}Rs, but not inhibitory Fc{gamma}RIIB, indicating that the activating Fc{gamma}Rs in NOD mice are critical elements in the development of T1D.


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

NOD/Shi Jic mice were purchased from CLEA Japan. FcR{gamma}–/– (20), Fc{gamma}RIII–/– (21), and Fc{gamma}RIIB–/– mice (22), originally developed to have a 129/SvJ and C57BL/6 (B6) hybrid background and backcrossed with B6 mice for 8 generations for FcR{gamma}–/– mice or at least 4 generations for Fc{gamma}RIII–/– and Fc{gamma}RIIB–/– mice, were further backcrossed with NOD/Shi Jic mice for 12 generations. The FcR{gamma}–/–Fc{gamma}RIIB–/– (FcRnull) mice were a gift from Dr. J. V. Ravetch (Rockefeller University, New York, NY), and had been generated by crossing between FcR{gamma}+/–Fc{gamma}RIIB+/– male and females obtained through the mating of FcR{gamma}–/– mice with a B6 background with Fc{gamma}RIIB–/– mice with a B6 background. FcR{gamma}–/–Fc{gamma}RIIB–/– mice were backcrossed with NOD/Shi Jic mice for 12 generations. In these backcrossing procedures, heterozygous mice harboring each Fc{gamma}R genetic deletion were identified among littermates at each cycle of backcross by PCR with appropriate primer pairs, and were used for the next cycle of backcross. The heterozygous mice obtained at the final backcross were intercrossed to obtain homozygous mutant NOD mice and the corresponding heterozygotes as well as the wild-type littermates. The mice were kept and bred in the Animal Unit of the Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan), an environmentally controlled and specific pathogen-free facility, according to guidelines for experimental animals defined by the facility, and animal protocols were reviewed and approved by the Institute of Development, Aging and Cancer Animal Studies Committee. All experiments were performed on age-matched female mice.

Assessment of diabetes

Blood glucose levels were measured every 2 wk with Precision QID measurement (Abbott Laboratories). The mice were diagnosed as having diabetes when the blood glucose level was >200 mg/dl.

Histological and immunohistochemical analyses

The pancreas was embedded in Tissue-Tec OCT Compound (Sakura Finetek), and then processed for H&E staining. Insulitis was scored according to the standard criteria as follows: grade 0, no infiltration; grade 1, perivascular/periductular infiltrates with leukocytes touching, but not penetrating, islet perimeters; grade 2, leukocytic penetration of up to 25% of islet mass; grade 3, leukocytic penetration of up to 75% of islet mass; and grade 4, end-stage insulitis with <20% of islet mass remaining. The insulitis index was calculated according to the following formula:

Formula
where n denotes the number of mice for the insulitis score for each grade. For immunohistochemical analysis, cryosections were fixed in 10% neutralized formalin (Wako Pure Chemical Industries) and then incubated with 0.3% H2O2 methanol for 30 min. After incubation with 3% skimmed milk, the cryosections were stained with the following mouse-specific Abs: CD4 (GK1.5; BD Biosciences), CD8 (53–6.7; BD Biosciences), CD11c (N418; Chemicon International), F4/80 (CI:A3–1; Serotec), CD49b/Pan-NK (DX5; BD Biosciences), Gr-1 (RB6–8C5; BD Biosciencess), and CD45R/B220 (RA3–6B2; BD Biosciences). After overnight incubation at 4°C, the sections were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG Abs (DakoCytomation) for 50 min at room temperature. Finally, the sections were incubated with diaminobenzidine (Wako Pure Chemical Industries), and then counterstained with hematoxylin. The sections were observed under an Olympus BX50 microscope (Olympus). For immunohistochemical analysis of pancreatic sections, ≥50 islets in total were examined in each age group tested.

Flow cytometric analysis of pancreatic islet-infiltrating cells

Islet cells including infiltrating cells were isolated according to the method described elsewhere (23). Pancreas islets were collected from 10 pancreas samples from 15-wk-old NOD.{gamma}–/– or WT NOD mice. For flow cytometry, the following mouse-specific Abs were used: FITC-conjugated anti-CD8 (53–6.7), anti-CD49b/Pan-NK (DX5), anti-CD11c (HL3), anti-CD3 (145–2C11), PE-conjugated anti-CD4 (GK1.5), anti-CD3 (145–2C11), anti-CD11b (Mac-1), and anti-mouse IgG1 (A85–1). All Abs were from BD Biosciences. {alpha}-galactosyl ceramide ({alpha}-GalCer, provided by Kirin Brewery, Gunma, Japan)-loaded CD1d:Ig dimeric protein was purchased from BD Biosciences. Cells were stained using standard techniques, and flow cytometric analysis was performed using FACS LSR and CellQuest software (BD Biosciences).

Cytokine measurement by ELISA

For evaluation of IFN-{gamma} and IL-4 production by islet-infiltrating T cells, pancreatic islet cells were harvested from 15- to 17-wk-old prediabetic WT NOD or NOD.{gamma}–/– mice. The bulk islet cells (5 x 105 cells/well) were stimulated with 5 µg/ml or 10 µg/ml anti-CD3 Ab (145–2C11; BD Biosciences) in the RPMI 1640 medium containing 10% FCS at 100 µl/well on 96-well tissue culture plates for 16 h at 37°C under 5% CO2. IFN-{gamma} and IL-4 protein were detected by sandwich ELISA (ELISA MAX Set Standard; BioLegend). In brief, the capture Ab anti-IFN-{gamma} or anti-IL-4 was bound to flat-bottom 96-well Nunc MaxiSorp plates at 4°C overnight. The plates were blocked with 200 µl/well of PBS containing 1.0% BSA at room temperature for 2 h. At 50 µl of cell culture, supernatants and cytokine standards were titrated and incubated at room temperature for 2 h on the plates that were then washed with PBS containing 0.02% Tween 20 and 0.05% BSA. The plate was incubated with biotinylated anti-IFN-{gamma} or anti-IL-4 detection Abs at room temperature for 1 h, and then washed and incubated with streptavidin-HRP solution at room temperature for 30 min. After washing, the assay was developed at room temperature for 15 min with 50 µl of TrueBlue Peroxidase Substrate (Bio FX Laboratories). The OD450 was read with a microplate reader (Biolumin 960; Molecular Dynamics). The concentration of cytokine in each sample was determined from the standard curve. The results were examined using nine mice in each group.

Measurement of glutamic acid decarboxylase (GAD) and insulin antibodies

Serum Ab titers were measured by ELISA. In brief, a 96-well microplate (Nalge Nunc International) was coated with 50 µl/well of a 30-µg/ml solution of GAD peptide (aa 509–528 or 524–543; Sigma-Aldrich), and was coated with 50 µl/well of a 0.1-ng/ml solution of mouse insulin (Morinaga Institute of Biological Science) in 0.1 M sodium bicarbonate at room temperature for 2 h, and then blocked with 200 µl/well of PBS containing 1.0% BSA at room temperature for 2 h. The serum diluted (1/100 or 1/200 for GAD or insulin Ab measurement, respectively) with PBS containing 0.1% BSA was added at 50 µl/well and allowed to react at 4°C overnight. The wells were washed three times with PBS containing 0.02% Tween 20 and 0.05% BSA, incubated with 50 µl of a 1/1000 dilution of goat anti-mouse IgG (Sigma-Aldrich) or IgG2c (Bethyl) coupled with HRP at room temperature for 2 h, washed four times with PBS containing 0.02% Tween 20 and 0.05% BSA, and then developed at room temperature for 15 min with 50 µl of TrueBlue Peroxidase Substrate (Bio FX Laboratories). The serum Ab titers were measured using five mice in each age group. The OD450 was read with a microplate reader (Biolumin 960; Molecular Dynamics).

Adoptive transfer of dendritic cells

Bone marrow cells were obtained from nondiabetic WT NOD or NOD.{gamma}–/– mice according to the method described elsewhere (24). After depleting RBC and lymphocytes, the bone marrow cells were cultured at 1 x 106 cells/ml in the presence of 20 ng/ml murine recombinant GM-CSF (PeproTech). The medium was replaced with a GM-CSF-containing medium on Day 4 of culture. On Day 6 of culture, the cells were collected and CD11c+ dendritic cells (DCs) were purified by MACS sorting (Miltenyi Biotec). Prediabetic WT NOD or NOD.{gamma}–/– mice were injected i.v. with 4 x 105 DCs twice a week from 4 wk to 6 wk of age.

Adoptive transfer of NK cells

NK cells were isolated from the spleen cells of 5- to 8-wk-old prediabetic WT NOD or NOD.{gamma}–/– mice using a BD IMagnet NK cell enrichment set (BD Biosciences). Prediabetic WT NOD or NOD.{gamma}–/– mice were injected i.v. with 1 x 106 purified NK cells (purity >85%, as assessed by DX5 staining) once a week from 4 to 6 wk of age.

Ab-dependent cell-mediated cytotoxicity (ADCC) assays

DX5+ NK-enriched cells were purified by MACS sorting (Miltenyi Biotec) from 5- to 8-wk-old nondiabetic WT NOD, NOD.{gamma}–/–, or C57BL/6 mice. These cells were incubated in the RPMI 1640 medium supplemented with 10% FCS, 50 µM penicillin, 50 µg/ml streptomycin, 50 µM 2-ME, 1 mM sodium pyruvate, and nonessential amino acids (Invitrogen Life Technologies) in the presence of 1000 IU/ml human rIL-2 for 7 days, and then used as effector cells. Cytotoxicity was measured by means of a 51Cr-release assay. The target cells were labeled with 100 µl Ci/ml Na251CrO4 (GE Healthcare) for 1 h at 37°C under 5% CO2 in RPMI 1640 containing 20% FCS. For the ADCC assay, 51Cr-labeled EL-4 lymphoma cells (American Type Culture Collection) were incubated for 15 min on ice with anti-Thy-1.2 Ab (30-H12; BD Biosciences), and then incubated with the stimulated NK cells (effector) for 4 h at 37°C under 5% CO2. The effector/target (E/T) ratio was 10:1 (effector: 1 x 105 cells/well, target: 1 x 104 cells/well). Cytotoxicity was measured with an autowell {gamma} system (Aloka).

Intravenous gamma globulin (IVIg) treatment

The IVIg preparation used was Venilon-I (Teijin), which is sulfonated human gamma globulin prepared from healthy volunteers. NOD mice were injected i.p. with 1 g/kg or lower doses of IVIg or saline twice or four times, once a week when they were 4–8 wk of age as indicated in each experiment.

Statistical analyses

Statistical analyses were performed using Student’s t test, the log-rank test, or the {chi}2 test, as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Activating Fc{gamma}R-deficient NOD lines are protected from diabetes

We generated Fc{gamma}R-less NOD mouse lines by backcrossing each type of gene-targeted mice with NOD mice for 12 generations. The NOD.{gamma}–/–, NOD.III–/–, NOD.IIB–/–, and NOD.null mice were genotyped at 139 microsatellite markers at the 12th generation (data not shown). All of the Fc{gamma}R-less NOD lines had a set of 114 microsatellite markers of NOD origin, but some lines did not have this set in terms of the 25 markers spanning from D1Mit194 (71.5 cM) to D1Mit459 (102.0 cM) on chromosome 1, including the genomic interval where fcgr2b, fcgr3, fcer1g, and Sle1b are located (Fig. 1). The 129/SvJ strain, from which the embryonic stem cells in the Fc{gamma}R targeting strategies were derived, has the same set of genes for SLAM/CD2 family molecules at the Sle1b locus as those in NOD mice (SLAM haplotype 2), which is related to autoantibody production in systemic lupus erythematosus (25). Sle1b from 129/SvJ mice is suggested to mediate autoimmunity in the context of the B6 genetic background (25). Because susceptibility to autoimmune diabetes might be influenced by the Sle1b haplotype but not by the neighboring Fc{gamma}R gene deletion itself, it was important to identify the origin of Sle1b in our Fc{gamma}R-less NOD lines. Our NOD lines carried the Sle1b region of 129/SvJ (or NOD) origin (SLAM haplotype 2), not B6 origin (SLAM haplotype 1), because the genetic interval at 93.3 cM in our Fc{gamma}R-less NOD lines was derived from either the 129/SvJ or NOD strain (Fig. 1). Thus, we considered that WT NOD mice served as a suitable control for our Fc{gamma}R-less NOD lines.


Figure 1
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  		FIGURE 1. Microsatellite marker analysis of chromosome 1 60–110 cM region from Fc{gamma}R-deficient NOD lines. Fc{gamma}R genes are located in a restricted area of chromosome 1 (92.3–93.3 cM), which is adjacent to the Sle1b locus harboring the genes for SLAM/CD2 family molecules that are related to autoantibody production in systemic lupus erythematosus (25 ). Our Fc{gamma}R-less NOD lines had 129/SvJ or NOD-derived genetic markers around 93.3 cM, indicating that the Sle1b region is the same haplotype (the SLAM haplotype 2) as that of NOD (25 ).

 
NOD mice lacking the expression of one or more activating Fc{gamma}R, namely NOD.{gamma}–/–, NOD.III–/–, and NOD.null mice, developed diabetes at an incidence of 28.6, 47.6, and 38.9%, respectively, at 50 wk of age, which was significantly lower (p < 0.05 or p < 0.01) than in WT NOD mice (69.4%) of the same age (Fig. 2A and Table I). In contrast, NOD.IIB–/– mice showed an incidence (56.5%) comparable to that in WT NOD mice. In Fc{gamma}R-mutant littermates heterozygous for each allele, reduction in disease incidence was not significant in each activating Fc{gamma}R-deleted line (Fig. 2B). Comparison of the incidence of diabetes among homozygous NOD.IIB–/–, heterozygous littermate NOD.IIB+/– and wild-type littermate NOD.IIB+/+ mice revealed no significant difference at 50 wk of age (data not shown).


Figure 2
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  		FIGURE 2. Reduced susceptibility to autoimmune type 1 diabetes in Fc{gamma}R-deficient NOD mice. A, Incidence of diabetes for NOD.{gamma}–/– ({circ} with solid line, n = 21), NOD.III–/– ({blacktriangleup} with dotted line, n = 21), NOD.IIB–/– ({triangleup} with solid line, n = 23), NOD.null ({square} with solid line, n = 18), and WT NOD (• with solid line, n = 36). B, Incidence of diabetes for Fc{gamma}R-mutant heterozygous littermates, NOD.{gamma}+/– (n = 20), NOD.III+/– (n = 25), NOD.IIB+/– (n = 31), and NOD.null ({gamma}+/–IIB+/–, n = 21) mice. The incidence of WT NOD mice is shown as a control. NS, Not significant. C, Survival rate. *, p < 0.05, **, p < 0.01 vs WT NOD mice by the log-rank test. All experiments were performed on female mice.

 

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  		Table I. Average onset and incidence of diabetes and survival of various NOD mice

 
Consistent with the reduced disease incidence, the activating Fc{gamma}R-less NOD lines exhibited significantly lower mortality than WT NOD and NOD.IIB–/– mice (p < 0.05 or p < 0.01) at 50 wk of age (Fig. 2C and Table I). In WT NOD mice, the blood glucose level rapidly increased after ~20 wk of age, whereas in NOD.{gamma}–/– mice this increase was suppressed (data not shown). Diabetes onset in the NOD lines lacking activating Fc{gamma}Rs was delayed by 7–9 wk compared with that in WT NOD mice (Table I; p < 0.05 or p < 0.01). Based on these data, we concluded that deletion of FcR{gamma} (Fc{gamma}RI, III, and IV), Fc{gamma}RIII, or all Fc{gamma}Rs (Fc{gamma}RI, IIB, III, and IV) rendered NOD mice diabetes resistant to a significant extent, whereas Fc{gamma}RIIB deletion did not affect disease development.

FcR{gamma}-deficient NOD line shows less severe insulitis

Insulitis beginning at 3–4 wk of age first involves DCs and macrophages, and later CD4+ and CD8+ T cells, and B cells. This early stage infiltration leads to T cell-mediated destruction of islet beta cells by 16–24 wk of age due to an unidentified sequence of events (1, 3, 4). To assess the severity of insulitis in NOD.{gamma}–/– mice, we determined the numbers of infiltrating cells in the islets of diabetic or nondiabetic WT NOD and NOD.{gamma}–/– mice at various ages. H&E staining of pancreatic sections revealed that the mean insulitis indices for prediabetic NOD.{gamma}–/– mice were significantly lower in all the age groups examined than those for prediabetic WT NOD mice, whereas after the onset of diabetes, NOD.{gamma}–/– and WT NOD mice showed comparable insulitis indices (Fig. 3, A and B). This was also the case for the insulitis grade, the severity of insulitis being graded as one of five levels in each islet, and the number of islets with each grade of insulitis being determined (Fig. 3C). Detailed immunohistochemical analyses of the population of infiltrated cells in pancreatic islet samples revealed less severe infiltration of cells with any of CD4, CD8, DX5, CD11c, or B220 in NOD.{gamma}–/– mice (Fig. 4, A and B). Comparison of the profiles for the absolute number of each inflammatory cell type in islets from WT NOD and NOD.{gamma}–/– mice in each age group did not reveal any dramatic difference between WT NOD and NOD.{gamma}–/– mice, although a notable reduction in infiltrating CD4+ and CD8+ T cell numbers was observed in NOD.{gamma}–/– mice at 10, 20, and 30 wk of age (Fig. 4C). Flow cytometric analysis confirmed the lower abundance of infiltrated cells in the pancreatic islets (Fig. 5A), but not in the spleens (Fig. 5B), of NOD.{gamma}–/– mice. Measurement of cytokine secretion from pancreatic islet-infiltrating cells stimulated with anti-CD3 Ab in vitro revealed that IFN-{gamma} secretion extremely dominated IL-4 release from these cells either from WT and NOD.{gamma}–/– mice (Fig. 5C), indicating that the reduced incidence of insulitis in NOD.{gamma}–/– mice was not due to an induction of type 2 helper T cells in the activating Fc{gamma}R-deficient line. In addition, IFN-{gamma} secretion was less significant in the cells from NOD.{gamma}–/– mice than those from WT mice (Fig. 5C), reflecting the reduced number of CD4+ and CD8+ T cells in pancreatic islet-infiltrating cells from NOD.{gamma}–/– mice. Thus, NOD.{gamma}–/– mice had less severe insulitis due to the decreased number of infiltrated cells than WT NOD mice, but the cellularity or Th1/Th2 balance in the infiltrated leukocytes was not markedly different between WT NOD and the activating Fc{gamma}R-deficient line.


Figure 3
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  		FIGURE 3. Histopathological analyses of Langerhans islets and insulitis in WT NOD and NOD.{gamma}–/– mice. Insulitis indices (A), cellular infiltration in islets (B), and insulitis grades in prediabetic WT NOD or NOD.{gamma}–/– mice at various ages as well as those in diabetic mice (C). In A, the insulitis index was calculated as described under Materials and Methods. In B, the arrowheads indicate areas of infiltrated cells. H&E stain. Original magnification, x 400. In C, the severity of insulitis was graded on a scale of 0–4; grade 0 (no infiltration), 1 (perivascular/periductular infiltrates with leukocytes touching, but not penetrating, islet perimeters,), 2 (leukocytic penetration of up to 25% of islet mass), 3 (leukocytic penetration of up to 75% of islet mass), and 4 (end-stage insulitis, <20% of islet mass remaining). A total of ≥50 islets from 5 to 10 mice in each age group and diabetic mice were examined. *, p < 0.05, **, p < 0.01 vs WT NOD mice by Student’s t test.

 

Figure 4
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  		FIGURE 4. Immunohistochemical findings in Langerhans islets in WT NOD and NOD.{gamma}–/– mice. A, Percentage of inflammatory islets containing cells with the indicated markers in prediabetic NOD mice. An inflammatory islet was defined as that containing at least one inflammatory cell. A total of at ≥50 islets from 5 to 10 mice were examined in each age group tested. *, p < 0.05, **, p < 0.01 by the {chi}2 test. B, Pancreatic sections from 20-wk-old mice were stained with the indicated markers followed by counterstaining with hematoxylin. A positive signal is brown. Original magnification, x 400. C, Average infiltrating cell numbers per islet. The absolute number of each inflammatory cell type in islets from WT NOD and NOD.{gamma}–/– mice in each age group were counted and expressed as mean infiltrating cell numbers. The profiles were not dramatically different between WT NOD and NOD.{gamma}–/– mice, but reduction in the numbers of CD4+ and CD8+ T cells was noted in NOD.{gamma}–/– mice at 10, 20, and 30 wk of age. A total of ≥50 islets from 5 to 10 mice were examined in each age group tested.

 

Figure 5
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  		FIGURE 5. Flow cytometric analyses and cytokine assay of pancreatic islet-infiltrating cells in WT NOD and NOD.{gamma}–/– mice. A, Populations of CD4+ or CD8+ T cells, DX5+ NK cells, CD1d/{alpha}-GalCer dimer-binding CD3+ NKT cells, and CD11c+CD11b+ DCs in pancreatic islets of prediabetic WT NOD or NOD.{gamma}–/– mice at 15 wk of age. Pancreatic islet cells were confirmed to contain less abundant infiltrated cells in NOD.{gamma}–/– mice. B, Flow cytometric analyses of CD4+ and CD8+ T cells in splenocytes in prediabetic WT NOD and NOD.{gamma}–/– mice at 10 or 20 wk of age. Data are presented as mean ± SD. The data are representative of three separate experiments with similar results. Flow cytometric analysis of splenocytes from WT NOD and NOD.{gamma}–/– mice at 10 and 20 wk of age did not reveal any profound decrease in any of CD4+CD25+ T regulatory cells, NK, NKT cells, macrophages or DCs (data not shown), or CD4+ or CD8+ T cells, indicating that the decreases in T cells and DCs in NOD.{gamma}–/– mice were pancreatic islet specific. C, IFN-{gamma} and IL-4 production of pancreatic islet-infiltrating cells from prediabetic WT NOD and NOD.{gamma}–/– mice at 15–17 wk of age. Pancreatic islet cells were separately stimulated in vitro with anti-CD3 Abs, and the cytokines released into the medium were estimated by ELISA. IL-4 production was scarcely detected. Data are presented as mean ± SD of three separate experiments with similar results using three mice in each group and in each experiment. **, p < 0.01 vs WT NOD mice by Student’s t test.

 
Anti-GAD65 and insulin autoantibody titers do not correlate with disease susceptibility

The direct or indirect role of autoantibodies against pancreatic islet beta cell-specific Ags, such as GAD65, insulin, proinsulin, and hsp60, in the pathogenesis of T1D remains uncertain (3, 4). However, the presence of these autoantibodies in the sera coincides with early insulitis in NOD mice (4, 26). To examine the link between autoantibody production and diabetes development in activating Fc{gamma}R-less NOD mice, we compared the anti-GAD65 and anti-insulin IgG Ab levels in sera from prediabetic as well as diabetic WT and Fc{gamma}R-less NOD mice. In the case of anti-GAD Abs, IgG2c, and total IgG reacting with major epitope peptides P509–528 and P524–543 in the sera were higher in NOD.{gamma}–/– mice than in WT NOD mice at 10 wk of age, and the levels were comparable or lower in the samples from 20-wk-old mice (Fig. 6A). This was also the case for anti-insulin IgG and IgG2c levels (Fig. 6B). In contrast, Fc{gamma}RIIB-deficient mice, in general, exhibit augmented autoantibody production in either induced or spontaneously developed autoimmune diseases (7, 15). The notion that Fc{gamma}RIIB expression is reduced in NOD mice (16, 17, 18) prompted us to examine whether complete elimination of the fcgr2b gene in NOD mice rendered the animals more active regarding autoantibody production. Five- and 10-wk-old NOD.IIB–/– mice had, in fact, increased levels of anti-GAD65 and anti-insulin total IgG as well as IgG2c, whereas at 20 wk of age, they showed levels comparable to those in age-matched WT NOD mice (Fig. 6). Because these observations were not apparently consistent with the delayed or comparable onset of diabetes in NOD.{gamma}–/– or NOD.IIB–/– mice, respectively, we concluded that these increases in the anti-GAD65 and anti-insulin IgG Ab levels themselves in juvenile Fc{gamma}R-less NOD lines are not correlated with the development of diabetes.


Figure 6
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  		FIGURE 6. Concentrations of anti-GAD65 and anti-insulin Abs in sera from WT NOD, NOD.{gamma}–/–, and NOD.IIB–/– mice. The levels of autoantibodies for GAD65 (aa 509–528 or 524–543) (A), and insulin (B) in sera taken from WT NOD, NOD.{gamma}–/–, and NOD.IIB–/– mice at various ages were measured by ELISA. We measured the serum levels of anti-GAD 65 and anti-insulin total IgG, and the IgG2c isotype instead of IgG2a, whose gene is lost in NOD and other strains of mice with the Igh1-b allele. In the case of anti-insulin measurement, the levels of IgG1 and IgG2b are also shown. Data are shown as mean ± SD. The data are representative of three separate experiments with similar results. *, p < 0.05, **, p < 0.01 vs WT NOD mice by Student’s t test. The higher levels of anti-GAD65 and anti-insulin IgG autoantibodies in juvenile NOD.{gamma}–/– mice may be partially explained by the lack of a clearance mechanism for IgG immune complexes through activating Fc{gamma}Rs on phagocytic cells such as macrophages, as was seen for autoimmune glomerulonephritis (67 ) and hemolytic anemia (68 ).

 
Adoptive transfer suggests participation of Fc{gamma}R(s) on DCs and NK cells in the development of diabetes

The major roles of activating Fc{gamma}Rs are the swift elimination of Ags through effective endocytosis or phagocytosis of IgG immune complexes, the enhancement of Ag presentation efficiency, and the activation of cellular signaling, leading to effector functions such as ADCC (13, 15). Thus, a possible reason for the attenuated insulitis in Fc{gamma}R-less NOD mice would be reduced Ag presentation efficiency. A possible scenario would be that IgG autoantibodies produced in juvenile WT and activating Fc{gamma}R-less NOD mice could bind to the autoantigens present in pancreatic islets. Such immune complexes would be taken up efficiently via Fc{gamma}R-mediated internalization and processed by local DCs in WT NOD mice, and then these cells should mature and present the autoantigens to T cells in the pancreatic lymph nodes. In activating Fc{gamma}R-less NOD lines, however, the DCs might not be able to efficiently take up the immune complexes, and thus cannot sufficiently present the antigenic peptides to T cells. Then, T cell priming could not take place in such mutant NOD mice, which would lead to attenuated insulitis. Another possible mechanism would be that the activating Fc{gamma}R-less NOD line could not mount effective target cell killing by macrophages and NK cells. The IgG autoantibodies bound to islet beta cells could activate these effector cells through activating Fc{gamma}Rs, thereby, leading to destruction of the cells by ADCC or initiation of inflammatory responses via secreted cytokines and chemokines.

To gain insight into these two possibilities, we conducted adoptive transfer experiments on DCs and NK cells. First, activating Fc{gamma}R-sufficient or -deficient DCs were adoptively transferred to prediabetic, juvenile WT NOD or NOD.{gamma}–/– mice. The transfer of activating Fc{gamma}R-less DCs to WT NOD mice exhibited an incidence similar to that in WT NOD mice, whereas the transfer of activating Fc{gamma}R-sufficient DCs to NOD.{gamma}–/– mice changed the incidence profile of diabetes from the NOD.{gamma}–/– type to the WT NOD type (Fig. 7A and Table II). This result may indicate that activating Fc{gamma}R-sufficient DCs had a dominant effect on the development of diabetes. In other words, DCs from WT NOD mice could convert NOD.{gamma}–/– mice into diabetes-prone animals.


Figure 7
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  		FIGURE 7. Adoptive cell transfer experiments and IVIg treatment of NOD mice. A, Adoptive transfer of DCs from WT NOD mice rendered NOD.{gamma}–/– mice more susceptible to diabetes. Incidence of diabetes in mice after adoptive transfer of DCs from WT NOD or NOD.{gamma}–/– mice at 5–8 wk of age. The transfer of DCs from WT NOD mice rendered NOD.{gamma}–/– mice remarkably susceptible to the development of diabetes ({square}). The transfer of DCs from NOD.{gamma}–/– mice did not suppress the incidence of diabetes in WT NOD mice ({blacktriangleup}). *, p < 0.05 by the log-rank test. The data are summarized in Table II. B, Natural cytotoxicity of NK cells from WT NOD and NOD.{gamma}–/– mice. NK cells from C57BL/6 (B6, {diamondsuit}), WT NOD (•) and NOD.{gamma}–/– ({circ}) mice were used in a 51Cr release assay targeting YAC-1 cells at the indicated E:T ratios. C, NK cells from C57BL/6 ({diamondsuit}), WT NOD (•), and NOD.{gamma}–/– ({circ}) were analyzed regarding killing activity against 51Cr-pulsed EL-4 target cells coated with (ADCC activity) or without (natural cytotoxicity against this target) anti-Thy1.2 mAb. 51Cr release was measured after a 4-h incubation period. D, Incidence of diabetes in mice after adoptive transfer of NK cells from WT NOD or NOD.{gamma}–/– mice at 5–8 wk of age. The transfer of NK cells from WT NOD mice rendered NOD.{gamma}–/– mice ({square}) more susceptible to the development of diabetes than those receiving NK cells from NOD.{gamma}–/– mice ({circ}). The transfer of NK cells from NOD.{gamma}–/– did not suppress the incidence of diabetes in WT NOD mice ({blacktriangleup}). *, p < 0.05 by the log-rank test. The data are summarized in Table II. E, Therapeutic effect of IVIg treatment on diabetes in WT NOD and NOD.IIB–/– mice. The incidences of diabetes in WT NOD mice after IVIg treatment (each dose: 1 g/kg {blacksquare}, 0.1 g/kg {blacktriangleup}, 0.01 g/kg •), or NOD.IIB–/– mice (dose: 1 g/kg {triangleup}) are shown. IVIg treatment was performed four times, once a week from 5 to 8 wk of age. **, p < 0.01 by the log-rank test. F, The therapeutic effect of IVIg treatment on diabetes was lost in NOD.{gamma}–/– mice. The incidence of diabetes in WT NOD (•) and NOD.{gamma}–/– mice ({blacksquare}) after IVIg treatment was compared with that of WT NOD ({circ}) and NOD.{gamma}–/– mice ({square}) not receiving IVIg. The IVIg treatment (1 g/kg) was performed twice at 4 and 5 wk of age.

 

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  		Table II. Incidence of diabetes in mice receiving adoptive transfer of DCs or NK cells from WT NOD or NOD.{gamma} –/– micea

 
Fc{gamma}RIII is the sole Fc{gamma}R expressed on murine NK cells (13). DX5+ and CD3– NK cells were prepared from splenocytes from WT NOD or NOD.{gamma}–/– mice for the adoptive transfer experiments. We first confirmed that both Fc{gamma}RIII-sufficient and -deficient NK cells were functional in terms of NK killing of YAC-1 cells (Fig. 7B), whereas Fc{gamma}RIII-deficient NK cells did not show efficient ADCC against IgG-coated EL-4 cells (Fig. 7C). These NK cells were transferred to juvenile WT NOD and NOD.{gamma}–/– mice. Fc{gamma}RIII-sufficient NK cell transfer to NOD.{gamma}–/– mice significantly changed the susceptibility of the mice to diabetes (Fig. 7D and Table II; p < 0.05), whereas Fc{gamma}RIII-less NK cell transfer to WT NOD mice failed to make the WT mice resistant to the disease. Thus, Fc{gamma}RIII-sufficient NK cells could convert, to a significant extent, NOD.{gamma}–/– mice into diabetes-prone animals, whereas Fc{gamma}RIII-deficient NK cells did not have a protective effect regarding diabetes development. Therefore, it is suggested that Fc{gamma}RIII on NK cells plays a dominant role in the development of diabetes.

IVIg therapy provides protection of NOD mice from diabetes

One therapeutic choice for autoimmune diseases in humans is IVIg therapy (27), in which pooled, normal poly-specific IgG is transfused into autoimmune patients such as those with immune thrombocytopenic purpura (ITP). IVIg may exhibit its therapeutic effect, at least in part, through the blocking of activating Fc{gamma}Rs (27) or the modulation of Fc{gamma}RIIB expression (28, 29) on APCs and effector cells. To gain insight into the possible efficacy of IVIg in T1D, especially in the context of Fc{gamma}RIIB involvement, we administered IVIg to juvenile NOD mice four times. We found it to be robustly effective in a dose-dependent manner in terms of delayed onset and reduced incidence in both WT NOD and NOD.IIB–/– mice (Fig. 7E), indicating that the therapeutic effect might be independent of Fc{gamma}RIIB, which is involved in the IVIg effect observed in animal models of ITP (28) and arthritis (29). In both WT NOD and NOD.IIB–/– mice that received the highest dose of IVIg four times, the earliest onset was delayed by >20 wk, and the incidence was less than half that in nontreated WT NOD mice. In addition, we examined whether the effect of IVIg in delaying the onset of diabetes could also be obtained in NOD.{gamma}–/– mice. Interestingly, IVIg administration into NOD.{gamma}–/– mice did not yield any further delay of disease onset, suggesting that the IVIg effect is dependent, at least in part, on FcR{gamma}. Collectively, these data suggest an important role of activating Fc{gamma}Rs in IVIg treatment of NOD mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Possible roles of Fc{gamma}Rs on DCs and NK cells in the development of diabetes

The results of adoptive transfer experiments led us to propose that activating Fc{gamma}Rs expressed on DCs, which would include Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV, may play a significant role in the development of diabetes, possibly due mainly to the facilitation of Ag presentation of diabetogenic autoantigens to T cells. We (30, 31) and others (32, 33, 34, 35, 36, 37, 38) have extensively studied the significant role of Fc{gamma}Rs on APCs in enhancing the efficiency of Ag presentation, the uptake of IgG immune complexes into the cells via Fc{gamma}Rs being 50–100 times more efficient regarding presenting antigenic peptides to MHC class I- or class II-restricted T cells than nonspecific internalization. It is conceivable that autoreactive T cells in NOD mice are activated upon autoantigen presentation by DCs taken up via Fc{gamma}Rs pancreatic beta cell-derived Ags complexed with IgG autoantibodies. In this regard, it was reasonable to observe that the reduction in the incidence of diabetes (Fig. 2A) was more significant in NOD.{gamma}–/– mice than that in NOD.III–/– mice, because FcR{gamma} deletion results in the loss of all three activating Fc{gamma}Rs, namely Fc{gamma}RI, Fc{gamma}RIII, and Fc{gamma}RIV on DCs (13, 20).

The adoptive transfer experiments also suggested a significant role of Fc{gamma}RIII on NK cells in the development of diabetes in NOD mice. It has been recently reported (39) that NK cells are crucial effector cells for the progression of insulitis to destructive diabetes in NOD mice. In that study, mouse strains with aggressive lesions in pancreatic islets had an increased proportion of NK cells among infiltrated leukocytes, and NK cell depletion resulted in impaired diabetes development in the context of CTLA-4 blockade (39). NK cells destroy virus-infected target cells through natural killing, and destroy Ab-coated targets through ADCC via the sole activating Fc{gamma}R expressed on NK cells, Fc{gamma}RIII, and release inflammatory cytokines such as IFN-{gamma}. Thus, it is possible that increases in these effector mechanisms in NK cells directly promote insulitis in coordination with CD4+ and CD8+ T cells. In this regard, our observations suggest the importance of ADCC and inflammatory cytokine release activity mediated by Fc{gamma}RIII on NK cells stimulated with IgG autoantibodies complexed with pancreatic beta cell components. Therefore, the reduced incidence of diabetes in NOD.III–/– mice shown in Fig. 2A could be due to the reduced Ag presentation of Fc{gamma}RIII-deficient DCs and the elimination of Fc{gamma}RIII-mediated NK cell effector functions.

Fc{gamma}R-independent mechanisms in NOD mice

Compared with the incidence in WT NOD mice at 40 wk of age, FcR{gamma} deletion halved the incidence (Fig. 2A). From another perspective, one-third of NOD.{gamma}–/– mice still develop diabetes even in the absence of activating Fc{gamma}Rs. The remaining susceptibility to diabetes is attributable to other mechanisms, which are not related to activating Fc{gamma}Rs. Such effector scenarios may include: 1) selection and activation of autoreactive, diabetogenic T cells; 2) Ag presentation of diabetogenic epitopes to these autoreactive T cells; and 3) insulitis provocation by effector cells such as macrophages and NK cells, which use various Fc{gamma}R-independent cytotoxic tools, such as proinflammatory cytokine production and natural killing. Diabetogenic T cells are selected preferentially by thymic stromal cells expressing NOD-specific MHC class II molecule I-Ag7, this process having been verified as being suppressed in the presence of the mutated I-Ag7 (40) or I-E molecules (41, 42), which is not expressed genetically in NOD mice. APCs mediate efficient conversion of naive, autoreactive T cells into diabetogenic effector T cells with the CD4 or CD8 coreceptor. These effector T cells, which do not express Fc{gamma}Rs, have the intrinsic potential to destroy islet beta cells (4, 43, 44, 45). Macrophages and NK cells, even though both cell types express Fc{gamma}Rs, have the ability to kill target cells through Fc{gamma}R-independent processes such as phagocytosis or perforin/granzyme-mediated natural killing. Thus, the residual susceptibility of activating Fc{gamma}R-less NOD mice leads us to the notion that T1D development comprises two distinct or mutually interacting cascades, namely Fc{gamma}R-mediated and Fc{gamma}R-independent processes.

Fc{gamma}RIIB in NOD mice

Our observation that Fc{gamma}RIIB-deficient NOD mice did not show higher susceptibility to diabetes than NOD mice was unexpected. With regard to the regulation of immune cells, Fc{gamma}RIIB was established as a unique inhibitory Fc{gamma}R, which binds IgG immune complexes and abrogates cellular activating signals initiated by activating-type receptors such as the BCR and Fc{gamma}RIII (15, 22, 46). Moreover, mice deficient in Fc{gamma}RIIB are prone to spontaneously developing diseases similar to systemic lupus erythematosus (47, 48), and are susceptible to the induction of various autoimmune diseases (49, 50, 51, 52, 53). In particular, NOD mice show reduced expression of Fc{gamma}RIIB possibly due to a mutation in the gene’s promoter sequence, as was the case for other autoimmune-prone mice such as NZB and MRL.lpr (11, 15, 16, 17, 18). These preceding observations led us to expect that Fc{gamma}RIIB-deficient NOD mice would develop more severe insulitis and diabetes than WT animals, but this was not the case. One may speculate that the maximum incidence of diabetes would be the maximum level that we could observe in NOD mice, and that Fc{gamma}RIIB deletion would not augment the incidence further. However, a recent observation in NOD mice with programmed cell death 1 deletion does not support this speculation because the incidence of diabetes in programmed cell death 1-deficient NOD mice reached 100% at 10–15 wk of age, which is much higher and earlier than that in WT animals (54). One plausible explanation for our observation is that the expression of Fc{gamma}RIIB in NOD mice is severely reduced so that the complete elimination of Fc{gamma}RIIB expression in the same animal does not have any further effect on diabetes development. Judging the validity of this scenario would require further analysis using other systems, such as the creation of a Fc{gamma}RIIB gene-transgenic NOD line, which might render the animals more resistant to the development of diabetes. In the current study, we obtained only circumstantial evidence regarding the effect of complete Fc{gamma}RIIB elimination in NOD mice, anti-GAD65 IgG autoantibody production being significantly enhanced in juvenile NOD.IIB–/– mice, as was the case for Fc{gamma}RIIB-deficient mice in general (15, 22, 46).

IVIg therapy for NOD mice

Therapeutic approaches for T1D have been tested extensively in NOD mice (55), aiming mainly at the modulation of T cells. These approaches included the administration of cytokines such as IL-10 (56), IL-4 (57), and TNF-{alpha} (58), the cell transfer approach such as with Ag-primed DCs (59), the modulation or blocking of important activating or inhibitory/regulatory receptors such as CD28 (60) and CD3 (61) expressed on T cells and NKG2D expressed on CD8+ T cells (62), and NK depletion (39), some of which have promising effects regarding blocking the development of diabetes in NOD mice.

The beneficial effect of IVIg on diabetes in NOD mice could be due, at least in part, to effective blocking of the activating Fc{gamma}Rs, although one cannot rule out the possible enhancement of autoantibody degradation by masking neonatal Fc{gamma}R, FcRn, which plays an important role in IgG homeostasis (63), or other Fab portion-associated effects (27, 64). In accordance with our speculation, the significance of activating Fc{gamma}Rs on DCs in the IVIg effect in a murine model of ITP was recently demonstrated (65), in which DCs stimulated with IVIg in vitro could suppress ITP after adoptive transfer into recipient mice. Interestingly, this suppressive effect of IVIg-treated DCs on ITP was dependent on the activating Fc{gamma}Rs on DCs. Although we should be careful because the autoimmune model differed between their system and ours, namely ITP and T1D, respectively, our observation in IVIg treatment of NOD matches their observation.

In addition, attempts have already been reported in which IVIg preparations were applied to NOD mice (64) as well regarding children with T1D (66) with some beneficial effects. The current study, however, now provides the rationale that blocking of activating Fc{gamma}Rs could also be effective for preventing T1D in humans if it is performed with careful control of the nonspecific side effects of IVIg therapy. The combination of IVIg with other T cell-targeted therapies would provide a more promising prophylactic effect against T1D in juvenile patients as well as successful control in elderly patients.


   Acknowledgments
 
We are grateful to Jeffrey V. Ravetch for providing the founder mice and Kazuo Goto for conducting the genetic marker analysis.


   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 in part by the Core Research for Evolutional, Science and Technology Program of the Japan Science and Technology Agency, a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the 21st-century Center of Excellence program "Center for Innovative Therapeutic Development Towards the Conquest of Signal Transduction Diseases." Back

2 Address correspondence and reprint requests to Dr. Toshiyuki Takai, Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo 4–1, Sendai, Japan. E-mail address: tostakai{at}idac.tohoku.ac.jp Back

3 Abbreviations used in this paper: T1D, type 1 diabetes mellitus; ADCC, Ab-dependent cell-mediated cytotoxicity; DC, dendritic cell; FcR{gamma}, Fc receptor common {gamma} subunit; GAD, glutamic acid decarboxylase; IVIg, intravenous gamma globulin; NOD.{gamma}–/–, FcR common {gamma}-chain-deficient NOD; NOD.IIB–/–, Fc{gamma}RIIB-deficient NOD; NOD.III–/–, Fc{gamma}RIII-deficient NOD; NOD.null, FcR common {gamma}-chain and Fc{gamma}RIIB double-deficient NOD; ITP, immune thrombocytopenic purpura. Back

Received for publication August 30, 2006. Accepted for publication May 4, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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