The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 6964 -6974
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Calderon, B.
Right arrow Articles by Unanue, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Calderon, B.
Right arrow Articles by Unanue, E. R.
Right arrowPubmed/NCBI databases
*GEO DataSet
*Substance via MeSH
Medline Plus Health Information
*Diabetes Type 1

IFN-{gamma}-Dependent Regulatory Circuits in Immune Inflammation Highlighted in Diabetes1

Boris Calderon, Anish Suri, Xiaoou O. Pan, Jason C. Mills and Emil R. Unanue2

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Aknowlegments
 Disclosures
 References
 
We demonstrate diverse roles of IFN-{gamma} in the induction and regulation of immune-mediated inflammation using a transfer model of autoimmune diabetes. The diabetogenic CD4+BDC2.5 (BDC) T cell clone upon transfer into NOD.scid mice induced destruction of islets of Langerhans leading to diabetes. Administration of a neutralizing Ab to IFN-{gamma} (H22) resulted in long-term protection (LTP) from diabetes, with inflammation but persistence of a significant, albeit decreased, number of β cells. BDC T cells were a mixture of cells expressing high, intermediate, and low levels of the TCR. Clonotypelow BDC T cells were required for LTP. Furthermore, islet-infiltrating leukocytes in the LTP mice contained Foxp3+CD4 T cells. Islet inflammation in both diabetic and LTP mice was characterized by heavy infiltration of macrophages. Gene expression profiles indicated that macrophages in diabetic mice were M1 type, while LTP mice contained M2 differentiated. The LTP was abolished if mice were treated with either Ab-depleting CD4 T cells or a neutralizing Ab to CTLA-4, in this case, only at a late stage. Neutralization of IL-10, TGF-β, glucocorticoid-induced TNF receptor (GITR), or CD25 had no effect. Transfer of only clonotypehigh- expressing BDC T cells induced diabetes; in contrast, H22 Abs did not inhibit diabetes. While clonotypehigh T cells induced diabetes even when IFN-{gamma} was neutralized, paradoxically there was reduced inflammation and no diabetes if host myeloid cells lacked IFN-{gamma} receptor. Hence, using monoclonal CD4 T cells, IFN-{gamma} can have a wide diversity of roles, depending on the setting of the immune process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Aknowlegments
 Disclosures
 References
 
The NOD mouse model is useful for testing strategies that modulate diabetogenic T cells: a number of manipulations in the NOD mouse can control the diabetic process (1, 2, 3). Although several proinflammatory cytokines may play a role in diabetogenesis, their precise mechanism and cellular targets are complex and remain to be defined. Indeed, the NOD mouse diabetes involves polyclonal sets of both CD8 and CD4 T cells, some of which develop into effector cells while others are regulatory in nature, making it difficult to dissect the effects of cytokines on any one defined pathogenic T cell.

The highly pleiotropic cytokine IFN-{gamma} plays a major role in immune-mediated inflammation (4, 5). Initially described as the major cytokine responsible for macrophage activation (6), IFN-{gamma}, the prototype Th1 cytokine, also mediates a number of effects on lymphocytes (4). To examine the role of IFN-{gamma} in immune inflammation, an acute transfer model of diabetes mediated by the diabetogenic BDC2.5 (BDC)3 T cell was examined (7, 8, 9). This accelerated model allowed us to focus exclusively on how activated CD4 T cells induced islet inflammation and pathology without the participation of B cells or CD8 T cells. In general, IFN-{gamma} appears to have a limited involvement in the spontaneous diabetes of NOD mice (10, 11, 12, 13, 14) but participates in the acute models induced by cyclophosphamide injection or diabetogenic cell transfers (14, 15, 16, 17). In this acute diabetes transfer model, the cell responsible for the killing of β cells is an activated macrophage (9): their selective depletion precludes β cell death despite the persistence of inflammation containing mostly neutrophils and NK cells (9). We report that diabetogenic CD4 T cells triggered inflammation that could be modulated by IFN-{gamma} with profound consequences on the development of diabetes.


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

The BDC2.5 (BDC) TCR transgenic mice on the NOD background and BDC2.5/NOD.scid (BDC.scid) mice were established in our mouse colony at Washington University School of Medicine. NOD mice on the scid genetic background (NOD.CB17-Prkdcscid/J) or on Rag-1–/– were obtained from The Jackson Laboratory and maintained in our mouse colony, referred to as NOD.scid and NOD.Rag-1–/– mice. To obtain NOD.Rag-1–/–.IFN-{gamma}R–/– mice, the NOD.Rag-1–/– and NOD.IFN-{gamma}R–/– (NOD.129S1(B6)-Ifngr2tm1Pbro/DvsJ) mice, obtained from The Jackson Laboratory, were intercrossed. IFN-{gamma}R–/– mice were identified by PCR using the primers described by Serreze et al. (11). All experimental mice were 6–8 wk old except BDC.scid, which were 3–4 wk of age by the time they developed diabetes. (The NOD.IFN-{gamma}R–/– came from The Jackson Laboratory, which provided the information on its development; see Serreze et al. (11).)

Adoptive transfer

Splenocytes from BDC mice were activated in culture with Con A (Sigma-Aldrich) as previously described (9). Results with either were identical and were not separated from each other. Usually mice were injected i.v. with a dose of 4 x 106 activated BDC T cells, known to transfer diabetes to all recipients by day 8 (9). For the rechallenge experiments, mice were injected i.v. with an equal dose of activated BDC T cells (4 x 106) or a higher dose (1.5 x 107), which transfers diabetes in 6 days when injected into NOD.scid recipients. For BDC.scid transfers, splenic T cells were harvested and 106 cells were transferred i.v. CD4 T cells were sorted from splenocytes by their clonotype expression using an anti-BDC mAb. The purity of BDC high clonotype (BDChigh) and BDC low clonotype (BDClow) cells was 98% and 78%, respectively (supplemental Fig. 1).4 Sorted cells were transferred i.v. (106) into recipients. The recipient mice were followed for diabetes incidence. Two consecutive readings of blood glucose of ≥250 mg/dl, measured by a glucometer (Bayer), were indicative of diabetes.

Ab treatments

For most experiments involving IFN-{gamma} neutralization in vivo, NOD.scid and rag-1–/– mice received 300 µg i.p. of H22 mAb (kindly provided by Dr. R. Schreiber, Washington University School of Medicine, St. Louis, MO) 1 day before and 2 days after cell transfer. Anti-CD25 (P61), anti-TGF-β (1D11), anti-glucocorticoid-induced TNF receptor (anti-GITR; DTA-1), anti-IL-10 (JES5-2A5), anti-CD4 (YTS191.1), and anti-CTLA-4 (4F10) mAbs were injected twice with a dose of 500 µg i.p. 3 days apart. A rat IgG isotype Ab (Sigma-Aldrich) was used as control.

Measurement of pancreatic insulin

Frozen pancreata from acute diabetes and LTP mice at different times after BDC T cell transfer were weighed before homogenization in prechilled acid alcohol (80% alcohol in 12 N sulfuric acid). One milliliter aliquots of the homogenate for each sample were stored at 4°C overnight and then microfuged at 10,000 rpm for 5 min at room temperature. Dilutions of 1/1000 to 1/5000 were prepared with Linco RIA buffer assay and measured with a Linco rat insulin kit (Linco Research). Samples were obtained from four to six mice per time point.

Isolation of cells from infiltrated mouse islets

Infiltrated mouse islets were isolated and dispersed by trypsinization as previously described (9). In brief, seven mice were sacrificed per islet preparation by cervical dislocation. Hanks’ solution (Invitrogen) was injected into the common bile duct and then the pancreas was resected, cut into small evenly sized pieces, and digested in collagenase at 39°C. The tissue suspension was then centrifuged through a Ficoll (Sigma-Aldrich) gradient of 23, 20.5, and 11%. All Ficoll interfaces were collected (except for the bottom pellet) and the cells dispersed by trypsin digestion in a 37°C water bath for 3 min and then washed in CMRL medium 1066 (Invitrogen) several times. Infiltrating islet leukocytes were obtained from LTP and diabetic NOD.scid mice that received BDC T cells.

Flow cytometry

Splenocytes and infiltrating islet leukocytes were stained for different cellular markers. Leukocytes were stained with a FITC-labeled anti-CD45 (Ly-5) Ab (BD Biosciences); T cells were stained with either a FITC-labeled anti-CD4 (L3T4) or FITC-labeled anti-CD3{epsilon} chain Ab (145-2C11); neutrophils were stained with a FITC-labeled anti-Gr-1 (RB6-8C5) Ab. Macrophages were analyzed with a FITC-labeled anti-F4/80 Ab (BM8), while dendritic cells were analyzed with a FITC-labeled anti-CD11c Ab (HL3). NK cell analysis was performed with biotinylated anti-CD49b/Pan-NK (DX5) and secondary stain with streptavidin-allophycocyanin. NK cells were defined as CD3{epsilon}-negative and CD49b/Pan-NK-positive. All Abs were obtained from BD Biosciences. For Foxp3 staining, surface-stained CD4 T cells were incubated in permeabilization buffer for 16–18 h at 4°C before performing intracellular staining (FJK-16s) (eBioscience). All FACS analysis was performed on a FACSCalibur (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

Histology

Mice were anesthetized with ketamine and sacrificed by cervical dislocation. The pancreata were fixed in 10% formalin. Slides were stained with H&E. For detection of insulin, 5-µm serial sections were deparaffinized and stained with a HistoMouse-SP kit (AEC, Broad Spectrum, Bulk) (Invitrogen) using guinea pig anti-insulin polyclonal Ab (1/100) (Linco Research). Insulitis scoring was performed according to the following criteria: extensive insulitis, >50% of the islet area is infiltrated; insulitis, <50% of the islet area is infiltrated; normal or peri-insulitis, islets are either intact or infiltration is restricted to the periphery.

Laser capture microdissection

Whole pancreata were dissected from mice immediately (within 1–2 min) following sacrifice with minimal direct handling of the pancreas itself. The pancreas was immediately immersed in OCT (Sakura Finetek) and frozen in Cytocool II (Richard-Allen Scientific). For laser capture, serial 7-µm-thick cryosections were cut onto Superfrost slides (Fisher Scientific) and immediately fixed in methanol (~1 min) precooled to –20°C and kept within the cryostat. After fixation, OCT was removed manually from sections in the cryostat, and sections were washed twice in 95% ethanol, stained with eosin (in 100% ethanol) at room temperature for 5 s, and then washed in several changes of 95% then 100% ethanol followed by multiple changes of tissue-grade xylenes. Slides were then incubated in desiccation chambers and microdissected within 3–4 h. Extensive initial testing of RNA quality following different treatments indicated that abundant endogenous RNases present in exocrine pancreas led to degradation of RNA: pancreata needed to be frozen within a few minutes of excision, laser capture performed within a few hours of sectioning, and sections fixed immediately after transfer to slides. Problems developed if pancreata blocks were stored for more than a few months, if sections were fixed in ethanol, acetone, or formaldehyde, if methanol was not thoroughly precooled and kept continuously at ≤–20°C, or if sections were exposed to aqueous solution at any time (hence, staining only in alcohol-dissolved eosin).

Laser capture microdisection was performed on an Arcturus PixCell IIe LCM (laser capture microdissection) microscope in a specially designated climate-controlled room. Islets were recognized histologically and were laser-captured whole. RNA from dissected islets was purified using the PicoPure kit (Arcturus), and quality was verified on an Agilent 2100 BioAnalyzer for assessment of picogram-level RNA. Dissection specificity was routinely verified by quantitative RT-PCR for acinar- (amylase), ductal- (carbonic anhydrase II), and islet-specific (insulin) markers.

Microarray analysis

For GeneChips, RNA from the laser-captured islets was pooled from untreated NOD.scid mice, diabetic, and LTP mice at days 3–8 (n = 5 mice/group). Pooled RNA (30 ng/sample) was amplified, labeled, and fragmented (by one round of Arcturus RiboAmp HS kit amplification followed by the RNA amplification and labeling kit from Enzo Life Sciences). The resulting biotinylated cRNA probes were hybridized to Affymetrix MOE430v2 GeneChips; expression patterns were analyzed using dChip (18, 19) and an in-house-developed analysis suite that annotates lists of genes by associated gene ontology (GO) terms and then clusters and analyzes the lists using the fractional representations of each GO term (20). As is standard for dChip analysis software, all GeneChips used to determine effects of time following transfer of T cells or experimental treatment were normalized to the GeneChip with total average intensity closest to the mean intensity of all chips in the experiment. Filtering of false-positives generated by multiple comparison error (21) was also performed as a standard part of the dChip analysis software in computing model-based expression levels. Time-course visualization and clustering of patterns of M1-related and M2-related gene expressions identified using dChip were further confirmed using several clustering algorithms in the SpotFire DecisionSite software package (TIBCO Software) and plotted using GraphPad Prism. Validation of selected gene expression changes was performed using quantitative RT-PCR as previously described (22), except that 18s primers were used as standards, and analysis was performed on a Stratagene MX3000P machine. Gene chip data Gene Expression Omnibus (GEO) accession number is GSE12389.

Bone marrow engraftment

Donor cells were isolated from IFN-{gamma}R–/– and IFN-{gamma}R+/+ NOD.Rag-1–/– mice treated with 5-fluorouracil (Sigma-Aldrich) for 3–5 days before mice were killed. Recipient mice (NOD.Rag.IFN-{gamma}R–/– and NOD.Rag.IFN-{gamma}R+/+) received a single lethal dose of 9.50 Gy using a cesium source and were engrafted with 5 x 106 bone marrow cells i.v. Transplanted mice were given water supplemented with trimethoprim-sulfamethoxazole (Hi-Tech Pharmacal) ad libitum for 2 wk and were allowed to engraft for a minimum of 70 days before transfer experiments. Bone marrow-engrafted mice were analyzed by flow cytometry to corroborate the effectiveness of engraftment by detecting intracellular phosphorylation of STAT-1 upon IFN-{gamma} administration (1000 U/ml for 15 min at room temperature) with an anti-STAT-1 mAb (BD Biosciences) (supplemental Fig. 2).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Aknowlegments
 Disclosures
 References
 
Abs to IFN-{gamma} induced LTP from diabetes

Transfer of BDC T cells into NOD.rag-1–/– or scid mice rapidly resulted in diabetes, with the time of development depending on the amounts of T cells transferred (results with either were identical and thus are not separated). In all of our transfer experiments BDC T cells were first activated for 3 days: pooling all experiments, all 135 injected mice became diabetic (Fig. 1A and supplemental Fig. 3A). When testing a total of 272 mice through several experiments, the mAb H22 to IFN-{gamma} (23) inhibited the development of diabetes by 93%. In most experiments H22, at 300 µg, was administered a day before the diabetogenic T cell transfer and a second dose 2 days after (Fig. 1A and supplemental Fig. 3A). The inhibition of diabetes persisted: most of the mice were tested for up to 140 days after the injection of BDC T cells, and these were still normoglycemic.


Figure 1
View larger version (82K):
[in this window]
[in a new window]

  		FIGURE 1. Treatment with Abs to IFN-{gamma} induces LTP from diabetes. A, Incidence of diabetes in NOD.Rag-1–/– or NOD.scid mice that received activated BDC T cells (4 x 106) alone or along with the anti-IFN-{gamma} (H22) mAb. Mice injected with BDC T cells had 100% diabetes incidence by day 7, while mice injected with BDC T cells plus the H22 Ab showed only 7% diabetes incidence over a period of 140 days. Results obtained were pooled from a total of 18 experiments. B, Distribution of islet-infiltrating leukocytes at day 7 from NOD.scid mice after BDC T cell transfer (Diabetic) and from H22-treated recipients (LTP). Leukocytes were recovered from pancreatic islets of six mice in each group and analyzed by flow cytometry for the various leukocyte markers. The total numbers of islet-infiltrating leukocytes recovered per mouse from diabetic vs H22 treated mice were 5.9 x 105 and 3.8 x 105, respectively. C, Histological analysis. Sections were stained with H&E and insulin from NOD.scid mice that were untreated (No Transfer) or that received activated BDC T cells alone (Acute Diabetes) or in conjunction with the H22 mAb (Protected). Magnification, x20. D, Insulitis scores from H&E pancreata sections of LTP mice. Infiltration started at the same time as the acute diabetic mice (day 3) and by day 6 there was 100% infiltration without diabetes. Data obtained from 1201 scored islets of 61 experimental mice. E, Upper and middle bars: rechallenge of LTP mice with 4 x 106 or 1.5 x 107 activated BDC T cells, respectively, at day 90 of LTP and followed for 60 days after rechallenge. Lower bar: transfer of bulk splenocytes from LTP mice (between 60 and 143 days of protection) into unmanipulated NOD.scid recipients (diabetes incidence of 100% by day 19).

 
The LTP was not dependent on the presence of circulating H22 Ab. ELISA measurements of H22 mAb in the sera of 31 mice showed a range between 0.3 and 2.8 µg/ml at days 72–89 postantibody injection. We then determined whether the levels of H22 found in LTP mice were enough to afford protection from diabetes. Administration of a dose <25 µg did not give protection (data not shown). In conclusion, the serum concentration of Ab in the protected mice at later time points was too far below the neutralizing levels, indicating that LTP depended only on neutralizing IFN-{gamma} during an early, critical window of time.

Both sets of mice that received the BDC T cells, that is, with or without injections of H22 mAb, showed a similar severe inflammatory response made up mostly of mononuclear phagocytes. Fig. 1B shows the distribution of cells in the islet exudates from each group: both mostly comprised F4/80+ cells, with a smaller fraction of neutrophils, dendritic cells, and NK cells. Histopathologically, the extensive inflammatory reaction in the diabetic mice was accompanied by complete disruption of islets with loss of insulin-positive cells (Fig. 1C). A recent study of ours, using depleting Abs, indicated that of the three main cells of the exudate (neutrophils, NK cells, and macrophages), macrophages were the only cells responsible for killing the β cells (9).

During the first week, the inflammatory reaction in the BDC T cells plus H22 mAb-injected mice disrupted the islets, which became lobulated with inflammatory cells separating the lobules. Insulin staining was not detected in the diabetic mice while LTP mice showed decreased insulin staining (Fig. 1C). The total islet mass as determined by the insulin content of the pancreas was absent in acute diabetic mice and substantially reduced in the LTP cohort (Fig. 2). In the LTP mice, the inflammatory reaction persisted but became less intense throughout the observation time (Fig. 1D).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 2. Insulin content from total pancreas measured by radioimmunoassay from acute diabetes and LTP mice at different times after BDC T cell transfer. Each point represents the average insulin content of four to six mice per time point.

 
H22-treated mice were more resistant to the induction of diabetes when a second dose of BDC T cells was administered, even if the second dose occurred well after H22 concentration had dropped below neutralizing levels. In these experiments, when 4 x 106 T cells were injected into untreated NOD.scid mice, all mice became diabetic by the sixth day. However only 2 of 10 mice previously injected with H22 mAb became diabetic, even though, in this case, the mice had been injected 90 days before with H22 and BDC T cells (Fig. 1E, upper bar and supplemental Fig. 3B). In another set of experiments, increasing the dose of T cells to 1.5 x 107 did induce diabetes after 25 days in six of six mice, while all six control mice were diabetic by the fourth day (Fig. 1E, middle bar and supplemental Fig. 3B). Hence, the H22-treated recipients were not conducive to induction of disease when limiting numbers of diabetogenic CD4 T cells were reintroduced.

The LTP mice, however, still harbored diabetogenic T cells in their spleens. Sixty to 143 days after injection of BDC T cells plus H22 mAb, the protected mice were sacrificed and their spleen cells isolated and transferred into unmanipulated NOD.scid recipients. All 21 mice from six different experiments became diabetic by 8–19 days (Fig. 1E, lower bar and supplemental Fig. 3C).

In brief, treatment with anti-IFN-{gamma} mAb protected mice from diabetes by reducing the mass of islet β cells that died following the injection of BDC T cells. Protection was prolonged and accompanied by a persistent inflammatory reaction predominantly made up of macrophages. Importantly, the LTP mice contained diabetogenic T cells, indicating an active immunomodulatory environment that kept the pathogenic T cells in check.

Gene expression analysis of islets from protected vs diabetic recipient mice showed different macrophage profiles

Initially, gene expression was assayed in the pancreas from control untreated mice as well as LTP and diabetic mice at day 3 following BDC T cell injection (n = 5 mice/condition, with RNA pooled for each GeneChip). We identified 1279 genes whose expression was increased in diabetic mice relative to untreated NOD.scid (lower bound of 90% confidence interval set at 1.3, intensity difference of ≥50), 1162 that increased in LTP vs NOD.scid mice, and 1098 that decreased from NOD.scid to either diabetic or LTP mice. GO analysis (20, 24) showed that the most significant patterns of changes in both diabetic and LTP islets relative to untreated mice at day 3 were increases in genes classified by the GO term "Immune response" (0.45% of genes preferentially expressed in control mice vs 4.8% of LTP and 5.9% of diabetic genes). Many of the genes that increased in both diabetic and LTP islets were associated with macrophages. (When comparing to 247 genes specifically increased in activated macrophages (25, 26), we found that 60 representing 4.7% of the diabetic transcripts and 48 representing 4.1% of the LTP transcripts were characteristic of activated macrophages vs only 0.5% of transcripts enriched in untreated NOD.scid islets.) Altogether, 31% of the selected group of genes representing macrophage activation was enriched in either the unprotected or protected islet population.

A distinct pattern was found in the profile of macrophage genes up-regulated in islets of diabetic mice compared with LTP mice. When the LTP and diabetic expression profiles were compared directly to each other, using a relatively high threshold for difference (fold change of ≥3.5, intensity difference of ≥75), those genes enriched in diabetic islets were characteristic of the activated M1 type associated with inflammatory responses, whereas genes enriched in LTP mice were characteristic of the M2 phenotype, activated by the alternative pathway (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). M1 macrophages are activated via IL-12- and IFN-{gamma}-induced pathways that depend on downstream signaling via Stat1 and Stat4. Stat1 gene expression was enriched in diabetic mice relative to islets from LTP mice (Fig. 3). Multiple target genes of Stat1-induced pathways were also expressed preferentially in islets from the diabetic mice. Examples include Ly6a, Igtp, Ifi47, Irf1, Tgtp, as well as the chemokines CXCL9 and CXCL10 (Fig. 3). In islets from LTP mice the following genes were expressed: chitinase 3-like 3 (Chi3l3, aka YM-1), CCL24, CCL9 (aka Scya9), Ear1, Timp1, IL4i1, and CCL6 (Fig. 3). Interestingly, expression of arginase I, a key M2-associated gene, was statistically high in LTP at day 3 but much higher at the fourth day (Fig. 4A). Finally, quantitative RT-PCR on laser-captured islets at day 3 confirmed for selected cytokines that the M1 cytokines CXCL9 and CXCL10 were increased in diabetic vs LTP mice and the M2 cytokine CCL6 was increased in LTP vs diabetic mice (Fig. 4B).


Figure 3
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 3. Laser-capture microdissected LTP islets preferentially express M2 macrophage genes, whereas diabetic islets express M1 genes. GeneChip expression profiles were generated by directly comparing day 3 LTP to day 3 diabetic GeneChips. Those genes with profoundly differing patterns of expression (3.5-fold difference between LTP and diabetic, expressed at >75 intensity level) were identified. All genes, increased in either direction, were then classified by literature search as M1 associated or M2 associated. A, All differentially expressed M1 (blue) and M2 (green) genes from this analysis are depicted in the heat map. Note how almost all the M2 genes have higher expression in LTP and vice versa; none of the genes is expressed at the highest level in control islets. B, The same genes are plotted according to intensity levels. Genes with high expression (upper) and low expression (lower) are separated for clarity. Follow-up experiments with additional cohorts of mice and using quantitative RT-PCR confirms the pattern of association of M1 genes with diabetic islets and M2 with LTP.

 

Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 4. Follow-up experiments with additional cohorts of mice and using quantitative RT-PCR confirms the pattern of association of M1 genes with diabetic islets and M2 with LTP. A, Plots of representative selected M1 (upper) and M2 (lower) genes at later time points. B, Plots of quantitative RT-PCR of laser-captured islets for selected M1 and M2 genes showing the same pattern of expression as on GeneChips. Results are means ± SD from duplicate, independent experiments. Note that the scale is log2.

 
Evidence for regulatory T cell-dependent protection upon neutralization of IFN-{gamma}

Flow cytometry analysis from five independent experiments from LTP mice showed expression of the BDC TCR at intermediate or low levels in 29–49% of BDClow T cells, while the remaining cells expressed high levels of the TCR BDChigh T cells (Fig. 5A). A previous report by Kanagawa et al. indicated that the BDClow T cells were regulatory in nature in that they either delayed or inhibited the onset of diabetes (36). Additonally, the BDC2.5 TCR transgenic mice genetically crossed into the NOD.scid mice contained a homogeneous set of T cells only expressing high levels of the clonotype TCR (Fig. 5A): all of these mice became spontaneously diabetic within 28 days after birth.


Figure 5
View larger version (63K):
[in this window]
[in a new window]

  		FIGURE 5. Anti-IFN-{gamma} treatment did not inhibit diabetes induced by the transfer of BDChigh T cells. A, Flow cytometry analysis of spleen CD4+ T cells from BDC preactivation, postactivation, BDC.scid, and LTP mice (day 70 of protection from diabetes) and their clonotype BDChigh and BDClow expression. B, Flow cytometry analysis (on gated CD4+) looking at BDC and Foxp3+ expression from splenocytes of BDC preactivation, postactivation, BDC.scid,and islet exudate (day 30) of LTP mice. Upper panels were stained with BDC plus the isotype IgG control Ab. Lower panels were stained with BDC plus the anti-Foxp3 mAb. C, Incidence of diabetes in mice that received BDC.scid T cells (1 x 106) alone or together with the H22 mAb treatment: both sets of mice developed diabetes 10–15 days posttransfer. D, Transfer of sorted CD4+ T cells for their high and low clonotype expression of BDC into NOD.scid recipients alone or with the H22 mAb treatment and followed for 30 days for diabetes incidence. The lower bar represents the combined transfer of BDChigh and BDClow that leads to LTP from Fig. 1A.

 
To ascertain whether H22-mediated protection required CD4 T cells that expressed endogenous TCRs in addition to the BDC2.5 TCR, transfer experiments were performed using only the BDChigh CD4 T cells. BDChigh T cells isolated from either BDC or BDC.scid mice transferred diabetes, but this transfer of diabetes was not affected by injection of H22 (Fig. 5C and supplemental Fig. 3D). Experiments were done administering once a very high dose of 1.5 mg of H22, or giving three injections in seven days with 300 µg each. The diabetic process was not inhibited; hyperglycemia was delayed in a few mice by 4–20 days compared with the untreated mice, but once diabetes developed it had the same features of severity and degree of inflammation (Fig. 5, C and D, and supplemental Fig. 3, D and E). BDClow CD4 T cells did not transfer diabetes, and a mixture of BDChigh and BDClow T cells was needed for the protective effect of anti-IFN-{gamma} (Fig. 5D). Flow cytometry analysis of BDChigh T cells showed their high expression of IFN-{gamma} but very limited expression of IL-17 (not shown).

Thus, neutralization of IFN-{gamma} did not directly control islet β cell destruction mediated by BDChigh T cells. The results pointed to a BDClow T cell regulating the response of BDChigh T cells: at face value in the absence of IFN-{gamma}, the negative effects of these cells predominated over those of the BDChigh T cells. The BDClow T cells directly or indirectly regulated the pathogenic potential of BDChigh T cells or could control the final effector-activated macrophage (e.g., by regulating its differentiation along M1 or M2 lineages) that kills islet β cells.

BDC T cells were examined for the expression of Foxp3. As shown in Fig. 5B, CD4 T cells from BDC.NOD mice but not BDC.scid mice contained Foxp3+ cells. The CD4+Foxp3+ population in the BDC.NOD mice pre- and postactivation predominated in the high and intermediate clonotype population. Moreover, flow cytometry analysis of islet-infiltrating T cells from H22-treated mice at day 30 of protection (i.e., from Fig. 1A that are protected from diabetes) showed the presence of CD4 T cells that expressed Foxp3 with about the same distribution of the BDC clonotype (Fig. 5B).

The mode of regulation was next examined by using a variety of neutralizing mAbs. Those that neutralized IL-10 or TGF-β had no effect; that is, LTP mice injected with H22 were still normoglycemic when given the neutralizing Abs (Fig. 6A). Also, injection of PC61, an Ab to CD25 that inhibits regulatory T cells and an Ab to the GITR had no effect (Fig. 6A). The efficacy of all of these Abs was ascertained in different experimental systems tested in the laboratory (37).


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 6. Mode of regulation in LTP mice by neutralizing Abs. A, LTP mice after 75 days were treated with an irrelevant IgG or with depleting Abs to CD25, TGF-β, GITR, or IL-10 and followed for 30 days for diabetes incidence. B, LTP mice at day 75 were treated with either an irrelevant IgG or anti-CD4-depleting mAb and followed for diabetes incidence. C, Insulitis score analysis from H&E pancreata sections of anti-CD4-treated LTP mice. The extensive infiltration increased after anti-CD4 mAb treatment correlated with diabetes onset. Data obtained from 212 scored islets of 14 experimental mice. D, LTP mice at different times were treated with either an irrelevant IgG or anti-CTLA-4-blocking Ab (4F10) and followed for 30 days. Treatment before day 35 did not show an increase in diabetes, while treatment performed after 45 days of protection increased diabetes incidence. E, Histogram analysis by flow cytometry for total expression of CTLA-4 in BDChigh and BDClow T cells isolated from the islet exudate or the spleen of LTP mice at day 60 of protection. The blue line represents CTLA-4 and the red line the isotype control stain. F, Flow cytometry analysis for total expression of CTLA-4 and Foxp3 (gated on BDChigh T cells) from islets of LTP mice after 60 days of protection. Left panel, Isotype control staining; right panel, CTLA-4 and Foxp3 staining.

 
Two Abs reversed the LTP. Injection of the YTS191.1 mAb, an Ab that depleted CD4 T cells, rapidly induced diabetes in LTP mice. LTP mice at day 75 posttreatment with H22 mAb were injected with the YTS191.1 mAb (two injections of 500 µg each, 3 days apart); 31 of 33 (94%) mice developed diabetes 10 days after the treatment (Fig. 6B and supplemental Fig. 3F). Histopathological analysis showed islets with an extensive leukocyte infiltration by day 10, time of diabetes onset (Fig. 6C). FACS analysis of YTS191.1-treated mice demonstrated depletion of both BDChigh and BDClow T cells (data not shown).

A neutralizing Ab to CTLA-4 also reversed the protected state induced by H22 mAb administration. Protected mice (those given BDC T cells plus H22 mAb) after 45 days, or later, reverted to diabetes when injected with anti-CTLA-4 mAb: 17 of 20 mice became diabetic, whereas 2 out of 17 injected with control Abs became diabetic, a difference of 85% vs 12% (Fig. 6D and supplemental Fig. 3G). However, the capacity of anti-CTLA-4 mAb to reverse LTP protection was not found in mice at days 9, 25, or 35 after administration of BDC T cells plus H22. From a total of 25 mice given anti-CTLA-4, only 4 became diabetic, in comparison with 2 of 24 given control Abs (Fig. 6D).

Thus, the early stages of H22 protection were independent of engagement of CTLA-4 while the later stage was influenced by CTLA-4-dependent inhibition. Note that flow cytometry analysis of CTLA-4 total expression in the exudates of LTP mice indicated noticeable levels in the BDChigh but not in the BDClow T cells (Fig. 6E). CTLA-4 expression was not found in the splenocytes of LTP mice.

We analyzed the correlation of CTLA-4 and Foxp3 expression in BDChigh T cells from the islet exudates of LTP mice. As shown in Fig. 6F, 30% of the BDChigh-gated T cells expressed CTLA-4 and, from this, nearly 50% were positive for the expression of Foxp3. Only 5.5% of the BDChigh T cells were positive for Foxp3 and negative for CTLA-4.

Transfer of diabetes requires host myeloid cells to express the IFN-{gamma} receptor

As noted above, the BDChigh CD4 T cells induced diabetes resistant to H22 mAb treatment, implying that IFN-{gamma} was not required to mediate the effector reactions. Surprisingly and paradoxically, BDC T cells sorted for clonotypehigh or from BDC.scid mice, which only express high clonotype, or BDC T cells from NOD mice bearing BDChigh and BDClow cells did not transfer diabetes into mice lacking the IFN-{gamma}R (Fig. 7A and Table I). By making bone marrow chimeras, mice were examined in which the bone marrow-derived cells or the β cells expressed or did not express IFN-{gamma}R. In agreement with the published work from Katz’s laboratory (38), BDC T cells induced diabetes in mice in which the β cells lacked the receptor. However, diabetes was not induced in mice where the myeloid compartment lacked the IFN-{gamma}R (Fig. 7B, Table I, and supplemental Fig. 3H). Mice were followed up to 60 days after transfers. All mice had an inflammatory reaction: while ~30% of islets were clean and not infiltrated, 40% showed a mixed infiltrate surrounding the islet which appeared normal, and the remaining 30% had inflammation penetrating the islets (Fig. 7C). In the case of the bone marrow chimeras their peripheral blood cells were confirmed to lack the IFN-{gamma}R by testing Stat1 movement to the nucleus after incubation with IFN-{gamma} (supplemental Fig. 2). Thus, the lack of IFN-{gamma}R in the myeloid lineage translates in a reduction in the inflammatory reaction that kills β cells, implying that some IFN-{gamma} interactions are amenable to H22 neutralization while others are not.


Figure 7
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 7. Transfer of diabetes requires host myeloid cells to express the IFN-{gamma}R. A, Transfer of 4 x 106 activated BDC T cells or BDC.scid T cells (106) into NOD.Rag.IFN-{gamma}R+/+ or NOD.Rag.IFN-{gamma}R–/– recipients and followed for diabetes incidence. While activated BDC T cells transferred diabetes into NOD.Rag.IFN-{gamma}R+/+, the transfer of either activated BDC.NOD T cells or BDC.scid cells into NOD.Rag.IFN-{gamma}R–/– did not increase diabetes incidence. Experimental mice were followed for 60 days after T cell transfer. B, Bone marrow chimeric mice in which the bone marrow-derived cells or the β cells expressed or did not express IFN-{gamma}R were transferred with activated BDC T cells (4 x 106) and followed for diabetes incidence for 30 days. Only recipient mice that contained bone marrow-derived cells that lacked IFN-{gamma}R and β cells expressing IFN-{gamma}R were free of diabetes. C, Islets from previous mice that were free of diabetes (day 30, last day of observation) were examined for insulitis by H&E sections. Data obtained from 24 experimental mice.

 

View this table:
[in this window]
[in a new window]

  		Table I. Summary of IFN-{gamma}R transfer experimentsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Aknowlegments
 Disclosures
 References
 
The acute transfer model pointed to several notable effects of IFN-{gamma} in autoimmune diabetes mediated by a clonal set of diabetogenic CD4 T cells. In general, our results, together with those of others, outline the various distinct and complex cellular interactions taking place in autoimmune diseases where there is chronic antigenic stimulation and involvement of practically all cells of the immune system. The experiments identified two situations in which IFN-{gamma} participated in the induction of the inflammatory response and the consequent diabetes mediated by BDC T cells (Fig. 8).


Figure 8
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 8. IFN-{gamma} effects in the induction of the inflammatory response and the consequent diabetes mediated by BDC T cells. As a result of the interaction between an APCs containing a diabetogenic Ag and diabetogenic CD4 T cells, two sets of activated CD4 T cells are generated, differing in the extent of expression of their TCR (noted by the numbers of "v" in red in the figure). In the islet tissue these cells generated an inflammatory exudate rich in M1-activated macrophages. IFN-{gamma} exerts an effect in this step, marked as no. 1, allowing the TCRhigh T cell to predominate. In the absence or blocking of IFN-{gamma} in the step 1, the TCRlow predominates and the resulting exudate has features of the M2-activated macrophages, and β cell function is preserved. The macrophage precursor differentiates in a process influenced by IFN-{gamma} (see no. 2) to generate macrophages that respond to inflammatory cues.

 
First, when inflammation was transferred by BDC T cells bearing high and low levels of the TCR, IFN-{gamma} was required to induce an M1 exudate that led to β cell death; in its absence, the BDC T cells entered into a regulatory pathway in which the exudate was typical of the M2 macrophages. The M1 exudate is typical of IFN-{gamma} mediated processes. Among the various genes modulated by IFN-{gamma}, that encoding for CXCL10 was previously implicated in the progression of diabetes: neutralizing Ab to CXCL10 in a cyclophosphamide-exacerbated NOD model suppressed diabetes (39). The M2 macrophages are associated with the alternative pathway activated by the cytokines such as IL-4/13, which can promote allergic inflammation and wound healing in target tissues, rather than cytotoxicity (40, 41). YM-1 and arginase epitomize the genes expressed in the alternative pathway. Arginase competes with inducible NO synthase for nitrogen, shunting it away from production of cytotoxic NO (a presumed mediator of β cell death in diabetes) (42) and toward production of extracellular matrix and/or proliferation-stimulating polyamines. Another transcript enriched in the LTP islets was CCL6 shown to be induced in macrophages by IL-4, inhibited by IFN-{gamma}, and a key chemotactic factor for fibrogenic (i.e., noncytotoxic) macrophages (40, 41).

Activated macrophages in the M1 exudate were in close contact with β cells and were responsible for their death. In culture the same cells killed β cells (9). The mechanism of action of the activated macrophages has not been determined. Activated macrophages produce peroxynitrate, and β cells have been shown to be highly sensitive to oxidative damage (42). In contrast, the alternative activated macrophages in the M2 exudates show considerably less cytocidal molecules. Note that the response to an M1 or M2 exudate is not all or none; indeed, LTP mice did have a reduced mass of β cells, but it is sufficient to maintain euglycemia. In unpublished experiments no proliferation of β cells was discerned in the LTP mice.

The key role of the clonotypelow BDC T cell was apparent: it did not transfer diabetes, yet it influenced profoundly the behavior of the clonotypehigh BDC T cells. Thus, in a situation in which T cells bearing different amounts of the TCR were together, IFN-{gamma} was a key cytokine that allowed the clonotypehigh T cells to dominate and to induce the M1 exudate and diabetes (Fig. 8, no. 1). Pari passu, in the absence of IFN-{gamma}, the modulatory action of the clonotypelow T cells predominated, influenced the type of exudate (M2), and diabetes was controlled. How the BDClow cells deviated the exudate to an M2 type is not apparent. We could not find evidence for the involvement of IL-10 or TGF-β. The finding that Foxp3 T cells distributed among the T cells bearing different amounts of the clonotype (Fig. 5B) does not allow us to determine which set, if any, was involved in the protection. Note that neither anti-CD25 nor anti-GITR mAbs affected the LTP. However, CTLA-4 was definitely involved but at a later stage of the chronic LTP.

CTLA-4 has been implicated in some of the effects mediated by regulatory T cells (43) and in NOD diabetes (44, 45); in our case, CTLA-4 expression was found within the Foxp3 population of the BDChigh T cells. CTLA-4 could be participating in two effects: 1) direct inhibition of the effector T cell (46, 47, 48) and 2) direct influence on the macrophage, as it has been suggested, for example, by increasing the production of IDO (44, 49, 50, 51, 52, 53, 54). Ongoing experiments are focused on solving these questions.

An important feature of the LTP pathway is that the effector diabetogenic T cells were not eliminated but were present not in islets but in spleen. In contrast to the BDC T cells in islets, which expressed CTLA-4, those in the spleen were negative. A comparable situation was previously reported in NOD mice given Freund’s adjuvant and protected from diabetes (55). All of this implies that much of the regulation is local.

Another finding of note is that of the rapid reversal of the LTP when BDC T cells were depleted. Our expectation was that the depletion would simply abrogate the inflammatory process; that is, elimination of the BDC-M2 axis would require new interaction leading to a BDC-M1 axis. However, reversal was fast, implying that the LTP is a continuous process of control, and that elimination of the regulatory pathway immediately converts the process back to the original M1 exudate.

Finally, a highly paradoxical result emerged from the analysis of the requirements for IFN-{gamma} signals in the host leukocytes. By performing bone marrow chimeras, we noted that the host APC compartment but not the islet β cells needed IFN-{gamma} signals to allow BDC T cells to induce the full inflammatory response that leads to diabetes (Fig. 8, no. 2). Thus, while diabetes in the presence of clonotype-high T cells could not be neutralized by H22, it still required APCs to express the IFN-{gamma} receptor. It could be that the H22 treatment is not effective at neutralizing the IFN-{gamma} signals transmitted between T cells and APCs at close proximity. Another possibility is that tonic levels of IFN-{gamma} signaling may be necessary for macrophages to differentiate into effector cells that kill islet β cells. If there is already a pool of such cells, lowering IFN-{gamma} at or around the time of the BDC transfer may not have any effect on the recruitment of these killer APCs into the islets.

Previous studies had shown that the natural NOD diabetogenesis was affected to a minor degree in situations where the IFN-{gamma} receptor or the structural genes for IFN-{gamma} were mutated: at most a slower development was found with a small reduction in incidence (10, 11, 12, 13, 14). This suggests other pathways of activation and effector functions taking place during this chronic process. On the other hand, in the various manipulations where the diabetogenic process was dissected, such as shown in these studies, a role of IFN-{gamma} is apparent (15, 16, 17). In the acute cyclophosphamide model, Abs to IFN-{gamma} were highly effective in stopping diabetogenesis (15, 16) as was the absence of the IFN-{gamma} receptor in the myeloid cell compartment (56). Gene expression profiles disclosed high expression of IFN-{gamma}-induced genes (57). Other systems have also shown other roles of IFN-{gamma} when involving other cells. In the experiments reported by Cain et al. (58), the BDC T cells derived from BDC.scid (i.e., with high clonotype) could be inhibited by NKT cells and, in this instance, IFN-{gamma} was required in a process where the target tissue appeared to be that of the host. The take-home message is that as more cells join the process, there are more diverse interactions taking place.


   Aknowlegments
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Aknowlegments
Disclosures
 References
 
We thank Katherine Frederick, Patrice Bittner, and Shirley Petzold for their technical assistance; members of the Unanue laboratory, particularly Javier A. Carrero and Roger Belizaire; members of the Mills laboratory; and Robert D. Schreiber for his valuable reagent and for critical analysis.


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


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

1 This work was supported by National Institutes of Health Grants DK058177 (to E.R.U.) and K08 DK066062 and DRTC (Diabetes Research and Training Center) 5 P60 DK20579 (to J.C.M.), the Juvenile Diabetic Research Foundation, the Kilo Diabetes and Vascular Research Foundation, and the Multiplex Gene Analysis Core of the Siteman Cancer Center (supported in part by National Cancer Institute Grant P30 CA91842). Back

2 Address correspondence and reprint requests to Dr. Emil R. Unanue, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: unanue{at}pathbox.wustl.edu Back

3 Abbreviations used in this paper: BDC, BDC2.5; BDC.scid, BDC2.5/NOD.scid; GITR, glucocorticoid-induced TNF receptor; GO, gene ontology; IFN-{gamma}R–/–, IFN-{gamma} receptor–/–; LTP, long-term protected; NOD.Rag, NOD.Rag-1–/–; NOD.scid, NOD.CB17-Prkdcscid/J. Back

4 The online version of this article contains supplemental material. Back

Received for publication June 9, 2008. Accepted for publication September 12, 2008.


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

  1. Tisch, R., H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell 85: 291-297. [Medline]
  2. Anderson, M. S., J. A. Bluestone. 2005. The NOD mouse: a model of immune dysregulation. Annu. Rev. Immunol. 23: 447-485. [Medline]
  3. Shoda, L. K., D. L. Young, S. Ramanujan, C. C. Whiting, M. A. Atkinson, J. A. Bluestone, G. S. Eisenbarth, D. Mathis, A. A. Rossini, S. E. Campbell, et al 2005. A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity 23: 115-126. [Medline]
  4. Schoenborn, J. R., C. B. Wilson. 2007. Regulation of interferon-{gamma} during innate and adaptive immune responses. Adv. Immunol. 96: 41-101. [Medline]
  5. Bach, E. A., M. Aguet, R. D. Schreiber. 1997. The IFN{gamma} receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15: 563-591. [Medline]
  6. Schreiber, R. D., J. L. Pace, S. W. Russell, A. Altman, D. H. Katz. 1983. Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon. J. Immunol. 131: 826-832. [Abstract]
  7. Katz, J. D., B. Wang, K. Haskins, C. Benoist, D. Mathis. 1993. Following a diabetogenic T cell from genesis through pathogenesis. Cell 74: 1089-1100. [Medline]
  8. Suri, A., J. D. Katz. 1999. Dissecting the role of CD4+ T cells in autoimmune diabetes through the use of TCR transgenic mice. Immunol. Rev. 169: 55-65. [Medline]
  9. Calderon, B., A. Suri, E. R. Unanue. 2006. In CD4+ T-cell-induced diabetes, macrophages are the final effector cells that mediate islet β-cell killing: studies from an acute model. Am. J. Pathol. 169: 2137-2147. [Abstract/Free Full Text]
  10. Hultgren, B., X. Huang, N. Dybdal, T. A. Stewart. 1996. Genetic absence of {gamma}-interferon delays but does not prevent diabetes in NOD mice. Diabetes 45: 812-817. [Abstract]
  11. Serreze, D. V., C. M. Post, H. D. Chapman, E. A. Johnson, B. Lu, P. B. Rothman. 2000. Interferon-{gamma} receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice. Diabetes 49: 2007-2011. [Abstract/Free Full Text]
  12. Kanagawa, O., G. Xu, A. Tevaarwerk, B. A. Vaupel. 2000. Protection of nonobese diabetic mice from diabetes by gene(s) closely linked to IFN-{gamma} receptor loci. J. Immunol. 164: 3919-3923. [Abstract/Free Full Text]
  13. Wang, B., I. Andre, A. Gonzalez, J. D. Katz, M. Aguet, C. Benoist, D. Mathis. 1997. Interferon-{gamma} impacts at multiple points during the progression of autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94: 13844-13849. [Abstract/Free Full Text]
  14. Nicoletti, F., P. Zaccone, R. Di Marco, M. Di Mauro, G. Magro, S. Grasso, L. Mughini, P. Meroni, G. Garotta. 1996. The effects of a nonimmunogenic form of murine soluble interferon-{gamma} receptor on the development of autoimmune diabetes in the NOD mouse. Endocrinology 137: 5567-5575. [Abstract]
  15. Debray-Sachs, M., C. Carnaud, C. Boitard, H. Cohen, I. Gresser, P. Bedossa, J. F. Bach. 1991. Prevention of diabetes in NOD mice treated with antibody to murine IFN gamma. J. Autoimmun. 4: 237-248. [Medline]
  16. Campbell, I. L., T. W. Kay, L. Oxbrow, L. C. Harrison. 1991. Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice. J. Clin. Invest. 87: 739-742. [Medline]
  17. Trembleau, S., G. Penna, S. Gregori, N. Giarratana, L. Adorini. 2003. IL-12 administration accelerates autoimmune diabetes in both wild-type and IFN-{gamma}-deficient nonobese diabetic mice, revealing pathogenic and protective effects of IL-12-induced IFN-{gamma}. J. Immunol. 170: 5491-5501. [Abstract/Free Full Text]
  18. Schadt, E. E., C. Li, B. Ellis, W. H. Wong. 2001. Feature extraction and normalization algorithms for high-density oligonucleotide gene expression array data. J. Cell. Biochem. (Suppl. 37): 120-125.
  19. Zhong, S., C. Li, W. H. Wong. 2003. ChipInfo: software for extracting gene annotation and gene ontology information for microarray analysis. Nucleic Acids Res. 31: 3483-3486. [Abstract/Free Full Text]
  20. Doherty, J. M., L. K. Carmichael, J. C. Mills. 2006. GOurmet: a tool for quantitative comparison and visualization of gene expression profiles based on gene ontology (GO) distributions. BMC Bioinform. 7: 151[Medline]
  21. Benjamini, Y., Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57: 289-300.
  22. Mills, J. C., N. Andersson, T. S. Stappenbeck, C. C. Chen, J. I. Gordon. 2003. Molecular characterization of mouse gastric zymogenic cells. J. Biol. Chem. 278: 46138-46145. [Abstract/Free Full Text]
  23. Schreiber, R. D., L. J. Hicks, A. Celada, N. A. Buchmeier, P. W. Gray. 1985. Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol. 134: 1609-1618. [Abstract]
  24. Harris, M. A., J. Clark, A. Ireland, J. Lomax, M. Ashburner, R. Foulger, K. Eilbeck, S. Lewis, B. Marshall, C. Mungall, et al 2004. The gene ontology (GO) database and informatics resource. Nucleic Acids Res. 32: D258-D261. [Abstract/Free Full Text]
  25. Lang, R., D. Patel, J. J. Morris, R. L. Rutschman, P. J. Murray. 2002. Shaping gene expression in activated and resting primary macrophages by IL-10. J. Immunol. 169: 2253-2263. [Abstract/Free Full Text]
  26. Pull, S. L., J. M. Doherty, J. C. Mills, J. I. Gordon, T. S. Stappenbeck. 2005. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proc. Natl. Acad. Sci. USA 102: 99-104. [Abstract/Free Full Text]
  27. Mosser, D. M.. 2003. The many faces of macrophage activation. J. Leukocyte Biol. 73: 209-212. [Free Full Text]
  28. Gordon, S.. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: 23-35. [Medline]
  29. Welch, J. S., L. Escoubet-Lozach, D. B. Sykes, K. Liddiard, D. R. Greaves, C. K. Glass. 2002. TH2 cytokines and allergic challenge induce Ym1 expression in macrophages by a STAT6-dependent mechanism. J. Biol. Chem. 277: 42821-42829. [Abstract/Free Full Text]
  30. Cormier, S. A., S. Yuan, J. R. Crosby, C. A. Protheroe, D. M. Dimina, E. M. Hines, N. A. Lee, J. J. Lee. 2002. TH2-mediated pulmonary inflammation leads to the differential expression of ribonuclease genes by alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 27: 678-687. [Abstract/Free Full Text]
  31. Mantovani, A., A. Sica, M. Locati. 2005. Macrophage polarization comes of age. Immunity 23: 344-346. [Medline]
  32. Rauh, M. J., V. Ho, C. Pereira, A. Sham, L. M. Sly, V. Lam, L. Huxham, A. I. Minchinton, A. Mui, G. Krystal. 2005. SHIP represses the generation of alternatively activated macrophages. Immunity 23: 361-374. [Medline]
  33. Herbert, D. R., C. Holscher, M. Mohrs, B. Arendse, A. Schwegmann, M. Radwanska, M. Leeto, R. Kirsch, P. Hall, H. Mossmann, et al 2004. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20: 623-635. [Medline]
  34. Zhu, Z., T. Zheng, R. J. Homer, Y. K. Kim, N. Y. Chen, L. Cohn, Q. Hamid, J. A. Elias. 2004. Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation. Science 304: 1678-1682. [Abstract/Free Full Text]
  35. Mantovani, A., A. Sica, S. Sozzani, P. Allavena, A. Vecchi, M. Locati. 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25: 677-686. [Medline]
  36. Kanagawa, O., A. Militech, B. A. Vaupel. 2002. Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice. J. Immunol. 168: 6159-6164. [Abstract/Free Full Text]
  37. Suri, A., J. Shimizu, J. D. Katz, S. Sakaguchi, E. R. Unanue, O. Kanagawa. 2004. Regulation of autoimmune diabetes by non-islet-specific T cells: a role for the glucocorticoid-induced TNF receptor. Eur. J. Immunol. 34: 447-454. [Medline]
  38. Pakala, S. V., M. Chivetta, C. B. Kelly, J. D. Katz. 1999. In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor {alpha}. J. Exp. Med. 189: 1053-1062. [Abstract/Free Full Text]
  39. Morimoto, J., H. Yoneyama, A. Shimada, T. Shigihara, S. Yamada, Y. Oikawa, K. Matsushima, T. Saruta, S. Narumi. 2004. CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced β cell proliferation without affecting insulitis. J. Immunol. 173: 7017-7024. [Abstract/Free Full Text]
  40. Orlofsky, A., E. Y. Lin, M. B. Prystowsky. 1994. Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. J. Immunol. 152: 5084-5091. [Abstract]
  41. Orlofsky, A., Y. Wu, M. B. Prystowsky. 2000. Divergent regulation of the murine CC chemokine C10 by Th1 and Th2 cytokines. Cytokine 12: 220-228. [Medline]
  42. Heitmeier, M. R., A. L. Scarim, J. A. Corbett. 1997. Interferon-{gamma} increases the sensitivity of islets of Langerhans for inducible nitric-oxide synthase expression induced by interleukin 1. J. Biol. Chem. 272: 13697-13704. [Abstract/Free Full Text]
  43. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, T. Nomura. 2006. Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212: 8-27. [Medline]
  44. Fallarino, F., R. Bianchi, C. Orabona, C. Vacca, M. L. Belladonna, M. C. Fioretti, D. V. Serreze, U. Grohmann, P. Puccetti. 2004. CTLA-4-Ig activates forkhead transcription factors and protects dendritic cells from oxidative stress in nonobese diabetic mice. J. Exp. Med. 200: 1051-1062. [Abstract/Free Full Text]
  45. Luhder, F., C. Chambers, J. P. Allison, C. Benoist, D. Mathis. 2000. Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells. Proc. Natl. Acad. Sci. USA 97: 12204-12209. [Abstract/Free Full Text]
  46. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164: 183-190. [Abstract/Free Full Text]
  47. Taylor, P. A., C. J. Lees, S. Fournier, J. P. Allison, A. H. Sharpe, B. R. Blazar. 2004. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J. Immunol. 172: 34-39. [Abstract/Free Full Text]
  48. Tang, Q., E. K. Boden, K. J. Henriksen, H. Bour-Jordan, M. Bi, J. A. Bluestone. 2004. Distinct roles of CTLA-4 and TGF-β in CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2996-3005. [Medline]
  49. Grohmann, U., F. Fallarino, R. Bianchi, C. Orabona, C. Vacca, M. C. Fioretti, P. Puccetti. 2003. A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice. J. Exp. Med. 198: 153-160. [Abstract/Free Full Text]
  50. Puccetti, P., U. Grohmann. 2007. IDO and regulatory T cells: a role for reverse signalling and non-canonical NF-{kappa}B activation. Nat. Rev. Immunol. 7: 817-823. [Medline]
  51. Keskin, D. B., B. Marshall, D. Munn, A. L. Mellor, D. A. Gearhart. 2007. Decreased protein nitration in macrophages that overexpress indoleamine 2,3-dioxygenase. Cell. Mol. Biol. Lett. 12: 82-102. [Medline]
  52. Mellor, A. L., P. Chandler, B. Baban, A. M. Hansen, B. Marshall, J. Pihkala, H. Waldmann, S. Cobbold, E. Adams, D. H. Munn. 2004. Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase. Int. Immunol. 16: 1391-1401. [Abstract/Free Full Text]
  53. Mellor, A. L., B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen, P. A. Koni, M. Iwashima, D. H. Munn. 2003. Cutting edge: Induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J. Immunol. 171: 1652-1655. [Abstract/Free Full Text]
  54. Mellor, A. L., D. H. Munn. 2004. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 4: 762-774. [Medline]
  55. Ulaeto, D., P. E. Lacy, D. M. Kipnis, O. Kanagawa, E. R. Unanue. 1992. A T-cell dormant state in the autoimmune process of nonobese diabetic mice treated with complete Freund’s adjuvant. Proc. Natl. Acad. Sci. USA 89: 3927-3931. [Abstract/Free Full Text]
  56. Mori, Y., T. Kato, T. Kodaka, E. M. Kanagawa, S. Hori, O. Kanagawa. 2008. Protection of IFN-{gamma} signaling-deficient NOD mice from diabetes by cyclophosphamide. Int. Immunol. 20: 1231-1237. [Abstract/Free Full Text]
  57. Matos, M., R. Park, D. Mathis, C. Benoist. 2004. Progression to islet destruction in a cyclophosphamide-induced transgenic model: a microarray overview. Diabetes 53: 2310-2321. [Abstract/Free Full Text]
  58. Cain, J. A., J. A. Smith, J. K. Ondr, B. Wang, J. D. Katz. 2006. NKT cells and IFN-{gamma} establish the regulatory environment for the control of diabetogenic T cells in the nonobese diabetic mouse. J. Immunol. 176: 1645-1654. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
Y. Mori, T. Kodaka, T. Kato, E. M. Kanagawa, and O. Kanagawa
Critical role of IFN-{gamma} in CFA-mediated protection of NOD mice from diabetes development
Int. Immunol., November 1, 2009; 21(11): 1291 - 1299.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. S. Turner, L. P. Kane, and P. A. Morel
Dominant Role of Antigen Dose in CD4+Foxp3+ Regulatory T Cell Induction and Expansion
J. Immunol., October 15, 2009; 183(8): 4895 - 4903.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Calderon, B.
Right arrow Articles by Unanue, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Calderon, B.
Right arrow Articles by Unanue, E. R.
Right arrowPubmed/NCBI databases
*GEO DataSet
*Substance via MeSH
Medline Plus Health Information
*Diabetes Type 1

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS