Diabetic Mice Regulatory T Cell Function in Nonobese TNF Receptor ...

The Journal of Immunology

TNF Receptor 1 Deficiency Increases Regulatory T Cell Function in Nonobese Diabetic Mice Jonathan Chee,*,†,1 Eveline Angstetra,*,†,1 Lina Mariana,* Kate L. Graham,* Emma M. Carrington,* Horst Bluethmann,‡ Pere Santamaria,x,{ Janette Allison,* Thomas W. H. Kay,*,† Balasubramanian Krishnamurthy,*,1 and Helen E. Thomas*,†,1 TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to b cell-specific, highly diabetogenic TCR transgenic I-Ag7–restricted NOD4.1 mice and Kdrestricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for b cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4+CD25+Foxp3+ T regulatory cells in TNFR1-deficient mice. TNFR1deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes. The Journal of Immunology, 2011, 187: 1702–1712.

T

he thymus purges the majority of the autoreactive T cells by a process called negative selection. However, this process is incomplete, with some autoreactive T cells escaping thymic negative selection and circulating in the periphery. Autoreactive T cells in the periphery are controlled by peripheral deletion–inactivation and dominant regulatory mechanisms that involve CD4+CD25+Foxp3+ T regulatory cells. Failure of any of these broad mechanisms can lead to autoimmune disease. Type 1 diabetes (T1D) results from an imbalance between the two functionally opposite cell types, autoreactive T effector cells, and T regulatory cells. This imbalance leads to destruction of b cells by autoreactive T effector cells (1). In T1D, both CD4+ and CD8+ T cells have been shown to have major roles in b cell destruction (2, 3), and mechanisms used by CD4+ and CD8+ T cells to kill target cells differ (4, 5). However, both cell types have been shown to produce cytokines that may *St. Vincent’s Institute, Fitzroy, Victoria 3065, Australia; †Department of Medicine, University of Melbourne, Victoria 3065, Australia; ‡Roche Center for Medical Genomics, Basel 4070, Switzerland; xJulia McFarlane Diabetes Research Centre, University of Calgary, Calgary, Alberta T2N 4N1, Canada; and {Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada 1

J.C., E.A., B.K., and H.E.T. contributed equally to this work.

Received for publication February 17, 2011. Accepted for publication June 4, 2011. This work was supported by grants from the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation. P.S. was supported by the Canadian Institutes of Health Research and the Canadian Diabetes Association. P.S. is a scientist of Alberta Innovates–Health Solutions and a Juvenile Diabetes Research Foundation scholar. The Julia McFarlane Diabetes Research Centre is supported by the Diabetes Association (Foothills). Address correspondence and reprint requests to Dr. Helen E. Thomas, St. Vincent’s Institute, 41 Victoria Parade, Fitzroy, VIC 3065, Australia. E-mail address: hthomas@ svi.edu.au The online version of this article contains supplemental material. Abbreviations used in this article: DC, dendritic cell; ILN, inguinal lymph node; PLN, pancreatic lymph node; T1D, type 1 diabetes. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100511

participate in b cell destruction. TNF is a pluripotent cytokine secreted by immune cells such as macrophages, T cells, and B cells and by other cells, such as fibroblasts (6, 7). In vitro, a combination of TNF with IFN-g has been shown to cause b cell death, an effect that is mediated primarily by intraislet IL-1 and NO production (8, 9), as well as direct caspase activation (10). Systemic administration or overexpression of TNF in islet b cells in neonatal C57BL/6 mice leads to massive insulitis and accelerates diabetes in NOD mice (11–18). Consistent with this finding, anti-TNF Ab prevents diabetes progression when given to neonatal NOD mice (15). In contrast, diabetes progression is delayed when TNF is systemically administered to adult NOD mice (19, 20). The effects of TNF when administered at different time points highlight the different functions of TNF with effects on the progression of T1D. TNF mediates its biologic functions through its receptors, TNFR1 and TNFR2. TNFR1 is the primary signaling receptor that initiates the majority of inflammatory responses classically attributed to TNF (21). One of the effects of TNF-TNFR2 signaling is the promotion of T regulatory cell expansion (22). It has been shown recently that activated autoreactive T effectors secrete TNF-a, which induces an expansion of T regulatory cells via TNF–TNFR2 interactions (23). This is one of the proposed mechanisms for fine tuning the balance between effector T cells and the regulatory T cells to control autoimmune diseases such as multiple sclerosis and diabetes. Complete protection from diabetes was observed when TNFR1 but not TNFR2 was deleted from NOD mice (24, 25). However, whether TNFR1 has a direct role in b cell death in vivo has not been defined clearly. The effects of TNF on b cells need to be separated from effects on the immune system. Studies using different experimental models have suggested that the absence of TNFR1 on b cells did not provide protection from destruction by activated T lymphocytes (24, 26). Transferred diabetogenic BDC2.5.scid splenocytes were able to destroy TNFR1-deficient islets transplanted into NOD.scid mice only when they were

The Journal of Immunology grafted together with wild type NOD islets, but not when TNFR1deficient islets were grafted alone (26). In addition, TNFR1 deficient islets were not protected from destruction in an accelerated diabetes model induced by expression of IL-10 in b cells (24). Both these studies suggest that the absence of diabetes in TNFR12/2 mice may not be due to protection from TNF-induced b cell destruction. Our study investigated the role of TNFR1 in NOD mice and in TCR transgenic NOD mice that have a T cell repertoire biased to islet-specific CD4+ or CD8+ T cells. Diabetes was significantly reduced by the absence of TNFR1 in NOD, NOD4.1, and NOD8.3 mice. TNFR1 deficiency did not protect islets from direct destruction by effector T cells; however, blockade of TNF signaling through TNFR1 advantageously increased regulatory T cell number and function.

Materials and Methods

1703 blood glucose of .15 mM on two consecutive days were considered diabetic.

Flow cytometry Dispersed islet cells were stained with mAbs using standard procedures. Abs used were anti-CD4 (GK1.5, RM4.4) conjugated to FITC or allophycocyanin, anti-CD25 (3C7) conjugated to allophycocyanin, anti-CD11c (HL3) conjugated to allophycocyanin, anti-CD86 (GL1) conjugated to FITC, anti-CD80 (16-10A1) conjugated to FITC, anti-CD69 (H1-2F3) conjugated to PE, anti-CD45 (3F11) conjugated to PerCp-Cy5.5 (BD Pharmingen, San Diego, CA), anti-TNFR1 (55R-286) conjugated to PE (BioLegend, San Diego, CA) and anti-TNFR2 (HM102) conjugated to FITC (Abcam, Cambridge, MA). When PerCpCy5.5 was used, propidium iodide was used at 3.3 mg/ml (Calbiochem, La Jolla, CA). Intracellular Foxp3 staining was done using the eBioscience Foxp3 staining kit according to manufacturer’s instructions (eBiosciences, San Diego, CA). The percentage of CD4+Foxp3+ T cells in islet infiltrates was calculated as the percentage of the CD45+ population that was positive for both Foxp3 and CD4. Analysis was performed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) using FlowJo (Treestar, Ashland, OR) software.

Mice

Anti-CD4+ Ab treatment

All mice used were bred and maintained at St. Vincent’s Institute of Medical Research. NOD/Lt mice were purchased from the Walter and Eliza Hall of Institute animal breeding facility, Melbourne, Australia. NOD4.1 mice express the TCRab rearrangements of the I-Ag7-restricted CD4+ T cell clone NY4.1 (27). NOD8.3 mice express the TCRab rearrangements of the H-2Kd-restricted, b cell-reactive, CD8+ T cell clone NY8.3 (28). TNFR1-deficient mice were made on a 129xC57BL/6 background using 129/OlaHsd ES cells as described previously (29). TNFR1deficient mice were backcrossed to NOD/Lt mice for 12 generations. DNA from the 12th generation backcross was genotyped by the Australian Genome Research Facility using fluorescently labeled polymorphic markers. Samples were processed for the mouse 5K targeted genotyping array run on the Affymetrix GeneChip Scanner 3000 7G MegAllele system. Data were analyzed using the GeneChip Targeted Genotyping System software. Regions of difference between strains were identified using data obtained from the Jackson Laboratories Mouse Genome Informatics SNP database and NCBI databases. Backcrossed TNFR1-deficient NOD mice were of NOD genotype across the whole genome, except for a region on chromosome 6 between and including ∼119.8 Mb (rs13479002) and 127.8 Mb (rs3023092) encompassing the Tnfrsf1a gene. No known 129 diabetes susceptibility or resistance alleles have been reported for loci within this region (30). Backcrossed TNFR1-deficient mice were then crossed to NOD4.1 mice (NOD4.1/TNFR12/2) and NOD8.3 mice (NOD8.3/ TNFR12/2). The institutional animal ethics committee approved all experiments.

Six-week-old NOD8.3 and NOD8.3/TNFR12/2 mice were injected i.v. with anti-CD4 (GK1.5) depleting Ab. The initial dose was 1 mg followed by 0.5 mg after 3 d. Thereafter, 0.5 mg was injected every week until the mouse became diabetic.

Islet isolation Islets of Langerhans were isolated from mice according to methods described previously (31). Islets were handpicked and cultured in Connaught Medical Research Laboratories medium-1066 (Invitrogen), containing antibiotics, 2 mmol/l glutamine, and 10% FCS at 37˚C with 5% CO2. For flow cytometry, islets were dispersed into single cells by a brief incubation with trypsin (342 U/ml; Calbiochem, La Jolla, CA) and 2 mM EDTA in PBS. Dispersed islets were then washed free of trypsin and allowed to recover in culture medium for 1–1.5 h before staining. Islets were analyzed on the day of isolation.

CFSE labeling and adoptive transfer Single-cell suspensions of splenocytes were resuspended in 0.1% BSA in PBS at 107 cells/ml. Cells were labeled with 5 mM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37˚C in the dark. After labeling, cells were washed three times in complete RPMI 1640. Cells (2–5 3 107) were resuspended in PBS before i.v. injection into the tail veins of recipient mice. Recipient mice were sacrificed 3, 4, or 6 d later. Pancreatic lymph nodes (PLNs), inguinal lymph nodes (ILNs), and islets were examined for CFSE+ cells by flow cytometry.

In vivo suppression of adoptive transfer of diabetes Either MACS purified CD4+CD252 or CD4+CD25+ T cells (5 3 105) from prediabetic NOD or TNFR12/2 mice were coinjected with 2 3 107 splenocytes from newly diabetic NOD mice into the tail vein of NOD.scid mice. Mice were monitored after 7 d for diabetes by blood glucose levels using Advantage II Glucose Strips (Roche, Basel, Switzerland). Mice with

Preparation of dendritic cells Dendritic cells (DCs) were prepared by digesting spleen and pancreatic lymph nodes (pooled from four to six mice) at room temperature for 30 min in a collagenase solution, followed by purification with anti-CD11c–coated beads (Miltenyi Biotec). Purity was .90% CD11c+ cells. Purified DCs were stimulated overnight with LPS (Sigma-Aldrich, St. Louis, MO; 1 mg/ ml).

Islet grafts and histology Newly diabetic NOD4.1 or streptozotocin-treated diabetic NOD.scid mice were grafted under the kidney capsule with 600 or 400 islets, respectively, from 6-wk-old donor mice. Mice were monitored for blood glucose levels using Advantage II Glucose Strips (Roche). Mice with blood glucose .15 mM on two consecutive days were considered diabetic. For histologic analysis, the pancreas and graft were placed in Bouin’s fixative and embedded in paraffin. Serial sections (5 mm) were cut and stained with H&E, or with anti-insulin followed by anti-guinea pig-HRP, or with anti-glucagon followed by anti–rabbit-HRP (all antisera from Dako, Carpinteria, CA). Staining was developed with diaminobenzidine (Sigma-Aldrich) and sections were counter-stained with hematoxylin. Islets were scored as follows: 0 = no infiltrate; 1 = peri-islet infiltrate; 2 = intra-islet infiltrate, ,50% islet destruction; 3 = intra-islet infiltrate, .50% islet destruction; 4 = complete islet destruction.

Fluorescent immunohistochemistry Cryosections were fixed and stained with primary Abs, purified anti-mouse MAdCAM-1 mAb (rat IgG2a, MECA 367), purified anti-mouse VCAM-1 mAb (rat IgG2a, clone 429), and the pan endothelial marker (clone MECA32; all from Biolegend, San Diego, CA). For isotype controls, purified antimouse KLH mAb was used (rat IgG2a, clone A110-2; BD Pharmingen). Staining was detected using Alexa Fluor 568 conjugated goat anti-rat IgG (Molecular Probes, Eugene, OR). Pancreatic islets were visualized by staining for insulin using guinea pig anti-mouse insulin mAb (Dako), followed by FITC-conjugated anti-guinea pig IgG mAb (ICN, Aurora, OH). Photographs of sections were taken using Olympus DP20 camera with Olympus software (Olympus, Center Valley, PA) and subsequently overlayed using Adobe Photoshop Elements 4.0 (Adobe, San Jose, CA).

Statistics Statistical analysis of incidence of diabetes was performed with the log-rank test (survival curve analysis). The percentage of CD4+CD25+ T cells was analyzed with two-tailed t test with Mann–Whitney’s posttest for comparison. Statistical analyses of in vitro and in vivo CD4+ T cell proliferation were performed with two-way ANOVA with Bonferroni’s posttest for comparison of multiple columns. All statistical analysis was done using the GraphPad Prism program (GraphPad Software, San Diego, CA).

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REGULATORY T CELL FUNCTION IN TNFR1-DEFICIENT NOD MICE

Results TNFR1 deficiency protects NOD mice from diabetes and insulitis Diabetes incidence and insulitis was studied in TNFR1-deficient NOD mice (NOD/TNFR12/2). Consistent with the findings of Ka¨gi et al. (25), NOD/TNFR12/2 mice were completely protected from diabetes (Fig. 1A; p , 0.0001). Histologic analysis of NOD/ TNFR12/2 mice showed reduced insulitis with the majority of islets being free of insulitis and no invasive or destructive insulitis (Fig. 1B, 1C; p , 0.001). To investigate whether the absence of TNF signaling has a predominant effect on CD8+ or CD4+ T cells, we studied TNFR1-deficient NOD-expressing TCRab transgenes of highly diabetogenic islet specific CD4+ T cells (NOD4.1 mice) or CD8+ T cells (NOD8.3 mice). The 4.1-TCR recognizes a yet unknown islet Ag, and NOD4.1 mice show a dramatic acceleration of the onset of diabetes, owing to massive recruitment of b cell-cytotoxic CD4+ T cells into islets (28). NOD4.1/TNFR12/2 mice were significantly protected from insulitis and diabetes (Fig. 1D–F). The 8.3-TCR recognizes the peptide IGRP206–214, and NOD8.3 mice show accelerated diabetes because of the infiltration

of islets with 8.3 CD8+ T cells (28, 32). NOD8.3/TNFR12/2 mice were also significantly protected from insulitis and diabetes (Fig. 1G–I). NOD4.1/TNFR12/2 and NOD8.3/TNFR12/2 mice displayed thymic, splenic, and lymph node cytofluorometric profiles that were comparable to those seen in NOD4.1 mice and NOD8.3 mice, respectively (Supplemental Fig. 1). Significant protection from diabetes in both NOD4.1 and NOD8.3 mice indicates that the absence of TNF signaling affects both CD8 and CD4 compartments. Activated NOD4.1 T cells destroy TNFR1-deficient islet grafts The protection from diabetes in TNFR1-deficient mice could be due to loss of the effects of TNF on b cells or on the immune system, or both. To determine whether TNFR1 signaling on islet cells is required for b cell death induced by NOD4.1 effector T cells, 600 NOD/TNFR12/2 islets were grafted under the kidney capsule of diabetic NOD4.1 mice. NOD/TNFR12/2 islets were rejected as rapidly as NOD islets in diabetic NOD4.1 mice (Fig. 2A). Both NOD/TNFR12/2 and NOD islets were only able to transiently restore blood glucose levels in the diabetic NOD4.1 mice. Islets isolated at the same time were able to restore

FIGURE 1. TNFR1-deficient mice have less severe insulitis and are protected from diabetes. A, Incidence of T1D in NOD/TNFR12/2 and NOD mice, p , 0.0001. B, Histologic grading of insulitis in pancreas sections from 18-wk-old female NOD or backcrossed NOD/TNFR12/2 mice (n = 4–5 per group; p , 0.001). C, Bouins’ fixed sections from 18-wk-old NOD and NOD/TNFR12/2 were stained with H&E. D, Incidence of diabetes in NOD4.1/TNFR12/2 and NOD4.1 mice. p , 0.0001. E, Histologic grading of insulitis in pancreas section from 5–6-wk-old female NOD4.1 or NOD4.1/TNFR12/2 mice (n = 3 per group; p , 0.0001) F, Bouins’ fixed sections from 6-wk-old NOD4.1 and NOD4.1/TNFR12/2 stained with H&E. G, Incidence of diabetes in NOD8.3/ TNFR12/2 and NOD8.3 mice. p , 0.0001. H, Histologic grading of insulitis in pancreas sections from 8-wk-old female NOD8.3 or backcrossed NOD8.3/ TNFR12/2 mice (n = 4–5 per group; p , 0.05). I, Frozen sections from 8-wk-old NOD8.3 and NOD8.3/TNFR12/2 were stained with H&E. Histologic figures shown are representative of at least three individual mice. Original magnification 3200.


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FIGURE 2. TNFR1 deficiency does not protect islets from destruction by activated NOD4.1 T cells. Six hundred of either NOD (n = 4 mice) or NOD/TNFR12/2 islets (n = 5 mice) were grafted under the kidney capsule of diabetic NOD4.1 mice. Four hundred of either NOD (n = 2 mice) or NOD/ TNFR12/2 islets (n = 2 mice) were grafted under the kidney capsule of diabetic streptozotocin-treated NOD.scid mice. A, Posttransplantation blood glucose level of diabetic NOD4.1 or diabetic streptozotocin (STZ)-treated NOD.scid recipient mice grafted with NOD or NOD/TNFR12/2 islets. B, Grafts from diabetic NOD4.1 mice were fixed and embedded in paraffin. Sections were stained with anti-insulin Ab and counterstained with hematoxylin. Representative sections are shown. Original magnification 3200.

normoglycemia in streptozotocin-treated NOD.scid mice, demonstrating that the islets were functional. Histologic analysis showed that islets grafts were equally destroyed regardless of TNFR1 expression (Fig. 2B). This result demonstrates that TNFR1 deficiency does not have a direct effect on b cell death. Similarly, Pakala et al. (26) showed that activated BDC2.5 CD4+ T cells could destroy TNFR12/2 islets. We also found similar results when NOD or NOD/TNFR12/2 islets were transplanted under the kidney capsule of diabetic NOD8.3 mice (data not shown). TNFR1 deficiency does not affect priming of Ag-specific T cells in the pancreatic lymph nodes We next studied the effects of TNFR1 deficiency on the immune cells. To determine whether protection from diabetes is due to aberrant maturation of DCs in the absence of TNFR1, the level of activation markers CD80, CD86, CD69, and MHC class II were compared in NOD/TNFR12/2 and NOD mice. CD11c+ DCs isolated from the pancreatic lymph nodes and spleens of NOD/ TNFR12/2 and NOD mice showed no difference in the expression level of activation markers either in the steady state or after activation with LPS (1 mg/ml; Fig. 3A), suggesting TNFR1 does not detectably affect the maturation of DCs. The absence of diabetes and decreased severity of insulitis in NOD/TNFR12/2 mice suggests that protection of from diabetes could be due to a defect in T cell priming in the regional lymph node. To address this question, we transferred CFSE-labeled wild type or TNFR1-deficient NOD4.1 or NOD8.3 T cells into NOD or NOD/TNFR12/2 hosts. Islet Ag-specific CD4+ or CD8+ T cells

proliferated specifically in NOD draining PLNs and in NOD/ TNFR12/2 PLN (Fig. 3B–E). Furthermore, Ag-specific T cells proliferated independently of TNFR1 expression. A trend of reduced proliferation was observed when both T cells and recipient APCs were TNFR1 deficient (Fig. 3B, 3C). NOD/TNFR12/2 mice have defective homing of T cells into islets We next analyzed homing and proliferation of transferred CFSElabeled NOD4.1 and NOD8.3 T cells in the islets of NOD and NOD/TNFR12/2 mice. Whereas the transferred cells homed and proliferated in the islets of NOD mice, significantly fewer cells homed to the islets of NOD/TNFR12/2 mice (Fig. 4A, 4B). TNFTNFR1 signaling upregulates the expression of important adhesion molecules such as MAdCAM-1 and VCAM-1 (33). Previous studies have shown that blocking MAdCAM-1 and VCAM-1 in NOD mice prevents diabetes, demonstrating the importance of these adhesion molecules in T1D progression (34). To investigate whether defective homing was due to effects of the TNFR1 deficiency on the expression of adhesion molecules on the vascular endothelial cells, we studied MAdCAM-1 and VCAM-1 expression and expression of the vascular endothelial cell marker MECA-32 in the pancreas of NOD and NOD/TNFR12/2 mice. MAdCAM-1 and VCAM-1 were similarly expressed on the pancreas vascular endothelial cells in age-matched NOD and NOD/ TNFR12/2 mice (Fig. 4C). TNFR1 deficiency results in defective homing of T cells into the islets, but it was not due to different levels of expression of adhesion molecules.

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FIGURE 3. CD4+ or CD8+ T cells proliferate in the PLN independent of TNFR1 on either T cells or APCs. A, CD11c+ DCs were MACS purified from pancreatic lymph nodes of 8–12-wk-old NOD or NOD/TNFR12/2 (n = 4) mice. The purified cells were pooled and cultured in vitro for 24 h either unstimulated or stimulated with 1 mg/ml LPS. The cells were analyzed with Abs to CD11c and CD80, CD86, CD69, or MHC class II. A, Histogram plot


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FIGURE 4. Migration of T cells into the islets is impaired in TNFR12/2 recipients. CFSE-labeled splenocytes from NOD8.3 and NOD 8.3/TNFR1 mice were adoptively transferred into NOD and NOD/TNFR1 recipient mice as described in Fig. 3. A, Representative plots showing number of CD8+CFSE+ T cells in islets of recipient mice. B, Graph representing pooled data from 5–16 experiments showing the number of CD8+CFSEhi T cells. Mean 6 SEM is shown. p , 0.01 when comparing NOD8.3→NOD and NOD8.3/TNFR1→NOD/TNFR1 and NOD8.3→NOD/TNFR1. C, Pancreatic sections from NOD and NOD/TNFR12/2 mice were stained with anti-insulin, anti–MAdCAM-1, anti–VCAM-1, anti-MECA32 (pan endothelial cell marker), and isotype control Abs followed by FITC-conjugated anti–guinea-pig and AlexaFluor568-conjugated anti-rat IgG2a secondary Abs. Insulin staining in representative islets is shown in green. MAdCAM-1, VCAM-1, MECA32, and isotype are shown in red. Arrows indicate areas of MAdCAM-1 or VCAM-1 staining. Original magnification 3200.

represents expression of the indicated marker on CD11c+ cells. Data are representative of three individual experiments. B and C, Bar graphs representing pooled data from 5–16 experiments showing percentage proliferating CD4+ CFSEhi T cells (D) and CD8+CFSEhi T cells (E). Mean 6 SEM is shown. p , 0.01 when comparing NOD8.3→NOD and NOD8.3/TNFR1→NOD/TNFR1; p . 0.05 when comparing other different transfer groups (one-way ANOVA with Bonferroni’s posttest). D, Representative plots of 2–5 3 107 CFSE-labeled NOD4.1 or NOD4.1/TNFR12/2 T cells injected into NOD or NOD/ TNFR12/2 mice. E, Representative plots of 2–5 3 107 CFSE-labeled NOD8.3 or NOD8.3/TNFR12/2 T cells injected into NOD or NOD/TNFR12/2 mice. On days 4–6 after transfer, PLNs and ILNs were collected and assessed for CFSE dilution by flow cytometry. The numbers within histogram plots indicate the percentage of proliferating CD4+CFSEhi T cells (D) or CD8+CFSEhi T cells (E).

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TNFR1 deficiency increases regulatory T cells +

+

TNF-TNFR2 signaling is required for CD4 Foxp3 regulatory T cell expansion. Absence of TNFR1 may preferentially direct TNF to signal through TNFR2 and expand regulatory T cells. To investigate this possibility, the percentage and absolute number of CD4+Foxp3+ T cells were determined in TNFR1-deficient NOD, NOD4.1, and NOD8.3 mice. There was a significant increase in the percentage of CD4+Foxp3+ cells in ILNs and PLNs of NOD4.1/TNFR12/2 and NOD8.3/TNFR12/2 (Fig. 5A, 5B) compared with NOD4.1 and NOD8.3 mice, respectively. However, there was no significant increase in regulatory T cells in NOD/ TNFR12/2 mice compared with NOD mice. There was a significant increase in the percentage of CD4+Foxp3+ T cells in the islets of NOD/TNFR12/2 mice compared with islets of NOD mice (Fig. 5A; p , 0.05). We studied expression of TNFR1 and TNFR2 on CD4+Foxp3+ cells and CD4+Foxp32 cells in TNFR12/2 and wild type NOD, NOD4.1, and NOD8.3 mice. TNFR1 was barely detectable on CD4+Foxp3+ and CD4+Foxp32 cells from NOD, NOD4.1, and NOD8.3 mice. Our data are similar to what has been reported in C57BL/6 mice (22). TNFR2 expression was higher in CD4+ Foxp3+ cells compared with CD4+Foxp32 cells from NOD, NOD4.1, and NOD8.3 mice. There was no difference in TNFR2 expression level in CD4+Foxp3+ cells between TNFR1-deficient and wild type NOD, NOD4.1, and NOD8.3 mice (Supplemental Fig. 2).

FIGURE 5. NOD/TNFR12/2 mice have relatively increased numbers of CD4+Foxp3+ T cells. A, Percentage of CD4+Foxp3+ T cells in 8-wk-old NOD4.1, NOD8.3, NOD4.1/ TNFR12/2, and NOD8.3/TNFR12/2 (n = 5– 8 per group) and 18–20-wk-old NOD and NOD/TNFR12/2 mice as measured by intracellular Foxp3 staining and flow cytometry. The percentage of CD4+Foxp3+ T cell in islet infiltrates was determined by calculating the percentage CD4+Foxp3+ T cells in the CD45+ population. *p , 0.05. B, Absolute number of CD4+Foxp3+ T cells in NOD, NOD4.1, NOD8.3, NOD/TNFR1, NOD4.1/ TNFR12/2, and NOD8.3/TNFR12/2 calculated from the percentage of cells and the total number of cells in spleen, thymus, PLNs, ILNs, or islets. *p , 0.05. C, Splenocytes (2 3 107) from newly diabetic NOD mice were cotransferred into NOD.scid recipients with either 5 3 105 splenic CD4+CD25+ or 5 3 105 splenic CD4+CD252 MACS purified T cells from either NOD or NOD/TNFR12/2 mice. +p = 0.006, comparing mice receiving NOD/TNFR12/2 CD4+CD252 versus NOD/ TNFR12/2 CD4+CD25+ T cells.

We next examined the suppressive function of TNFR12/2 CD4+ CD25+ T cells by comparing the regulatory function of CD4+ CD25+ T cells from NOD and TNFR12/2 mice using an in vivo diabetes transfer study. Splenocytes from diabetic NOD mice transferred diabetes to all NOD.scid recipients beginning at 3 wk after post transfer. Diabetes was suppressed and delayed in NOD. scid recipients of TNFR12/2 CD4+CD25+ T cells cotransferred with diabetogenic NOD splenocytes (Fig. 5C). (At 80 d after transfer, four of seven mice cotransferred with TNFR12/2 CD4+ CD25+ T cells developed diabetes.) However, diabetes was delayed but the incidence was not altered in NOD.scid recipients of diabetogenic NOD splenocytes together with NOD CD4+ CD25+ T cells (Fig. 5C). (At day 63 after transfer, six of six mice cotransferred with NOD CD4+CD25+ T cells developed diabetes.) This finding demonstrates that TNFR12/2 CD4+CD25+ T cells are functionally superior in vivo suppressors of cotransferred diabetogenic NOD splenocytes in NOD.scid mice compared with NOD CD4+CD25+ T cells. To further investigate whether protection from diabetes in NOD8.3/TNFR12/2 mice is abrogated when CD4+CD25+ T regulatory cells are depleted, we treated NOD8.3 and NOD8.3/ TNFR12/2 mice with CD4-depleting GK1.5 Ab. CD8 T cells are the primary mediators of T cell destruction in the NOD8.3 mouse model; therefore, depleting all CD4 T cells would deplete the CD4+ regulatory T cells. Three of 4 NOD8.3/TNFR12/2 mice treated with GK1.5 developed diabetes within 4 wk, whereas


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NOD8.3/TNFR12/2 mice treated with PBS did not develop diabetes (Table I). Flow cytometric analysis of NOD8.3/TNFR12/2 mice treated with GK1.5 showed complete depletion of CD25+ Foxp3+ cells (Fig. 6A), and pancreas histology of the treated mice showed only CD8+ cells but not CD4+ cells infiltrating islets from mice given GK1.5 (Fig. 6B). Our data further suggest that TNF has an important role in the regulation of CD4+CD25+Foxp3+ cells and that the lack of TNFR1 has advantageous effects on the expansion of CD4+CD25+Foxp3+ cells, which subsequently prevents diabetes in both NOD4.1/TNFR12/2 and NOD8.3/TNFR12/2 mice.

Discussion

We found that NOD/TNFR12/2 mice are completely protected from diabetes and have significantly less insulitis than agematched NOD mice. When NOD/TNFR12/2 mice were crossed to TCR transgenic NOD4.1 or NOD8.3 mice, TNFR1 deficiency also provided significant protection from diabetes and insulitis. Our finding is significant because NOD8.3 and NOD4.1 mice develop accelerated diabetes and have been shown to contain T cell clones, which are highly diabetogenic. Our data suggest that TNFR1 signaling on b cells is not required for diabetes, but the loss of TNFR1 increased the number of Foxp3-expressing regulatory T cells. In addition, the protection from insulitis and diabetes may be in part due to increased regulatory T cell number and function. The environment in TNFR1-deficient NOD mice fails to provide the adequate signals to permit diabetogenic T cells to migrate to the pancreas. Studies have shown that vascular adhesion molecules are important for T cell migration into islets, and TNF-TNFR1 signaling is important for upregulating the expression of adhesion molecules (33, 34). However, we did not find decreased expression of vascular adhesion molecules in TNFR1-deficient mice. We found a relative increase in regulatory T cells in the islets of NOD/ TNFR12/2 and peripheral lymphoid organs of NOD4.1/TNFR12/2 and NOD8.3/TNFR12/2 mice. TNF has been shown to have a role in the control of regulatory T cells in rheumatoid arthritis patients, children with Crohn’s disease and NOD mice (35–38). Neutralizing anti-TNF Ab (infliximab) increased the number and suppressive function of CD4+CD25+Foxp3+ T cells in patients with rheumatoid arthritis (35–38). A similar observation was made in NOD mice treated with anti-TNF Ab (38). Although the TNFR1-deficient NOD8.3 and NOD4.1 mice had increased regulatory T cells in the PLNs to account for decreased homing of T cells to islets, we saw increased regulatory T cells in only the pancreas of TNFR1-deficient NOD mice. However, TNFR12/2 regulatory T cells had increased suppressive function compared with wild type regulatory T cells. In transgenic NOD mice expressing TNF in b cells, repression of TNF expression at 25 d of age delayed the normal rapid onset of diabetes (12, 39). In agreement with our data, this delay correlated with an increase in Table I. Diabetes protection in TNFR12/2 is reversed when regulatory T cells are depleted with GK1.5 mAb

Mouse Strain

NOD8.3 NOD8.3/TNFR1

GK 1.5 Treatment

No. of Diabetic Mice after 4 wk

2 + 2 +

1 2 0 3

Table I shows the strain and number of mice in each group that underwent GK1.5 treatment and developed T1D. Six-week-old NOD8.3 and NOD8.3/TNFR1 mice were treated with GK1.5 mAb (+) or PBS (2) (n = 4 in each group). Blood glucose levels were measured weekly.

CD4+CD25+ T cells in the islets and PLNs (39). Thus, TNF, through TNFR1 signaling, has a suppressive effect on regulatory T cell number and function. Regulatory T cells can mediate their suppressive function either by secreting cytokines like IL-10, TGF-b, or IL-35 or by direct cell–cell contact. These cells can act by suppressing the effector T cells directly at the target site (40), by suppressing DC in the regional lymph nodes and thereby preventing priming of T cells in the regional lymph nodes (41), or by recruiting mast cells to the site (42). In TNFR1-deficient mice, DC maturation markers in the PLNs and the priming of autoreactive T cells are normal. In this study, we show that increased regulatory T cells by the absence of TNF-TNFR1 signaling mice seem to prevent diabetes by preventing homing of T cells into islets. NOD BDC2.5 mice are protected from diabetes, whereas the BDC2.5 TCR transgene bred onto the NOD.scid background resulted in a much accelerated onset of diabetes, suggesting that regulatory cells prevent diabetes induction in the normal NOD setting. Transfer of NOD BDC2.5 T cells into immunodeficient NOD mice does not lead to the development of insulitis or diabetes (43). However, insulitis and diabetes develop efficiently if CD4+ T cells from NOD BDC2.5. scid or NOD BDC2.5 after depleting CD4+CD25+ T cells are transferred, indicating that regulatory T cells can prevent homing of effector T cells into islets (26). In experimental autoimmune encephalomyelitis, regulatory T cells have been shown to prevent migration of effectors T cells to the target organs (44), possibly by trapping the effector cells at the site of priming and preventing their egress to the target organ (45). In vitro experiments have shown that regulatory T cells prevent expansion of effector T cells. In vivo experiments have also shown that regulatory T cells can prevent the expansion of effector T cells in the draining lymph nodes by inhibiting the maturation and Agpresenting capacity of DCs (41). We did not find decreased proliferation of effector T cells in the pancreatic lymph nodes. This result is consistent with results of others showing that regulatory T cells can prevent homing into islets without affecting the priming of effector T cells in the draining lymph nodes (46). IGRP-specific transgenic CD8+ T cells mediate diabetes in NOD8.3 mice. IGRP-specific T cells require CD4+ T cell help for diabetes, because the incidence of diabetes is reduced in NOD8.3. rag22/2 mice and the incidence is restored upon transfer of CD4+ T cells from NOD mice (28). The requirement of CD4+ T cell help is in the early stages of the disease process because depletion of CD4+ T cells at 4 wk of age does not prevent or reduce the incidence of diabetes in NOD8.3 mice (T. Kay and B. Krishnamurthy, unpublished observations). We have used GK1.5 mAb to deplete CD4+ T cells starting at 6 wk of age. In this way, we have allowed CD4+ T cell help to be established and then depleted CD4+ Foxp3+ T cells. In NOD8.3 mice expressing proinsulin in the APC, a model where protection from diabetes is not due to dominant regulatory tolerance, we have shown that depletion of CD4+ T cells does not restore diabetes (47). In TNFR1-deficient NOD8.3 mice, depletion of CD4+ T cells restores diabetes, indicating a dominant regulatory mechanism for diabetes protection. Our results demonstrate that TNFR1 deficiency does not affect the priming of autoreactive T cells. We propose that primed autoreactive T cells secrete TNF that signals through TNFR2, which is constitutively expressed at higher levels on regulatory T cells than on effector T cells (22). Signaling through TNFR2 then leads to expansion of regulatory T cells that protect NOD mice from diabetes. Our data are compatible with the recent study showing that blockade of TNF reduced the proliferation of regulatory T cells and that TNF secreted by effector T cells boosted the expansion of regulatory T cells (23). To prove our hypothesis,

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FIGURE 6. Depleting T regulatory cells reverses protection against T1D in TNFR12/2 mice. A, Representative FACS plots showing proportion of CD4+ and CD8+, CD4+Foxp3+ splenocytes after treatment of mice. B, Fixed frozen sections from mice after GK1.5 or PBS treatment were stained with antiinsulin, anti-CD4, or anti-CD8 Abs and counterstained with hematoxylin. Representative sections from each treatment group are shown. Original magnification 3200.

future experiments are required to examine whether treatment of NOD/TNFR12/2 mice with TNF antagonist Ab releases mice from diabetes protection. In conclusion, we have demonstrated TNF is not required for b cell death or expression of adhesion molecules in islets, but is essential in diabetes progression in the NOD mouse, and in NOD4.1 and NOD8.3 TCR transgenic mice. This result demon-

strates TNF has significant effects on both CD4+ and CD8+ T cells. In NOD islets, TNF gene expression has been reported to peak at or before the onset of diabetes (48–50). We hypothesize that increased TNF expression in mice deleteriously effects regulatory T cell number and function and the deficiency in TNFR1 on regulatory T cells prevents this effect. This study further highlights the potential benefits of targeting TNF in T1D.


Acknowledgments We thank Rochelle Ayala, Sarah Emmett, Melanie Rowe, Kylie Tolley, and Stacey Fynch for technical assistance.

Disclosures The authors have no financial conflicts of interest.

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