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Bim deficiency protects NOD mice from diabetes by diverting thymocytes to regulatory T cells
Balasubramanian Krishnamurthy1,4, Jonathan Chee1, Gaurang Jhala1, Prerak Trivedi1, Tara Catterall1, Claudia Selck1, Esteban N. Gurzov1, Thomas C. Brodnicki1, Kate L. Graham1, Jibran A. Wali1, Yifan Zhan2, Daniel Gray2, Andreas Strasser2,3, Janette Allison1, Helen E. Thomas1,4,5 and Thomas W.H. Kay1,4,5 1
St. Vincent’s Institute, 41 Victoria Parade, Fitzroy, 3065, Victoria, Australia.
2
The Walter and Eliza Hall Institute of Medical Research, 2G Royal Parade, Parkville,
Victoria, 3050, Australia 3
Department of Medical Biology, The University of Melbourne, Melbourne, Australia
4
The University of Melbourne Department of Medicine, St Vincent’s Hospital,
Fitzroy, 3065, Victoria, Australia. 5
These authors contributed equally to the work
Running title: Bim deficiency promotes self-reactive Tregs Word count: 3936 Number of figures: 6 Correspondence and reprint requests should be addressed to: Dr Thomas W.H. Kay St Vincent’s Institute 41 Victoria Parade Fitzroy, VIC, 3065 Email:
[email protected]
Phone: 61-3-9288-2480 Fax: 61-3-9416-2676
1 Diabetes Publish Ahead of Print, published online May 6, 2015
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Abstract:
Because regulatory T (Treg) cell development can be induced by the same agonist self antigens that induce negative selection, perturbation of apoptosis will impact both negative selection and Treg cell development. But how the processes of thymocyte deletion versus Treg cell differentiation bifurcate and their relative importance for tolerance has not been studied in spontaneous organ-specific autoimmune disease. We addressed these questions by removing a critical mediator of thymocyte deletion, Bim, in the non-obese diabetic (NOD) mouse model of autoimmune diabetes. Despite substantial defects in the deletion of autoreactive thymocytes, Bim deficient NOD (NODBim-/-) mice developed less insulitis and were protected from diabetes. Bim deficiency did not impair effector T cell function, however, NODBim-/mice had increased numbers of Tregs, including those specific for proinsulin, in the thymus and peripheral lymphoid tissues. Increased levels of Nur77, CD5, GITR and phosphorylated I-κB in thymocytes from NODBim-/- mice suggest that autoreactive cells receiving strong TCR signals that would normally delete them ‘escape’ apoptosis and are diverted into the Treg pathway. Paradoxically, in the NOD model, reduced thymic deletion ameliorates autoimmune diabetes by increasing Tregs. Thus modulating apoptosis may be one of the ways to increase antigen specific Tregs and prevent autoimmune disease.
Autoreactive T cells / Apoptosis / Bim / NOD / type 1 diabetes
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The immune system has evolved to allow robust responses against a diverse range of pathogens while preserving self-tolerance to prevent immuno-pathology. Developing self-reactive thymocytes that express T cell receptors (TCR) that bind with high affinity to MHC/self-peptide complexes are usually deleted by apoptosis in the thymus, a process termed deletion or recessive tolerance (1). A complementary mechanism of immune tolerance, termed dominant tolerance, involves the generation of FoxP3-expressing regulatory T cells (Tregs) capable of suppressing the activation of autoreactive, conventional T cells that fail to undergo negative selection in the thymus (for example because the cognate self-antigen is not expressed and presented in the thymus) (2). The importance of Tregs and thymic deletion in preventing autoimmunity is illustrated in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome and in autoimmune polyglandular syndrome-1 patients, respectively. Both diseases result in widespread autoimmune disease including type 1 diabetes (3; 4). Tregs have considerable therapeutic potential in autoimmune diseases such as type 1 diabetes. Tregs that express TCR specific for self antigen can home, along with autoreactive effector T cells, to antigen-expressing target peripheral tissues such as pancreatic islets and their draining lymph nodes. At these sites they can suppress autoreactive effector cells with TCR specificities for the same antigen they recognize but also others - a phenomenon that is called bystander suppression. The signaling events that regulate and presumably distinguish between thymic deletion and Treg cell development remain incompletely defined. Apoptosis is critical for deletion of self-reactive T cells in both the thymus and peripheral lymphoid tissues and thymic Treg development. BIM is a proapoptotic BH3-only protein required for
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deletion of autoreactive thymocytes (5). BIM-deficient mice not only have defects in T cell deletion(6) but also have increased numbers of FoxP3 expressing Tregs (7). How BIM controls the development of Tregs in BIM-deficient mice and the functional significance of increased Tregs in BIM deficient mice have not been studied. Although it has been demonstrated that high affinity TCR interaction with selfantigen is required to trigger both thymic deletion and Treg cell differentiation, these studies involved transgenic expression of antigen or peptide administration, or utilized TCR transgenic T cells present at high clonal frequencies (4; 8-10). However, recent data suggest that thymic Treg cell development utilizes a limited antigenic niche (11; 12), implying that models utilizing ubiquitous antigen presentation or TCR transgenic T cells at high clonal frequencies may not reflect normal events. Therefore, it is important to study autoreactive T cell deletion and Treg cell differentiation in a disease with a polyclonal T cell repertoire and natural self-antigens that are expressed at physiological levels. To this end, we studied the impact of removing a critical mediator of thymocyte apoptosis, BIM, on thymic deletion and Treg cell differentiation in the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes (13). In this model, mice express natural self-antigens and have a polyclonal T cell repertoire. Our data show that reducing thymic deletion by disabling thymic apoptosis ameliorates autoimmune diabetes by increasing Tregs.
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Research design and methods: Mice: BIM-deficient C57BL/6 mice(6) were backcrossed onto the NOD/Lt genetic background for more than 10 generations. NOD.BDC2.5 mice were provided by Drs D. Mathis and C. Benoist (Harvard University). NOD.Rag1-/- and NOD mice expressing GFP under control of the FoxP3 promoter were obtained from The Jackson Laboratory. C57BL/6 and NOD/Lt mice were obtained from the Walter and Eliza Hall Institute animal breeding facility (Kew, Victoria, Australia). Diabetes monitoring and insulitis scoring were performed as described before(14). Mice with two consecutive blood glucose readings >15 mM were considered diabetic. All animal studies were conducted under specific pathogen free conditions at St Vincent’s Institute (Melbourne, Australia) following the guidelines of the institutional animal ethics committee. Genotyping: The BIM gene (Bcl2l11) lies within the Idd13 diabetes susceptibility locus. To confirm the genetic background of NOD.BIM-/- mice and define the congenic interval encompassing the Bcl2l11 null allele, DNA was isolated from tail biopsies from 10th generation backcross mice and genotyped by the Australian Genome Research Facility using the mouse 5K targeted genotyping array run on the Affymetrix GeneChip Scanner 3000 7G MegAllele system. To refine the 129/Svderived congenic interval encompassing the Bcl2l11 null allele, additional polymorphic markers (nucleotide repeats and single nucleotide polymorphisms [SNPs]) were identified using the Mouse Genome Informatics and NCBI databases. DNA was genotyped by standard PCR and gel electrophoresis methods (selected nucleotide repeats) and by The Centre for Applied Genomics (The Hospital for Sick Kids, Canada) using the Illumina medium density linkage panel (1,449 SNPs), which contained SNPs that further refined the congenic interval. NOD.BIM-/- mice were of
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NOD genotype across the whole genome, except for a region on chromosome 2 encompassing the Bcl2l11 locus. The congenic interval is 129-derived between, but not including D2mit277 (Chr2:123,255,831) and D2mit338 (Chr2:130,655,046), and no B6 alleles were detected within this interval. The 129-congenic interval is unlikely to contribute to diabetes protection because 129 mice are predicted to harbor a susceptibility, not a resistance allele in this region of chromosome 2 (15). Antibodies and flow cytometry Antibodies used were anti-CD4-PerCp-Cy5.5 (RM4-5), anti-CD3 (145-2C11), antiCD5 (clone 53-7.3), anti-CD24 (clone M1/69) all conjugated to FITC, anti-CD44AlexaFluor
700
(1M7),
anti-CD69-allophycocyanin
(H1.2F3),
anti-62L-
allophycocyanin-Cy7 (MEL-14) (all from BD Biosciences, San Jose, CA): antiCD11c (N418), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-F4/80 (BM8) all conjugated to efluor450 (all from BioLegend, San Diego, CA) and anti-CD8a-Pacific Orange (5H10) (Invitrogen, Carlsbad, CA). FITC-conjugated antibodies specific for individual TCR Vβ-chains were purchased as a kit from BD Biosciences. Anti-FoxP3 (FJK-16s), anti-Nur77 (12.14) conjugated to PE, anti-Helios-FITC (22F6) and antiGITR-allophycocyanin (DTA-1) were purchased from eBioscience (San Diego, CA). Analysis was performed on a LSRFortessa (Becton Dickinson, Franklin Lakes, NJ) using FlowJo (Treestar, Ashland, OR) software. Intracellular staining was performed according to the manufacturer's specifications using the Cytofix/Cytoperm Plus kit (BD Biosciences, San Diego, CA). MHC tetramers and magnetic bead based cell enrichment: The PE-conjugated I-A (g7) tetramers, Ins B9-23 (HLVERLYLVCGGEG) (16) and control CLIP (AMKRHGLDNYRGYSL), were provided by the NIH tetramer Core facility.
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Tetramer staining and magnetic bead based cell enrichment were performed as described previously (17). Western blotting Whole thymus or sorted thymocyte subsets were homogenized in lysis buffer containing: 50 mmol/LTris-HCl pH 8, 150 mmol/L NaCl, 0.5% Triton X-100 and protease inhibitor cocktail (Sigma). Proteins (8 µg/lane) were resolved by SDS– PAGE and transferred to nitrocellulose, using standard procedures. Monoclonal mouse antibodies to NF-κB p65, phosphorylated IκB-α and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish peroxidase conjugated antimouse Ig antibodies were used. Cell proliferation and Treg cell suppression assays For CFSE dilution assay, T cells were labeled with CFSE as previously described (18). For Treg cell suppression assays, sorted CD25-GITR-CD4+ or CD4+FoxP3CD25- were used as responder T cells and CD4+GITR+ or CD4+FoxP3+ cells were used as Treg cells. CFSE-labeled responder T cells (5x104) were cultured for 72 h with γ-irradiated (4000 Rads) splenocytes (5x104) and anti-CD3 monoclonal antibody (1 µg/mL, 145-2C11, BioXCell) in the presence or absence of the indicated ratio of Treg cells.
Statistical analysis: Data are presented as the mean +/- the standard deviation. Analysis of data was performed using GraphPad Prism (GraphPad Prism Software, San Diego, CA) and the Mann-Whitney test used to assess statistical significance. The Log-Rank test was used to perform survival curve analysis.
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Results: NODBIM-/- mice have impaired deletion of autoreactive T cells. NODBIM-/- mice had higher percentages of CD4+CD8- and CD8+CD4- thymocytes and a reduction in CD4+CD8+ thymocytes compared to control NOD mice (Fig. 1A, B, E and G). The total splenocyte number was significantly increased in NODBIM-/- mice while the proportions of CD4+ T cells, CD8+ T cells and B cells were not statistically different from control NOD mice (Fig. 1A, B, E and G). These findings are similar to BIM-/mice on other genetic backgrounds (5; 6; 19), and suggest impaired thymocyte deletion. In NOD mice, T cells bearing the TCR Vβ3 chain represent only 0.4% of the CD4+ T cells. This is because of clonal deletion of most Vβ3+ immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen presented by class II MHC (20). T cells bearing the TCR Vβ3 chain were significantly increased in the spleen and thymus of NODBIM-/- mice (Fig. 1C and F and supplementary figure 1A), indicating that loss of BIM causes a defect in superantigen-mediated deletion within a polyclonal T cell repertoire on the NOD background. We then assayed the numbers of insulin B9-23 specific CD4+ T cells, a population of T cells known to play a central role in the pathogenesis of type 1 diabetes (21). Proinsulin 2 is expressed in the mouse thymus (22; 23) and NOD mice deficient in proinsulin 2 develop accelerated diabetes, suggesting that high affinity insulinspecific T cells are tolerized in the normal NOD thymus (24). Using the method of staining with tetramer followed by enrichment using magnetic beads (17), we detected rare insulin B9-23 specific CD4+ T cells in the peripheral lymphoid organs (PLO) and thymus of NOD mice. The small number of insulin B9-23 specific CD4+ T cells (16) in the thymus of control NOD mice suggests that deletion of thymocytes with this
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specificity is stringent, but incomplete. This population was significantly increased (P85 % of the Treg population in NODBIM-/- mice expressed Helios (Fig. 3D). Next we investigated whether thymocytes bearing TCR Vβ3 chains and self-reactive insulin B9-23 specific CD4+ T cells that are normally deleted after encountering selfantigen in NOD mice differentiate preferentially into Tregs in NODBIM-/- mice. We examined GFP+ (Foxp3 expressing) insulin B9-23 specific CD4+ T cells and CD4+ T cells bearing TCR Vβ3 chains in transgenic NOD and NODBIM-/- mice that express GFP under control of the FoxP3 promoter (NODBIM-/-FoxP3-GFP mice) (29). Firstly, we found FoxP3 (GFP) expressing T cells bearing TCR Vβ3 and insulin B9-23 specific CD4+ thymocytes were increased in NODBIM-/-FoxP3-GFP mice as compared to NODFoxP3-GFP mice (Fig 4A and B). Secondly, as the majority of Foxp3+ T cells also express the cell surface marker GITR, we also used GITR as a marker of regulatory T cells. We found that GITR expressing Tregs were enriched in the fraction of T cells bearing TCR Vβ3 chains, suggesting that “autoreactive” T cells are enriched within the Treg population in NODBIM-/- mice (supplementary figure 2A). Incomplete deletion of insulin B9-23 specific CD4+ thymocytes in NOD mice yielded both GITR+ and GITR- insulin specific T cells (supplementary figure 2B). Interestingly, insulin B9-23 specific CD4+ T cells were significantly increased (P
