Autoimmune Diabetes

THE AUTOIMMUNE DIABETES PROGRAM

Our investigations focus mostly on examining pancreatic islets at the early start of the diabetic process- we do this by isolating islets and examining the resident myeloid cells, the endothelial and mesenchymal cells, as well as the first entering T cells. Islets are mini-organs where the initiating events and interactions among  cells can be examined in great detail. Cells can be isolated and examined by different functional and gene expression signatures. In the NOD mouse, diabetogenesis is already evident by the third week of life. Our goals are to identify the initial cellular and molecular events that initiate and perpetuate this autoimmune disease. Islets normally have a population of macrophages that is distinct from those that inhabit the exocrine pancreas.These  macrophages have been extensively analyzed by us since they are vital for islet homeostasis and in the case of NOD, in the initiation of diabetes. The autoimmune process starts in islets with the entrance of CD4+ T cells and a small number of dendritic cells many of which are DC1 expressing the XCR1 protein. Many of the early T cells react to insulin peptides. We have extensively examined the insulin reactive T cells and characterized the insulin epitopes that are recognized by them.  We examine how insulin peptides are presented to these insulin T cells, the role of the unique MHC class II molecules of the NOD [I-Ag7] and the various cellular interactions.

We are always looking for talented graduate students and post-docs interested in studying the nuances of autoimmune diabetes disease initiation

The following are key publications that review our major findings.

Reviews

Unanue ER. Antigen presentation in the autoimmune diabetes of the NOD mouse. Annu Rev Immunol. 2014;32:579-608. PMID: 24499272

Calderon B, Carrero JA, Unanue ER. The central role of antigen presentation in islets of Langerhans in autoimmune diabetes. Curr Opin Immunol. 2014, 26:32-40 PMID: 24556398

Unanue ER, Ferris ST, Carrero JA: The role of antigen presenting cells and the
presentation of insulin in the initiation of autoimmune diabetes in the NOD mouse
Immunol Rev 2016; 272:183-201 PMID: 27319351

Carrero JA, Ferris ST, Unanue ER: Macrophages and dendritic cells in islets of
Langerhans in diabetic autoimunity: a lesson on cell interactions in a mini- organ. Curr Op in Immunol 2016; 43: 54-59

Wan X, Unanue ER: Unique features in the presentation of insulin epitopes in autoimmune diabetes: an update. Curr Opin Immunol 2017; 46: 30-37

Beta cell biology:interactions with the immune system

Xiaoxiao Wan, Bernd H. Zinselmeyer, Pavel N. Zakharov, Anthony N. Vomund, Ruth Taniguchi, Laura Santambrogio, Mark S. Anderson, Cheryl F. Lichti & Emil R. Unanue. Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides, Nature, 2018, in press, 10.1038/s41586-018-0341-6  Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice. A major set of pathogenic CD4 T cells specifically recognizes the 12–20 segment of the insulin B-chain (B:12–20), an epitope that is generated from direct presentation of insulin peptides by antigen presenting cells. These T cells do not respond to antigen presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13–21. CD4 T cells that recognize B:12–20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13–21 have only a minor role in autoimmunity. Although presentation of B:12–20 is evident in the islets, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in -cell granules of mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

The specificity of T cells: T:B interactions

Levisetti MG, Suri A, Petzold SJ, Unanue ER:  The insulin-specific T cells of NOD mice recognize a weak MHC-binding segment in more than one register.  J Immunol 2007;178: 6051-6057. PMID17475829

Levisetti MG, Lewis DM, Suri A, Unanue ER: Weak proinsulin peptide-MHC complexes are targetted in autoimmune diabetes in mice. Diabetes 2008. 57: 1852-1860. PMC2453633

Mohan JF, Levisetti MG, Calderon B, Herzog JW, Petzold SJ, Unanue ER:  Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes.  Nat Immunol  2010;11:350-354.  PMC3080751  Besides the genetic framework, there are two critical requirements for the development of tissue-specific autoimmune diseases. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic NOD mice. A set of autoreactive CD4+ T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by the antigen presenting cells (APC) escape thymic control, participate in diabetes and can cause disease. We also find that APCs situated in close contact with the beta cells in the islets of Langerhans bear vesicles with the antigenic insulin peptides and activate the peptide-specific T cells. These findings may be relevant for other cases of endocrine autoimmunity.

Mohan JF, Petzold SJ, Unanue ER:  Register shifting of an autoimmune insulin peptide-MHC II complex allows for the escape of diabetogenic T cells from negative selection.  J Exp Med 2011;208:2375-2383.  PMC3256971  In nonobese diabetic (NOD) mice, two sets of autoreactive CD4+ T cells recognize the B:9–23 segment of the insulin B chain. One set, type A, recognizes insulin presented by antigen-presenting cells (APCs). These T cells are highly deleted in the thymus. The second set, type B, does not recognize insulin protein but reacts with soluble B chain peptide. This set is not deleted in the thymus but is activated in the islets of Langerhans. In this study, we examine the specificity of these two types of T cells. The protein-reactive set recognizes the stretch of residues 13–21 of the insulin B chain. The set reactive to peptide only recognizes the stretch from residues 12–20. A single amino acid shift of the B chain peptide bound to I-Ag7determines whether T cells recognize peptides generated by the processing of insulin, and consequently their escape from thymic purging. Biochemical experiments indicate that peptides bound in the 13–21 register interact more favorably with I-Ag7 than peptides that bind in the 12–20 register. Thus, self-reactive T cells can become pathogenic in the target organ where high concentrations of antigen and/or differences in intracellular processing present peptides in registers distinct from those found in the thymus.

Mohan JF, Calderon B, Anderson MS, Unanue ER:  Pathogenic CD4 T cells recognizing an unstable peptide of insulin are directly recruited into islets bypassing local lymph nodes.  J Exp Med 2013;210:2403-2414.  PMC3804950  In the nonobese diabetic mouse, a predominant component of the autoreactive CD4+ T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12–20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4+ T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on β cells and along the vessel walls.

Nayak DK, Calderon B, Vomund AN, Unanue ER:  In NOD mice ZnT8 reactive T cells are weakly pathogenic but can participate in diabetes under inflammatory conditions.  Diabetes 2014;63:3438-3448.  PMCID4171664

Wan X, Thomas JW, Unanue ER: Class-switched anti-insulin antibodies originate from unconventional antigen presentation in multiple lymphoid sites.  J Exp Med 2016:213: 967-978.  PMCID27139492. Autoantibodies to insulin are a harbinger of autoimmunity in type 1 diabetes in humans and in non-obese diabetic mice. To understand the genesis of these autoantibodies, we investigated the interactions of insulin-specific T and B lymphocytes using T cell and B cell receptor transgenic mice. We found spontaneous anti-insulin germinal center (GC) formation throughout lymphoid tissues with GC B cells binding insulin. Moreover, because of the nature of the insulin epitope recognized by the T cells, it was evident that GC B cells presented a broader repertoire of insulin epitopes. Such broader recognition was reproduced by activating naive B cells ex vivo with a combination of CD40 ligand and interleukin 4. Thus, insulin immunoreactivity extends beyond the pancreatic lymph node–islets of Langerhans axis and indicates that circulating insulin, despite its very low levels, can have an influence on diabetogenesis.

Characterization of the islet macrophages

Calderon B, Carrero JA, Ferris ST, Sojka DK, Moore L, Epelman S, Murphy KM, Yokoyama WM, Randolph GJ, Unanue ER. The pancreas anatomy conditions the origin and properties of resident macrophages.  J Exp Med. 2015;212:1497-512.  We examine the features, origin, turnover, and gene expression of pancreatic macrophages under steady state. The data distinguish macrophages within distinct intrapancreatic microenvironments and suggest how macrophage phenotype is imprinted by the local milieu. Macrophages in islets of Langerhans and in the interacinar stroma are distinct in origin and phenotypic properties. In islets, macrophages are the only myeloid cells: they derive from definitive hematopoiesis, exchange to a minimum with blood cells, have a low level of self-replication, and depend on CSF-1. They express Il1b and Tnfa transcripts, indicating classical activation, M1, under steady state. The interacinar stroma contains two macrophage subsets. One is derived from primitive hematopoiesis, with no interchange by blood cells and alternative, M2, activation profile, whereas the second is derived from definitive hematopoiesis and exchanges with circulating myeloid cells but also shows an alternative activation profile. Complete replacement of islet and stromal macrophages by donor stem cells occurred after lethal irradiation with identical profiles as observed under steady state. The extraordinary plasticity of macrophages within the pancreatic organ and the distinct features imprinted by their anatomical localization sets the base for examining these cells in pathological conditions.

Carrero JA, McCarthy DP, Ferris ST, Wan X, Hu H, Zinselmeyer BH, Vomund AN, Unanue ER. Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice.  Proc Natl Acad Sci U S A. 2017 ;114: E10418-E10427. PMID 29133420

Zinselmeyer BH, Vomund AT, Saundrs B, Johnson M, Carrero JA, Unanue    ER The resident pancreatic macrophages in pancreatic islets are constantly probing their local environment capturing beta cell granules and microparticles. Diabetologia, 2018, 61: 1374-1383

Importance of dendritic cells

Ferris ST, Carrero JA, Mohan JF, Calderon B, Murphy KM, Unanue ER:  A minor subset of Batf3-dependent antigen presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes.  Immunity 201441:657-669 PMCID: PMC4220295 Autoimmune diabetes is characterized by inflammatory infiltration; however the initiating events are poorly understood. We found that the islets of Langerhans in young non-obese diabetic (NOD) mice contained two antigen presenting cell (APC) populations: a major macrophage and a minor CD103+ dendritic cell (DC) population. By four weeks of age, CD4+ T cells entered islets coincident with an increase of CD103+ DCs. In order to examine the role of the CD103+ DCs in diabetes, we examined Batf3-deficient NOD mice that lacked the CD103+ DCs in islets and pancreatic lymph nodes. This led to a lack of autoreactive T cells in islets and, importantly, no incidence of diabetes. Additional examination revealed that presentation of major histocompatibility complex (MHC) class I epitopes in the pancreatic lymph nodes was absent with a partial impairment of MHC class II presentation. Altogether, this study reveals that CD103+ DCs were essential for autoimmune diabetes development.

Cellular Interactions in islets-Diabetes progression

Calderon B, Carrero JA, Miller MJ, Unanue ER: Entry of diabetogenic T cells into islets induces changes that lead to amplification of the cellular response.  Proc Natl Acad Sci USA 2011;108:1561-1566.  PMC3029745 Understanding the entry of autoreactive T cells to their target organ is important in autoimmunity because this entry initiates the inflammatory process. Here, the events that lead to specific localization of diabetogenic CD4 T cells into islets of Langerhans resulting in diabetes were examined. This was evaluated in two models, one in which T cells specific for a hen-egg white lysozyme (HEL) peptide were injected into mice expressing HEL on β cells and the other using T cells in the nonobese diabetic mouse strain, which develops spontaneous diabetes. Only T cells specific for β-cell antigens localized in islets within the first hours after their injection and were found adherent to intraislet dendritic cells (DCs). DCs surrounded blood vessels with dendrites reaching into the vessels. Localization of antigen-specific T cells did not require chemokine receptor signaling but involved class II histocompatibility and intercellular adhesion molecule 1 molecules. We found no evidence for nonspecific localization of CD4 T cells into normal noninflamed islets. Thus, the anatomy of the islet of Langerhans permits the specific localization of diabetogenic T cells at a time when there is no inflammation in the islets.

Carrero JA, Calderon B, Towfic F, Artyomov MN, Unanue ER:  Defining the transcriptional and cellular landscape of type 1 diabetes in the NOD mouse.  PLoS One 2013;8:e59701.  PMC3608568

Vomund AN, Zinselmeyer BH, Hughes J, Calderon B, Valderrama C, Ferris ST, Wan X, Kanekura K, Carrero JA, Urano F, Unanue ER. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.  Proc Natl Acad Sci U S A. 2015;112:5496-502.  Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

Carrero JA, McCarthy DP, Ferris ST, Wan X, Hu H, Zinselmeyer BH, Vomund AN, Unanue ER. Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice.  Proc Natl Acad Sci U S A. 2017 ;114: E10418-E10427. 29133429  Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti-CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20-40 wk of age. Diabetes occurred in the anti-CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

Early investigations on the characterization of the class II-MHC, I-Ag7

Suri A, Walters JJ, Gross M, Unanue ER:  Natural peptides selected by diabetogenic DQ8 and murine I-Ag7 molecules show common sequence specificity.  J Clin Invest 2005;115:2268-2276. PMID16075062 In this study, a large number of naturally processed peptides was isolated and identified from the human diabetes-susceptible class II MHC molecules HLA-DQ8 (DQA1*0301,DQB1*0302) and from murine I-Ag7 species, both of which are expressed in genetically identical APC lines. The peptides presented during the processing of autologous proteins were highly selective in showing sequence specificity, mainly consisting of 1 or more acidic residues at their C terminus. Testing for binding to the MHC molecules revealed that the position 9 (P9) acidic residues of the peptides contributed decisively to binding. For HLA-DQ8, the P1 residue, which was also an acidic amino acid, influenced binding positively. Both HLA-DQ8 and I-Ag7 selected for common peptides that bound in the same register. There was no evidence for selection of peptides having nonspecific or promiscuous binding. Thus, diabetogenic class II MHC molecules are highly selective in terms of the peptides presented by their APCs, and this is governed by the features of their P9 anchor pocket. These results are in striking contrast to those from studies examining synthetic peptide or phage display libraries, in which many peptides were shown to bind.

Chang KY, Suri A, Unanue ER:  Predicting peptides bound to I-Ag7 class II histocompatibility molecules using a novel expectation-maximization alignment algorithm.  Proteomics 2007;7:367-377. PMID17211830

Suri A, Walters JJ, Rohrs HW, Gross ML, Unanue ER: First signature of islet -cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules.  J Immunol 2008;180:3849-3856. PMID18322192

Chang KY, Unanue ER: Prediction of HLA-DQ8 cell peptidome using a computational program and its relationship to autoreactive T cells.  Int Immunol 2009;21:705-713. PMC2686615

Initial investigations

Levisetti MG, Suri A, Vidavsky I, Gross ML, Kanagawa O, Unanue ER:  Autoantibodies and CD4 T cells target a beta-cell retroviral envelope protein in NOD mice.  Int Immunol 2003;15:1473-1483. PMID14645156

Suri A, Shimizu J, Katz JD, Sakaguchi S, Unanue ER, Kanagawa O:  Regulation of autoimmune diabetes by non-islet-specific T cells - A role for the glucocorticoid-induced TNF receptor.  Eur J Immunol 2004;34:447-454. PMID14768049

Levisetti MG, Suri A, Frederick K, Unanue ER:  Absence of lymph nodes in NOD mice treated with lymphotoxin- receptor immunoglobulin protects from diabetes.  Diabetes 2004;53:3115-3119. PMID15561941

Suri A, Calderon B, Esparza TJ, Frederick K, Bittner P, Unanue E:  Immunological reversal of autoimmune diabetes without hematopoietic replacement of beta cells.  Science 2006;311:1778-1780. PMID16556846