The Insulin-Specific T Cells of Nonobese Diabetic Mice Recognize a Weak MHC-Binding Segment in More Than One Form (original) (raw)
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Proceedings of the National Academy of Sciences, 2011
In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4 + T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA g7 . This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4 + T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA g7 tetramers that bind to most insulin-specific Tcell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4 + T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA g7 in register 3 may explain the escape of insulin-specific CD4 + T cells from the mechanisms that usually eliminate self-reactive T cells.
F1000 - Post-publication peer review of the biomedical literature, 2010
Besides the genetic framework, there are two critical requirements for the development of tissuespecific 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. Although autoimmune diabetic NOD mice exhibit a wide range of autoreactivities 1 , the one directed against the insulin molecule is prominent 2. T cells reactive to insulin were identified in NOD mice and shown to transfer diabetes into non-diabetic mice 3-7. T cells from T cell receptor (TCR) transgenic mice specific for insulin were also diabetogenic 8. Additional findings point to insulin as an important primary autoantigen for disease initiation. The amplitude of insulin expression in the thymus was linked to diabetes incidence 9-12 , and high expression of insulin in APCs using a transgene ablated diabetes development 13 , 14. Moreover, mice expressing a mutant insulin gene product not recognized by T cells did not develop diabetes 7. It is noteworthy that the T cell response to the insulin molecule is highly focused on a segment of the β-chain, encompassing residues 9-23 (B: 9-23) 15-18. This peptide binds poorly (μM affinity and has a high dissociation rate) to the class II major histocompatibility complex (MHC) molecule I-A g7 (ref 18 , 19). How a small protein that yields a very weak binding peptide and circulates at ηM concentrations can function as a significant autoantigen is surprising, and raises a number of important Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Diabetes, 1997
A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic p-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Thl-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cp transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-7 transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-7 characteristic of islet-infiltrating lymphocytes in vehicleinjected NOD controls. Hence, Thl-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.
Proceedings of the National Academy of Sciences of the United States of America, 2014
Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA(g7) in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell cl...
Diabetes, 1997
A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic p-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Thl-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cp transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-7 transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-7 characteristic of islet-infiltrating lymphocytes in vehicleinjected NOD controls. Hence, Thl-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.
Priming and effector dependence on insulin B:9–23 peptide in NOD islet autoimmunity
Journal of Clinical Investigation, 2007
NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients' pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin-positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9-23 (insulin B:9-23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9-23 sequence in their pancreata. Additionally, CD4 + T cells from B16:A-dKO mice immunized with native insulin B:9-23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9-23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9-23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9-23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity. Nonstandard abbreviations used: B16:A, alanine at position 16 of insulin B chain; B16:A-dKO, double Ins1 and Ins2 knockout NOD mice with a mutated B16:A preproinsulin transgene; B16:Y, tyrosine at position 16 of insulin B chain; B16:Y-dKO, double Ins1 and Ins2 knockout NOD mice with a mutated B16:Y preproinsulin transgene; IAA, insulin autoantibody; insulin B:9-23, insulin B chain amino acids 9-23. Conflict of interest: The authors have declared that no conflict of interest exists.
CD4 T cells and their antigens in the pathogenesis of autoimmune diabetes
Current Opinion in Immunology, 2011
Pathogenesis of type 1 diabetes (T1D) is mediated by effector T cells and CD4 Th1 and Th17 T cells have important roles in this process. While effector function of Th1 cells is well established, due to their inherent plasticity Th17 cells have been more controversial. Th17 cells contribute to pathogenicity, but several studies indicate that Th17 cells transfer disease through conversion to Th1 cells in vivo. CD4 T cells are attracted to islets by β-cell antigens which include insulin and the two new autoantigens, chromogranin A and islet amyloid polypeptide, all proteins of the secretory granule. Peptides of insulin and ChgA bind to the NOD class II molecule in an unconventional manner and since autoantigenic peptides may typically bind to MHC with low affinity, it is postulated that post-translational modifications of β-cell peptides could contribute to the interaction between peptides, MHC, and the autoreactive TCR.
A Self MHC Class II -Chain Peptide Prevents Diabetes in Nonobese Diabetic Mice
The Journal of Immunology, 2000
We explored T cell responses to the self class II MHC (I-A g7) -chain-derived peptides in diabetic and prediabetic nonobese diabetic (NOD) mice. We found that one of these immunodominant epitopes of the -chain of I-A g7 molecule, peptide 54-76, could regulate autoimmunity leading to diabetes in NOD mice. T cells from prediabetic young NOD mice do not respond to the peptide 54-76, but T cells from diabetic NOD mice proliferated in response to this peptide. T cells from older nondiabetic mice or mice protected from diabetes do not respond to this peptide, suggesting a role for peptide 54-76-specific T cells in pathogenesis of diabetes. We show that this peptide is naturally processed and presented by the NOD APCs to self T cells. However, the peptidespecific T cells generated after immunization of young mice regulate autoimmunity in NOD mice by blocking the diabetogenic cells in adoptive transfer experiments. The NOD mice immunized with this peptide are protected from both spontaneous and cyclophosphamide-induced insulin-dependent diabetes mellitus. Immunization of young NOD mice with this peptide elicited T cell proliferation and production of Th2-type cytokines. In addition, immunization with this peptide induced peptide-specific Abs of IgG1 isotype that recognized native I-A g7 molecule on the cell surface and inhibited the T cell proliferative responses. These results suggest that I-A g7 (54-76) peptide-reactive T cells are involved in the pathogenesis of diabetes. However, immunization with this peptide at young age induces regulatory cells and the peptide-specific Abs that can modulate autoimmunity in NOD mice and prevent spontaneous and induced diabetes.
Frontiers in Immunology
Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other β-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I...