Rocky L. Baker

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Diabetogenic T-Cell Clones Recognize an Altered Peptide of Chromogranin A

Chromogranin A (ChgA) has been identified as the antigen target for three NOD-derived, diabetogenic CD4 T-cell clones, including the well-known BDC-2.5. These T-cell clones respond weakly to the peptide WE14, a naturally occurring proteolytic cleavage product from ChgA. We show here that WE14 can be converted into a highly antigenic T-cell epitope through treatment with the enzyme transglutaminase (TGase). The WE14 responses of three NOD-derived CD4 T-cell clones, each with different T-cell receptors (TCRs), and of T cells from BDC-2.5 TCR transgenic mice are increased after TGase conversion of the peptide. Primary CD4 T cells isolated from NOD mice also respond to high concentrations of WE14 and significantly lower concentrations of TGase-treated WE14. We hypothesize that posttranslational modification plays a critical role in the generation of T-cell epitopes in type 1 diabetes.

CD40 on NOD CD4 T cells contributes to their activation and pathogenicity

Our goals in this study were to investigate conditions under which T cells from NOD mice express CD40 and to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity in T1D. Using CD40-positive diabetogenic T cell clones and CD4 T cells from NOD mice, we examined expression of CD40 upon activation through the TCR and costimulation through either CD28 or CD40. Our results indicate that CD40 expression is increased upon activation with antigen/MHC and that activation of NOD CD4 T cells through TCR/CD40 rapidly induced CD40 expression. Furthermore, CD40 costimulation promoted T cell proliferation to the same extent as costimulation through TCR/CD28. Importantly, costimulation of CD4 T cells through CD40 also interfered with T cell homeostasis by altering regulation of CTLA-4 expression. Through CD40-CD154 blocking studies, we demonstrated that signaling between T cells through CD40 and its ligand contributes to activation of pathogenic T cells and that blocking CD40 on T cells abrogates their ability to transfer diabetes. Thus, costimulation through CD40 on NOD T cells contributes to their pathogenicity by providing additional pathways for activation and by inhibiting upregulation of CTLA-4 during T cell activation.

Islet Amyloid Polypeptide Is a Target Antigen for Diabetogenic CD4+ T Cells

OBJECTIVE To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9. RESEARCH DESIGN AND METHODS To purify β-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of β-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice. RESULTS Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9. CONCLUSIONS IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.

An insulin-IAPP hybrid peptide is an endogenous antigen for CD4 T cells in the non-obese diabetic mouse.

BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule-insulin C-peptide and IAPP-donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.

Cutting Edge: Nonobese Diabetic Mice Deficient in Chromogranin A Are Protected from Autoimmune Diabetes

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the β cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA−/− mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA+/+ mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

Cutting Edge: CD4 T Cells Reactive to an Islet Amyloid Polypeptide Peptide Accumulate in the Pancreas and Contribute to Disease Pathogenesis in Nonobese Diabetic Mice

We previously reported a peptide KS20 from islet amyloid polypeptide (IAPP) to be the target Ag for a highly diabetogenic CD4 T cell clone BDC-5.2.9. To track IAPP-reactive T cells in NOD mice and determine how they contribute to the pathogenesis of type 1 diabetes, we designed a new I-Ag7 tetramer with high affinity for BDC-5.2.9 that contains the peptide KS20. We found that significant numbers of KS20 tetramer+ CD4 T cells can be detected in the pancreas of prediabetic and diabetic NOD mice. To verify pathogenicity of IAPP-reactive cells, we sorted KS20 tetramer+ cells and cloned them from uncloned T cell lines isolated from spleen and lymph nodes of diabetic mice. We isolated a new KS20-reactive Th1 CD4 T cell clone that rapidly transfers diabetes. Our results suggest that IAPP triggers a broad autoimmune response by CD4 T cells in NOD mice.

T cells interact with T cells via CD40-CD154 to promote autoimmunity in type 1 diabetes

We have investigated the role of CD40 signaling in islet‐reactive, diabetogenic CD4+ Th1 T‐cell clones. Using multispectral flow cytometry, we showed that CD40 and CD154 are co‐expressed and form complexes on the surface of activated T cells. We also demonstrate that activated Tcells can transactivate CD4+CD40+ T cells through the CD40‐CD154 pathway. To investigate the role of CD40 signaling on Th1 cells, we used the diabetogenic clone BDC‐5.2.9 retrovirally transduced with a truncated form of the CD40 molecule to produce a CD40 dominant‐negative T‐cell clone. Upon challenge with antigen in vitro, the production of IFN‐γ by BDC‐5.2.9 CD40DN was greatly reduced and, in vivo, the dominant‐negative variant was unable to induce diabetes. Transduction with the CD40DN vector was also effective in preventing transfer of disease by primary NOD CD4+ T cells. Ex vivo analysis of pancreatic infiltrates after transfer of BDC‐5.2.9 CD40DN cells revealed an overall reduction of cell numbers and cytokine production by both T cells and macrophages. These data indicate that CD40 is an important signaling molecule on autoreactive CD4+ T cells and contributes to their pathogenic effector function.

Novel autoantigens for diabetogenic CD4 T cells in autoimmune diabetes

Autoreactive CD4 T cells play a central role in the development of type 1 diabetes. The BDC panel of diabetogenic T cell clones was originally isolated from non-obese diabetic mice and has been used to study the role of autoreactive CD4 T cells and T cell autoantigens in the development of diabetes. Recent studies by our group have led to the identification of two new target antigens for clones of this panel. This review describes the proteomic strategy used for antigen identification, the antigens identified, and the potential contribution of post-translational modification to autoantigen generation. In addition, we compare peptide epitopes for the T cell clones and discuss their potential applications in investigating the role of T cell autoantigens in the pathogenesis and regulation of disease.

The T Cell Repertoire-Diversifying Enzyme TSSP Contributes to Thymic Selection of Diabetogenic CD4 T Cell Specificities Reactive to ChgA and IAPP Autoantigens.

Multiple studies highlighted the overtly self-reactive T cell repertoire in the diabetes-prone NOD mouse. This autoreactivity has primarily been linked to defects in apoptosis induction during central tolerance. Previous studies suggested that thymus-specific serine protease (TSSP), a putative serine protease expressed by cortical thymic epithelial cells and thymic dendritic cells, may edit the repertoire of self-peptides presented by MHC class II molecules and shapes the self-reactive CD4 T cell repertoire. To gain further insight into the role of TSSP in the selection of self-reactive CD4 T cells by endogenous self-Ags, we examined the development of thymocytes expressing distinct diabetogenic TCRs sharing common specificity in a thymic environment lacking TSSP. Using mixed bone marrow chimeras, we evaluated the effect of TSSP deficiency confined to different thymic stromal cells on the differentiation of thymocytes expressing the chromogranin A–reactive BDC-2.5 and BDC-10.1 TCRs or the islet amyloid polypeptide–reactive TCR BDC-6.9 and BDC-5.2.9. We found that TSSP deficiency resulted in deficient positive selection and induced deletion of the BDC-6.9 and BDC-10.1 TCRs, but it did not affect the differentiation of the BDC-2.5 and BDC-5.2.9 TCRs. Hence, TSSP has a subtle role in the generation of self-peptide ligands directing diabetogenic CD4 T cell development. These results provide additional evidence for TSSP activity as a novel mechanism promoting autoreactive CD4 T cell development/accumulation in the NOD mouse.