A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4+ T cell clones not only respond to proinsulin C-peptide (PI40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI40-47-IAPP74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognize self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4+ T cell clones reactive to native PI40-54 epitope and multiple HIPs fused at the same N-terminus (PI40-54) to the degradation products of two highly expressed pancreatic islet proteins, neuropeptide Y (NPY68-74) and amyloid polypeptide (IAPP23-29 and IAPP74-80). We observed that five out of the seven selected SKW3 T cell lines expressing TCRs isolated from CD4+ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI40-54 and HIP epitopes. Crystal structures of two TRAV26-1+-TRBV5-1+ T cell receptors (TCRs) in complex with PI40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI40-54 and HIPs determined that the P2, P7, and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.

Optimal allogeneic islet dose for transplantation in insulin-dependent diabetic Macaca fascicularis monkeys.

Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3+ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen.

We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.

Probing the Aggregation and Immune Response of Human Islet Amyloid Polypeptides with Ligand-Stabilized Gold Nanoparticles.

The use of nanomaterials has recently become an emerging strategy against protein amyloidosis associated with a range of metabolic and brain diseases. To facilitate research in this area, here we first demonstrated the use of hyperspectral imaging (HSI) and COMSOL simulations for reporting the aggregation of human islet amyloid polypeptides (IAPPs), a hallmark of type 2 diabetes, as well as the physical interactions between the peptide and gold nanoparticles (AuNPs) grafted with citrate and poly(ethylene glycol) (PEG400 and PEG3000). We found a distinct anticorrelation between increased IAPP aggregation and decreased spectral red shifts incurred in the AuNP plasmonic resonance. Moreover, Jurkat cells exposed to IAPP and AuNPs were characterized by quantifying their cytokine secretions with a localized surface plasmon resonance (LSPR) immunoassay, where a peak response was registered for the most toxic IAPP oligomers and most suppressed by citrate-coated AuNPs. This study demonstrated the potential of using HSI and LSPR as two new platforms for the facile examination of protein aggregation and their induced immune response associated with amyloid diseases.

Ultraeffective Inhibition of Amyloid Fibril Assembly by Nanobody-Gold Nanoparticle Conjugates.

Amyloid fibril assembly is associated with many human disorders, and to approach an inhibitor of amyloid formation that is effective at ultralow stoichiometric concentrations remains a big challenge. Taking fibril assembly of human islet amyloid polypeptide (IAPP) as a model system, we demonstrate here that conjugating a rationally designed sequence-specific nanobody inhibitor M1 with gold nanoparticles (AuNPs) can significantly enhance the inhibition potency of M1, leading to complete inhibition of IAPP amyloid fibrillation at very low M1:IAPP molar ratios. Thioflavin T kinetics fluorescence assays, dynamic light scattering measurements, far-UV circular dichroism, and transmission electron microscopy all indicate that M1-AuNP conjugates prevent IAPP fibrillation at M1:IAPP molar ratios of as low as 1:50, while free M1 is unable to prevent fibrillation at the same substoichiometric concentrations. This strategy represents a prototype of the facile development of a variety of highly potent amyloid inhibitors with enhanced therapeutic effects.

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole.

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H2O2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at -200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL-1 to 50 pg∙mL-1, with a detection limit of 0.92 fg∙mL-1. This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents. Graphical abstract Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

The toxic nature of murine amylin and the immune responsivity of pancreatic islet to conformational antibody in mice.

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.

Localization of amylin-like immunoreactivity in the striped velvet gecko pancreas.

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactive cells in the pancreas of gecko Homopholis fasciata. Four types of endocrine cells were distinguished: insulin immunoreactive (B cells), pancreatic polypeptide immunoreactive (PP cells), glucagon and pancreatic polypeptide immunoreactive (A/PP cells) and somatostatin immunoreactive cells (D cells). Pancreatic islets contained B, A/PP and D cells, whereas extrainsular regions contained B, D and PP cells. In the pancreatic islets, amylin-like immunoreactive cells corresponded to B cells, but not to A/PP or D cells. In the extrainsular regions, amylin-like immunoreactive cells corresponded to either B or PP cells. Amylin secreted from intrainsular B cells may regulate pancreatic hormone secretion in an autocrine and/or a paracrine fashion. On the other hand, amylin secreted from extrainsular PP and B cells, and/or intrainsular B cells may participate in the modulation of calcium homoeostasis in an endocrine fashion.

Human amylin induces CD4+Foxp3+ regulatory T cells in the protection from autoimmune diabetes.

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet ß cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic ß cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24 h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n = 25) or solvent for control (n = 25) for 7 months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p < 0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10 nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p = 0.045) in the 6th month and 57% (p = 0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p = 0.0013). Expression of transforming growth factor-ß (TGF-ß) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.

Active Immunization Against hIAPP Oligomers Ameliorates the Diabetes- Associated Phenotype in a Transgenic Mice Model.

Type 2 diabetes is characterized by insulin tolerance in target cells followed by a reduction of pancreatic ß-cell mass. Islet amyloid polypeptide oligomeric assemblies were shown to contribute to ß-cell apoptosis by forming discrete pores that destabilize the cellular membrane. We previously characterized α-helical cytotoxic islet amyloid polypeptide oligomers which interact with cell membranes, following a complete internalization that leads to cellular apoptosis. Moreover, antibodies which bind the oligomers and neutralize the cytotoxicity were exclusively identified in the serum of type 2 diabetes patients. Here, we examined the usage of the newly characterized oligomers as an active immunization agent targeting amyloid self- assembly in a diabetes-associated phenotype transgenic mice model. Immunized transgenic mice showed an increase in hIAPP-antibody serum titer as well as improvement in diabetes-associated parameters. Lower fasting blood glucose levels, higher insulin, and lower islet amyloid polypeptide accumulation were observed. Furthermore, antibodies derived from the immunized mice reduced hIAPP oligomers cytotoxicity towards ß-cells in a dose-dependent manner. This study highlights the significance of targeting the early amyloid self-assembly events for potential disease management. Furthermore, it demonstrates that α-helical oligomers conformers are valid epitope for the development of future immunization therapy.

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.

Differential Activation of Innate Immune Pathways by Distinct Islet Amyloid Polypeptide (IAPP) Aggregates.

Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1ß secretion by stimulating both the synthesis and processing of proIL-1ß, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1ß synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2- (TLR2-) dependent stimulus for NF-κB activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Non-amyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1ß secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1ß secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPP-induced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.

The Anti-Amyloid-ß Monoclonal Antibody 4G8 Recognizes a Generic Sequence-Independent Epitope Associated with α-Synuclein and Islet Amyloid Polypeptide Amyloid Fibrils.

Recently we reported that several monoclonal antibodies that recognize linear segments of amyloid-ß (Aß) also recognize amyloid fibrils, but not monomers of unrelated sequences, indicating that recognition of a linear sequence segment is not a reliable indicator of sequence specificity. We asked whether any of the commonly used commercially available Aß antibodies also recognize fibrils of unrelated sequence. Here we report that 4G8, which recognizes residues 18-23 of the Aß sequence and is widely believed to be sequence-specific, also recognizes fibrils formed from α-synuclein and islet amyloid polypeptide (IAPP). The recognition of amyloid fibrils is aggregation-dependent because 4G8 does not recognize α-synuclein or IAPP monomer. 4G8 also stains fibrillar α-synuclein aggregates in human multiple system atrophy brain where it colocalizes with anti-α-synuclein monoclonal antibody LB509 immunoreactivity. We also found that LB509 recognizes Aß fibrils, but not monomer, indicating that generic epitope-reactive antibodies are also produced in response to α-synuclein immunization. Taken together, our results indicate that generic fibril conformational epitope specificity may be a pervasive property among monoclonal antibodies raised against amyloid-forming antigens and that the specificity of their immunoreactivity should be rigorously established and otherwise interpreted with caution.

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.

Amylin-like immunoreactivity in pancreatic X cells of the black-spotted frog Rana (Pelophylax) nigromaculata.

tIn this study, we investigated the presence of ovoid or ellipsoidal amylin-immunoreactive cells of the pancreatic islets of the black-spotted frog Rana (Pelophylax) nigromaculata. Using double immunofluorescent staining, all amylin-immunoreactive cells were shown to be immuno-negative for insulin, glucagon, and somatostatin, and they were often observed in peripheral regions of clusters of insulin-immunoreactive cells. Under immunoelectron microscopy, amylin-immunoreactive signals were detected on the secretory granules in a specific type of endocrine cells. From our results, we conclude that the amylin-immunoreactive cells correspond to X cells among the 4 distinct types of endocrine cells (B, A/PP, D, and X) previously identified in the frog. Amylin secreted from X cells may regulate the hormone secretion from A/PP cells and/or B cells through a paracrine mechanism.

Islet amyloid polypeptide is not a target antigen for CD8+ T-cells in type 2 diabetes.

BACKGROUND: Type 2 diabetes (T2D) is a chronic metabolic disorder in which beta-cells are destroyed. The islet amyloid polypeptide (IAPP) produced by beta-cells has been reported to influence beta-cell destruction. OBJECTIVE: To evaluate if IAPP can act as an autoantigen and therefore, to see if CD8+ T-cells specific for this protein might be present in T2D patients. METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from human leukocyte antigen (HLA)-A2+ T2D patients and non-diabetic healthy subjects. Cells were then screened for peptide recognition using ELISPOT assay for the presence of IFN-γ producing CD8+ T-cells against two HLA Class I-restricted epitopes derived from IAPP (IAPP5-13 and IAPP9-17) and common viral antigenic minimal epitopes Flu MP 58-66, CMV495-503, EBV280-288 and HIV77-85 as controls. RESULTS: A total of 36.4% of patients and 56.2% of healthy subjects showed a response against IAPP5-13 peptide. No significant difference in response against this peptide was noted between the patients and the healthy donors. With respect to peptide IAPP9-17, although healthy subjects showed a higher mean number of spot forming cells than the patients, the difference was not significant; 36.4% of patients and 37.5% of controls responded to this peptide. The response of healthy subjects to the common viral peptides was stronger than that of the patients, though the result was not significant. CONCLUSIONS: It is unlikely that IAPP would be a target for CD8+ T-cells in diabetic patients; however, the trend observed toward a lower response of T2D patients against IAPP and common viral peptides may imply a decreased immune response in these patients.

High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus.

AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. RESULTS: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels. CONCLUSIONS/INTERPRETATION: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies.

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

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.

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

AIM: To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. METHODS: The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. RESULTS: A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. CONCLUSION: The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.