Angela M. Alexander

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Recovery of the Endogenous β Cell Function in the NOD Model of Autoimmune Diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of β cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft‐bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous β cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity‐free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

Restricted TCR Vβ gene expression and enterovirus infection in Type I diabetes: a pilot study

Aims/hypothesis. High frequencies of T-cell receptor (TCR) Vβ7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR Vβ gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients. Methods. T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes. Results. T-cell receptor Vβ7 gene family values were higher in recently-diagnosed diabetic patients (10.5 % ± 3.7) than in age-matched non-diabetic control subjects (5.1 % ± 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i. e., converters), T-cell receptor Vβ7 gene expression showed values consistently above 10 % (p < 0.0005). Long-standing diabetic patients showed lower percentage of Vβ7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of Vβ7 gene transcripts. Conclusion/interpretation. The deviation in the T-cell receptor Vβ repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes. [Diabetologia (2000) 43: 1484–1497]

Gene Combination Transfer to Block Autoimmune Damage in Transplanted Islets of Langerhans

Islet transplantation therapy would be applicable to a wider range of diabetic patients if donor islet acceptance and protection were possible without systemic immunosuppression of the recipient. To this aim, gene transfer to isolated donor islets ex vivo is one method that has shown promise. This study examines the combined effect of selected immunomodulatory and anti-inflammatory genes known to extend the functional viability of pancreatic islet grafts in an autoimmune system. These genes, indoleamine 2,3-dioxygenase (IDO), manganese superoxide dismutase (MnSOD), and interleukin (IL)-1 receptor antagonist protein (IRAP), were transferred to isolated NOD donor islets ex vivo then transplanted to NODscid recipients and evaluated in vivo after diabetogenic T-cell challenge. The length of time the recipient remained euglycemic was used to measure the ability of the transgenes to protect the graft from autoimmune destruction. Although the results of these cotransfections gave little evidence of a synergistic relationship, they were useful to show that gene combinations can be used to more efficiently protect islet grafts from diabetogenic T cells.

Distinct characteristics and features of allogeneic chimerism in the NOD mouse model of autoimmune diabetes.

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.

IMMUNOLOGY OF TYPE 1 DIABETES: Intervention and Prevention Strategies

Type 1 diabetes is the outcome of a progressive and selective destruction of insulin-producing cells in the pancreatic islets of Langerhans. The precise cause and mechanism(s) that trigger the insulin-producing cell destruction are still unclear, although it is well accepted that an autoimmune process plays a central role in diabetes development among genetically susceptible children. Additionally, certain viral infections, especially those caused by Coxsackievirus B, have been associated with the onset of type 1 diabetes. Possible gene therapy-based prevention and intervention strategies are discussed, based on the most accepted models of type 1 diabetes pathogenesis.

An expert system for evaluating risk in type-1 diabetes

Type-1 diabetes is a life-long disorder that often occurs in children or young adults. Susceptibility to type-1 diabetes is believed to be dependent on both genetic and environmental causes. Genetic screening is becoming a common tool for evaluating risk, potentially before the onset of the disease. However, interpreting the results of genetic tests involves many rules taken from the literature on the correlation between risk and genetic markers. We introduce an expert system based approach in which the evaluation of risk for type-1 diabetes can be automated. This method can act as a tool for medical professionals and provides the crucial explanatory component, including citations to the relevant literature. The expert system was developed in conjunction with geneticists. As part of the corresponding clinical trial, the expert system correctly identified the at-risk genetic profiles in a set of 253 subjects evaluated by human experts.