Relationship of Islet Autoantibodies to Tcell Responses in Type 1 Diabetes

The analysis of islet autoantibodies provides a means by which individuals may be identified at early stages of pancreatic autoimmunity and gives an indication of the risk of development of type 1 diabetes. T cells are likely to be more directly related to diabetes development than are antibodies, but it is unclear to what extent autoantibody secretion reflects a pathogenic T-cell response. Measures of disease-related T-cell responses in diabetes and an understanding of the relationships of these to autoantibodies are essential for further advances in our understanding of the etiology of type 1 diabetes and to provide an accurate assessment of autoimmune status. Indicators of T-cell autoimmunity will become particularly valuable for monitoring signs of disease recurrence following immune intervention and islet transplantation.

Analysis of T-cell reactivity in diabetes has proven to be an extremely complex task. Assessment of these responses requires that the lymphocyte population be isolated from the host individual and maintained in a viable state in culture while being stimulated with the antigen of interest. The outcome is then measured in some form of readout. The fundamental sensitivity of the T-cell population to external factors may result in disease-specific responses being distorted when attempts are made to assess them in vitro. Accordingly, the modulating effects on T-cell responses of nearly every facet of T-cell culture and assay continue to be hotly debated in international T-cell workshops. Factors likely to be important include the source and purity of antigen, serum supplements, culture medium, cell numbers and density in culture, the nature of the antigen-presenting cell (APC), the use of fresh versus cryopreserved cells, and the inclusion of response-potentiating factors [140-146]. Meaningful interpretation of T-cell responses in human diabetes has been hampered by a lack of appropriate controls. An added complication is that the T-cell population of most interest in type 1 diabetes resides in the pancreas, but this population is normally inaccessible. Any reliable method for measuring antigen-specific T-cell responses in diabetes must therefore have sufficient sensitivity to detect those islet-reactive T cells that migrate to the periphery, where they occur at extremely low frequencies, typically one in 105 cells [147, 148].

Initial studies to detect islet antigen-specific cells among the peripheral T-cell population from diabetic patients relied on the capacity of these cells to proliferate in response to exogenous antigen. Evidence of mitotic activity was typically provided by assessing changes in cellular DNA content through the incorporation of radiolabeled nucleotides [149]. Knowledge of autoantibody targets led to purified preparations of recombinant GAD65, insulin, and IA-2 (expressed in E. coli, yeast, or insect cells) being investigated as potential targets of pathogenic T cells. Increased proliferative responses to these autoantigens were detected in peripheral blood mononuclear cells (PBMC) isolated from type 1 diabetic patients [150-152]. Similarly high T-cell reactivity was detected in PBMC from relatives of diabetic patients identified as being at-risk for diabetes through possession of islet autoantibodies, but not from healthy, islet autoantibody-negative controls. A primary or dominant target of the T-cell response in type 1 diabetes was not identified among these candidates but, as with autoantibody reactivity, those most at risk of developing disease have T-cell autoreactivity to multiple autoantigens [153, 154].

Proliferation assays for T-cell responses to islet-cell antigens also suggested an inverse correlation with autoantibody levels [155, 156], leading to the suggestion that individuals who exhibit higher concentrations of antibodies might be expected to progress more slowly to clinical disease. Such observations have even thrown into question the use of islet autoantibodies as markers for predicting type 1 diabetes [157]. However, the reliability of T-cell response data obtained using recombinant autoantigen preparations, which are probably contaminated with highly antigenic bacterial proteins, has been questioned, and disease-associated responses to such preparations have not been reproduced in T-cell workshops [145, 158]. Furthermore, T-cell proliferation assays yield little information on the nature of the response to the antigen of interest, and results obtained may be clouded by effects of regulatory cells present in the culture [159]. As a result of these problems, the emphasis of T-cell studies has turned to the monitoring of specific cytokines secreted in response to peptide antigen stimulus.

Early assays to detect cytokine secretion by T cells relied upon ELISA of culture supernatants following the incubation of T cells with antigen and APC [160]. While being adequate for assessing strong responses that result from viral or bacterial challenges, these assays unfortunately lack the sensitivity required to detect the activity of rarer autoantigen-specific T cells. Instead, the enzyme-linked immunospot assay (ELISPOT) has become the method of choice for analyzing autoreactive T-cell responses in diabetes. This technique represents a significant advance on standard ELISA methodology in that cytokine production is assessed at the single-cell level and is able to provide an estimate of frequency of individual T cells secreting a specific cytokine in response to antigen or pep-

15.4 Relationship of Islet Autoantibodies to T-cell Responses in Type 1 Diabetes | 341

tide [161]. ELISPOT is currently sensitive to a level of one in 105-106 cells [162164]. The assay involves the capture of cytokine directly onto the surface of an antibody-coated microtiter plate as it is secreted. Further treatment with a combination of anti-cytokine antibodies and detection reagents then allows individual cells secreting cytokine to be visualized in the form of spots on the plate, where the size and intensity are proportional to the amount of cytokine secreted per cell.

A number of strategies have been employed to identify the immunodominant T-cell epitopes within the known type 1 diabetes autoantigens for use in these assays. These include the screening of naturally processed peptides eluted from type 1 diabetes-associated HLA [165-168] or synthetic peptides spanning an autoantigen [152, 169-171] for their ability to stimulate responses in either T-cell lines generated against purified antigen or peripheral blood lymphocytes isolated from type 1 diabetes subjects. Using such approaches, T-cell responses have been identified in type 1 diabetes that target the (pro)insulin residues B9-23, B11-C24, B20-C4, C18-A1, C28-A21, and C35-50 [172-174]. For GAD65, a number of T-cell epitopes have been identified spanning residues 115-130, 206220, 247-285, 481-495, and 555-570 [165, 166, 168-171]. Meanwhile, multiple independent studies have identified a focus of T-cell reactivity to IA-2 against two central regions of the PTP domain, residues 787-817 and 831-869 [152, 167, 175, 176].

Comparison of these T-cell determinants with previously identified autoantibody epitopes (Fig. 15.1) suggests that these may frequently overlap, raising the question as to whether T- and B-cell responses at the epitope level are related [102, 177]. Antigen-specific B cells and the soluble antibodies that they secrete are able to play a major role in shaping T-cell responses through their ability to act as highly efficient APCs [178]. Examples of both B cell-mediated enhancement and suppression of T-cell epitopes in type 1 diabetes have been reported. In one study, stimulation of a T-cell hybridoma recognizing the GAD 274-286 epitope was greatly enhanced when the APCs were exposed to GAD65 protein complexed with anti-GAD serum antibodies, rather than the recombinant antigen alone [179]. In another study, GAD65-specific B cells were able to enhance the processing and presentation of T-cell determinants when they resided outside the antibody epitope region, but any overlap between the antibody and T-cell epitopes led to a dominant suppression of antigen presentation [177]. The possible suppression of diabetogenic T-cell responses by autoantibodies has led to the suggestion that recombinant Fabs may represent a novel tool by which to modulate the disease process [77].

When T-cell responses were determined against proinsulin peptides in antibody-positive, first-degree relatives of type 1 diabetes patients, a significant positive correlation was seen between insulin autoantibody levels and the T-cell response [154]. Further refinement of these experiments revealed that T cells from individuals with increased levels of IAAs prior to the onset of clinical disease characteristically secreted cytokines of a Th2 or regulatory (Tr) phenotype (IL-4, IL-5, and/or IL-10) in response to synthetic proinsulin peptides [180-182]. By contrast, T cells from individuals already undergoing insulin therapy for type 1 diabetes, and therefore responding to exogenous injected insulin, exhibited increased production of the Th1 cytokine IFN-y [181]. Recent results from our own laboratory also reveal a positive association between T-cell responses to peptides representing residues 831-860 of IA-2 and autoantibodies to the 96/3 epitope region of the molecule (residues 795-889 of the IA-2 PTP; Fig. 15.1) that overlaps this region (S. Weenink, unpublished observations). Diabetic patients with higher levels of autoantibodies to the 96/3 epitope region were found by ELISPOT to have increased frequency of T cells secreting IL-10 in response to synthetic peptides spanning the 831-860 region.

Together, these observations challenge the belief that proinflammatory Th1 cy-tokine responses (IFN-y) exclusively drive disease progression in type 1 diabetes, while anti-inflammatory Th2 responses (IL-4, IL-10) offer protection [183]. Instead, the Th2 predominance of T-cell reactivity that is associated with the presence of antibodies strongly linked with diabetes progression suggests that elevated Th2 or regulatory responses continue in the presence of destructive auto-immunity. Similar observations of an early Th2-type response have been reported in pre-diabetic NOD mice and in T cells that protect against diabetes isolated from islet infiltrates from diabetic mice [184, 185]. Such a Th2 environment would account for the activation of the humoral immune response (auto-antibody production). However, the critical factor that tips the balance into a Th1 state towards destructive insulitis and overt disease remains unknown.

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