Antigen presentation of detergent-free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

Abstract

SummaryThe smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 complexed with HSA were incubated with Priess B cells (HLA DRB1*0401) and antigen presentation was tested with HLA DRB1*0401-restricted and epitopespecific T33.1 (GAD65 epitope 274-286) and T35 (GAD65 epitope 115-127) T cell hybridomas. Specific epitope recognition by T33.1 (274-286) and T35 (115-127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation were detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274-286, but the lowest 115-127 presentation. This suggests that HSA-GAD65 complexes qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274-286 epitope presentation, while suppressing the 115-127 epitope.Keywords antigen presentation; GAD65; Human serum albumin; T cell assay; Type 1 diabetes; T cells; Diabetes; Autoimmunity; Antigen Presentation/Processing IntroductionType 1 diabetes mellitus (TID) is an autoimmune disease that results in the specific destruction of the pancreatic islet beta cells. The smaller isoform of glutamate decarboxylase (GAD65) is implicated as a major contributing autoantigen in the pathogenesis of beta cell destruction [1,2]. Autoantibodies directed to GAD65 (GAD65Ab) are present in the majority of new onset T1D patients [3,4] and GAD65-specific T cells have been identified in T1D patients and in spontaneously diabetic NOD mice and BB rats (for review see [5,6] [9]. The establishment of a standard T cell assay remains critical for the understanding of GAD65 antigen uptake, processing and presentation.GAD65, which catalyzes the generation of the neurotransmitter GABA, is associated with intracellular membranes [10,11]. In vitro, the highly hydrophobic GAD65 requires detergents to remain in solution [12]. However, because detergents are extremely cytotoxic, it is critical that the detergent is removed prior to incubation with antigen-presenting cells (APC) and responder T cells.In the present study, we tested the well known property of human serum albumin (HSA) to maintain proteins of hydrophobic character in solution (for review see [13,14]). The aim of our study was to test whether GAD65 complexed with HSA affected antigen presentation by human Priess B cells to two GAD65-and one HSA-specific specific HLA-matched T cell hybridoma as readouts of antigen presentation. The specific epitope response of the two ...

Immune therapy in type 1 diabetes mellitus

“…Although CD4+ T cells expressing TCR that recognize autoantigenpeptides presented on HLA-DR or -DQ heterodimers remain in undisputable target for prevention, it seems equally important that B cells expressing BCR reactive an islet autoantigen are targeted ( Figure 6). As shown in ex vivo experiments, B cells are effective antigen presenting cells 58,59 . Recent studies in the mouse provide evidence that there is a so-called cross-talk between B lymphocytes and CD8+effector T cells that may lead to diabetes (ref).…”

Vaccination against type 1 diabetes

“…The results with Rituximab underscore the importance of B-lymphocytes as possible antigen-presenting cells in the pathogenesis of islet autoimmunity and type 1 diabetes. Indeed, B-lymphocytes as antigen-presenting cells may be responsible for the chronic anti-islet autoantigen response as exemplified in in vitro experiments with human B-lymphocytes presenting GAD65 on HLA Class II antigens [23, 24]. Taken together, immunosuppressive agents seem do not seem to be able to block the autoimmune process that is associated with loss of the pancreatic islet beta cells.…”