Objective
The goal of this grant is to identify antigens targeted by CD4 T cells specific to islets, and we will pursue the following two aims. Aim 1. Identify (neo)epitopes for human pancreatic islet derived CD4 T cells. Post translational and transcriptional modifications may produce key antigens in beta cells that elicit breaking tolerance. We will test TCRs expressed by CD4 T cells in the islets of organ donors having T1D for the response to islet proteins that received PTMs such as deamidation, citrullination, and fusion peptide formation as well as aberrant gene expression products. Aim 2. Determine tri-molecular complexes targeted by islet-reactive CD4 T cells in the early stage of T1D. We will determine TCR repertoires of islet-specific CD4 T cells at early stages of T1D in two resources: (1) islets of prediabetic organ donors who developed T1D-associated autoantibodies (AAb) but not diabetes, and (2) islet lysates-reactive TCRs enriched from CD4 T cells in pancreatic lymph nodes (PLNs) of multiple AAb+ organ donors. TCR sequences expressed by those CD4 T cells will be expressed on T-hybridoma cells to determine response to preproinsulin and GAD65.
Background Rationale
Type 1 diabetes (T1D) results from the immune mediated targeting of pancreatic beta cells with T cells playing a central role in disease pathogenesis. Tissue specificity and strong genetic association with the HLA locus suggest that antigen specificity is necessary for T cells to attack beta cells and cause T1D. Therefore, enormous efforts have been expended to identify antigens for disease-associated T cells to understand pathogenesis and develop therapies to prevent disease onset. However, antigen specificity for the majority of CD4 T cells is still unknown, and there may be common and major antigens that are targeted by subsets of T1D patients but have not been identified. Appearance of autoantibodies against one islet antigen typically precedes those against other antigens, suggesting the spread of antigen specificity from limited antigens to others during the disease development. Studies using animal models support this notion and demonstrated a hierarchy of antigens in which autoreactivity to one antigen is necessary for activation of T cells specific for other antigens. Thus, while there are dozens of antigens targeted by islet-specific CD4 T cells, it will be important to identify those that are involved in triggering or driving T1D to develop effective immunotherapies.
Description of Project
Type 1 diabetes (T1D) results from the immune mediated targeting of pancreatic beta cells with T cells playing a central role in disease pathogenesis. Tissue specificity and strong genetic association with the HLA locus suggest that antigen specificity is necessary for T cells to attack beta cells and cause T1D. Therefore, enormous efforts have been expended to identify antigens for disease-associated T cells to understand pathogenesis and develop therapies to prevent disease onset. However, antigen specificity for the majority of CD4 T cells is still unknown, and there may be common and major antigens that are targeted by subsets of T1D patients but have not been identified. Appearance of autoantibodies against one islet antigen typically precedes those against other antigens, suggesting the spread of antigen specificity from limited antigens to others during the disease development. Studies using animal models support this notion and demonstrated a hierarchy of antigens in which autoreactivity to one antigen is necessary for activation of T cells specific for other antigens. Altogether, while there are dozens of antigens targeted by islet-specific CD4 T cells, it will be important to identify those that are involved in triggering or driving T1D to develop effective immunotherapies. Thus, the goal of this grant is to identify antigens targeted by CD4 T cells specific to islets, and we will pursue the following two aims. Aim 1. Identify (neo)epitopes for human pancreatic islet derived CD4 T cells. Post translational and transcriptional modifications may produce key antigens in beta cells that elicit breaking tolerance. We will test TCRs expressed by CD4 T cells in the islets of organ donors having T1D for the response to islet proteins that received PTMs such as deamidation, citrullination, and fusion peptide formation as well as aberrant gene expression products. Aim 2. Determine tri-molecular complexes targeted by islet-reactive CD4 T cells in the early stage of T1D. We will determine TCR repertoires of islet-specific CD4 T cells at early stages of T1D in two resources: (1) islets of prediabetic organ donors who developed T1D-associated autoantibodies (AAb) but not diabetes, and (2) islet lysates-reactive TCRs enriched from CD4 T cells in pancreatic lymph nodes (PLNs) of multiple AAb+ organ donors. TCR sequences expressed by those CD4 T cells will be expressed on T-hybridoma cells to determine response to preproinsulin and GAD65. In aim 1, We anticipate identifying a number of islet-derived CD4 TCR clonotypes that are reactive to neoepitopes with various posttranslational and transcriptional modifications. It is important to determine epitopes that induce robust responses since such epitopes may be common between patients, and likely differ by age of T1D onset and HLA-DR-DQ types. In aim 2, hundreds of islet-specific TCR clonotypes are expected to be identified from multiple at risk donors. A portion of these TCRs may be reactive to preproinsulin and GAD65. Since antigen spreading in AAb+ donors may be limited, only a few epitopes but targeted by a number of T cells may be detected. Such epitopes are particularly important because they may be involved in the early stage of T1D pathogenesis. Also, epitopes inducing robust T cell responses in both AAb+ and T1D donors may be beneficial for antigen-specific immunotherapies that can be effective for all stages of T1D patients. Overall, the impact of this work provides a foundation for using these identified epitopes in antigen specific immunotherapies.
Anticipated Outcome
We anticipate identifying a number of islet-derived CD4 TCR clonotypes that are reactive to neoepitopes with various posttranslational and transcriptional modifications. It is important to determine epitopes that induce robust responses since such epitopes may be common between patients, and likely differ by age of T1D onset and HLA-DR-DQ types. Also, hundreds of islet-specific TCR clonotypes are expected to be identified from multiple at risk donors. A portion of these TCRs may be reactive to preproinsulin and GAD65. Since antigen spreading in AAb+ donors may be limited, only a few epitopes but targeted by a number of T cells may be detected. Such epitopes are particularly important because they may be involved in the early stage of T1D pathogenesis. Also, epitopes inducing robust T cell responses in both AAb+ and T1D donors may be beneficial for antigen-specific immunotherapies that can be effective for all stages of T1D patients. Overall, the impact of this work provides a foundation for using these identified epitopes in antigen specific immunotherapies.
Relevance to T1D
Having optimal CD4 T cell epitopes specific for each stage of T1D enables the design of antigen specific immunotherapy and the ability to select patients having these reactivities to receive these therapies.