Objective
A broadly applicable assay to monitor autoreactive T cells in the blood of individuals with T1D is needed to assess the response to drug treatment and enable studies to understand underlying mechanisms. Activation induced marker (AIM) assays have been implemented recently to detect autoreactive T cells in T1D and have several advantages compared to other methods. However, rigorous optimization of AIM assay conditions and variability has not been undertaken in T1D and is essential for widespread application of the assay. The objective of this proposal is to undertake central optimization of the AIM assay conditions and inter-laboratory validation of the AIM assay for the detection of autoreactive T cells in T1D. Specific Aim 1 will optimize AIM assay conditions for the most sensitive detection of autoreactive T cells in three expert laboratories using a shared set of peripheral blood mononuclear cells (PBMC) samples that have been spiked with autoreactive CD4 T cells known to recognize pancreatic β-cells. Next, the variability of the optimized AIM assay to detect autoreactive T cells will then be evaluated by testing replicate PBMC samples from donors with T1D. Specific Aim 2 will validate the sensitivity and reproducibility of the optimized AIM assay to detect autoreactive T cells in six independent laboratories using standardized protocols and shared reagents and PBMC samples. Centralized analysis of the data will be performed to determine the technical variability across laboratories.
Background Rationale
T1D results from destruction of pancreatic β-cells by infiltrating autoreactive T cells. T cells recognizing numerous β-cell proteins have been identified in the pancreas, nearby lymph nodes, and blood using several different methods. These studies have demonstrated the presence of autoreactive T cells before and after T1D diagnosis, in clinical trials, and in relation to disease progression, pointing to the importance of monitoring autoreactive T cells in these settings. In clinical trials for T1D, a widely applicable assay to monitor autoreactive T cells is needed to assess the response to drug treatment and enable experiments to understand the drug mechanism. This is particularly relevant for drugs that target T cells that recognize specific β-cell proteins (antigen specific drugs). Previous studies have rigorously tested several methods to detect autoreactive T cells in peripheral blood, the most readily available specimen in T1D. However, these methods can only be utilized in a subset of individuals with T1D who carry high risk HLA genes, whereas a widely applicable method that can be used to test for autoreactive T cells in all T1D subjects is lacking. Activation induced marker (AIM) assays have been implemented recently to detect autoreactive T cells in T1D and have several advantages over other methods. First, they can be used to test all subjects with T1D regardless of their HLA genes. Second, they allow detection of different types of autoreactive T cell types in the same blood sample, saving precious samples. Third, AIM assays are compatible with other experimental methods to assay the cells, making it an attractive assay for wide application. However, rigorous optimization of AIM assay experimental conditions and variability has not been undertaken in T1D and is essential for widespread application of the assay to monitor autoreactive T cells during disease progression and in response to drug treatment.
Description of Project
Autoreactive T cells mediate destruction of the insulin producing β-cells in the pancreas in T1D, leading to loss of insulin production. These T cells are detected in the blood both before and after T1D diagnosis and vary with T1D progression. Thus, monitoring autoreactive T cells in T1D is important to understand how β-cell destruction starts, progresses, and how autoreactive T cells respond to drugs being tested for T1D prevention and treatment. However, a widely applicable assay to monitor autoreactive T cells in all individuals with T1D is lacking. Activation induced marker (AIM) assays have been implemented recently to detect autoreactive T cells in T1D and have several advantages over other methods. First, they can be used to test all subjects with T1D regardless of their genetic background. Second, they allow detection of different types of autoreactive T cell types in the same blood sample, saving precious samples. Third, AIM assays are compatible with other experimental methods to assay the cells, making it an attractive assay for wide application. A rigorous testing of AIM assay experimental conditions and variability has not been undertaken in T1D and is essential for widespread application of the assay. The objective of this proposal is to optimize AIM assay conditions and validate the AIM assay for the detection of autoreactive T cells in multiple different labs. In Specific Aim 1, we will first optimize AIM assay experimental conditions for the best detection of autoreactive T cells in three expert laboratories using a shared set of peripheral blood mononuclear cells (PBMC) samples that have been spiked with autoreactive CD4 T cells known to recognize pancreatic β-cells. Next, the variability of the optimized AIM assay to detect autoreactive T cells will then be evaluated by testing replicate PBMC samples from donors with T1D. In Specific Aim 2, we will validate the sensitivity and reproducibility of the optimized AIM assay to detect autoreactive T cells in six independent laboratories using standardized methods and shared reagents and PBMC samples. Successful completion of these Specific Aims will produce a validated AIM assay for use by the T1D community. The study will lay the groundwork for future assessment of variation in AIM assay detection of autoreactive T cells over time to determine enrollment of T1D subjects for studies of the initiation and progression of β-cell destruction and in clinical trials.
Anticipated Outcome
The overall outcome of the proposed study is 1) the development of the most specific and sensitive AIM assay, and 2) an assessment of the sensitivity and reproducibility of the optimized assay to detect autoreactive T cells in T1D. This will determine the applicability of the AIM assay for widespread implementation in studies of disease progression and clinical trials. Anticipated outcomes for each Specific Aim are as follows. Specific Aim 1a will identify the AIM assay with the highest sensitivity and the lowest background, based on the detection of spiked autoreactive T cells that recognize known pancreatic β-cell proteins. This includes the best culture conditions, the optimal set of activation markers to define autoreactive T cells, and the best method to isolate and detect the autoreactive T cells. Specific Aim 1b will determine the intra- and inter-experimental reproducibility of the optimized AIM assay in a single lab using replicate samples tested over multiple experiments. If the assay doesn’t reach <35% technical variation as specified in recent T cell assay validation efforts, conditions will be optimized further. The result will be a harmonized AIM assay for use in Specific Aim 2. We expect to achieve an average coefficient of variation (COV) of <35% for detection of autoreactive CD4 and CD8 T cells based on short-term longitudinal sampling from 10 T1D subjects performed in the Cerosaletti lab (2023 Immunology of Diabetes Society Conference). However, the COV for autoreactive regulatory T cells may be higher as these cells are more rare. Specific Aim 2 will determine the sensitivity and reproducibility of the optimized AIM assay to detect autoreactive T cells in six independent laboratories. Experimental variation will be minimized through the used of standardized protocols, shared reagents and PBMC samples (including several samples spiked with autoreactive T cells that recognize known pancreatic β-cell proteins) so that the sensitivity and variation in ability of the AIM assay to detect autoreactive T cells can be accurately assessed. The benchmark will be a COV <35%. The sensitivity and reproducibility of the AIM assay in the multi-laboratory testing will be compared to other methods for detecting autoreactive T cells in T1D that have undergone validation by the Immunology of Diabetes T cell Workshop group to assess the suitability of the AIM assay for broad applicability.
Relevance to T1D
Successful completion of the proposed project is highly relevant to ongoing efforts to predict, prevent, and treat T1D. The demonstration of the presence of autoreactive T cells before and after T1D diagnosis, in clinical trials, and in relation to disease progression, points to the importance of monitoring autoreactive T cells in these settings. In clinical trials for T1D, a widely applicable assay to monitor autoreactive T cells is needed to assess the response to drug treatment and enable experiments to understand the drug mechanism. This is particularly relevant for drugs that target T cells that recognize specific β-cell proteins (antigen specific drugs) that are in development for T1D and don’t cause broad immunosuppression. Development of a sensitive and reproducible AIM assay that can be implemented by many different labs to study questions of how β-cell destruction starts, progresses, and how autoreactive T cells respond to drugs has implications for 1) predicting when at-risk individuals will develop T1D to allow intervention while sufficient β-cells remain in the pancreas, 2) refining existing drugs or developing new drugs to prevent and treat T1D, and 3) matching the right patients for the right drug through precision medicine approaches to treatment of T1D.