Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System.

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts (i.e., screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction.

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System.

Recent advancements in single cell sequencing technologies allow for identification of numerous immune-receptors expressed by T cells such as tumor-specific and autoimmune T cells. Determining antigen specificity of those cells holds immense therapeutic promise. Therefore, the purpose of this study was to develop a method that can efficiently test antigen reactivity of multiple T cell receptors (TCRs) with limited cost, time, and labor. Nuclear factor of activated T cells (NFAT) is a transcription factor involved in producing cytokines and is often utilized as a reporter system for T cell activation. Using a NFAT-based fluorescent reporter system, we generated T-hybridoma cell lines that express intensely fluorescent proteins in response to antigen stimulation and constitutively express additional fluorescent proteins, which serve as identifiers of each T-hybridoma expressing a unique TCR. This allows for the combination of multiple T-hybridoma lines within a single reaction. Sensitivity to stimulation is not decreased by adding fluorescent proteins or multiplexing T cells. In multiplexed reactions, response by one cell line does not induce response in others, thus preserving specificity. This multiplex assay system will be a useful tool for antigen discovery research in a variety of contexts, including using combinatorial peptide libraries to determine T cell epitopes.

The economic costs of undiagnosed diabetes.

The objective is to estimate the national economic costs associated with undiagnosed diabetes mellitus (UDM). UDM is defined as unknowingly having an elevated glucose level that meets the definition of diabetes. National Health and Nutrition Examination Survey (NHANES) data are used to estimate the prevalence of UDM. Because UDM cannot be directly observed in medical claims for analyzing per capita patterns of health care use, we analyze annual medical claims from a proxy population--people within 2 years of first diagnosis of diabetes. For a commercially insured population first diagnosed with diabetes in 2006 (n = 29,770), we compare their annual health care use in 2004 and 2005 to that of patients with no history of diabetes between 2004 and 2006 (n = 3.2 million). We combine estimates of UDM prevalence from NHANES with health care use patterns from the proxy population to estimate etiological fractions that reflect the portion of national health care use associated with UDM. Approximately 6.3 million adults in the United States have UDM in 2007. Annual per capita use of health care services for the UDM proxy population is higher than for a comparable group with no history of diabetes, but lower than for a comparable group with a history of diabetes. The estimated economic costs of UDM in 2007 is $18 billion ($2864 per person with UDM), including medical costs of $11 billion and indirect costs of $7 billion. Although the high prevalence of UDM makes it an important health issue to be studied, data limitations have contributed to a dearth of information on the health care use patterns and economic costs of UDM. By omitting UDM, estimates of the total national cost of diabetes are underestimated.