Gluten-challenge in subjects with celiac disease, results in a transient upregulation of gliadin-specific CD4+ T cells in the blood. Despite the increase, these cells are quite rare requiring a selective and sensitive assay for detection. We have developed a 12-color tetramer flow assay to enable detection and immunophenotyping of the gliadin α-I/α-II CD4+ T cells in the blood.
The assay was validated using healthy cryopreserved AccuCell™ PBMCs and CD4+ T cells spiked with T cells specific for gliadin α-I (QLQPFPQPELPY) or gliadin α-II (PQPELPYPQPE). The assay conditions were optimized for sensitivity, optimal signal:noise ratio, and detection of the gliadin α-I/α-II tetramers. The assay identifies gluten-specific (tetramer+) T cells and immunophenotypes the cells as either naïve or memory (CD4/CD3/CD45RA/CD62L/CCR7), activated (CD38), regulatory (CD39) and gut-homing (b7/a4). For validation, pre-specified criteria were used to assess inter-assay, intra-assay, inter-operator precision and post-staining stability. Technical validation was successfully met; and the assay performs within acceptable precision parameters.
Tetramer positive T cells in the blood are rare events; therefore, this assay was developed and validated to be sensitive and selective. This assay will enable characterization of the gliadin α-I/α-II CD4+ T cells in the blood for celiac patients, providing a more comprehensive evaluation of response to new therapies and may reduce invasive biopsy-based measurements.