T-Cell Proliferation Assay: Determination of Immunodominant T-Cell Epitopes of Food Allergens

Abstract

Characterization of allergen-specific T cells is critical to understand their contribution to disease pathogenesis. The identification of immunodominant T-cell epitopes is crucial for development of T-cell-based vaccines. Peptide-specific T-cell proliferation studies are usually performed in a library of short synthetic peptides (15mer or 20mer) with 3 or 5 offset spanning the entire length of the allergen. T-cell peptide epitopes lack the primary and tertiary structure of the native protein to cross-link IgE, but retain the ability to stimulate T cells. The peptides sequences can also be obtained either by in silico approaches and in vitro binding assays. The efficacy of T-cell epitope-based peptide immunotherapy has been proven in certain allergies. The present methodology describes T-cell proliferation assays using whole blood sample from allergic subjects.

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

“…Indeed, ELIspot has been used extensively as the "gold standard" immune assay given its sensitivity and has been optimized and validated as part of the global HIV/AIDS Comprehensive T Cell Vaccine Immune Monitoring Consortium (26)(27)(28). However, these protocols are limited by the relatively high number of cells required, especially when considering targets with low frequencies (24), when collection of large blood volumes is challenging (29,30), or when there are many experimental variables [e.g., vaccine/peptide (14,17,31,32) or epitope testing (33)(34)(35)]. Therefore, there is an unmet need for a robust RNA extraction and transcriptomic analysis protocol from limited input cell numbers (e.g., PBMCs) with high analytical and diagnostic sensitivity that meets or exceeds that of proteinlevel immuno-assays.…”