Medullary Thymic Epithelial Cell Antigen-presentation Assays

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3(+) regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4(+) T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4(+) T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4(+) T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4(+) T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA(323–339), to assess mTEC antigen presentation to CD4(+) T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS(®) Pro Separator, a BD FACSAria(TM) III cell sorter, and a BD(®) LSR II flow cytometer.