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 OTIIx-/- mice and the cognate peptide OVA, to assess mTEC antigen presentation to CD4 T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS Pro Separator, a BD FACSAria III cell sorter, and a BD LSR II flow cytometer.