Immunology

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.

Graphical overview

Dendritic cells have been investigated for cell-based immunotherapy for various applications. The low abundance of dendritic cells in blood hampers their clinical application, resulting in the use of monocyte-derived dendritic cells as an alternative cell type. Limited knowledge is available regarding blood-circulating human dendritic cells, which can be divided into three subsets: type 2 conventional dendritic cells, type 1 conventional dendritic cells, and plasmacytoid dendritic cells. These subsets exhibit unique and desirable features for dendritic cell-based therapies. To enable efficient and reliable human research on dendritic cell subsets, we developed an efficient isolation protocol for the three human dendritic cell subsets, resulting in pure populations. The sequential steps include peripheral blood mononuclear cell isolation, magnetic-microbead lineage depletion (CD14, CD56, CD3, and CD19), and individual magnetic-microbead isolation of the three human dendritic cell subsets.

Graphical overview

Scheme of the dendritic cell (DC) isolation protocol. Starting material for this process is human blood (buffy coat or aphaeresis). From that, peripheral blood mononuclear cells (PBMCs) are isolated by using ficoll gradient centrifugation. Then, an enrichment for DCs is performed using semi-automated equipment. From the enriched fraction, DC subsets are obtained by magnetic cell sorting.