Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was possible to induce T cell priming to most of the moderate/weak allergens, including lipophilic molecules highly insoluble in culture media. Therefore, the present optimized in vitro human T cell priming assay is a valuable method to detect the sensitizing properties of chemical allergens.