Relative contribution of medullary thymic epithelial cells and dendritic cells to thymocyte central tolerance as assessed by multi-photon microscopy

Abstract

Abstract Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce self-tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce negative selection of autoreactive thymocytes, but their relative contribution remains unresolved. Previous studies using genetic models to impair antigen presentation by mTECs or DCs demonstrated both cell types could induce negative selection. However, because interactions between thymocytes and stromal cells maintain a properly differentiated stromal compartment, these studies may not reflect physiologic interactions or relative contributions of mTECs and DCs when both are intact. Thus, we developed a 2-photon microscopy approach to directly observe interactions between thymocytes and DCs or mTECs during negative selection, while monitoring T cell receptor (TCR) activation. Despite earlier studies indicating DCs are required for negative selection to a soluble TRA, we find that mTECs and DCs contribute equivalently to TCR stimulation of CD4 and CD8 single positive (SP) thymocytes responding to membrane-bound or secreted TRAs. Interactions with mTECs and DCs generally accounted for all TCR activation events; however, a significant fraction of CD4SP activation events were not accounted for in response to a soluble TRA, suggesting presentation by other medullary stromal subsets. Initial analysis of polyclonal thymocytes also indicates equal contributions of mTECs and DCs to medullary TCR activation. In summary, mTECs and DCs contribute equivalently to presentation of soluble and membrane-bound mTEC-expressed TRAs on MHC-I and MHC-II during negative selection.