Real time visualization of the differential mTEC and DC contributions to T cell central tolerance

T cell central tolerance for peripherally expressed tissues is established by thymocyte:stromal cell interactions that occur within the thymic medulla. However, the roles of the medullary thymic epithelial cell (mTEC) and dendritic cell (DC) subsets in mediating negative selection against tissue-restricted antigens are unclear. While genetic models provide evidence regarding the selecting potential of either stromal cell subset in the absence of the other, these models interfere with the normal thymocyte:stromal cell crosstalk necessary to establish a properly differentiated thymic microenvironment; this leaves open the relative contribution of either stromal cell type in mediating negative selection when both subsets are intact. Here, using two-photon fluorescence microscopy, we observe thymocyte:stromal cell interactions at the onset of TCR signaling as they occur in real time in the thymic medulla. We have quantitated CD8+ and CD4+ thymocyte contacts with either Aire+ mTECs or DCs in the presence of membrane-bound or secreted soluble forms of antigen, and find that both thymocyte MHC-restriction and subcellular localization of the antigen modulate the contributions of mTECs and DCs to negative selection. These experiments provide insight into the cellular mechanisms of central tolerance that deter autoimmunity, as they occur in a physiological environment.