Two-photon imaging reveals distinct contributions of Aire+ medullary thymic epithelial cells and dendritic cell subsets to central tolerance induction

Abstract

Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce central tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce thymocyte negative selection and regulatory T cell induction. However, their relative contributions to tolerance remain unresolved, partially because the genetic models that have been used to address this question can interfere with thymocyte-stromal cell crosstalk, potentially resulting in secondary defects in stromal differentiation that independently alter central tolerance. Thus, we developed a 2-photon microscopy approach to directly observe thymocytes as they undergo tolerance induction in response to interactions with DCs versus Aire+ mTECs presenting various forms of a TRA in an intact thymic stromal environment. We find that both mTECs and DCs present transmembrane TRAs with equivalent efficiency to CD8SP and CD4SP thymocytes. However, in the context of a secreted TRA, DCs play a greater role than mTECs in presenting antigen to CD4SP, but not CD8SP thymocytes. Furthermore, distinct DC subsets differentially contribute to TRA presentation to CD4SP versus CD8SP thymocytes, with a major role for Sirpα+DCs in cross-presentation of TRAs. In the context of a polyclonal T cell repertoire responding to endogenous antigens, DCs play a more significant role than mTECs in activating CD4SP cells. In summary, we find that in an intact thymic microenvironment, multiple DC subsets and Aire+ mTECs cooperate to present self-antigens to auto-reactive thymocytes during the establishment of tolerance against diverse medullary TRAs.