Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE + mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APC). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APC; thus, thymocytes must efficiently enter the medulla and scan APC to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APC in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DC) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DC. EBI2 deficiency altered the TCR repertoire, but did not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impaired negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency resulted in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.

Following positive selection, thymocytes migrate into the medulla where they encounter diverse self-antigens that induce central tolerance. Thymocytes expressing T cell receptors (TCRs) with high affinity for self-antigens displayed by medullary antigen presenting cells (APCs) undergo either negative selection or diversion to the regulatory T cell (Treg) lineage, thus ensuring maturation of non-autoreactive T cells. Because many self-antigens are expressed by only a small percentage of medullary thymic epithelial cells, thymocytes must enter the medulla and efficiently scan APCs therein to encounter the full array of self-antigens that induce central tolerance. Chemokine receptors play a critical role in promoting medullary entry and rapid motility of post-positive selection thymocytes. We found that the chemokine receptor CCR8 is expressed by post-positive selection CD4+ single positive (SP) thymocytes in mice, while the corresponding chemokine ligands are expressed by medullary APCs, and thus hypothesized that CCR8 would promote thymocyte medullary entry and/or rapid motility to induce negative selection. However, despite a subtle decline in thymocyte medullary accumulation and the presence of autoantibodies in aged CCR8-deficient mice, CCR8 was not required for thymocyte differentiation, rapid motility, or negative selection.

Experimental autoimmune encephalomyelitis (EAE) is a well-described mouse model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). While the role of CD4 T cells is well established, other immune cell types, particularly CD8 T cells, are key contributors to disease pathogenesis. Our study aims to categorize the functional contributions of CD8 T cell subsets to EAE kinetics and disease outcomes. The use of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide alongside an adjuvant to inoculate mice, provides a reproducible disease model. Demyelinating disease severity was analyzed at defined timepoints including, onset (d10–14), peak (d17–21), early chronic (d28–35) and late chronic (d80–120) stages. The total number of CD3+ T cells in the CNS remained consistent during EAE. However, it goes from a CD4 T cell dominated response at peak EAE to an equalized 1:1 ratio of CD4 and CD8 T cells at late chronic stage. Furthermore, differentiation of CD8 T cell subsets based on CD8α and CD8β protein expression revealed that CD8αα T cells numbers increase to comprise ~40% of the CD8+ population at late chronic EAE. In addition, CD8βLO cells become a prominent subset during late chronic stage, with a concomitant decrease in CD8αβHI T cells. Moreover, CD8αα T cells are activated and exhibit a central memory phenotype along with downregulation of exhaustion markers by FACS analysis, and yet have significantly reduced cytokine expression. These results are consistent with single cell RNA sequencing data obtained at the peak and late chronic time points. Thus, CD8αα T cells constitute a phenotypically distinct class of CD8 T cell subsets, with a blunted effector role in demyelinating disease outcome.

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.