Central tolerance, or the establishment of self-tolerance during T cell development in the thymus, is critical for the prevention of autoimmunity. Developing T cells (thymocytes) are exposed to a wide array of self-peptides presented by a variety of thymic resident cells. Thymocytes bearing T cell receptors (TCR) that strongly engage self-ligands are deleted from the T cell repertoire during negative selection. Alternately, strong TCR signals can direct the development of thymocytes into unconventional T cell lineages that play regulatory or immunosuppressive roles, a process termed agonist selection. Given that strong TCR signaling can result in either of these divergent thymocyte fates, the factors that govern autoreactive thymocyte fate remain unclear. Here, we used an experimental system, in which thymocytes of a defined specificity are overlaid onto thymic slices and undergo a synchronized wave of development, to address this question.

First, we focused on factors that promote autoreactive thymocyte death. We have previously observed that thymocyte death and phagocytosis are concurrent in a ubiquitous model of negative selection. Here, we observed that these interactions are also prevalent in an additional model of negative selection, indicating that the close coupling of cell death and phagocytosis is a general feature of autoreactive thymocyte death in situ. We further explored the role of phagocytes in negative selection, and demonstrated that phagocytosis is critical for the induction of autoreactive thymocyte death. We also found that negative selection is most efficient when peptide is presented by phagocytic cells. Taken together, our data demonstrate that phagocytes play a more active role in negative selection than previously appreciated, both as peptide-presenting cells and as active inducers of autoreactive thymocyte death. Moreover, our findings indicate that the phagocytic ability of a peptide-presenting cell is an important determinant of autoreactive thymocyte fate.

One example of an agonist-selected lineage is TCRαβ CD8αα intraepithelial lymphocytes (CD8αα IEL), which reside in the small intestine. CD8αα IELs have been shown to play a protective role in colitis, suggesting that they are important for maintaining tolerance and homeostasis in the gut. However, the cellular and molecular interactions required to mediate CD8αα IEL development are not well defined. Here, we demonstrate that the selection of CD8αα IELs requires higher affinity TCR signals than negative selection. Additionally, we demonstrate that access to cytokines influences autoreactive thymocyte fate, as the cytokine IL-15 impairs negative selection, while IL-15 and a related cytokine, IL-2, both promote CD8αα IEL development. Furthermore, our data indicate that peptide presentation by hematopoietic cells is required for efficient CD8αα IEL development.

Taken together, these studies define factors that play important roles in determining autoreactive thymocyte fate, including the strength of TCR signal, phagocytic ability of the peptide-presenting cell, and access to cytokines. Our data contributes to mounting evidence that the context in which a thymocyte encounters peptide, shaped largely by the characteristics of the peptide-presenting cell, is a key determinant of thymocyte fate.