Quantitative dissection of T cell negative selection mechanisms in the thymus
Creators: name: Yap, Jin Yan
Publisher: name: The Australian National University
Year: 2017

description: While many factors can influence the fate of developing T cells in the thymus, among the most influential are the strength and timing of signals transmitted by the T cell antigen receptor (TCR). Weak TCR binding to peptide/major histocompatibility complex (MHC) ligands induces thymocytes to develop into naïve T cells, a process called positive selection. Mechanisms that prevent naive T cell development are termed “negative selection” and include apoptotic deletion and agonist selection into non-naive T cell lineages. Strong TCR binding to pMHC ligands induces negative selection of thymocytes. Thymocytes interact with two broad types of antigen-presenting cells (APCs): bone marrow (BM)-derived APCs and thymic epithelial cells (TECs). The traditional view of thymic selection is that positive selection occurs in the thymic cortex via interaction with TECs, followed by negative selection in the thymic medulla via interaction with BM-APCs. However, recent studies reveal that thymocytes can undergo either cortical or medullary negative selection, which differ in terms of the phenotypes of the thymocytes involved and their timing during T cell development. It remains unclear which APC types are required to mediate these two negative selection processes, which are called “Wave 1” and “Wave 2” of negative selection. In addition, the contributions of MHC class I (MHCI) and MHC class II (MHCII) to the two waves are unclear. To dissect thymic selection, two assays were used in this study. First, the transcription factor, Helios, was used in a flow cytometry assay as a marker of negatively selected cells, in conjunction with chemokine receptor-7 (CCR7) to distinguish thymocyte maturation stages. Apoptosis-defective mice were used to inhibit death of negatively selected cells, allowing direct quantification of negative selection. Genetic ablation of MHCII expression within BM-APCs or autoimmune regulator (Aire–/–) within TECs was used to examine the roles of APC types in thymic selection. Mice lacking expression of MHCI and/or MHCII were also examined. Second, a TCR sequencing assay was used to examine the characteristics of positively and negatively selected TCR repertoires in mice lacking expression of MHCI and/or MHCII. In TCR transgenic and polyclonal models, MHCII+ BM-APCs were required to induce wave 1 negative selection. Ablation of MHCII+ BM-APCs either abrogated negative selection completely or delayed negative selection from wave 1 to wave 2. Although MHCI and MHCII were found to induce similar frequencies of TCR-signalled thymocytes, MHCII was found to make a greater contribution to negative selection than MHCI. Sequencing data revealed that TCRs that provoke negative selection at wave 1 are enriched with hydrophobic amino acids in the region expected to interact with the peptide component of pMHC ligands. Interestingly, hydrophobic amino acids are also enriched in the same region of TCRs that transmit a signal in mice lacking MHC expression. These results provide new insight into the determinants of thymic negative selection.