Final Report Summary - SELF-TOLERANCE (Generating self-antigen diversity in the thymus: from gene expression patterns in single cells to the system level, an integrative approach)
We addressed various aspects of the role medullary thymic epithelial cells (mTECs) play in central T cell tolerance with a focus on the regulation of promiscuous gene expression (pGE) in this cell type. Since pGE in mTECs (and cTECs for that matter) is developmentally controlled with little knowledge of its regulation, we set out to more precisely map mTEC differentiation from its tissue-specific stem cells to the terminally differentiated stage both ex vivo and in vitro. We identified a bipotent TEC stem cell, which can give rise to both the mTEC and cTEC lineage in so-called sphere cultures (“thymospheres”), but not to fully mature TEC lineages. In complementation to the sphere assay we developed a 3D organotypic culture model, which allows the differentiation of immature mTECs, first to mature mTECs and then to the terminal keratinizing stage. This culture model is the first one, which sustains pGE in vitro. We further revised the in vivo roadmap of mouse and human mTEC development on the basis of newly identified cell surface markers. This led to the further subdivision of both the immature and mature mTEC compartments into additional developmental stages including a pre- and post-Aire stage.
With regard to the regulation of pGE we elaborated the concept that the patterning of pGE in single mTECs is governed by co-regulation rather than being a stochastic process. We could document this finding at the population, subset and single cell level. Based on our data we proposed the model of
“sliding co-expression groups” implying that individual mTECs scan a sizeable portion of the genome during their lifetime thereby increasing local antigen diversity within micro-domains of the
medulla.
Though encompassing including more than 3000 tissue-restricted antigens (TRAs), pGE has inherent pitfalls, which lead to an incomplete representation of the immunological self in the thymus versus the periphery. We identified yet another mechanism by which central tolerance can be breached, namely mis-initiation of TRA transcripts specifically in mTECs thus leading to truncated mRNA isoforms missing out T cell epitopes, as recently shown for the human melanoma antigen MART-1.
Taken together, our findings represent a substantial advance in our understanding mTEC
development, the principles of how self-antigen diversity is generated in mTECs and how loss of
self-tolerance can be traced back to subtle faults in in pGE.
With regard to the regulation of pGE we elaborated the concept that the patterning of pGE in single mTECs is governed by co-regulation rather than being a stochastic process. We could document this finding at the population, subset and single cell level. Based on our data we proposed the model of
“sliding co-expression groups” implying that individual mTECs scan a sizeable portion of the genome during their lifetime thereby increasing local antigen diversity within micro-domains of the
medulla.
Though encompassing including more than 3000 tissue-restricted antigens (TRAs), pGE has inherent pitfalls, which lead to an incomplete representation of the immunological self in the thymus versus the periphery. We identified yet another mechanism by which central tolerance can be breached, namely mis-initiation of TRA transcripts specifically in mTECs thus leading to truncated mRNA isoforms missing out T cell epitopes, as recently shown for the human melanoma antigen MART-1.
Taken together, our findings represent a substantial advance in our understanding mTEC
development, the principles of how self-antigen diversity is generated in mTECs and how loss of
self-tolerance can be traced back to subtle faults in in pGE.