A Spatiotemporal Study of Thymic Selection and Lineage Commitment

T cells are one of the main effectors of the adaptive immune response and as such, their development must be tightly regulated. Despite being well studied, the processes that produce mature T cells are not fully understood. The ultimate goal in T cell development biology is to understand the in vivo cues well enough to be able to recapitulate development in vitro. Such knowledge would unleash the therapeutic potential of stem cell biology. Although there are many gaps in our current knowledge, I have chosen to focus on two extreme ends of the developmental spectrum, from the earliest determinants in hematopoietic fate in human embryonic stem cells to the final processes of T cell development, positive and negative selection, which are ultimately responsible for the generation of a useful and self-tolerant T cell repertoire./nAlthough T cells can be derived in vitro from cord blood and fetal liver, a robust development from human embryonic stem cells into the T lineage has not yet been achieved in vitro. It's known that there are different developmental potentials among human embryonic stem cell lines. Therefore, we performed a comparison of the T lineage developmental potential of a panel of six independently derived human embryonic stem cell lines for their ability to differentiate into the known T lineage progenitors, CD34+ hemangioblasts or CD34+CD45+ hematopoietic precursors by embryoid body formation or OP9 co-cultures. Not only did we find indeed, there are differences in hematopoietic potentials among lines, but the passage conditions through which the stem cell lines were maintained also dramatically affected hematopoietic potential. None of the stem cell-derived progenitors could develop into T cells, unlike those found in cord blood. Differences between individual lines should allow for the future study of genetic or epigenetic factors that determine lineage commitment as a cell transitions from a stem cell to a T cell./nSecond, I focused on the final stages of T cell development, specifically, the processes of positive and negative selection. Most of our current understanding of signaling behavior comes from in vitro studies that fail to fully recapitulate thymic selection events. Therefore, we expanded upon a relatively new model of selection, the thymic slice system, which supports positive selection and can be used to introduce a synchronized population of thymocytes. By adding a cognate peptide antigen or overlaying slices with peptide-loaded DCs, we were able to induce negative selection. Using the thymic slice system, we visualized the migration and signaling behavior of thymocytes undergoing positive versus negative selection. Additionally, we characterized the effect of the affinity of the peptide-TCR interaction on migration, signaling, and developmental outcome. We found that thymocytes undergoing positive selection formed transient, serial interactions that lasted only a few minutes while thymocytes undergoing negative selection formed long lasting, monogamous contacts. The APC type plays an important role in selection outcome as dendritic cells and not epithelial cell cells presenting OVA most efficiently promoted negative selection and sustained calcium levels. Co-stimulatory and adhesion molecules did not appear to be required for selection outcome but contributed to sustained calcium signaling during negative selection and may therefore play a role in determining the threshold of affinity. We also found that thymocytes undergoing positive selection accumulate TCR signals over time, increase their speed, and appear to change their responsiveness to a TCR-induced stop signal over time.

Advisor: Robey, Ellen A