Project 1: Impaired iNKT generation on deletion of chemokine receptors homing to the thymic medulla and Project 2: The requirement for co-stimulation in generation and homeostasis of conventional and memory-phenotype regulatory T-cells

摘要

Project 1: Invariant natural killer T-­cells (iNKT) are a subset of unconventional T-­cells arising from the same pool of CD(^4)+8(^+) double-­‐positive (DP) precursors as conventional T-­cells, but are distinct in their T-­cell receptor (TCR) specificity -­ recognizing CD1d-­bound glycolipids -­ as well as constitutive expression of memory cell markers and cytokine mRNA. Development of iNKT within the thymus is drastically different in terms of both the molecular requirements, and the transcriptional processes involved. However, like conventional thymocytes, they are known to undergo positive and negative selective processes to screen TCR functionality and auto-­reactivity, albeit on contrasting cell types – DP thymocytes and dendritic cells respectively. Progressive maturational stages are defined by expression of activation and NK-­ cell markers. Conventional thymocytes up-­regulate the chemokine receptors CCR4 and CCR7 post-­positive selection, which control re-localization to the medulla, where they are negatively selected on self-­‐antigens promiscuously expressed by medullary epithelial cells. On deletion of these chemokine receptors, development is unaffected, but auto-­reactive T-­ cells escape deletion, and cause auto-­immunity in the periphery. Our work has demonstrated that deletion of either receptor severely impedes thymic generation of iNKT, although only CCR4 is expressed on developing iNKT, suggesting localization of other CCR7(^+) cell types with iNKT is vital to their development. ududProject 2: Natural regulatory T-­cell (nTreg) are a T-­cell subset developed in the thymus which possess the unique capability to actively suppress antigen-­specific CD4 responses in the periphery, which have been shown to be essential in the prevention of auto-­immunity through deletion of their lineage-­defining transcription factor Foxp3. Foxp3(^+) nTreg possess T-­cell receptors (TCRs) with high auto-reactivity relative to conventional cells, and are believed to require strong TCR-­ligation and co-stimulation during negative selection for the production of CD25(^+) precursors. More recently, it has been demonstrated that peripheral Treg are a heterogenous population, consisting of death-­prone, as well as highly suppressive effector-­memory subsets, which can be separated on the basis of expression of the activation markers ICOS and CD44. In this study, we have demonstrated ICOS(^+)CD44(^+) memory Treg to have unique homeostatic requirements from conventional CD4 memory cells, which depend on provision of co-­stimulation by TNF superfamily members CD30L and OX40L for survival. We did however discover a role for OX40 signaling in development of nTreg, as well as establishing redundancy in mTEC expression of the co-stimulatory molecules CD80/86 and ICOSL, the former of which has been identified as a key molecule for generation of both nTreg precursors and ICOS(^{hi})CD44(^{hi}) Treg. ud