The integrated hypothesis of this Program proposal is that age-associated cellular and molecular features of the mouse thymus microenvironment, mirrored by similar features in the human thymus, play a major role in shaping the distinct functional potential of T cells that emigrate from the thymus during the perinatal versus juvenile periods. Currently, there is little information in mice, and even less in humans, on changes in the composition and function of thymic epithelial cells (TECs), hematopoietic antigen presenting cells (HAPCs), and other stromal cell subsets during this transition. Given that T cells with effector and regulatory functions are vital for newborn health, the Program research objectives are to: 1) identify changes in the composition and organization of TECs, HAPCs and other thymus stromal cells over the perinatal to juvenile transition in both mice and humans; 2) map transcriptional changes in these cellular subsets that regulate their proliferation, differentiation, function and cross-talk; and 3) determine how such changes impact the ability of the perinatal thymic environment to generate T cells with distinct functional capabilities. The Program is structured to use scRNA-seq and multiplex imaging to collaboratively identify candidate cell populations and molecular mechanisms underlying functionally significant changes in mouse and human thymus microenvironments across the perinatal to juvenile transition. In the discovery phase, RPs will generate scRNA- seq datasets of TECs (RP1), stromal cells (RP2) and HAPCs (RP3) from mouse and human (Core C) thymus. These datasets will be analyzed by Core B to identify cellular and molecular candidates. In the prioritization phase, RPs will determine which candidates are conserved across species with Core B. RPs will then collaborate to use multiplex imaging to visualize changes in location and/or interactions of these candidates during the transition that may mediate important biological functions. Both species conservation and imaging results will provide a rationale to prioritize cell subsets and molecular pathways for functional testing. In the testing phase, each RP will assess novel, as well as previously identified candidates, for functional significance according to Project specific goals. Collectively, these parallel scRNA-seq and imaging experiments in the three RPs will synergize to: 1) identify changes in the transcriptional profiles, cellular composition and organization of the thymus microenvironment over the perinatal to juvenile transition in both mice and humans; and 2) enable us to test which changes regulate proliferation, differentiation, and function of these subsets across the transition with downstream consequences for perinatal immune function. Program components are: RP1 (Richie): Molecular mechanisms controlling TEC dynamics and lineage hierarchies in the perinatal thymus RP2 (Manley): The role of Foxn1 in controlling the transition from thymus expansion to homeostasis RP3 (Ehrlich): Differential contribution of thymic APCs to central tolerance during the perinatal to adult transition Core A (Richie): Admin. Core; Core B (Yi): Bioinformatics/Biostats Core; Core C: (Hale) Human Thymus Core
The neonatal thymus microenvironment consists of multiple stromal cell types which are critical for the development of T cells that protect newborns from infectious diseases and autoimmunity. The cellular and molecular differences between neonatal and adult thymic stromal cells and the mechanisms regulating the transition from neonatal thymus growth to adult homeostasis are not understood. This program project will use single cell RNA sequencing, advanced imaging and other cutting edge approaches to identify cellular and molecular changes in stromal cell subsets in the mouse and human thymus during the neonatal to adult transition, determine the molecular pathways that regulate these changes and evaluate the ensuing effects on T cell function.