Understanding the role of the blood vascular system and endothelial cells in early thymus regeneration after cytotoxic treatments using novel intravital imaging techniques - PROJECT SUMMARY Hematopoietic cell transplantation (HCT) remains an important treatment for hematologic malignancies such as multiple myeloma, lymphoma, acute leukemias, and myelodysplastic syndromes.1 A key component of HCT is the cytotoxic preconditioning required to reduce the burden of malignancy, suppress the host immune system, and enable engraftment and tolerance of the donor hematopoietic cells.2 Unfortunately, conditioning regimens invariably damage the tissues where donor cells engraft and expand (e.g., bone marrow and thymus), leading to significant morbidity and mortality.1 Recent data suggests that radio-resistant thymus vascular endothelial cells (ECs) are critical cells in endogenous thymus regeneration as they provide the conduit for progenitor cell entry into the thymus, express ligands important for early thymic seeding, and secrete regenerative associated factors (RAFs) such as BMP4.3–5 Understanding how the vascular network, and in particular ECs, respond to cytotoxic therapy may give us insights that will enable the development of improved therapies and restoration of adaptive immunity after acute injury.4,5 But how do these regimens change the microenvironment of these tissues (especially the thymus) and impact the recovery of the hematopoietic and adaptive immune systems? To address this question, we need new methods to directly analyze the live thymus in its native site.6 Our previous intravital imaging work demonstrated that cytotoxic therapies drastically change the bone marrow (BM) vascular integrity, oxygen tension (pO2), and cell behavior.7,8 In the thymus, we also observe widespread changes in vascular structure suggesting functional damage. We hypothesize (a) that the native cortical and CMJ vascular compartment is heterogenous with functionally distinct blood vessels; (b) that these distinct vessels are made up of specific sub-types of ECs; (c) that this heterogeneity diminishes as the thymus matures and/or after cytotoxic conditioning; and (d) that cytotoxic conditioning causes abnormal hemodynamics in the blood vascular system leading to spatially and temporally restricted changes in blood flow, vessel permeability, and tissue oxygenation. To test these hypotheses, we propose the following aims: (1) Utilize novel high-resolution intravital imaging and two-photon oxygen microscopy to directly image and functionally characterize the native thymic cortical and CMJ blood vascular physiology; (2) Define EC heterogeneity and subtypes using flow cytometry, single cell RNA sequencing, and immunostaining and identify age dependent functional and anatomical alterations in EC and vascular heterogeneity; and 3) Utilize high-resolution imaging and two-photon phosphorescence oxygen microscopy to characterize the thymus microenvironment over time after myeloablative (MAC) and reduced intensity conditioning (RIC) to identify physiological changes impacting the recruitment of progenitors to the thymus in the context of HCT. The studies outlined in this proposal have the potential to elucidate the microenvironmental effects of cytotoxic preconditioning on the thymus in ways that have not been previously possible through use of novel imaging techniques for direct thymus evaluation.
