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.
