ABSTRACT
Lymphatic vessels are a critical component of the immune response to pathogens because they transport
leukocytes carrying antigens to lymph nodes (LNs) to facilitate antigen presentation. Now known to actively
shape the innate and adaptive immune responses, lymphatic endothelial cells (LECs) express chemokines and
receptors, secrete mediators such as sphingosine-1-phosphate (S1P) and C-C motif ligand 21 (CCL21),
modulate dendritic cell (DC) function, and regulate trafficking of immune cells into LNs. Lymphatic dysfunction
also plays a significant role in many diseases, yet little is known about how lymphatic dysfunction impacts
response to infection. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G-protein coupled receptor highly
expressed on lymphocytes and endothelial cells that is important for angiogenesis, barrier function, and immune
cell trafficking. Others report decreased S1PR1 on lymphatic endothelial cells (LECs) is associated with
lymphedema and skewed CD4 T cell activation in mice and humans. Our collaborators in the Srinivasan lab
discovered that mice lacking S1PR1 on LECs (S1pr1ΔLEC mice) have enhanced VEGF receptor 3 (VEGFR3)
signaling that, in multiple organs, leads to increased lymphatic vessel density and spontaneous formation of
tertiary lymphoid organs (TLOs), which are organized accumulations of immune cells that form in non-lymphoid
tissues with infection or inflammation. The impact that these altered CD4 T cell responses and aberrant TLOs
found with deficiency of LEC-expressed S1PR1 have on the immune response to infection is unclear. I
hypothesize that, in S1pr1ΔLEC mice with influenza A infection (IAV), increased VEGFR3-mediated
lymphangiogenesis will enhance the lymphatic CCL21 gradient and increase dendritic cell (DC) migration from
the lung to the draining mediastinal lymph node (mLN) and that pulmonary TLO formation will enhance viral
clearance compared to infected controls, but deficient S1PR1 signaling in LECs will alter mLN architecture and
impair generation of B and T lymphocyte memory. To test this, I will infect S1pr1ΔLEC mice with IAV in the following
aims. In aim 1, I will examine viral clearance, lymphatic branching, pulmonary TLO formation, DC migration from
the lungs to the mLN and spleen, and the transcriptome of S1pr1-deficient LECs. To determine whether
phenotypes in S1pr1ΔLEC mice are due to excess VEGFR3, I will also perform these studies in S1pr1ΔLEC mice
with a reduced gene dosage of Vegfr3. In aim 2, I will investigate lymphocyte memory responses in S1pr1ΔLEC
mice with IAV by quantifying antigen-specific T cells and memory B cells, measuring IAV-specific antibodies,
examining mLN architecture, and studying response to challenge IAV infection. Preliminary data from my studies
show S1pr1ΔLEC mice have reduced morbidity, increased pulmonary TLO formation, altered DC migration, and
impaired antibody production following IAV infection. The successful completion of these studies will provide
novel insight into the interactions of immune cells with the lymphatic endothelium during infection and have
implications for development of therapeutics that act on lymphatic pathologies.
