Pleiotrophic roles of VEGF during neonatal thymic development

In this report, we show that VEGF-blockade in neonates induces rapid changes in all three stromal compartments of the thymus, and that these changes recapitulate the maturation of these cell types seen during the transition from the neonatal to the young adult thymus. These changes in the microenvironment occur prior to the loss of thymocytes, and include not only a loss of cTECs relative to mTECs but also depletion of CD146+CD140aneg cells, a novel population of pericytes we have identified in the neonatal thymus. Importantly, we show that based on receptor expression and receptor blockade, VEGF drives the fetal/neonatal phenotype of the thymus through distinct VEGFR2- and NRP1-dependent pathways in thymic endothelial and mesenchymal cells respectively. In addition, receptor signaling induces changes in critical pathways affecting the function of all three compartments, with consequences on thymopoieisis. To investigate the role of VEGF in the neonatal thymic microenvironment, we treated P1 mice with either Aflibercept (aka VEGF-Trap), a VEGFR1/R2 fusion protein which acts as a decoy molecule for all VEGF-A isoforms, or an hFC control antibody. Thymi were harvested 2 days post injection (P3), digested and depleted from thymocytes (CD45 magnetic depletion). Thymic stromal cells were isolated by fluorescence activated cell sorting as follows: within the stromal cell gate (CD3-CD4-CD8-CD45-Ter119- cells), thymic epithelial cells (TECs) were identified by their expression of the epithelial cell adhesion molecule (EpCAM) and were further subdivided into cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) based on high to low/negative Ly51 expression, respectively. Platelet endothelial cell adhesion molecule (PECAM-1 aka CD31) expression allowed the isolation of endothelial cells. By excluding endothelial, epithelial and hematopoietic cells, we next examined the mesenchymal population of the thymus. CD146+ and CD140a+ mesenchymal cells were isolated within the EpCAMnegCD31neg stromal cell gate.