ABSTRACT
Trans-endothelial migration (TEM) is essential for leukocyte circulation. While much is known about trans-blood endothelial migration, far less is known about trans-lymphatic endothelial migration. We established an in vitro system to evaluate lymphatic TEM for various cell types across primary mouse and human lymphatic endothelial cells (LEC), and validated the model for the murine LEC cell line SVEC4-10. T cells exhibited enhanced unidirectional migration from the basal (abluminal) to the apical (luminal) surface across LEC, whereas for blood endothelial cells (BEC) they migrated similarly in both directions. This preferential, vectorial migration was chemotactic toward many different chemoattractants and dose-dependent. Stromal protein fibers, interstitial type fluid flow, distribution of chemokines in the stromal layer, and inflammatory cytokines influenced LEC phenotype and leukocyte TEM. Activated and memory CD4 T cells, macrophages, and dendritic cell (DC) showed chemoattractantΔdriven vectorial migration, while CD8 T cell migration across LEC was not. The system was further validated for studying cancer cell transmigration across lymphatic endothelium. This model for lymphatic TEM for various migrating and endothelial cell types possesses the capacity to be high-throughput, highly reproducible and integrate the complexities of lymphatic biology, stromal variability, chemoattractant distribution, and fluid flow.
Subject(s)
Cell Movement , Endothelial Cells/cytology , Endothelium, Lymphatic/cytology , Models, Biological , Animals , CD4-Positive T-Lymphocytes/cytology , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cellular Microenvironment , Chemokines/metabolism , Chemotaxis , Humans , Inflammation/pathology , Mice, Inbred C57BL , PhenotypeABSTRACT
BACKGROUND AIMS: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. METHODS: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. RESULTS: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. DISCUSSION: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.
