Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions

Since vascular endothelium is now recognized as an immunologically active t issue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited b y methodology. We developed a flow cytometric method allowing for concurren t investigation of multiple cell populations within the same culture that c an be applied to these complex interactions. Allogeneic CD8(+) or CD4(+) T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFS E) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle- blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing ce llular division, we minimized erroneous interpretation of the data encounte red by previous studies, which utilized H-3-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilita ted the study of CD8(+) lymphocyte activation, differentiation, and acquisi tion of effector function. By gating on the endothelial cell population, ph enotypic changes such as upregulation of surface MHC molecules or immune-me diated apoptosis could be detected. In conclusion, we present a flow cytome tric approach that could have important applications for clinical immunolog ical monitoring in allogeneic or xenogeneic transplantation, and might prov ide the requisite information to better tailor immunotherapy to prevent chr onic rejection. (C) 2001 Wiley-Liss, Inc.