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

Alexander S. Krupnick, Daniel Kreisel, Wilson Y. Szeto, Sicco H. Popma, Kunjlata M. Amin, Jonni S. Moore, Bruce R. Rosengard

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Since vascular endothelium is now recognized as an immunologically active tissue, 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 by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycleblocking 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 cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized 3H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.

Original languageEnglish
Pages (from-to)271-280
Number of pages10
JournalCommunications in Clinical Cytometry
Volume46
Issue number5
DOIs
StatePublished - Oct 15 2001

Keywords

  • 5,6-Carboxyfluorescein diacetate succinimidyl ester
  • Cytometry
  • Endothelial cells
  • Mouse

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