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Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8⁺ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes¹

Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8⁺ T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8⁺ T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8⁺ T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8⁺ T cells by either BLC or HUVEC derived from the same donor. CD8⁺ T cells expanded by allostimulation were identified as CD8⁺, CFSE^low cells and were categorized as CTL by the expression of intracellular perforin and IFN-. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8⁺ T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8⁺ T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.

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