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 lymphocytes

We have previously shown that human endothelial cells (EC) are less efficie nt than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulatin g allogeneic CD8(+) T cells to develop into CTL. In this study we describe FAGS-based limiting dilution analyses using the dilution of the intracellul ar dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion w ith significantly increased sensitivity compared with conventional, cytotox icity-based assays. In addition, this assay permits the relative size of cl onal CTL populations that are generated in individual CD8(+) T cell culture s to be determined (clonal burst size). We have applied this method to quan titatively compare the generation of CTL at the clonal level following stim ulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from th e same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSElow cells and were categorized as CTL by the expression of int racellular perforin and IFN-gamma, 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-stimuls ited CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes e xclusively with BLC stimulation, Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large bu rst sizes of CTL were observed from both naive and memory CD8(+) T cell pre cursors. These data establish that both a lower frequency of reactive precu rsors and more limited clonal expansion, but not regulatory T cells, contri bute to the reduced capacity of EC to promote alloreactive CTL differentiat ion compared with that of professional APC.