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 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-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-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.
Dengler
T J
TJ
Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.
Johnson
D R
DR
Pober
J S
JS
D015703:Antigens, CD / Q000096:biosynthesis; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002455:Cell Division / Q000276:immunology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002999:Clone Cells; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay / Q000379:methods; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007156:Immunologic Memory; D007399:Interphase / Q000276:immunology; D007519:Isoantigens / Q000276:immunology; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D013234:Stem Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D015703:Antigens, CD; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007519:Isoantigens; D013388:Succinimides