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.
J Immunol
; 166(6): 3846-54, 2001 Mar 15.
Article
en En
| MEDLINE
| ID: mdl-11238628
ABSTRACT
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.
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Colección:
01-internacional
Asunto principal:
Células Madre
/
Succinimidas
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Pruebas Inmunológicas de Citotoxicidad
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Endotelio Vascular
/
Activación de Linfocitos
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Subgrupos de Linfocitos B
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Subgrupos de Linfocitos T
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Linfocitos T CD8-positivos
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Fluoresceínas
Límite:
Humans
Idioma:
En
Revista:
J Immunol
Año:
2001
Tipo del documento:
Article
País de afiliación:
Estados Unidos