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. 2001 Mar 15;166(6):3846-54. doi: 10.4049/jimmunol.166.6.3846.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, CD / biosynthesis
  • B-Lymphocyte Subsets / cytology
  • B-Lymphocyte Subsets / immunology*
  • B-Lymphocyte Subsets / metabolism
  • CD8-Positive T-Lymphocytes / cytology
  • CD8-Positive T-Lymphocytes / immunology*
  • CD8-Positive T-Lymphocytes / metabolism
  • Cell Division / immunology
  • Cell Line, Transformed
  • Cells, Cultured
  • Clone Cells
  • Coculture Techniques
  • Colony-Forming Units Assay / methods
  • Cytotoxicity Tests, Immunologic / methods*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / immunology*
  • Flow Cytometry / methods
  • Fluoresceins / metabolism*
  • Fluorescent Dyes / metabolism
  • Humans
  • Immunologic Memory
  • Interphase / immunology
  • Isoantigens / immunology
  • Lymphocyte Activation*
  • Lymphocyte Count
  • Stem Cells / cytology
  • Stem Cells / immunology*
  • Stem Cells / metabolism
  • Succinimides / metabolism*
  • T-Lymphocyte Subsets / cytology
  • T-Lymphocyte Subsets / immunology*
  • T-Lymphocyte Subsets / metabolism
  • T-Lymphocytes, Regulatory / immunology
  • T-Lymphocytes, Regulatory / metabolism

Substances

  • 5-(6)-carboxyfluorescein diacetate succinimidyl ester
  • Antigens, CD
  • Fluoresceins
  • Fluorescent Dyes
  • Isoantigens
  • Succinimides