Bibliographic Metadata

Ki-67 staining and CFSE labelling for determination of HCV specific proliferative CD4 T-cell responses in acute HCV infection / submitted by Rene Manhart
AuthorManhart, Rene
CensorHolzmann, Heidemarie
Description68 Bl. : Ill., graph. Darst.
Institutional NoteWien, Med. Univ., Diss., 2010
Zsfassung in dt. Sprache
Bibl. ReferenceOeBb
Document typeDissertation (PhD)
Keywords (DE)CFSE / T-Zellen / Proliferation / HCV / Ki-67
Keywords (EN)CFSE / T-cells / proliferation / HCV / Ki-67
URNurn:nbn:at:at-ubmuw:1-3510 Persistent Identifier (URN)
 The work is publicly available
Ki-67 staining and CFSE labelling for determination of HCV specific proliferative CD4 T-cell responses in acute HCV infection [4.67 mb]
Abstract (English)

Hepatitis C virus (HCV) specific CD4 T-cell proliferation has been shown to be a decisive factor for resolving acute HCV infection, and impairment of CD4 T-cell proliferative capacity is an early predictor of chronically evolving HCV infection. Several methods exist to evaluate HCV specific T-cell proliferation. Methods compatible with Flow Cytometry include the detection of intranuclear Ki-67 expression in cells during the active phases of the cell cycle, and the use of the intracellular dye CFSE, which detects cells that actually have undergone cell division. We have previously used early Ki-67 expression after 48 hours of stimulation to detect HCV specific CD4 T-cell proliferation.

This short culture period made the simultaneous detection of cytokine production and proliferation at the single cell level possible. However, questions arose whether early Ki-67 expression really indicates proliferation in terms of completed divisions. The comparability of Ki-67 and CFSE data has previously only been investigated in monkey PBMC with contradicting results. We therefore aimed to correlate both markers for the detection of HCV specific proliferative responses in human PBMC.

PBMC samples from HCV patients were assessed for CD4 T-cell proliferative responses in vitro. Therefore PBMC were stimulated with mitogen or viral antigens. Ki-67 expression and CFSE dilution were simultaneously measured using Flow cytometry, and their correlation was investigated at different timepoints post stimulation. Early Ki-67 upregulation, detectable after 48 hours of stimulation time, was observed after mitogenic and antigenic stimulation. Cell division as measured by CFSE dilution was only detectable in mitogen-stimulated cells at this early timepoint. However we observed cell division at later timepoints in all antigen stimulated cells that did show early Ki-67 upregulation. Stimulating PBMC for 120 hours showed the highest sensitivity in both tests, however the CFSE assay was more sensitive than the Ki-67 assay in detecting proliferating CD4 T-cells. We found that Ki-67 is a very early marker for CD4 T-cell proliferation and predicted later cell division. However highest sensitivity was observed using longer stimulation times of 120 hours with CFSE, while Ki-67 is rapidly downregulated in divided cells and does not correlate with CFSE data. Incorporating certain cellular morphological changes into the analysis of Ki-67 expression yields results comparable to CFSE data.