Characterization of regulatory T cells in ex vivo expansion of human umbilical cord blood

Jung, Eui; Kim, Yong Man; Song, Ha Young; Won, Hye Sung; Kim, Ahm; Kang, Byung Moon

Abstract

OBJECTIVE
Regulatory T cells, which expressing CD4 and CD25, have a crucial role in suppressing immune systems to self-antigens and preventing autoimmune diseases. This study aims to evaluate the role of regulatory T cells in maternal tolerance to the fetus by comparing the proportion of regulatory T cells in peripheral blood of pregnant and non-pregnant women with those in umbilical cord blood. Also we analyze the changes of proportions of regulatory T cells of umbilical cord blood according to ex vivo expansion.
METHODS
An immunophenotypic study on 10 peripheral blood of pregnant and non-pregnant women and 10 cord blood was performed by means of FACSort flow cytometry using anti-CD4, anti-CD25 and anti-CD152 antibodies. Fresh cord blood mononuclear cells (MNCs) were isolated by Ficoll-Hypaque density centrifugation. The MNCs were cultured in anti-CD3 Ab-coated flasks for 4 days. The cells were then transferred to non-coated flasks with IL-2 175 U/mL and were cultured for another 17 days. The expression of CD4, CD25, CD152 and FoxP3 PCR were analyzed in accordance with days in culture. We also performed FoxP3 PCR in isolated CD25+ and CD25- T cells using MACSTM kit.
RESULTS
Umbilical cord blood had a higher proportion of CD4+CD25++ regulatory T cells than adults, and term-pregnant women had a lower proportion than non-pregnant women (p<0.05). After ex vivo expansion in anti-CD3 Ab coated flask with IL-2, we observed a significantly increased expression of CD4, CD25, CD152 at 4th day after culture and decrease thereafter. The result was the same as the expression of FoxP3 PCR.
CONCLUSION
Umbilical cord blood contained a high proportion of CD4+CD25++ regulatory T cells and regulatory T cells increased on culture day 4 and declined thereafter. Umbilical cord blood may serve as a readily available source of regulatory T cells for immunotherapy.