Supernatant Of Human Epithelial Ovarian Carcinoma Cell Culture Induces CD4+CD25-CD45RA+ Na(?)ve T Cells To Develop Into Regulatory T Cells In Vitro

PARTⅠThere are different FOXP3 expression profiles of human CD4+CD25-CD45RA+T cells and CD4+CD25-CD45 RA-T cellsObjective To study the forkhead box protein 3 (FOXP3) expression in activated or unactivated different subtypes of CD4+T cells, and to explore the latent relation between these cells and regulatory T cells.Methods T cells were isolated over the autoMACS Separator. CD4+ T cells, CD4+CD25+T cells, CD4+CD25-CD45RA+T cells and CD4-CD25-CD45RA-T cells were isolated with human CD4+T cell isolation kit II, CD25 microbead and CD45RA microbead respectively. The isolated T cells were cultured in complete RPMI 1640 with or without anti-CD3/CD28 dual-signal activation. The purity of all isolated populations was routinely controlled by flow cytometry. The FOXP3 expression of cultured T cells was determined by single-cell analysis using flow cvtometrv after 3 days. Results High purity CD4+CD25-CD45RA+ naive T cells didn't contain any cells with FOXP3 expression by single-cell analysis. Only a few of these CD4+CD25-CD45RA+T cells will express FOXP3 when they are activated with anti-CD3/CD28 dual-signal for 3 days. However, a few CD4+CD25-CD45 RA-T cells express FOXP3 without activation with anti-CD3/CD28 dual-signal, and about half of all these cells will express FOXP3 when they are activated with anti-CD3/CD28 dual-signal for 3 days. FOXP3 expression was observed in a considerable part of CD4+CD25+T cells.Conclusion There are different forkhead box protein 3 expression profiles in different subtypes of CD4+T cells, especially when the cells were activated with anti-CD3/CD28 dual-signal. Foxp3 expression is absent in unstimulated CD4+CD25-CD45RA+T cells, and few cells express FOXP3 in activated CD4+CD25-CD45RA+ naive T cells. The CD4+CD25-CD45RA+ naive T cells are competent precursors for the study of inducing regulatory t cells in vitro.PARTⅡThe activated human CD4+CD25-CD45RA-T cells express FOXP3 and exhibit suppressive ability in vitro.Objective To determine the suppressive ability of activated CD4+CD25-CD45RA-T cells.Methods The isolation and culture of T cells were shown in the part I. The FOXP3 expression of cultured T cells was determined by single-cell analysis using flow cytometry as shown in part I. The suppressive ability of activated CD4+CD25-CD45RA -T cells was determined with coculture suppression assay using a BrdU proliferation ELISA kit.Results More than half of CD4+CD25-CD45RA-T cells will express FOXP3 when they are activated with anti-CD3/CD28 dual-signal for 3 days in the presence or absence of exogenous IL-2. There are high rate of cells will express FOXP3 even these cells are activated just with anti-CD3 single-signal. Neutralization assays revealed that neutralizing antibody against TGF-βor interleukin-10 did not abrogate the expression of FOXP3. Furthermore, an in vitro coculture suppression assay showed that these cells could suppress the proliferation of autologous CD4+CD25-CD45RA+T cells T cells. The suppressive ability of activated CD4+CD25-CD45RA-T cells decreased accompanying with increased apoptosis as prolong of activation duration.Conclusion There is a subset of regulatory T cell precursors in human memory CD4+ T cells, which does not express FOXP3 in rest state, quickly express FOXP3 and exhibit suppressive ability once exposed to its cognate antigen, but experience promptly apopatosis. [Key words] regulatory T cells; memory T cells; forkhead box protein 3; apoptosisPART III Human epithelial ovarian carcinoma cell-derived cytokines cooperatively induce activated CD4+CD25-CD45RA+ naive T cells to express FOXP3 and exhibit suppressive ability in vitro.Objective To study whether the culture supernatant of human epithelial ovarian carcinoma cells could induce activated CD4+CD25-CD45RA+ naive T cells to express FOXP3 and exhibit suppressive ability in vitro and explore the involved mechanismMethods We collected high-purity human CD4+CD25-CD45RA+ naive T cells by microbead cell separation. The CD4+CD25-CD45RA+ naive T cells were cultured in the absence or presence of the culture supernatant of human epithelial ovarian carcinoma cells for 3 days. The FOXP3 expression of cultured T cells was determined by single-cell analysis using flow cytometry as shown in part I. The suppressive ability of induced FOXP3+ T cells was determined with coculture suppression assay using a BrdU proliferation ELISA kit. In neutralization experiments.10 ug/mL of neutralizing anti-TGF-beta. anti-IL-10, anti-IL-4, anti-LIF antibody or a combination of neutralizing anti-TGF-beta and anti-IL-10 antibody was used. In induction experiments, hrLIF or IL-6 at 10 ng/mL was used. The FOXP3 expression of cultured T cells was determined by single-cell analysis using flow cytometry as shown in part I.Results High-purity human CD4+CD25-CD45RA+ naive T cells did not express FOXP3 by single-cell analysis, and few cells expressed FOXP3 when they were activated with anti-CD3/CD28 dual signal. However, more cells expressed FOXP3 when the supernatant of human epithelial ovarian carcinoma cell culture was added, yet not the supernatant of normal human ovarian surface epithelia cell culture. A further coculture suppression assay showed that these cells could suppress the proliferation of autologous CD4+CD25-CD45RA-T cells. Neutralizing antibody against transforming growth factor beta (TGF-beta), interleukin-10, and interleukin-4 did not abrogate elevated FOXP3 expression induced by carcinoma cell culture supernatant, whereas neutralizing leukemia inhibitory factor (LIF) partially abrogated FOXP3 expression, but LIF alone could not increase FOXP3 expression in activated naive T cells.Conclusion Human epithelial ovarian carcinoma cells are able to induce expression of FOXP3 and exhibit suppressive ability in activated CD4+CD25-CD45RA+ naive T cells, which may be related with the increased regulatory T cells in the patient of ovarian carcinoma. Multiple human epithelial ovarian carcinoma cell-derived cytokines could be involved in the FOXP3 expression induction of activated CD4+CD25-CD45RA+ naive T cells. There should be a complicated interaction among these cytokines, which cooperatively induces the expression of FOXP3 in activated naive T cells and differentiates these cells into regulatory T cells.