Proportion of CD4+CD25+ regulatory T lymphocyte in peripheral blood of patients with gynecologic cancer / 대한산부인과학회지

OBJECTIVE: Recently the existence of a CD4+CD25+ regulatory (Treg) population has been described in rodents and humans. It is unclear how the immune response cells interact to tumor cells effectively, but the malignant tumor cell growth was suppressed by the main effect of T lymphocytes and natural killer cells in experimental studies using various biologic response modifier. This study was performed to investigate the proportion of CD4+CD25high Tregs and expression of Foxp3 in Peripheral blood (PBL)s in patients with cervical, ovarian or uterine cancers. METHODS: Blood samples were collected from 10 healthy women and a total of 40 patients with gynecologic cancer at department of Obstetrics and Gynecology, Asan Medical Center, Seoul, Korea, from March 2005 to September 2005, were enrolled in study group. Information regarding patient history and tumor stage was recorded. They were diagnosed at same center at first, and never been treated any therapy. The population of CD4+CD25+high Tregs as a percentage of total CD4+cells was evaluated by flow cytometric analysis. We measured the proportion of Treg cell that co-express CD4 and CD25 in the peripheral blood lymphocytes form patients with either cervical, ovarian uterine cancer or carcinoma in situ of cervix. Expression of Foxp3 in the CD4+subsets defined by electrophoresis. RESULTS: The following tumor entities were included cervical cancer (n=10. 7 in stage I, 1 in stage II, 1 in stage III, 1 in stage IV); ovarian cancer (n=10. 4 in stage I, 0 in stage II, 5 in stage III, 1 in stage IV), ; uterine cancer (n=10. 9 in stage I, 0 in stage II, 0 in stage III, 1 in stage IV). In cervical cancer patient, ovarian cancer patients, uterine cancer patients and healthy women, the proportion of CD4+CD25high Tregs was 4.53% (SD 2.30), 6.89% (SD 7.81), 4.37% (SD 2.43) and 0.87% (SD 0.57) of the total CD4+cells respectively. The proportion of CD4+CD25+high T cells was significantly higher in cervical cancer patients (p=0.016), ovarian cancer patients (p=0.001) and uterine cancer patients (p=0.038) when compared with healthy women. But there was no significant difference in proportion of CD4+CD25+ Tregs comparing with healthy women. Expression of Foxp3 was significantly thicker in tumor-associated lymphocytes than control T cells by electrophoresis. CONCLUSION: In conclusion, our data suggested that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against cervical, ovarian, uterine cancer could be important in design of immunotherapeutic approaches.

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

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.

Comparison of characteristics of Dendritic Cells derived from CD 14+ Monocyte and CD34+ Hematopoietic Stem Cell in Human Umbilical Cord Blood / 대한산부인과학회지

OBJECTIVE: To compare the morphological, phenotypical, and functional characteristics of dendritic cells (DCs) generated from two precursor cell sources (CD 14+ monocyte and CD34+ hematopoietic stem cell) of human umbilical cord blood (UCB) under identical ex vivo generation conditions and to determine the best cellular source for DCs-based anticancer immunotherapy. METHODS: CD14+ monocytes and CD34+ hematopoietic stem cells were isolated from human UCB and induced to differentiate into DCs under identical culture conditions using granulocyte macrophage colony stimulating factor (800 U/mL) and IL-4 (500 U/mL). Then maturation of DCs was induced with IFN-gamma (1000 U/mL) and LPS (1 microgram/mL). Morphology was compared with confocal microscopy and phenotypical analysis was done with FACS. The level of IL-12p70 production was determined with ELISA kit and T cell proliferation capacity was assessed with mixed lymphocyte reaction (MLR). RESULTS: Eight-day-old mature DCs from CD14+ monocytes or CD34+ hematopoietic stem cells had identical morphology. Flow cytometric analysis revealed that CD14+ monocyte-derived DCs (M-DCs) and CD34+ hematopoietic stem cell-derived DCs (CD34-DCs) showed similar enhanced expression of CD80, CD83, and CD86 (P>0.05). The level of IL-12p70 production was significantly higher in mature DCs (8-day-old) than immature DCs (6-day-old) of either source (P<0.05), but it was significantly highly elevated in CD34-DCs (P<0.05). DCs of both precursors showed potent stimulation capacity in MLR, with CD34-DCs having a maximum effect at 1:2 ratio and M-DCs at 1:20 ratio, although CD34-DCs had significantly greater proliferative effects at all ratios (P<0.05). CONCLUSION: These findings suggest that CD34-DCs may be a more attractive source for DC-based immunotherapy. But because of morphologic, phenotypical, and functional similarities between the two cellular sources, both are good sources for generating DCs and one is not superior to the other. Thus cellular availability could be an important factor in determining DC-generation protocol to be used if all other issues are equal. Further comparative testing for these two precursors is in need.