The Effects Of Ovarian Carcinoma Cells On The Function And Differentiation Of The Peripheral T Lymphocytes

PART â… Ovarian Carcinoma Cell Supernatants Inhibit Proliferation andIL-2R Expression of CD 8⁺ T cellsObjective To study the effect of supernatants derived from three ovarian carcinoma cell lines on proliferation and function of CD8⁺ T cell and to investigate the mechanism by which ovarian carcinoma escapes from immune recognition.Methods CD8⁺ T cell was cultured in complete RPMI 1640 with or without supematants of three ovarian carcinoma cell lines(OVCAR₃, CAOV₃ and SKOV₃). Proliferation of CD8⁺T cell was determined by MTT. Flow cytometry was used to detect the expressions of IL-2R α ,β and γ_c on CD8⁺T cell surface. ELISA was used to detect the concentration of sIL-2R in the CD8⁺T cell supernatants.Results 1. All culture supematants(OVCAR₃, CAOV₃ and SKOV₃) derived from three ovarian carcinoma cell lines inhibited the proliferation of CD8⁺ T cell. When the concentration of supernatants was 25%, P value was 0.004, 0.006, 0.013 respectively;When the concentration of supernatants was 50%, P value was 0.002, 0.003, 0.005 respectively;When the concentration of supematants was 75%, P value was 0.001. The degree of inhibition was dose dependent 2. The expressions of IL-2R β and γ_c on CD8⁺T cell surface were down-regulated significantly by all ovarian carcinoma cell culture supernatants;P value was 0.002, 0.013, 0.011 and 0.015, 0.039,0.040 respectively. The expression of IL-2R a was also down-regulated, but not significantly, P value was 0.235, 0.224 and 0.498 respectively. 3. The concentration of sIL-2R elevated significantly in the supernatant of CD8+ T cell that was cultured with ovarian carcinoma cell supernatants;P value was 0.008,0.010 and 0.005 respectively.Conclusion Soluble factors derived from ovarian carcinoma may inhibit the proliferation and function of CD8+ T cell, which may be one of the mechanisms by which the local immunity in the peritoneal cavity of ovarian carcinoma was suppressed.EARTHProportion of CD4+CD25+ Regulatory T Cells Is Increased in ThePatients with Ovarian CarcinomaObjective To study the frequency of the CD4+CD25+ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism.Methods The percentages of CD4+CD25+ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4+ PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, and men the percentage of CD4+CD25+ T cells was detected.Results CD4+CD25+ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4+CD25+ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4+PBLs from the patients was negative. The percentages of CD4+CD25+ T cells inPBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P=0.001 and 0.001 respectively). Conclusion There is an increasing of the proportion of CD4+CD25+ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.part inOvarian Carcinoma Cell Supernatants Enhance the Proliferation and Function of CD4+CD25* Regulatory T CellsObjective To investigate whether supernatants derived from cultured ovarian carcinoma cell line SKOV3 can enhance the proliferation and inhibitory capacity of CD4+CD25+ regulatory T cells. Methods CD4+CD25+ regulatory T cells were incubated in 1640 culture medium in presence of supernatant or not and supplemented or not with anti-CD3 and CD28 for 72 hours. Cell proliferation was determined by BrdU labeling. Expressions of GITR and CTLA-4 were detected by flow cytometry. Foxp3 mRNA expression was detected by semi-quantitative RT-PCR. Results SKOV3 supernatants could induce the proliferation of CD4+CD25+ regulatory T cells and enhance the inhibitory capacity whether CD3 and CD28 mAb existed or not. After treated with supernatant, the expressions of GITR and CTLA-4 of CD4+CD25+ regulatory T cells were significantly increased;and the expressions of Foxp3 mRNA didn't change significantly. Conclnsion Supernatants from SKOV3 culture can enhance the proliferation and inhibitory capacity of CD4+CD25+ regulatory T cells by up-regulating CTLA-4 and GITR expression, which may be responsible for the immune inhibitory of ovarian cancer.PARTIVConversion of Peripheral CD4+CD25 T Cells to CD4+CD25+Regulatory T Cells by Ovarian Carcinoma Cell SupernatantsObjective To investigate whether the supernatant from cultured ovarian carcinoma cell line SKOV3 could convert peripheral CD4+CD25" T cells to CD4+CD25+ regulatory T cells. Methods Culture supernatant from SKOV3 was collected. CD4+CD25" T cells isolated from peripheral blood of healthy woman were activated with plate-bound anti-CD3 and anti-CD28 in the absence or presence of supernatant for 72 hours. Cells were harvested and further sub-populated into CD25" and CD25+ T cells. Cell proliferation was determined by BrdU labeling. Expressions of GITR and CTLA-4 were detected by flow cytometry. Foxp3 mRNA expression was detected by RT-PCR. Then Supernatant was fractionated into three different molecular weight fractions (MWFs) and the above experiments were repeated with the supernatant instead of three different MWFs.Results In contrast to activated CD4+CD25+ T cells, supernatant-induced CD25+ T cells exhibited decreased TCR-triggered proliferation, acquired the capacity to suppress CD4+CD25" T proliferation and expressed the same phenotype (GITR and CTLA-4) and Foxp3 mRNA as natural CD4+CD25+ regulatory T cells. Among the three different MWFs, the low MWF have the same function in the transformation process as the whole supernatant. Whereas the medium and high MWFs induced CD4+CD25+ T cells exhibited low proliferation and no immunosuppressive capacity.Conclusion Low MWF of supernatant secreted by SKOV3 converts CD4+CD25' T cells into CD4+CD25+ regulatory T cells, which is probably responsible for the increasing number of CD4+CD25+ regulatory T cells in the patients with ovarian carcinoma.PARTVHuman Ovarian carcinoma Cells Generates CD4CD25+ RegulatoryT Cells from peripheral CD4+CD25" T Cells through SecretingTGF-pObjectives: The increase of CD4+CD25+ regulatory T cells is responsible for the immunosuppressive state in patients with ovarian carcinoma, but there is still little study about the mechanism. To explore this, we investigated the effects of TGF-P secreted by ovarian carcinoma cell line SKOV3 on the CD4+CD25" T cells isolated from healthy women. Materials and Methods: Supernatant from SKOV3 was collected and fractionated into low molecular weight fractions (3-50kDa). The concentration of TGF-P in supernatant was determined by ELISA assay. CD4+CD25" T cells were activated by anti CD3 and CD28 mAb and cultured with or without TGF-P or fee low MWF. In some experiments, anti-TGF-p (lOug/ml) was added to the low MWF. CD4+CD25+ T cells were isolated after culture for 72h. The phenotype of glucocorticoid-induced tumor necrosis factor receptor (GITR) and Cytotoxic T lymphocyte antigen-4 (CTLA-4) was detected by flow cytometry. The proliferation and inhibitory ability of individual isolated CD4+CD25+ T cells were assayed. Foxp3 mRNA expression was detected by RT-PCR. Results: The concentration of TGF-P in the SKOV3 supernatant was 2.347 ± 0.169 ng/ml. Similar to TGF-p, the low MWF could convert part of freshly isolated CD4+CD25'T into CD25+ population. These CD25+ T cells showed similar characteristics as natural CD4+CD25+ regulatory T cells, as they also exhibited decreased TCR-triggered proliferation, possessed the capability to suppress CD4+CD25" T proliferation and demonstrated the same phenotype (GITR and CTLA-4) and produced Foxp3 mRNA. Depleted TGF-P in the low MWF by neutralizing anti-TGF-p was able to eliminate most of this transformation phenomenon. Conclusions: TGF-P secreted by ovarian carcinoma cells is likely to play a key role in generating CD4+CD25+ regulatory T cells from CD4+CD25' T cells.