| Part1. The immunosuppression of CD14+HLA-DR-/low Myeloid derived suppressor cells to T lymphocytes in gastric cancerObjective:To investigate the frequency of myeloid-derived suppressor cells denoted here as CD14+HLA-DR-/low in gastric cancer patients, and explore the suppressive function to immunity of CD4+T lymphocytes.Methods:Mononuclear cells suspension was isolated from19pairs of gastric cancer tissues and adjacent normal tissue, then we detected the CD14and HLA-DR phenotype expression and analysed the differences between them by flow cytometry. For immunosuppression analysis, immunomagnetic bead separation was used in CD14+mononuclear cells isolation from tumor infiltrating tissue and adjacent normal tissue, which were subsequently cocultured with healthy donor CD4+T lymphocytes in the presence of anti-CD3/anti-CD28stimulation.At last, IFN-Y and IL-2production of CD4+T lymphocytes were analysed by flow cytometry.Results:Flow cytometry results of mononuclear cells showed there was a significant increase in the frequency of CD14+HLA-DR-/low cells in tumor-infiltrating tissue (13.2±8.0%,P<0.01), as compared with adjacent normal tissue (8.3±2.8%,P<0.01). Furthermore, the frequency of CD14+HLA-DR-/low cells in tumor-infiltrating tissue is associated with the depth of tumor invasion and TNM staging (p<0.05). In immunosuppression analysis, CD14+mononuclear cells from tumor infiltrating tissue are more suppressive in IFN-y production of healthy donor CD4+T lymphocytes, comparing with CD14+mononuclear cells isolated from non-tumor infiltrating tissue (8.1±2.3%vs.16.2±1.3%,P<0.05), as the same as IL-2production of healthy donor CD4+T lymphocytes (8.2±2.5%vs.13.7±2.1%, P<0.05).Conclusion:The immunosuppression to CD4+T lymphocytes immunity in tumor microenvironment of gastric carcer is mediated by CD14+HLA-DR-/low myeloid-derived suppressor cells. Part2. Involvement of Vasoactive intestinal polypeptide induction in human CD14+HLA-DR-/low MDSC differenciationObjective:To investigate the capacity of VIP in inducing CD14+mononuclear cells into CD14+HLA-DR-/low MDSC which acquired suppression function to CD4+T lymphocytes.Methods:CD14+mononuclear cells were isolated from healthy donors’ PBMC by immunomagnetic bead separation.Then the acquired cells were incubated with different concentration of VIP. Later, pretreated CD14+mononuclear cells were harvested and cocultured with CD4+T cells.which were also isolated from allogeneic healthy donors’ PBMC,in the presence of anti-CD3/anti-CD28stimulation.Then IFN-y and IL-2production of CD4+T lymphocytes were analysed by flow cytometry. IL-10production in coculture supernatant removed from the VIP induction assay were tested by ELISA, anti-IL-10was added to the coculture system to investigate the involvement of IL-10production in VIP mediated CD4+T cells immune tolerance.Results:Comparing to none-VIP pretreatment group, IFN-y production of CD4+T lymphocytes was down-regulated after cocultured with10-7M VIP pretreated CD14+PBMC (9.8±1.4%vs.15.1±3.1%, P<0.05), as same as IL-2production (30.4±4.3%vs.37.7±7.2%, P<0.05). At the same time, higher concentration of VIP was applied and pretreated CD14+PBMC also acquiered suppression function towards CD4+T lymphocytes. However, a dose-independent phenomenon was observed between VIP pretreatment groups in both IFN-y (9.2±1.5%vs.9.8±1.4%, P>0.05) and IL-2(29.9±3.7%vs.30.4±4.3%,P>0.05)production. Production of IL-10was elevated in the coculture system of VIP pretreated CD14+PBMC and CD4+T lymphocytes as compared to none-VIP pretreatment group. Therefore, anti-IL-10was added into the coculture system and the suppression progress was reversed as effector cytokines IFN-y and IL-2production was recovered.Conclusion:VIP is involved in the induction of human CD14+HLA-DR-/low MDSC which subsequently suppress CD4+T lymphocytes response. Moreover, increased production of IL-10get involved in the suppression mechanism. |
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