The Role Of The Exsome From Glioma Mediated Bregs Cell In Immune Escape

Invasion and metastasis are the important properties of malignant tumor. They also are main factors of recurrence of cancer. Glioma is the most aggressive brain tumor with h igh invasiveness and poor prognosis. One of the most lethal characteristics of gliomas is their capability of invading the side normal brain tissues of glioma.The Glioma is the most common intracranial malignant tumors. The incidence of brain glioma patients accounts for 35% ~ 60% of intracranial tumor patients in China. The 5-year survival rate of brain glioma patients is only 20% ~ 30%.There are the malignant degree is higher, the prognosis is poorer and the recurrence rate is higher that made the glioma to one of the most serious tumor. in clinical neurosurgery.The operation is the most important i for glioma treatment. As a result of the glioma is infiltrating grew, it is easy to recurrence after single surgical treatment. As glioma cells resisted, postoperative radiotherapy is not kill residual tumor cells. How the glioma cells to avoid or reduce radiation damage? It is great necessity to improve the curative effect of radiotherapy in glioma.The immune escape of tumor cells refer to the many ways to escape the body’s immune recognition and attacks, such as the change of cell surface markers, including tumor cells on the surface of the MHCⅠmolecular expression decreased or lost, the variation of tumor antigen, the effect of FAS system and abnormal expression of B7 molecules, etc. Source of tumor immunosuppression factor: such as IL- 10, VEGF, TGF-beta, etc. It can affect the DC and T cell mature or activation. The microenvironment of tumor, through the receptor/ligand binding way release inhibitory signals, induce d the T cell dysfunction. While T cell function is obstacle, the body’s immune is weak.PIGF will be high expressed in cancer cases, and the related receptor VEGFR1 expression was improved either. PIGF is apparent rise in many tumors, which can be used as the indexes in some cancers. Pl GF not only stimulate source cells to form new blood vessels, but Pl GF can act directly on the tumour cells, damage DC cells, so as to promote the growth of tumors cells. Pl GF also stimulate angiogenesis progenitor cells metastasis to the tumor lesions in the bone marrow. The antibody of PIGF is used not only prevent the generation of new blood essels, also can stimulate the degradation of blood vessels which has been generated. Recent studies have reported PlGF also participates in the regulation of the pathology of and immune process in glioma.We assume that the glioma secreted Pl GF, angiogenesis factors, give play to function through exsome. The exsome induced the B cells in the glioma microenvironment. While the B cells carried the exsome with Pl GF can express TGF – beta and transform into TGF-beta(+) Bregs cells. The Bregs cells can inhibited specificity CD8(+) T cells proliferation and activity. Thereby glioma cells can escape immune surveillance to spread and invade the normal tissue.To prove by the experiment1. We isolated and purified the malignant glioma cells(WHO Ⅲ to Ⅳ) from glioma tissues and culture in vitro. The glioma cells can expressed Pl GF m RNA and protein, when using different concentrations PMA stimulation, the PlGF increase with the increase of PMA. The data show that malignant glioma cells can express Pl GF.2 The supernatant of glioma cells secrete exsome by density gradient centrifugation. The LAMP1 is the specific marker of exsome. The PlGF protein could be detected in exsome. The results suggest that glioma cells can express Pl GF, and its secreted exsome also with Pl GF protein.3 The normal Naive B cells(CD19 + IL-7R + CD45 +) were isolated in whole blood. The exsome from glioma cell was labeled CSFE and with Naive B cell co-culture. Naive B cells captured the exsome can be found by immunofluorescence staining laser confocal microscope. when using different concentrations exsome stimulation, the B cells secrete TGF-beta mRNA and protein increase with the increase of exsome. While signal PlGF induced the Na?ve B cells, The B cell can express the TGF-beta. The data show that the exsome which carried Pl GF can be captured by Naive B cell, and the B cell secreted TGF-beta after the capture of exsome. The exsome which carried PlGF can be Naive B cell captured, TGF-beta(+) Bregs cells, in glioma microenvironment.4 isolated B cells from glioma tissues, the B cells isolated from PBMC in glioma patients and TGF-beta(+) Bregs cells were labeled with CSFE, respectively. extracted glioma tissues inclusions, 10 mg/ml of glioma tissue extracts stimulate three types B cells, and 10 mg/ml BSA as negative control. After three days in 37℃ 5% CO2 incubator, flow cytometry tested three types B cell proliferation. The results show that the source of 91.2% of the group B cell proliferation and 8.14% of peripheral blood B cell proliferation, about 60.8% of Bregs cell proliferation. The data show that the proliferation of B cells with glioma specificity. The TGF-beta(+)Bregs may exist glioma cells and the peripheral circulation with glioma patients.5 Isolated the PBMC from whole blood of glioma patients by density gradient centrifugation. inoculated the PBMC in the 75 cm bottle in 2 h. The suspension cell is separated from the adherent cells culture supernatant. Add to different stimulating factor to develop 7 days for DC cells and T cells, respectively. and purified CD8(+) CD25(-) T cells by the magnetic beads. The CD8(+) CD25(-) T cells were labeled with CSFE. The T cells mixed with DC cell(300000:200000) co-culture in 96-well cell plates. 10 mg/ml glioma tissue extract induced the T mix cells, and the negative add 10 mg/ml BSA. There were three B cells, add 200000 induced Bregs cells, Naive B cells and Bregs stimulated T mix cells in 3 days. Without B cells as negative control. The flow cytometry results showed that, CSFE labeled cells set door, 2.18% BSA stimulate CD8 + T cell prolife ration, 23.0% glioma extracts stimulate CD8 + T cell proliferation, which included some specific CD8 + T cells of glioma; The glioma specific CD8 + T cells can be specific inhibited activity by Bregs. 2.11%glioma CD8 + T cell proliferation induced by Bregs, and 30.2% CD8 + T cell proliferation induced by the natural B cells. Induction Bregs cell and T/DC separate training again after mixing the same conditions can also be suppressed, CD8 + T cell proliferation by 3.33%.Glioma extract training stimulus gliomas CD8 + T cells can secrete particles enzyme B and perforin, when there is Bregs cells also can inhibit the secretion.Showed that carrying Pl GF secrete body induced TGF- beta Bregs(+) cells has a specific inhibition glioma CD8(+) T cell proliferation, perforin and granular enzyme B tests showed Bregs cells also inhibits CD8(+) T cell killing activity.Anyhow, The Pl GF should be expressed in malignant glioma cells, and the exsome,which was glioma cells secreted, carried PlGF could be captured by the Naive B cells. The exsome with Pl GF changed the Naive B cells to TGF- beta(+) Bregs cells in the tumor microenvironment. the TGF-beta(+) Bregs cells, was specificity in glioma, can inhibit the proliferation and activity of specificity glioma CD8(+) T. It may be one of the reason that the glioma cells escaped the immune detection attack and invaded the normal brain cells.