B7-H1Affects The Differentiation Of Treg Cell In Glioma

Tumor is one of the most important diseases threatening to human health nowadays.It is important to find new treatment methods. Because of proactive immune escapemechanism, a variety of immune treatments show little efficiency at present. Variousmethods are adopted to evade immune clearance by anti-tumor immunity of tumor cells.And the PD-L1(B7-H1)/PD-1signaling and regulatory T cells are two important wayshelping tumor to evade immune clearance. B7-H1is highly expressed on the surface oftumor cells and other related cells, and the proportion of tumor-infiltrating Treg cellsincreases abnormally in all T lymphocytes. The combination of B7-H1and PD-1conductssuppressive immune signal. Treg cells inhibit the activity of tumor-specific T lymphocytesby directly or indirectly immunosuppressive effect that helps the tumor to evade immuneclearance. The latest study found that as an important molecule, B7-H1maintains thedevelopment and function of inducible regulatory T lymphocyte. The B7-H1signalingalone can convert naive (CD4+CD25-) T cells into Treg cells which play animmunosuppressive effect. Yet, there are no answers that whether the B7-H1regulates immunosuppressive effect by Treg cells or how much effect the induction of Treg cellswork during the process of tumor immune escape. Our study intends to find out how thetumor-associated B7-H1achieves tumor immune escape via regulating Treg cells by theco-culture of glioma cells and na ve T cells. And establish new ideas of suppressing tumorimmune escape by interfering tumor cell’s expression of B7-H1. This research can deeplyunderstand the mechanism of tumor immune escape in order to find out ways inovercoming tumor immune escape and propose new therapeutic strategies. So there is avery important theoretical and practical value for improving tumor immune clearance andcuring cancer.Objective:Clarify the relationship between the immunosuppressive effects of tumor associatedB7-H1and induced Treg cells in order to further clarify the tumor immune suppressionmechanism of B7-H1molecule. Explore the effect of interfering tumor associated B7-H1molecule on tumor immune escape.Methods:1. Immunohistochemistry and immunofluorescence were used to detect theexpression of B7-H1molecule in clinical glioma and Treg cells infiltrating in the tumor,respectively.2. The expression of B7-H1molecules in glioma cell lines was detected by flowcytometry, and t a cell line which expresses highly B7-H1molecules was selected.3. Lentiviral expression vector containing shRNA targeting B7-H1molecules wasconstructed and used to infect high expression B7-H1molecules cell line. The interferenceeffect was detected by utilizing Western blot and qRT-PCR.4. PBMCs were isolated from normal human blood. Na ve T cells were selected withimmunomagnetic beads and sorting purity was detected.5. Na ve T cells were activated by anti-CD3and anti-CD28stimulating.6. Na ve T cells were co-cultured with glioma cells expressing low B7-H1moleculesby interfering and wild type glioma cells expressing normal B7-H1molecules. The degreeof Treg cells differentiation was detected. Results:1. In10glioma specimens B7-H1molecules expression was observed in7cases byimmunohistochemical staining. In3cases there are more Treg cells infiltrating andpositive B7-H1expression.2. The expression of B7-H1was detected in5human glioma cell lines using flowcytometry and there was higher expression in3cases. U251cells were used for futhertests.3. After co-stimulation by anti-CD3and anti-CD28proliferation of na ve T cells wasobserved which means na ve T cells were successfully activated.4. TGF-β played an important role in Treg cells differentiation.5. B7-H1and TGF-β showed a synergistic effect on promoting differentiation ofnaive T cells towards Treg cells.Main conclusions:This experiment confirmed the effect of B7-H1molecules in promoting thedifferentiation of na ve T cells towards Treg cells at the cellular level. Treg cellsnegatively regulate all of adaptive immune, so they contribute more to tumor escape.B7-H1which is highly expressed in tumor tissue promotes the differentiation of na ve Tcells towards Treg cells. Because Treg cells secrete several cytokines which cause localimmunosuppression, they reduce the scavenging effect of the immune system against thetumor. The experiment helps us further understanding the interaction of the tumor-relatedB7-H1molecules and Treg in tumor immunity suppression, for proposing new treatmentstrategies and breaking through the obstacles of tumor immune escape.