The Molecular Mechanism Of HAb18G/CD147 In The Formation Of Immune Synapse And T Cell Activation

HAb18G/CD147 is a newly found hepatocellular carcinoma associated antigen screened in human hepatoma tissues cDNA library by HAb18, a liver cancer specific antibody made in our laboratory. HAb18G/CD147 belongs to the immunoglobulin superfamily with a molecular weight of 35~65 kDa. It has been demonstrated that HAb18G/CD147 is highly expressed in many tumor cells and is participated in the regulation of tumor cell growth, invasion and metastasis. Further researches also show that highly expression of HAb18G/CD147 has a direct impact on the treatment and prognosis of cancer. In recent years, more and more attention is focused on the study of CD147 and immune-related diseases, especially researches on the relationship between CD147 and T cell-mediated immune and inflammatory diseases. Many experimental results show that the up-regulation of CD147 has a significant correlation in inflammatory diseases related to abnormal activation of T cells.Immune synapse is a supra-molecular activation cluster structure formed between T cells and antigen presenting cells, which is involved in the process of T cell activation and proliferation. Previous studies in our laboratory showed that, HAb18G/CD147 was up-regulated in TCR/CD3 activated T cells, and colocalized with TCR co-receptor CD4/CD8 and CD48, a marker of lipid rafts, in the immunological synapse, suggesting that CD147 is participated in the formation of the immune synapse. Taking into account that many chronic inflammatory autoimmune diseases are related to the abnormal activation of T cells, which accompanied by a significant increase in CD147, we proposed a question that, is CD147 capable of promoting the occurrence and development of autoimmune diseases by activating T cells which is mediated by immune synapse formation?Our research aims to clarify the role of HAb18G/CD147 in the immune synapse formation and the mechanism in T cell activation, which will provide a theoretical basis for the treatment of T cell-mediated immune diseases by targeting CD147. The research work is divided into the following four parts:Part I: Distribution and expression of HAb18G/CD147 in T cells. CD147 has long been identified as a lymphocyte activation associated antigen in T lymphocytes. However, the distribution of CD147 in different T cell subsets and the expression under different stimuli conditions is still not clear. Therefore, this study first analyzed the expression of CD147 in different subsets of CD4+ T cells and the changes in the expression of CD147 under different activated conditions. It was found that the non-activated CD4+ T cells expressed a relatively lower level of CD147. However, the expression of CD147 significantly increased under different activation conditions, in which CD3 plus CD28 antibody induction increased most obviously, and was time-dependant. CD147 expression in memory T cells was much higher than that in naive T cells, and the expression of CD147 after activated in memory T cells was also higher than that in naive T cells, suggesting that CD147 may play a more important role in memory T cells, as its specific function still needs further studies. In view of this, we choose memory T cells to perform the following experiments in the formation of immune synapse and T cell activation.Part II: The location of CD147 in immune synapse and its function. After confirming that the expression of CD147 increased in activated T cells, we further observed whether CD147 is involved in the formation of the immune synapse and its exactly location in it. Jurkat T-Raji B cell model was used to mimic the immune synapse formed between T-APC cells in vivo. Both confocal microscopy and immunogold electron microscopy results showed that CD147 localized in the immune synapse. Through the threedimensional reconstruction of cross-sectional structure of the immunological synapse, we found that about 90% of CD147 co-localized with CD2, which confirmed that CD147 are mainly distributed in p-SMAC. We further down-regulated the expression of CD147 in both Jurkat T and Raji B cells by lentiviral vector technology to observe its effect on the immune synapse formation. Interestingly, no significant immune synapse formation was found after CD147 knocked down in T cells, while, the formation of immune synapse was not influenced after CD147 knocked down in B cells. Therefore, we draw the conclusion that CD147 did play an important role in the immune synapse formation, and it is mainly associated with CD147 on T cells.Part III: CD147 participated in IS formation and mediated T cell activation in vivo. In this part, we used T-APC cells isolated from human body to replace in vitro Jurkat T- Raji B as an immune synapse model, which is a more realistic reflection of CD147 involved in immune synapse in vivo. Monocytes were isolated from PBMC and differentiated into mature macrophages and dendritic cells. After that, confocal microscopy was used to observe the immune synapse between memory T cells and APC cells. It was found that the up-regulated CD147 in activated T cells also accumulated in the immune synapse, which prompted that CD147 is participated in the formation of IS. CD147-specific monoclonal antibody HAb18 could not only inhibit the expression of early T cell surface activation marker CD69, but also reduce the expression of late T cell activation marker CD25 and the secretion of IL-2. These results strongly suggested that CD147 molecule played an important role in T cell activation.Part IV: The molecular mechanism of CD147 in the formation of immune synapse and T cell activation. CD147 accumulated in the immune synapse and played a critical role in T cell activation. However, due to its short structure of the intracellular segment, CD147 is still not reported to be capable of transducing signals. We considered that CD147 may interact with other molecules which involved in synapse formation and T cell activation. It was found that the down-regulation of CD147 did not affect the membrane expression of CD3, CD2, LFA-1 and CD48. However, the expression of CD98 was significantly reduced. And through the three-dimensional structure reconstruction technology, we could observe more than 90% of CD147 and CD98 molecules were co-localized together, suggesting that the interaction between CD98 and CD147 may participate in the formation of the immune synapse and its subsequent signals. To further clarify the signals of CD147 involved in T cells, we used two high-throughput detection technologies, CBA and Luminex, to test the molecules involved in T cell signal transduction and verified by Western Blot. The results showed that after blocking with HAb18, the levels of p-ZAP70, p-ERK, pC-JUN, p-CREB and p-LAT were significantly decreased, suggesting that these molecules might involved in T cell activation. CD147 could participate in the activation of T cells from MAPK, PI3K-AKT and other signaling pathways.In summary, CD147 is highly expressed on activated T cells and is involved in the formation of the immune synapse between a T-APC cells, the exact location of CD147 in IS is p-SMAC. Down-regulation of CD147 in T cells can significantly reduce the rate of immune synapse formation. CD147 specific monoclonal antibody HAb18 has an inhibitory effect on T cell activation from multiple signaling pathways. CD147 may interact with CD98 and participate in the immune synapse formation and activation of T cells. This is the first study to clarify the important role of CD147 molecule in the immune synapse formation and T cell activation, and reveals the molecular mechanism of CD147 in T cell signaling transduction, providing a new theory of anti-inflammatory treatment targeting CD147.