Involvement Of HAb18G/CD147 In T Cell Activation And Immunological Synapse Formation

The human cell surface molecule CD147 was designated at the Sixth International Workshop and Conference of Human Leukocyte Differention Antigens. It is a 50-60 kDa type I transmembrane glycoprotein that was categorized as a member of the Ig superfamily. HAb18G is a new hepatoma-associated antigen recently cloned by hepatoma monoclonal antibody HAb18 screening from human hepatocellular carcinoma (HCC) cDNA library in our lab. The sequence of HAb18G is highly homology with CD147 molecule. Functional studies revealed that CD147 is a pleiotropic molecule and plays very important roles in fetal, neuronal, lymphocyte development and in pathologic conditions including heart disease, Alzheimer's disease, stroke, tumor, inflammation and autoimmune disease. In T cells, CD147 was identified very early as a lymphocyte activation-associated antigen, it's expression depends on the differentiation state and activation level. Within the thymus, CD147 expression is highest on late stage and correlates with cycling of immature thymocytes even in the absence of TCRbeta selection. Treatment immature fetal lymphocytes with an anti-CD147 monoclonal antibody can arrest any further development. Within circulating immune cell populations, CD147 is expressed on the surface of all immune cells, but increased on activated T cells. Over-expressed CD147 was also found on CD3+ T lymphocytes from systemic Lupus Erythematosus (SLE) patients when compared with CD3+ T lymphocytes from healthy donors. Moreover, studies conducted in CD147–/– mice have also shown that the absence of this protein leads to hyperproliferation of T cells during mixed lymphocyte reactions. To date, the exact role of CD147 in T cell activation still remains unknown. In this study, the molecular function of HAb18G/CD147 in regulating T cell activation was investigated.For this purpose, we choose the classical CD3-mediated T cell activation model to elucidate the functions of CD147 during this process. The CD147 expression level of mRNA and membrane protein was measured by Real-Time PCR and flow cytometry independently. Anlysis of mRNA level in both resting and activated CD3+ T cells revealed a 10.8-fold increase in CD147 transcription in the activated population. As demonstrated by flow cytometry, the expression of membrane CD147 was significantly upregulated in both CD4+ and CD8+ activated T cells in contrast to resting ones. The above data confirmed the notion that CD147 expression level correlates with the state of T cell activation, which is based on studies in PHA activated or tumor-specific T cells. Here, our data for the first time revealved the expression of CD147 upon TCR/CD3 stimulation and provides more detailed evidence of it's expresion in different T cell subsets. At the early stage of T cell activation, it has been proposed that the sustained and close apposition of cell surfaces required for TCR: antigen peptide-MHC interaction relies on the formation of immunological synapse. A number of T cell activation related signaling molecules are enriched into immunological synapse during T cell activation. Therefore, we studied whether unregulated CD147 translocates to the cap structure triggered by TCR/CD3 in activated T cells or to the immunological synapse between APC and T cell. Images by confocal microscope clearly show that CD147 was recruited to the cap structure or the immunological synapse together with CD4/CD8 TCR-coreceptor and lipid raft marker CD48 and GM1. Isolation lipid raft fractions and Western blot analysis further confirmed the immunological synapse enrichment distribution feature of CD147 during T cell activation. These observations prove the requirement of CD147 for T cell activation and promote us to analyze the effect of a TCR/CD147 costimulation on T cell activation.In order to investigate whether a CD147/TCR costimulation would affect T cell proliferation, we tested the influence of cross-linking HAb18G/CD147 with specific mAbs generated in our lab. Among four mAbs 3B3,5A12,6H8 and HAb18, we found that mAb 5A12 can strongly inhibit the proliferation of T cell induced by CD3 stimulation or CD3 plus CD28 costimulation. Cell cycle analysis demonstrated that the proliferation inhibition induced mAb 5A12 ligation is associated with a deceleration of G1 S/G2/M phase cell cycle progression. Annexin V binding analysis by flow cytometry revealved that increased apoptosis was also associated with mAb 5A12 cross-linking. In agreement with above results, impaired IL-2 secretion and CD25 expression were also observed when blockade of CD147 with mAb 5A12. Th1 and Th2 cytokines also modulate an appropriate and specific immune response,we found here that cross-linking HAb18G/CD147 with mAb 5A12 results in significantly higher production of Th1 cytokine IFN-γ, but decreases secretion of Th2 cytokine IL-4. Taken together, the above results show that CD147 engagement attenuates the T cell response to CD3 ligation, therefore, strongly suggest that CD147 possibly functions as a negatively regulatory costimulatory receptor during cell activation. Previous studies show that the cross-linking of CD147 with antibodies can block TCR-induced T cell proliferation, an effect probably derived from the displacement of CD48 and CD59 from the lipid raft. Starting from this evidence, we then investigated the effect of mAb 5A12 on the organization of lipid raft. We found that ligation CD147 with mAb 5A12 interferes with formation of the cap structure. Remarkably, mAb 5A12 disturbed the accumulation of CD48 and reorganization of cap structur. Together with the finding that mAb 5A12 neutralize T cell homotypic aggregation during activation process, it shows that the regulation role of CD147 in T cell actiovation partly through reorganizing lipid rafts and involving the regulation of cell adhension.Ligation of the T cell antigen receptor (TCR) stimulates protein tyrosine kinases (PTKs), which regulate intracellular calcium and control the activity of protein kinase C (PKC) isozymes. As the results shown, cross-linking CD147 slightly decreased tyrosine phosphorylation level upon TCR stimulation. Moreover, downstream Ca2+ release from the intracellular store and extracellular Ca2+ influx triggered by TCR stimulation was also inhibited by mAb 5A12.In conclusion, although CD28 is the principal T cell costimulatory molecule for the TCR, a number of other cell surface proteins have costimulatory functions and perform specific roles in different contexts. Here we analyzed the mechanism of CD147 costimulation of the TCR-mediated signaling. Our results demonstrate that CD3/CD147 costimulation can induce an inhibition of T cell activation. These studies provide evidences that CD147 aggregation induces reorganization of immunological synapse and downregulates intracellular global tyrosine phoshorylation of substrates and intracellular Ca2+ concentration involved upon TCR signaling and suggets that CD147 is involved in the control of activation of T cells and that, more importantly, it could act as a negative costimulatory molecule for T cell activation.