MOLECULAR MEDICINE REPORTS 16: 4593-4602, 2017 CD147 blockade as a potential and novel treatment of graft rejection JING LUAN1,2*, YU ZHAO1,2*, YANG ZHANG1,2, JINLIN MIAO1,3, JIA LI1,3, ZHI-NAN CHEN1,2 and PING ZHU1,3 1National Translational Science Center for Molecular Medicine, The Fourth Military Medical University; 2Cell Engineering Research Center and Department of Cell Biology, The Fourth Military Medical University; 3Department of Clinical Immunology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China Received March 16, 2017; Accepted August 1, 2017 DOI: 10.3892/mmr.2017.7201 Abstract. Cluster of differentiation (CD)147 is highly involved Furthermore, the level of inflammatory cell infiltration in in the T cell activation process. High CD147 expression is transplanted skin was reduced. CD147 blockade decreased observed on the surfaces of activated T cells, particularly the serum levels of interleukin (IL)-17 and the proportions of CD4+ T cells. In organ transplantation, it is important to peripheral blood CD4+ and CD8+ memory T cells. The data prevent graft rejection resulting from the excessive activa- demonstrated that CD147 blockade suppressed skin graft tion of T cells, particularly CD4+ T cells, which exhibit a rejection, primarily by suppressing CD4+T and memory key role in amplifying the immune response. The present T cell proliferation, indicating that CD147 exhibits great study aimed to investigate the effects of CD147 blockade potential as a target of immunosuppressant drugs. in vitro and in vivo and used a transplant rejection system to assess the feasibility of utilizing CD147 antibody-based Introduction immunosuppressant drugs for the treatment of graft rejec- tion. The effects of CD147 antibodies were evaluated on Transplantation remains the best treatment option for lymphocyte proliferation stimulated by phytohemagglutinin correcting certain types of organ failure and tissue damage, or CD3/CD28 magnetic beads and in a one-way mixed e.g., that of the kidneys, heart, and lungs (1). In organ transplan- lymphocyte reaction (MLR) system in vitro. For the in vivo tation, it is always a main goal to prevent rejection. However, analysis, an allogeneic skin transplantation mouse model was the long‑term use of globally nonspecific immunosuppressant used. CD147 antibodies were effective against lymphocytes, drugs to prevent rejection is associated with serious risks, particularly CD4+T lymphocytes, and were additionally including a weakened immune system, infection, and cancer, effective in the one-way MLR system. In the allogeneic skin as well as graft loss due to their toxicity (1,2). Thus, identifying transplantation mouse model, the survival of transplanted safe, target‑specific immunosuppressive agents for promoting skin was extended in the CD147 antibody-treated group. allograft survival is a high priority. Acute rejection is clinically the most common and is mainly mediated by cellular immune responses; this type of rejection can occur any time from a week to months after Correspondence to: Dr Zhi-Nan Chen or Professor Ping Zhu, transplantation. T cells, especially CD4+ T cells from the National Translational Science Center for Molecular Medicine, The recipient, play a key role in acute transplantation rejection. Fourth Military Medical University, 169 Changle West Road, Xi'an, As a trigger, CD4+ T cells amplify the downstream signal. Shaanxi 710032, P.R. China By recognizing HLA‑Ⅱ antigens and interacting with them, E-mail: [email protected] CD4+ T cells cause antigen-presenting cells to release inter- E-mail: [email protected] leukin (IL)-1, which could promote CD4+ T cells releasing IL-2. IL-2 can, in turn, promote CD4+ T cell proliferation * Contributed equally and provide an auxiliary signal for CTL cell differentiation. Furthermore, CD4+ T cells produce IL-4 and IL-5 to promote Abbreviations: CCK-8, Cell Counting kit 8; FITC, fluorescein B cell differentiation and the secretion of antibodies against isothiocyanate; FK506, tacrolimus; IFN-γ, interferon γ; IL-17, the graft to participate in the rejection reaction. Different interleukin 17; IL-4, interleukin 4; mAb, monoclonal antibody; MHC, major histocompatibility complex; MLR, mixed lymphocyte CD4+ T cell subsets execute different functions during reaction; MS, multiple sclerosis; PBMCs, peripheral blood transplant rejection. There have been studies supporting mononuclear cells; PE, phycoerythrin; PI, propidium iodide; RA, the notion that the balance of the Th1/Th2 cell proportion rheumatoid arthritis is a determining factor in this process. Th1 cells mainly participate in graft rejection, especially in acute rejection; Key words: CD147 antibody, immunosuppressant, graft rejection, in contrast, Th2 cell activation leads to the formation of treatment transplantation tolerance (3,4). Furthermore, cytokines such as Th1-related interferon (IFN)-γ and Th2-related IL-2, IL-4 4594 LUAN et al: CD147 BLOCKADE SHOWS GREAT POTENTIAL FOR THE TREATMENT OF GRAFT REJECTION and IL-10 participate in the development of rejection (5). As not meet the criteria for FDA approval; however, it did research continues, CD4+ T cell subsets, e.g., Th17 and Treg, show activity against CD147, which suggests that CD147 is are considered to be the two main types involved in allograft an effective target for the treatment of GVHD (25-27). The rejection and survival (6,7). effect of CD147 blockade on allograft rejection has never In addition to the above-mentioned mechanisms of CD4+ been investigated before. Thus, this study was performed to T cell subsets, memory T cells (both CD4+ and CD8+) have investigate whether the blockade of CD147 can inhibit the been found to be involved in development of the rejection rejection reaction and to determine whether CD147 antibodies reaction (8-10). In a primate model, the elimination of could be developed as specific immunosuppressors for the memory T cells has been shown to induce the body's immune graft rejection response. tolera nce (11). It has also been reported that infusion of sensitized memory CD4+ T cells produces acute rejection Materials and methods in mice that have undergone abdominal heterotopic heart transplantation. In addition, infusion of sensitized memory Antibodies and reagents. Humanized mAbs (5A12) against CD4+ T cells has been shown to significantly shorten the CD147 were generated and identified in our laboratory. survival times of transplanted hearts, and this effect cannot A purified human IgG1 isotype control antibody (Clone: be reversed by commonly used immunosuppressants (12,13). ET901) was purchased from BioLegend, Inc. (San Diego, Clinical studies have shown that the prognoses of patients CA, USA). An anti‑mouse CD147 functional‑grade purified who have undergone liver transplantation are dependent on antibody (Clone: RL73) and its isotype control (Clone: eBR2a) patient CD8+ T memory cell status (14). The results of an were purchased from eBioscience (San Diego, CA, USA). in vivo study have shown that endogenous donor-reactive Tacrolimus (FK506) was purchased from KeHao (Wuhan, CD8+ memory T cells infiltrate the transplanted hearts Hubei, China). Fluorescein isothiocyanate (FITC)-conjugated of mice within a few h after reperfusion and secrete mouse anti-human CD4 and phycoerythrin (PE)-conjugated IFN-γ, causing inflammation (15). It is well known that mouse anti-human CD3 antibodies, Foxp3 staining buffer antibody-mediated T cell depletion is one of the most potent and a cell activation cocktail used for intracellular staining immunosuppressant therapies. This therapy is increasingly were purchased from BioLegend. PE-Cy7-conjugated rat used as an induction therapy in organ transplantation (16). anti-mouse IFN-γ, PE-conjugated rat anti-mouse IL-4, However, T cell homeostasis after depletion therapy leads to Alexa 647-conjugated rat anti-mouse IL-17, FITC-conjugated a predominance of memory T cells, which are more potent rat anti-mouse CD4, allophycocynanin-H7-conjugated rat than naïve T cells in mediating graft rejection and present a anti-mouse CD8, PE-Cy7-conjugated rat anti-mouse CD44, major obstacle to achieving tolerance (17,18). BUV737-conjugated rat anti-mouse CD62 L, allophyco- CD147 is a cell-surface glycosylated transmembrane cyanin-conjugated rat anti-mouse CD25, and PE-conjugated protein that belongs to the immunoglobulin superfamily. This rat anti-mouse Foxp3 antibodies were purchased from BD protein serves multiple biological functions and is widely Biosciences (San Diego, CA, USA). expressed in many tissues and cell types, such as normal brain tissue, tracheal, lung, and breast tissues, as well as Cell isolation. Human peripheral blood was obtained from lymphocytes and neutrophils (19,20). High CD147 expression healthy volunteers who provided informed consent. In brief, is involved in many different diseases. In the immune system, lymphocytes were isolated by density gradient centrifugation CD147 participates in different stages of T cell activity, over Lymphocyte Separation Medium (MP Biomedicals, including development, activation, proliferation, migration, LLC, Santa Ana, CA, USA) according to the manufacturer's and adhesion (19,21,22). It is worth mentioning that CD147 instructions. CD4+ T cells were isolated by negative selec- has been identified as a T cell activation‑related antigen (M6) tion using a magnetic cell separation system according to expressed in phytohemagglutinin (PHA)-activated T lympho- the manufacturer's instructions (Miltenyi