Tetrameric Far-red Fluorescent Protein Used As A Model Antigen For Visualizing Tumor Immunity

Cancerisone of the most difficult diseases to overcome since the20th century,and the scientific researchson tumor growth and elimination, have been the focus and hotspot inthe life sciences, which formed a specialized subjectnamedtumor immunology.People extremly areeager to understand the process details of tumor growth, development and eliminationdue to the complexity of the immune system in tumor immunology.However, the tumor-associated antigens derived from tumors often fail toelicit effective immune responses to inhibit tumor growth, to solve this issue, some foreign proteins, such as ovabumin (OVA), β-galactosidase and some viral antigen foreign protein,were used as model antigens in tumor immunology.It is well known that, OVA is one of the most widely used model antigens in recent researchs, which allows researchers to better understand the process details of tumor growth, development and elimination in immune response and guide researchers to design a reasonable tumor vaccine or immunological tumor therapystrategy.Although certain knowledge on cells and molecules related to tumor immunity had been obtained by the traditional biochemistry method, it still need to verify these informations in vivo. With the development of in vivo optical imaging technology, it is easy to label different proteins at the same time through genetic engineering methods,which makes convenient to visualizeimmume cell movements dynamically in tumor growth, development andelimination. In the traditional intravital microscopy research, it often needs to introduce a model antigen for eliciting immune response and one fluorescent protein as the marker for imaging. If we can find an exogenous protein, which not only can be used as model antigen to induce immune response but also possess fluorescence characteristics forintravital microscopy, it will play an important role in the intravital microscopy of tumor immunity.A tetrameric protein KatushkaS158A was chosen as the represented fluorescent protein in this study toinvestigate the characteristics of KatushkaS158A used as an antigen and to figure out whetherthis protein can induce the specific immuneprotection in C57BL/6mice.Furthermore, we also investigated its applications in DC vaccine incubated with KatushkaS158A or its modified fusion antigen. We aim to establish a tumor immnunology model by using KatushkaS158A as a model antigen. Detailed results are listed as follows:1) Sucessfully, five H2-Db-specific epitope peptides derived from KatushkaS158A antigen were predicted via an on-line software, based on this results we further validated that the peptide TSLQNGCLI was an epitope peptide by using affinity assay with RMA-S cells and lymphocytes proliferaion experiments.2) The immune response elicited by KatushkaS158A had been verified in vitro. The KatushkaS158A-specific IgG antibody in serum collected from KatushkaS158A immunized C57BL/6mice had been detected by ELISA assay. And the proportion of CD8+cells in splenocytes from immunized micewas found slightly higher when compared to the control group.Then the splenocytes collected form immunized mice were re-stimulated with KatushkaS158A antigen,obvious higher IFN-y production compared to unimmunized mice had been observed.3) By using the stable expression cell line B16-KatushkaS158A, the fluorescent signal obtainedby the wholebody fluorescence imaging system was found had a good correlation to the tumor size changes.Nearly20days after B16-KatushkaS158A tumor i.v. inoculation, meatastasis tumor was found in several visceral organ, such as lung, liver, kidneys, eyes and even brain. Thereby,we sucessfully established anopticalB16melanoma tumor growth and metastasis model in C57BL/6mice with KatushkaS158A fluoresecent protein expressing. By further in vivo experiments, we found that KatushkaS158A as antigen could induce the body to produce immunoprotection. After C57BL/6mice immunized with the KatushkaS158A antigenand then inoculated subcutaneously with B16-KatushkaS158A,immunized mice showed decreased tumor growth rate compared to control mice. And the results also showed the immunoprotection was specific to KatushkaS158A expressing B16tumor, while not to the parental B16tumor.4) Sucessfully visualized the movements of immune cells and B16-KatushkaS158A tumor cells in tumor microenviroment. We found that there were various distinct differences exist in the movement of immune cells between KatushakS158A immunized mice and control mice in tumor microenviroment, when using a window chamber model in EGFP-actin transgenic C57BL/6mice. These data indicated that KatushkaS158A protein used as a model antigen would play an important roles in the opitacal imaging of tumor immnunology.5) Sucessfully screened and obtained an optical molecular probe for the detection of calcium,3xCFP-TnC-cpVenus, which is suitable for visualizing in vivo. It had the largest dynamic range compared to the reported calcium sensors so far, which provided an important tool for the detection of calcium signal in tumor microenviroment.6) We demonstrated that DC vaccine incubated with KatushkaS158A or its modified fusion protein，gp10025-33-KatushkaS158A-gp10025-33(denoted Octa-gp100), coupled with gp10025-33(KVPRNQDWL),an antigen epitope peptide derived from B16tumor associated antigen gp100, could elicit effective immunoprotection effectspecific to B16-KatushkaS58A tumor. And as expected, Octa-gp100incubated DC vaccine had better immunoprotection effect in C57BL/6mice, presumably due to the fusion epitope peptide gp10025-33.This study reported tht KatushkaS158A protein can be used as a model antigen to visualize tumor elimination in the tumor immunology research. The fluorescent protein not only can be used as fluorescence labeling moleculars for optical imaging, but also can be used as a model antigen to elicit immune responses, without additional model antigen like OVA in optical imaging of tumor immunology research. Using KatushkaS158A as a model antigen, it would simpify the experiment protocol, and more importantly, it would avoid the potential effects in traditional model antigen research because of the additionlal fluorescent protein needed. Contrary, we can make utilize of the immunogenicity of KatushkaS158A protein. This research are important to the optical imaging of tumor immunology research.