Killerartificial Antigen-presentingcells Treating Experimental Autoimmune Encephalomyelitis In A Murine Model

Multiple sclerosis(MS) is an autoimmune, chronic, demyelinating disease of the central nervous system(CNS).The main pathological changes are that the lateral ventricle of cerebral hemisphere, brain stem and spinal cord have myelin depigmentation and different degree of inflammatory cell infiltration. The disease is mainly mediated by its reactive T cells to autoantigen CNS myelin, in which CD4+T cells play the key role and CD8+ T cells also have a certain pathogenic. There are no very effectively cure for MS, sometimes patients even cannot tolerate which terribly impact patients’ quality of life. The selective elimination or inhibition of antigen-specific T cells is one of the fundamental strategies for the treatment of transplant rejection and autoimmune diseases because it does not suppress the overall immune systems. Therefore, numerous specific immune therapies have been explored during decades. Killer artificial antigen-presenting cells (KaAPCs), which are genetically engineered to overexpress the apoptosis-inducing Fas ligand (FasL) onto dendritic cells (DCs) and macrophages, have been studied in animal and human models of allograft transplantation, autoimmunity or allergy by various investigators.Nowadays people began to use KaAPCs to remove or inhibit the autoantigen specific T cells. Based on our preliminary work,KaAPCs have been generated by covalently co-coupling H-2Kb-Ig dimers and anti-mouse Fas mAbs onto poly lactic-co-glycolic acid (PLGA) microspheres with a diameter of 4.5μm, and then administrated into alloskin recipient mice followed by overall immune function inspection.The results show that KAPCs treatment can significantly prolong the skin allograft survival without impairment of host overall immune function.Experimental Autoimmune Encephalomyelitis(EAE) is the ideal animal model of multiple sclerosis.lt has greatly advanced our understuding of T-cell mediated neuroinflammation,which in turn has lead to the development of MS therapies. processes that may be involved in MS. In this report, killer artificial antigen-presenting cells have been generated by covalently co-coupling H-2Db/MOG40-54 dimmer, H-2IAb/MOG35-55 multimer, and anti-mouse Fas mAbs,PD-L1-lg,TGF-βlas T cell negative regulation molecules and CD47-lg as antiphagecytic molecules onto poly lactic-co-glycolic acid (PLGA) microspheres with a diameter of 4.5μm,to make the new multifunctional KaAPCs. It was found that KAPCs treatment can significantly relieve the symptoms of EAE. The results are summarized as following:1. C57BL/6 mice have been randomly divided into 3groups:PBS group, BCGgroup and MOG group. MOG35-55 should be diluted in PBS to a concentration of 5mg/ml.Then an equal volume of complete Freund’s adjuvant(CFA) is added to be thoroughly mixed to form a thick emulsion on ice.Ideally,the mice receive approximately 200ul of emulsion subcutaneously divided equally across four sites on the dorsal flank.At the same time, 500ng of pertussis toxin must be administered intraperitoneally(i.p.).All subjects are pathogenetic as mice are injected for about 7 days. The time is determined 11th day for early onset,18 days for peaks, to 40 days for chronic phase;HE staining showed the brain and spinal cord area more inflammatory cells increased significantly in the early onset and the peak be the most significant one. Immunohistochemical displays that MBP protein was reducing from the onset of the early start, incidence the least at the peak. Whereas, GFAP protein shows the opposite:the early onset of positive cells increased, peak most and chronic plateau;Flow test showed that the lymph nodes MOG35-55 specific CD4+T cells in lymph nodes frequency and MOG40-54 frequency specific CD8+T cells increased at the beginning of the disease, the peak highest, chronic phase times not very much. ELISPOT shows that:the spleen cells of MOG35-55 and MOG40-54 polypeptide stimulus has the highest frequency of T cells in the early onset of reactivity, the peak not that much, chronic phase lowest, do not match the specific CD4+ T and CD8+ T numbers.2. Cell-sized cationic PLGA microparticles are successfully fabricated using a double-emulsion-solvent evaporation method. The PLGA microspheres displayed a heterogeneous size distribution under the SEM. Approximately 61.2% of these microspheres were 4.0-5.0μm in diameter. The mean zeta potential was 36.3±6.11 mV, as detected by the PALS zeta instrument, implying their strong capacity to covalently couple protein molecules. The BSA protein loaded onto 5×106 PLGA microspheres reached up to 54.2μg. The killer PLGA microspheres were prepared by immobilizing H-2Db-Ig dimmers, H-2IAb-Ig and anti-mouse Fas mAbs onto PLGA microspheres, and displayed correct phenotype as detected by flow cytometry.3. KaAPCs were used in EAE model:multifunctional KaAPCs were used fortail intravenous injection at the incidence of EAE 11 days,14 days and17 days,1×107 PLGA/ml/mouse. Preliminary clinical observation and neurological function score showed that KaAPCs treatment group after the third treatment began to appear in mice significantly alleviate clinical symptoms, whereas,PBS treatment group, blank PLGA treatment group has nothing to do with the antigen peptide has not been specific KaAPCs treatment group showed no signs of relief, reminding KaAPCs’ effectiveness of the initial therapy.Conclusion:EAE mice model was successful preparation and the dynamic pathological indexes and antigen were analyzed to know the changing rule of the specific T cells’number and reactivity;KaAPCs were Successful preparation using PLGA microspheres, and in the treatment of EAE model showed the effectiveness preliminary.