PD-L1 Genetically Engineered Macrophages For The Treatment Of Transplant Rejection

Part Ⅰ:Preparation and properties of bionic glucan particlesObjective:Macrophage imaging plays an important role in the diagnosis and treatment of diseases such as transplant rejection.Glucan particles（GPs）are specifically recognized by Dectin-1,a pathogen recognition receptor expressed by macrophages,giving them the potential to target macrophages.Moreover,GPs exist mainly as hollow vesicles and right-handed triple helix structures,which provide a potential restricted domain space for a novel aggregation-induced emission（AIE）molecule（HBTTPIP）with positive charge.Therefore,in this study,we propose to construct bionic probes（HBTTPIP/GPs）using GPs loaded with positively charged HBTTPIP,Then,the properties of HBTTPIP/GPs will be examined and their biosafety will be evaluated in vitro and in vivo.Methods:（1）GPs were extracted from Anchorage yeast,and then co-incubated with HBTTPIP to construct HBTTPIP/GPs.The basic properties of HBTTPIP/GPs were examined by laser confocal microscopy,transmission electron microscopy,fluorescence spectrophotometer,and benchtop rapid powder diffractometer.（2）After oral administration of HBTTPIP/GPs,their biosafety was assessed based on immunogenicity,blood routine,liver and kidney function,and pathological histology of vital organs.Results:（1）Successful preparation of HBTTPIP/GPs:The particle size of HBTTPIP/GPs is about 3.1μm.Transmission electron microscopy images show that the cavities of GPs are filled with HBTTPTP.（2）HBTTPIP/GPs exhibit excellent light emission performance:Laser confocal imaging results showed distinct red light emission from HBTTPIP visible in the core of GPs.x-ray diffraction（XRD）,fourier transform infraredspectroscopy（FTIR）and solid-state fluorescence spectroscopy results showed that the fluorescence intensity was significantly enhanced after incubation of GPs with HBTTPIP,and the characteristic infrared absorption peaks of HBTTPIP/GPs were similar to those of GPs.（3）HBTTPIP/GPs possess good biosafety:After oral administration of HBTTPIP/GPs,no significant changes in immunogenicity,blood routine and liver and kidney function indexes were observed compared with the control group;H&E staining results of major organs showed no changes in heart,lung,liver,spleen and kidney;bioavailability results after oral administration of HBTTPIP/GPs showed that the C_max,T_1/2 and AUC_（0-t）of HBTTPIP were（17.61±2.44）μg/L,（2.38±0.38）h and（145.63±20.06）μg/L·h,respectively.In vivo fluorescence results showed that HBTTPIP/GPs were mainly distributed in the liver and lung after oral administration.Conclusion:In this study,HBTTPIP/GPs were successfully developed with good imaging performance,stability and biosafetyPart Ⅱ: In vivo imaging,transport mechanism and biological function study of bionic glucan particlesObjective: Glucan particles can be specifically recognized by dectin-1 receptors on the surface of macrophages in gut-associated lymphoid tissues by mimicking the yeast intestinal infection pathway,thus enabling targeting of macrophages in vivo.Therefore,this study aimed to explore the performance of HBTTPIP/GPs imaging for tracing macrophages and to evaluate their transport mechanisms and biological functions.Methods:（1）After incubated with HBTTPIP/GPs,the phagocytic ability of macrophages on HBTTPIP/GPs was observed by TEM,CLSM and FCM,and the effect of HBTTPIP/GPs on the biological function of macrophages was also detected by CCK8 and cell migration assay.（2）The mouse inflammatory bowel disease（IBD）model was constructed,and the successful construction of the IBD model was verified by body weight change,DAI score,colon weight and pathological histological indexes.The correlation of fluorescence intensity with IBD severity and the monitoring of FK506 treatment effect were examined based on in vivo and ex vivo imaging.（3）After oral administration of HBTTPIP/GPs,the biological functions of HBTTPIP/GPs were evaluated by weight change,DAI score,colonic weight,inflammatory mediators,intestinal mucosal barrier and pathological histological indices.Results:（1）Macrophages were able to recognize and phagocytose HBTTPIP/GPs: HBTTPIP/GPs were able to be phagocytosed by macrophages in vitro with a phagocytosis rate of nearly 100%,and macrophages retained their phagocytic activity and migration function after phagocytosis.（2）HBTTPIP/GPs can track the migration of macrophages in vivo:The mouse IBD model was successfully constructed.After oral administration of HBTTPIP/GPs,the results of in vivo and ex vivo imaging showed that the fluorescence intensity increased with the severity of IBD in a positive correlation,and decreased significantly after treatment with FK506,indicating that oral administration of HBTTPIP/GPs could not only monitor the course of IBD but also evaluate the efficacy.The results of peyer’s patches（PPs）,mesenteric lymph nodes（MLNs）and colon tissue sections showed that after oral administration,HBTTPIP/GPs were phagocytosed by macrophages in PPs,then transferred to MLNs and finally migrated to the site of intestinal inflammation.（3）HBTTPIP/GPs could alleviate IBD: Oral administration of HBTTPIP/GPs inhibited the secretion of pro-inflammatory cytokines（IL-1β and IL-6）,decreased the levels of NO,MDA and MPO activity,and also improved the barrier function of colonic mucosa,and histological and TUNEL staining results showed that HBTTPIP/GPs attenuated intestinal inflammation and reduced apoptosis in colonic tissues.Conclusion: HBTTPIP/GPs can be specifically recognized by macrophages,and the oral administration of HBTTPIP/GPs can track the transport behavior of macrophages in the IBD model,monitor the progress of IBD,and reflect the efficacy of IBD.This study broadens the method of tracking macrophages and provides a new strategy for the diagnosis and treatment of macrophage-related diseases.Part Ⅲ: Bionic glucan particle/PD-L1 engineered macrophages for transplant rejection treatmentObjective: Programmed death ligand 1（PD-L1）plays an important role in the treatment of transplant rejection,but single or systemic application of it would lack targeting,leading to inadequate efficacy and nonspecific immune response and immune-related toxicity.Therefore,targeted treatment of rejection by PD-L1 is essential to improve the prognosis of transplant patients.Macrophage infiltration into the graft is a natural pathological feature of transplant rejection,which possesses a great potential to mediate PD-L1 targeting to the graft.The aim of this study was to prepare PD-L1 gene-modified and HBTTPIP/GPslabeled engineered macrophages(MΦ_PD-L1 @HBTTPIP/GPs),to trace their performance in targeting grafts,to evaluate the efficacy of combined CTLA4-Ig for the treatment of transplant rejection,and to explore the related mechanisms.Methods:（1）After RAW264.7 was modified with PD-L1 gene,the overexpression of PD-L1 was detected by WB,qPCR,FCM,and immunofluorescence,respectively.（2）Macrophages were incubated with HBTTPIP/GPs followed by TEM,CLSM and FCM to detect their phagocytic ability.（3）The mouse skin graft model was constructed,and after intravenous administration of engineered macrophages,their ability to target grafts was examined by live imaging and the mechanism was explored.（4）The mouse skin graft model was constructed and treated with intravenous administration of engineered macrophages combined with CTLA4-Ig for rejection.The therapeutic effects were evaluated based on weight change and survival time of the graft skin.The therapeutic mechanisms were explored with H&E staining,immunohistochemistry,FCM,immunofluorescence,and ELISA.Results:（1）Successful preparation of MΦ_PD-L1@HBTTPIP/GPs: Engineered macrophages overexpressing PD-L1 and loaded with HBTTPIP/GPs(MΦ_PD-L1@HBTTPIP/GPs)were successfully constructed.After RAW264.7 was modified with PD-L1 gene,the results of WB,qPCR,FCM and immunofluorescence showed that PD-L1 was highly expressed by macrophages.After co-incubation of MΦ_PD-L1 with HBTTPIP/GPs in vitro,TEM,CLSM and FCM results showed that HBTTPIP/GPs were successfully phagocytosed by macrophages with a phagocytosis rate close to 100% and without affecting the overexpression of PD-L1 protein and the biological function of macrophages and without triggering the immune response of macrophages.（2）HBTTPIP/GPs are able to migrate to grafts:At 12 h after intravenous reinfusion of MΦ_PD-L1@HBTTPIP/GPs,strong fluorescent signals were specifically distributed in the graft skin of allogeneic mice,and quantitative results showed that the intensity of fluorescent signals was positively correlated with the severity of graft rejection.（3）MΦ_PD-L1@HBTTPIP/GPs combined with CTLA4-Ig can synergistically treat transplant rejection: The survival time of grafted skin was significantly prolonged after intravenous infusion of engineered macrophages and combined with CTLA4-Ig in the allogeneic mouse skin graft model.On postoperative day 7,H&E staining showed reduced lymphocyte infiltration in the grafted skin,and immunohistochemical results showed reduced T-lymphocyte infiltration and decreased granzyme B secretion in the grafts.FCM results showed a drop in the proportion of cytotoxic T lymphocytes（CD8+ T）cells and an increase in regulatory T cells（Treg）in the lymph nodes and spleen.Immunofluorescence results showed increased expression of Foxp3 in the grafts.ELISA results showed the reduction of pro-inflammatory cytokines IL-2,IL-6,and IFN-γ and the increase of anti-inflammatory cytokine IL-10 in the peripheral blood.Conclusion: In this study,MΦ_PD-L1 @HBTTPIP/GPs were successfully constructed,which could specifically migrate to transplant skin after intravenous infusion,and the combination with CTLA4-Ig could synergistically inhibit the effect of transplant rejection and elucidate the related mechanism.This study is expected to provide a potential new strategy for cell therapy in the treatment of organ transplant rejection.