Non-invasive Evaluation Of PD-L1 By NIRFI And Therapeutic Effects Of PD-L1 MAb In Type 2 Diabetic Stroke Mice

Part I Synthesis of PD-L1-IR640 B probe and non-invasive NIRF imaging analysis in WT and diabetic ischemic stroke modelsObjective To synthetize one near-infrared fluorescence(NIRF)imaging probe targeting program-death ligand 1(PD-L1)and characterize the probe in vitro and in vivo to visualize the biodistribution of PD-L1-positive immune cells in infarction zones and quantify PD-L1 expression levels among the diabetic and WT stroke mice in vivo.Materials and Methods The PD-L1-targeting NIRF imaging agent was synthetized by the conjugation of functional anti-mouse PD-L1 antibody with IR640 B and then characterized by immunohistochemistry and co-location staining.WT and diabetic mice with intravenous IR640B-anti-PD-L1 injection post-ischemia were subjected to NIRFIs and the brains,livers,hearts,kidneys,spleens and lungs were dissected for ex vivo imaging for detecting expression levels and biodistribution of PD-L1.Results Fluorescence intensities and microdistribution features of IR640B-PD-L1 probe were detected by in vivo Near-infrared fluorescence(NIRF)images.Compared with control agent,significant higher fluorescence intensities of targeted agent were seen in infarct area over the ipsilateral side 6h after the injection of IR640B-PD-L1 probe.Moreover,fluorescence intensities of PD-L1-tageting agent were higher in diabetic group than WT group.The results of Near-infrared fluorescence intensities detected in excised brain tissue and other organs showed more apparent accumulation of probe in the ischemic hemisphere other than liver、kidneys and other organs.The accumulation of fluorescent signals in livers and kidneys further indicted the excretory pathway.The well targeting property of IR640B-PD-L1 probe to the peri-infarction zone have been tested in immunohistochemistry and co-location staining.Conclusion Specificity and affinity of PD-L1-targeting probe were confirmed by NIRF imaging and immunohistochemistry and co-location staining.This study shows feasibility of molecular imaging by radiolabeled NIRFI probe in evaluating dynamic expression of PD-L1 in vivo,with significant increase in diabetic group post-stroke as evidence of involvement of PD-L1 in worse prognosis with diabetes mellitus.Part II Effects of anti-PD-L1 m Ab treatment in Type 2 Diabetic Ischemic Stroke ModelsObjective Basis on the immunomodulatory properties in WT mice after MCAO,PD-L1 checkpoint blockage was applied as promising target in diabetic stroke.This study showed further exploration and verification of therapeutic potency of anti-PD-L1 m Ab treatment in the prognosis of diabetic ischemic stroke via the assessment of MRI、behavioral scores and WB.Materials and Methods Same concentration of functional anti-PD-L1 m Ab was injected intravenously at 4h after successfully establishing diabetic and WT stroke models.Magnetic resonance imaging(MRI)was performed at different time points to prognosticate the verify changes of infarct volume.The nervous functional status were further evaluated by modified neurological severity score(m NSS)and diffusion tensor magnetic resonance imaging(DTI),the expression levels of inflammation factors were investigated via Western Blot(WB)on day 7.Results Remarkably decreased ischemia-induced infarct volumes in WT and diabetic stroke models with anti-PD-L1 treatment were determined by T2 WI on day 7.The recovery of motor function and nerve fibers integrity of two anti-PD-L1 treatment groups were obviously better than those of the control groups respectively in subacute phase,with significant improvement on day 7.WB analysis showed the down-regulation of pro-inflammatory cytokines(TNF-α)the increased expression of anti-inflammatory factors(IL-10、 IL-4).Conclusion Application of anti-PD-L1 m Ab post-stroke decreased cerebral infarct volumes,promoted neurologic behavioral recovery,ameliorated the nervous integrity and made down-regulation of ischemia-induced inflammation response.