Photo Controlled Phosphatidylserine/Tim-3 Pathway Regulates NK And T Cell Function

Blockage of important immune molecules/signals by antibody or inhibitor has been widely used in clinic and has achieved good therapeutic effect in inflammation-related diseases such as tumors and autoimmune diseases.However,immunotherapy can lead to sever side effects,limiting its clinical application.Therefore,it is urgently required to dynamically regulate the immune response and immunotherapy.T cell immunoglobulin domain and mucin domain-3(Tim-3),an immunosuppressive receptor,is widely expressed on a variety of immune cells such as NK cells,T cells and macrophages.It regulates the functions of these immune cells and is involved in chronic viral infection,tumor and other diseases.Preclinical studies have shown that intervening Tim-3 affects a variety of disease processes and is a potential target for immunotherapy.We previously found that Tim-3 binds to its ligand phosphatidylserine(PS),leading to inhibition of AKT-mTOR pathway and consequently exhaustion of tumor infiltrating NK cells.As a small molecule,PS is easy to be synthesized and modified.To get a tool for dynamically regulate Tim-3 pathway,by cooperation,we got a small molecule probe,photophosphatidylserine(phoPS),which exhibits cis-conformation as native PS under 365 nm light(cis-phoPS)and trans-conformation under 455 nm light(trans-phoPS).Similar to PS,cis-phoPS binds to Tim-3 with high binding affinity,while trans-phoPS barely binds to Tim-3.Here,in this study we aim to explore the potential of phoPS in dynamically immune regulation which would provide new strategies for photo-controlled immunotherapy.The main research methods and results are divided into the following parts:Ⅰ.Photo-controlled phosphatidylserine regulates NK cell function in a Tim-3-dependent mannerIn order to further determine the regulatory effect of PS on NK cells,we firstly used liposome-encapsulated PS to treat activated human NK92 cells and mouse primary NK cells in vitro.Flow cytometry showed that PS significantly inhibited the cytokine secretions and target cells killing ability of NK cells from different sources.The above experiments were repeated with NK cells derived from Tim-3 knockout(Tim-3-/-)mice,and it was found that PS did not inhibit the function of NK cells derived from Tim-3-/-mice.The above results proved our original conclusion that PS as a ligand is involved in Tim-3-mediated NK cell regulatory functions.In order to verify the regulatory effect of phoPS on NK cells,we firstly evaluated the cytotoxic effects of phoPS and irradiation treatments to NK92 cells and target cells.It was found that treatments with 365 nm and 455 nm light did not affect NK cell viability,and treatment with 20 μg/mL or less phoPS had little cytotoxicity to NK92 cells and target cells.We therefore treated NK cells with 20 μg/mL phoPS together with irradiation under 365 nm or 455 nm in the following studies.The results of flow cytometry showed that,similar to PS,cis-phoPS(irradiation under 365 nm)significantly inhibited the cytokine secretions and cytotoxicity of NK92 and murine spleen NK cells,while trans-phoPS(no irradiation or 455 nm irradiation)did not affect the functions of NK cells.While on the contrast,although Tim-3-/-NK cells exhibited higher cytokine secretion and cytotoxicity to target cells than NK cells derived from WT mice,cis-phoPS did not regulate Tim-3-/-mice NK cells function.These results suggest that phoPS under 365 nm irradiation regulates NK cell function in a Tim-3-dependent manner.To determine whether light could control the phoPS-mediated regulation of NK cells,NK92 cells treated with phoPS were exposed to different wavelengths of lights for several times,and NK cell functions were detected by flow cytometry.The results showed that phoPS could be dynamically regulated by lights and act as a switch to regulate NK cell functions:cis-phoPS activated by 365 nm irradiation could be turned off by 455 nm irradiation(which could not regulate NK cell functions),and trans-phoPS treated by 455 nm light could re-inhibit NK cell functions after activated by 365 nm irradiation.These results indicated that NK cell functions could be regulated by phoPS under 365 nm irradiation.To verify whether phoPS could be used to dynamically regulate NK cells function in vivo,poly(I:C)induced acute hepatitis mice were treated with phoPS by gavage,following with irradiation with light of specific wavelength every 12 hours.Consistent with the in vitro results,phoPS gavage combined with 365 nm light treatment effectively alleviated poly(I:C)-induced acute hepatitis in mice,but 455 nm light treatment had little effect.Ⅱ.Photo-controlled phosphatidylserine regulates T cell functions in a Tim-3-dependent mannerTim-3 is selectively expressed on activated CD8+T cells and Th1 cells and inhibits their functions.However,whether Tim-3 can bind to PS and mediate Thl and CD8+T cell dysfunction have not been reported.To determine whether PS regulates T cell function in a Tim-3-dependent manner,CD8+cytotoxic T lymphocyte(CTL)and Th1 cells were induced from splenic mononuclear cells isolated from OT-Ⅰ and OT-Ⅱ transgenic mice respectively.Flow cytometry and cell killing assay showed that PS not only significantly inhibited secretion of cytokines and killing activities of CTL,but also inhibited IFN-γ production by Th1 cells.Expectedly,the phoPS mediated inhibition on CTL were disrupted by Tim-3 blockage.Constantly,PS did not affect CTL cell functions derived from Tim-3-/-OT-Ⅰ mice.CTL with and without PS were collected for RNA-seq.KEGG analysis combined with Western blotting results showed that PS treatment inhibited TCR signaling pathway and mTOR signaling pathway in CTL.Taken together,PS regulated CD8+T and Th1 cell functions in a Tim-3-dependent manner with changes in signaling pathways.To determine whether phoPS could dynamically regulate T cell function by light control,CTL cells derived from OT-Ⅰ mice were treated with phoPS under 365 nm or 455 nm of irradiation.Consistent with the results of NK cells,phoPS inhibited the effector function of CTL only after a single irradiation at 365 nm or multiple irradiations which were ended with 365 nm irradiation,but not after a single irradiation at 455 nm or multiple irradiations ended by 455 nm.That is,T cell functions can be dynamically regulated by phoPS under specific wavelength irradiations.To further determine the regulatory role of phoPS in vivo,Concanavalin A(ConA)induced acute hepatitis mice were pretreated with phoPS together with 365 nm or 455 nm irradiation.As expected,phoPS combined with 365 nm light irradiation significantly alleviated acute hepatitis in mice,displayed as decreased level of AST,reduced expression of inflammatory cytokines and alleviative liver inflammation.Conclusion:phoPS under 365 nm/455 nm irradiation regulates NK cells and T cells function dynamically.Our data provides a new strategy for controlled immunotherapy.