Research On The Immunogenicity Of HESC-derived RPE Cells

Background:Retinal pigmented epithelium（RPE）is a monolayer of polygonal cells located between the choroidal capillaries and the photoreceptor cells of the retina,which is extremely important to maintain the stability of retinal microenvironment.The onset of blinding diseases such as diabetic retinopathy（DR）,age-related macular degeneration（AMD）and retinitis pigmentosa（RP）are associated with dysfunction of RPE cells.Unfortunately,there is no clearly effective treatment to cure such diseases at present.A large number of studies in recent years revealed that RPE cell transplantation may be a promising approach to ameliorate the deterioration of blinding diseases mentioned above.Therapeutic RPE cells have been successfully induced from embryonic stem cells（ESCs）and induced pluripotent stem cells（iPSCs）,which showed short-term curative effects in animals and clinical trials.However,the survival of transplanted cells in the retinal microenvironment was far from satisfactory.One of the keys to the problem is the allograft rejection.Studies have indicated that most of the subretinal RPE grafts will undergo immune rejection after a certain period of time.It is well known that the the retina is immune privileged with the help of blood-retinal barrier and a series of immunoregulatory factors secreted by the retinal innate cells.Stable immune microenvironment is important for the function of neural retina,and the occurrence of RP is closely related to inflammation and autoimmune response.The destruction of the blood-retinal barrier and the activation of microglia deteriorate the immune microenvironment of the retina.In addition,the cell transplantation surgery itself may disturb the barrier and cause inflammation,which poses a serious threaten to the survival and function of the graft in the retina.To overcome the immune rejection of the transplanted RPE cells,a variety of immunosuppressive agents are used in clinical treatment during perioperative period.However,the immunosuppressive may impair the function of liver and kidney of patients and lead to opportunistic infections.Therefore,researchers are looking for a"universal cell"that can evade immune attack from host.Researchers showed that,immune response to therapeutic cells could be effectively reduced by upregulating the expression of immune inhibitory ligands such as PDL1 and CTLA-4 in transplanted cells.Therefore,we raise questions for our research direction:Which pathway plays the leading role in regulating the immunogenicity of human embryonic stem cells derived retinal pigmented epithelium（hESC-RPE）cells?Could transplanted cells be pretreated by certain means to protect them from immune attack?Object:To study the immune microenvironment of RP and investigate the key factors regulating the immunogenicity of hESC-RPE.To explore the immunogenic regulation mechanism of hESC-RPE and pretreat hESC-RPE cells with specific method.To evaluate the effect of pretreatment on function and immune characteristics of RPE cells in the immune microenvironment.Methods:The study was divided into three parts:Part I:The immune microenvironment of retinitis pigmentosa1.Using liquid suspension chip to investigate the changes of retinal immune microenvironment in RCS rats at late degeneration stage.The retinal proteins were extracted from retinae of P60d RCS and RDY rats.The original fluorescence detection value and the standard curve formula were calculated to obtain the sample concentration.2.The retina of RCS rats at early degeneration stage（P20d）,intermediate（P40d）,and late（P60d）and the retinae of RDY rats at the corresponding age were extracted to get the total mRNA,which were reversed to cDNA next.The mRNA expression of lymphocyte-associated cytokines IL-2 and IFN-γ,chemokines CCL2,CXCL9,CXCL10 and CXCL11 were evaluated by RT-PCR.2.Using flow cytometry to quantitatively analyze the quantity and activation of lymphocytes in the retina of RCS rats at late degeneration stage.Retinal tissues of P60d RCS and RDY rats（n=3）were extracted to make single cell suspension.Lymphocyte surface markers（CD4/CD8/CD161）and intracellular cytokine IFN-γwere co-stained and detected by flow cytometry.3.Using immunohistochemical staining to study the distribution of lymphocytes in the retina of RCS rats at late degeneration stage.Frozen retinal sections of P60d RCS and RDY rats were prepared.CD4,CD8 and CD161 were used to mark helper T lymphocytes,cytotoxic T lymphocytes and natural killer cells in retina,respectively.Analyze the expression of IFN-γin the above cells to determine whether they were activated.4.Using ELISA to quantitatively analyze the IFN-γconcentration in retina of RCS rats at different degeneration stages.The retinae of RCS and RDY rats were collected at P20d,P40d,and P60d.IFN-γconcentration in the retina were calculated according to the standard curve and the original absorbance value of the sample.Part II:The induction of hESC-RPE1.Observe the morphological changes from hESC to hESC-RPE during the induction.After complete fusion of hESCs,medium was replaced with a differentiation medium without BFGF.The pigmentation was observed about 20 days later and the pigmented colony was picked and sub-cultured to get cobble stone-like hESC-RPE cells.A phase contrast inverted microscope was used to record process of induction.2.Using immunofluorescence staining and flow cytometry to identify the expression of pigment epithelial cell-specific markers Bestrophin,CRALBP,MITF and RPE65 in hESC-RPE cells on day 70 of induction.3.Using RT-PCR to detect the expression of embryonic stem cell-specific genes including OCT4,Nanog,SOX2 and pigment epithelial cell-specific genes including PAX6,MITF,Besttrophin,RPE65 and CRALBP in hESC-RPE on the day 70 of induction.4.Using flow cytometry to detect the expression of human leukocyte antigen（HLA）in hESCs and hESC-RPE,and the expression of HLA with and without IFN-γtreatment in hESC-RPE.Part III:The immunogenic regulatory mechanism of hESC-RPE1.Pretreat hESC-RPE cells with 4μM Ruxolitinib,and then stimulate the cells with 100ng/mL IFN-γ.Using RNA-Sequence（RNA-Seq）technology based on Illumina HiSeq platform to analyze the mRNA expression levels.2.Pretreat hESC-cells with Ruxolitinib at concentrations of 0,2,4,6,8,10,12 and 14μM,followed by stimulation with 100 ng/mL IFN-γ.Using flow cytometry to detect the expression levels of HLA-ABC,HLA-DR,HLA-E and HLA-G after pretreatment with Ruxolitinib at different concentrations,and explore the dose-effect relationship between Ruxolitinib and HLA expression.Using RT-PCR to compare the expression of classical HLA-I antigen-associated genes（HLA-A,HLA-B,HLA-C）,classical HLA-II antigen-related genes（HLA-DRA,HLA-DRB1,HLA-DRB3,HLA-DRB4 and HLA-DRB5）,and non-classical HLA-I antigen-related genes（HLA-E and HLA-G）.Using Western-Blot to detect the intracellular phosphorylation transcription factor p-STAT1.3.Pretreat hESC-RPE cells with 4μM Ruxolitinib,and then stimulate the cells with 100ng/mL IFN-γ.Using immunofluorescence staining to analyze the effect of Ruxolitinib on HLA expression and cell tight junction of hESC-RPE cells.Using flow cytometry to explore the protective effects of Ruxolitinib on hESC-RPE phagocytosis.4.The hESC-RPE cells were pretreated with Ruxolitinib at concentrations of 0,2,4,6,8,10,12 and 14μM,and were co-cultured with CD4⁺T helper cells CD8⁺cytotoxic T lymphocytes or NK cells sorted from human peripheral blood,respectively.Using ELISA kit to detect the IFN-γconcentration of supernatant medium co-cultured with CD4⁺T lymphocyte,and evaluate the activation of CD4⁺T lymphocytes under different Ruxolitinib concentrations.Using LDH assay to explore the level of lactate dehydrogenase in the supernatant medium co-cultured with CD8⁺T lymphocytes or NK cells,and evaluate the immune cells attack to hESC-RPE cells under different Ruxolitinib concentrations.Using immunofluorescence staining to compare the chemotaxis of hESC-RPE cells for CD8⁺T lymphocytes and NK cells with and without 4μM Ruxolitinib treatment.5.Luc Lentiviral-labeled hESC-RPE cells were pretreated with 4μM Ruxolitinib,and were transplanted subcutaneously in humanized mice.Using IVIS Spectrum in vivo imaging system to record the bioluminescence signal generated by subcutaneous hESC-RPE cells on2^nd,4^th,6^thh and 8^th day after transplantation,and compare the survival of hESC-RPE cells with and without Ruxolitinib treatment in the simulated human immune microenvironment.6.4μM Ruxolitinib pretreated hESC-RPE cells and untreated hESC-RPE cells were transplanted into the subretinal space of RCS rats respectively;wherein the rats transplanted with untreated hESC-RPE cells were further divided into two groups:oral administration of water（Vehicel group）and oral administration of immunosuppressive drug（Control group）.Using fERG to detect the electrophysiological activity of rat retina on 2^nd week,4^th week,6^thh week,8^th week and 10^th week after transplantation,and evaluate the survival and function of hESC-RPE cells.Results:Part I:The immune microenvironment of retinitis pigmentosa1.The liquid suspension microarray confirmed the microglia activation in RCS rats retina at late degeneration stage.Besides,we found that the concentration of lymphocyte-associated cytokines such as IL-2 and IFN-γwas significantly increased,suggesting that there was infiltration and activation of lymphocytes in the retina at late degeneration stage as well.2.The RT-PCR analysis revealed that expression level of chemokines including CCL2,CXCL9,CXCL10 and CXCL11,and cytokines including IFN-γand IL-2,were significantly higher in degenerative retina than control one.In addition,the expression levels of these genes were up-regulated with the age,reaching a peak in the late stage of degeneration.3.Flow cytometry and immunohistochemistry studies showed that a large number of lymphocytes infiltrated in the inner retina of RCS rats in the late degeneration stage.CD4⁺T lymphocytes and NK cells were the main sources of IFN-γin the retina.4.Quantitative detection of IFN-γby ELISA showed that the IFN-γconcentration tended to increase with the development of degeneration in the retina of RCS rats.There was a significant increase of IFN-γconcentration in the retina of RCS rats compared to RDY rats.Part II:The induction of hESC-RPE1.Immunohistochemistry and flow cytometry confirmed the high expression of pigment epithelial cell-specific markers Bestrophin,CRALBP,MITF and RPE65 in hESC-RPE cells on day 70 of induction.Bestrophin,CRALBP and RPE65 were mainly expressed in the cell membranes and MITF is mainly expressed in the nucleus.2.RT-PCR analysis confirmed that the expression of stem cell-specific genes OCT4,Nanog and SOX2 were significantly down-regulated during hESC differentiation into hESC-RPE,while the expression of pigment epithelial cell-specific genes were significantly up-regulated.3.Flow cytometry showed that T lymphocyte immune-activated surface antigen（HLA-ABC,HLA-DR）and NK cell immunosuppressive surface antigen（HLA-E and HLA-G）were low-expressed both in hESCs and hESC-RPE cells.However,after 24-hour treatment with 100 ng/mL IFN-γ,the expression of all HLA related antigen was significantly up-regulated on hESC-RPE cells.Part III:The immunogenic regulation mechanism of hESC-RPE1.The cell transcriptome and bioinformatics analysis found that the main biological effect of IFN-γon hESC-RPE cells was to increase the expression of HLA to promote cell antigen presentation and immune rejection.However,Ruxolitinib could block HLA up-regulation by IFN-γ,and maintain low immunogenicity of hESC-RPE cells.2.Flow cytometry study of the dose-effect relationship between Ruxolitinib concentration and HLA expression of hESC-RPE cells showed that 2μM to 10μM was an effective and safe concentration range for blocking HLA expression.3.RT-PCR and immunocytochemistry showed that 4μM-Ruxolitinib pretreatment could effectively block the HLA expression of mRNA and protein in hESC-RPE cells induced by IFN-γ.4.Western-Blot assay confirmed that Ruxolitinib could block the phosphorylation of STAT1 and inhibit HLA expression on the surface of hESC-RPE cells.It also confirmed that IFN-γphosphorylates transcription factor STAT1 by activating JAK kinase coupled to IFN-γreceptor.Phosphorylatd STAT1 enhanced the immunogenicity by up-regulating the expression of HLA on hESC-RPE cells,which could lead to immune rejection.It was also found that IFN-γhad a detrimental effect on the tight junction and phagocytosis function of hESC-RPE cells,while 4μM Ruxolitinib could alleviate the side effect of IFN-γ.5.Co-culture of hESC-RPE cells with human peripheral blood lymphocytes showed that the immunogenicity was up-regulated after treated with IFN-γ,which was characterized with the chemotaxis and activation on immune cells.After pretreatment with Ruxolitinib,hESC-RPE cells were co-cultured with immunocytes,or stimulated with IFN-γbefore co-culture.We found that the response of immune cells to hESC-RPE cells both could be reduced by Ruxolitinib.6.Subcutaneous transplantation in humanized mice showed that:Ruxolitinib could reduce the recognition and attack by immune cells in mice with humanized immune system,and prolong the survival time of hESC-RPE cells in vivo.7.Subretinal transplantation in RCS rats revealed that compared with the systemic administration of immunosuppressive drugs,pretreatment of hESC-RPE cells with Ruxolitinib showed no significant difference in fERG at early post-transplantation stage（within 6 weeks）.Ruxolitinib also showed protective effects on transplanted cells and delayed the decline of retinal function in degenerative rats.