Allogeneic Antigen-specific Memory Stem T Cells Generated From Human Peripheral Blood Effectively Eradicate Targets In Mice

The naive T cells proliferating and differentiating into memory T cells and eventually effector T cells,is initiated by contacting with antigen.In the process of T cell differentiation,they gain their effector functions while constantly lose their self renewal and survival ability.T memory stem cells(T_SCM)are of both stem cell and memory T cell properties,which are considered as the earliest stage of CD8+T cell differentiation from the T_N.As the implantation and long-term existence of tumor-specific T cells in host are the prerequisite for adoptive immunotherapy,Tscm with self-renewal and differentiation capacity show the greatest potential to implant and long-term exhibit function in vivo,compared with other T cells of differentiation stages.Allo-antigen is the initiator of allo-rejection.T cell responses to alloantigen are of peptide-MHC complex（pMHC）specificity in the exact same way as to nominal antigen,but show much higher precursor frequencies.For the convenience of preparation and study of T_SCM,we set up an in vitro coculture to generate allogeneic antigen-specific T_SCM cells using strategy of suppression of T-cell differentiation.The prepared T_SCM cells exhibited the stem cell and memory T cell-characteristic behavior in vitro and in vivo experiments.1.Preparation of allo-antigen-specific T_SCM cells from peripheral PBMC in vitroTo prepare allo-antigen-specific T_SCM,this study began with a coculture of a simulator cells and allogeneic PBLs.The stimulator was a lymphoblastoid cell line（LCL）with defined HLA allotyping,ie.E007.The PBL cocultured with E007 was going to stimulate the allo-specificfic T_N develop into T_SCM.By the adding of T_SCM differentiation inhibitor TWS119 into the coculture,the T_SCM were enriched.As the allo-antigen-specific T cells showed proliferation in the coculture,sorting of proliferation cells ensured the antigen-specificity of the perepared lymphocytes.The sorted lymphocytes underwent further expansion by cytokines IL-7 and IL-15,making the allo-specific T_SCM expanded in number.The T_SCM in the coculture were able to be also difined by the phenotype,CD3+CD8+CD45RA+CD62L+CD95+CCR7+CD28+.Our results indicated that the TWS119 inhibition of T_SCM differentiation enriched the T_SCMCM numbers by 100 folds for the first 7 days.E007-specific T cells underwent proliferation in the coculture,proliferation sorting reach above 98%purity of the prolifeative cells.The sorted cells were further expanded by IL-7 and IL-15 for the next 7 days,the allo-specific T_SCM increased by another 150 folds.For the 2 weeks preparation,our protocol began with 1x10⁷ PBLs and resulted in 2′10⁷ E007-specific T_SCM cells,the number of the allo-specific T_SCM cells was enough to meet the need of the following studies.2.The prepared T_SCM cells exhibit stem cell and memory T cell properties in vitroCharacteristic of stem cells was self-renewing and differentiation potential,which can be reflected by maintaining their phenotype and properties after division.Self-renewing of the T_SCMCM is able to be observed by their daughter cell phenotype and the TREC levels.The latter is for T cell receptor rearrangement excision circles,which is highest in T_N,and graduately dilutded as the T cell division.Characteristic of memory T cells was rapid differentiation into effector T cells in response to the same allo-antigen stimulation,mediating cytokine production and allo-antigen-specific cytotoxicity.In ordr to check the antigen specificity of the prepared T_SCM cells,we used an E001cell line of HLA class I fully mismatched with the E007 for the control.The T_SCM cells cultured with the pro-differentiation cytokine IL-2,the proliferation of the T_SCM cells was observed at each CFSE dilution peak on day 10.Each generation of partial daughter cells kept the identical T_SCM cell phentype to the parent cells.The TREC level analysis showed the T_SCM cells were differentiated at a stage between T_N and T_CM.The results demonstrated that the T_SCMCM cells were of self-renewing ability.As the T_SCMCM cells were gererated by the E007 stimulatioin,they were able to differentiate into effector cells,produce cytokines and kill targets in response to the E007 restimulation.The control stimulator E001 showed no ability to induce such response.These observations indicated the T_SCMCM cells were allo-antigen specific.The T_SCMCM cells-derived daughter cells,especially the T_EMM and T_EFF subsets,were the main effector cells to produce the cytokines.3.The prepared T_SCM cells were able to implant and effectively eradiacate the targets.Implantaion is refered to as the T_SCMCM cells survive and function in the host for long term.This property is due to continuous self-renewing and differentiation into effectors,providing a long-term T cell response to eradicate the targets.LCL cell lines E001 or E007 were infused into the NOD-SCID mice,and the E007-specific T_SCM cells were adoptively transferred into the LCL-burden mice on day 0.Transferring of the E007-specific T_EM and T_EFF was also set up for comparison of implantation and target-killing with the T_SCM.Blood sampling was taken once a week for 5 weeks after the E007-specific T cells transferring.On the day 35,the mice were euthanized,the spleen and bone marrow specimens were observed for the transferred T cells and residual LCL cells detected by the LMP1.These parameters would reflect the human T cell survival,differentiation and eradication of the targetsHuman T cells were found in all blood samples of the mice transferred with the Tscm cells,but only in the day 7 blood samples with the T_EM and T_EFF cells,and dispeared on day 14.The number of T_SCMCM cells in the blood samples was observed consistent after the T_SCMCM transferring.The LCL cells mainly entered the spleen and bone marrow after infusion into the mice.In the E007-burden mice,the T_SCM-transferred mice showed more T_EM and T_EFF cells in the blood,spleen and bone marrow.The residual LCL cells in spleen and bone marrow were similar to that of the control without LCL infusion.The residual LCL in the T_EM and T_EF-transferred mice were more than that of the control.In the E001-burden mice,the T_SCM-transferred mice showed less T_EM and T_EFF cells,and the residual LCL cells were similar to that of the LCL infusion mice without T cell transferring.These results indicated the prepared T_SCM cells were able to implant and long-term exist in the mice.In case of the same allo-antigen infusion into the mouse,the T_SCM cells quickly differentiate into effector T cells,eradicating the target bearing the same allo-antigen.Our study provided a practical protocol for allo-antigen-specific T_SCM cell preparation.This method could be adapted to prepare T_SCM cells specific for antigen of interesting.As T_SCMCM cells show the implantation and long-term existence in the host after transferring,the preparation of antigen-specific T_SCM cells is crucial for T-cell adoptive immunotherapy.