The Study On Combination Of TCF-1~+CD8~+ Stem-like Memory T Cell And Eomes Deficiency In Tumor Immunotherapy

The emergency of immunotherapy brings new hope and opportunity for the treatment of tumor,among which immune checkpoint therapy and cell therapy have achieved exciting therapeutic effects and have been well applied in various types of tumors.With the crossdevelopment of molecular biology and immunology,the continuous emergence of new technologies has also provided more possibilities for the development of tumor immune therapy.Although it has been reported that CD4+helper T cells also has direct antitumor effects,the dominant role of CD8+T cells in anti-tumor immune effect is still significant.At the same time,with the continuous in-depth understanding of CD8+T cell subsets,people have a more precise understanding of the role of different T cell subsets in tumor immune response.In addition to antigen-specific cytotoxic T lymphocytes(CTL),memory cells also play an important role,among which stem-like memory cells(TSCM)have received extensive attention due to their characteristics of both stemness and memory,including self-renewal,high proliferative ability,and differentiation potential.It has been well documented that TSCM has a significant ability to respond to immune checkpoint blockade therapy(ICB).Therefore,the phenotype,effector function and application of the cell subset are worth further exploration.And combining it with the existing adoptive cell transfer therapy(ACT),giving full play to the characteristics of TSCM,so that the cells can exist in vivo for a long time,and exert the superior anti-tumor effect,which can be prepared for better clinical application,it has also become one of the research hotspots in the field.Numerous studies have shown that TSCM expresses T cell factor 1(TCF-1),which is also highly expressed in naive T cell(TN).As a transcription factor,TCF-1 can regulate the downstream gene EOMES,which is closely related to the regulation of early memory and the formation of central memory T cell(TCM).Our research previously found that Eomes deficiency can delay tumor growth and improve survival of tumor-bearing mice,meanwhile,it can affect the differentiation and function of the cells with stem-like and memory characteristics by affecting the expression of TCF-1+CD8+T cells in peripheral immune organs and tumor microenvironment(TME).In addition,Eomes deficiency combined with anti-PD-1 monoclonal antibody therapy can significantly inhibit tumor growth,so we speculate that the inhibitory effect is closely related to the expression and function of TCF-1+CD8+T cells.Therefore,in this paper,EKO mice(CD4cre×Eomesflox/flox Eomes conditional knockout)was used to construct tumor bearing mouse models,WT_LLC and EKO_LLC,and PD-1 monoclonal antibody therapy was performed.Take stem-like memory cells as the entry point,to investigate the role and possible mechanism of stem-like memory CD8+T cell with high expression of TCF-1(or Ly108)in immune response.At the same time,the stem-like memory CD8+T cell was induced in vitro and combined with ACT to further explore,providing a new idea for tumor immunotherapy.[Objective]To investigate the role of stem cell-like memory T cells(Tscm)with high expression of TCF-1(or Ly108)in anti-tumor immune response mediated by Eomes deficiency,and its role in tumor immunotherapy.[Methods]C57BL/6J WT and EKO mice were subcutaneously inoculated with lung cancer cells LLC,and anti-PD-1 monoclonal antibody treatment was performed on day 4,8 and 12 after tumor inoculation,respectively.Meanwhile,the IgG injection group was used as the control group to monitor the tumor growth and survival of mice.On the day 13,the expressions of CD44,CD62L,Ly108,PD-1,Eomes and IFN-y in spleen,lymph nodes and tumor were detected by flow cytometry.CD8+T cells were sorted from spleen cells of C57BL/6J WT and EKO mice by mouse CD8a microbeads,and the expression of Ly108,PD-1,Eomes,CD62L and IFN-γ were detected at different time points in vitro under the Tc1 conditions with or without GSK-3βinhibitor,TWS119.Tumor cell LLC were inoculated in the right flank of CD45.1(B6.SJL-Ptprca Pepcb/BoyJ)mice to construct tumor-bearing mouse model.On the day 4 of tumor cell inoculation,CTX was injected to eliminate endogenous lymphocytes of mice,and on the day 5,induced CD8+T cells from different mice were adoptively to the tumor-bearing mice.At the same time,the mice were injected with PBS as control,and the tumor growth and mice survival were measured.Meanwhile,flow cytometry was used to detect the expression of CD8,Ly108,PD-1 in different tissue samples of each group mice on the day 26.[Results]1.Compared with WT mice,anti-PD-1 monoclonal antibody further inhibited tumor growth in EKO mice;2.The main target cell population of anti-PD-1 monoclonal antibody therapy may be Ly108+/TCF-1+PD-1+CD8+T cell subsets with stem-like memory cell characteristics;3.Eomes deficiency and anti-PD-1 monoclonal antibody treatment can significantly upregulate the expression of CD8+TN in tdLN and CD8+Tcm in TME;4.Anti-PD-1 monoclonal antibody synergized with Eomes deficiency significantly upregulated the expression of TSCM(Ly108+CD62L+CD8+)in peripheral immune organs tdLN,and improved its effector function;5.TWS119,a GSK-3β inhibitor,can induce CD8+T cells to exhibit stemness and memory characteristics in vitro.In addition,TWS119 synergized with Eomes deficiency upregulation of TSCM/TCM expression in vitro induction;6.TWS119 can change the expression pattern of the effector molecule IFN-γ and improve the effect function of TSCM;7.Induced EKO-CD8+T cells can inhibit tumor growth and prolong the survival of tumorbearing mice;8.TSCM(TCF-1+/Ly108+CD62L+CD8+)was significantly associated with prolonged overall survival of human LUAD and SKCM patients.[Conclusion]Eomes deficiency,in collaboration with anti-PD-1 monoclonal antibody therapy,significantly upregulates the expression of TSCM(Ly108+CD62L+CD8+T cells)in peripheral immune organs tdLN,improves its effect function,stores a large number of favorable cells in tdLN,and delivers cells with superior anti-tumor effects to the tumor microenvironment,to maintain the long-term antitumor effect of CD8+T cells.Tscm was successfully induced in vitro by TWS119,an inhibitor of GSK-3β,a key factor in Wnt signaling pathway.Ly108,a key molecule related to stem-like and memory,was up-regulated expression in coordination with Eomes deficiency.Tscm also showed superior antitumor effects in adoptive transfer treatment,and was more significant in EKO mice.