Study On Antitumor Effect Of Tumor-Infiltrating CD8~+T Cells With High Expression CCR2 In Mouse Lung Adenocarcinoma And Glioblastoma

Objective:In recent years,traditional surgery combined with chemoradiotherapy and complementary immunotherapy have made great progresses in lung adenocarcinoma（LUAD）and glioblastoma（GBM）therapy,among which immunotherapy strategies still face challenges and urgently need to be addressed.In this study,considering tumor heterogeneity,a single-cell sequencing database was used to perform bioinformatics analysis of the tumor immune microenvironment and the results were verified by flow cytometry.Based on the results of bioinformatics analysis,CD8⁺tumor-infiltrating T lymphocytes（TILs）were transfected with lentiviral vectors with high expression chemokine receptor 2（CCR2）,and their antitumor effects were investigated in vivo and vitro of mouse LUAD and GBM.This study will provide new targets and theoretical support for tumor immunotherapy.Method:1.LUAD data in TCGA database was used for bioinformatics analysis.（1）CD8⁺T cells in the database were divided into 4 cell subsets in the reduced dimensionality of 13670immune microenvironments.（2）Screening of immune checkpoint molecules of cell subsets verified by flow cytometry.（3）Pseudotime trajectory analysis of the depletion trend of cell subsets.（4）The correlation between cell subsets and overall survival was analyzed using clinical data in the database and verified by clinical specimens.2.In vivo and vitro,the antitumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 were studied in LUAD and GBM.2.1.The antitumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 were studied in LUAD.（1）A mouse Lewis lung cancer（LLC）model was constructed and CD8⁺TILs from tumor tissue were purified by magnetic cell sorting.（2）Lentiviral transfection of CD8⁺TILs.（3）Co-culture of CD8⁺TILs and LLC cells,four groups were divided according to different treatment of CD8⁺TILs in vitro:empty vector transfection CD8+TILs group（CD8⁺T）,high expression CCR2 lentiviral transfected CD8⁺TILs group(CD8⁺T^CCR2+),anti-PD1 group(CD8⁺T^anti-PD1),high expression CCR2 lentiviral transfection CD8⁺TILs and combination treatment group(CD8⁺T^{anti-PD1/CCR2+}).CD69,CD107A,IFN-γand Granzyme B expression of CD8⁺TILs were detected by flow cytometry.（4）Flow cytometry was used to detect apoptosis of LLC cells,CCK8 was used to detect LLC cell proliferation.（5）Different groups of CD8⁺TILs were injected to the para-tumor,mouse tumor volume was measured;and mouse tumor frozen sections were prepared for detection of the chemotaxis of theinfused CD8⁺TILs by confocal microscopy.2.2.The anti-tumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 were studied in GBM.（1）The CCR2 expression of human GBM clinical specimens and mouse glioma were detected by immunohistochemistry.（2）Mouse glioma 261（GL261）glioma model was constructed,tumor tissue,spleen and peripheral blood CD8⁺T cells were extracted by magnetic cell sorting,and CD69,CD107A and CCR2 expression were detected by flow cytometry.（3）CCR2 high expression lentivirus was transfected,CD8⁺TILs（groups same to 2.1（3））were co-cultured with GL261 cells for 24 h,48 h and 72 h,respectively,and CD69,CD107A,IFN-γand Granzyme B expression were detected by flow cytometry.（4）Apoptosis of GL261 cells was detected by flow cytometry,and GL261 cell proliferation was detected by CCK8.（5）Different groups of CD8⁺TILs were injected to the para-tumor,mouse tumor volume was measured.Result:1.Database bioinformatics analysis results showed four subpopulations of C1,C2,C2and C4.The C1 of CD8⁺T cells with killing activity had better prognostic correlation compared to other subpopulations;The C2 that was highly enriched in the tumor microenvironment was depleting CD8⁺T cells,and anti-PD1 may reverse the depletion.2.The antitumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 in LUAD and GBM.2.1.Results of antitumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 in LUAD.（1）Successfully constructed a mouse LLC model,extracted and purified CD8⁺TILs from tumor tissues.（2）CCR2 high expression lentivirus was successfully transfected into CD8⁺TILs.（3）Build different processing groups,the co-culture results showed that CD69expression in the CD8⁺T^CCR2+group was higher than CD8⁺T group and CD8⁺T^anti-PD1 group（P<0.0001）;and there was no difference in CD69 expression between CD8⁺T^CCR2+group and CD8⁺T^{anti-PD1/CCR2+}group（P>0.05）.The expression of IFN-γand Granzyme B was higher in CD8⁺T^CCR2+group than the CD8⁺T group and CD8⁺T^anti-PD1 group（P<0.05）;IFN-γand Granzyme B expression was higher in CD8⁺T^{anti-PD1/CCR2+}group than in CD8⁺T^CCR2+group（P<0.05）.（4）Compared with other groups,LLC cells in the CD8⁺T^{anti-PD1/CCR2+}group had the highest apoptosis rate（P<0.05）and the lowest cell proliferation（P<0.05）.（5）The results of in vivo experiments in mice showed that compared with other groups,the tumor volume in the combination treatment group was the smallest;The results of frozen sections of tumor tissue showed that compared with the CD8⁺T group,the CD8⁺T^CCR2+group had more fluorescence labeled lentiviral transfected cell infiltration.2.2.Results of antitumor effects of CD8⁺TILs with high expression CCR2,combination of both CD8⁺TILs with high expression CCR2 and anti-PD1 in GBM.（1）Immunohistochemical results showed that there was differential expression of CCR2between GBM tissue and adjacent tissues in human and mouse specimens（P<0.0001）.（2）Successfully constructed a mouse GL261 model,extracted and purified CD8⁺T cells from tumor tissue,peripheral blood and spleen,compared peripheral blood and spleen,and the expression of CD69,CD107A and CCR2 was the most in CD8⁺TILs at the tumor site.（3）CCR2 high expression lentivirus was successfully transfected and different processing groups were constructed,the co-culture results showed that CD69 and CD107A expression of CD8⁺T^CCR2+group was higher than CD8⁺T group and CD8⁺T^anti-PD1 group（P<0.0001）,and CD69 and CD107A expression in CD8⁺T^CCR2+group and CD8⁺T^{anti-PD1/CCR2+}group was not different（P>0.05）.The expression of IFN-γand Granzyme B of CD8⁺T^CCR2+group was higher than the CD8⁺T group and CD8⁺T^anti-PD1 group（P<0.05）.IFN-γexpression were higher in CD8⁺T^{anti-PD1/CCR2+}group than the CD8⁺T^CCR2+group（P<0.01）.（4）Compared with other groups,GBM cells in the CD8⁺T^{anti-PD1/CCR2+}group had the highest apoptosis rate（P<0.05）and the lowest cell proliferation（P<0.05）.（5）The results of in vivo experiments in mice showed that compared with other groups,the tumor volume in the combination treatment group was the smallest.Conclusion:1.Depleted CD8⁺TILs are potential immunotherapeutic cell subsets,and immune checkpoint blockade may reverse the depletion of CD8⁺TILs.2.High expression CCR2 exerts antitumor effect by promoting the early activation of CD8⁺TILs,promoting apoptosis of tumor cells,inhibiting tumor cell proliferation and promoting the migration of CD8⁺TILs to tumor tissues in LUAD.3.High expression CCR2 exerts antitumor effect by promoting the activation and killing effects of CD8⁺TILs,promoting apoptosis of tumor cells,inhibiting the proliferation of tumor cells and promoting the migration of CD8⁺TILs to tumor tissues in GBM.4.CD8⁺TILs with high expression CCR2 combined with anti-PD1 blockade have highly effective antitumor effect in both LUAD and GBM.