Study On The Mechanism And Clinical Application Of PD-1 And TIM-3 In The Immune Evasion Mediated By Exhausted T-cells In B-cell Lymphoma

Background and ObjectivesAt present,lymphoma is the most common hematological malignant tumor,with the incidence rising in the last decade,which endangers human health.Lymphoma is highly heterogeneous and has nearly 100 distinct and independent types.Diffuse large B-cell lymphoma(DLBCL)is the most common subtype,accounting for about 30% of all lymphoma.In recent years,R-CHOP regiment has become a standard first-line therapy,which significantly improves the efficacy and survival of patients with DLBCL.However,refractory,and recurrent(R/R)DLBCL remains a thorny problem in clinical practice.Clinical applications of immunotherapy,especially the breakthroughs of immunocheckpoint therapy and Chimeric Antigen Receptor T-Cell Immunotherapy(CAR-T)therapy,bring new hope to R/R DLBCL patients.Programmed cell death 1 receptor(PD-1)is one of the key immune checkpoint molecules.PD-1 monoclonal antibody(m Ab)targeted blocking therapy in R/R Hodgkin’s lymphoma has achieved a total response rate up to 90%.However,at present,the clinical data and experience of DLBCL treated with PD-1 m Ab are not enough,which need to be further explored and studied.T cell immunoglobulin mucin-3(TIM-3)is another important immune checkpoint molecule.Literature reports that the induced up-regulation of TIM-3 on effector T cells is associated with the secondary resistance of PD-1 m Ab.This study focuses on the immunotherapy of R/R DLBCL,and intends to explore the clinical efficacy,safety,prediction of biomarkers and drug resistance mechanism of PD-1 targeted blocking strategies(PD-1 m Ab and PD-1 gene knockdown CD19 CAR-T cells)applied in R/R DLBCL patients.And further explore the combination of PD-1 monoclonal antibody and TIM-3 monoclonal antibody in the treatment of B-line lymphoma,laying a foundation for future clinical application.Methods1)Flow cytometry was used to detect the distribution of T cell subsets in peripheral blood(40 cases)and lymph nodes(30 cases)and the expression of PD-1 on the T cell surface of DLBCL patients.The levels of soluble PD-1 and PD-L1 in plasma(40 cases)were determined by ELISA.The above parameters were compared with the control group(normal population and patients with reactive hyperplasia of lymph nodes)to analyze the role of PD-1(both modular and soluble expression)and soluble PD-L1 in the pathogenesis of DLBCL.Subgroup analysis and survival analysis of clinical characteristics of DLBCL patients were conducted to further explore the influence of PD-1/PD-L1 on clinical characteristics and survival of DLBCL patients.2)The PD-1 targeted blocking strategy(PD-1 monoclonal antibody and PD-1 gene knockdown CD19 car-t cells)was used to treat R/R DLBCL patients,and the efficacy and adverse reactions were observed.Exon gene capture second generation sequencing,oligonucleotide array chip and immunohistochemistry were used to detect patients’ lymph node tissues,to explore the predictive biomarkers related to the efficacy of PD-1 blocking therapy.Flow cytometry was used to monitor the dynamic changes of PD-1 and TIM-3 expression in peripheral blood T cells before and after treatment,to further explore the drug resistance mechanism related to the exhausted T cells in PD-1 targeted blocking therapy.3)Established a subcutaneous model of lymphoma with A20 cells in BALB/c mice and observed the tumorigenesis rate and growth of tumors;Then,the blocking PD-1 monoclonal antibody or PD-1 monoclonal antibody + TIM-3 monoclonal antibody were used to treat tumor-forming mice,and the therapeutic effect and survival of each group were observed.The immunological mechanism of PD-1+TIM-3 monoclonal antibody was further investigated by CD8+ magnetic bead separation,flow cytometry,ELISA,CCK8 and other methods.Results1)Compared with the control group,the proportion of T cell subgroups in peripheral blood of DLBCL patients was significantly different(CD4+/CD3+: 45.47±10.07% VS.61.91±10.52%,P=0.036;CD8+/CD3+: 43.99±9.32% VS.31.94±6.50%,P=0.045),the expression of PD-1 on T cells was up-regulated significantly(CD4+PD-1+: 16.30±4.58% VS.8.96±2.95%,P=0.017;CD8+PD-1+: 18.12±5.43% VS.10.13±3.30,P=0.023),and the plasma levels of soluble PD-1 and PD-L1 were also significantly increased at the initial diagnosis(s PD-1: 4.49 ± 3.53ng/ml VS.0.47± 0.36ng/ml,P=0.002;s PD-L1: 1.21 ±1.03ng/ml VS.0.26±0.24ng/ml,P=0.039).Subgroup analysis of clinical characteristics of DLBCL patients showed that PD-1(both modular expression and soluble expression)and soluble PD-L1 level were correlated with clinical characteristics of patients(pathological subtypes,IPI scores).Survival analysis showed that the OS and PFS of the group with high expression of PD-1(both modular expression and soluble expression)and soluble PD-L1 were significantly lower than that of the group with low expression(P<0.05).Dynamic analysis of patients’ plasma soluble PD-1 and PD-L1 levels showed that they were closely related to the patient’s condition.2)The PD-1 targeted blocking strategy(PD-1 monoclonal antibody and PD-1 gene knockdown CD19 CAR-T cells)was used to treat R/R DLBCL,and some patients obtained obvious efficacy(ORR 33% and 72%),overall good tolerance and mild toxic and side effects.The next generation sequencing(NGS)of exon trapping and single nucleotide polymorphism(SNP)array suggested that patients with high tumor mutations were more sensitive to PD-1 therapy(P<0.05).Immunohistochemistry suggested that patients with high number of tumor infiltrating T cells or/and high expression of PD-L1 had better response to PD-1 treatment(P<0.05).The expression of PD-1 and TIM-3 on peripheral blood T cells showed a dynamic change before and after treatment.With the treatment,the expression of PD-1 was down-regulated,but the expression of TIM-3 was up-regulated,and the expression of TIM-3 was significantly higher in the group with poor efficacy(CD4+TIM-3+: 29.12±3.52% VS.20.10±2.71%,P=0.001;CD8+ TIM-3+: 30.12±3.65% VS.19.93±3.52%,P=0.001).3)Established the subcutaneous model of A20 cell lymphoma in BALB/c mice;The efficacy and survival of PD-1 m Ab+TIM-3 m Ab group were significantly better than that of PD-1 m Ab group and control group.The study of immunological mechanism suggested that the infiltration of T cells in the tumor was increased in the PD-1+TIM-3 m Ab group(30.12±2.15% VS.14.94±1.42% VS.4.99±0.95%,P=0.001),and the expression of TIM-3on T cells was significantly down-regulated(20.55±1.46% VS.60.14±2.98% VS.40.01 ± 2.52%,P=0.001).In vitro T cell functional experiments suggested that the proliferation and killing ability of CD8+T cells in the tumor of the PD-1+TIM-3 m Ab group was significantly stronger than that of the PD-1 m Ab group and the control group(P<0.05);Apoptosis of PD-1+TIM-3 m Ab group was significantly lower than the other two groups(P<0.05).Plasma levels of IL-2,IFN-γ and TNF-α in PD-1+TIM-3 m Ab group were also significantly higher than those in the other two groups(P<0.05).Conclusion1)The expression of PD-1 on T cells and plasma soluble PD-1/PD-L1 in DLBCL patients played an important role in the pathogenesis of DLBCL.These indicators were closely related to patients’ clinical characteristics(pathological subtypes,IPI scores)and survival(OS/PFS).The dynamic changes of plasma soluble PD-1 and PD-L1 in patients were closely related to their disease status,which could be used as biomarkers for disease monitoring.2)The PD-1 targeted blocking strategy(PD-1 monoclonal antibody and PD-1 gene knockdown CD19 CAR-T cells)was used to treat R/R DLBCL,which was generally well tolerated and mild in toxic and side effects,but only effective in some patients.The exon trapping NGS,SNP-array and immunohistochemical test suggested that the degree of tumor mutation,the number of tumor infiltrating T cells and the expression of PD-L1 were related to the PD-1 response,which could be used as biomarkers for PD-1 targeted blocking therapy.After treatment with PD-1 monoclonal antibody,the induced expression of TIM-3 on the effector T cells in patients was up-regulated,which was related to the secondary resistance of PD-1 monoclonal antibody.3)In the subcutaneous model of A20 cell lymphoma in BALB/c mice,the efficacy and survival of PD-1+TIM-3 m Ab group was significantly better than that those of PD-1 m Ab group and control group.Immunological mechanism studies suggested that the combination of PD-1+TIM-3 m Ab could inhibit the TIM-3 induced expression of effector T cells,avoid the depletion of T cells,and enhance the anti-tumor effect of T cells.