The Mechanism Of CD200~+ Cytotoxic T Lymphocytes Mediated Anti-tumor Immune Response In The Tumor Microenvironment Summary

Research backgroundAntitumor immunity is that the hosts use own immune system to recognize and eliminate tumor cells,thereby protecting themselves from tumors.However,tumor cells incline to domesticate the host’s immune system while expanding and progression,resulting in immune cells in the tumor microenvironment(TME)moving towards immunosuppression and losing the ability of killing tumor cells.T cell dysfunction with high expression of programmed cell death-1(PD-1)signaling in TME is a defining feature of cancer,which are induced by TME factors,including persistent tumor antigen stimulation,chronic inflammation,and hypoxia.The immune checkpoint blockades(ICB),such as anti-PD-1 and anti-programmed cell death ligand-1(PD-L1)can reinvigorate dysfunctional CD8~+tumor-infiltrating lymphocytes(TILs)and exert its anti-tumor role.So far,anti-PD-1/PD-L1 therapy have achieved a great success in a subset of cancer patients,inducing their T cells to mount a successful antitumor response.However,the fundamental mechanisms underlying different efficacy of PD-1/PD-L1 blockade therapy remains poorly understood.Infiltration of CD8~+T cells into TME is crucial for the efficacy of anti-PD-1/PD-L1therapy.However,single-cell RNA sequencing(sc RNA-seq)data of CD8~+TILs has revealed that CD8~+TILs are highly heterogeneous population in liver cancer and non-small cell lung cancer.Increasing evidence indicates that the heterogeneity of CD8~+TILs is one main reason to hinder the maximum efficacy of PD-1/PD-L1 blockade therapy,as anti-PD-1/PD-L1therapy does not exert equivalent effects on the heterogeneous CD8~+TILs population.Clinical evidence indicates that different subsets of CD8~+TILs can be used as biomarkers to predict the efficacy of anti-PD-1/PD-L1 therapy.For example,in patients with advanced melanoma,TCF1~+CD8~+TILs are shown better able to control tumor growth than terminally exhausted T cells and their abundance in baseline tumor tissues was associated with efficacy of the anti-PD-1 therapy.Conversely,in preclinical models of Non-alcoholic steatohepatitis-induced hepatocellular carcinoma,anti-PD-1 therapy resulted in an increased tumor burden,which correlated with increased infiltration of hepatic CD8~+PD-1~+CXCR6~+TOX~+TNF~+T cells.So far,the different subset of CD8~+TILs,which is crucial for the efficacy of anti-PD-1/PD-L1therapy remains poorly defined.Different subsets of CD8~+T cells with specific functional status are usually distinguished by the expression patterns of immune checkpoint molecules.For example,the co-expression of PD-1 and T cell immunoglobulin and mucindomain3(TIM-3)identifies a subset of terminal exhausted CD8~+TILs,which does not respond to PD-1 blockade therapy.In addition,lymphocyte-activation gene 3(LAG-3)expression as an exhaustion phenotype on peripheral CD8~+T cells predicts a poor outcome for anti-PD-1 therapy.Moreover,a recent study suggested that CD28~+CD8~+T cells were necessary for effective PD-1 therapy in tumor-bearing mouse models.Based on these studies,we hypothesized that dissecting CD8~+TILs into different subsets based on the expression of various immune checkpoint molecules might further identify CD8~+T cells responding to anti-PD-1/PD-L1 therapy.In order to explore newly CD8~+T cell subsets that respond to anti-PD-1/PD-L1 therapy,we analyzed the TILs sc RNA-seq data in the public clinical immunotherapy cohort.By classifying tumor-infiltrating T cells into different subsets based on their expression pattern of various immune checkpoint molecules,we identified an increase in proportion of CD200~+T cells after anti-PD-1 treatment and found that a higher proportion of CD200~+T cells was positively correlated with a favorable clinical efficacy of PD-1/PD-L1 blockade therapy.By using transplanted tumor models(MC38,LLC),we explore the impact of anti-PD-L1 therapy on the proportion of CD200~+T cells and CD200~+cytotoxic T lymphocytes(CTLs).And whether the absence of CD200~+CTLs in a transplanted tumor model affect the anti-PD-L1efficacy.Secondly,we investigated the anatomical location distribution and biological function of tumor-infiltrating CD200~+CTLs.At last,we explore the correlation between CD200~+CTLs infiltration in TME and the efficacy and survival prognosis of immunotherapy in multiple patient cohorts with various tumor types.Through this study,a new subset of CD8~+T cells responding to the effective PD-1/PD-L1 blockade therapy was discovered,and the mechanism of its antitumor effect and its relationship with the long-term response to anti-PD-1/PD-L1 treatment were analyzed,which provided a new strategy for clinical biomarkers to predict immunotherapy efficacy and adoptive T cell therapy.Methods1.Explore the relationship between CD200~+T cells and anti-PD-1 therapy in tumor patients1.1 Collect and organize the sc RNA-seq data of T cells and clinical information in the public immunotherapy cohort;1.2 The Wilcoxon test was used to compare the changes in the proportion of each immune checkpoint molecule positive T cells before and after immunotherapy;1.3 Explore the relationship between the proportion of CD200~+T cells and the efficacy of anti-PD-1 therapy;2.Construct experimental model to verify the relationship between CD200~+T cells and anti-PD-L1 therapy2.1 Construct wildtype(WT)mice MC38 tumor-bearing model,and explore the effect of anti-PD-L1 therapy on the infiltration of CD200~+T cells and CD200~+CTLs;2.2 Investigate whether CD200~+CTLs are necessary for effective anti-PD-L1 treatment;3.Explore the anatomical location distribution and biological function of CD200~+CTLs3.1 Detect and analyze the proportion of CD200~+CTLs in tumors,tumor-draining lymph nodes(Td LNs),spleens and peripheral blood;3.2 Detect the proliferation ability of intra-tumoral CD200~+CTLs;3.3 Detect the effector function of intra-tumoral CD200~+CTLs;3.4 Detect the ability of CD200~+CTLs to killing tumor antigen-expressing tumor cells;4.Explore the relationship between the infiltration of CD200~+CTLs and the long-term response to ICB therapy in tumor patients4.1 The log-rank test and the Kaplan-Meier method were used to test the difference in overall survival between the high and low CD200~+CTLs infiltration groups;4.2 The univariate cox model was used to evaluate the effect of CD200~+CTLs infiltration on the ICB prognosis.Results:1.CD200~+T cells expand after PD-1 blockade therapy in human tumors1.1 The sc RNA-seq data of immunotherapy cohort suggests that the proportion of CD200~+T cell infiltration increases in response to anti-PD-1 therapy;1.2 Patients with a higher proportion of CD200~+T cells is positively related to a favorable clinical outcome to anti–PD-1 therapy;2.CD200~+CTLs cells increased in experimental model after anti-PD-L1 treatment,and are necessary for efficacious anti–PD-L1 therapy2.1 In MC38 tumor-bearing mice,CD200~+CTLs infiltration increase after anti-PD-L1therapy;2.2 In MC38 tumor-bearing mice,CD200~+CTLs depletion reduce the efficacy of anti-PD-L1 in tumor control and abolish the survival benefit;3.CD200~+CTLs mainly enrich in the TME and exhibit superior antitumor capacity3.1 CD200~+CTLs mainly exist in the TME;3.2 CD200~+CTLs have superior proliferative potential than CD200~-CTLs;3.3 CD200~+CTLs exhibit enhanced production of multiple effector molecules than CD200~-CTLs;3.4 CD200~+CTLs are much more effective in killing tumor antigen–expressing tumor cells than CD200~-CTLs;4.The infiltration of CD200~+CTLs can predict efficacy of immunotherapy4.1 High CD200~+CTLs infiltration are corelated with favorable overall survival;4.2 Higher CD200~+CTLs infiltration are associated with favorable depth of response to ICB therapy.Conclusion:1.CD200~+T cells expand after PD-1/PD-L1 blockade therapy,which is positively related to the efficacy of ICB treatment;2.CD200~+CTLs mainly enrich in TME and have superior antitumor capacity;3.High infiltration of CD200~+CTLs in the TME can predict the efficacy of immunotherapy.