Immunotherapeutic Effect And Mechanism Of Interleukin-2 Complex In Mouse Melanoma And Bladder Cancer

Objective:Immune checkpoint inhibitors are available for clinical treatment of patients with advanced melanoma and bladder cancer,but most patients with advanced or metastatic disease do not response in clinical treatment.Developing new immunotherapy strategies is an urgent clinical problem to be solved.Interleukin-2 complex(IL-2c)is a cytokine preparation that selectively activates anti-tumor effector cells,which can improve the efficacy of immunotherapy,but the specific mechanism is still unclear.The purpose of this project was to evaluate the immunotherapeutic effect of IL-2c on mouse models of melanoma and bladder cancer,and to identify the immunotherapeutic mechanism and effector cells of IL-2c.At the same time,we compared the differences in the efficacy and mechanism of IL-2c and αPD-L1 immunotherapy to determine if there is a nonoverlapping therapeutic mechanism between IL-2c and αPD-L1.This project also evaluated the efficacy and effector cells of IL-2c plus αPD-L1 combination in immune checkpoint inhibitor-refractory lung metastases,providing preclinical evidence for the optimization of existing immunotherapy strategies.Methods:We used the mouse melanoma cell line B16 to construct an intraperitoneal melanoma model and a lung metastatic melanoma model by injecting tumor cells through the abdominal cavity and tail vein,respectively.Using the mouse bladder cancer cell lines MB49 and MBT2,we constructed an orthotopic and lung metastatic bladder cancer model by orthotopic bladder perfusion and tail vein injection of tumor cells.We use intraperitoneal injection to give mice model IL-2c or αPD-L1 therapy,and evaluate the efficacy of drug treatment by weighing the tumor weight,measuring the fluorescence signal intensity in vivo and mice survival.The immune microenvironment is analyzed via flowcytometry analysis.Results:IL-2c reduced the weight of the main attachment tumor tissue of abdominal melanoma omentum(average weight:IL-2c 1334 mg vs.Isotype 2644 mg,p=0.0001)and prolonged the survival time of tumorbearing mice(log-rank,p=0.001);IL-2c inhibited the increase of the fluorescence signal of melanoma lung metastases tumor(mean value of fluorescence signal:IL-2c 1.7×106 vs.Isotype 8.1×06,p=0.002),prolonged the lung metastasis tumor-bearing mice Survival time(median survival time:IL-2c 37.5 days vs.Isotype 27 days,log-rank,p=0.04);for the mouse MB49 bladder cancer orthotopic model,IL-2c inhibited the fluorescence signal of the bladder cancer orthotopic model Intensity(mean fluorescence signal:IL-2c 7.8×106 vs.Isotype 2.1×107,p=0.004),decreased tumor-bearing bladder weight(mean weight:IL-2c 169.3 mg vs.Isotype 100.9 mg,p=0.03),Prolonged survival in tumor-bearing mice(median survival time:IL-2c 33 days vs.Isotype 21 days,log-rank,p=0.02);IL-2c inhibition in mouse MBT-2 bladder cancer orthotopic model Tumor fluorescence signal intensity(mean fluorescence signal:IL-2c 7.1×106 vs.Isotype 2.6×107,p=0.021),decreased tumor-bearing bladder weight(weight mean:IL-2c 23.5 mg vs.Isotype 39.6 mg,p=0.008),prolonging the survival time of tumor-bearing mice(median survival time:IL-2c 33 days vs.Isotype 22 days,log-rank,p=0.007);IL-2c could not prolong the survival time of the mouse model of cystic carcinoma lung metastasis Survival time(median survival time:IL-2c 18 days vs.Isotype 22 days,log-rank,p=0.63).The results of flow cytometry analysis of melanoma and bladder cancer tissues responded to IL-2c therapy showed that:IL-2c increased the proportion of NK cells(%NK1.1+of CD45+),enhanced NK cell immune activation(%CD69+of CD3-NK1.1+),reduce immune exhaustion of NK cells(%PD-1+of CD3-NK1.1+)and promote functional maturation of NK cells(%CD27-of CD3-NK1.1+,%KLRG-1+of CD3NK1.1+);IL-2c did not alter the proportion of CD8+T cells(%CD8+of CD45+CD3+),immune activation(%CD69+of CD8+),immune depletion(%PD-1+of CD8+)and expression of effector molecules(%Perforin+of CD8+,%Granzyme B+of CD8+).αPD-L1 reduced intraperitoneal melanoma weight(weight mean:αPD-L1 2644 mg vs.Isotype 2052 mg,p=0.0001)and prolonged intraperitoneal melanoma mice survival(median survival:αPD-L1 19 days vs.Isotype 16 days,log-rank,p=0.003).Flow cytometry results showed that αPD-L1 increased the proportion of CD8+T cells(%CD8+of CD45+CD3+),enhanced immune activation(%CD69+of CD8+),decreased immune exhaustion(%PD-1+of CD8+)and increased effector molecules The expression of NK cells(%Perforin+of CD8+,%Granzyme B+of CD8+),but did not change the immune indicators of NK cells.IL-2c plus αPD-L1 reduces the number of lung tumor foci in mice with lung metastases from melanoma and prolongs the survival time of mice with lung metastases from melanoma and bladder cancer.Conclusion:IL-2c has immunotherapy effect on abdominal melanoma model,lung metastatic melanoma model,and bladder cancer orthotopic model.The therapeutic mechanism is to promote the immune activation and functional maturation of NK cells,independent of CD8+T cell-mediated anti-tumor immunity.αPD-L1 has immunotherapy effect on abdominal melanoma,and the therapeutic mechanism is to promote CD8+T cell immune activation and effector function.IL-2c plus αPD-L1 combination therapy has immunotherapy effects on melanoma lung metastases and bladder cancer lung metastases that are resistant to immune checkpoint inhibitors.The therapeutic mechanism involves anti-tumor immunity mediated by NK cells and CD8+T cells.Expand tumor types and metastatic sites for further study.