Study On The Mechanism Of PD-1 McAb Combined With Cytokine IL-33 In Antitumor Immune Response

The tumor microenvironment is the internal environment for tumor cells to produce and live,including tumor cells,fibroblasts,immune and inflammatory cells and glial cells,as well as the surrounding environment interstitial cells,microvessels,and biomolecules infiltrating them.The immune inflammatory features produced by the complex tumor microenvironment are conducive to the growth,metastasis and angiogenesis of tumor cells and promote the generation and development of malignant tumors.The number and function of immune cells infiltrating the tumor microenvironment are the basis of the body's anti-tumor immune response.Among them,the infiltration of CD8+ cytotoxic T lymphocytes(CTLs)and CD4+ helper T lymphocytes(Th)is the main factor.Infiltrating CD4+ T lymphocytes and CD8+ T lymphocytes exert anti-tumor immune responses by secreting various effector molecules such as IFN-? and perforin,which are closely related to the prognosis of patients.However,the tumor-infiltrating CD4+ Th1 and CD8+ CTLs can be exhausted due to the complex immunosuppressive mechanisms of the tumor microenvironment,such as the expression of immune checkpoint molecules PD-1,PD-L1,CTLA-4,Tim-3 etc.As a result,immune cells can not carry out immune surveillance and killing of tumor cells.Therefore,to change the immunosuppressive state of the tumor microenvironment,to reactivate the infiltrating immune cells and to reverse the state of incompetence,it has become a hot topic in tumor immunotherapy.The research on PD-1/PD-L1 immune checkpoint molecules on tumor immunotherapy is a hot topic at home and abroad.PD-1/PD-L1 binds to receptors/ligands in the tumor microenvironment to exhaust lymphocyte and inhibit lymphocyte activation and proliferation,produces immunosuppressive molecules such as IL-10,and induces antigen-specific T lymphocyte apoptosis,also,induces the differentiation of naive CD4+ T cells to CD4+Foxp3+ Treg cells,resulting in immunosuppression and immune escape of tumor cells.Therefore,in recent years,many preclinical studies have blocked PD-1/PD-L1 to rescue the lymphocytes effector functions,allowing them to re-stimulate activation to produce an anti-tumor immune response.The current clinical trials have shown that anti-PD-1/PD-L1 therapy can reverse the state of advanced and metastatic tumors,significantly improve the survival rate of cancer patients.The treatment lasting effect,have a significant effect on a variety of solid tumors.Although PD-1/PD-L1 monotherapy has been a significant effect,but only in some patients showed clinical benefits,the effective rate of about 20%,some patients on the treatment effect is not significant,so much as invalid.So looking a combination therapy of PD-1 antibodies has become a crucial issue.The cytokine Interleukin 33(IL-33)is a 30 k Da protein molecular,was first found in 1999,was originally called "high endothelial micro-venous nuclear factor".In 2005,Schmitz confirmed IL-33 is a novel member of the IL-1 family,whose receptors are dimers of ST2 and IL-1 receptor accessory proteins.The original literature reports that IL-33/ST2 is mainly involved in Th2-type inflammatory and anti-viral immune responses.Later studies showed that IL-33/ST2 pathway can promote Th1-type immune response and affect the function of antigen-specific CD8+ T lymphocytes and play an important role in anti-tumor immune response.Based on our previous study,IL-33,in combination with TCR signaling and interleukin-12(IL-12)to stimulate the activation of CD4+ T lymphocytes and CD8+ T lymphocytes in vitro,and promotes its secretion of IFN-?.In the mouse model,the expression of IL-33 in the tumor microenvironment can increase the infiltration of T lymphocytes and enhance the ability of secreting IFN-?,significantly inhibiting tumor growth and metastasis.Therefore,this topic aims to study the effect of the combination of PD-1 monoclonal antibody and cytokine IL-33 in the tumor-bearing mouse model,to observe the growth of the tumor and the survival of mice,and further analysis anti-tumor immune response mechanism to provide a new theory and ideas for tumor immunotherapy.?Objective?To investigate whether blockade immune checkpoint molecular PD-1 combined with cytokine IL-33 can inhibit tumor growth,prolong the survival of tumor-bearing mice.?Methods ? 1.2×105 B16 and B16-IL33 melanoma cells were injected intradermally to Humanized PD-1 KI/KO mice.Tumor-bearing mice of each group were injected intraperitoneally with humanized PD-1 m Ab or control normal Ig G(200 ?g/100 ?l)four times after tumor inoculation.Size of the tumor and survival of tumor-bearing mice were monitored every two days,and the growth curves and survival curves were drawn.2.The infiltration of CD45+ cells,CD4+ T lymphocytes and CD8+ T lymphocytes were analyzed by immunofluorescence labeling and flow cytometry in the early,middle and late stages of tumor growth,and the expression of IFN-?,Foxp3 and Ki-67 in tumor microenvironment were detected with antibody.3.The expression of IFN-?,GZ-B,perforin were detected by real-time PCR.4.Immunofluorescence was used to observe the infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes for frozen section of tumor tissue.?Results?1.PD-1 m Ab combining with cytokine IL-33 significantly inhibited tumor growth in B16 tumor-bearing mice model,and compared with PD-1 monoclonal antibody monotherapy,the difference was more significant,P<0.05;2.PD-1 m Ab combining with cytokine IL-33 significantly prolonged the survival of tumor-bearing mice,P <0.05;3.The infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes in the tumor microenvironment of PD-1 m Ab combined with cytokine IL-33 treatment increased significantly,P <0.05;4.In the early stage of tumor growth,the proportion of IFN-?+CD8+ T cells in the treatment group was significantly more than that in the control group,and the combination group effect was more obvious(P<0.01);the proportion of IFN-?+CD4+ T cells no significant difference.The proportion of IFN-?+CD4+ T cells and IFN-?+CD8+ T cells in each group increased in the middle and late stages,especially in the PD-1 m Ab group and the combination group,P< 0.05;5.In the early stage of tumor growth,the expression of Ki-67 on CD4+ T lymphocytes and CD8+ T lymphocytes increased in the tumor microenvironment of PD-1 m Ab combined with cytokine IL-33 treatment group,the difference was statistically significant(P<0.05);In the middle and late stages,there was no difference in the expression level of Ki-67 between the groups;6.The proportion of MDSC immunosuppressive cells in the tumor microenvironment of PD-1 m Ab combined with cytokine IL-33 treatment group was significantly decreased in the middle and late stages of the tumor,and the level of MHC class II molecules was increased,the difference was statistically significant,P< 0.05;7.In early and late stages of tumor growth,to analyze the expression of IFN-?,GZ-B and perforin at the m RNA level on real-time PCR.PD-1 m Ab combined with cytokine IL-33 treatment group was significantly increased,the difference was statistically significant,P <0.05,P <0.01,P <0.001.?Conclusion? PD-1 immune checkpoint molecule blocking therapy combined with cytokine IL-33 can enhance the proliferation of CD4+ T lymphocytes and CD8+ T lymphocytes in the tumor microenvironment,and promote the infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes effector function(IFN-?,GZ-B,perforin),and enhance the body's anti-tumor immune response;In addition,the combination therapy can inhibit the proliferation of bone marrow-derived MDSC cells to stimulate the reactivation of T lymphocytes,and rescue effector function,which significantly inhibited tumor growth and prolong the survival of tumor-bearing mice.