PPARδ Inhibition Blocks Tumor-induced Immunosuppressed B Cells And Enhances CD40 Agonist Immunotherapy

Background and Objective:In recent years,revolutionary progress has been made in tumor immunotherapy.Drugs represented by immune checkpoint blockade(PD-1/PDL-1 mAb,CTLA-4 mAb)have achieved remarkable results in advanced solid tumors such as advanced melanoma,lung cancer,and urothelial carcinoma.However,blocking immune checkpoints to restore or reactivate preexisting antitumor immune responses is a strategy that is effective only in "hot tumors," which represent a small percentage of patients with cancer and a limited population of patients who can benefit.The activation of antitumor immune response by dendritic cell(DC)and T cell activation agonists can transform cold tumors into hot tumors.Drugs represented by CD40 agonist mAb are the key to turn the tumor immune response from defective to effective and are the hotspots of immunotherapy drug development.However,its effective treatment window is narrow and its clinical application is limited.It has been reported that PPARy or PPARδ inhibitors can promote the transformation of macrophages from a pro-tumor M2 phenotype to an anti-tumor M1 phenotype,and it is speculated that PPARy or PPARδ inhibitors may have a synergistic anti-tumor effect with low-dose agonizing anti-CD40 antibody.The aim of this study is to enhance the anti-tumor efficacy of CD40 without enhancing hepatotoxicity through combination therapy,In addition,we unexpectedly found that tumor-induced immunosuppressive B cells reduce the efficacy of CD40 monoclonal antibody,and PPARδ inhibitor blocks this group of B cells and reduces their inhibitory function,which provides a direction and feasible strategy for studying tumor-related immunosuppressive B cells and improving the efficacy of immunotherapy.Methods:We first evaluated the anti-tumor effects of a PPARδ inhibitor and a PPARy inhibitor combined with anti-CD40 mAb in mouse melanoma(B16)and bladder cancer(MB49)models,respectively.We further confirmed that PPARδ inhibitor synergistically enhanced the anti-tumor immune response of CD40 agonistic antibody by flow cytometry,immunohistochemistry,and anti-tumor immune memory model.Then,the expression of PPARδ and PPARy in the spleen,peripheral blood and draining lymph nodes of naive mice and tumor-bearing mice in each treatment group was detected by magnetic bead sorting,RNA sequencing,Western blot,and flow cytometry.Next,the inhibitory function of B cells in draining lymph nodes of tumor-bearing mice in each treatment group was analyzed by magnetic bead sorting and flow cytometry.Finally,the effect of B cell depletion on the efficacy of anti-CD40 and anti-PD-1 monoclonal antibodies was verified in a mouse melanoma model.Results:1.The anti-tumor effect of CD40 agonistic antibody is dose-dependent,and the effect of low dose is limited.CD40 agonist-type antibody has dose-dependent hepatotoxicity,and the hepatotoxicity is significantly increased at the effective dose.PPARδ inhibitors enhance the anti-tumor activity of CD40 agonist mAb without increasing the hepatotoxicity of treatment.2.PPARδ inhibitors synergistically enhanced the anti-tumor immune response of CD40 agonizing mAb and promoted the infiltration of CD8+T cells into the tumor,especially in the deep part of the tumor;It also enhanced the anti-tumor immune memory response.3.PPARδ inhibitors synergistically enhanced the anti-tumor immune response of CD40 mAb,and the target cells were not macrophages as originally envisaged.4.PPARδ expression of B cells in draining lymph nodes of tumor-bearing mice is increased,which has T cell suppressive function,and PPARδ blockade can reduce this inhibition.The limited efficacy of low-dose CD40 mAb is due to the proliferation of these immunosuppressive B cells,and B-cell depletion enhances the antitumor efficacy of low-dose CD40 monoclonal antibody.5.PPARδ inhibitors enhance the anti-tumor activity of CD40 monoclonal antibody and PD-1 monoclonal antibody by blocking B cells with immunosuppressive function.Conclusion:1.PPARδ inhibitors synergistically enhanced the anti-tumor immune response of CD40 agonist mAb without increasing hepatotoxicity.2.The combination of a PPARδ inhibitor and a CD40 agonist mAb targets tumorinduced B cells,not macrophages,as originally intended.3.The expression of PPARδ in tumor-induced B cells is up-regulated and has T cell inhibitory function,which reduces the efficacy of anti-tumor immunotherapy.Depletion of B cells can enhance the efficacy of immunotherapy.4.Blockade of tumor-induced suppressor B cells with a PPARδ inhibitor synergistically enhances the antitumor effect of PD-1 mAb.