The Screening For Regulators Of Tumor Immunity And The Effect Of ZDHHC9 In Anti-tumor Immune Response

With the development of tumor biology and immunology,immunotherapy has become a new approach of tumor treatment.T cell-mediated immune response plays a crucial role in tumor recognition and clearance.Whether CTL cells can launch effective immune attacks against tumor cells or not,to some extent,even determines the final outcome of the immune fighting against cancer.In recent years,significant breakthroughs have been made in the clinical research and application of T cells as therapeutic tools or targets.The problem of immune tolerance in immunosuppressive TME,however,remains to be considered as an important cause of the failure of immunotherapy.Elucidating the mechanism of immune escape is still a challenge as well as a focus in the field of tumor immunity.In order to further clarify the underlying mechanism of tumor immune evasion,improve the regulatory network of tumor immunity and find the promising potential targets to transform the suppressive state of immune tolerance in the TME,we use human membrane proteins CRISPRi and CRISPRa screen to identify previously undescribed targets in regulation of immune response to cancer.Here we discover ZDHHC9,a member of DHHC protein family,as an important regulator in T cell mediated immunity against cancer.The CRISPRi and CRISPRa based sg RNA lentivirus libraries of membrane proteins were transduced into the human colon cancer cell line DLD1 with the exogenous expression of CD19.CD19-CAR T cells were used as effectors to kill DLD1-CD19 cells,and variations of sg RNA abundance in the genome of surviving target cells were analyzed by high-throughput sequencing.We found some membrane molecules,such as CD58 and CMTM6,which have definite effects on tumor immunity.Therefore,the presence of these genes with definite roles in sg RNA-enriched/depleted candidate genes,in turn,validates the rationality of the screening system and conditions.Additionally,among the genes ranked high in sg RNA abundance variations,we also found multiple genes of DHHC protein family,gene encoding potassium ion channel protein and some CD molecules whose functions have not been fully illuminated.We thus focused on the regulatory roles of ZDHHC9 in tumor immunity.According to statistic analysis of TCGA and GTEx,the expression level of ZDHHC9was significantly upregulated in a variety of digestive system cancers;meanwhile,disease-free survival of colon cancer patients was negatively correlated with the expression of ZDHHC9.ZDHHC9 was found to repress the proliferation of tumor cells in vitro,whereas in vivo,ZDHHC9 promotes tumor growth.In vitro T cell killing assay showed that ZDHHC9 can protect tumor cells from CTL cells.In vivo experiments,IFN-γ~+CD8~+and TNF-α~+CD8~+cells in subcutaneous xenografts were significantly increased in the ZDHHC9 deficient group.Correlation analysis based on TCGA database indicated that ZDHHC9 was negatively correlated with expression of IFN-γ,granzymes and perforin in colon cancer samples.We concluded that ZDHHC9 can facilitate tumor growth via inhibiting T cell-mediated tumor immune response.In addition,we also found that ZDHHC9 can positively regulate the expression of PD-L1 by affecting the activation of the IFN-γinduced JAK/STAT1 signaling pathway,thus modulating the immune response of T cell tumors.Toll-like receptors(TLRs)recognize invading pathogens and various danger,thereby triggering intracellular signaling that gives rise to an inflammatory response.Inflammation is generally considered as a protective response that eliminates the initiating factors of cell damage,removes injurious cells and impaired tissues,and initiates tissue repair.In order to avoid inflammatory deregulation and unfavorable overreaction,the signal transduction of TLR pathway is strictly regulated by a substantial quantity of positive and negative regulators.We probed the regulatory role of TAOK1,a member of the serine/threonine protein kinase family,in TLR-induced inflammatory response,providing a potential target for the treatment of inflammatory diseases and new insight into regulatory mechanism of TLR4 in tumor immunity.We conclude three aspects regarding cancer immunomodulation and innate immunomodulatory mechanisms.We identify the Ste20-like kinases TAOK1 as a positive regulator of TLR4-triggered inflammatory responses in macrophages.TAOK1 constitutively interacts with TRAF6 and TLP2,which selectively enhances LPS-induced activation of ERK1/2 and facilitates the production of pro-inflammatory cytokine such as IL-6,TNF-?and IL12p40.TAOK1deficient mice showed decreased susceptibility to endotoxin shock,with less pro-inflammatory cytokine production than control mice.These finding unravel the important role of TAOK1 as a positive regulator of TLR4-induced inflammatory responses.