The Immune Function Changes Of SHP-1-knockout T Cells And Its Effect On The Treatment Outcome Of Hepatocellular Carcinoma

Background and Purpose:Hepatocellular carcinoma(HCC)is a common malignant tumor.Immunotherapy based on immune checkpoint blockade can improve survival in HCC patients compared to sorafenib.SHP-1 is a key intracellular inhibitory molecule in T cell signaling,and blocking its signaling can affect T cell function in multiple ways.Studies in cell lines and mouse models have shown that blocking SHP-1significantly enhances T cell immune killing function.However,phase I clinical trials using anti-SHP-1 antibodies have not yielded promising results,possibly due to difficulties in penetrating the cell membrane.In this study,we utilized CRISPR/Cas9 editing technology to knock out SHP-1 in T cells and examined its anti-tumor function and mechanism in both in vivo and in vitro liver cancer models.Materials and Methods:CD8~+T cells were edited using CRISPR/Cas9technology to knock out SHP-1.The knockout efficiency was confirmed through Sanger sequencing,while the expression levels of SHP-1 and its downstream molecules were assessed using Western blot to validate the success of the knockout.Flow cytometry was utilized to analyze the changes in immune subsets between the SHP-1 knockout group(KO)and non-knockout group(wild type,WT)before and after CD3/CD28stimulation.In vitro killing assays were conducted to compare the killing function of the KO and WT groups.Bulk RNA-seq was employed to investigate the pathways altered in T cells following SHP-1 gene knockout.To establish PDX models,tumor tissues were obtained from liver cancer patients.T cell aggregation was verified through local injection of T cells into the tumor and T cells injected through the tail vein.The phenotype and functional changes of tumor-infiltrating T cells were examined in the KO group,WT group,and medium group in PDX models with and without immune system reconstruction.Bulk RNA-seq was used to analyze the pathways altered in the knockout group,while protein interaction analysis was utilized to identify the effective targets of combined cell therapy.Results:In vitro experiments revealed that CD8~+T cells in the SHP-1knockout(KO)group displayed lower TIM3 expression compared to the wild-type(WT)group(P=0.0014).Conversely,CD69,an early T cell activation marker,showed a significant increase in expression after stimulation in the KO group(P=0.0019),while no significant change was observed in the WT group.Additionally,effector memory T cells(TEM)increased significantly in the KO group after stimulation(P=0.0322),with TEM in the KO group surpassing that of the WT group(P=0.0387).T cell proliferation was unaffected by CRISPR/Cas9 editing,while the secretion of IFN-γ(P=0.0301),Granzyme B(P=0.0148)and Perforin(P=0.0454)by CD8~+T cells was higher in the KO group than in the WT group.In the killing assay,the KO group showed superior efficacy against Hep3B(P=0.0146),Huh7(P=0.0047),and Hep G2(P=0.049)compared to the WT group.Furthermore,bulk RNA-seq analysis indicated that the KO group enhanced pathways related to cell-extracellular matrix adhesion.Using patient-derived xenograft(PDX)models,we observed that intratumoral injection of SHP-1-KO-T cells exhibited the strongest tumor-killing effect(with P values below 0.05)in non-immune regeneration-PDX and humanized PDX models.TEM was the primary T cell subpopulation in the tumor,and there was a significant increase in IFN-γsecretion(with P values below 0.01).RNA-seq sequencing in non-immune refactor-PDX and immune refactor-PDX models demonstrated that intratumoral injection of SHP-1-KO-T cells substantially reduced oxidative phosphorylation in the tumor while increasing lipid metabolism.Moreover,protein interaction analysis identified HMGCR as the gene with the largest weight in the pathway related to lipid metabolism.In the immune recombination-PDX model,the combination of SHP-1-KO T cells and HMGCR specific inhibitor(simvastatin)was significantly more effective than either treatment alone(P=0.0052 for the combination,P=0.0043for simvastatin alone).Conclusion:Our study highlights the anti-tumor effect of SHP-1knockout in T cells by CRISPR/Cas9 in vitro,in PDX models of HCC,and in humanized PDX models.The addition of simvastatin further enhanced this effect.