Secretion Of Human SPD1 By Chimeric Antigen Receptor–Modified T Cells Enhances Anti-Tumor Efficacy

Objective:In recent years,CAR T cells targeting CD19 have achieved significant results in the treatment of B-cell lymphoma,but the complex tumor microenvironment leads to tumor immune escape.The PD1/PDL1 signaling pathway plays an important role in this process.Soluble PD1(sPD1)can bind to PDL1,reducing the immunosuppressive effect on CAR T cells.This study aims to explore the anti-tumor effect of CAR T cells secreting sPD1,and provide a new idea for tumor immunotherapy.Methods:1.Lentiviruses were prepared for T cell and tumor cell line transduction.2.In vitro functional experiment:flow cytometry was used to detect the transduction efficiency and the tumor killing efficacy by 2nd CAR T and 2nd-sPD1 CAR T cells.ELISA was used to detect the secretion of sPD1 and the expression of cytokines such as IFN-?,TNF-?and IL-2.3.NALM6-PDL1 mouse model was constructed,2nd CAR T and 2nd-sPD1 CAR T cells were reinjected,and live imaging was used to detect tumor burden in mice.4.Flow cytometry was used to detect the expression of activation markers such as CD25,CD107a,differentiation markers such as CD62L,CD45RA and anti-apoptotic marker molecule Bcl2,etc.,and the anti-tumor mechanism of 2nd-sPD1CAR T cells was analyzed from different perspectives.5.The glycolysis and oxidative phosphorylation levels of CAR T cells were detected by bioenergy analyzer,and the function of 2nd-sPD1 CAR T cells was analyzed from a metabolic point of view.Results:1.2nd-sPD1 CAR T cells were successfully prepared,and the K562,NALM6,786o tumor cell lines expressing CD19 and/or PDL1 were constructed.2.The2nd CAR T and 2nd-sPD1 CAR T cells were co-incubated with tumor cell lines expressing CD19 and/or PDL1,respectively.The results showed that CD19~+PDL1~+tumor cells caused increased secretion of cytokines,such as IFN-?,TNF-?and IL-2,and enhanced anti-tumor ability of 2nd-sPD1 CAR T cells.3.In vivo imaging showed that NALM6-PDL1 mice in the 2nd-sPD1 CAR T group had a lower tumor burden,indicating that the 2nd-sPD1 CAR T cells had a stronger tumor suppressing effect.4.After co-incubation,there is an increase in CD25 and CD107a levels stimulated by the tumor-specific antigen CD19;On days 8 and 15 of cell culture alone,the total proportion of CD45RA~+CD62L~+naive T cells and CD62L~+central memory T cell in the 2nd-sPD1 CAR T group(82.7%,88.8%)was greater than that in the 2nd CAR T group(68.9%,69.4%).The expression level of Bcl2 is also higher than that in the 2nd CAR T group.5.Compared with the control group,the 2nd-sPD1 CAR T cells always maintained a lower glycolysis level regardless of tumor antigen stimulation,while the oxidative phosphorylation level rose after co-incubation with tumor cells.6.RNA-seq results show that compared with the control group,2nd-sPD1 CAR T cells have higher levels of gene expression related to early memory T cells,non-depleted T cells and naive T cells,and lower levels of gene expression related to glycolysis and cell apoptosis.Conclusion:1.2nd-sPD1 CAR T cells only produce positive results for tumor cells expressing CD19 and PDL1,presumably sPD1 blocks the inhibition of CAR T cell function by PDL1 on the surface of tumor cells,thereby enhancing the killing effect on tumor cells.2.2nd-sPD1 CAR T cells had a better tumor control effect in the mouse model inoculated with NALM6-PDL1 tumor cells,indicating that 2nd-sPD1 CAR T can enhance the control of tumor cells in complex conditions in vivo.3.When cultured alone,the proportion of naive T cells and central memory T cells in the 2nd-sPD1 CAR T group is larger,and the Bcl2 level is higher,indicating that the sPD1 secreted by2nd-sPD1 CAR T cells was helpful in maintaining the stemness and resisting apoptosis caused by PDL1.After being stimulated by CD19,CD25 and CD107a expression levels of the 2nd-sPD1 CAR T cells were higher,indicating that sPD1 also increased the pre-activation intensity of CAR T cells.4.In cell energy metabolism analysis,the lower glycolysis level of 2nd-sPD1 CAR T cells and the higher oxidative phosphorylation level after being stimulated by tumor antigens verified the enhanced stemness of the2nd-sPD1 CAR T cells.5.The RNA sequencing results and the former cell experiments results are mutually confirmed.The use of CAR T cells to secrete soluble PD1 provides a new idea for future tumor immunotherapy research.