Co-expression Of A PD-L1-specific Chimeric Switch Receptor Augments The Efficacy And Persistence Of CAR T Cells Via The CD70-CD27 Axis

【Background】 Programmed cell death receptor-1(PD-1)/ Programmed cell death ligand-1(PD-L1)axis is considered to be one of the important mechanisms leading to immune escape of carcinoma cells during T cell killing.PD-L1 expression is up-regulated in multiple solid tumors,and PD-L1 on carcinoma cells interacts with PD-1 on T cells to mediate immunosuppressive function,ultimately leading to T cell depletion.PD-L1 is also expressed on T cells,and several studies have shown that expression of PD-L1-specific chimeric switch receptors(CSRs)on T cells can improve the anti-tumor effect of CAT T cells.However,the mechanism by which trans-binding of CSRs targeting PD-L1 activates PD-L1 on the surface of CAR-T cells,and the effect of its activation on the anti-tumor activity of CAR-T cells,is currently unknown.The CD70-CD27 axis belongs to the tumor necrosis factor(TNF)superfamily.CD70 is abnormally expressed in carcinoma cells and promotes immune escape and tumor progression through the tumor microenvironment(TME).CD70 is temporarily up-regulated on activated T cells,while its receptor,CD27,is physiologically expressed on T cells.CD27/CD70 co-stimulated T cell proliferation and survival,and promoted the differentiation of initial T cells into antigen-specific cytotoxic and memory T cells.The CD70-CD27 axis also promotes Th1 cell differentiation.Th1 cells can produce abundant proinflammatory factors.Instead,Th2 cells secrete anti-inflammatory cytokines.【Purpose】 We aim to find the possible mechanism in trans-recognition between CSRs and PD-L1 in activated CAR T cells on anti-tumor activity of CAR T cells.【Methods】 We designed a CSR specific for PD-L1(CARP),containing the trans-membrane and cytoplasmic signaling domains of CD28 but not the CD3 ζchain.We also constructed a CARMz(MSLN).Then,we tested the cytotoxicity of co-cultured CARP and CARMz with target cells in vitro and in vivo.The secretion of cytokines in the supernatant was detected by ELISA.The contact between CARP T cells and CARMz T cells was photographed using confocal microscopy.The effects of CARP T cells on CARMz T cells were demonstrated by RNA sequencing and single cell sequencing.【Results】 We show that CARP T cells enhance the anti-tumor activity of anti-mesothelin CAR(CARMz)T cells in vitro and in vivo.In addition,confocal microscopy indicates that PD-L1 molecules on CARMz T cells accumulate at cell-cell contacts with CARP T cells.Using single-cell RNA sequencing analysis,we reveal that CARP T cells promote CARMz T cells differentiation into central memory-like T cells,up regulate genes related to Th1 cells,and down regulate Th2-associated cytokines through the CD70-CD27 axis.Moreover,these effects are not restricted to PD-L1,as CAR19 T cells expressing anti-CD19 CSR exhibit similar effects on anti-PSCA CAR T cells with truncated CD19 expression.【Conclusion】 These findings suggest that target trans-recognition by CSRs on CAR T cells may improve the efficacy and persistence of CAR T cells via the CD70-CD27 axis.