Peripheral Clonal Deletion Induces Immune Tolerance By Targeting Programmed Death-1

Objective:Organ transplantation is the most effective treatment for end-stage organ failure,but the side effects of existing immunosuppressants greatly limit the quality of life and survival of recipients.Finding more ideal targets for intervention is key to reducing the side effects of immunosuppressants.The alloreactive T cells are the primary mediators of transplant rejection.Intrathymic clonal clearance of alloreactive T cells can be induced by injecting donor cells into newborn mice,achieving acquired immune tolerance.However,this strategy is difficult to translate to the clinic,and further research on peripheral clonal clearance of alloreactive T cells is needed.In this study,we intend to screen intervention targets against alloreactive T cells and explore whether and how peripheral clonal deletion of alloreactive T cells induces transplant tolerance.Research routes:1)Multiomics analysis screens for ideal intervention targets against alloreactive T cells.2)Verify the effectiveness of peripheral clonal clearance of alloreactive T cells and induction of immune tolerance through this intervention target.3)Develop a depleting antibody against this target and evaluate the effectiveness in inhibiting graft rejection.Research contents and methods:1)The identification and verification of the marker of alloreactive T cells:(1)Screening of characteristic surface marker expressed on the transferred TEa cells with transcriptome sequencing and flow cytometry analysis.(2)Phenotypic detection and multi-omics analysis of the endogenous alloreactive T cells with flow cytometry analysis and single-cell transcriptome combined with TCR sequencing.(3)Functional verification of the marker of alloreactive T cells with MLR and ELISpot assay.2)The effects and mechanisms of PD-1-positive cells on transplant rejection:(1)Mice selectively expressed by DTR following PD-1 were constructed,and cells expressing PD-1 were specifically deleted by DT.(2)The effect of depleting PD-1-positive cells on transplant rejection was explored with murine skin and heart transplantation.(3)The underlying mechanisms were detected by bioluminescence,MLR,ELISpot,TCR sequencing and adoptive transfer model.3)The development of nonblocking anti-PD-1 depleting antibody and the evaluation of its in vivo effects:(1)The effects of murine anti-PD-1 depleting antibody on alloreactive T cells were explored with TEa cells transfer and heart transplantation model.(2)Construct nonblocking human anti-PD-1 depleting antibody by hybridoma technique.(3)The effects on transplant rejection were evaluated in a PD-1 humanized mouse heart transplantation model.Results:1)The identification and verification of the marker of alloreactive T cells:(1)PD-1 was significantly upregulated on the surface of transferred TEa cells and Pdcd1 was highly expressed in endogenous alloreactive T cells.(2)During transplant rejection,the graft-infiltrated T cells were almost all PD-1-positive,and PD-1-positive T cells in recipient spleen were significantly increased.(3)The PD-1-positive T cells in recipient spleen simultaneously expressed activation,proliferation and effector molecules.Moreover,the alloreactive T cells detected by MLR and ELISpot were almost exclusively present in PD-1-positive T cells.2)The effects and mechanisms of PD-1-positive cells on transplant rejection:(1)DT almost completely depleted PD-1-positive cells from local skin grafts and draining lymph nodes.(2)Ablation of PD-1-positive cells by DT significantly prolonged survival of skin and heart grafts,while long-term survival of partial grafts(>100 days)was achieved with thymectomy.(3)Mechanistically,deleting PD-1-positive cells remodelled the TCR repertoire,significantly reduced the number of donor-specific alloreactive T cells with high clonal proliferation in rejection,but the reactive T cells against other antigens were reserved.3)The development of nonblocking anti-PD-1 depleting antibody and the evaluation of its in vivo effects:(1)Anti-mPD-1 depleting antibody selectively eliminated alloreactive T cells.(2)Nonblocking anti-h PD-1 depleting antibody was successfully constructed.(3)Nonblocking anti-h PD-1 depleting antibody significantly reduced the number of alloreactive T cells and prolonged allograft survival in PD-1 humanized mouse heart transplant model.Conclusions:1)PD-1 is a surface hallmark of alloreactive T cells.2)Ablation of PD-1-positive cells promotes the peripheral clonal deletion of the alloreactive T cells and the induction of immune tolerance.3)Nonblocking anti-h PD-1 depleting antibody ameliorates transplant rejection in PD-1 humanized mouse heart transplant model.