The Role And Mechanism Of Coinhibitory Molecule TIGIT In Preventing Solid Organ Transplant Rejection

Objective:Transplantation is a curative,life-saving therapy for end-stage organ failure.However,the incidence of significant morbidity and graft loss due to immunosuppression-induced toxicities remain unacceptably high.The CD28costimulation blocker belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitor-based immunosuppression,but is associated with an increased rate of acute rejection.In recent immunophenotypic studies comparing pre-transplant T cell phenotypes of patients who go on to reject vs.those that remain stable on belatacept,three potential“risky”memory T cell subsets have been identified to potentially underlie belatacept-resistant rejection:CD4⁺CD28⁺T_EM,CD8⁺CD28^nulland CD4⁺CD57⁺PD1^-subsets.Meanwhile,Th17 cell subset has also been conceived as a potential belatacept-resistant rejection mediator.The similarities and differences,as well as potential overlap,between these memory T cell subsets has not been investigated.Moreover,identification of molecules and pathways that regulate their activation and functionality in the setting of CD28 blockade remains an important goal for immunotherapy in clinical transplantation.The novel coinhibitory molecule TIGIT is currently being targeted therapeutically in several ongoing clinical trials in cancer.However,the role of TIGIT signaling in regulating alloreactive immune responses,especially these risky memory T cell subsets implicated in belatacept-resistant rejection in the context of solid organ transplantation has not been fully elucidated.Materials and methods:1.Human study approvalPBMCs were collected from healthy volunteers and renal transplant patients treated with belatacept following protocols approved by the Emory University Institutional Review Board after informed consent was obtained.2.In vitro stimulation assay（1）Polyclonal stimulation assayFresh PBMCs were isolated and incubated in 24 well flat-bottomed plates in culture medium（R10）.Cells were unstimulated or stimulated with anti-CD3/CD28 Dynabeads（beads to cell ratio 1:1）or 3μg/m L plate-bound functional grade anti-CD3 for 3 d at37°C in a 5%CO₂,humidified atmosphere.Some cultures were treated with 10μg/m L agonisticαTIGIT or mouse Ig G2a isotype control in the presence of belatacept or not.（2）Alloreactive stimulation assayOne-way mixed lymphocyte reactions（MLRs）were performed using human PBMCs from stimulator-responder pairs.Irradiated stimulator PBMCs（8×10⁵）were labeled with Cell Trace Violet dye and then cultured with responder PBMCs（4×10⁵）in culture medium R10 with 10%plasma from the responders for 6 d at 37°C in a 5%CO₂,humidified atmosphere.Some cultures were treated with agonisticαTIGIT or isotype control in the presence of belatacept or not.3.Preclinical murine skin transplantation modelSac donor mice to use for OVA-expressing skin grafts.Take ears and tails and place in PBS on ice.Wipe down recipient mice with gauze wetted with 70%ethanol.Cut holes in skin.Full-thickness tail and ear skin grafts were then sutured as 1 cm²grafts and transplanted onto the dorsal thorax of recipient mice.Wrap in bandaid and transfer mice to clean cages on heating pads.Skin grafts were monitored and scored daily.4.Methods（1）Single cell suspension preparation;（2）Cell surface staining;（3）Intracellular cytokine staining（ICCS）;（4）CD4⁺T_convcell sorting（MACS sorting）;（5）CD8⁺T cell sorting;（6）Cell thawing and recovery;（7）Fgl2 measurement in cell culture supernatant（ELISA）;（8）Cell specific stainingResults:1.CD4⁺CD28⁺T_EMand CD4⁺CD57⁺PD1^-subsets exhibited similar proliferation potential（Ki-67⁺）,pro-inflammatory cytokine production（TNF⁺,IL-2⁺）,and T cell activation（CD69⁺）upon polyclonal stimulation.2.CD4⁺CD28⁺T_EMsubset displayed more active proliferation potential（Ki-67⁺）,stronger effector function（TNF⁺,IFN-γ⁺,IL-17⁺）and much fewer apoptotic cells（Caspase3/7⁺7-AAD⁺）upon alloreactive antigen stimulation.3.CD4⁺CD28⁺T_EMand CD4⁺CD57⁺PD1^-subsets had minimal overlapping repertoires.4.Coinhibitory molecule TIGIT was expressed on all the risky memory T cell subsets in vivo and in vitro in the presence of belatacept and might therefore be a potential target in these populations.5.TIGIT expression significantly increased following skin graft transplantation,while markedly decreased in the setting of belatacept.6.The skin graft survival rate was higher in recipient mice treated with belatacept plus TIGIT agonist relative to those treated with belatacept alone following skin transplantation.7.TIGIT agonism significantly increased apoptotic cell death（Caspase3/7⁺7-AAD⁺）of all the risky memory T cell subsets.8.TIGIT signaling markedly blocked Th17 cell cytokine production,transcription and differentiation.9.TIGIT signaling markedly decreased IL-17⁺T_regpopulation.10.TIGIT did not impact sorted risky memory T cell or Th17 cell immune responses.Conclusion:1.CD4⁺CD28⁺T_EMand CD4⁺CD57⁺PD1^-subsets are phenotypically and functionally similar,and perform stronger effector function relative to CD8⁺CD28^nullsubset.2.CD4⁺CD28⁺T_EMsubset performs stronger effector function relative to other subsets in alloreactive immune response and may therefore be the possible risky factor in mediating belatacept-resistant rejection.3.CD4⁺CD28⁺T_EMand CD4⁺CD57⁺PD1^-subsets are two distinct subsets,and perform their effector function individually in alloreactive immune response.4.Belatacept plus TIGIT agonist significantly improve allo-graft survival.5.TIGIT regulates risky memory T cell response by inducing apoptotic cell death.6.TIGIT modulates Th17 cell response.7.TIGIT controls T_reginstability induced by belatacept and lulizumab.8.TIGIT regulates risky memory T cells and Th17 cells in a cell-extrinsic effect.