Gasdermin E-mediated Target Cell Pyroptosis By CAR T Cells Triggers Cytokine Release Syndrome

Objective:Adoptive T cell transfer(ACT),also known as cell therapy,is to modify the autologous T cells into specific T cells through gene modification that can mediate tumor immunity:recognize tumor antigens and kill tumor cells.ACT therapy has made breakthrough progress in the treatment of hematoma.However,CAR T cell therapy is no exception to have side effects as a drug.The most common side effect is cytokine release syndrome(CRS),which can cause death in severe cases,and severe CRS is the most common cause of death in CAR T cell therapy.Although hindered the application of CAR T cell therapy,so far,there is no well documented markers could predict CRS clinically,and the underlying mechanisms that induce CRS are still unclear.Therefore,it is important and urgent to study the pathogenesis of CRS.Methods:Firstly,constructed CAR T cells targeted human CD19(CD19-CAR)molecules then co-incubated with target primary tumor cells of patients or Raji or NALM-6 that express CD 19 molecules.After co-incubation,took photos to observe tumor cells death,tested ATP cell viability,assessed the release of LDH and the percentage of PI positive cells in tumor cells.Meanwhile,the Western blotting was used to confirm the target cell pyroptosis at the molecular level.Secondly,tumor cells GSDME gene was knockout by using CRISPR/Cas9 technology,point-mutated or restored after knockout to confirm the key role of GSDME in CAR T cells inducing target cell pyroptosis.Furthermore,by inhibiting or silencing caspase-3,perforin(PRF1)or granzyme B(GZMB)to explore the pathway that CAR T cells induced tumor cell pyroptosis.Thirdly,co-incubated mouse CAR T cells targeted human CD 19(hCD19-mCAR T)or HER2(hHER2-mCAR T)with B16 cells overexpressed human CD 19(CD19-B16)or HER2(HER2-B16)molecule.Then compared the amount of mouse perforin(Prf1)/granzyme B(Gzmb)released by CAR T cells with that released by specific T cells or rejection T cells.And further explored the role of Prfl/Gzmb in inducing pyroptosis by adding purified Prfl/Gzmb in vitro.Construction of mCAR T cells containing different costimulatory signals to study the role of costimulatory signals in promoting CAR T cell killing and inducing target cell pyroptosis.Fourthly,isolated and cultured mononuclear macrophages from normal human whole blood and stimulated by the supernatants of CAR T cells co-incubated with wild type(WT)or GSDME knockout targeted tumor cells.Then the inflammatory factor IL-1? and IL-6 in the macrophage supernatant were detected.At the same time,WT,caspase-1 or GSDMD gene knockout and NLRP3 silenced THP1 were treated with the supernatant of CAR T cell treated target cells to detect the changes of IL-1? and IL-6.Moreover,the damage-associated molecule patterns of ATP,HMGB1 and HSP70 in the supernatant of CAR T cell treated target cells were detected.Finally,a model of CAR T cell therapy induced severe CRS was established in mice,then the incidence of severe CRS,mortality related to severe CRS and changes of inflammatory factors were compared after infusion with CAR T cell to mice burdened with WT or GSDME gene knockout tumor cell line.Furthermore,after inhibiting caspase-1,ATP or its receptors or clearing monocyte macrophages in mice,the changes of CRS were detected.Results:1.CAR T cells induces target cell pyroptosis rather than apoptosis.Tumor cells isolated from the blood of patients were co-incubated with CD 19-CAR T cells.The photographs of target cells showed that the tumor cells membrane swelling and dark vesicles blowing on the membrane.Meanwhile,the ATP cell viability decreased,the LDH released and the percentage of PI positive tumor cells increased.The same results were obtained after incubating CD19-CAR T cells with Raji or NALM-6 and HER2-CAR T cells with MCF-7 or SGC-7901.2.GSDME mediates CAR T cells inducing target cell pyroptosis.GSDME instead of GSDMD is widely expressed.CAR T cells interacted with target cells to induce GSDME but not GSDMD or MLKL activation.Then knockout or point-mutated of GSDME,the target cells changed from pyroptosis to apoptosis.Consistently,after recovering GSDME expression,tumor cells could enter pyroptosis again.3.CAR T cells induce target cells pyroptosis through perforin/granzyme B/caspase-3 pathway.CAR T cells interacted with target cells to induce the cleavage of caspase-3.Both inhibition of caspase-3,GZMB or silence of PRF1,GZMB could block CAR T cell-induced tumor cell death.4.The strong affinity of CAR T cells to target cells and the co-stimulatory signals are key factors that induce target cells pyroptosis.Compared with OT-I T cell mediated specific killing and rejection reaction in vitro,CAR T cells induced target cells stronger pyroptosis.Meanwhile,CAR T cells released more Prfl/Gzmb and cleaved more caspase-3 in target cells.Moreover,silencing Prfl/Gzmb blocked the killing effect of T cells.Results from co-cultured T cells with target cells showed that linearly connecting different co-stimulatory signals significantly enhanced CAR T cell killing and induced target cells pyroptosis.5.Damage-associated molecule patterns released from target cell pyroptosis strongly stimulated macrophages to release inflammatory factors.The supernatant of CAR T cells co-incubated with target cells activated monocyte macrophages to release inflammatory factors IL-1? and IL-6,while the specific T cell killing supernatant failed to activate macrophages to release inflammatory factors.Western results showed that the supernatant from CAR T cells co-incubated with GSDME deficient target cells reduced caspase-1 activation and GSDMD cleavage in macrophages.Meanwhile,knockout of caspase-1 or GSDMD in macrophage significantly reduced inflammatory factors release.Furthermore,ATP and HMGB1 in the supernatant of CAR T cells co-incubated with target cells were significantly increased.Degrading ATP or inhibiting ATP receptors on macrophages could significantly inhibit macrophage activation and reduce the release of inflammatory factors.Knockout of HMGB1 also significantly reduced IL-6 elevation.6.Large number of tumor cells enter GSDME-mediated pyroptosis is the inherent cause of CRS induced by CAR T cells therapy.The knockout of GSDME gene in target cells showed that the increased inflammatory factors and body temperature in mice were significantly reduced,and the mortality due to severe CRS was also significantly reduced.Degrading ATP or inhibiting ATP receptors also significantly reduced the incidence and severity of CRS in mice.The same results were obtained after inhibiting caspase-1 or eliminating macrophages in mice.Severe CRS appeared in mice eliminated macrophages after rebuilding WT macrophages,but the reconstruction of macrophages from caspase-1 or GSDMD knockout mice did not show the same phenomenon.Analysis of LDH and ATP in patients' plasma revealed a correlation with the severity of CRS in patients,and the same as the GSDME expression in primary leukemia cells.Conclusion:1.CAR T cells induced targeted cells pyroptosis rather than apoptosis.2.Through releasing much more PRF1/GZMB than specific T cell,CAR T cells activated excessive caspase-3 to cleave enough GSDME to induce targeted cells pyroptosis.3.Lots of content such as ATP and HMGB1 leaked from a mass of pyroptosis cells could activate mononuclear macrophages to release cytokines,which elicited CRS in patients.4.The expression level of GSDME in leukemia cells can be used as a marker to predict the occurrence of severe CRS induced by CAR T treatment.