Study On The Mechanism Of Human-derived CD19 CAR-T Cell Therapy In Rescuing Relapsed/Refractory Diffuse Large B-cell Lymphoma Unresponsive To Mouse-derived CD19 CAR-T Cell Therapy

Objective: Diffuse large B-cell lymphoma(DLBCL)is a highly invasive form of lymphoma.Despite the availability of various standardized treatments,including chemotherapy,radiotherapy,and immunotherapy,a significant proportion of patients progress to relapse or refractory stages.In recent years,CAR-T cell therapy,or chimeric antigen receptor T-cell therapy,has been highly anticipated due to its personalized treatment plans and significant efficacy against certain malignant tumors.However,in the treatment of relapsed/refractory DLBCL with murine CD19 CAR-T cells,some patients still cannot be completely cured.Humanized CAR-T cells,with their characteristics closer to the natural immune environment of the human body,may provide new options for the treatment of such difficult-to-treat diseases.Therefore,this study explores the role and mechanism of human CD19 CAR-T cells in rescuing relapsed/refractory DLBCL patients who do not respond to murine CD19CAR-T cell therapy.Methods:1.The first part: Comparison of the immune phenotype and in vitro cytotoxic ability of human-derived CD19 CAR-T cells and mouse-derived CD19 CAR-T cellsThis includes the following methods: constructing lentiviral vectors and preparing lentiviruses,preparing peripheral blood mononuclear cells,isolating and activating T cells,preparing Senl＿H19 cells,detecting the transfection efficiency of CAR-T,multicolor flow cytometry,and cytotoxicity experiments.2.The second part: Experimental study on the cytotoxic ability of CAR-T cells regulated by PD-L1 secretion from Notch-1 signaling pathway activated macrophages in diffuse large B-cell lymphomaThis includes the following methods: constructing myeloid-specific Notch1 knockout mice,extracting primary bone marrow cells from mice,differentiating mouse primary bone marrow cells,co-culturing BMDM and mouse leukemia lymphoma A20 cells,separating exosomes,Western blotting,co-culturing BMDM,mouse leukemia lymphoma A20 cells and mousederived CAR-T cells,detecting IFN-γ and CD4 in CAR-T cells by flow cytometry,CCK8 cell viability test,clonal proliferation experiment,and detecting CAR-T cell apoptosis by flow cytometry.3.The third part: Animal and clinical research on the treatment of relapsed/refractory diffuse large B-cell lymphoma unresponsive to mousederived CD19 CAR-T cell therapy using human-derived CD19 CAR-T cells.Four patients with R/R DLBCL who were treated in our Hematology Department from August 2018 to March 2023 were selected for the study.These four patients had all previously received mouse-derived CAR-T cell therapy,but all experienced disease progression within a short time(1 to 3months)and subsequently received human-derived CD19 CAR-T cell therapy.q PCR was used to monitor the proliferation of peripheral blood CD19 CAR-T cells,and the treatment effects on the four patients were observed and followed up.Lymphoma models were established in immunodeficient mice,which were then treated with either solvent(normal saline+20% human serum albumin),blank T cells,human-derived CAR-T cells,or mouse-derived CAR-T cells.In vivo imaging technology was used to evaluate the effects of mouse-derived and human-derived CAR-T cells on tumors in mice.Results:1.Comparison of the immune phenotype and in vitro cytotoxicity of human-derived CD19 CAR-T cells and mouse-derived CD19 CAR-T cells1)There is no difference in the proliferative capacity of human-derived CD19 and mouse-derived CD19 CAR-T cells.2)There is no difference in the transfection efficiency of human-derived CD19 and mouse-derived CD19 CAR-T cells.3)There is no difference in the memory phenotype of human-derived CD19 and mouse-derived CD19 CAR-T cells.4)There is no difference in the cytotoxicity of human-derived CD19CAR-T and mouse-derived m CD19 CAR-T cells against tumor cell lines.This section confirms in vitro that CD19 CAR-T cells of different origins have similar characteristics and cytotoxic effects on tumor cells.2.Experimental study on the regulation of CAR-T cell cytotoxicity by PD-L1 secretion from the Notch-1 signaling pathway activated by macrophages in diffuse large B-cell lymphoma1)Conditional knockout mice for Notch-1 show a high knockout efficiency for Notch-1.These mice can be used for subsequent experimental verification.The expression of Notch-1 in the bone marrow mononuclear macrophages of the conditional knockout mice for Notch-1 is continuously suppressed.Co-cultivation with lymphoma cells and the use of the downs-tream IRE1/XBP1 s activator IXA4 cannot change the expression of Notch-1.2)Knockout of Notch-1 does not change the expression levels of t-STAT3,t-STAT6,p-STAT3,and p-STAT6.Lymphoma cells do not change the protein content of t-STAT3 and t-STAT6,but can increase the expression levels of p-STAT3 and p-STAT6.IXA4 does not change the expression levels of t-STAT3 and t-STAT6,but it can relieve the inhibitory effect of Notch-1knockout on the expression of p-STAT3 and p-STAT6.3)Knockout of Notch-1 does not change the expression levels of p-PERK,p-IRE1α,and ATF6.It does not influence the downstream endoplasmic reticulum stress signaling pathway.Lymphoma cells enhance the expression levels of p-PERK,p-IRE1α,and ATF6.IXA4 can relieve the inhibitory effect of Notch-1 knockout on p-IRE1α expression.4)Knockout of Notch-1 does not change the expression levels of IL-4and IL-6.Lymphoma cells can significantly promote the expression of IL-4and IL-6.IXA4 can relieve the inhibitory effect of Notch-1 knockout on the expression of IL-4 and IL-6.5)Knockout of Notch-1 does not change the expression levels of p-AKT,t-AKT,and the expression levels of CD9,CD63,and PD-L1 in exosomes.Lymphoma cells significantly promote the expression of the aforementioned proteins.IXA4 can relieve the inhibitory effect of Notch-1knockout on the expression of these proteins.6)Knockout of Notch-1 weakens the effects of PD-L1 on the cytotoxic activity,proliferative capacity,and apoptosis of CAR-T cells.PD-L1 antibodies can significantly restore the cytotoxic activity,proliferative capacity,and apoptosis of CAR-T cells.3.Animal and clinical research on the treatment of relapsed/refractory diffuse large B-cell lymphoma unresponsive to mouse-derived CD19 CAR-T cell therapy using human-derived CD19 CAR-T cells1)Human-derived CD19 CAR-T cells are effective in treating relapsed/refractory diffuse large B-cell lymphoma that does not respond to treatment with mouse-derived CD19 CAR-T cells.2)Mouse-derived CAR-T cells exhibit good tumor cell killing effects in mice.3)This section confirms that homologous CD19 CAR-T cells have better therapeutic effects.In clinical practice,human-derived CD19 CAR-T cells have more advantages,while in animal experiments,mouse-derived CD19CAR-T cells perform better.This section confirms that homologous CD19 CAR-T cells have better therapeutic effects.In clinical settings,human CD19 CAR-T cells have an advantage.However,in animal experiments,mouse CD19 CAR-T cells have better effects.Conclusions:1.There is no significant difference in the memory phenotype and in vitro cytotoxicity towards target cells between human-derived CD19 CAR-T cells and mouse-derived CD19 CAR-T cells.2.Diffuse large B-cell lymphoma cells can activate the Notch-1 signaling pathway in macrophages and affect the release of PD-L1 exosomes,thereby inhibiting the cytotoxicity of CAR-T cells.3.Homologous CD19 CAR-T cells have better therapeutic effects.4.The use of human-derived CD19 CAR-T cells in the treatment of relapsed/refractory diffuse large B-cell lymphoma patients unresponsive to mouse-derived CD19 CAR-T cell therapy has significant therapeutic effects and is worth further clinical application.