[Establishment of a cytokine release syndrome associated with chimeric antigen receptor T cell treatment in SCID/Beige mice model].

Objective: To establish a cytokine release syndrome (CRS) mouse model related to CAR-T cell therapy and provide a research model for the clinical phenomena. Methods: CAR-T cells targeting human CD19 molecule were constructed by molecular cloning and lentiviral transfection. Flow cytometry (FACS) was used to detect the transfection efficiency of CAR-T cells. The tumor-killing efficiency of CAR-T cells was detected by ELISA and flow cytometry. The CAR-T cells were injected into the tumor-bearing SCID/Beige mice through tail vein, and divided into phosphate buffered solution (PBS) group, low-burden group (1×10(5) Raji-Luc2 cells) and high-burden group (5×10(5) Raji-Luc2 cells). The tumor treatment effect was detected by animal in vivo imaging. Serum levels of cytokines including human IFN-γ, human IL-2, mouse IL-6, and mouse GM-CSF were measured by ELISA. The health status of the mice was evaluated by pathological examination. Results: The health scores of T cell group and T cell+ OKT-3 group were (1.15±0.08) and (2.90±0.15), respectively, after the injection of human T cell and T cell + OKT-3 antibody through tail vein, and the difference was statistically significant (P<0.001). The serum levels of human IL-2, human IFN-γ, human IL-15, mouse IL-6 and mouse GM-CSF in T cell+ OKT-3 group were (1 064.00±50.14), (1 285.00±193.90), (202.4±18.76), (1 478.00±289.20) and (350.70±42.27) pg/ml, respectively, higher than (22.67±6.36), (23.67±3.71), (44.33±14.45), (147.30±36.20), (138.00±22.74) pg/ml in T cell group (P<0.05). OKT-3 combined with human T cells caused a rapid increase in serum levels of human IL-2, human IFN-γ, mouse IL-6 and mouse GM-CSF, accompanied by an increase in body temperature and weight loss. CD19-targeting CAR-T cells were successfully constructed, and the positive rate of CAR-T cells was >30% detected by flow cytometry. ELISA results showed that in the presence of CD19 antigen, IL-2 and IFN-γ secreted by CAR-T19 cells co-incubated with Raji and Nalm were (561.00±37.07), (680.30±71.27), (369±25.71) and (523.00±26.31) pg/ml, respectively, higher than (55.00±20.53) and (64.00±7.55) pg/ml in the co-incubated with K562 group (P<0.001). Activated CAR-T19 cells were reinjected through the tail vein on the seventh day after tumor formation. Imaging experiments in mice showed that on the thirteenth day after tumor formation, the fluorescence intensities of tumors in the low-burden and high-burden groups were lower than on the seventh day of tumor inoculation, and the fluorescence intensity of tumors in the high-burden group decreased from 144.00±24.69 to 5.02±2.35 (P=0.005). The fluorescence intensity of low burden group decreased from 58.47±9.36 to 3.48±1.67 (P=0.004). The serum levels of T cell activation related cytokines IL-2, IL-15 and IFN-γ increased rapidly, and the secretion of monocyte related cytokines IL-16 and GM-CSF increased, accompanied by the typical characteristics of CRS such as increased body temperature and weight loss at 72 hours after injection of CAR-T19 cells. Conclusions: CAR-T cells targeting CD19 molecule are successfully constructed, and CRS phenomenon is verified in tumor-bearing mice by CAR-T cell re-infusion, providing an animal model for the mechanism of CAR-T treatment-related CRS and CRS prevention strategies.

[Effect of Δ40p53 isoform on enhancing the pro-apoptotic function of p53 in tumor cells].

Objective: To investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53. Methods: The wild-type p53 was ectopically expressed in HCT116-p53(-/-) (endogenous Δ40p53 expression), HCT116-p53(+ /+) (wild-type p53) and H1299 (p53-null) cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide (PI) staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group. Results: HCT116-p53(-/-) cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 could bind to each other. Calcein-AM/PI staining showed that the apoptotic rates of H1299-Control, HCT116-p53(-/-) -Control, H1299+ p53, HCT116-p53(-/-)+ p53, H1299+ oxaliplatin (Oxa), HCT116-p53(-/-)+ Oxa, H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups were (2.50±0.47)%, (2.40±0.32)%, (5.20±0.58)%, (4.10±0.18)%, (22.40±1.73)%, (19.30±1.11)%, (29.90±1.15)% and (39.30±2.26)%, respectively. It was statistically significant between H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups (t=3.721, P=0.0205). Moreover, the apoptotic rates of H1299-Control, H1299+ Δ40p53, H1299+ p53, H1299+ p53+ Δ40p53, H1299+ Oxa, H1299+ Δ40p53+ Oxa, H1299+ p53+ Oxa and H1299+ p53+ Δ40p53+ Oxa groups were (2.60±0.35)%, (2.20±0.17)%, (4.80±0.49)%, (4.90±1.10)%, (20.30±1.10)%, (19.60±1.45)%, (27.90±1.39)%, (35.20±1.43)%, respectively. Furthermore, flow cytometry assay showed that the apoptotic rates of above cells were (2.70±0.32)%, (2.20±0.24)%, (4.60±0.48)%, (3.90±0.67)%, (19.30±1.11)%, (17.70±0.66)%, (28.30±2.76)% and (37.50±1.51)%, respectively. H1299+ p53+ Δ40p53+ Oxa cells showed higher cell apoptosis than H1299+ p53+ Oxa cells (t=2.930, P=0.042). Conclusion: Δ40p53 isoform can bind to full-length p53, and enhance its pro-apoptotic function in tumor cells.