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Establishment and observation of a mouse model of IL-12-CAR-T cell-induced cytokine release syndrome
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Abstract:
Objective：To discuss the method of establishing a cytokine release syndrome (CRS) model by constructing murineoriented chimeric antigen receptor-modified T (CAR-T) cells expressing IL-12 and infusing them back into mice via the tail vein.Methods: CAR molecules targeting murine-derived CD19 were constructed, in which the retroviral vectors were packaged, and then the constructed molecules were used to infect mouse T cells to prepare mCD19-CAR-T and mCD19/IL-12-CAR-T cells. The anti-tumor activity of mCD19/IL-12-CAR-T cells was measured by constructing pancreatic cancer Panc02-CD19 cell transplanted tumor model in mice, and the levels of IL-12 and IFN-γ secreted by two CAR-T cells were detected by ELISA; CRS model was constructed by infusing mCD19/IL-12-CAR-T cells back into the tail vein of the mice. The levels of IL-6, MCP-1, IL-1, IL-10, TNF- α, IFN- γ, and other cytokines in the serum of mice were detected by flow cytometry, and the histopathological changes of liver, spleen, lung, and kidney of the mice were observed by H-E staining. Results: After culture expansion, mCD19/IL-12-CAR-T cells could effectively secrete IL-12,and the CAR positive rate reached (56.9±5.4)%; the modified T cells could efficiently secrete IFN-γ no matter co-cultured with nontargeted Panc02 cells or targeted Panc02-CD19 cells. A mouse pancreatic cancer Panc02-CD19 cell transplanted tumor model was successfully constructed, and the tail vein infusion of 1×106 mCD19/IL-12-CAR-T cells significantly inhibited the growth of the transplanted tumor, but failed to induce severe CRS; after the infusion of 2×106 mCD19/IL-12-CAR-T cells, a series of typical CRS manifestations such as reduced body mass, elevated serum inflammatory factor levels, tissue damage and even death were observed in the mice. Conclusion: The IL-12-CAR-T cell-induced CRS model in mice was successfully constructed, and it is stable and reproducible with wide application prospects.
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