CD19 CAR-T细胞培养过程中PD-1蛋白、mRNA水平及细胞杀伤活性变化

OBJECTIVE: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. METHODS: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. RESULTS: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05). ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05). ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001). There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05). ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05). There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05). ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001). The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05). CONCLUSION: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.