Abstract:

Objective ·To investigate the immunotherapeutic effects of exosomes derived from leukemia cells with high expression of B7 molecules (co-stimulatory molecules CD80 and CD86) in tumor-bearing mice. Methods ·L1210 leukemia cells were transfected with lentiviral vectors containing the CD80 and CD86 genes, and thereby the leukemia cells with high expression of CD80 and CD86 molecules were obtained. Exosomes LEX-8086 with high expression of CD80 and CD86 molecules were then isolated from the culture supernatant. The biological characteristics of LEX-8086 were analyzed by using transmission electron microscopy (TEM) and Western blotting. A DBA/2-leukemia tumor-bearing mouse model was established. The tumor-bearing mice were divided into LEX-8086 group and PBS group. The LEX-8086 group received intravenous injections of 500 μL LEX-8086 (150 μg/mL) via the tail vein 7 d and 12 d after tumor cell inoculation, while the PBS group received injections of equal volumes of PBS at the same time points. Differences in body weight, tumor volume, tumor mass, and survival of the mice between the two groups were observed. Flow cytometry was used to analyze the changes in the immune cells in spleens and lymph nodes of the mice in both groups. Western blotting and real-time fluorescent quantitative PCR (qPCR) were used to detect the expression of markers for M1 and M2 macrophages in the spleens of the mice in both groups. Results ·The exosomes LEX-8086 were isolated from the supernatant of L1210 cells overexpressing CD80 and CD86 molecules. TEM revealed that LEX-8086 exhibited a disc-shaped vesicular structure with a diameter of approximately 100 nm. Western blotting demonstrated the expression of exosome-specific markers. In the animal experiments, compared with the PBS group, the body weight of the LEX-8086 group showed no significant change, while both the tumor volume and the relative tumor mass ratio decreased significantly, and the survival was significantly prolonged (all P<0.05). Flow cytometry results indicated that the proportions of natural killer (NK) cells and CD4⁺ T cells in the lymph nodes and spleens of the LEX-8086 group significantly increased (all P<0.05); as for CD8⁺ T cells, they only increased in the lymph modes (P=0.012). Moreover, flow cytometry results showed that the proportion of M1 macrophages in the spleens of mice in the LEX-8086 group was significantly elevated (P=0.003), while the proportion of M2 macrophages remained unchanged. Western blotting and qPCR also revealed that the mRNA and protein expression levels of M1 macrophage markers interleukin-6 and tumor necrosis factor-α in the spleens of mice in the LEX-8086 group increased significantly (all P<0.05). Conclusion ·Exosomes derived from leukemia cells with high expression of co-stimulatory molecules CD80 and CD86 can induce a therapeutic anti-leukemic effect, which may be achieved by increasing the proportions of NK cells, M1 macrophages, CD4⁺ T cells, and CD8⁺ T cells.

Key words: leukemia, exosome, costimulatory molecule, gene modification, immunotherapy