Abstract: Objective: To explore the antitumor effect and mechanism of spleen lymphocytes in mice with B16 melanoma, and provide new ideas for adoptive immunotherapy. Methods: Spleen lymphocytes of normal mice were extracted from B16 cells (NL) and splenic lymphocytes of tumorbearing mice (ML) were co-cultured according to 1:20, and ConA was added to stimulate cells for 24h. CCK8 was used to detect cell viability of all groups, and Calcein/PI fluorescent staining was performed simultaneously. The proportion of CD4+ and CD8+ in NL/ML was detected by flow cytometry. ConA stimulated NL/ML for 24h, the expression levels of PD-1,Granase B and perforin were detected by Western blot, and the expression levels of TNF-α and IFN-γ were detected by ELISA. Results: CCK8 and Calcein/PI fluorescence staining indicated that ML had more obvious tumor suppressive effect. Compared with NL, the level of CD8+ expression proportion was higher in ML, and PD-1 expression was lower (P<0.05). After ConA stimulation, the expression of Granase B, perforin, TNF-α, IFN-γ in ML was higher than that in NL (P<0.05). Conclusion: Compared with normal mice, spleen lymphocytes of B16 cells have more obvious antitumor effect on melanoma.

Key words: melanoma, Tumor-bearing mice, PD-1, adoptive cell therapy