Abstract: Hu Bin, Ni Nana, Lyu Yalin, Chen Hao, Liu Yi, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （the current affiliation of the first author was Department of Dermatology, Wuhan No. 1 Hospital, Wuhan 430222, China） Corresponding authors: Sun Jianfang, Email: Sunjf57@163.com; Liu Yi, Email: dr.liuyi@gmail.com 【Abstract】 Objective To evaluate in vitro effects of specific small interfering RNA （siRNA）-silencing of the casitas B-lineage lymphoma b （Cbl-b） gene on immunocompetence of primary murine lymphocytes. Methods Spleens were resected from C57BL/6 mice, and splenic lymphocytes were sterily isolated and cultured in vitro. These lymphocytes were divided into 3 groups: silence group transfected with a Cbl-b-specific siRNA using the EntransterTM-R 4000 reagent, negative control group transfected with a negative control siRNA using the EntransterTM-R4000 reagent, blank control group receiving no treatment. After additional culture for 72 hours, ELISA was performed to measure levels of interferon γ （IFN-γ） and tumor necrosis factor α （TNF-α） in culture supernatants of lymphocytes. In addition, the Cbl-b gene-silenced lymphocytes were co-cultured with B16F10 melanoma cells to evaluate their immunocytotoxic effects on melanoma cells. Results Splenic lymphocytes were successfully isolated from C57BL/6 mice and cultured in vitro, and the Cbl-b-specific siRNA was also successfully transfected into the primary murine lymphocytes and effectively down-regulated the expression of Cbl-b gene in them. Compared with the negative control group and blank control group, the silence group showed significantly increased supernatant levels of IFN-γ and TNF-α （all P < 0.05）. The immunocytotoxic effect of lymphocytes on melanoma cells was significantly stronger in the silence group than in the negative control group. Conclusions Cbl-b gene silencing can promote secretion of IFN-γ and TNF-α by murine lymphocytes, and enhance their immunocytotoxic effects on B16F10 melanoma cells in vitro.