title: Silencing the expression of Cbl-b enhances the immune activation of T lymphocytes against RM-1 prostate cancer cells in vitro.
abstract: BACKGROUND
  The ubiquitin ligase Cbl-b potently modulates T lymphocyte immune responses and is critical in modulating tumor-induced immunosuppression. The influence of Cbl-b in modulating T lymphocyte activity against prostate cancer remains ill defined. We have determined the effects of silencing Cbl-b expression in T lymphocytes and their subsequent cytotoxic activity against prostate cancer cells.
  
  
  METHODS
  T lymphocytes were isolated from the spleens of C57BL/6 mice. Lipofectamine-directed transfection of T lymphocytes with specific small interfering RNA (siRNA) silenced Cbl-b expression, which was confirmed by Western immunoblotting. The siRNA species were chosen that promoted the greatest transfection efficiency and dampened Cbl-b expression in T lymphocytes. The expression of CD69, CD25, and CD71 by the transfected T lymphocytes was determined by flow cytometry. T lymphocyte proliferation was assessed by CCK-8 assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-β. The objective was to compare the cytotoxic activity of transfected T lymphocytes and nontransfected (i.e., negative control) T lymphocytes against the murine prostate cancer cell line target RM-1 in vitro.
  
  
  RESULTS
  We selected a specific siRNA that decreased T lymphocyte Cbl-b expression to 15%. The siRNA-transfected T lymphocytes showed higher proliferation; higher CD69, CD25, and CD71 expression (p < 0.001); and higher IL-2, IFN-γ, and TNF-β secretion (p < 0.05), compared to the nontransfected cells. Transfected T lymphocytes were also more potent at killing RM-1 prostate cancer cells, compared to the negative control in vitro.
  
  
  CONCLUSIONS
  Silencing Cbl-b significantly enhanced T lymphocyte function and T lymphocyte cytotoxicity activity against a model prostate cancer cell line in vitro. This study suggests a potentially novel immunotherapeutic strategy against prostate cancer.
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Shu-Kui
authors.initials: SK
authors.lastname: Zhou
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Chen
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Zheng-Duo
authors.initials: ZD
authors.lastname: Shi
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Shun-Ping
authors.initials: SP
authors.lastname: Wang
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China.
authors.forename: Xiao-Fei
authors.initials: XF
authors.lastname: Wen
authors.affiliation: Department of Urology, East Hospital, Tongji University, Shanghai, China. Electronic address: wangyuemin@hotmail.com.
authors.forename: Yue-Min
authors.initials: YM
authors.lastname: Wang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D037181:Lectins, C-Type / Q000032:analysis; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011471:Prostatic Neoplasms / Q000276:immunology* / Q000473:pathology; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D011990:Receptors, Transferrin / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology*; D000099192:CD69 Antigens
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D016207:Cytokines; D037181:Lectins, C-Type; D050721:Proto-Oncogene Proteins c-cbl; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D000099192:CD69 Antigens; C068126:CD71 antigen; C118387:Cblb protein, mouse
keywords: Cbl E3 gene silencing; T lymphocytes; adoptive immunotherapy; prostate cancer; ubiquitin protein ligase