Silencing the expression of Cbl-b enhances the immune activation of T lymphocytes against RM-1 prostate cancer cells in vitro

BackgroundThe 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.MethodsT 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.ResultsWe 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.ConclusionSilencing 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.