Abstract: Objective: To examine the anti-tumor mechanism involved when 4-1BBL/CD20and diabody are used in combination. Methods:Human peripheral blood lymphocytes (PBLs) were isolated by Ficoll–Hypaque density-gradient centrifugation. Cell proliferation was assessed by measuring the conversion of the trazolium salt WST- 8. Concentrations of IL-2 were measured by Enzyme-linked immunosorbent assay (ELISA). Transcript expression of perforin and Grb was inves-tigated by reverse transcription - polymerase chain reaction (RT-PCR). Cytotoxicity analysis was performed using calce -in-AM retention assays. Results: The proliferation of PBLs in the group that was treated with4-1BBL/CD20plus the diabody was significantly higher than that of cells treated with either4-1BBL/CD20or diabody alone. Combination treatment with the 4-1BBL/anti-CD20fusion protein and the anti-CD 3/anti-CD 20diabody led to significantly increased T cell cytotoxicity to human B lymphoma cells in vitro. Mechanistic studies revealed that significantly higher levels of granzyme B and perforin were detected in PBLs treated with the diabody plus 4-1BBL/CD20, compared to those levels in PBLs treated with the di -abody alone. The level of IL- 2 expression in PBLs treated with the diabody plus 4 1BBL/CD20 was significantly higher than that in cells treated with either the diabody or 4-1BBL/CD20alone. Conclusion:Diabody plus 4-1BBL/CD20is a more effec -tive combination for lymphocyte activation than diabody alone. The combined administration of 4-1BBL/CD20and diabody augments the antitumor activity of the diabody. Such a reaction may have significance for clinical application in the treat-ment of human CD20-positive B cell malignancies.