An ADCC-Independent Mechanism of Rituximab-Mediated B-NHL Cells Depletion Via Sensitization to Cell Death through the Expression of TRAIL on NK Cells

Abstract 4919

Rituximab, a chimeric anti-CD20 mAb, has being used, alone or in combination with chemotherapy, in the treatment of patients with B-NHL and rheumatoid arthritis. It is also being tested clinically in the treatment of other B cell malignancies. The mechanisms by which the antibody depletes the B cells have been shown to be mediated via ADCC, CDC, and apoptosis. In addition, the antibody also signals the cells and modifies various survival pathways and sensitizes the resistant tumor cells to various apoptotic stimuli (Jazirehi and Bonavida, Oncogene 24:2121, 2005). The role of the host innate cytotoxic cells, such as NK cells, in cooperation with rituximab in the depletion of B-NHL cells has been poorly explored. Studies by us and others have reported that rituximab sensitizes resistant B-NHL tumor cells to both Fas ligand and TRAIL-induced apoptosis (Bonavida, Oncogene 26:3629, 2007; Daniel, D. et al., Blood 110:4037, 2007). Since NK cells express on the surface TRAIL, we hypothesized that rituximab may also sensitize the TRAIL-resistant tumor cells to NK-mediated cytotoxicity. Accordingly, we have examined various TRAIL-resistant B-NHL cell lines and used peripheral blood-derived purified human NK cells. Treatment of various B-NHL cell lines with rituximab sensitized the cells to TRAIL-induced apoptosis. The mechanism of TRAIL-induced cytotoxicity was found to be the result of TRAIL-induced inhibition of NF-κB and downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) as well as inhibition of anti-apoptotic gene products such as Bcl_xl. Treatment of various B-NHL cell lines with rituximab, unlike treatment with control IgG1, resulted in significant cytotoxicity in the presence of purified NK cells. The extent of the cytotoxic activity was a function of the E:T ratios used. We then examined the contribution of TRAIL expressed on the NK cell surface for its role in NK-mediated cytotoxicity of rituximab-pretreated B-NHL cells. We used a neutralizing TRAIL antibody that was added in the reaction mixture and demonstrated that the NK cytotoxic activity was significantly reduced compared to controls. These studies with rituximab were also confirmed with other CD20 mAbs. We are currently examining the sensitization of freshly-derived B-NHL and CLL cells that are treated with rituximab and other anti-CD20 mAbs to NK-mediated cytotoxicity for validation of the findings with cell lines. The present findings suggest that, in vivo, patients who are treated with rituximab may recruit NK and other effector cells to mediate, independently of ADCC, cytotoxicity via the TNF-family ligands (e.g. TNF-α, Fas-L, TRAIL). The studies also suggest that this B cell-depletion mechanism by NK cells may contribute to the mechanism of rituximab- mediated depletion of B-NHL cells in vivo. Noteworthy, the proposed host cytotoxic mechanism may not be functional if the therapeutic treatment consists of the combination of rituximab and immunosuppressive chemotherapeutic drugs that may lead to depletion or inactivation of host cytotoxic cells.