Within Peripheral Blood Mononuclear Cells, Antibody-Dependent Cellular Cytotoxicity of Rituximab-Opsonized Daudi cells Is Promoted by NK Cells and Inhibited by Monocytes due to Shaving

Abstract

Treatment of chronic lymphocytic leukemia patients with anti-CD20 mAb rituximab (RTX) leads to substantial CD20 loss on circulating malignant B cells soon after completion of the RTX infusion. This CD20 loss, which we term shaving, can compromise the therapeutic efficacy of RTX, and in vitro models reveal that shaving is mediated by effector cells which express FcγRI. THP-1 monocytes and PBMC promote shaving, but PBMC also kill antibody-opsonized cells by antibody-dependent cellular cytotoxicity (ADCC), a reaction generally considered to be due to NK cells. We hypothesized that within PBMC, monocytes and NK cells would have substantially different and competing activities with respect ADCC or shaving, thereby either enhancing or inhibiting the therapeutic action of RTX. We measured ADCC and RTX removal from RTX-opsonized Daudi cells promoted by PBMC, or mediated by NK cells and monocytes. NK cells take up RTX and CD20 from RTX-opsonized B cells, and mediate ADCC. PBMC depleted of NK cells show little ADCC activity, whereas PBMC depleted of monocytes have greater ADCC than the PBMC. Pre-treatment of RTX-opsonized B cells with THP-1 cells or monocytes suppresses NK cell-mediated ADCC, and blockade of FcγRI on monocytes or THP-1 cells abrogates their ability to suppress ADCC. Our results indicate NK cells are the principal cells in PBMC that kill RTX-opsonized B cells, and that monocytes can suppress ADCC by promoting shaving. These results suggest that RTX-based immunotherapy of cancer may be enhanced based on paradigms which include infusion of compatible NK cells and inhibition of monocyte shaving activity.

The Direction of Plasma Membrane Exchange between Lymphocytes and Accessory Cells by Trogocytosis Is Influenced by the Nature of the Accessory Cell

“…This observation, which can only be due to shaving of the RTX/CD20 complexes and cannot be due to phagocytosis, suggests that the small regions of fluorescence within the J774 macrophages are likely caused by internalized transferred fragments of plasma membrane via shaving/trogocytosis and not by the downstream products of a phagocytic reaction. In addition, the demonstration of trogocytosis using the Al488/ PKH26 dyes in these experiments is in good agreement with our previous observations, which documented trogocytosis by THP-1 cells and human monocytes (26,39).…”

“…For example, trogocytosis mediated by FcgR + cells appears to be critically involved in antigenic modulation (i.e., the loss of mAb-targeted Ag at the surface of cells) (23,25,26). Indeed, FcgR + monocyte/macrophages remove CD20-RTX complexes associated with other membrane components from the surface of leukemia cells in vitro, and it is likely that this reaction contributes to the escape of leukemia cells that have lost CD20 in vivo as a consequence of RTX treatment (24,26,27,39,40). In other studies, membrane fragments were shown to be captured by the mAb-targeted cells, making the direction of membrane movement somewhat uncertain (29).…”

Immune recovery after fludarabine–cyclophosphamide–rituximab treatment in B-chronic lymphocytic leukemia: implication for maintenance immunotherapy

“…15 PBLs derived from B-CLL patients have much lower cellular cytotoxic capacity, compared with those from healthy donors. As NK cells support most, if not all, PBMC natural cytotoxicity, 16 this observation suggests that, before treatment, NK basal function is profoundly altered in B-CLL. Decrease in NK-mediated B-CLL lysis has been largely described.…”

Fractionated subcutaneous rituximab is well-tolerated and preserves CD20 expression on tumor cells in patients with chronic lymphocytic leukemia

“…There was no evidence of C3dg deposition on circulating cells promoted by subcutaneous RTX; it is, therefore, likely that malignant cells were cleared based on recognition mechanism promoted by Fcγ receptors on effector cells, such as NK cell-meditated ADCC and phagocytosis by tissue macrophages. 9,10 Compared to requirements for CDC, far less IgG needs to be bound to target cells to promote these later reactions. 11,12 Interestingly, a recent study reported that complement deposition on target cells can inhibit interaction between RTX and NK-cell CD16, thereby antagonizing ADCC.…”