Since macrophages have been implicated as major players in the mechanism of action of Rituximab (Mabthera®) in vivo, we have investigated the factors that modulate their tumour cell killing potential in vitro. Human macrophages expressing CD16, CD32 and CD64, were differentiated from CD14+ peripheral blood monocytes by culture for 5–7 days in presence of M-CSF. Binding of rituximab opsonised target cells was measured after 5 minutes incubation of macrophages with CFSE labelled B-CLL cells and FACS analysis. Phagocytosis was quantified after 2 hours at 37°C by microscopic count of stained slide preparations. ADCC was measured by standard release assays. Rituximab induced specific binding of CD20+ target cells to macrophages and this was followed by phagocytosis, but not ADCC. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels on target cells. The CD16A polymorphism at amino acid 158 (Val/Phe) that affects IgG binding did not significantly modify the efficacy of phagocytosis at different rituximab doses, possibly due to the role of additional Fcγ receptors. Indeed phagocytosis was blocked by excess human immunoglobulins. Since macrophages can be differentiated to M1 type or M2 type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by pro-inflammatory stimuli (IFNγ + LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analysed the effect of these different polarisation programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2–3 fold greater phagocytic capacity compared to GM-CSF induced cells. Furthermore addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF and GM-CSF differentiated macrophages. LPS/IFNγ had little effect. Expression of CD16, CD32A/C and CD64 correlated with the phagocytic capacity of the different polarised populations, suggesting that M-CSF and IL-10 induced maximal phagocytosis through upregulation of several activating FcγRs. Several B lymphoma cell lines were observed to secrete 400–1300 pg/ml IL-10 in vitro and co-culture of human macrophages with lymphoma supernatant increased significantly their phagocytic capacity. These data suggest that IL-10 produced in the lymphoma microenviroment may activate macrophages to become M2 type cells with high phagocytic capacity. Thus usually tumour promoting M2 type macrophages within the lymphoma would become tumour suppressing in presence of rituximab.

Disclosures: Golay:Roche Italia: Honoraria, Research Funding. Introna:Roche Italia: Honoraria.

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