Tocilizumab (Anti-IL-6R) Suppressed TNFα Production by Human Monocytes in an In Vitro Model of Anti-HLA Antibody-Induced Antibody-Dependent Cellular Cytotoxicity

BACKGROUND We previously demonstrated that natural killer (NK) cells activated via FcγRIIIa (CD16) interactions with anti-HLA antibodies binding to peripheral blood mononuclear cells (PBMCs) in the in vitro antibody-dependent cellular cytotoxicity (ADCC) assay produced IFNγ. Here we investigate if other CD16 bearing cells are responsive to alloantigen via alloantibody in the in vitro ADCC and if the ADCC-induced cytokine reactions and cytotoxicity can be modified by the anti-interleukin 6 receptor (IL-6R) monoclonal antibody, Tocilizumab (TCZ). METHODS Whole blood from a normal individual was incubated overnight with irradiated allo-PBMCs pretreated with anti-HLA antibody positive (in vitro ADCC) or negative sera (mixed lymphocyte reaction [MLR]), with or without TCZ or control IgG. IFNγ+, TNFα+ or IL-6+ cell% in NK cells, monocytes and CD8+ T cells were enumerated by cytokine flow cytometry. ADCC using PBMCs (effector) and Farage B cells (FB, target) with anti-HLA antibody positive sera, with or without TCZ, was measured by flow cytometry. RESULTS IFNγ+ and/or TNFα+ cell% in NK cells, monocytes and CD8+ T cells were elevated in the ADCC compared to the MLR condition. IL-6+ cells were significantly increased in ADCC versus MLR (10.2 ± 4.8% vs 2.7 ± 1.5%, P = 0.0003), but only in monocytes. TCZ treatment significantly reduced TNFα+ cell% in monocytes in ADCC, but had no effect on other cytokine+ cells. TCZ showed no effect on cytotoxicity in ADCC. CONCLUSIONS IFNγ, TNFα, and IL-6 production induced by HLA antibody-mediated CD16 bearing cell activation in NK cells, monocytes, and CD8+ T cells suggests a potential role for ADCC and these inflammatory cytokines in mediation of antibody-mediated rejection. TCZ suppressed TNFα production in monocytes in the ADCC condition, suggesting a role of IL-6/IL-6R pathway in monocytes activation. Inhibition of this pathway could reduce the inflammatory cascade induced by alloantibody, although the inhibitory effect on cytotoxicity is minimal.