title: Tocilizumab (Anti-IL-6R) Suppressed TNFα Production by Human Monocytes in an In Vitro Model of Anti-HLA Antibody-Induced Antibody-Dependent Cellular Cytotoxicity.
abstract: 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.
authors.affiliation: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA.
authors.forename: Bong-Ha
authors.initials: BH
authors.lastname: Shin
authors.affiliation: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA.
authors.forename: Shili
authors.initials: S
authors.lastname: Ge
authors.affiliation: Biostatistics Core, Research Institute, General Clinical Research Center, Cardiothoracic Surgery, Cedars-Sinai Medical Center/UCLA School of Medicine Los Angeles, CA.
authors.forename: James
authors.initials: J
authors.lastname: Mirocha
authors.affiliation: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA.
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Jordan
authors.affiliation: Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA.
authors.forename: Mieko
authors.initials: M
authors.lastname: Toyoda