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

BackgroundWe 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).MethodsWhole 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.ResultsIFNγ+ 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.ConclusionsIFNγ, 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.

Cytokine Profiles Associated With Angiotensin II Type 1 Receptor Antibodies

“…HLA class II binding has been associated with increased IL-6 production in endothelial cells and promotion of TH17 lymphocyte expansion 63 . In peripheral blood mononuclear cells isolated from transplant patients, some studies have shown HLA antibodies increase cytokines (TNF-α, IFN-γ, and IL-6 64 ) whereas others have not 65 . We found an association between IL-6 and HLA mismatch, but not HLA DSA.…”

Small-Molecule Immunosuppressive Drugs and Therapeutic Immunoglobulins Differentially Inhibit NK Cell Effector Functions in vitro

“…On the other hand, fingolimod (FTY720) increases NK cytotoxicity by up-regulation of NKp30, NKp44 and NKG2D expression (79). Furthermore, the new generation of anti-inflammatory biologicals such as tocilizumab (anti-IL6R) (80), anti-TNF drugs (81), B cell depleting monoclonal antibodies such as rituximab (82), or kinase inhibitors (83) can also influence NK cells.…”

A Human Osteochondral Tissue Model Mimicking Cytokine-Induced Key Features of Arthritis In Vitro

“…Simulating such features in a preclinical drug screening tool would be transformative for the translational process necessary for optimizing rheumatological care. Here, we have developed a human MSC-derived OTM and described its capacity to create a RA-like phenotype, which replicated in part the immunomodulatory effect of well-known anti-rheumatic drugs such as adalimumab [ 30 , 31 ] and tocilizumab [ 32 , 33 ], but also the antineoplastic agent milatuzumab [ 34 ].…”

“…Finally, we provide first evidence that effects of immunomodulatory drug candidates can be demonstrated in our MSC-derived OTM on mRNA level ( Figure 7 ). We used adalimumab [ 30 , 31 ], tocilizumab [ 32 , 33 ] and milatuzumab [ 34 ] for proof of concept, as these are clinically used monoclonal antibodies directed against TNFα, IL-6 and MIF. Recent work has indicated that TNFα may mediate its angiogenic effect in RA via IL-8 and VEGF [ 65 ].…”

“…To mimic chronic cytokine-mediated joint inflammation, stimulation was performed using CM (CTRL) supplemented with 10 ng/mL recombinant human macrophage migration inhibitory factor (MIF) [ 60 ], 30 ng/mL recombinant human IL-6 [ 62 ], 10 ng/mL recombinant human tumor necrosis factor alpha (TNFα; all ImmunoTools GmbH, Friesoythe; Germany) [ 61 ] using concentrations as reported from synovial fluid of patients with RA and 23 µg/mL Flebogamma (Grifols, Barcelona, Spain) in the following referred as STIM. To evaluate the impact of specific therapeutic approaches, CM was supplemented with cytokines and well-known clinically available drugs in their therapeutic dosage: 10 µg/mL adalimumab (Amgen, Thousand Oaks, CA, USA) [ 30 , 31 ], 8 µg/mL tocilizumab (Roche, Basel, Switzerland) [ 32 , 33 ] and 5 µg/mL milatuzumab (Immunomedics, Morris Plains, NJ, USA) [ 34 ], in the following referred to as TREAT. Medium was changed every 3 days including the respective supplements resulting in a repetitive chronic cytokine stimulation in case of STIM and a repeated counter-treatment in case of TREAT.…”