Background: Antibody-mediated rejection (ABMR) is a major obstacle for successful transplantation in HLA-sensitized patients. ADCC is one of the primary mechanisms for ABMR. IdeS cleaves all 4 human IgG subclasses at the hinge region of IgG heavy chains, critical for ADCC and CDC. Here we examined the effect of IdeS on in vitro ADCC to assess its utility to prevent or treat ADCC-mediated ABMR.

Methods: Allo-CFC by intracellular flow cytometry (CFC) and ADCC assay by flow cytometry were performed to assess the effect of IdeS on antibody-mediated NK activation and cell cytotoxicity, respectively. In the Allo-CFC, normal whole blood was incubated overnight w/irradiated allo-peripheral blood mononuclear cells (PBMCs) pre-treated w/anti-HLA antibody+ (HS) or – (NS) serum. IFNγ+ NK were enumerated by CFC. To assess the effect of IdeS, serum-treated PBMCs (Allo-CFC-1) or serum used for pre-treatment of PBMCs (Allo-CFC-2) were incubated w/Ides at 37[deg]C for 1 hour, and then used for the assays. In the ADCC, normal PBMCs (effector cells) were incubated at 37[deg]C for 1 hour w/pre-labeled CD19+ Farage B cells (FB, target cells) together w/NS (control) or HS pre-treated w/IdeS, followed by addition of 7AAD, then 7AAD+FB cell% was enumerated.

Results: The results are shown in the table. IFNγ+ NK cell% was significantly elevated when blood was incubated w/HS-coated PBMCs in Allo-CFC-1 (11±8 vs. 2±2) and 2 (26±9 vs. 4±2) compared to NS-coated PBMCs. These elevated IFNγ+ NK cell% were significantly reduced by treatment w/IdeS in a dose dependent manner in both assays. 7AAD+ FB cell% was also significantly elevated when HSs was added in the ADCC assay compared to addition of NS (27±13 vs. 13±4), and this cell cytotoxicity was significantly reduced by treatment of HS w/IdeS in dose dependent manner.

Conclusions: IdeS inhibited antibody-mediated NK cell activation and ADCC by 50% even at 0.1-1.0 [mu]g/ml and these levels went back to control (NS) levels at 10 [mu]g/ml, the max therapeutic concentration. These suggest that IdeS could contribute to the reduction of ABMR resulted from ADCC mechanism.

CITATION INFORMATION: Ge S, Chu M, Choi J, Jordan S, Toyoda M. The Bacterial Enzyme IdeS, Inhibits HLA Antibody-Mediated NK Cell Activation and Cell Cytotoxicity (ADCC) In Vitro. Am J Transplant. 2016;16 (suppl 3).

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