Background: Viral infections represent significant morbidity factors for transplant recipients. Tools to assess the efficacy of viral-specific responses are lacking. ADCC is the major antibody-mediated anti-viral activity and is mediated primarily by NK cells. Here, we report on an in vitro assay to measure anti-CMV-antibody-mediated NK cell responses. Methods: Whole blood from 20 normal individuals (4 CMV sero[-]) were submitted for the CMV-specific T and NK cell-CFC assay (CMV-T/NK-CFC) where whole blood was incubated with CMV lysate with brefeldin A overnight followed by staining intracellular IFNγ and/or degranulation marker, CD107a surface expression. Results were expressed as IFNγ+ or CD107a+ cell% in CD4+ T and CD56+ NK cells. In some experiments, the CFC assay was performed with or without IdeS (10mg/ml),an IgG-degrading enzyme that cleaves 4 human IgG subclasses at the hinge region of IgG heavy chains, critical for ADCC. >0.2% for IFNγ+ and >0.5% for CD107a+ cells were considered (+). Results: Most CMV sero(+) individuals (14/16) showed CMV-T (+) as assessed by IFNγ positivity, and all 4 sero(-) individuals were (-). Similarly, all 16 CMV sero(+) individuals were (+) for CMV-NK, and all sero(-) showed (-). To determine if the observed CMV-T and NK positivity is antibody-dependent, the assay was performed with IdeS in 5 sero(+) blood samples. The elevated IFNγ+ (1.5±0.7% vs. 1.2±0.6%, NS) and CD107a+ cell% (0.4±0.3% vs. 0.4±0.4%, NS) in T cells in response to CMV lysate were similar w/o vs. w/ IdeS. In contrast, the elevated IFNγ+ (18±8% vs. 0±1.1%, p<0.01) and CD107a+ cell% (14±9% vs. 0±0.6%, p<0.03) in NK cells was completely abrogated by IdeS. Conclusions: These results suggest that in the CMV-T/NK-CFC, CD4+ T cell activation in response to CMV lysate is anti-CMV antibody-independent and is memory T cell response specific to CMV antigens via T cell receptor, while NK cell activation is anti-CMV antibody-dependent via CD16 (FcγIIIa receptor) and the involvement of other NK cell receptors is unlikely as NK cell activation became the background level after IdeS treatment. The CMV-NK-CFC assay should be helpful in assessing ADCC-mediated anti-CMV-immunity in transplant recipients.

CITATION INFORMATION: Ge S, Maggie C, Shin B.-H, Karasyov A, Jordan S, Toyoda M. NK Cells Respond to CMV Lysate by Inducing IFNγ and CD107a Surface Expression in an Antibody-Dependent Manner: This Represents a Cytokine Flow Cytometry (CFC) Assay That Could Assess Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) as a Marker of CMV Immunity in Transplant Recipients. Am J Transplant. 2017;17 (suppl 3).

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