Abstract
CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes virus entry mediator (HVEM) activates NK cells for cytokine production and cytolytic function via CD160. Fc-fusions are a well-established class of therapeutics, where the Fc domain provides additional biological and pharmacological properties to the fusion protein including enhanced serum t 1/2 and interaction with Fc receptor-expressing immune cells. We evaluated the specific function of HVEM in regulating CD160-mediated NK cell effector function by generating a fusion of the HVEM extracellular domain with human IgG1 Fc bearing CD16-binding mutations (Fc*) resulting in HVEM-(Fc*). HVEM-(Fc*) displayed reduced binding to the Fc receptor CD16 (i.e., Fc-disabled HVEM), which limited Fc receptor-induced responses. HVEM-(Fc*) functional activity was compared with HVEM-Fc containing the wild type human IgG1 Fc. HVEM-(Fc*) treatment of NK cells and PBMCs caused greater IFN-γ production, enhanced cytotoxicity, reduced NK fratricide, and no change in CD16 expression on human NK cells compared with HVEM-Fc. HVEM-(Fc*) treatment of monocytes or PBMCs enhanced the expression level of CD80, CD83, and CD40 expression on monocytes. HVEM-(Fc*)-enhanced NK cell activation and cytotoxicity were promoted via cross-talk between NK cells and monocytes that was driven by cell-cell contact. In this study, we have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-γ production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor-induced effects. Soluble Fc-disabled HVEM-(Fc*) may be considered as a research and potentially therapeutic reagent for modulating immune responses via sole activation of HVEM receptors.
Highlights
Herpes virus entry mediator (HVEM)-(Fc*) had significantly reduced mean fluorescence intensity (MFI) (1267 6 421) compared with HVEM-Fc MFI (67266 6 5232) (Fig. 1B). These data show that the loss-of-function Fc* stalk of HVEM-(Fc*) does not bind efficiently to recombinant human CD16a (rhCD16a)-conjugated beads compared with HVEMFc containing the wild type Fc stalk
NK cells from healthy donor PBMC (n = 6) were cocultured without purified monocytes (MN) or with purified monocytes (NK cells + MN) at a ratio of 1:1 in the presence of media or HVEM-(Fc*) (1 mg/ml) for 44 h. (A and B) After 44 h, NK cells were analyzed for cell surface (A) CD69 and (B) CD56 expression by flow cytometry
After 44 h of HVEM-(Fc*) treatment of NK cells and NK cells + MN cocultures, prelabeled K562 target cells were added at an E:T ratio of 1.5:1 in the presence of anti-CD107a Ab for 5 h
Summary
We have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-g production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor–induced effects. (I and J) PBMC were cultured with PBS or HVEM-(Fc*) for 44 h and were washed, counted, and further cocultured with prelabeled K562 cells with fluorescent dye at an E:T ratio of 20:1 for 5 h in the presence of GolgiStop and anti-CD107a Ab. CD69 expression on NK cells and (H) IFN-g secretion in culture supernatants were determined via flow cytometry and ELISA, respectively.
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