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. 2019 Apr 1;202(7):2057-2068.
doi: 10.4049/jimmunol.1801449. Epub 2019 Feb 15.

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Qinglai Meng  1   2 , Asifa K Zaidi  2 , John Sedy  3 , Armand Bensussan  4 , Daniel L Popkin  5   6   7
Affiliations

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes

Qinglai Meng et al. J Immunol. .

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.

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Figures

Figure 1:
Figure 1:. Soluble Fc-disabled HVEM-(Fc*) structure, sequence and binding capacity.
(A) Schematic diagrams of HVEM-Fc and HVEM-(Fc*) fusion proteins. The HVEM-Fc fusion protein includes the extracellular domain of HVEM protein (amino acid residues from L39 to S199) and the CH1 deleted Fc stalk (amino acid residues T223-K447) of human IgG1. Soluble Fc-disabled HVEM-(Fc*) fusion protein was generated by mutating six amino acid residues at position E233P, L234V, L235A, ΔG236, A327G, A330S in the CH2/CH3 Fc stalk (from T223-K447) and then fusing this loss of function stalk to the same extracellular region of HVEM (amino acid residues from L39 to S199). (B) Recombinant human CD16a (Fc receptor) pre-conjugated beads were incubated with 10 μg/ml of human IgG1 (hIgG), HVEM-Fc (from PeproTech or G&P) or HVEM-(Fc*) (from G&P) on ice for 45 min. After washing, beads were stained with PE-conjugated anti-HVEM mAb (1 μg/ml) and analyzed by flow cytometry. X-axis shows the fluorescence intensity of beads incubated with hIgG1, HVEM-Fc (PeproTech and G&P) or HVEM-(Fc*) (G&P). (C) CD160-expressing CHO cells (CHO-CD160) were incubated with 10 μg/ml of hIgG1, HVEM-Fc (PeproTech and G&P) or HVEM-(Fc*) (PeproTech) on ice bath for 45 min. After washing, cells were incubated with PerCP-Cy5.5 conjugated mouse anti-human IgG-Fc mAb (2 μg/ml) and then cells were analyzed by flow cytometry. Percentages of HVEM+ cells are shown.
Figure 2:
Figure 2:. HVEM-(Fc*) activates NK cells and induces IFN-γ production and cytotoxicity.
(A-F) Purified NK cells from PBMC of healthy controls (n=9) were cultured with 20 µg/ml of human IgG (hIgG), HVEM-(Fc*) or HVEM-Fc in the presence of IL-2 (100 IU/ml) for 16 h. After 16 h, cells were examined by flow cytometry for surface expression of (A) CD69, (B) IFN-γ was quantitated from supernatants via ELISA, (C) viability was measured via 7-AAD staining, (D) CD16 cell surface expression was examined on CD56dimCD16+ NK cells. (E-F) To evaluate (E) spontaneous killing and (F) reverse ADCC activity, NK cells, cultured with 20 µg/ml hIgG, HVEM-(Fc*), or HVEM-Fc for 16 h, were further cocultured with target K562 cells or P815 cells, respectively, pre-labeled with fluorescent dye at effector (E) to target (T) ratio of 1:1 and 0.5:1 for 5 h. For reverse ADCC assays, each coculture was treated with 10 µg/ml of mouse IgG1 or anti-CD16 mAb. Dead targets cells were gated as 7-AAD+ cells. (G-J) PBMC from healthy controls (n=9 except panel H where n=5) were cultured with PBS or HVEM-(Fc*) (1µg/ml) for 44 h. Then, cell surface (G) CD69 expression on NK cells and (H) IFN-γ secretion in culture supernatants were determined via flow cytometry and ELISA respectively. Next, (I-J) PBMC were cultured with PBS or HVEM-(Fc*) for 44 h were washed, counted and further cocultured with pre-labeled K562 cells with fluorescent dye at an E to T ratio of 20:1 for 5 h in the presence of Golgi stop and anti-CD107a antibody. CD69 expression on NK cells and (H) IFN-γ secretion in culture supernatants were determined via flow cytometry and ELISA, respectively. Degranulation and cytotoxicity of NK cells were quantified by cell surface (I) CD107a expression on NK cells and (J) dead K562 target cells (7-AAD+Annexin-V+ cells) using flow cytometry. * p<0.05; ** p<0.01; *** p<0.001.
Figure 3:
Figure 3:. HVEM-(Fc*)-induced cytokines in NK cells.
NK cells purified from PBMC of healthy controls (n=5) were cultured in wells pre-coated with 20 µg/ml of human IgG1 (hIgG1) or HVEM-(Fc*) in the presence of (A) media only vs. (B-F) media, IL-2 (100 IU/ml), IFN-β (50 IU/ml), IL-12 (1 ng/ml), or IL-15 (5 ng/ml) for 16 h. Cell free supernatants were collected and analyzed for 65 cytokines via Luminex-based multiplex assay. Panels A-F shows the cytokines whose concentrations were significantly changed by HVEM-(Fc*) treatment compared to hIgG. Panel E-F shows the HVEM-(Fc*)-induced cytokines minus hIgG-induced cytokines. * p<0.05; ** p<0.01; *** p<0.001.
Figure 4:
Figure 4:. IL-2 priming upregulates LIGHT and BTLA expression on CD56bright NK cells and co-treatment with HVEM-His downregulates expression of LIGHT and CD160 on NK cells.
(A-D) Purified NK cells from healthy donor PBMC (n=6) were incubated with IL-2 (100 IU/ml) for 18 h and then cells were analyzed for (A) LIGHT, (B) BTLA, (C) CD160 or (D) CD69 expression on CD56bright vs. CD56dimCD16+ subsets of bulk NK cells using monoclonal antibodies (clones: T5-39, J168-540, 688327 and FN-5 respectively) by flow cytometry. The gating strategy for assessment of LIGHT, BTLA, CD160 and CD69 expression on CD56bright NK cells and CD56dim NK cells is shown in Fig. S2. (E-G) Purified NK cells from healthy donor PBMC (n=6) were incubated with media or plate bound HVEM-His (20-μg/ml, 20μl/well) in the presence or absence of IL-2 (100 IU/ml) for 18 h and then cells were analyzed for (E) LIGHT or (F) BTLA expression on CD56bright NK cells and (G) CD160 expression on CD56dim NK cells using monoclonal antibodies (clones: T5-39, J168-540, 688327 and FN-5 respectively) by flow cytometry. * p<0.05; ** p<0.01; *** p<0.001.
Figure 5:
Figure 5:. Ligation of HVEM-(Fc*) with CD160 and LIGHT can contribute to IFN-γ production from NK cells.
The KHYG1 NK cell line was analyzed for (A) LIGHT (left panel), BTLA (middle panel) and CD160 (right panel) expression using monoclonal antibodies (clones: T5-39, J168-540 and 688327 respectively) by flow cytometry. Representative flow histograms show fluorescence intensity of receptors on the X-axis. In each panel: red histogram = isotype control antibody staining; light blue histogram = receptor antibody staining. (B) KHYG1 cells were incubated with media or plate bound HVEM-His (20 μg/ml) for 18 h. After 18 h, cell free supernatants were analyzed for IFN-γ production via ELISA. Mean ± SEM. shown (C) Purified NK cells from healthy controls (n=4), pre-incubated with isotype control antibody (mouse IgG2b) or anti-human CD160 blocking antibody at 20 μg/ml, were cultured with HVEM-(Fc*) pre-coated on a 384-well plate at the indicated concentration shown on the X-axis for 18 h. After 18 h, IFN-γ production was quantified from cell free supernatants via ELISA.
Figure 6:
Figure 6:. Monocytes express LIGHT greater than BTLA amongst potential HVEM receptors.
PBMCs from healthy donors (n=8) were stained for (A) CD160, (B) LIGHT or (C) BTLA and gated as shown in panel (D) for monocytes (CD14+/SSChigh), NK cells (CD14−/CD19−/CD3−/CD56+), conventional dendritic cells (cDC; CD3-CD14-HLA-DR+CD11c+), and plasmacytoid DC (pDC; CD3-CD14-HLA-DR+CD123+). Stained and washed cells were analyzed by flow cytometry. (E) Representative flow histograms show fluorescence intensity of receptors (CD160, BTLA, or LIGHT) on the X-axis for monocytes, NK cells, cDCs and pDCs. In each panel: red histogram = isotype control antibody staining; light blue histogram = receptor antibody staining.
Figure 7:
Figure 7:. HVEM-(Fc*) promotes activation of monocytes.
Monocytes (A) purified by positive selection or (B) gated as CD3-CD14+/SSChigh cells within PBMC cultures from healthy donors (n=6) were incubated with media or HVEM-(Fc*) (1 μg/ml) for 22 h and subsequently analyzed for cell surface activation markers (CD80, CD83, CD86 and CD40) via flow cytometry. * p<0.05; ** p<0.01.
Figure 8:
Figure 8:. HVEM-(Fc*) promotes crosstalk between monocytes and NK cells, enhancing activation and cytotoxicity of NK cells.
(A-D) Purified 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 μg/ml) for 44 h. (A-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, pre-labeled K562 target cells were added at an E:T ratio of 1.5:1 in the presence of anti-CD107a Ab for 5h. HVEM-(Fc*) treatment enhanced NK cell cytotoxicity was evaluated as (C) percent dead 7-ADD+-Annexin V+ K562 cells (HVEM-induced minus media-induced) and (D) NK cell degranulation was estimated by cell surface CD107a expression (HVEM-induced minus media-induced) for both NK cells alone and the mixture of NK cells + monocytes as the effector cells. * p<0.05; ** p<0.01.
Figure 9:
Figure 9:. HVEM-(Fc*)-induced IFN-γ production and cytotoxicity of NK cells is enhanced by monocyte-NK cell-cell contact.
(A-D) NK cells and monocytes were purified from PBMC by negative selection and positive selection respectively. The purified NK cells and monocytes were either cocultured in a flat bottom well or separately cultured in a transwell in the presence or absence of HVEM-(Fc*) (1 μg/ml) for 44 h. After 44 h, cell surface (A) CD69 expression on NK cells was measured by flow cytometry and (B) IFN-γ production in cell free supernatants was quantified via ELISA. (C-D) Cells were treated as above with HVEM-(Fc*) for 44 h and subsequently cocultured with pre-labeled K562 cells in the presence of anti-CD107a Ab for 5 h. (C) Dead 7AAD+Annexin-V+ K562 cells were quantified and (D) NK cell CD107a expression was quantified, all by flow cytometry.
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