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

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 t1/2 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.

N atural killer cells, a subset of lymphoid cells, are an essential component of the innate immune system that protects against viruses (e.g., human CMV, HIV, and hepatitis C virus), tumor cells, and other pathogens (1)(2)(3)(4)(5). NK cell innate immune responses are tightly regulated by multiple activating and inhibitory receptors. Unlike typical activating and inhibitory receptors on NK cells, CD160 is tightly regulated in two alternative splice variants: a GPI-anchored (CD160-GPI) form and a differentially spliced transmembrane form of the protein (CD160-TM) that is unique to NK cells. CD160 is part of the Ig superfamily of receptors and it is predominantly expressed in peripheral blood NK cells, gd T (6), and CD8 T lymphocytes (7,8) with cytolytic effector activity. In circulating cells, the highest expression of CD160 RNA is identified in peripheral blood CD56 dim CD16 + NK cells greater than CD8 T cells (9). CD160 signals upon engagement of the widely expressed molecules herpes virus entry mediator (HVEM) and/or HLA-C (10)(11)(12). The engagement of CD160 by soluble HVEM (HVEM conjugated to the Fc portion of IgG1) or HVEM expressed on the cell surface was shown to activate NK cells (10). Genetic deficiency of CD160 in mice specifically impairs NK cell production of IFN-g, which is an essential component of the innate response to control tumor growth (13).
HVEM is a member of the TNFR superfamily and is expressed on many immune cells, including NK cells, T and B cells, monocytes, and neutrophils (14)(15)(16)(17)(18). HVEM is an immune regulatory molecule (15,18) that signals bidirectionally both as a receptor and a ligand. HVEM interacts with three cell surface molecules, CD160, LIGHT (homologous to lymphotoxins [LT], shows inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), and B and T lymphocyte attenuator (BTLA) and in humans with LT-a or TNF-b (14)(15)(16)(17)(18). HVEM generates bidirectional signals, and recent literature provides evidence of signaling induced by interaction between HVEM and CD160, LIGHT, BTLA, or LT-a in different immune cells (7,15,(19)(20)(21)(22)(23)(24)(25). The extracellular domain of HVEM was fused to the Fc portion of human IgG1 (hIgG1) in previous studies to produce a soluble protein used to detect HVEM ligands or alternatively to specifically activate BTLA or CD160 receptors (10,26,27). Because hIgG1 Fc binds to Fc receptor expressed on innate cells, including NK cells, HVEM-Fc fusion proteins may engage receptors for both the HVEM domain and the Fc domain. Fc fusion proteins have been widely used to interrogate the activities of cell surface proteins or soluble molecules and are widely used in immunotherapies such as etanercept, alefacept, and abatacept. The Fc domain of these fusion proteins may contribute to biological activities unrelated to the fusion partner and which can be removed through mutation of the Fc domain. To determine how HVEM engagement of NK cells may specifically function to activate NK cells in the absence of Fc receptor binding, we generated fusion proteins constructed of the extracellular domain of HVEM conjugated to a mutant hIgG1 Fc that does not bind to Fc receptor (HVEM-[Fc*]) (28).
LIGHT, a member of the TNF ligand superfamily, is mainly expressed on T cells, monocytes, NK cells, and immature dendritic cells (DC) (29) and binds to HVEM and LTbR, two membrane receptors (30). LIGHT-HVEM interactions are thought to regulate a variety of immune responses, for example, costimulation of T cell proliferation, polarizing CD4 T cells into Th1 cells and associated cytokine production (31), inducing DC maturation (31), stimulating Ig production in B cells (32), and activating NK cells (19). This interaction enhances phagocytosis of monocytes and neutrophils and contributes to antibacterial activity via production of ROS, NO, other proinflammatory cytokines, and direct bactericidal activity (33). In contrast, engagement of HVEM with BTLA on T cells inhibits anti-TCR-induced activation and cytokine secretion (34).

Cells
Fresh human blood was collected from healthy donors giving written informed consent at Case Western Reserve University, and the Institutional Internal Review Board approved all handling. PBMCs were isolated by Lymphoprep (STEMCELL Technologies) density gradient centrifugation as per manufacturer's description from fresh human blood and fresh leukocyte reduction filters obtained from Red Cross, Cleveland, and cryopreserved where indicated. NK cells were isolated from PBMC by negative selection using the NK Cell Enrichment Kit (STEMCELL Technologies). Monocyte isolation was performed by positive selection using CD14 microbeads and MACS columns (Miltenyi Biotec). HVEMexpressing CHO cells (HVEM-CHO) were obtained from K. G. Kousoulas. CD160-CHO and HVEM-CHO were maintained in Ham's F12 medium plus modified Eagle's minimal essential medium (1:1) supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin.
We used CD160-his and hIgG1 as controls for HVEM-his and HVEM-(Fc*)/HVEM-Fc, respectively. CD160-his binds to HVEM-expressing CHO cells but NK cells treated with CD160-his did not cause IFN-g production compared with HVEM-his (data not shown). Because CD160his did not induce any functional responses similar to observations with hIgG control or media control, we have used hIgG1 as the control for HVEM-(Fc*). However, we acknowledge that the appropriate control would be a fusion protein bearing an irrelevant protein fused with (Fc*) and/or production of an HVEM-(Fc*) fusion protein mutated for loss of HVEM function to assess HVEM-(Fc*)-induced responses. However, this was not synthesized because we observed the same response from CD160-His and hIgG1 and were resource limited.

Cytokine expression analysis
Supernatants from PBMC, NK cells, monocytes, and NK cell and monocyte coculture were analyzed for IFN-g by ELISA (capture Ab [clone M700A] and biotin-conjugated secondary Ab [clone M701B] were from Thermo Fisher Scientific) and were also analyzed by 65-plex Luminex array (Eve Technologies).

NK cell cytotoxicity assay
Frozen PBMCs from healthy controls were thawed and cultured overnight in RPMI 1640 supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin. PBMCs were then incubated with or without 1 mg/ml HVEM-(Fc*) for 42-44 h. Activation marker CD69 on NK cells was measured after 44 h by flow cytometry. PBMC were further cocultured with prelabeled K562 cells at an E:T ratio of 20:1 in the presence of anti-CD107a-allophycocyanin-Cy7 for 5 h. K562 cells were stained with 7AAD and annexin-V-PE to quantitate dead cells (7AAD + annexin-V + ) by flow cytometry.
Where described, cytotoxicity assays were performed using purified NK cells (purity $95%) and monocytes (purity $95%) in cocultures. Briefly, NK cells were purified from thawed overnight-cultured PBMC by negative selection. Monocytes were purified by CD14-positive selection from the same donor. Purified NK cells (0.3 3 10 6 ), purified monocytes (0.3 3 10 6 ), or purified NK cells plus purified monocytes coculture at a ratio of 1:1 (0.3 3 10 6 + 0.3 3 10 6 ) was performed in the presence or absence of HVEM-(Fc*) (1 mg/ml) for 44 h. After 44 h, cells were then stained for activation markers and analyzed by flow cytometry. NK cells, monocytes, or NK cells plus monocyte mixture were further cocultured with labeled K562 cells at a ratio of NK cells to K562 cells of 1.5:1 for 5 h in the presence of anti-CD107a mAb-allophycocyanin-Cy7 or its isotype control Ab. The cocultured cells were then stained with 7-AAD and annexin-V-PE to quantitate the dead cells (7AAD + annexin-V + ) by flow cytometry. For transwell cultures (96-well plate), NK cells (0.3 3 10 6 cells per well) were seeded into the lower chamber, and monocytes (0.3 3 10 6 cells per well) were seeded in the upper chamber or both NK cells plus monocyte mixture (1:1) were seeded in the lower chamber and were cultured in the absence or presence of HVEM-(Fc*) for 44 h. Activation markers and cytotoxicity were measured identically to above in nontranswell cultures.
We performed reverse Ab-dependent cellular cytotoxicity (ADCC) assay using the well-characterized P815 mouse mastocytoma cell line that abundantly expresses mouse CD16 (mouse Fc receptor) as a target cell to evaluate the activity of HVEM-(Fc*)-induced activation of NK cells. This is a well-studied model, similar to K562 killing assays for spontaneous killing, to ascertain reverse ADCC assay with a defined system. A mouse anti-human CD16 monoclonal is used to bridge human NK cell effectors with mouse P815 mastocytoma targets resulting in reverse ADCC assay (49). Briefly, the NK cells were pretreated with either HVEM-Fc, HVEM-(Fc*), or IgG1 for 16 h. Cells were washed to remove the unbound stimuli (HVEM-Fc, HVEM-[Fc*], or IgG1) and then were cocultured with P815 target cells in the presence of mouse anti-human CD16 Ab (containing a murine Fc stalk) or its isotype control Ab to evaluate the effect of HVEM-(Fc*) on NK cell reverse ADCC assay.

Statistical analysis
Student paired t test was performed to determine the difference between two groups unless otherwise indicated. All tests were considered statistically significant at p , 0.05.

Results
Soluble HVEM-(Fc*) activates NK cells and induces IFN-g production and cytotoxicity independent of Fc binding HVEM-Fc fusion proteins have been used to study the interaction between HVEM and its ligands on NK cells and immune responses (10, 48, 50-52). We sought to determine how a soluble HVEM-Fc fusion may function independent of Fc receptor interactions through mutation of residues required for Fc receptor binding (28,53,54). The entire HVEM extracellular domain was conjugated to a loss-of-function Fc stalk (Fc*) of hIgG1 (HVEM-[Fc*]) that has been previously shown to lack Fc receptor functionality (Fig. 1A). Six amino acids were mutated at position E233P, L234V, L235A, DG236, A327G, A330S on the Fc* stalk of hIgG1 as described (28). The binding capacity of the Fc* stalk of HVEM-(Fc*) was tested using rhCD16a-conjugated beads by flow cytometry. 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 rhCD16a-conjugated beads compared with HVEM-Fc containing the wild type Fc stalk. The binding capacity of HVEM-Fc and HVEM-(Fc*) to CD160 was tested using CD160-expressing CHO cells. As shown in Fig. 1C, both HVEM-Fc and HVEM-(Fc*) had comparable MFI (7814 6 587 and 6900 6 469, respectively) and percentage of HVEM + cells (93 6 3% versus 88 6 4%). Taken together, these data demonstrate that HVEM binding to CD160 is comparable for both HVEM-Fc and HVEM-(Fc*), but binding to the Fc receptor of HVEM-(Fc*) is reduced 50-fold compared with HVEM-Fc. As an additional control, we used HVEM-His (which does not contain an Fc binding stalk) to determine HVEM ligand function uncoupled from Fc-Fc receptor effects in subsequent experiments.
To evaluate the effect of HVEM on general activation of NK cells, we cultured purified NK cells with HVEM-(Fc*) and compared its effect with HVEM-Fc-induced responses. The percentage of NK cells expressing CD69 that were treated with HVEM-(Fc*) was similar to the percentage of NK cells that were treated with hIgG1 ( Fig. 2A). In contrast, HVEM-Fc treatment increased 3.4-fold and 4-fold percentage of NK cells expressing CD69 compared with HVEM-(Fc*) and IgG1 treatment, respectively ( Fig. 2A). Despite absent percentage of CD69 induction on NK cells by HVEM-(Fc*), this Fc-disabled HVEM was a more potent inducer of IFN-g in the presence of IL-2 from NK cells than HVEM-Fc ( Fig. 2B) but not in the absence of IL-2 (Supplemental Fig. 1A). These data suggested that HVEM-induced NK cell production of IFN-g required IL-2 priming.
Additionally, we observed increased NK cell death with HVEM-Fc versus HVEM-(Fc*) and hIgG1. This is likely attributable to NK fratricide via Fc-Fc receptor binding (Fig. 2C) resulting in the observed reduction in CD16 (Fc receptor) expression on NK cells after HVEM-Fc treatment (Fig. 2D). Furthermore, we suspect that apparent IFN-g secretion induced by CD16 and/or CD160 ligation of HVEM-Fc would be attenuated as compared with HVEM-Fc* (Fig. 2B) secondary to HVEM-Fc-induced fratricide (i.e., HVEM-Fc-Fc receptor cross-linking likely occurs before these NK cells fully produced and/or released IFN-g from HVEM-Fc engagement [ Fig. 2C]).
Given the somewhat unexpected findings of increased IFN-g production and decreased NK cell death with HVEM-(Fc*) versus HVEM-Fc, we then measured NK cell-induced cytotoxicity of K562 target cells. HVEM-(Fc*) enhanced NK cell-induced cytolysis of K562 cells (Fig. 2E). Surprisingly, HVEM-Fc caused significantly reduced spontaneous killing of K562 cells by NK cells (Fig. 2E) compared with HVEM-(Fc*) and hIgG1. We also tested the effect of HVEM-(Fc*)-activated NK cells in a classic reverse ADCC assay using the well-characterized P815 mouse mastocytoma cell line that abundantly expresses mouse CD16 (mouse Fc receptor) as described in the Materials and Methods section. HVEM-(Fc*) but not HVEM-Fc enhanced killing of P815 target cells via NKmediated ADCC (Fig. 2F). In addition, HVEM-(Fc*)-induced spontaneous killing and ADCC did not require IL-2 priming (Supplemental Fig. 1B, 1C) as was required for IFN-g production ( Fig. 2B, Supplemental Fig. 1A). IL-2 priming trended to, but did not significantly, enhance soluble HVEM-induced spontaneous killing of K562 cells (p = 0.074) nor reverse ADCC of P815 cells (p = 0.21) (Supplemental Fig. 1D, 1E). These data demonstrate that HVEM-(Fc*) induces greater NK IFN-g production in addition to spontaneous killing and ADCC as compared with HVEM-Fc. Taken together, we demonstrate that CD16 engagement with the Fc stalk of HVEM-Fc can interfere with the ability for a soluble HVEM reagent to augment NK effector function. This has important therapeutic applications. Therefore, we focused on characterizing HVEM-(Fc*) for all subsequent studies described below.
Several recent studies suggest that NK cell function can be modulated via APCs such as monocytes, macrophages, and DC (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). These cells also express HVEM receptors. Therefore, we tested the effect of soluble HVEM-(Fc*) on NK cell function when cocultured in the presence of the physiological complement of immune cells present in circulation. As shown in Fig. 2G-J, in contrast to purified NK cells, soluble HVEM-(Fc*)-treated PBMCs caused CD69 induction on NK cells (Fig. 2G). Similar to purified NK cells, soluble HVEM-(Fc*) significantly increased IFN-g production (Fig. 2H) and increased lysis of K562 cells (Fig. 2I) in treated PBMCs. However, given the difficulty in characterizing the effector component of bulk PBMCs, a direct comparison cannot be made with purified NK cell data. We observed a mild increase in NK cell degranulation as assessed by surface CD107a expression (Fig. 2J) consistent with our direct cytotoxicity measurements (Fig. 2I). These data suggest that HVEM-(Fc*) stimulates NK cells intrinsically for IFN-g production and spontaneous killing. However, CD69 and thus potentially other metrics of HVEM-(Fc*) activation may be further augmented by accessory cell(s). We pursued this line of investigation in Figs. 3,4,5 and 6 below.
HVEM-(Fc*) drives broad NK cytokine production that can be further augmented by IL-2 more so than other NK cell-stimulating cytokines Previously, it was reported that CD160 signaling induces NK production of IFN-g, IL-6, IL-8, TNF-a, MIP-1b, and lower amounts of IL-4 and IL-10 (12,55). To more comprehensively evaluate the effects of soluble HVEM on cytokine production in a relatively unbiased manner, we performed 65-plex cytokine protein array analysis using the Luminex platform. We repeated this analysis in the presence of media 6 the best characterized NK-activating cytokines (IL-2, IFN-b, IL-12, and IL-15) to more fully capture the stimulating potential of soluble HVEM given that IFN-g production required the presence of IL-2 (Fig. 2B,  Supplemental Fig. 1A). We reasoned that other NK-activating cytokines might be required for the production of additional cytokines similarly to what has been observed for NK cell IFN-g production. HVEM-(Fc*)-stimulated NK cells produced significant levels of GM-CSF, GRO-a, MIP-1a, TNF-a, I-309, IL-1a, IL-1b, IL-6, IL-8, and IL-10 without IL-2 priming compared with hIgG1 ( Fig. 3A), whereas HVEM-(Fc*)-induced IL-5 and IL-13, similar to IFN-g, required IL-2 priming of NK cells (Fig. 3B, 3C). During infection, type I IFN, IL-12, and IL-15 production promotes NK cell effector function (56,57). When we evaluated the effect of these cytokines in 65-plex Luminex-based assays, we found that only IFN-g (Fig. 3D) production was significantly augmented by IFN-b, IL-12, and IL-15 versus media alone and that HVEM-(Fc*)-induced production of other cytokines (e.g., I-309 and GM-CSF, Fig. 3E, 3F) were distinctly affected by the presence of IFN-b, IL-12, and IL-15 (Fig. 3). IL-2 and IL-15 treatment significantly enhanced but IFN-b treatment significantly decreased HVEM-(Fc*)-induced GM-CSF and I-309 production (Fig. 3E,  3F). Overall, we uncovered the production of additional cytokines (GM-CSF, GRO-a, MIP-1a, I-309, IL-1a, and IL-1b) that, to our knowledge, were not previously published, and that were elaborated directly by soluble HVEM treatment and did not require the presence of IL-2 or other NK-activating cytokines. Moreover, we identified that IL-5 and IL-13 could be induced by HVEM-(Fc*) in the presence of IL-2, similarly to IFN-g. These data highlight the potential therapeutic application of HVEM-(Fc*).
IL-2 priming upregulates LIGHT and BTLA but not CD160 expression on CD56 bright NK cells NK cells are divided into two major subtypes: CD56 dim and CD56 bright . CD160 is mostly expressed on CD56 dim NK cells, which make up the vast majority of circulating NK cells. In contrast, LIGHT and BTLA are expressed at low levels on both CD56 dim and CD56 bright NK cells (10,35). IL-2 priming enhances HVEM-(Fc*)-stimulated cytokine production and might enhance it by increasing expression of NK cell HVEM receptors. Therefore, we determined whether IL-2 priming enhanced CD160, LIGHT, and/or BTLA (receptors of HVEM) expression on NK cells. As shown in Fig. 4, Supplemental Fig. 2, IL-2 priming of purified bulk NK cells upregulated expression of LIGHT (Fig. 4A) and BTLA (Fig. 4B) on CD56 bright but not CD56 dim NK cells. IL-2 priming of purified bulk NK cells did not change CD160 expression on either CD56 dim or CD56 bright subsets of NK cells (Fig. 4C). We confirmed IL-2 activation of purified bulk NK cells by demonstrating upregulation of CD69 expression on both subsets of NK cells (Fig. 4D). Interestingly, HVEM-His treatment of purified bulk NK cells reduced cell surface expression of LIGHT on CD56 bright NK cells in the presence of IL-2 ( Fig. 4E) but did not change BTLA expression on CD56 bright NK cells either in the presence and absence of IL-2 (Fig. 4F). CD160 expression on CD56 dim CD16 + NK cells that was not affected by IL-2 priming but was reduced by HVEM-His treatment and HVEM-his-induced reduction in CD160 was further enhanced by the presence of IL-2 on CD56 dim CD16 + NK cells (Fig. 4G). These data suggest that IL-2 priming distinctly enhanced the cell surface of expression HVEM receptors on different NK cell subsets and may enhance NK cell function differentially by HVEM activation. Because CD160 expression was not enhanced by soluble HVEM treatment of NK cells and BTLA was identified as an inhibitory receptor (10), we tested whether HVEM activates NK cells via LIGHT engagement. Human CD56 dim NK cells only express CD160. However, both CD56 bright and CD56 dim NK cells express LIGHT at low levels (Fig. 4). To test the potential sole effect of HVEM via LIGHT, we made use of the KHYG1 NK cell line, which we discovered only expressed LIGHT and not BTLA (Fig. 5A). The surface expression of CD160 on KHYG1 was similar to background isotype control (Fig. 5A). HVEM-His  treatment of KHYG1 NK caused significant production of IFN-g (Fig. 5B). Given this finding, we evaluated HVEM-induced activation via LIGHT on purified NK cells by incubating them with HVEM-(Fc*) in the presence of anti-CD160 blocking Ab (clone 688327; R&D Systems). We found that HVEM-(Fc*) caused significant production of IFN-g from NK cells after blocking of CD160 receptor (Fig. 5C). We hypothesize that CD160 blockademediated enhancement of IFN-g production from HVEM-(Fc)*treated NK cells resulted from more HVEM-(Fc)* binding of other HVEM ligands, such as LIGHT. The IL-2-pretreated NK cells express high levels of CD160 (the majority is localized on the CD56 dim subset and the minority is localized on the CD56 hi subset) and low levels of LIGHT and BTLA, which are mostly localized on the CD56 hi subset. In the physiological condition (absence of CD160 blocking Ab), CD160 will compete with LIGHT, BTLA, or other unknown ligand(s) on the NK cells to bind with plate-bound HVEM-(Fc)*. Therefore most IFN-g is likely released from NK cells by ligation of HVEM and CD160. Conversely, in the presence of anti-CD160 blocking Ab, the other HVEM ligands, such as LIGHT, will have a greater likelihood to bind with HVEM-(Fc)*, and this binding might elicit greater IFN-g production than CD160-HVEM binding. These data support the notion that HVEM may activate NK cells via LIGHT in addition to CD160.

HVEM-(Fc*) promotes activation of monocytes
Several reports have demonstrated that monocytes and macrophages regulate NK cell effector function(s) (36,(38)(39)(40)(41)(43)(44)(45)(46). Monocytes also express receptors of HVEM (18,34). Thus, we determined the level of expression of all three HVEM receptors on monocytes as a readily available cell type that may explain why PBMC cultures showed different results than isolated NK cells with respect to soluble HVEM treatment (Fig. 2). We found that monocytes expressed LIGHT and BTLA and there was no CD160 expression (Fig. 6). Among monocytes, NK cells, and DC, LIGHT expression was greatest on monocytes. Next, we tested whether HVEM-(Fc*) activated monocytes similarly to NK cells. Activation of monocytes was measured by surface expression of CD80, CD83, CD86, and CD40 (Fig. 7). Expression of activation markers CD80, CD83, and CD40 but not CD86 was enhanced by HVEM-(Fc*) treatment of both purified monocytes (Fig. 7A) and flow-gated monocytes in PBMC cultures (Fig. 7B), consistent with Schwarz et al. (58) who also found upregulation of CD80, CD83, and CD40 but downregulation of CD86 in response to LPS. Similar to HVEM-(Fc*), HVEM-His also activated both monocytes and NK cells in PBMC cultures (Supplemental Fig. 3), confirming that soluble HVEM could directly activate monocytes in addition to NK cells.

HVEM-(Fc*) promotes cross-talk between NK cells and monocytes
Next, we reasoned that because monocytes can modulate NK activity, were activated by HVEM-(Fc*), and NK cell activation was different in PBMC cultures versus enriched NK cells incubated with HVEM-(Fc*), that monocytes may play a role in HVEM-(Fc*)-induced NK activation and subsequent effector functions. Thus, we compared purified NK cells cultured with and without purified monocytes in the presence or absence of HVEM-(Fc*). Activation of NK cells was quantified via cell surface CD69 expression, degranulation via CD107a, cytotoxicity to K562 target cells, and IFN-g production. When purified NK cells were cocultured with purified monocytes in the presence of HVEM-(Fc*), there was significant enhancement of CD69 (Fig. 8A) and CD56 (Fig. 8B) cell surface expression on the NK cells. There was also enhanced NK cell degranulation (increased cell surface expression of CD107a) and enhanced cytotoxicity toward K562 cells when NK cells were cocultured with monocytes in the presence of HVEM-(Fc*) (Fig. 8C, 8D) compared with NK cells cultured with HVEM-(Fc*). These data demonstrate that monocytes can augment HVEM-(Fc*)-induced NK cell activation and effector function.
To test whether monocytes augment NK cell effector function via cell-cell contact or soluble mediators, NK cells were cocultured with monocytes in the presence or absence of HVEM-(Fc*) in a transwell tissue culture plate. Monocytes or NK cells were either cultured separately in the upper or lower chambers respectively separated by transmembrane or cultured together in the bottom chamber in a transwell culture plate. Transwell separation of purified NK cells and monocytes resulted in a slightly lower basal expression of CD69 (Fig. 9A); however, this was not statistically significant. Otherwise, HVEM-(Fc*) similarly induced CD69 expression in NK cell and monocyte transwell separated cultures as compared with culturing both cell types together in the bottom chamber (Fig. 9A). In contrast, we observed that HVEM-(Fc*)-induced IFN-g production was enhanced by coculture of NK and monocytes as compared with separation of these cells by transwell membrane (Fig. 9B). Additionally, there was more significant HVEM-(Fc*)-induced K562 cytotoxicity in cocultures of NK and monocytes as compared with separation of these cells by transwell membrane (Fig. 9C). This mild difference in cytotoxicity was not observed in NK cell degranulation as measured by CD107a expression (Fig. 9D). HVEM-(Fc*)-induced activation of monocytes in transwell cocultures was assessed by cell surface expression of CD80, CD86, CD83, and CD40 (Supplemental Fig. 4). Similar to the HVEM-(Fc*)-treated purified monocytes (Fig. 7), there was significant enhancement of CD80, CD83, and CD40 but not CD86 (Supplemental Fig. 4) expression on monocytes in the transwell cocultures. Taken together, these transwell experiments indicated that HVEM-(Fc*) activation of NK via NK-monocyte cross-talk required cell-cell contact mechanism(s) for maximal enhancement of IFN-g production and potentially mild enhancement of spontaneous killing.

Discussion
NK cells play a key role in the protection from pathogens and cancer. NK cell-mediated effector function depends on the type and number of receptor/ligand interactions occurring between NK cells  and their targets. In this context, it has been shown that CD160 drives NK cell activation, cytokine production, and cytotoxicity via engagement with HLA-C (12) and HVEM (10,16,18,48). In this study, we have shown that HVEM conjugated to the wild type sequence of Fc (HVEM-Fc) activates NK cells; however, the Fc portion can cause interaction and activation of other Fc receptorexpressing immune cells via Fc-Fc receptor ligation in addition to HVEM (Figs. 1, 2). This is notable given this form of HVEM-Fc is the reagent readily available "off the shelf" from multiple vendors including BioLegend, R&D Systems, Sigma-Aldrich, SBH Sciences, AB Biosciences, BPS Bioscience, Enzo, PromoCell, Tonbo Biosciences, GenScript, G&P Biosciences, and others. We designed and evaluated a soluble Fc-disabled HVEM to interrogate the specific function of HVEM-mediated NK cell responses and provide an alternative reagent for potential therapeutic application. To do this, we fused the HVEM extracellular domain to a mutant Fc stalk that poorly binds the Fc receptor HVEM-(Fc*). HVEM-(Fc*) binding to CD160 on primary NK cells resulted in increased inflammatory cytokine expression, degranulation (enhanced CD107a expression), and cytolysis by NK cells as compared with wild type HVEM-Fc.
We have also shown for the first time, to our knowledge, that HVEM induces a broader array of cytokines than previously demonstrated, which occurred in both IL-2-dependent and -independent manners. Previously, it has been reported that engaging CD160 via its agonist monoclonal anti-CD160 Ab clone CL1R2 (CL1R2 Ab) or HLA-C binding on peripheral blood NK cells drove a large amount of IFN-g, IL-6, and TNF-a (12) as well as IL-8 and MIP-1b but marginal amounts of IL-4 or IL-10 (55). The same authors reported their unpublished data that IL-1b, IL-5, IL-7, IL-12, IL-13, IL-17, G-CSF, and MCP-1 production was not detectable by NK cells via CL1R2 Ab or HLA-C (55). We have shown that HVEM-(Fc*) treatment of NK cells can drive proinflammatory cytokines including GM-CSF, GRO-a, MIP-1a, TNF-a, I-309, IL-1a, IL-1b, IL-6, IL-8, and IL-10 that did not require IL-2 priming, and IFN-g, IL-5, and IL-13 required IL-2 priming. We did not uncover any significant additional synergy of HVEM-(Fc*) with type 1 IFN (IFN-b), IL-12, or IL-15 beyond  what we observed for IL-2 in 65-plex Luminex-based cytokine quantitation. It is plausible that previous reports of undetectable levels of several cytokines reported is due to nonpriming of NK cells with IL-2.
The expression of HVEM ligands on NK cells can be regulated by stimulation with cytokines and tumor cells (59,60). For example, long-term stimulation of NK cells with high concentration of IL-2 or IL-15 downregulates the CD160-GPI isoform expression and simultaneously upregulates the CD160 transmembrane (CD160-TM) isoform expression (60). The data of Giustiniani et al. (60) suggests complex CD160 signaling specific to NK cells. Specifically, the GPI isoform lacks any intracellular domain. In contrast, the CD160-TM possesses an intracellular domain that induces phosphotyrosine-dependent Erk activation signaling via Src family kinase p56 lck . These data suggest that cytokines can modulate the expression of CD160 isoforms via chronic stimulation. Similarly, stimulation of NK cells with K562 tumor cells and IL-2 or IL-15 upregulates LIGHT expression, another HVEMinteracting receptor implicated in the enhancement of antitumor responses (59). Together, these data support the notion that stimulated NK cells become uniquely receptive to HVEM engagement. Secretion of cytokines via HVEM-activated NK cells (novel members identified in this paper) might further regulate the cascade of events leading to a specific and efficient recruitment of these receptors for their respective signaling pathways and the function(s) of NK cells and other immune cells. In our study, we found IL-2 induced expression of LIGHT and BTLA only on the CD56 bright NK cells (Fig. 4A, 4B). CD56 bright NK cells are mainly cytokine producers, more so found in lymphoid organs and considered to be precursors of CD56 dim NK cells, the latter mainly perform cytotoxicity in addition to cytokine production. Holmes et al. (59) have shown that tumor-activated NK cells enhanced the expression of LIGHT in human NK cells and are linked to the initiation of adaptive immunity via LIGHT-mediated NK-DC cross-talk. In this context, the modulation of LIGHT and BTLA expression on CD56 bright NK cells could regulate NK cell activity and subsequently participate in the shaping of adaptive immune responses. Overall, these findings support a mechanism for directing specific NK action within the HVEM-LIGHT-BTLA-CD160-LTb signaling network. This work serves as a basis for better understanding and ultimately adjusting immune responses for therapeutic benefit. When pursuing this application, it will be important to test in future studies additional potential mechanisms that might govern NK cell activation via the HVEM-LIGHT-BTLA-CD160-LTb signaling network including potentially counterregulatory mechanisms.
Interestingly, costimulation of bulk NK cells with HVEM-(Fc*) and IL-2 downregulates expression of CD160 and LIGHT on CD56 dim and CD56 bright NK cells, respectively (Fig. 4E, 4G). These data suggest that IL-2 priming modulates the expression of HVEM receptors distinctly on the two subsets of NK cells and cross-talk may occur between both cell types, resulting in a potentially additional layer of complexity in HVEM-stimulated NK responses.
We reasoned that the Fc portion of HVEM-Fc found in commercially available reagents could contribute to effects beyond HVEM receptor engagement on innate immune cells (i.e., via Fc receptor engagement). Recent literature suggests that NK cells also interact with other immune cells (e.g., monocytes, macrophages, or DC) for optimal cytokine production, cytotoxicity, and control of virally infected or tumor cells. It has been shown that both TLR ligands and pathogens can promote cross-talk between monocytes/ macrophages and NK cells via cell-cell contact and/or cytokines (36). We found that HVEM-(Fc*) activated monocytes and enhanced expression of activation markers CD40, CD80, and CD83 but not CD86 (Fig. 7). Our finding is consistent with previous reports demonstrating that LPS, similar to HVEM-(Fc*), enhanced the expression of activation markers CD40, CD80, and CD83 but not the expression of CD86 (58,61). Recent human and mouse studies have shown that CD80 and CD86 have differential roles in different disease states (62)(63)(64)(65). However, the significance of CD80 upregulation and CD86 downregulation on monocytes for subsequent immune responses is unknown.
We have also shown that HVEM-(Fc*)-induced NK cell cytotoxicity was enhanced when NK cells were cocultured with monocytes and is reduced when NK cells were separated by monocytes in a transwell coculture system. Likewise, HVEM-(Fc*)-induced IFN-g production in NK cell plus monocyte cocultures was significantly reduced when monocytes were separated by NK cells in transwells. Our data show that HVEM-(Fc*) promotes cross-talk between NK cells and monocytes in vitro that requires cell-cell contact for maximal enhancement of IFN-g secretion and optimum killing of K562 target cells.
In this study, we have reported for the first time, to our knowledge, a reagent HVEM-(Fc*) (which negligibly binds to the Fc receptor) activates monocytes and promotes cross-talk between NK cells and monocytes largely via cell-cell contact. Based on the expression levels of HVEM receptors on monocytes, we propose that it is most likely that HVEM-(Fc*) engages LIGHT for the activation of monocytes (versus CD160 on NK cells), which subsequently may provide for enhanced overall NK function.
In conclusion, we report in this study that HVEM-(Fc*) activates NK cells and monocytes and promotes NK cell and monocyte cross-talk for enhanced activation of NK cells and NK cell cytotoxicity. We have also shown that the Fc portion of HVEM-Fc but not HVEM-(Fc*) can interfere with augmenting NK function by reducing CD16 expression on NK cells and NK cell cytotoxicity via Fc-Fc receptor engagement. Our work supports the potential use of soluble HVEM as a therapeutic agent by eliminating the potentially counteracting effects of the Fc portion of conventional HVEM-Fc fusion proteins, consistent with Boice et al. (22).