Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes This information is current as of October 2, 2021. Qinglai Meng, Asifa K. Zaidi, John Sedy, Armand Bensussan and Daniel L. Popkin J Immunol published online 15 February 2019 http://www.jimmunol.org/content/early/2019/02/14/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 15, 2019, doi:10.4049/jimmunol.1801449 The Journal of Immunology

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 Asifa K. Zaidi,†,1 John Sedy,‡ Armand Bensussan,x and Daniel L. Popkin†,{,‖

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 therapeu- tics, 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 Downloaded from (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-g 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 http://www.jimmunol.org/ 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. 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. The Journal of Immunology, 2019, 202: 000–000.

atural killer cells, a subset of lymphoid cells, are an es- CD8 T cells (9). CD160 signals upon engagement of the widely sential component of the innate immune system that expressed molecules herpes virus entry mediator (HVEM) and/or

N protects against viruses (e.g., human CMV, HIV, HLA-C (10–12). The engagement of CD160 by soluble HVEM by guest on October 2, 2021 and hepatitis C virus), tumor cells, and other pathogens (1–5). NK (HVEM conjugated to the Fc portion of IgG1) or HVEM expressed cell innate immune responses are tightly regulated by multiple ac- on the cell surface was shown to activate NK cells (10). Genetic tivating and inhibitory receptors. Unlike typical activating and in- deficiency of CD160 in mice specifically impairs NK cell produc- hibitory receptors on NK cells, CD160 is tightly regulated in two tion of IFN-g, which is an essential component of the innate re- alternative splice variants: a GPI-anchored (CD160-GPI) form and a sponse to control tumor growth (13). differentially spliced transmembrane form of the protein (CD160-TM) HVEM is a member of the TNFR superfamily and is expressed on that is unique to NK cells. CD160 is part of the Ig superfamily of many immune cells, including NK cells, T and B cells, monocytes, receptors and it is predominantly expressed in peripheral blood NK and neutrophils (14–18). HVEM is an immune regulatory molecule cells, gd T (6), and CD8 T lymphocytes (7, 8) with cytolytic effector (15, 18) that signals bidirectionally both as a receptor and a ligand. activity. In circulating cells, the highest expression of CD160 RNA is HVEM interacts with three cell surface molecules, CD160, LIGHT identified in peripheral blood CD56dimCD16+ NK cells greater than (homologous to lymphotoxins [LT], shows inducible expression,

*Institute of Biomedical Sciences, Shanxi University, Xiaodian District, Taiyuan Research, the National Institute of Allergy and Infectious Diseases, and the Inter- City, Shanxi Province 030006, China; †Department of Dermatology, Case Western national AIDS Society. These contents are solely the responsibility of the authors Reserve University School of Medicine, Cleveland, OH 44106; ‡Infectious and In- and do not necessarily represent the official views of the National Institutes of flammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, Health or other funding sources. La Jolla, CA 92037; xINSERM UMR 976, Hoˆpital Saint-Louis, 75475 Paris Cedex { Address correspondence and reprint requests to Dr. Daniel L. Popkin, Case Western 10, France; Department of Pathology, Case Western Reserve University School of ‖ Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail address: Medicine, Cleveland, OH 44106; and Department of Molecular Biology and Micro- [email protected] biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 The online version of this article contains supplemental material. 1Q.M. and A.K.Z. equally contributed. Abbreviations used in this article: 7-AAD, 7-amino-actinomycin D; ADCC, ORCIDs: 0000-0002-6428-1054 (J.S.); 0000-0002-0409-2497 (A.B.). Ab-dependent cellular cytotoxicity; BTLA, B and T lymphocyte attenuator; DC, Received for publication November 2, 2018. Accepted for publication January 20, dendritic cell; hIgG1, human IgG1; HVEM, herpes virus entry mediator; LIGHT, 2019. homologous to lymphotoxins, shows inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes; LT, This work was supported by the Skin Diseases Research Center (P30AR039750 to D.L.P.), lymphotoxin; MFI, mean fluorescence intensity; rhCD16a, recombinant human Veterans Affairs Merit Award IBX002719A (to D.L.P.), a Doris Duke Charitable CD16a; SSC, side scatter. Foundation clinical scientist developmental award (to D.L.P.), and by the Creative and Novel Ideas in HIV Research Program through a supplement to the University of Ó Alabama at Birmingham Center for AIDS Research funding (P30 AI027767 to D.L.P.). Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 This funding was made possible by collaborative efforts of the Office of AIDS

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801449 2 HVEM AUGMENTS NK EFFECTOR FUNCTION BY NK–MONOCYTE CROSS-TALK and competes with HSV glycoprotein D for HVEM, a receptor Lymphoprep (STEMCELL Technologies) density gradient centrifugation expressed by T lymphocytes), and B and T lymphocyte attenuator as per manufacturer’s description from fresh human blood and fresh leu- (BTLA) and in humans with LT-a or TNF-b (14–18). HVEM kocyte reduction filters obtained from Red Cross, Cleveland, and cryopreserved where indicated. NK cells were isolated from PBMC by generates bidirectional signals, and recent literature provides ev- negative selection using the NK Cell Enrichment Kit (STEMCELL idence of signaling induced by interaction between HVEM and Technologies). Monocyte isolation was performed by positive selec- CD160, LIGHT, BTLA, or LT-a in different immune cells (7, 15, tion using CD14 microbeads and MACS columns (Miltenyi Biotec). HVEM- 19–25). The extracellular domain of HVEM was fused to the Fc expressing CHO cells (HVEM-CHO) were obtained from K. G. Kousoulas. CD160-CHO and HVEM-CHO were maintained in Ham’s F12 medium portion of human IgG1 (hIgG1) in previous studies to produce a plus modified Eagle’s minimal essential medium (1:1) supplemented with soluble protein used to detect HVEM ligands or alternatively to 10% FBS, 1% glutamine, and 1% penicillin and streptomycin. specifically activate BTLA or CD160 receptors (10, 26, 27). Be- Reagents and mAbs cause hIgG1 Fc binds to Fc receptor expressed on innate cells, including NK cells, HVEM-Fc fusion proteins may engage re- Soluble Fc-disabled HVEM-(Fc*) (product ID: HVEM-FcKO, SKU#: ceptors for both the HVEM domain and the Fc domain. Fc fusion FCL0096K; available at: https://www.gnpbio.com/index.php/products/ proteins have been widely used to interrogate the activities of cell 1050/11/recombinant-proteins-enzymes/human-hvem-ecd-extracellular- domain-protein-fc-fusion-adcc-ko-mutant-recombinant-detail) was custom surface proteins or soluble molecules and are widely used in im- synthesized by G&P Biosciences, Santa Clara, CA, and demonstrated having munotherapies such as etanercept, alefacept, and abatacept. The binding activity to recombinant BTLA and LIGHT protein and also to inhibit Fc domain of these fusion proteins may contribute to biological LIGHT-mediated signaling activities including cytotoxicity in L-929 activities unrelated to the fusion partner and which can be re- mouse fibroblast. Soluble-CD160 and HVEM-His were purchased from SinoBiological, Beijing, China. HVEM-Fc was purchased from G&P moved through mutation of the Fc domain. To determine how

Biosciences and PeproTech (Rocky Hill, NJ). The purity of fusion pro- Downloaded from HVEM engagement of NK cells may specifically function to ac- teins by SDS-PAGE were as follows: HVEM-(Fc*) .95% pure, en- tivate NK cells in the absence of Fc receptor binding, we gener- dotoxin level ,0.1 endotoxin unit/mg protein; HVEM-Fc .95% pure, ated fusion proteins constructed of the extracellular domain of endotoxin level ,0.1 endotoxin unit/mg protein; HVEM-his .90% , . HVEM conjugated to a mutant hIgG1 Fc that does not bind to Fc pure, endotoxin level 1 endotoxin unit/mg protein; CD160-his 95% pure, endotoxin level ,1 endotoxin unit/mg protein. Alefacept was a receptor (HVEM-[Fc*]) (28). generous gift from K. D. Cooper, Case Western Reserve University, LIGHT, a member of the TNF ligand superfamily, is mainly Cleveland, OH. Anti-CD3–PE, AF700 or allophycocyanin-Cy7, anti- expressed on T cells, monocytes, NK cells, and immature dendritic CD56–BV785 or PE-Cy7, anti-CD16–allophycocyanin (Southern http://www.jimmunol.org/ cells (DC) (29) and binds to HVEM and LTbR, two membrane Biotech) or BUV395 (BD Bioscience), anti-CD19–PE, anti-CD20–PE anti-CD80–FITC, anti-CD83–BV421, anti-CD86–PE-Cy7, anti-CD14–AF700, receptors (30). LIGHT–HVEM interactions are thought to regulate anti-CD69–allophycocyanin or allophycocyanin-Cy7, anti-CD107a– a variety of immune responses, for example, costimulation of allophycocyanin-Cy7 mAb, anti-LIGHT-PE, anti-BTLA-BV510, and the T cell proliferation, polarizing CD4 T cells into Th1 cells and corresponding isotype controls were from BioLegend. AF488-labeled anti- associated cytokine production (31), inducing DC maturation (31), CD160 and unlabeled anti-CD160 mAbs (clone 688327) were from R&D stimulating Ig production in B cells (32), and activating NK cells Systems. The anti-CD160 mAb (688327) was reported as a CD160 blocking Ab (48). We confirmed that 20 mg/ml anti-CD160 mAb (clone 688327) (19). This interaction enhances phagocytosis of monocytes and could completely block binding of 4 mg/ml HVEM-Fc to CD160 on neutrophils and contributes to antibacterial activity via production the CD160-expressing CHO (CD160-CHO) cells (data not shown). Hence, by guest on October 2, 2021 of ROS, NO, other proinflammatory cytokines, and direct bacte- 20 mg/ml anti-CD160 mAb was used in the blocking experiments. Annexin- ricidal activity (33). In contrast, engagement of HVEM with BTLA V–PE and 7-amino-actinomycin D (7-AAD) were purchased from eBioscience and BioLegend respectively. Cytokines IL-2, IL-12, IL-15, on T cells inhibits anti-TCR–induced activation and cytokine and IFN-b were purchased from PeproTech. secretion (34). We used CD160-his and hIgG1 as controls for HVEM-his and HVEM- The expression of CD160, LIGHT, and BTLA varies greatly (Fc*)/HVEM-Fc, respectively. CD160-his binds to HVEM-expressing between different cell types, activation, and differentiation states CHO cells but NK cells treated with CD160-his did not cause IFN-g (14, 18, 34, 35). NK cells mainly express CD160 and little to no production compared with HVEM-his (data not shown). Because CD160- his did not induce any functional responses similar to observations with expression of LIGHT or BTLA (14, 18, 34, 35). In contrast, hIgG control or media control, we have used hIgG1 as the control for monocytes, bone marrow–derived DC, and other immune cells HVEM-(Fc*). However, we acknowledge that the appropriate control express mainly LIGHT and little to no expression of BTLA or would be a fusion protein bearing an irrelevant protein fused with (Fc*) CD160 (14, 18, 34). Thus, dynamic regulation of these receptors and/or production of an HVEM-(Fc*) fusion protein mutated for loss of HVEM function to assess HVEM-(Fc*)–induced responses. However, this especially on NK cells and cells interacting with NK cells via was not synthesized because we observed the same response from CD160-His HVEM signaling provide a potential mechanism for control of ac- and hIgG1 and were resource limited. tivating and inhibitory signals depending on cellular context. NK cells interact with other immune cells for optimal cytokine Immunophenotyping of cells by flow cytometry production, cytotoxicity, and control of virally infected or tumor PBMCs within the live gate (7-AAD–negative population) were analyzed for 2 2 cells (36–47). There are several reports showing that monocytes, expression of HVEM and its receptors on CD56dim NK cells (CD14 /CD19 / 2 dim bright 2 2 2 bright DC, and macrophages interact with NK cells and cooperate in the CD3 /CD56 ), CD56 NK cells (CD14 /CD19 /CD3 /CD56 ), and CD14+ monocytes (CD14+/side scatter [SSC]high) by flow cytometry (BD innate immune response for protection against pathogens (36–47). LSRII), and data were analyzed by FlowJo (version 10.1; Tree Star). However, the role of HVEM-CD160 interactions in the activation of NK cells by other accessory cells expressing HVEM, its li- Cytokine expression analysis gands, or Fc receptors is not known. In this study, we investigated Supernatants from PBMC, NK cells, monocytes, and NK cell and monocyte the role of soluble Fc-disabled HVEM-(Fc*)–induced activation coculture were analyzed for IFN-g by ELISA (capture Ab [clone M700A] of NK cells and regulation of NK-mediated effector responses. and biotin-conjugated secondary Ab [clone M701B] were from Thermo Fisher Scientific) and were also analyzed by 65-plex Luminex array (Eve Technologies). Materials and Methods Cells Assessment of Fc binding to CD16 and HVEM binding to CD160 Fresh human blood was collected from healthy donors giving written in- formed consent at Case Western Reserve University, and the Institutional To assess the binding of the Fc stalk of HVEM-Fc and HVEM-(Fc*) to Internal Review Board approved all handling. PBMCs were isolated by CD16, beads conjugated with anti-His mAb (Miltenyi Biotec, Auburn, The Journal of Immunology 3

CA) were treated by His-tag–conjugated recombinant human CD16a binding (28, 53, 54). The entire HVEM extracellular domain was 6 (rhCD16a) at 4˚C for 2 h. Prebound rhCD16a beads (0.3 3 10 ) were conjugated to a loss-of-function Fc stalk (Fc*) of hIgG1 (HVEM- incubated with hIgG1 (10 mg/ml), HVEM-Fc (10 mg/ml; PeproTech), [Fc*]) that has been previously shown to lack Fc receptor func- HVEM-Fc (10 mg/ml; G&P Biosciences), or HVEM-(Fc*) (10 mg/ml; G&P Biosciences) on ice for 45 min. Then, beads were stained with tionality (Fig. 1A). Six amino acids were mutated at position anti-HVEM–PE mAb (1 mg/ml) at 4˚C for 30 min and were analyzed by E233P, L234V, L235A, DG236, A327G, A330S on the Fc* stalk flow cytometry. of hIgG1 as described (28). The binding capacity of the Fc* stalk To assess the binding capacity of HVEM-Fc and HVEM-(Fc*) to of HVEM-(Fc*) was tested using rhCD16a-conjugated beads by CD160, CHO cells expressing CD160 (CD160-CHO cells) were incubated with hIgG1 (10 or 40 mg/ml), HVEM-Fc (5 or 10 mg/ml; PeproTech), or flow cytometry. HVEM-(Fc*) had significantly reduced mean HVEM-Fc and HVEM-(Fc*) (10, 20, or 40 mg/ml; G&P Biosciences) on fluorescence intensity (MFI) (1267 6 421) compared with ice for 45 min. After washing, cells were incubated with PE-conjugated HVEM-Fc MFI (67266 6 5232) (Fig. 1B). These data show that goat anti-hIgG Fc secondary Ab on ice for 45 min. Then, cells were washed the loss-of-function Fc* stalk of HVEM-(Fc*) does not bind ef- and stained with mouse anti-human HVEM–PerCP-Cy5.5 (2 mg/ml) Ab at ficiently to rhCD16a-conjugated beads compared with HVEM- room temperature for 15 min. Cells were analyzed by flow cytometry. Fc containing the wild type Fc stalk. The binding capacity NK cell cytotoxicity assay of HVEM-Fc and HVEM-(Fc*) to CD160 was tested using Frozen PBMCs from healthy controls were thawed and cultured overnight CD160-expressing CHO cells. As shown in Fig. 1C, both in RPMI 1640 supplemented with 10% FBS, 1% glutamine, and 1% HVEM-Fc and HVEM-(Fc*) had comparable MFI (7814 6 587 penicillin and streptomycin. PBMCs were then incubated with or without and 6900 6 469, respectively) and percentage of HVEM+ cells 1 mg/ml HVEM-(Fc*) for 42–44 h. Activation marker CD69 on NK cells (93 6 3% versus 88 6 4%). Taken together, these data demon- was measured after 44 h by flow cytometry. PBMC were further cocultured strate that HVEM binding to CD160 is comparable for both with prelabeled K562 cells at an E:T ratio of 20:1 in the presence of anti- Downloaded from CD107a–allophycocyanin-Cy7 for 5 h. K562 cells were stained with HVEM-Fc and HVEM-(Fc*), but binding to the Fc receptor of 7AAD and annexin-V–PE to quantitate dead cells (7AAD+annexin-V+)by HVEM-(Fc*) is reduced 50-fold compared with HVEM-Fc. As an flow cytometry. additional control, we used HVEM-His (which does not contain an Where described, cytotoxicity assays were performed using purified Fc binding stalk) to determine HVEM ligand function uncoupled NK cells (purity $95%) and monocytes (purity $95%) in cocultures. Briefly, NK cells were purified from thawed overnight-cultured PBMC from Fc–Fc receptor effects in subsequent experiments. by negative selection. Monocytes were purified by CD14-positive se- To evaluate the effect of HVEM on general activation of lection from the same donor. Purified NK cells (0.3 3 106), purified NK cells, we cultured purified NK cells with HVEM-(Fc*) and http://www.jimmunol.org/ 3 6 monocytes (0.3 10 ), or purified NK cells plus purified monocytes compared its effect with HVEM-Fc–induced responses. The coculture at a ratio of 1:1 (0.3 3 106 +0.33 106) was performed in the presence or absence of HVEM-(Fc*) (1 mg/ml) for 44 h. After 44 h, cells percentage of NK cells expressing CD69 that were treated with were then stained for activation markers and analyzed by flow cytometry. HVEM-(Fc*) was similar to the percentage of NK cells that were NK cells, monocytes, or NK cells plus monocyte mixture were further treated with hIgG1 (Fig. 2A). In contrast, HVEM-Fc treatment cocultured with labeled K562 cells at a ratio of NK cells to K562 cells of increased 3.4-fold and 4-fold percentage of NK cells expressing 1.5:1 for 5 h in the presence of anti-CD107a mAb–allophycocyanin-Cy7 CD69 compared with HVEM-(Fc*) and IgG1 treatment, respec- 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+) tively (Fig. 2A). Despite absent percentage of CD69 induction on by flow cytometry. For transwell cultures (96-well plate), NK cells NK cells by HVEM-(Fc*), this Fc-disabled HVEM was a more by guest on October 2, 2021 (0.3 3 106 cells per well) were seeded into the lower chamber, and potent inducer of IFN-g in the presence of IL-2 from NK cells than 3 6 monocytes (0.3 10 cells per well) were seeded in the upper chamber or HVEM-Fc (Fig. 2B) but not in the absence of IL-2 (Supplemental 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 Fig. 1A). These data suggested that HVEM-induced NK cell pro- 44 h. Activation markers and cytotoxicity were measured identically to duction of IFN-g required IL-2 priming. above in nontranswell cultures. Additionally, we observed increased NK cell death with HVEM- We performed reverse Ab-dependent cellular cytotoxicity (ADCC) assay Fc versus HVEM-(Fc*) and hIgG1. This is likely attributable to using the well-characterized P815 mouse mastocytoma cell line that NK fratricide via Fc–Fc receptor binding (Fig. 2C) resulting in the abundantly expresses mouse CD16 (mouse Fc receptor) as a target cell to evaluate the activity of HVEM-(Fc*)–induced activation of NK cells. observed reduction in CD16 (Fc receptor) expression on NK cells This is a well-studied model, similar to K562 killing assays for spon- after HVEM-Fc treatment (Fig. 2D). Furthermore, we suspect that taneous killing, to ascertain reverse ADCC assay with a defined system. apparent IFN-g secretion induced by CD16 and/or CD160 ligation A mouse anti-human CD16 monoclonal is used to bridge human NK cell of HVEM-Fc would be attenuated as compared with HVEM-Fc* effectors with mouse P815 mastocytoma targets resulting in reverse ADCC assay (49). Briefly, the NK cells were pretreated with either (Fig. 2B) secondary to HVEM-Fc–induced fratricide (i.e., HVEM- HVEM-Fc, HVEM-(Fc*), or IgG1 for 16 h. Cells were washed to remove Fc–Fc receptor cross-linking likely occurs before these NK cells the unbound stimuli (HVEM-Fc, HVEM-[Fc*], or IgG1) and then were fully produced and/or released IFN-g from HVEM-Fc engage- cocultured with P815 target cells in the presence of mouse anti-human ment [Fig. 2C]). CD16 Ab (containing a murine Fc stalk) or its isotype control Ab to In contrast, HVEM-(Fc*) poorly engaged CD16 (Fig. 1B) and did evaluate the effect of HVEM-(Fc*) on NK cell reverse ADCC assay. not cause a reduction in CD16 expression (Fig. 2D) nor increased Statistical analysis NK cell death (Fig. 2C). Thus, HVEM-(Fc*) treatment of NK cells Student paired t test was performed to determine the difference between significantly preserved NK cell viability and CD16 expression two groups unless otherwise indicated. All tests were considered statisti- compared with HVEM-Fc and hIgG1 treatment (Fig. 2D). cally significant at p , 0.05. Given the somewhat unexpected findings of increased IFN-g production and decreased NK cell death with HVEM-(Fc*) versus Results HVEM-Fc, we then measured NK cell–induced cytotoxicity of K562 Soluble HVEM-(Fc*) activates NK cells and induces IFN-g target cells. HVEM-(Fc*) enhanced NK cell–induced cytolysis of production and cytotoxicity independent of Fc binding K562 cells (Fig. 2E). Surprisingly, HVEM-Fc caused significantly HVEM-Fc fusion proteins have been used to study the interaction reduced spontaneous killing of K562 cells by NK cells (Fig. 2E) between HVEM and its ligands on NK cells and immune re- compared with HVEM-(Fc*) and hIgG1. We also tested the effect sponses (10, 48, 50–52). We sought to determine how a soluble of HVEM-(Fc*)–activated NK cells in a classic reverse ADCC HVEM-Fc fusion may function independent of Fc receptor in- assay using the well-characterized P815 mouse mastocytoma cell teractions through mutation of residues required for Fc receptor line that abundantly expresses mouse CD16 (mouse Fc receptor) 4 HVEM AUGMENTS NK EFFECTOR FUNCTION BY NK–MONOCYTE CROSS-TALK Downloaded from

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- http://www.jimmunol.org/ deleted Fc stalk (amino acid residues T223-K447) of hIgG1. Soluble Fc-disabled HVEM-(Fc*) fusion protein was generated by mutating six amino acid residues at position E233P, L234V, L235A, DG236, 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) rhCD16a- (Fc receptor) preconjugated beads were in- cubatedwith10mg/ml hIgG, HVEM-Fc (from PeproTech or G&P Biosciences), or HVEM-(Fc*) (from G&P Biosciences) on ice for 45 min. After washing, beads were stained with PE-conjugated anti-HVEM mAb (1 mg/ml) and analyzed by ﬂow cytometry. X-axis shows the ﬂuorescence intensity of beads incubated with hIgG1, HVEM-Fc (PeproTech and G&P Biosciences), or HVEM-(Fc*) (G&P Biosciences). (C) CD160-expressing CHO cells (CHO-CD160) were incubated with 10 mg/ml hIgG1, HVEM-Fc (PeproTech and G&P Biosciences), 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 mg/ml), and then cells were analyzed by +

flow cytometry. Percentages of HVEM cells are shown. by guest on October 2, 2021 as described in the Materials and Methods section. HVEM-(Fc*) comparison cannot be made with purified NK cell data. We observed a but not HVEM-Fc enhanced killing of P815 target cells via NK- mild increase in NK cell degranulation as assessed by surface CD107a mediated ADCC (Fig. 2F). In addition, HVEM-(Fc*)–induced expression (Fig. 2J) consistent with our direct cytotoxicity measure- spontaneous killing and ADCC did not require IL-2 priming ments (Fig. 2I). These data suggest that HVEM-(Fc*) stimulates NK (Supplemental Fig. 1B, 1C) as was required for IFN-g production cells intrinsically for IFN-g production and spontaneous killing. (Fig. 2B, Supplemental Fig. 1A). IL-2 priming trended to, but did However, CD69 and thus potentially other metrics of HVEM- not significantly, enhance soluble HVEM-induced spontaneous (Fc*) activation may be further augmented by accessory cell(s). killing of K562 cells (p = 0.074) nor reverse ADCC of P815 cells We pursued this line of investigation in Figs. 3,4,5 and 6 below. (p = 0.21) (Supplemental Fig. 1D, 1E). These data demonstrate that HVEM-(Fc*) induces greater NK IFN-g production in addi- HVEM-(Fc*) drives broad NK cytokine production that can tion to spontaneous killing and ADCC as compared with HVEM- be further augmented by IL-2 more so than other Fc. Taken together, we demonstrate that CD16 engagement with NK cell–stimulating cytokines the Fc stalk of HVEM-Fc can interfere with the ability for a soluble Previously, it was reported that CD160 signaling induces NK HVEM reagent to augment NK effector function. This has impor- production of IFN-g, IL-6, IL-8, TNF-a, MIP-1b, and lower tant therapeutic applications. Therefore, we focused on character- amounts of IL-4 and IL-10 (12, 55). To more comprehensively izing HVEM-(Fc*) for all subsequent studies described below. evaluate the effects of soluble HVEM on cytokine production Several recent studies suggest that NK cell function can be in a relatively unbiased manner, we performed 65-plex cytokine modulated via APCs such as monocytes, macrophages, and DC protein array analysis using the Luminex platform. We repeated (36–47). These cells also express HVEM receptors. Therefore, we this analysis in the presence of media 6 the best characterized tested the effect of soluble HVEM-(Fc*) on NK cell function NK-activating cytokines (IL-2, IFN-b, IL-12, and IL-15) to more when cocultured in the presence of the physiological comple- fully capture the stimulating potential of soluble HVEM given ment of immune cells present in circulation. As shown in Fig. 2G–J, that IFN-g production required the presence of IL-2 (Fig. 2B, in contrast to purified NK cells, soluble HVEM-(Fc*)–treated Supplemental Fig. 1A). We reasoned that other NK-activating PBMCs caused CD69 induction on NK cells (Fig. 2G). Similar to cytokines might be required for the production of additional cy- purified NK cells, soluble HVEM-(Fc*) significantly increased tokines similarly to what has been observed for NK cell IFN-g IFN-g production (Fig. 2H) and increased lysis of K562 cells production. HVEM-(Fc*)–stimulated NK cells produced significant (Fig. 2I) in treated PBMCs. However, given the difficulty in levels of GM-CSF, GRO-a, MIP-1a, TNF-a, I-309, IL-1a, IL-1b, characterizing the effector component of bulk PBMCs, a direct IL-6, IL-8, and IL-10 without IL-2 priming compared with hIgG1 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. HVEM-(Fc*) activates NK cells and induces IFN-g production and cytotoxicity. (A–F) Purified NK cells from PBMC of healthy controls (n =9) were cultured with 20 mg/ml 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-g was quantitated from supernatants via ELISA, (C) viability was measured via 7-AAD staining, (D) and CD16 cell surface expression was examined on CD56dimCD16+ NK cells. (E and F)Toevaluate(E) spontaneous killing and (F) reverse ADCC activity, NK cells cultured with 20 mg/ml hIgG, HVEM-(Fc*), or HVEM-Fc for 16 h were further cocultured with target K562 cells or P815 cells, respectively, prelabeled with fluorescent dye at effector E:T ratio of 1:1 and 0.5:1 for 5 h. For reverse ADCC assays, each coculture was treated with 10 mg/ml mouse IgG1 or anti-CD16 mAb. Dead target cells were gated as 7-AAD+ cells. (G–J) PBMC from healthy controls (n =9except[H] where n = 5) were cultured with PBS or HVEM-(Fc*) (1 mg/ml) for 44 h. Then, cell surface (G) CD69 expression on NK cells and (H)IFN-g secretion in culture supernatants were determined via flow cytometry and ELISA, respectively.

Next, (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 by guest on October 2, 2021 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. Degranulation and cytotoxicity of NK cells were quantified by cellsurface (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.

(Fig. 3A), whereas HVEM-(Fc*)–induced IL-5 and IL-13, similar vast majority of circulating NK cells. In contrast, LIGHT and BTLA to IFN-g, required IL-2 priming of NK cells (Fig. 3B, 3C). During are expressed at low levels on both CD56dim and CD56bright NK cells infection, type I IFN, IL-12, and IL-15 production promotes (10, 35). IL-2 priming enhances HVEM-(Fc*)–stimulated cytokine NK cell effector function (56, 57). When we evaluated the effect production and might enhance it by increasing expression of NK cell of these cytokines in 65-plex Luminex-based assays, we found HVEM receptors. Therefore, we determined whether IL-2 priming that only IFN-g (Fig. 3D) production was significantly augmented enhanced CD160, LIGHT, and/or BTLA (receptors of HVEM) ex- by IFN-b, IL-12, and IL-15 versus media alone and that HVEM- pression on NK cells. As shown in Fig. 4, Supplemental Fig. 2, IL-2 (Fc*)–induced production of other cytokines (e.g., I-309 and priming of purified bulk NK cells upregulated expression of LIGHT GM-CSF, Fig. 3E, 3F) were distinctly affected by the presence of (Fig. 4A) and BTLA (Fig. 4B) on CD56bright but not CD56dim NK IFN-b, IL-12, and IL-15 (Fig. 3). IL-2 and IL-15 treatment sig- cells. IL-2 priming of purified bulk NK cells did not change CD160 nificantly enhanced but IFN-b treatment significantly decreased expression on either CD56dim or CD56bright subsets of NK cells HVEM-(Fc*)–induced GM-CSF and I-309 production (Fig. 3E, (Fig. 4C). We confirmed IL-2 activation of purified bulk NK 3F). Overall, we uncovered the production of additional cytokines cells by demonstrating upregulation of CD69 expression on both (GM-CSF, GRO-a, MIP-1a, I-309, IL-1a, and IL-1b) that, to our subsets of NK cells (Fig. 4D). Interestingly, HVEM-His treatment knowledge, were not previously published, and that were elabo- of purified bulk NK cells reduced cell surface expression of rated directly by soluble HVEM treatment and did not require the LIGHT on CD56bright NK cells in the presence of IL-2 (Fig. 4E) presence of IL-2 or other NK-activating cytokines. Moreover, we but did not change BTLA expression on CD56bright NK cells either identified that IL-5 and IL-13 could be induced by HVEM-(Fc*) in the presence and absence of IL-2 (Fig. 4F). CD160 expression on in the presence of IL-2, similarly to IFN-g. These data highlight CD56dimCD16+ NK cells that was not affected by IL-2 priming the potential therapeutic application of HVEM-(Fc*). but was reduced by HVEM-His treatment and HVEM-his–induced reduction in CD160 was further enhanced by the presence of IL-2 IL-2 priming upregulates LIGHT and BTLA but not CD160 dim + bright on CD56 CD16 NK cells (Fig. 4G). These data suggest that expression on CD56 NK cells IL-2 priming distinctly enhanced the cell surface of expression NK cells are divided into two major subtypes: CD56dim and CD56bright. HVEM receptors on different NK cell subsets and may enhance CD160 is mostly expressed on CD56dim NK cells, which make up the NK cell function differentially by HVEM activation. 6 HVEM AUGMENTS NK EFFECTOR FUNCTION BY NK–MONOCYTE CROSS-TALK Downloaded from http://www.jimmunol.org/ FIGURE 3. HVEM-(Fc*)–induced cytokines in NK cells. NK cells purified from PBMC of healthy controls (n = 5) were cultured in wells precoated with 20 mg/ml hIgG1 or HVEM-(Fc*) in the presence of (A) media only versus (B–F) media, IL-2 (100 IU/ml), IFN-b (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. (A)–(F) show the cytokines whose concentrations were significantly changed by HVEM-(Fc*) treatment compared with hIgG. (E) and (F) show the HVEM-(Fc*)–induced cytokines minus hIgG-induced cytokines. *p , 0.05, **p , 0.01, ***p , 0.001.

Because CD160 expression was not enhanced by soluble HVEM LIGHT at low levels (Fig. 4). To test the potential sole effect of treatment of NK cells and BTLA was identiﬁed as an inhibitory HVEM via LIGHT, we made use of the KHYG1 NK cell line, receptor (10), we tested whether HVEM activates NK cells via which we discovered only expressed LIGHT and not BTLA by guest on October 2, 2021 LIGHT engagement. Human CD56dim NK cells only express (Fig. 5A). The surface expression of CD160 on KHYG1 was CD160. However, both CD56bright and CD56dim NK cells express similar to background isotype control (Fig. 5A). HVEM-His

FIGURE 4. IL-2 priming upregulates LIGHT and BTLA expression on CD56bright NK cells, and cotreatment 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 versus CD56dimCD16+ subsets of bulk NK cells using mAbs (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 Supplemental Fig. 2. (E–G) Purified NK cells from healthy donor PBMC (n = 6) were incubated with media or plate-bound HVEM-His (20 mg/ml, 20 ml/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 mAbs (clones T5-39, J168-540, 688327, and FN-5, respectively) by flow cytometry. *p , 0.05, ***p , 0.001. The Journal of Immunology 7 Downloaded from

FIGURE 5. Ligation of HVEM-(Fc*) with CD160 and LIGHT can contribute to IFN-g production from NK cells. The KHYG1 NK cell line was http://www.jimmunol.org/ analyzed for (A) LIGHT (left panel), BTLA (middle panel), and CD160 (right panel) expression using mAbs (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 Ab staining; light blue histogram, receptor Ab staining. (B) KHYG1 cells were incubated with media or plate-bound HVEM-His (20 mg/ml) for 18 h. After 18 h, cell-free supernatants were analyzed for IFN-g production via ELISA. Mean 6 SEM shown (C) Purified NK cells from healthy controls (n = 4), preincubated with isotype control Ab (mouse IgG2b), or anti-human CD160 blocking Ab at 20 mg/ml, were cultured with HVEM-(Fc*) precoated on a 384-well plate at the indicated concentration shown on the x-axis for 18 h. After 18 h, IFN-g production was quantified from cell-free supernatants via ELISA. *p , 0.05, **p , 0.01.

treatment of KHYG1 NK caused significant production of IFN-g why PBMC cultures showed different results than isolated NK by guest on October 2, 2021 (Fig. 5B). Given this finding, we evaluated HVEM-induced acti- cells with respect to soluble HVEM treatment (Fig. 2). We found vation via LIGHT on purified NK cells by incubating them with that monocytes expressed LIGHT and BTLA and there was no HVEM-(Fc*) in the presence of anti-CD160 blocking Ab (clone CD160 expression (Fig. 6). Among monocytes, NK cells, and 688327; R&D Systems). We found that HVEM-(Fc*) caused DC, LIGHT expression was greatest on monocytes. Next, we significant production of IFN-g from NK cells after blocking of tested whether HVEM-(Fc*) activated monocytes similarly to CD160 receptor (Fig. 5C). We hypothesize that CD160 blockade– NK cells. Activation of monocytes was measured by surface mediated enhancement of IFN-g production from HVEM-(Fc)*– expression of CD80, CD83, CD86, and CD40 (Fig. 7). Ex- treated NK cells resulted from more HVEM-(Fc)* binding of pression of activation markers CD80, CD83, and CD40 but not other HVEM ligands, such as LIGHT. The IL-2–pretreated NK CD86 was enhanced by HVEM-(Fc*) treatment of both purified cells express high levels of CD160 (the majority is localized on monocytes (Fig. 7A) and flow-gated monocytes in PBMC cul- the CD56dim subset and the minority is localized on the CD56hi tures (Fig. 7B), consistent with Schwarz et al. (58) who also subset) and low levels of LIGHT and BTLA, which are mostly found upregulation of CD80, CD83, and CD40 but downregu- localized on the CD56hi subset. In the physiological condition lation of CD86 in response to LPS. Similar to HVEM-(Fc*), (absence of CD160 blocking Ab), CD160 will compete with HVEM-His also activated both monocytes and NK cells in LIGHT, BTLA, or other unknown ligand(s) on the NK cells to PBMC cultures (Supplemental Fig. 3), confirming that solu- bind with plate-bound HVEM-(Fc)*. Therefore most IFN-g is ble HVEM could directly activate monocytes in addition to likely released from NK cells by ligation of HVEM and CD160. NK cells. Conversely, in the presence of anti-CD160 blocking Ab, the HVEM-(Fc*) promotes cross-talk between NK cells other HVEM ligands, such as LIGHT, will have a greater like- and monocytes lihood to bind with HVEM-(Fc)*, and this binding might elicit greater IFN-g production than CD160-HVEM binding. These Next, we reasoned that because monocytes can modulate NK data support the notion that HVEM may activate NK cells via activity, were activated by HVEM-(Fc*), and NK cell activation LIGHT in addition to CD160. was different in PBMC cultures versus enriched NK cells incubated with HVEM-(Fc*), that monocytes may play a role in HVEM-(Fc*) promotes activation of monocytes HVEM-(Fc*)–induced NK activation and subsequent effector Several reports have demonstrated that monocytes and macro- functions. Thus, we compared purified NK cells cultured with phages regulate NK cell effector function(s) (36, 38–41, 43–46). and without purified monocytes in the presence or absence of Monocytes also express receptors of HVEM (18, 34). Thus, we HVEM-(Fc*). Activation of NK cells was quantified via cell determined the level of expression of all three HVEM receptors surface CD69 expression, degranulation via CD107a, cytotoxicity on monocytes as a readily available cell type that may explain to K562 target cells, and IFN-g production. 8 HVEM AUGMENTS NK EFFECTOR FUNCTION BY NK–MONOCYTE CROSS-TALK Downloaded from

FIGURE 6. Monocytes express LIGHT greater than BTLA among potential HVEM receptors. PBMCs from healthy donors (n = 8) were stained for (A) 2 2 2 CD160, (B) LIGHT, or (C) BTLA and gated as shown in (D) for monocytes (CD14+/SSChigh), NK cells (CD14 /CD19 /CD3 /CD56+), conventional DC http://www.jimmunol.org/ (cDC; CD32/CD142/HLA-DR+/CD11c+), and plasmacytoid DC (pDC; CD32/CD142/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 Ab staining; light blue histogram, receptor Ab staining. *p , 0.05, ***p , 0.001.

When purified NK cells were cocultured with purified monocytes was enhanced by coculture of NK and monocytes as compared in the presence of HVEM-(Fc*), there was significant enhancement with separation of these cells by transwell membrane (Fig. 9B). of CD69 (Fig. 8A) and CD56 (Fig. 8B) cell surface expression on Additionally, there was more significant HVEM-(Fc*)–induced the NK cells. There was also enhanced NK cell degranulation (in- K562 cytotoxicity in cocultures of NK and monocytes as com- creased cell surface expression of CD107a) and enhanced cyto- pared with separation of these cells by transwell membrane by guest on October 2, 2021 toxicity toward K562 cells when NK cells were cocultured with (Fig. 9C). This mild difference in cytotoxicity was not observed monocytes in the presence of HVEM-(Fc*) (Fig. 8C, 8D) compared in NK cell degranulation as measured by CD107a expression with NK cells cultured with HVEM-(Fc*). These data demonstrate (Fig. 9D). HVEM-(Fc*)–induced activation of monocytes in that monocytes can augment HVEM-(Fc*)–induced NK cell acti- transwell cocultures was assessed by cell surface expression of vation and effector function. CD80, CD86, CD83, and CD40 (Supplemental Fig. 4). Similar to To test whether monocytes augment NK cell effector function via the HVEM-(Fc*)–treated purified monocytes (Fig. 7), there was cell–cell contact or soluble mediators, NK cells were cocultured significant enhancement of CD80, CD83, and CD40 but not with monocytes in the presence or absence of HVEM-(Fc*) in a CD86 (Supplemental Fig. 4) expression on monocytes in the transwell tissue culture plate. Monocytes or NK cells were either transwell cocultures. Taken together, these transwell experiments cultured separately in the upper or lower chambers respectively indicated that HVEM-(Fc*) activation of NK via NK–monocyte separated by transmembrane or cultured together in the bottom cross-talk required cell–cell contact mechanism(s) for maximal chamber in a transwell culture plate. Transwell separation of purified enhancement of IFN-g production and potentially mild enhance- NK cells and monocytes resulted in a slightly lower basal expression ment of spontaneous killing. of CD69 (Fig. 9A); however, this was not statistically significant. Otherwise,HVEM-(Fc*)similarlyinducedCD69expressioninNK Discussion cell and monocyte transwell separated cultures as compared with NK cells play a key role in the protection from pathogens and culturing both cell types together in the bottom chamber (Fig. 9A). In cancer. NK cell–mediated effector function depends on the type and contrast, we observed that HVEM-(Fc*)–induced IFN-g production number of receptor/ligand interactions occurring between NK cells

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 mg/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. The Journal of Immunology 9

FIGURE 8. HVEM-(Fc*) promotes cross-talk between monocytes and NK cells, enhancing activation and cytotoxicity of NK cells. (A–D) Purified Downloaded from 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. HVEM-(Fc*) treatment–enhanced NK cell cytotoxicity was evaluated as (C) percentage of 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 + MN as the effector cells. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ and their targets. In this context, it has been shown that CD160 (enhanced CD107a expression), and cytolysis by NK cells as drives NK cell activation, cytokine production, and cytotoxicity compared with wild type HVEM-Fc. via engagement with HLA-C (12) and HVEM (10, 16, 18, 48). In We have also shown for the first time, to our knowledge, that this study, we have shown that HVEM conjugated to the wild type HVEM induces a broader array of cytokines than previously sequence of Fc (HVEM-Fc) activates NK cells; however, the Fc demonstrated, which occurred in both IL-2–dependent and portion can cause interaction and activation of other Fc receptor– –independent manners. Previously, it has been reported that en- expressing immune cells via Fc–Fc receptor ligation in addition to gaging CD160 via its agonist monoclonal anti-CD160 Ab clone HVEM (Figs. 1, 2). This is notable given this form of HVEM-Fc is CL1R2 (CL1R2 Ab) or HLA-C binding on peripheral blood NK the reagent readily available “off the shelf” from multiple vendors cells drove a large amount of IFN-g, IL-6, and TNF-a (12) as well by guest on October 2, 2021 including BioLegend, R&D Systems, Sigma-Aldrich, SBH Sci- as IL-8 and MIP-1b but marginal amounts of IL-4 or IL-10 (55). ences, AB Biosciences, BPS Bioscience, Enzo, PromoCell, Tonbo The same authors reported their unpublished data that IL-1b, IL-5, Biosciences, GenScript, G&P Biosciences, and others. We IL-7, IL-12, IL-13, IL-17, G-CSF, and MCP-1 production was designed and evaluated a soluble Fc-disabled HVEM to interro- not detectable by NK cells via CL1R2 Ab or HLA-C (55). We gate the specific function of HVEM-mediated NK cell responses have shown that HVEM-(Fc*) treatment of NK cells can drive and provide an alternative reagent for potential therapeutic ap- proinflammatory cytokines including GM-CSF, GRO-a, MIP-1a, plication. To do this, we fused the HVEM extracellular domain to TNF-a, I-309, IL-1a, IL-1b, IL-6, IL-8, and IL-10 that did not a mutant Fc stalk that poorly binds the Fc receptor HVEM-(Fc*). require IL-2 priming, and IFN-g, IL-5, and IL-13 required IL-2 HVEM-(Fc*) binding to CD160 on primary NK cells resulted priming. We did not uncover any significant additional synergy of in increased inflammatory cytokine expression, degranulation HVEM-(Fc*) with type 1 IFN (IFN-b), IL-12, or IL-15 beyond

FIGURE 9. HVEM-(Fc*)–induced IFN-g 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 mg/ml) for 44 h. After 44 h, cell surface (A) CD69 expression on NK cells was measured by flow cytometry, and (B) IFN-g production in cell-free supernatants was quantified via ELISA. (C and D) Cells were treated as above with HVEM-(Fc*) for 44 h and subsequently cocultured with prelabeled K562 cells in the presence of anti- CD107a Ab for 5 h. (C) Dead 7AAD+annexin-V+ K562 cells were quantified, and (D)NKcell CD107a expression was quantified, all by flow cytometry. *p , 0.05, **p , 0.01. 10 HVEM AUGMENTS NK EFFECTOR FUNCTION BY NK–MONOCYTE CROSS-TALK what we observed for IL-2 in 65-plex Luminex-based cytokine but not CD86 (Fig. 7). Our finding is consistent with previous reports quantitation. It is plausible that previous reports of undetectable demonstrating that LPS, similar to HVEM-(Fc*), enhanced the ex- levels of several cytokines reported is due to nonpriming of NK pression of activation markers CD40, CD80, and CD83 but not the cells with IL-2. expression of CD86 (58, 61). Recent human and mouse studies have The expression of HVEM ligands on NK cells can be regulated shown that CD80 and CD86 have differential roles in different by stimulation with cytokines and tumor cells (59, 60). For ex- disease states (62–65). However, the significance of CD80 upregu- ample, long-term stimulation of NK cells with high concentration lation and CD86 downregulation on monocytes for subsequent im- of IL-2 or IL-15 downregulates the CD160-GPI isoform expres- mune responses is unknown. sion and simultaneously upregulates the CD160 transmembrane We have also shown that HVEM-(Fc*)–induced NK cell cytotoxicity (CD160-TM) isoform expression (60). The data of Giustiniani was enhanced when NK cells were cocultured with monocytes and is et al. (60) suggests complex CD160 signaling specific to NK cells. reduced when NK cells were separated by monocytes in a transwell Specifically, the GPI isoform lacks any intracellular domain. In coculture system. Likewise, HVEM-(Fc*)–induced IFN-g production contrast, the CD160-TM possesses an intracellular domain that in NK cell plus monocyte cocultures was significantly reduced when induces phosphotyrosine-dependent Erk activation signaling via monocytes were separated by NK cells in transwells. Our data show Src family kinase p56lck. These data suggest that cytokines can that HVEM-(Fc*) promotes cross-talk between NK cells and mono- modulate the expression of CD160 isoforms via chronic stimula- cytes in vitro that requires cell–cell contact for maximal enhancement tion. Similarly, stimulation of NK cells with K562 tumor cells of IFN-g secretion and optimum killing of K562 target cells. and IL-2 or IL-15 upregulates LIGHT expression, another HVEM- In this study, we have reported for the first time, to our knowledge, interacting receptor implicated in the enhancement of antitumor a reagent HVEM-(Fc*) (which negligibly binds to the Fc receptor) responses (59). Together, these data support the notion that activates monocytes and promotes cross-talk between NK cells and Downloaded from stimulated NK cells become uniquely receptive to HVEM en- monocytes largely via cell–cell contact. Based on the expression gagement. Secretion of cytokines via HVEM-activated NK cells levels of HVEM receptors on monocytes, we propose that it is most (novel members identified in this paper) might further regulate the likely that HVEM-(Fc*) engages LIGHT for the activation of cascade of events leading to a specific and efficient recruitment of monocytes (versus CD160 on NK cells), which subsequently may these receptors for their respective signaling pathways and the provide for enhanced overall NK function. function(s) of NK cells and other immune cells. In our study, we In conclusion, we report in this study that HVEM-(Fc*) activates http://www.jimmunol.org/ found IL-2 induced expression of LIGHT and BTLA only on the NK cells and monocytes and promotes NK cell and monocyte CD56bright NK cells (Fig. 4A, 4B). CD56bright NK cells are cross-talk for enhanced activation of NK cells and NK cell cyto- mainly cytokine producers, more so found in lymphoid organs and toxicity. We have also shown that the Fc portion of HVEM-Fc but considered to be precursors of CD56dim NK cells, the latter mainly not HVEM-(Fc*) can interfere with augmenting NK function by perform cytotoxicity in addition to cytokine production. Holmes reducing CD16 expression on NK cells and NK cell cytotoxicity et al. (59) have shown that tumor-activated NK cells enhanced via Fc–Fc receptor engagement. Our work supports the potential the expression of LIGHT in human NK cells and are linked to use of soluble HVEM as a therapeutic agent by eliminating the the initiation of adaptive immunity via LIGHT-mediated NK–DC potentially counteracting effects of the Fc portion of conventional by guest on October 2, 2021 cross-talk. In this context, the modulation of LIGHT and BTLA HVEM-Fc fusion proteins, consistent with Boice et al. (22). expression on CD56bright NK cells could regulate NK cell activity and subsequently participate in the shaping of adaptive immune Disclosures responses. Overall, these findings support a mechanism for The authors have no financial conflicts of interest. 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 References for therapeutic benefit. When pursuing this application, it will be 1. Fauci, A. S., D. Mavilio, and S. Kottilil. 2005. NK cells in HIV infection: par- important to test in future studies additional potential mechanisms adigm for protection or targets for ambush. [Published erratum appears in 2005 that might govern NK cell activation via the HVEM-LIGHT- Nat. Rev. Immunol. 5: 969.] Nat. Rev. Immunol. 5: 835–843. 2. Mikulak, J., F. Oriolo, E. Zaghi, C. Di Vito, and D. Mavilio. 2017. Natural killer BTLA-CD160–LTb signaling network including potentially cells in HIV-1 infection and therapy. AIDS 31: 2317–2330. counterregulatory mechanisms. 3. Pittari, G., L. Vago, M. Festuccia, C. Bonini, D. Mudawi, L. Giaccone, and B. Bruno. 2017. Restoring natural killer cell immunity against multiple myeloma Interestingly, costimulation of bulk NK cells with HVEM-(Fc*) in the era of new drugs. Front. Immunol. 8: 1444. and IL-2 downregulates expression of CD160 and LIGHT on 4. Pahl, J., and A. Cerwenka. 2017. Tricking the balance: NK cells in anti-cancer CD56dim and CD56bright NK cells, respectively (Fig. 4E, 4G). 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