Herpes Simplex Virus Directly Activate NK Cells via TLR2, Thus Facilitating Their Presentation to CD4 T This information is current as of September 27, 2021. Min Kim, Naomi R. Osborne, Weiguang Zeng, Heather Donaghy, Kay McKinnon, David C. Jackson and Anthony L. Cunningham J Immunol 2012; 188:4158-4170; Prepublished online 30 March 2012; Downloaded from doi: 10.4049/jimmunol.1103450 http://www.jimmunol.org/content/188/9/4158 http://www.jimmunol.org/ References This article cites 80 articles, 42 of which you can access for free at: http://www.jimmunol.org/content/188/9/4158.full#ref-list-1

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Herpes Simplex Virus Antigens Directly Activate NK Cells via TLR2, Thus Facilitating Their Presentation to CD4 T Lymphocytes

Min Kim,*,† Naomi R. Osborne,*,† Weiguang Zeng,‡ Heather Donaghy,* Kay McKinnon,x David C. Jackson,‡ and Anthony L. Cunningham*,†

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on -derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T IFN-g responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to

the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ Downloaded from and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as http://www.jimmunol.org/ targets for adjuvants during HSV vaccine development. The Journal of Immunology, 2012, 188: 4158–4170.

n cutaneous herpes simplex lesions of humans and in mu- MHC class I expression by keratinocytes. This is reversed by rine models, keratinocytes, dendritic cells (DCs), and infil- IFN-g, mainly from early infiltrating CD4 T lymphocytes, thus I trating lymphocytes, especially HSV-specific CD4 and CD8 allowing CD8 T lymphocytes to recognize infected keratinocytes T lymphocytes, were shown to play a central role in control- (1, 4). IFN-g also stimulates MHC class II (MHC II) expression on ling primary and recurrent HSV infections (1). In human recur- keratinocytes, allowing recognition by CD4 T lymphocytes. Thus, rent lesions, and CD4 T lymphocytes infiltrate first, Th1, rather than Th2, patterns of response are important for im- followed by CD8 T lymphocytes, which appear to clear HSV in- mune and vaccine control of HSV (6). Furthermore, resident by guest on September 27, 2021 fection (1–3). HSV infection of keratinocytes in vitro and in vivo memory CD8 T lymphocytes can be induced to proliferate directly induces the secretion of a sequence of chemokines and cytokines in (murine) skin without the requirement to migrate to the lymph (first IFN-b and b chemokines and then IL-12, followed by IL-1b nodes. This requires DC interactions with CD4 and CD8 T lym- and IL-6) (4). The b chemokines probably attract monocytes and phocytes (7). Infrequent “sentinel” CD4 and CD8 T lympho- CD4 and CD8 T lymphocytes into lesions. IFN-a/b and IL-12 cytes are present in the dermis between lesions. Residual dermal may entrain Th1 patterns of cytokine response from HSV Ag- DCs, plasmacytoid DCs (pDCs), and CD4 and CD8 T lympho- stimulated CD4 (and CD8) T lymphocytes, especially IFN-g. cytes persist in the dermis between lesions. CD8 T lymphocytes IFN-a and IFN-g synergize to inhibit infection of keratinocytes are most superficial, with some present at the dermo–epidermal after transmission from axon termini (5). HSV1/2 downregulate junction (8). Mucocutaneous HSV infection is usually confined to the epi- dermal layer. Therefore, Langerhans cells (LCs) are likely to be *Centre for Virus Research, Westmead Millennium Institute, Westmead, New South involved in initial HSV Ag uptake (9), as shown by older murine Wales 2145, Australia; †Sydney Medical School, University of Sydney, Sydney, New ‡ studies (10). However, recent studies in mice demonstrated that South Wales 2006, Australia; Department of Microbiology and Immunology, Uni- + versity of Melbourne, Parkville, Victoria 3010, Australia; and xParramatta Sexual migrating dermal CD103 , -expressing DCs are the major Health Clinic, Parramatta, New South Wales 2150, Australia transporters of HSVAg out of skin and, together with resident CD8+ Received for publication December 9, 2011. Accepted for publication February 16, DCs, are the major presenters of HSVAg to CD8 T lymphocytes in 2012. lymph nodes (11, 12), probably after Ag transfer from LCs (13). No This work was supported by the National Health and Medical Research Council of human equivalent of the CD103+ dermal DC subset has been de- Australia (Program Grant 358399 and Project Grant 632638). scribed, nor has a similar sequence of events, although it was shown Address correspondence and reprint requests to Prof. Anthony L. Cunningham, West- mead Millennium Institute, Darcy Road, Westmead, NSW 2145, Australia. E-mail that HSV-infected monocyte-derived DCs (MDDCs) undergo apo- address: [email protected] ptosis and can be taken up by bystander uninfected MDDCs, which Abbreviations used in this article: DC, ; gD, HSV glycoprotein D; gD2, can then present HSV Ag to CD8 T lymphocytes (14). HSV2 glycoprotein D; gD2-24-4, HSV2 glycoprotein D peptide 24-4; gD2-30-5, More recently, the important role of other innate immune ef- HSV2 glycoprotein D peptide 30-5; hTLR2, human TLR2; IS, immunological syn- apse; LC, Langerhans cell; MDDC, monocyte-derived dendritic cell; MHC II, MHC fectors, both humoral and cellular, such as , pDCs, class II; MOI, multiplicity of infection; Pam2Cys, dipalmitoyl-S-glyceryl cysteine; NK cells, and gd T lymphocytes has been re-emphasized, either in pDC, plasmacytoid dendritic cell; RH10, RPMI 1640 supplemented with 10% human direct immune control or via modulation of adaptive immune AB serum; RT, room temperature; UV-HSV, UV-inactivated HSV. responses (15, 16). Recently, pDCs were demonstrated to infiltrate Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the upper dermis of recurrent herpes lesions where they show www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103450 The Journal of Immunology 4159 evidence of IFN-a production and are in contact with activated general, short peptides are weak immunogens, so they need to be NK cells and lymphocytes (17). They produce IFN-a in response administered with adjuvants. To enhance the immunogenicity of to stimulation with HSV, to limit viral spread (18–21), and can our gD2 peptides, they were conjugated to dipalmitoyl-S-glyceryl present Ag to naive effector and regulatory lymphocytes (21–24). cysteine (Pam2Cys) (42), which binds specifically to and stim- Like DCs, NK cells can bridge the innate and adaptive immune ulates via TLR2 and its heterodimeric partner TLR6 and induces responses. In humans, there are two main NK subsets: the first and maturation in DCs (43–46) (some HSV strains also target TLR2 well-characterized CD56dimCD16+ NKs make up .95% of those naturally). in blood and readily lyse malignant or virally infected cells but are In preliminary experiments, we determined that these lipopep- low cytokine secretors. In contrast, the more recently described tides markedly enhanced HSV-specific CD4 T lymphocyte IFN-g CD56brightCD162 NK cells produce large amounts of cytokines, responses. Therefore, we examined the mechanism of these re- such as TNF-a and IFN-g, upon stimulation, but they only acquire sponses and then compared them with those of endogenous cytotoxicity after prolonged activation. DCs and NK cells often TLR2 stimulation by whole HSV as UV-inactivated Ag. Initi- colocalize and are able to interact both at sites of inflammation ally, the HSV2 186 strain, previously shown to stimulate se- and in lymph nodes (25, 26). In DC–NK cell interactions, cyto- quentially via TLR2 and 9, was used, followed by an HSV1 strain kines, such as IL-12, IFN-a, and IL-15, are important, but direct also shown to stimulate via TLR2. Both types of Ags, UV- cell-to-cell contacts also seem to be essential in promoting full NK inactivated HSV (UV-HSV) and Pam2Cys–gD2 peptides, are activation by DCs (27). The CD56brightCD162 subset can also act also known to stimulate maturation of human MDDCs (47, 48). as accessory APCs via upregulated HLA-DR and secretion of Because of the prominent infiltration of NK cells into herpetic

IFN-g, thus enhancing CD4 T lymphocyte responses (28, 29). lesions, we also examined the role of NK cells in modulating the Downloaded from Interactions among DCs, NK cells, and CD4 T lymphocytes can immune response of CD4 T lymphocytes to these Ags. We found enhance such responses (22, 30). IFN-g production by NK cells that NK cells contributed significantly to the unseparated PBMC can be induced by IL-12 secreted by TLR-stimulated DCs (31). IFN-g responses to HSV Ag and enhanced MDDC–CD4 Cross-talk between DCs and NK cells in viral infection was re- T lymphocyte responses to UV-HSV and Pam2Cys-conjugated ported (32). However, it has not been fully explored how the trio HSV peptides. Furthermore, NK cells alone were capable of di-

of NK cells, DCs, and CD4 T lymphocytes interact with each rect presentation of these HSV Ags to CD4 T lymphocytes in the http://www.jimmunol.org/ other, especially physically. absence of DCs. The mechanism was determined to be through TLRs on the surface and in endosomes of dendritic, NK, and other both direct NK cell–CD4 T lymphocyte physical interaction and innate immune cells are key sensors of viral pathogens leading to cytokines. These studies are relevant to such DC–NK cell–CD4 activation of the innate and adaptive immune responses. Human LCs T lymphocyte interactions in pairs and triplets in herpes lesions, as express TLRs 1, 2, 3, 5, 6, and 10 (33, 34); dermal DCs and MDDCs well as to adjuvant choice and their mechanisms of action in HSV express TLRs 1, 2, 3, 4, 6, 8, and 10 (23); and NK cells express vaccine development. TLRs 1, 2, 3, 5, 6, 7, 8, and 9 (31, 35). Some strains of HSV1 and HSV2, as well as human CMV, interact with TLR2 at the surface Materials and Methods of many cell types via an unknown mechanism, as well as with Synthesis of gD2 peptides by guest on September 27, 2021 TLR9 in the endosomes (particularly of pDCs) via unmethylated gD2-24-4 and -30-5 were synthesized using Fmoc chemistry, as previously CpGs in viral DNA. UV-inactivated HSV2 requires initial interac- described (42). The amino acid sequences of the immunodominant CD4 tion with TLR2 prior to full activation via TLR9, whereas infecti- T lymphocyte epitopes in gD2 were QQGVTVDSIGML and PEDPED- ous HSV2 can interact directly with TLR9 (18). IFN-a production SALLED for peptides gD2-24-4 and gD2-30-5, respectively. The two by pDCs via TLR9 (18–20) also activates NK cells, which, in turn, Pam2Cys-conjugated gD peptides (Pam2Cys–gD2-24-4 and Pam2Cys– gD2-30-5) were synthesized by covalently coupling of Pam2Cys lipid contribute to the Th1 adaptive immune response (36). TLRs 2 moiety through two serine residues to each parental peptide (42). For and 9 also synergize in control of HSV in the mouse brain (37). carboxyfluorescein conjugation onto gD2-30-5 and Pam2Cys–gD2-30-5, HSV glycoprotein D (gD) is a highly immunogenic molecule the N-terminal of the Fmoc group was removed following the last acyla- containing both B and epitopes capable of inducing neu- tion reaction, and 5(6)-carboxyfluorescein (Fluka Biochmika) was then tralizing Abs and CD4 T lymphocyte Th1 responses in the majority coupled to the exposed amino group using a 4-fold molar excess in the presence of equimolar amounts of O-benzotriazole-N,N,N9,N9-tetramethyl- of the human outbred population. Therefore, it has been used as uronium-hexafluorophosphate (Merck) and 1-hydroxybenzotriazole an immunogen in human vaccine trials. However, gD alone does (Merck) and a 1.5-fold excess of diisopropylethylamine (Fluka Biochmika) not appear to interact with surface TLRs on innate immune cells for 4 h. All peptides were cleaved from the solid support, and side chain (38). In consort studies of couples discordant for genital herpes, a protecting groups were simultaneously removed before being purified and characterized, as described previously (42). All peptide samples vaccine consisting of HSV2 gD (gD2) and the TLR4 agonist d- were dissolved at a concentration of 10 mM in DMSO and kept at monophosphoryl lipid A showed 73–74% efficacy in preventing 280˚C. genital herpes in HSV-seronegative women whose partners had genital herpes (39), but apparently this was not confirmed in im- PBMC preparation munizing a general population of HSV-seronegative women (40). Blood was collected from HSV1- and/or HSV2-seropositive healthy donors The reasons for these differences are unclear and indicate the need and donor serotype-matched HSV1 or HSV2 Ags in individual experiments. for increased understanding of the human immune response and Informed consent was obtained from all of the blood donors, and the study was approved by the Ethics Committee for Sydney Western Area Health control of HSV infection, including innate immunity and its key Service Research. PBMCs were prepared by Ficoll-Hypaque (GE Health- role in directing the adaptive immune response. care Biosciences) density gradient. Recently, a high density of promiscuous immunodominant Depletion of CD8+ and CD56+ lymphocytes from PBMCs peptide epitopes in gD2 for CD4 T lymphocytes was identified in our laboratory (41). The identified immunodominant gD2 peptide To examine the immunogenicity of Pam2Cys–gD2 peptides and whether 24-4 (gD2-24-4) and gD2 peptide 30-5 (gD2-30-5) promiscuously Pam2Cys–gD2 peptides target only CD4 T lymphocytes as intended, CD8 T lymphocytes and CD56+ NK cells were positively depleted using CD8 bound to a broad range of HLA-DR molecules and were recog- and CD56 MicroBeads (Miltenyi Biotec), respectively. The same starting nized by both HSV1- and HSV2-infected individuals, providing number of PBMCs was used as undepleted PBMC control for CD8 de- an explanation for the broad T cell recognition in humans. In pletion as well as for CD56 depletion. After cell depletion, the resulting 4160 HSV Ag-STIMULATED NK–CD4 T CELL INTERACTIONS cells for each depletion were resuspended in an equal volume of RPMI tained by gating on live lymphocytes and then CD4 and CD3 double- 1640 (Invitrogen) supplemented with 10% human AB serum (RH10; positive cells. NK cells were gated on live lymphocytes and then CD56+ Sigma-Aldrich) to a final concentration of 1 3 106 cells/ml based on the CD32 cells. The purity of NK and CD4 T lymphocytes was .98%. number of undepleted PBMCs. Cells were cultured for 40 h in a 96-well PerCP–anti-HLA-DR (BD) or PE–Cy7–anti-HLA-DR (BioLegend) and ELISPOT plate with 5 mg/ml PHA, UV-HSV1 (strain F) or UV-HSV2 FITC–anti-HLA-DQ (BioLegend) Abs were used to assess MHC II ex- (strain 186) at multiplicity of infection (MOI) 0.5, 10 mM peptide, or pression on stimulated NK cells. PE–Cy7–anti-TLR2 (eBioscience) was DMSO (Sigma-Aldrich) as negative control. UV-HSV was prepared as used to determine whether TLR2 is expressed on MHC II-expressing NK previously described (14). Viral infectivity was not observed after UV cells. Because of the low percentage of CD56brightCD162 NK cells and inactivation by plaque assay. To measure the immune response after the peptide-specific CD4 memory T cells, $200,000 cells were acquired per incubation, IFN-g ELISPOT was performed, as previously described (41). sample on a BD FACSCanto. Data were analyzed by FlowJo software.

TLR2-binding assay using HEK-Blue TLR2 cells Preparation of peptide-specific CD4 T cell line To investigate the mechanism of UV-HSV binding to NK cells, TLR2 was To increase the frequency of immunological synapse (IS) formation be- tested as one of the receptor candidates for UV-HSV. HEK-Blue human tween Pam2Cys–gD2-30-5–stimulated NK cells and CD4 T lymphocytes, TLR2 (hTLR2) cells and HEK-Blue Null cells (parental cell line of HEK- a gD2-30-5–specific CD4 T cell line was generated. PBMCs of HSV1– Blue hTLR2 cells) were purchased from InvivoGen. They were cultured infected individuals were pulsed with 10 mM gD2-30-5 in 100 ml serum- and used for assays, according to the manufacturer’s instructions. Before free RPMI 1640 for 1 h at 37˚C, 5% CO2 and cocultured with autologous the assay, TLR2 expression on both cells was examined by flow cytometry PBMCs at a 1:2 ratio at a cell concentration of 1 3 106 cells in RPMI 1640 using FITC-conjugated hTLR2 mAb. Pam2CSK4 (10 mM; InvivoGen), supplemented with 10% FBS, penicillin, and streptomycin (50 U/ml and Pam2Cys–gD2-30-5 (10 mM), UV-HSV1 (MOI 0.5), UV-HSV2 (MOI 50 mg/ml, respectively; Gibco-BRL). After 1 wk, cells were restimulated 0.5), and water and TLR7/8 agonist Resiquimod (5 mg/ml; InvivoGen) as with irradiated (3000 rad) peptide-sensitized autologous PBMCs, as above.

negative control were added to the cells and cultured for 20 h at 37˚C, 5% Restimulated cells were cultured in RPMI 1640 supplemented with 10% Downloaded from 6 CO2. Supernatants were collected for measuring NF-kB–induced secreted FBS, IL-2 (10 U/ml; Roche), and penicillin/streptomycin at 1 3 10 cells/ embryonic alkaline phosphatase resulting from TLR2 binding by tested ml. Restimulation was repeated once a week until enough cells were ob- stimuli and incubated with QUANTI-BLUE medium (InvivoGen) for 1 h at tained. Medium was replenished twice a week. 2 37˚C. After the incubation, OD was measured at 650 nm (Victor TM 1420 Multilabel Counter; PerkinElmer). Detection and quantification of NK cell–CD4 T lymphocyte conjugates Generation of MDDCs

To confirm that NK cells can directly contact CD4 T lymphocytes, purified http://www.jimmunol.org/ Monocytes were isolated from PBMCs using CD14 MicroBeads (Miltenyi and stimulated NK cells were cocultured with purified CD4 T lymphocytes Biotec), according to the manufacturer’s instructions. Isolated CD14+ 3 5 or gD2-30-5–specific CD4 T cell line as follows. Purified NK cells were monocytes were incubated at a cell concentration of 5 10 cells/ml for pulsed with 10 mM Pam2Cys–gD2-30-5 or UV-HSV1 at MOI 0.5, or 5–6 d in RH10 supplemented with IL-4 (250 U/ml; Biosource) and GM- without Ag as negative control, in 100 ml serum-free RPMI 1640 for 1 h at CSF (200 U/ml; Biosource) at 37˚C, 5% CO2. After the incubation, cell 37˚C, 5% CO2. After the incubation, cells received a 103 volume of morphology was examined under the microscope, and flow cytometry was serum-free RPMI 1640 and were centrifuged for 5 min at 300 3 g. After performed to check the phenotypes of immature MDDCs. To culture with decanting the supernatant, NK cell pellet was resuspended and added to NK cells and/or CD4 T lymphocytes in RH10 media in ELISPOT plate, the CD4 T lymphocytes at a 2:1 ratio. The combined NK cells and CD4 ratio of MDDCs, NK cells, and CD4 T lymphocytes was kept at 1:1:10. T lymphocytes were briefly centrifuged at 10,000 3 g and incubated for 5 min at 37˚C. After the conjugation, cells were gently resuspended; serum- Isolation and culture of NK cells and CD4 T lymphocytes by guest on September 27, 2021 free RPMI 1640 was added to the cells, and they were loaded onto the NK cells and CD4 T lymphocytes were negatively isolated using EasySep prepared Cell Tak (BD)-coated cover slips through “gummy squares” human NK Enrichment and EasySep human CD4 T Enrichment kit (eight-well flexiPERM removable and reusable TC chambers; ISC Bio- (StemCell Technologies), according to the manufacturer’s instructions. Express). Cells were centrifuged for 3 min at 300 3 g. The gummy square RH10 was used to culture cells at a concentration of 106 cells/ml. For was removed before cells were fixed and labeled. IFN-g ELISPOT, cells were cultured for 40 h in a 96-well ELISPOT plate (Millipore), with or without peptide. For the rest of the experiments, NK Immunofluorescence microscopy cells and CD4 T lymphocytes were cocultured overnight with 10 mM Cells loaded on cover slips were fixed with 3% paraformaldehyde (Pro- peptides or with DMSO as negative control. The ratio of NK cells/CD4 SciTech) for 20 min at room temperature (RT) and then neutralized with 50 T lymphocytes for coculture was 1:4, unless otherwise indicated. mM NH4Cl three times for 3 min each. Cells were permeabilized with Coculture of HSV Ag-pulsed NK cells with CD4 T lymphocytes 0.05% Triton X-100 (Sigma-Aldrich) for 1 min at RT and washed once with PBS. After permeabilization, cells were left for 15 min in PBS sup- in Transwells plemented with 1% fish skin gelatin (Sigma-Aldrich) and 0.02% saponin To investigate whether NK cells activate CD4 T lymphocytes by cell-to-cell (Sigma-Aldrich) and then for 30 min in Image-iT FX signal enhancer contact after being stimulated with peptides, 0.4-mM pore-sized Transwells (Invitrogen). For visualizing ISs, the actin-binding molecule talin was la- (Corning) were used for cell culture to block cell migration. Purified NK beled with mouse anti-human talin mAb (Abcam) as primary Ab for 30 cells were placed with purified CD4 T lymphocytes in the lower chamber min at RT and then with Alexa Fluor 633-conjugated goat anti-mouse IgG or placed in the upper chamber with CD4 T lymphocytes in the lower (Molecular Probes) for 30 min as secondary Ab. For the secondary staining chamber. CD4 T lymphocytes were cultured in the lower chamber, with or to differentiate NK cells and CD4 T lymphocytes, mouse anti-human CD4 without NK cells. Peptide (10 mM) and DMSO of equivalent volume as Ab (BD Pharmingen) was used to label CD4 T lymphocytes for 30 min, negative control were added to the well in which NK cells were placed, followed by Alexa Fluor 546-conjugated goat anti-mouse IgG (Molecular because NK cells were suspected APCs. Maximum cell number in the Probes) for 30 min. Mouse anti-human IgG (Sigma-Aldrich) was used as upper chamber was 1 3 105 in 100 ml RH10, and that in the lower chamber primary Ab for negative control staining for primary and secondary was 6 3 105 in 600 ml RH10. Cells were incubated overnight at 37˚C, 5% staining. All washes between steps were carried out with 10% goat serum (Sigma-Aldrich) in PBS. After staining, ProLong Gold Antifade reagent CO2. The following day, cells were collected separately from the bottom or upper chamber for flow cytometry. (Invitrogen) was added to the stained cells, and glass slides were mounted onto cell-loaded coverslips. Slides were visualized through an 3100 1.35 Flow cytometry NA oil-immersion lens with an inverted Olympus IX-70 microscope (DeltaVision Image Restoration Microscope; Applied Precision/Olympus) To check the phenotype of immature DCs, allophycocyanin–anti-CD14, and a Photometrics CoolSnap QE camera. PE–anti-CD86, and PE–Cy7–anti-HLA-DR Abs were purchased from BD. For cell depletion, allophycocyanin–anti-CD8 (BD), FITC–anti-CD3 (BD Immunostaining of histologic sections of herpetic lesion Pharmingen), and PE–anti-CD56 (Miltenyi Biotec) Abs were used to as- biopsies sess the efficiency of CD8 and CD56 cell depletion. To measure the ac- tivation of NK cells and CD4 T lymphocytes, the early activation marker Punch biopsies were taken from recurrent genital herpes lesions #4 d after CD69 was labeled with allophycocyanin–anti-CD69 Ab (BD Pharmingen). onset. Samples were obtained under a protocol approved by the ethics CD4 T lymphocytes labeled with PE–Cy7–anti-CD4 Ab (BD) were ob- committee for Sydney West Area Health Service. Written informed con- The Journal of Immunology 4161 sent was obtained prior to sample donation. The biopsies were snap-frozen whether they were stimulated with Pam2Cys–gD2-24-4 (p = 0.005, in OCT, cut into 5-mm sections, and placed on slides. Slides were kept at ANOVA) or UV-HSV1 or UV-HSV2 Ag (p = 0.050 and 0.030, 2 80˚C until required for immunofluorescent staining. Slides were fixed respectively) (Fig. 1B). This suggests that NK cells contribute with ice-cold methanol/acetone (1:1) prior to staining. Abs used for mi- croscopy were mouse mAb for CD56 (1:200; Invitrogen) and goat poly- directly or indirectly to PBMC IFN-g production. clonal Ab for CD4 (1:100; R&D Systems). Slides were incubated at 37˚C UV-HSV1 and UV-HSV2 strains bound to TLR2 with primary Abs for 45 min. Following washing, the secondary Abs Alexa Fluor 546 donkey anti-goat (1:400; Molecular Probes) and Alexa Fluor UV-HSV Ag was used because certain clinical strains of HSV2 488 donkey anti-mouse (1:400; Molecular Probes) were added for 30 min (including HSV2 strain 186) were also shown to stimulate cells via at 37˚C. Excess secondary Ab was washed away, cells were mounted using ProLong Gold Antifade supplemented with DAPI (Invitrogen), coverslips TLR2 (18). Therefore, we first determined the TLR2-stimulating were applied, and slides were refrigerated until imaged with an Olympus activity of UV-HSV1 strain F and HSV2 strain 186 with a TLR2- IX-70 microscope. transfected HEK293 responder cell line in comparative studies with Pam2Cys–gD2 peptides. To examine the physical binding Statistical analysis of UV-HSV to NK cells and subsequent stimulation, HEK-Blue Outcome variables were log transformed to stabilize the variance prior to hTLR2 cells were used with HEK-Blue Null cells as negative analysis. Repeated measures ANOVAwith multiple pair-wise comparisons control. Surface TLR2 expression was detected on HEK-Blue was used to assess the effects of treatment within a group. SPSS version 20 was used to analyze data. A 5% significance level with two-sided tests was hTLR2 cells but not on HEK-Blue Null cells (Fig. 2A). When used throughout. a TLR2 agonist binds to TLR2 on HEK-Blue hTLR2 cells, NF- kB–induced embryonic alkaline phosphatase is secreted into the

Results supernatant, which is detected by spectrophotometry. UV-HSV1 Downloaded from and UV-HSV2 bound to TLR2 expressed on HEK-Blue hTLR2 Conjugation of immunodominant gD2 peptides to the TLR2 cells, as did Pam2Cys–gD2-30-5 and Pam2CSK4 as positive con- agonist Pam2Cys enhanced IFN-g responses by CD4 trol, but not on HEK-Blue Null cells. Negative controls included T lymphocytes in MDDC–CD4 T lymphocyte cocultures and the TLR7/8 agonist Resiquimod (Fig. 2B). contributed to the better effect in PBMCs and NK cells Two 12 mer analogs from gD2-24-4 and -30-5, previously de- The presence of NK cells enhanced DC–CD4 T lymphocyte http://www.jimmunol.org/ fined as MHC II restricted, immunodominant and broadly recog- responses to Pam2Cys-conjugated gD2 peptides nized by CD4 T lymphocytes of HSV1-and HSV2-seropositive To directly examine whether NK cells contribute to CD4 T lym- subjects across multiple HLA-DR types, were selected for conju- phocyte IFN-g responses to gD2-24-4 Ag presentation by DCs, gation to Pam2Cys, a TLR2 agonist. The effects of lipidated and MDDCs were cultured with autologous NK cells and CD4 T nonlipidated peptides on PBMC and CD4 T lymphocyte IFN-g lymphocytes with lipidated or nonlipidated gD2-24-4. The lipo- responses were examined and compared. After conjugation to peptides enhanced responses by all three paired combinations of Pam2Cys, the immunogenicity of gD2-24-4 and -30-5 peptides NK cells, MDDCs, and CD4 T lymphocytes, as well as by NK for CD4 T lymphocytes in MDDC–CD4 T lymphocyte culture cells alone but not by CD4 T lymphocytes alone. The immune

was significantly enhanced, as measured by IFN-g ELISPOT (Fig. response was the greatest when all three cell types were cultured by guest on September 27, 2021 1A). As expected, this enhancement of PBMC IFN-g responses together (Fig. 3A, 3B). To further investigate the specific role of was not generally diminished by CD8 T lymphocyte depletion NK cells in the NK cell–CD4 T lymphocyte IFN-g response to alone (n =3,p = 0.256, ANOVA, followed by multiple pair- Pam2Cys–gD2-24-4 and UV-HSV2 strain 186, both subsets were wise comparisons); however, unexpectedly, this immune response purified from PBMCs of HSV1/2-seropositive subjects and cul- was significantly decreased by depletion of CD56 lymphocytes tured alone or together in the presence of gD2-24-4, Pam2Cys–

FIGURE 1. HSV Ags stimulate CD4 T lymphocyte (IFN-g) responses through DCs and NK cells. (A) Conjugation of two immunodominant HSV gD2 pep- tides (gD2-24-4, left panel, gD2-30-5, right panel) to Pam2Cys enhanced CD4 T lymphocyte IFN-g re- sponses when cocultured with MDDCs. Mean 6 SD of triplicates. Representative of three different donors. (B) Immune response of HSV-seropositive PBMCs, CD8 lymphocyte-depleted PBMCs, or CD56 lym- phocyte-depleted PBMCs to HSV Ags. Stimulation of CD4 T lymphocyte responses with HSV Ags requires NK cell participation. The immune responses were measured by IFN-g ELISPOT. Mean 6 SD of tripli- cates. Representative experiments from three donors. Negative control, no Ag; Pam24-4, Pam2Cys–gD2- 24-4; Pam 30-5, Pam2Cys–gD2-30-5; positive control, PHA-stimulated PBMCs; UV-1, UV-HSV1; UV-2, UV- HSV2; 24-4, gD2-24-4; 30-5, gD2-30-5. 4162 HSV Ag-STIMULATED NK–CD4 T CELL INTERACTIONS

FIGURE 2. Binding of UV-HSV1 and UV- HSV2 to TLR2. (A) HEK-Blue Null cells show no expression of TLR2 on the surface, whereas HEK-Blue hTLR2 cells do. (B) HEK-Blue Null cells and HEK-Blue hTLR2 cells were stimu- lated with Pam2CSK4 (10 mM), Pam2Cys–gD2- 30-5 (10 mM), UV-HSV1 (MOI 0.5), UV-HSV2 (MOI 0.5), and water and TLR7/8 agonist Resi- quimod (5 mg/ml) as negative control for 20 h at

37˚C, 5% CO2. NF-kB–induced secreted embry- onic alkaline phosphatase activity in culture su- pernatants was measured using QUANTI-BLUE 2 medium and spectrophotometry (Victor TM 1420 Multilabel Counter). OD was checked at 650 nm. *p , 0.05, versus negative control. gD2-24-4, or UV-HSV2. However, the NK cell–CD4 T lymphocyte ELISPOT) (Fig. 4 compared with Fig. 3). Both CD56brightCD162 combination again produced more IFN-g than NK cells alone and and CD56dimCD16+ NK cell subsets were activated, but activation in the absence of other APCs (Fig. 3C). Thus, NK cells produce was greater in the former (data not shown). Downloaded from IFN-g alone and probably stimulate CD4 T lymphocytes to enhance Pam2Cys–gD2 peptide and UV-HSV Ag-pulsed NK cells their production with both UV-HSV2 and Pam2Cys–gD2-24-4. activated CD4 T lymphocytes Both Pam2Cys–gD2 peptide and UV-HSV directly activated To confirm the activation of CD4 T lymphocytes by NK cells pulsed NK cells with Pam2Cys–gD2 peptide and UV-HSV, the expression of CD69 Whether NK cells are activated by Pam2Cys–gD2 peptide or UV- on CD4 T lymphocytes was examined by flow cytometry after HSV Ag was determined by measuring expression of the early coculturing with Ag-stimulated NK cells. The activation of CD4 http://www.jimmunol.org/ activation marker CD69 on the surface of NK cells, after stimu- T lymphocytes was significantly enhanced by Pam2Cys conju- lation by these HSV Ags. Both types of Ags directly and specif- gation of the gD2 peptides (Fig. 5A) and by UV-HSV1 stimulation ically activated NK cells, similar to the IFN-g responses (by (Fig. 5C), although there was considerable individual variation by guest on September 27, 2021

FIGURE 3. NK cells augment HSV Ag-induced IFN-g responses of CD4 T lymphocytes alone or with MDDCs. (A) Immune responses of MDDCs, NK cells, and CD4 T lymphocytes in different combinations (MDDC–CD4 T, MDDC–CD4 T–NK, NK alone, and NK–CD4 T lymphocytes) to gD2 peptide, with or without Pam2Cys. The response was measured by IFN-g ELISPOT. The predetermined ratio of MDDCs, NK cells, and CD4 T lymphocytes was 1:1:10. (B) Immune responses of MDDC–CD4 T, MDDC–NK cell, and MDDC–NK cell–CD4 T lymphocytes to gD2 peptides, with or without Pam2Cys conjugation. (C) NK cells alone and NK cell–CD4 T lymphocytes were cultured in the presence or absence of HSVAgs and in the absence of MDDCs. The immune response was measured by IFN-g ELISPOT. Mean 6 SD of triplicates. Representative experiments from three donors in (A)–(C). Pam24-4, Pam2Cys–gD2-24-4; 24-4, gD2-24-4. The Journal of Immunology 4163

FIGURE 4. NK cells alone can be activated with HSV Ags. (A and C) NK cells were pulsed or not with UV-HSV1 and incubated for 2 d at

37˚C, 5% CO2. NK cell activation was measured as surface CD69 expression by flow cytometry (A; shaded curve: isotype control; dashed line: no Ag; solid line: UV-HSV1). (B) NK cells were also cultured overnight in the presence of gD2- 30-5, Pam2Cys–gD2-30-5, or DMSO as nega- tive control. (B and C) Left panels, Percentage of Downloaded from CD69-expressing NK cells. Right panels, Mean fluorescence intensity (MFI; geometric means) of CD69 expressed on NK cells. Box-and-whisker plots show median and 25th and 75th percentiles of proportions of CD69+ NK cells. Pam30-5, Pam2Cys–gD2-30-5; 30-5, gD2-30-5. http://www.jimmunol.org/ by guest on September 27, 2021

(Fig. 5). CD4 T lymphocytes were not directly activated by the Ag and UV-HSV (Fig. 5), the expression of TLR2, HLA-DR, and (Fig. 5B), as also shown in Fig. 3B. HLA-DQ on resting blood NK cells in PBMCs was examined by flow cytometry (Fig. 6A). All three molecules were expressed at Expression of HLA-DR, HLA-DQ, and TLR2 on blood NK cells low and variable levels (1–8%) on resting NK cells and on both and their upregulation by HSV Ags the CD56bright and CD56dim NK subsets (Fig. 6B). A variable To examine the mechanism of direct stimulation of CD4 T lym- proportion (up to 4%) of these cells coexpressed TLR2, HLA-DR, phocytes by NK cells stimulated with Pam2Cys–gD2 peptide or HLA-DQ. However, after NK cell isolation and culture, HLA-

FIGURE 5. CD4 T lymphocyte activation by HSV Ags presented by NK cells. (A) NK cells were cultured overnight with CD4 T lymphocytes in the presence of gD2-30-5, Pam2Cys–gD2-30-5, or DMSO as negative control. (B) Effects of naked peptide and lipopeptide on CD4 T lymphocytes alone. As a control, CD4 T lymphocytes were also incubated with the above Ags overnight. (C) NK cells were pulsed with UV-HSV1 for 1 h and washed before NK and CD4 T lymphocytes were cocultured for 2 d at 37˚C, 5% CO2. Activation of CD4 T lymphocytes was measured as CD69 expression by flow cytometry. Box-and-whisker plots show median and 25th and 75th percentiles of proportions of CD69+ CD4 T lymphocytes. Pam30-5, Pam2Cys–gD2-30-5; 30-5, gD2-30-5. 4164 HSV Ag-STIMULATED NK–CD4 T CELL INTERACTIONS

FIGURE 6. Coexpression of HLA-DR, HLA-DQ, and TLR2 on fresh unstimulated NK cells in PBMCs. (A) Cells were gated on live lymphocytes (left panel), then on CD56+ CD32 cells (middle panel), and then on resting cells expressing TLR2, HLA-DR, or HLA-DQ (far right panels). The near right panels show isotype controls. TLR2, HLA- DR and DQ were often coexpressed on the same NK cells. (B) Proportion of CD56bright Downloaded from or CD56dim NK subsets expressing TLR2, HLA-DR, HLA-DQ, or combinations. http://www.jimmunol.org/

DR and HLA-DQ expression decreased, reducing the proportion 8A), indicating that there was little feedback effect of activated of TLR2+MHC II+ NK cells (M. Kim and N. Osborne, unpub- CD4 T lymphocytes on activation of NK cells. by guest on September 27, 2021 lished observations). Thus, cell-to-cell contact between NK and CD4 T lymphocytes The expression of HLA-DR and HLA-DQ molecules on the varied between donors with regard to the magnitude of its im- surface of cultured NK cells was then examined, after stimulation portance for CD4 T lymphocyte activation, being complemented by with both types of HSV Ags, and shown to be significantly in- the humoral (or cytokine) effects (similar results were observed creased (Fig. 7). When NK cell subsets were examined, the lev- with UV-HSV Ag stimulation; data not shown). els of HLA-DR and HLA-DQ and the proportion of MHC II- NK cells formed ISs with CD4 T lymphocytes after stimulation expressing NK cells were significantly increased and to a greater with Pam2Cys–gD2 peptide and UV-HSV1 degree by lipopeptide on the CD56bright CD162 NK subset com- pared with the CD56dim CD16+ NK subset (Fig. 7A). To examine the physical contact between NK cells and CD4 T lymphocytes, highly purified NK cells were pulsed with Pam2Cys–gD2 peptide and UV-HSV Ag-stimulated NK cells Pam2Cys–gD2-30-5, UV-HSV1 Ag, or controls for 1 h and then activated CD4 T lymphocyte responses through cell-to-cell washed prior to examining for conjugate formation with CD4 contact and cytokine effects T lymphocytes. After 5 min of coculture, NK cells and CD4 To determine whether CD4 T lymphocytes needed to directly T lymphocytes were fixed/permeabilized and labeled for immu- contact NK cells to be activated in an Ag-dependent manner, they nofluorescence microscopy. A very low background level of ho- were cultured separately or together in the same compartment in motypic and heterotypic cell-conjugate formation was observed Transwells. Because NK cells were postulated to be APCs, the between NK cells and CD4 T lymphocytes in control cultures peptide was always added to the chamber containing NK cells. After (i.e., CD4–CD4, NK–NK, and NK–CD4 T lymphocyte conjugates overnight culture, cells were harvested and labeled for flow formed) (Fig. 9A). However, the proportion of NK cell–CD4 cytometry. In all five donors, CD69 expression was enhanced to T lymphocyte conjugates in the Pam2Cys–gD2-30-5–stimulated a greater degree on CD4 T lymphocytes when they were cocultured cocultures was significantly greater than in controls. As expected, with NK cells in the same chamber (Fig. 8A); there was an overall these proportions were very low in the lipopeptide-stimulated NK significant decrease in expression when the two cell types were cells. Therefore, to increase the frequency of IS formation be- separated, although this showed marked interdonor variation in tween NK cells and CD4 T lymphocytes, gD2-30-5–stimulated magnitude (1.69-fold decrease; 95% confidence interval, 1.20– CD4 T cell lines were generated. As a result, ISs were more 2.37; p = 0.013, paired t test) (Fig. 8B). There was no significant frequent and recognized by polarization of the actin-binding reduction in naked peptide-stimulated NK cell–CD4 responses by molecule talin and also the CD4 molecule to the CD4 T lym- separate cultures of the two cultures in Transwells (p = 0.273, phocyte–NK cell contact region with the Pam2Cys–gD2-30-5– paired t test). Separate cultures of NK cell and CD4 T lymphocytes stimulated CD4 T cell line (1 IS/104 cell pair for gD2-specific had no consistent or significant effect on NK cell activation (Fig. peptide). Similar ISs were observed in UV-HSV1–pulsed NK The Journal of Immunology 4165 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. MHC II expression by NK cells is upregulated with HSV Ags. (A) NK cells were incubated with CD4 T lymphocytes overnight in the presence of gD2-30-5, Pam2Cys–gD2-30-5, or DMSO as negative control. The proportion of HLA-DR–expressing cells (Aa) and HLA-DQ–expressing cells (Ab) (total NK cells [left panels], CD56bright NK subset [middle panels], and CD56dim NK subset [right panels]) were measured by flow cytometry. (B)

UV-HSV1–stimulated NK cells were incubated with CD4 T lymphocytes for 2 d at 37˚C, 5% CO2, and the proportion of NK cells expressing HLA-DR (left panel) or HLA-DQ (right panel) was measured by flow cytometry. Medium was used as negative control for UV-HSV1. Box-and-whisker plots show median and 25th and 75th percentiles. Pam30-5, Pam2Cys–gD2-30-5; 30-5, gD2-30-5.

cells with CD4 T lymphocytes (1 IS/3 3 105 cell pair) (Fig. 9B, NK cells and CD4 T lymphocytes were in contact in the 9C, respectively). Although the cells partnering CD4 T lympho- mononuclear dermal infiltrates in recurrent herpetic skin cytes were of similar size and CD42 and, therefore, very un- lesions likely to be contaminating DCs, this possibility was excluded To determine whether these direct NK cell–CD4 T lymphocyte by their quantification in the purified NK population. Contami- interactions may occur in vivo, biopsies from three recurrent + 2 + 2 nating CD11c lin myeloid DCs or BDCA2 lin pDCs were herpetic lesions at #4 d after appearance were stained for the two + 2 undetectable in the 2% of non-CD56 CD3 NK cells (as were cell types. CD4 T lymphocytes were highly abundant in the in- + 26 CD19 B lymphocytes; i.e., a maximum concentration ,10 ). If filtrate. CD56+ NK cells were also detected, with up to 27 NK the maximum concentrations of Ag-specific CD4 T lymphocytes cells/field of view (340 magnification). For each biopsy, the 2 2 were ,5 3 10 2 in the cell lines and ,2 3 10 3 in UV-HSV number of CD56+ NK cells was counted in six to eight fields of Ag-stimulated CD4 T lymphocytes, then the frequency of any view. An average of 57.5% of the CD56+ NK cells was found to be IS formed by potential contaminating cells with CD4 T lym- contacting a CD4 T lymphocyte in the lesions (Fig. 10). In normal phocytes was calculated to be .3 logs lower than that actually skin control biopsies, no infiltrating CD4 T lymphocytes or CD56+ observed. NK cells were observed in the dermis. 4166 HSV Ag-STIMULATED NK–CD4 T CELL INTERACTIONS Downloaded from

FIGURE 8. Pam2Cys–gD2 peptide-stimulated NK cells activated CD4 T lymphocytes through cell-to-cell contact and through cytokine effects. CD4 T lymphocytes from five donors were cocultured with autologous NK cells in the lower chamber of Transwells, or NK cells were cultured sep- arately from CD4 T lymphocytes in the upper chamber. Pam2Cys–gD2- FIGURE 9. HSV Ag-stimulated NK cells and CD4 T lymphocytes in-

24-4 or the control gD2-24-4 peptides were always added to the chamber teract and form ISs. (A) NK–CD4 cell pairs without or before IS formation. http://www.jimmunol.org/ containing NK cells. The pore size of the Transwell membrane was 0.4 (B) Pam2Cys–gD2-30-5–stimulated NK cell–gD2-30-5–specific CD4 T mM to inhibit cell migration between the upper and lower chambers. After lymphocyte conjugates showing IS. (C) UV-HSV1–stimulated NK cell– overnight culture, cells were harvested and labeled. Activation of both NK CD4 conjugates showing IS. Purified CD4 T lymphocytes from stimulated cells (A) and CD4 T lymphocytes (B) was measured by CD69 expression. autologous PBMCs were used in (A) and (C). A gD2-30-5–specific CD4 T Pam24-4, Pam2Cys–gD2-24-4; 24-4, gD2-24-4. cell line was used in (B). NK cells were isolated from PBMCs and pulsed with Pam2Cys–gD2-30-5 (B) or UV-HSV1 (C) for 1 h; Ag was washed out A Discussion prior to coculture with CD4 T lymphocytes for 5 min at 37˚C. ( ) Non- specific conjugate of NK and CD4 T lymphocytes. Talin was expressed on Both CD4 and CD8 T lymphocytes are key effectors in human both cells and was evenly distributed around the membrane, with more on by guest on September 27, 2021 cutaneous herpetic lesions and persist as sentinels between lesions: NK cells. (B and C) IS formed between NK and CD4 T lymphocyte CD8 T lymphocytes at the dermo–epidermal junction and CD4 conjugates. Talin and CD4 were polarized at the contact site of NK and T lymphocytes deeper in the dermis (3, 49). They show different CD4 T lymphocytes. Representative images of five ISs each observed in kinetics of recruitment into clinical recurrent lesions and appar- three individuals (original magnification 3100). Scale bar, 5 mM(A, B); ently different, but overlapping, functions. Early in inflammation, 20 mM(C). (D) Frequency of ISs between lipopeptide or UV-HSV1 Ag- CD4 T lymphocytes are the major IFN-g–producing cells, al- pulsed NK cells and CD4 T lymphocyte cells or cell lines. though they are capable of cytolytic function against infected keratinocytes in vitro (1). IFN-g plays an important role in HSV- infected sites by restoring MHC class I downregulation of HSV- a pivotal role in interacting with CD4 T lymphocytes, as well as infected cells and upregulating MHC II molecules, allowing the DCs, to augment their response to HSV Ag and, specifically, to later-infiltrating CD8 T lymphocytes to recognize and clear in- gD2 peptides, especially after TLR2 stimulation on both DCs and fected cells. However, they also can secrete IFN-g. Recently, the NK cells. load of HSV2 shed in vaginal secretions was shown to be inversely In general, short peptides, such as the immunodominant gD2 12 proportional to the density of lesional CD8 T lymphocytes, al- mers, gD2-24-4 and gD2-30-5 used in this study, are poor im- though this could just represent the density of all key immune munogens and need to be administered with adjuvants to max- effectors (3). imize their immunogenicity. Such adjuvants are often TLR ago- NK cells are also present in the dermis of cutaneous herpetic nists, administered concurrently with Ag, which act on DCs and lesions, in contact with other infiltrating immune cells, especially macrophages via TLRs (e.g., d-monophosphoryl lipid A and CpG pDCs (17), as well as in cells expanded from genital sites of HSV2 oligonucleotides) (52). However, the TLR2 agonist Pam2Cys acts infection ex vivo (2). An important role for NK cells in HSV in- as a self-adjuvant when directly conjugated to peptides. It then fection was suggested previously: Dalloul et al. (50) observed binds specifically to TLR2 on APCs and directs peptide uptake by severe HSV2 infection in patients with myelodysplasia and un- the same cells (53–55). After uptake of these in PBMCs, pre- detectable NK cells and pDCs in the blood. Nandakumar et al. sumably by TLR2-expressing NK cells, monocytes, and myeloid (51) demonstrated that mice lacking or depleted of NK cells were DCs, Pam2Cys conjugation to the two gD2 peptides markedly susceptible to HSV-induced lesions and that NK cell supplemen- enhanced CD4 T lymphocyte IFN-g responses, as shown by IFN-g tation enhanced the function of anti-HSV CD8 T lymphocytes. ELISPOT. As expected from previous reports indicating that CD4 Nevertheless, the role of NK cells in human HSV immunology has T lymphocytes are the main IFN-g producers in this setting (1, 56, been underinvestigated, despite the likely contribution of both NK 57), CD8 T lymphocyte depletion did not affect these responses. cell subsets to control of HSV. The minor CD56brightCD162 However, unexpectedly, CD56 lymphocyte depletion markedly subset produces IFN-g, and the major CD56dimCD16+ subset is decreased such responses to Pam2Cys–gD2 peptides, as well as to cytotoxic. In this study, we determined that NK cells may play UV-HSV1 and UV-HSV2 shown to bind to TLR2 in a reporter cell The Journal of Immunology 4167

well as both NK subsets (although MHC II expression decreased on culture in the absence of HSVAgs). Thus, HSVAg binding to TLR2 could upregulate HLA-DR and HLA-DQ on the same NK cells or induce their expression de novo to a greater extent on the CD56bright CD162 NK subset. To determine whether direct contact between NK cell and CD4 T lymphocytes was required for CD4 T lymphocyte activation, a Transwell system was used. In all five donors, cell-to-cell contact enhanced CD4 T lymphocyte activation; although there was a considerable variation in the magnitude of this effect between donors, overall it was significant. Where the effect of contact was minor, activation of CD4 T lymphocytes probably occurred through NK cytokine production, in particular IFN-g, as previously de- scribed (64). Cytokines, such as IL-12 and IL-15 from DCs, IFN-g from NK cells, and IL-2 from CD4 T lymphocytes, may all contribute to these interactions. There may be several reasons for these differences in the relative importance of contact and cyto- kines between individual donors. These may include individual

variations in cytokine production, TLR2 expression on NK cells, Downloaded from FIGURE 10. Direct contact of CD4 T lymphocytes and NK cells in or expression of cell-adhesion molecules between NK cells and dermal infiltrate of recurrent herpes lesions. NK cells (green) are juxta- CD4 T lymphocytes. Separation of NK cells and CD4 T lym- posed and in contact with CD4 T lymphocytes (red) in recurrent herpetic phocytes had little effect on HSV Ag/TLR2-induced NK activa- 3 A 3 lesions, biopsied 4 d after onset at 40 magnification ( ) and 100 tion, arguing against a significant role for activated CD4 T lym- B C 6 magnification ( ). Scale bar, 10 mM. ( ) Mean proportion ( SD) of NK phocyte feedback on NK activation. cells in contact with CD4 T lymphocytes as a ratio of total NK cells in six

To define the mechanism of Ag presentation to CD4 T lym- http://www.jimmunol.org/ to eight high-power fields (340)/biopsy from three donors. phocytes by contacting NK cells pulsed with lipopeptide or whole UV-HSV Ag, their HLA-DR and HLA-DQ expression was line; this is important because not all HSV strains bind TLR2. Two examined. Both MHC II molecules were significantly upregulated possible explanations for the IFN-g production were considered: on NK cells, and HLA-DR expression was upregulated to the direct IFN-g secretion by NK cells or interactions between DCs, greatest extent on the CD56brightCD162 NK cell subset (data not NK cells, and CD4 T lymphocytes, resulting in the enhancement shown). Expression of MHC II molecules on human NK cells as of CD4 T lymphocyte IFN-g production, or most likely, both may mRNA and was reported (67, 68) but not previously shown occur in combination. to be upregulated by lipopeptide or a viral pathogen, although To examine the relative contributions of each of the three cell similar lipopeptides induce maturation and upregulate MHC II on by guest on September 27, 2021 types, they were studied in pairs and triplets after stimulation with DCs (71). the gD2 lipopeptides and UV-HSV. The presence of NK cells Direct cell-to-cell contact between NK cells and CD4 T lym- significantly enhanced CD4 T lymphocyte responses primed by phocytes, resulting in Ag presentation, is likely to occur via MDDCs stimulated with Pam2Cys–gD2-24-4 or by UV-HSV. Such ISs. MHC–peptide complexes reach a peak ∼5 min after coculture cross-talk by direct contact between DCs and NK cells (17, 58– and remain maximally concentrated for 10–20 min (72). Adhesion 61), as well as between CD4 T lymphocytes and NK cells through molecules in the clusters increase the avidity of the APC–T cell OX40–OX40 ligand or 2B4–CD48 interactions (62, 63), has been interaction, thus facilitating more efficient TCR engagement. They described, especially in murine systems. Fehniger et al. (64) also are polarized to the contact region between APCs and lymphocytes reported an interaction among DCs, NK cells, and CD4 T lym- (73). The actin-binding molecule talin is one of the molecules that phocytes through proinflammatory cytokines, such as IL-12, polarizes to ISs. In DC–NK cell ISs, F-action polymerization IFN-g, and IL-2. NK cells can mature DCs and induce the Th1 occurs within DCs, whereas in cytolytic NK cell–K562 conjugates, transcription factor T-bet in CD4 T lymphocytes through IFN-g F-actin polymerization occurs in the NK cells (74). Talin polari- production (65, 66), both of which promote Th1 responses. Hanna zation is present in ISs formed by noncytolytic and cytolytic NK et al. (28) reported that NK cells (especially the CD56brightCD162 cells but is transient in noncytolytic ISs (∼1 min) when interact- subset) activated by PHA and IL-2 can present Ag, and they de- ing with target cells (75). If true for interactions with CD4 fined distinct proteomic and genomic profiles of the two NK cell T lymphocytes, this could lead to underestimates of ISs in our subsets (67, 68). system. CD4 molecules are also involved in IS formation within 5 In this study, we showed that isolated NK and CD4 T lymphocytes min (76). In our study, ISs were observed as colocalization of CD4 in coculture could also respond to Pam2Cys–gD2 peptides and to and talin at the contact region with the expected kinetics, 5 min UV-HSV by IFN-g production, but the NK cell–CD4 T lymphocyte after coculture, of CD4 T lymphocytes and NK cells stimulated interaction produced significantly more IFN-g than NK cells alone. with Pam2Cys–gD2-30-5 or UV-HSV Ag. To our knowledge, this NK cells alone were directly activated, as shown by CD69 ex- is the first report of physical contact between NK cells and CD4 pression and IFN-g production, reflecting expression of TLR2 on T lymphocytes through ISs. human NK cells (35, 69, 70) but not on resting CD4 T lymphocytes. At all time points, significantly more NK–CD4 cell conjugates Furthermore, CD4 T lymphocyte activation by HSV Ag-pulsed NK were observed with Pam2Cys–gD2 peptide or UV-HSV Ag stim- cells was confirmed by upregulated CD69 expression, strongly ulation than in control unstimulated cultures. Low proportions suggesting that both cell types were sources for IFN-g.Wefurther of NK–NK and CD4–CD4 cell conjugates were also found in showed that TLR2, HLA-DR, and HLA-DQ were expressed and Pam2Cys–gD2 peptide-stimulated samples and are probably non- also coexpressed on a small proportion of unstimulated NK cells, as specific. As previously reported, unstimulated naive lymphocytes 4168 HSV Ag-STIMULATED NK–CD4 T CELL INTERACTIONS and DCs can form conjugates in the absence of Ag, which may play Acknowledgments a major role in the maintenance of naive lymphocytes in vivo (77). We thank Dr. Stuart G. Turville for training M.K. and N.R.O. for Delta- Specific NK cell–CD4 T lymphocyte IS formation, shown by Vision Microscopy and Fabienne Brillot-Turville for providing the proto- talin and CD4 polarization to the IS, was rare and reflects the low col for conjugation experiments. We also thank Dr. Karen Byth-Wilson frequency of circulating memory CD4 T lymphocytes specific for for statistical analysis. single gD2 peptides. The mean frequency of whole-HSV Ag- specific memory CD4 T lymphocytes is reported to be ∼0.2% Disclosures of total circulating CD4 T lymphocytes (78). Less than 5% of The authors have no financial conflicts of interest. A.L.C. has served as bright 2 NK cells (probably mostly the CD56 CD16 subset) were in- a consultant to GlaxoSmithKline on the development of vaccines for genital volved in ISs, also correlating with the proportion of NK cells herpes, but no commercial funding was received for this project. expressing HLA-DR (data not shown). Calculation of the ap- proximate specificities of whole-HSV Ags for human T lym- phocytes in seropositive patients suggests that 1 in 10,000 cells References should form specific ISs, consistent with our experimental re- 1. Cunningham, A. L., R. R. Turner, A. C. Miller, M. F. Para, and T. C. Merigan. 1985. Evolution of recurrent herpes simplex lesions. An immunohistologic study. sults. In an attempt to increase the frequency of IS formation, J. Clin. Invest. 75: 226–233. UV-HSV1 or a gD2-30-5–specific CD4 T cell line was used to 2. Koelle, D. M., C. M. Posavad, G. R. Barnum, M. L. Johnson, J. M. Frank, and L. Corey. 1998. Clearance of HSV-2 from recurrent genital lesions correlates study IS formation with NK cells. IS formation between UV- with infiltration of HSV-specific cytotoxic T lymphocytes. J. Clin. Invest. 101: HSV1 or Pam2Cys–gD2 peptide-stimulated NK cells and CD4 1500–1508.

T lymphocytes was observed in different HSV-seropositive do- 3. Schiffer, J. T., L. Abu-Raddad, K. E. Mark, J. Zhu, S. Selke, D. M. Koelle, Downloaded from A. Wald, and L. Corey. 2010. Mucosal host immune response predicts the se- nors. The mechanism of processing of particulate viral Ags, like verity and duration of herpes simplex virus-2 genital tract shedding episodes. HSV, by NK cells is unclear and is being investigated. Pre- Proc. Natl. Acad. Sci. USA 107: 18973–18978. sumably, this involves endocytosis, cathepsin-mediated peptide 4. Mikloska, Z., and A. L. Cunningham. 1998. Herpes simplex virus type 1 gly- coproteins gB, gC and gD are major targets for CD4 T-lymphocyte cytotoxicity in cleavage, and combination with MHC II molecules in vesicular HLA-DR expressing human epidermal keratinocytes. J. Gen. Virol. 79: 353–361. compartments, as with other APCs (79). 5. Mikloska, Z., and A. L. Cunningham. 2001. Alpha and gamma interferons in- bright 2 dim + hibit herpes simplex virus type 1 infection and spread in epidermal cells after Both CD56 CD16 and CD56 CD16 NK cell subsets are axonal transmission. J. Virol. 75: 11821–11826. http://www.jimmunol.org/ found in lung, liver, and skin and circulate through peripheral 6. Mikloska, Z., M. Ru¨ckholdt, I. Ghadiminejad, H. Dunckley, M. Denis, and blood. However, the minor subset CD56brightCD162 NK cells are A. L. Cunningham. 2000. Monophosphoryl lipid A and QS21 increase CD8 T lymphocyte cytotoxicity to herpes simplex virus-2 infected cell 4 and more abundant in secondary lymphoid organs, accounting for up 27 through IFN-gamma and IL-12 production. J. Immunol. 164: 5167–5176. to 75% of NK cells in lymph nodes and 50% in the spleen (25). 7. Wakim, L. M., J. Waithman, N. van Rooijen, W. R. Heath, and F. R. Carbone. CD56brightCD162 NK cells are found in T cell-enriched areas in 2008. 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