CLINICAL AND VACCINE IMMUNOLOGY, Dec. 2009, p. 1738–1749 Vol. 16, No. 121556-6811/09/$12.00 doi:10.1128/CVI.00280-09

Natural Killer Cell Dysfunction during Acute Infection withFoot-and-Mouth Disease Virus�

Felix N. Toka,1,2 Charles Nfon,1 Harry Dawson,3 and William T. Golde1*Plum Island Animal Disease Center, Agricultural Research Service, USDA, Greenport, New York 119441; Department of

Preclinical Science, Faculty of Veterinary Medicine, Warsaw University of Life Science, Warsaw, Poland2; andBeltsville Area Research Center, Agricultural Research Service, USDA, Beltsville, Maryland 207053

Received 7 July 2009/Returned for modification 18 August 2009/Accepted 6 October 2009

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, inparticular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infectionis unknown. Previously, we characterized the phenotype and function of NK cells from swine (F. N. Toka et al.,J. Interferon Cytokine Res. 29:179–192, 2009). In the present study, we report the analysis of NK cells isolatedfrom animals infected with FMDV and tested ex vivo and show that NK-dependent cytotoxic activity againsttumor cells as targets was impaired. More relevantly to this infection, the killing of target cells infected withFMDV also was inhibited. Further, the proportion of NK cells capable of producing gamma interferon andstoring perforin was reduced. Peripheral blood mononuclear cells isolated from infected animals are notproductively infected, but virus exposure in vivo resulted in the significant induction of NKp30 and Toll-likereceptor 3 expression and the moderate activation of SOCS3 and interleukin-15 receptor mRNA. However,there was little alteration of mRNA expression from a number of other receptor genes in these cells, includingSH2D1B and NKG2A (inhibitory) as well as NKp80, NKp46, and NKG2D (activating). These data indicate thatthis virus infection influences the ability of NK cells to recognize and eliminate FMDV-infected cells. Inaddition, a reduction in NK cell cytotoxicity coincided with the increase in virus titers, indicating the virusblocking of NK cell-associated innate responses, albeit temporarily. These effects likely culminate in brief buteffective viral immune evasion, allowing the virus to replicate and disseminate within the host.

Innate immunity is a vital part of the overall host immuneresponse to invasion by pathogens, particularly during virusinfections. Natural killer (NK) cells occupy a critical position inthe initial host responses against infection. Originally, NK cellswere discovered on the basis of their capability to kill certaintumors without prior activation. Now the role of NK cells hasbeen defined in virus infections such as human cytomegalovi-rus (8, 11, 55), murine cytomegalovirus virus (2, 32), influenzavirus (28, 35), herpes simplex virus (44, 52), ectromelia virus(16, 41), and human immunodeficiency virus (HIV) (14, 50,56). In two of these infections, a lack or deficiency in NK cellfunction leads to increased susceptibility to infection (6, 10).

The initiation of NK cell responses is thought to originatefrom signals delivered by the professional pathogen-sensingsystem, which is comprised mainly of dendritic cells (DC) (21,47, 57). Although the evidence is not yet definitive, the directactivation of NK cells also may occur through pathogen rec-ognition receptors expressed by NK cells (49, 51). The cross-talk between NK cells and DC leads to the activation of NKcells, after which they operate in a manner that is dependenton the sensing expression of specific molecules induced onvirus-infected cells through receptors present on the NK cellsurface. In part, the recognition of an infected cell by NK cellsrelies on the detection of the missing self, i.e., the lack of majorhistocompatibility complex class I expression on the infectedcell surface. Ultimately, the balance between signals from both

inhibiting and activation receptors (34) on NK cells control NKcell function in response to infection. Subsequently, NK cellsengage in cytokine secretion and, upon the encounter of avirus-infected cell, release cytotoxic granule contents or induceapoptosis. These mechanisms lead to the elimination of virus-infected cells. Whereas the discovery of activating or inhibitoryreceptors on NK cells has progressed tremendously, the iden-tification of respective ligands on infected or transformed cellshas been difficult (reviewed in reference 12).

Although much is known about the function of NK cells inhumans and mice, NK cell activity in swine or cattle remainspreliminary, and their role in animal viral diseases still is ob-scure. The recent progress in these animal species has beenreviewed by Boysen and Storset (9) and Gerner et al. (20). Inpigs, NK cells may account for a total of 5 to 10% of circulatinglymphocytes and currently are identified as belonging to asubset of cells that coexpress CD2 and CD8 molecules (17).Although mRNAs of many activating and inhibitory receptorshave been detected, no studies have been conducted to definetheir role in the generic function of porcine NK cells. But it isknown that porcine NK cells can secret gamma interferon(IFN-�), store perforin, and kill in vitro targets (54). Theirfunction can be modulated by direct stimulation with cytokinessuch as interleukin-2 (IL-2), IL-12, IL-15, IL-18 (42, 54), orIFN-�, or Toll-like receptor (TLR) agonists such as poly-inosinic:poly(C) (pI:C), CpG, imiquimod, and resiquimod inhumans (23).

In this study, we examine NK cell responses during infectionwith foot-and-mouth disease virus (FMDV). FMDV is a con-tagious disease of cloven-hoofed animals caused by a picorna-virus (25). Infection with FMDV presents as an acute disease

* Corresponding author. Mailing address: PIADC, ARS, USDA,P.O. Box 848, Greenport, NY 11944-0848. Phone: (631) 323-3249. Fax:(631) 323-3006. E-mail: william.golde@ars.usda.gov.

� Published ahead of print on 14 October 2009.

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characterized by fever, short-lived viremia, and the occurrenceof lesions on feet and tongue (reviewed in reference 25). Thecontrol of FMDV in certain regions of the world depends onthe use of inactivated vaccines. The use of these as emergencyvaccines, however, is compromised by the time from vaccina-tion to protection (7 days in cattle) (22) and the difficulty indistinguishing infected animals from vaccinated animals. InFMDV-free countries vaccination is not practiced, leading tooutbreak responses relying mostly on the elimination of allsusceptible animals within the areas of outbreaks (15).

The immune response to FMDV infection in pigs has notbeen fully dissected and remains an area of speculation.Whereas B-cell responses are activated early and neutralizingantibody titers correlate with protection (36), there is no clearpicture regarding the induction of functional T-cell immunityin infected animals. Bautista et al. (4) found the inhibition ofT-cell responses to mitogens during the infection of pigs withFMDV. Some groups have reported the induction of antigen-specific T cells reactive with nonstructural proteins of the virus(7). More recently, antigen-specific, major histocompatibilitycomplex-restricted CD8� T-cell responses were detected fol-lowing infection with FMDV (26).

The importance of innate immunity in FMDV infection isperhaps the most overlooked systemic response to FMDV insusceptible species and remains largely undefined. To date,there is no comprehensive study addressing the role of NK cellsduring infection with FMDV in either swine or cattle. A moresophisticated understanding of the innate response mechanismsinvolved in infection with FMDV is pertinent to the design ofpotent emergency vaccines that can protect against infection.

In these studies, we characterized the response of NK cellsderived from swine peripheral blood following infection withthe O1 Campos strain of FMDV. Results demonstrate thatthese cells are compromised in the killing of either tumor cellsor FMDV-infected target cells ex vivo. Moreover, this dysfunc-tion includes a reduction in the proportion of IFN-�-producingand perforin-storing cells and an alteration in the expressionpattern of activating and inhibitory receptor genes. These datasuggest a profound effect of FMDV infection on porcine NKcell function. Taken together, this abnormal functional statuslikely contributes to the acute nature of FMDV infection inthis species and toward making it one of the most contagiousviral infections of swine.

MATERIALS AND METHODS

Animals and viruses. Yorkshire pigs were purchased from Animal Biotech Inc.(Danboro, PA) at the age of 3 to 4 months and acclimated before use in theexperiments described here. All procedures performed on these animals wereapproved by the Plum Island Animal Disease Center Institutional Animal Careand Use Committee. Pigs were infected in the heel bulb intradermally with 104

PFU/ml of FMDV strain 01 Campos A12 or 105 PFU/ml LL-A12 (an attenuatedderivative of A12 achieved by removing the leader protease [13]). Body temper-ature was taken daily.

Preparation of PBMC and serum. Blood samples from animals before infec-tion and after infection were drawn into heparin-containing vacuum tubes, di-luted with phosphate-buffered saline (PBS) 1:1, and layered onto Lymphoprep(Axis-Shield; PoC AS, Oslo, Norway). Cells were centrifuged for 20 min at 20°C.Peripheral blood mononuclear cells (PBMC) were collected and washed in PBStwice followed by one was with RPMI 1640, and finally they were suspendedin RPMI 1640 supplemented with 10% fetal bovine serum (FBS), HEPES,L-glutamine, and antibiotic/antimycotic solutions. Whole-blood analysis was per-

formed on fresh blood samples in an AcT hematology analyzer (BeckmanCoulter, Hialeah, FL) according to the manufacturer’s procedures.

Blood for serum was collected into separate tubes containing clot-activatingfactor and later centrifuged for 20 min at room temperature. Serum was sepa-rated and immediately stored at �70°C until use. All cell-based assays describedin subsequent experiments were performed with freshly isolated cells.

Enrichment of porcine NK cells. NK cells were enriched based on the char-acterization by Denyer et al. (17), Pintaric et al. (42), and Gerner et al. (20), inwhich porcine NK cells are defined as CD3�/CD2�/CD8a�/CD8b�. Negativeselection was performed by removing adherent macrophages (M�) and CD172�,CD3�, CD4�, and CD21� cells. Magnetic-activated cell sorting (Miltenyi Bio-tech, Germany) was used to separate the cells according to the manufacturer’sinstructions. Mouse primary antibodies against porcine CD3 (clone PPT3), CD4(clone 74-12-4), CD21 (clone BB6-11C9.6), and CD172 (clone 74-22-15) werepurchased from Southern Biotech (Burmingham, AL). Briefly, PBMC werelabeled with primary antibodies for 10 min at 4°C. Subsequently, cells were bound bymicrobeads coated with goat anti-mouse immunoglobulin G and further incu-bated for 15 min at 4°C. Cells were washed and passed through a magnetic fieldfor separation. This procedure was repeated twice to ensure the complete re-moval of labeled cells. After separation, cells were washed twice and resus-pended in RPMI 1640 supplemented with 10% FBS. Purity was checked bystaining with CD2 (clone MSA4; Accurate Chemicals) and CD8 (clone 76-2-11;Southern Biotech). Enrichment usually reached 85 to 95% of CD2�/CD8�/CD3� cells containing the majority of porcine NK cells (Fig. 1A).

Virus isolation and quantification. The viral load in serum was determined bya standard assay for isolating virus (40). Tenfold serial dilutions of serum sampleswere prepared and added to monolayers of BHK21 cells in 12-well plates, andadsorption was carried out for 1 h at 37°C. An overlay of 2 ml tragacanth wasadded later, and plates were incubated further for 24 h. Finally, the monolayersin the plates were stained with crystal violet in a fixative for 5 min and thenwashed and dried at room temperature. Plaques were counted, and results areexpressed as PFU/milliliter.

NK cell lytic activity assay. A flow cytometry-based assay was employed toanalyze the NK cell cytotoxicity. K562 tumors cells (human erythroleukemia cellline), stably transfected with the green fluorescent protein (GFP) (referred tohere as K562-GFP), were kindly provided by Michael Olin (University of Min-nesota, School of Veterinary Medicine, St. Paul, MN). Bulk assays or assays withsorted CD2�/CD8�/CD3� cells were performed by incubating cells from healthyor FMDV-infected animals with target cells (K562-GFP) at effector:target (E:T)ratios of 50:1 to 12:1, followed by the addition of 7AAD (the dead/live discrim-inating dye 7 aminoactinomycin D; BD Biosciences, San Diego CA). Cells thenwere mixed, spun briefly at 233 � g, and placed in 5% CO2 for 3 to 4 h at 37°C.Data were acquired using a FACSCalibur flow cytometer and later analyzed withCellQuest Pro software (BD Biosciences, San Jose, CA) by establishing a gate onK562-GFP to display cells reflecting the killed population (cells stained by bothGFP and 7AAD). The lysis level was determined by the formula R1/(R1�R2) �100 � % lysis, where R1 is GFP-positive cells plus 7AAD-positive cells and R2is GFP positive cells.

Stimulation of NK cells within PBMC. NK cells within PBMC preparationswere stimulated with TLR agonists. pI:C was purchased from Amersham Bio-sciences, Piscataway, NJ, and used at 2 �g/ml. CpG2216 was purchased fromInvivoGen (San Diego, CA) and used at 10 �g/ml. The TLR7/8 agonist 3M-011was a gift from Richard Miller of 3M Pharmaceuticals, Inc. (Minneapolis, MN),and was used at 1 �M. PBMC were stimulated with these agonists for 18 h andlater used to assess the NK cell cytotoxicity.

NK cell cytotoxicity against FMDV-infected targets. A genetically attenuatedstrain of FMDV was used in this assay. LL-KGE virus is O1 Campos structuralproteins inserted into the A12 strain backbone with a positive-charge mutationand another mutation in the receptor binding (RGD) sequence to KGE to allowfor the binding of the heparin sulfate receptor so as to increase the cell hostrange. Additionally, the leader protease, which determines the in vitro attenua-tion, has been removed. To measure the cytotoxicity against an in vitro-infectedtarget, SK6 cells (a porcine kidney fibroblast cell line) were infected with LL-KGE virus at a multiplicity of infection of 10 for 4 h. Later, the cells were labeledwith 5 �M of carboxy fluorescein succinimidyl ester (CFSE) and used as targetcells in the 4-h killing assay at an E:T ratio of 50:1. CFSE here was used toidentify the target cells by flow cytometry.

Expression of SLA-I on SK6 and VP1 detection. SK6 cells were grown inminimum essential medium (MEM) to confluence in culture flasks and thendetached with cell dissociation medium for 5 min at 37°C. The cells were furtherpipetted repeatedly to allow for thorough dissociation into single-cell suspension.Cells (1 � 107) were infected with LL-KGE at a multiplicity of infection of 10 inMEM with 0.5% FBS and incubated for 4 h with shaking every 15 min. At the

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end of the incubation period, cells were washed four times in fluorescence-activated cell sorting (FACS) buffer and surface stained with SLA-I–fluoresceinisothiocyanate for 30 min at 4°C, followed by three washes in FACS buffer. Cellsthen were permeabilized with BD Cytoperm/Cytofix (BD Biosciences) for 30 minat 4°C, washed in BD Perm/Wash three times, and finally stained intracellularlywith 12FB2.2.2 monoclonal antibody against VP1 of FMDV for 45 min at 4°C.After three washes in BD Perm/Wash, the cells were stained with a secondaryantibody against mouse immunoglobulin G2a conjugated to phycoerythrin (PE)and incubated for 45 min at 4°C. Cells then were washed three times with BDPerm/Wash and three times with FACS buffer, and the fluorescence intensity wasdetermined in a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA).

CD107a flow cytometry. To determine if the PBMC contained cells that re-cently had been engaged in cytolysis, we stained for CD107a on the cell surface.Cells that have released their cytotoxic molecules (perforin, granulysin, or gran-zymes, which previously were stored in endosomes) express CD107a. We ana-lyzed cells from infected and noninfected animals stained with mouse anti-porcine CD107a-FITC (AbD; Serotec, Oxford, United Kingdom) in the presenceor absence of K562 cells for 4 h. Data were acquired using a FACSCalibur andanalyzed in CellQuest software (BD Biosciences, San Jose, CA).

Intracellular staining for IFN-� and perforin. PBMC from infected or non-infected animals were placed in 96-well plates at 1 � 106 per well and incubatedfor 5 h with 3 �g/ml brefeldin A (eBioscience, San Diego, CA). Cells for positivecontrols additionally were treated with a mixture of phorbol myristate acetateand ionomycin (Sigma, St. Louis, MO) at 100 and 10 ng/ml, respectively. Later,cells first were stained with CD2, CD3, and CD8a and then fixed and permeab-ilized, followed by staining with anti-porcine IFN-�–PE (clone P2G10, BD Bio-science, San Diego CA) at 1 �g/ml or anti-perforin-PE at the concentrationsuggested by the manufacturer (clone G9; BD Biosciences, San Diego CA).Data were acquired and analyzed on a FACSCalibur using CellQuest Pro soft-ware (BD Biosciences, San Diego CA).

Serum IL-12, IL-15, IL-18, and IFN-�. We determined the serum levels of thecytokines by antigen capture enzyme-linked immunosorbent assay (ELISA).Capture and detecting antibodies against porcine or human cytokines werepurchased from Biosource (human IL-15; Camarillo, CA), R&D Systems Inc.(porcine IL-12; Minneapolis, MN), Endogen/Pierce (porcine IFN-�; Rockford,IL), and Bender MedSystems (porcine IL-18; Vienna, Austria). ELISAs wereperformed as described by the manufacturers of the coating and detecting anti-

bodies. Optical densities were read on an ELISA reader (Bio-Tek, Winooski,VT), and data were corrected with a linear regression equation in MicrosoftExcel.

qrtRT-PCR. RNA were isolated from sorted CD2�/CD8�/CD3� cells origi-nating from noninfected or FMDV-infected pigs, treated with DNase, and thentranscribed into cDNA using the following reaction mixture: 5� reaction buffer,10 mM deoxynucleoside triphosphates, 50 ng/�l random primers, 0.1 M dithio-threitol, 40 U RNase inhibitor, and 200 U Moloney murine leukemia virus RT.The cDNA templates were used later in the quantitative real-time RT-PCRs(qrtRT-PCRs) in duplicate. The TaqMan RT-PCR system was used for detec-tion. Primers and probes were designed at the Beltsville Human Nutrition ResearchCenter (http://www.ars.usda.gov/Services/docs.htm?docid�6065; ARS, USDA), andsequences are detailed elsewhere (53, 54). PCRs were run in the 7700 ABI PrismSequence Detector (Applied Biosystems, Foster City, CA) with the followingcycling parameters: hold at 50°C for 2 min, hold at 95°C for 10 min, and then 40cycles at 95°C for 15 s (denature) and 60°C for 1 min (anneal and extension).Ubiquitin or cyclophilin was used as the reference gene to normalize geneexpression. Relative expression was calculated by 2�CT, and data are expressedas ratios relative to the calibrator (CD2�/CD8�/CD3� cells from noninfectedpigs).

Statistics. Statistical evaluation was applied using a two-tailed t test. Values ofP � 0.05 were considered significant.

RESULTS

Clinical disease following FMDV O1 Campos infection. Toput into perspective the conditions under which porcine NKcells are called to function during infection with FMDV, wemonitored multiple clinical parameters from days 0 to 7. Fig-ure 1B, C, and D summarize fever, peripheral blood lympho-cyte count profiles (lymphopenia) and virus titers on a sampleset of animals infected with FMDV O1 Campos. In all exper-iments, peak viremia and fever were observed between days 2and 3, after which vesicular lesions formed on the feet and

FIG. 1. Enrichment of porcine NK cells and clinical parameters during FMDV infection in swine. (A) PBMC were isolated and depleted ofCD3, CD4, CD172, and CD21 by a magnetic-activated cell sorting system. A different set of clones of anti-CD3, anti-CD4, and anti-CD21(BB238E6; Southern Biotech; and PT90A VMRD and B-ly4; BD Biosciences, respectively) were used to check the accuracy of depletion, andrepresentative plots are shown. (B) Body temperature was taken daily after infection until 7 days. (C) Blood was drawn daily, and lymphocytecounts were performed in a Coulter AcT hematology analyzer. Data are presented as percentages of lymphocytes. (D) Serum samples werecollected and virus isolation done as described in Materials and Methods. Virus titers are given as log10 values. The data shown represent a singleexperiment from a total of three performed. Day 0 depicts data from noninfected animals. Numbers in the graph legend denote individual animalidentification tags.

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snout (data not shown). These symptoms were concurrent withsignificant lymphopenia as previously reported (4). All param-eters analyzed appeared only transiently and had resolved byday 4 or 5. As reviewed elsewhere (25), during infection withFMDV, lesions appear rapidly on the feet and snout, and thevirus titers rise and fall quickly. Such conditions have beenreported to be limiting for the function of NK cells (27, 46).

FMDV infection of swine inhibits NK cell lytic activity. Toassess the influence of FMDV infection on NK cell function inswine, we examined the capability of NK cells harvested fromYorkshire pigs infected with the O1 Campos strain of FMDVto lyse target cells ex vivo. The inability of porcine NK cells tolyse a tumor target in vitro is shown in Fig. 2A and B. This wasevident beginning 24 h postinfection, when lytic levels droppedat least 1.5-fold compared to baseline levels (day 0). The re-duction in cytotoxic activity against K562-GFP on day 2 or 3was consistent in all animals examined but usually reboundedby day 5. By day 14 (data not shown), most animals had re-covered the cytolytic activity of NK cells. Occasionally, NKcells from a donor pig reacted by increasing the NK cell activity24 h after infection but abruptly declined by day 2.

Because earlier studies (24) have reported increased levelsof NK cell cytotoxicity when incubations were prolonged be-yond the 4 h of the standard assays, in the second experiment

(Fig. 2B) we also compared killing at 4 to that at 18 h. Al-though the level of killing was higher at 18 h, relative killing bycells at different days following FMDV infection were verysimilar regardless of how long we incubated effector cells withtumor cell targets.

We were curious to know if this inability to kill a tumortarget also was applicable to in vitro FMDV-infected targets.To test this, first SK6 cells were examined for the level ofSLA-I expression after infection with the attenuated FMDVstrain, LL-KGE. Figure 2C shows the reduction of SLA-I fol-lowing a 4-h infection. Later, SK6 cells were incubated withsorted CD2�/CD8�/CD3� cells (a population that constitutesporcine NK cells) from infected animals, and the NK cell lyticactivity was analyzed after 4 h. A similar trend of NK celldysfunction was observed but was more prominent on the thirdday postinfection (Fig. 2D). Therefore, the inability of NK cellsfrom infected animals to engage in lytic activity was consistentwhen using either FMDV-infected porcine fibroblasts or hu-man leukemia target cells.

Furthermore, the inhibition of cytolytic activity could not berestored with additional in vitro stimulation. We tested theaddition of TLR agonists (pI:C/TLR3, CpG2216/TLR9, or3M-011/TLR7 and 3M-011/TLR8 agonists) to PBMC fromFMDV-infected or noninfected animals and cultured them for

FIG. 2. Cytotoxic activity of porcine peripheral blood NK cells. PBMC were isolated from noninfected pigs or pigs infected with O1 Camposstrain of FMDV, and NK cell lytic assays against K562-GFP cells in a 4-h assay were performed daily until day 7. (A and B) Two different setsof animal groups were analyzed in two different experiments out a total of three separate experiments. (C) SK6 cells infected (inf.) with LL-KGE,VP1, and SLA-I were assessed by flow cytometry. The shaded area of the upper panel is VP1 staining in noninfected cells, and the green line isVP1 staining in infected cells; the shaded area of the lower panel is the isotype monoclonal antibody control, the green line is the SLA-I stainingof noninfected cells, and the red line is the SLA-I staining of virus-infected cells. (D) CD2�/CD8�/CD3� cells were purified from PBMC on days0, 1, 3, and 5, and the killing assay was performed on SK6 cells previously infected with LL-KGE virus. Data represent a single experiment froma total of two separate determinations. Data represent a single experiment from a total of two. Percent lysis at an E:T ratio of 50:1 is shown.Numbers in the graph legend denote individual animal identification tags.

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18 h before the NK assay. It was not possible to completelyrestore the NK cell activity with pI:C or CpG2216, particularlyon days 1 and 2 of infection (Fig. 3A and B). NK cell activationwith both agonists (pI:C and CpG2216) could be demonstratedfrom day 3 onwards. A slightly different pattern of activation

was observed for the TLR7/8 agonists, with increased activityon day 1 followed by stagnation on days 2 and 3 and a rapidincrease thereafter (Fig. 3C). These data indicate that theinfection with FMDV induces a profound defect in the func-tion of NK cells during the acute phase of the disease.

Effect of FMDV infection on NK cell degranulation. The factthat we did not detect the cytolytic activity of NK cells fromFMDV-infected animals could indicate an already exhaustedpopulation of peripheral blood NK cells that no longer wasreactive to targets in vitro. When NK cells engage in cytotoxicactivity, they degranulate to release their cytotoxic arsenal, whichleaves a trace on the cell surface in the form of a lysosome-associated membrane protein (LAMP-1), or CD107a. This pro-tein is expressed mainly in endosome-lysosome membranesbefore degranulation (1). Initially, we isolated PBMC fromhealthy pigs, stimulated them with IL-15, and assessed CD107aexpression after incubation with or without K562 cells. Todetermine the level of degranulation in vivo, PBMC from pigsinfected with FMDV were isolated and directly stained forCD107a or restimulated with K562 and then examined forCD107a expression.

Results presented in Fig. 4 show virtually no constitutiveexpression of CD107a detected on cells from healthy animals(Fig. 4a) and low levels of expression following stimulationwith a porcine IL-15 (Fig. 4c). However, incubation with K562cells increased the surface expression of CD107a both in cyto-kine-stimulated cells and in resting cells (Fig. 4b and d). Incontrast, the direct staining of cells isolated from infectedanimals showed little difference in the expression of CD107athrough the 7 days of assessment compared to that of cellsisolated from noninfected donors (Fig. 4e to k). Furthermore,in vitro restimulation with K562 cells did not induce the de-granulation of porcine NK cells from infected animals (datanot shown). However, beginning on day 1 postinfection, theexpression of CD107a could be detected on another popula-tion of lymphocytes, which was neither CD2� nor CD8� (datanot shown). This pattern of CD107a expression currently isunder investigation to identify the CD107a-expressing cellsduring FMDV infection. Overall, we could not detect a signif-icant presence of CD2�/CD8�-expressing CD107a in the pe-ripheral blood of FMDV-infected pigs. The result stronglysuggests the likelihood that NK cells did not engage in lyticactivity during the acute phase of infection.

IFN-� production and perforin storage are influenced byFMDV infection. The increased secretion of IFN-� in NK cellsis said to coincide with the enhanced activity of these cells. Weexamined this in CD2�/CD8�/CD3� cells from O1 Campos-infected pigs. Interestingly, three phases of the response wereseen in cells from infected animals. The first phase was theinitial transient increase of the total percentage of NK cellsproducing IFN-� after 24 h of infection, followed by a rapiddecline to below the levels detected before infection by day 2(Fig. 5A). The reduction on day 2 after infection was concur-rent with the reduction in cytolytic activity (Fig. 2A and B), theoccurrence of lymphopenia, and an increase in virus titer (Fig.1). These cells recovered IFN-� expression on day 3 in mostanimals but began to decline in expression by day 4 or 5. Thesame CD2�/CD8� cells were examined for the storage of per-forin, an important effector molecule in the function of NK

FIG. 3. Restimulation of PBMC does not fully restore the NK cellcytotoxic activity against K562-GFP. PBMC were isolated from in-fected or noninfected cells and restimulated with either pI:C (A), CpG(B), or TLR7/8 agonists (C) for 18 h and then incubated with K562-GFP cells for 4 h to assay the lytic capability of NK cells. Data are froma single experiment, and lytic levels are presented at an E:T ratio of50:1. Numbers in the graph legend denote individual animal identifi-cation tags.

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cells. A similar pattern was observed in the total percentages ofperforin-positive cells (Fig. 5B).

After examining the IFN-� production and perforin storage,it appeared that following infection with FMDV, the totalpercentage of porcine NK cells positive for IFN-� and perforinwas reduced on day 2, a time at which most of the responses inthese cells were low. The increase and decrease in cytokineproduction after day 2 did not correlate with the cytolyticactivity of the NK cells. Similar measurements performed oncells restimulated with TLR agonists also did not change thepattern of response (data not shown). These results suggestthat FMDV influences the function of porcine NK cells byaffecting the number of cells in the circulation and their capa-bility to secrete cytokines or store cytolytic molecules such asperforin.

Serum cytokine profile in FMDV-infected pigs. IL-2, IL-12,IL-15, IL-18, and IFN-� play an important role in activatingporcine NK cells. After infection, serum samples were col-lected throughout the 7-day period of assessment and wereanalyzed for the levels of cytokines that are critical to thefunction of NK cells. ELISA was performed to detect IL-2,IL-12, IL-15, IL-18, and IFN-�. Indeed, the levels of IFN-�were appreciably higher on days 2 and 3 after infection with O1Campos (Fig. 6). This result confirms our other studies show-

ing the induction of IFN-� that was detectable in serum ofswine infected with FMDV regardless of which serotype (A, O,or C) was analyzed (39a). The reduced NK cell function couldresult from a lack of NK cell-activating cytokines or from initialhigh levels of IFN-� early in the FMDV infection of swine, ashas been reported by others (18). Levels of IL-12, IL-I5, andIL-18 in serum of O1 Campos-infected pigs were below detec-tion (data not shown). Also, we could not detect any IL-2 in theserum of any of the experimentally infected animals. Takentogether, this result indicates that FMDV affects the cells re-sponsible for innate cytokine production during infection,which leads to inadequate NK cell activation, therefore ren-dering them dysfunctional.

Differential expression of NK cell-associated genes inCD2�/CD8�/CD3� cells from infected pigs. NK cells expressmultiple activating and inhibitory receptors, as well as effectormolecules, that direct the activity of NK cells in a given envi-ronment. Therefore, it would be assumed that in a situationwhere the function of NK cells is hampered, the expression ofNK cell receptors is skewed in favor of an inhibitory pheno-type. Ideally, the measurement of the surface expression of thereceptors would be more informative, but reagents are notavailable for the porcine homologues of these molecules onNK cells, and furthermore, not all molecules are known in the

FIG. 4. Degranulation of NK cells. To show evidence of NK cell release of cytotoxic granule contents, cells were stained for CD107a followingincubation with K562 cells or were left unstained not. (a) PBMC from noninfected (non-inf.) animals stained for CD107a; (b) PBMC fromnoninfected animals incubated with K562 cells and stained for CD107a; (c) PBMC from noninfected animals stimulated with IL-15 for 18 h andstained for CD107a; and (d) IL-15-stimulated PBMC from noninfected animals incubated with K562 and stained with CD107a. (e to k) PBMCfrom O1 Campos FMDV-infected pigs days 1 to 7 after infection, respectively. Histograms are from a single determination from two separateexperiments.

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porcine model. To learn if FMDV infection alters the expres-sion of NK cell receptor genes, we used qrtRT-PCR to mea-sure the mRNA expression of a selected set of inhibitory andactivating receptor genes and effector molecules, such as gran-zymes and perforin. RNA was isolated from sorted CD2�/CD8�/CD3� cells of FMDV-infected or noninfected pigs. Ofthe six activating receptors examined, only NKp30 was consid-erably upregulated 24 h after infection with FMDV, and Ly49was moderately upregulated (Fig. 7A, B, and C). NKp30 andLy49 gradually reduced expression at all three time pointsanalyzed, although expression still remained at an appreciablelevel. NKG2D, NKp80, and granzyme B were little changed atall time points examined, whereas the expression of two otherimportant effector molecules, granzyme A and perforin, grad-ually increased. One inhibitory receptor, NKG2A, was moder-ately increased by day 3 in all animals tested. Results show thevaried expression of NK cell receptor mRNA during infection,which does not correlate with the dysfunctional status of theNK cells.

The inability of NK cells to function also could result frominadequate cytokine receptor expression. Therefore, we inves-tigated the mRNA expression of IL-12R�2, IL-15R�, IL-18R�, and IFN-�R1 on the CD2�/CD8�/CD3� cell subsetduring infection with FMDV. In cells isolated from infectedanimals, all five cytokine receptors were moderately upregu-lated at all time points examined compared to results for cellsfrom noninfected animals. IL-15R� mRNA showed the high-est increase in expression, increasing each day to nearly 10-fold(Fig. 7D). There also was significant upregulation of TLR3mRNA and more moderate activation of CCR5 (Fig. 7C, D).Of interest was the nearly 20-fold induction of SOCS3 mRNAby 24 h, which rapidly waned thereafter. These results mini-mally demonstrate that porcine NK cells do transcribe thegenes for the receptors for these cytokines. The cytokine re-ceptor mRNA expression pattern shown here appears to beappropriate for the activated environment such that of as aviral infection, therefore cytokine receptors may not partici-pate in the aberrant function of porcine NK cells during infec-tion with FMDV.

Influence of viral proteins on function of porcine NK cells.We investigated whether the observed dysfunction of NK cellscould be attributed to virulence factors encoded by the virus.Using a genetically attenuated strain of FMDV, LL-A12 virus,and the parent strain, wild-type A12, we compared the activityof NK cells isolated from pigs infected with these viruses. Theattenuation is due to a lack of the leader protease encoded bythe virus, thus the designation leaderless (LL) for this attenu-ation. The leader protease blocks cap-dependent mRNA trans-lation in infected cells by cleaving elongation factor 4G in theribosomal assembly (19).

NK cell activity from animals infected with the wild-typevirus showed a trend between days 1 and 3 (Fig. 8A) that isdysfunctional similarly to the previous experiments using FMDVstrain O1 Campos. In contrast, NK cells derived from LL-A12-infected pigs increased the cytolytic activity within 24 h ofinfection (Fig. 8B). The supposition that some viral proteinsare involved in impairing the functions of porcine NK cells alsowas evident in the serum cytokine profile of IL-15 and IL-18 ofthe same animals. IL-15 was detected at a higher level inanimals infected with LL-A12 than in A12-infected pigs fromday 3 onwards (Fig. 9A and B). However, the difference be-

FIG. 6. Serum level of IFN-�. Serum was prepared from bloodsamples of infected animals and tested for the presence of IFN-� usingELISA. Data represent a single determination from three separateexperiments. Numbers in the graph legend denote individual animalidentification tags.

FIG. 5. IFN-� and perforin profile in infected animals. PBMC wereisolated on 5 consecutive days from infected animals and stained in-tracellularly for IFN-� and perforin as described in Materials andMethods. CD3� cells were gated out, and another gate was created oneither IFN-�- or perforin-positive cells and then applied to CD3�/CD2�/CD8� cells to give the percentage of CD2�/CD8� positive cellsfor either IFN-� (A) or perforin (B). Data represent a single examplefrom five separate experiments. Day 0 represents data derived fromnoninfected animals and serves as a point of reference. Numbers in thegraph legend denote individual animal identification tags.

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tween the two groups was not statistically significant (P �0.065) due to high variability among individual animals. Also,there was no significant difference observed for IL-18 (Fig. 9Cand D) between wild-type and attenuated virus-infected ani-mals (P � 0.071). Surprisingly, we could not detect IL-12 andIFN-� in either group of animals. Also, the difference in theinduction of IL-15 and IL-18 or a lack thereof during infectionwith 01 Campos and A12 viruses is not understood and may bevirus strain dependent. These results indicated that the leaderprotease is involved in downregulating the NK cell activity

following infection with FMDV, possibly by blocking cytokine-inducing signals from infected cells.

DISCUSSION

These studies have characterized the activity of NK cellsfrom animals infected with FMDV. We show that NK-depen-dent cytotoxicity against FMDV-infected cells as the killingtarget or a standard tumor cell target is impaired. Also, theproportion of NK cells capable of producing IFN-� and storing

FIG. 7. mRNA expression by qrtRT-PCR. CD2�/CD8�/CD3� cells were purified from PBMC of infected animals on days 1, 3, and 5. RNAwas isolated, and qrtRT-PCR was performed on the transcribed cDNA. (A to C) NK cell-associated genes; (D) cytokine receptor genes. Data arepresented as the mean expression levels relative to those of CD2�/CD8�/CD3� cells from noninfected animals. The experiment was repeated threetimes.

FIG. 8. Comparison of NK cell activity of two strains of FMDV. Two groups of three pigs each were infected with either A12 or LL-A12 (alaboratory genetically attenuated strain). PBMC were isolated on 7 consecutive days and incubated with K562-GFP cells to assess the lytic activity.(A) A12; (B) LL-A12. Data are representative of a single experiment. Day 0 denotes samples from the same animals before infection. Numbersin the graph legend denote individual animal identification tags.

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perforin is reduced. In vivo, the virus replicating in susceptibletissue appears to create an activated environment for porcineNK cells. The loss of function therefore is not anticipated, asthere is no detectable infection of the circulating NK cells,although that does not rule out a low level of abortive infec-tion. Few NK cell receptors are modulated in expression, butinitially, there is a significant induction of one receptor, NKp30,which then decreases through day 5. This is known to be anactivating receptor in other species.

Reduction in NK cell cytotoxicity coincides with the eleva-tion in virus titer in peripheral blood. For instance, cells werenot reactive to an in vitro-infected target, a target cell type thatmost reflected an in vivo encounter. Further, prolonging theincubation of these NK cells with targets did not improve thecytolytic activity, and additional stimulation with exogenouscytokines could not rescue the killing capability of NK cells. Anexception was observed only when TLR7/8 agonist was used torestimulate the cells, illuminating the potential of these ago-nists as biotherapeutics.

In analyzing the dysfunction of porcine NK cells duringFMDV, it is worth considering at least three factors that con-verge on the role of these cells in infection, i.e., viremia, lym-phopenia, and high fever. All of the functions assessed on theNK cells were at their lowest at the time of high virus load incirculation, a lower number of lymphocytes in the blood, andhigh body temperature. The effects of these three factors couldgive way to defects in generic NK cell functions, such as cyto-toxicity, cytokine and chemokine secretion, and NK cell pro-liferation. Viremia is associated with poor NK cell perfor-mance in HIV infection in humans as well (37). In fact, thecapability of NK or CD8� T cells to suppress endogenous HIVreplication inversely correlated with virus load such that highviral titers coincide with less NK cell suppression of viral rep-

lication. Also, the viral load negatively influenced the secretoryfunction of NK and CD8� T cells from viremic individuals asopposed to that of aviremic patients (30).

An increase in temperature led to a reduction in the activityof human NK cells incubated in vitro at 42°C for 1 h, just asintentional hyperthermia for cancer therapy at 42°C produceda similar reduction of NK cell cytotoxicity. In both instancesNK cell function was restored after the temperatures werereturned to normal (27). In those studies, the authors attrib-uted the perturbation in NK cell function to the downregula-tion of perforin and granzyme B (29) by CD56dim cells, apopulation of NK cells in humans that is characterized by highcytotoxicity. A similar example of multifactorial influence onearly immune responses is described for Ebola hemorrhagicfever virus infection in cynomologous macaques, where thenumbers as well as function of NK cells were low, and therewas no indication of a robust immune response to infection(46).

Previously, we reported the induction of lymphopenia dur-ing the acute phase of FMDV infection (4), but this report isthe first to connect the impact of lymphopenia in FMDV to thedysfunction of NK cells in the infected pigs. A recent report byMohan et al. (38) indicates the occurrence of a similar phe-nomenon in cattle and buffaloes and indicates the correlationbetween an increase in body temperature and leukocyte reduc-tion. Regarding lymphocyte kinetics after infection, FMDVdiffers from other viral infections in that NK cell numbersappear to be reduced already in the acute phase. In HIV,initially there is a selective increase of CD3�/CD56dim/CD16�

cells and a depletion of CD3�/CD56bright/CD16� cells, but asthe infection progresses, CD3�/CD56dim/CD16� cells are de-pleted while CD3�/CD56bright/CD16� cells become anergic(29). Therefore, these three factors, viremia, lymphopenia, and

FIG. 9. Serum levels of IL-15 and IL-18. Serum was prepared from blood samples collected from either infected or noninfected pigs, andELISA was performed for IL-15 and IL-18. Shown are levels of IL-15 in A12 (A)- and LL-A12 (B)-infected pigs as well as levels of IL-18 in A12(C)- and LL-A12 (D)-infected pigs. Representative data are from a single experiment. Day 0 denotes samples from the same animals beforeinfection. Numbers in the graph legend denote individual animal identification tags.

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fever, induced concurrently during acute FMDV infection, ap-pear to exert a strong inhibitory effect on the cytolytic andsecretory function of porcine NK cells.

The activation of NK cells is said to require cytokines such asIFN-� (56). Indeed, earlier in infection, FMDV induces theproduction of IFN-�, which is detectable in serum on days 2and 3 postinfection in swine. However, it is surprising that eventhough this cytokine is present in the peripheral blood, the NKcells from FMDV-infected pigs fail to function. This observa-tion is not attributable simply to the loss of NK cells fromperipheral blood, as NK cells (CD2�/CD8�/CD3�) isolated onthese days following infection are inactive. Apparently, othermechanisms are required to fully restore and maintain thefunctional status of these cells. On the other hand, the higherlevels of IFN-� may be incapacitating for NK cells due tostrong stimulation. The repeated administration of pI:C in piglets,which was intended to stimulate NK cells, only rendered themhyporeactive due to higher levels of IFN-� in the serum (18).However, the mechanism leading to hyporeactivity remainsundefined.

In the case of FMDV, a negative feedback mechanism couldbe implicated in this event, such as the upregulation of theSOCS genes. Increased levels of IFN-� could signal throughIFN-�R1, inducing the upregulation of SOCS3 and leading toa blockade in IFN-�-induced gene expression programs (33).Alternatively, since FMDV is an RNA virus, an increase inSOCS3 mRNA could be mediated through RIG-I or TLR3sensing, as suggested by Pothlichet et al. (43). In our results,both TLR3, and more transiently, SOCS3 mRNAs were up-regulated, indicating the possibility of such a mechanism forthe inhibition of NK cell response.

Regarding other cytokines of importance to NK cell activa-tion, we could not detect IL-12, IL-15, and IL-18 in the serumof pigs infected with FMDV strain O1 Campos, but the cyto-kines could be detected in the serum of animals infected withthe A12 strain and the attenuated derivative, LL-A12. Presum-ably, the efficiency with which each strain effects host cellprotein synthesis was different. The lack of these cytokinescould further contribute to the lack of the cytotoxic potential ofNK cells after infection with FMDV. Barnett et al. (3) reportedno elevation of IL-12 in pigs challenged with FMDV strain O1Lausanne. The lack of NK cell-activating cytokines in serummay indicate an expected lack of protein synthesis due to themanner in which FMDV alters the cell protein synthesis ma-chinery.

The leader protease cleaves elongation factor 4G (elF-4G),therefore shutting down the cap-dependent translation of cel-lular mRNA (19). Consequently, the infected cells may fail totranslate new proteins (48). However, this mechanism is likelyonly in the case of an infected cell. There is no clear evidencethat innate cytokine-producing cells are infected with FMDV,yet the inhibition of cytokine secretion has been reported (5,39). The infection of pigs with the attenuated so-called lead-erless virus is marked by the activation of NK cell activity,while the parent wild-type virus inhibits NK cell function invivo. Kottilil et al. (31) found that HIV envelope proteinsderived from R5 or X4 subtype isolates altered the expressionof genes associated with NK cell function. They upregulatedgenes involved in apoptosis and downregulated genes involved

in cell proliferation. Such NK cells were dysfunctional regard-ing NK cell cytotoxicity and cytokine secretion.

One alternative explanation for a lack of NK cell cytotoxicactivity when testing cells isolated from infected pigs is that NKcells encountered target cells in vivo, preceding isolation andanalysis ex vivo. NK cells from HIV-infected individuals dis-play a substantial expression of CD107a ex vivo without in vitrostimulation with target cells (45). We did a similar assessmentof CD107a to discriminate several populations of NK cells (1)beyond the killing assays and cytokine secretion. We wereunable to detect CD107a expressed at significant levels on cellsfrom infected animals, meaning that the cells likely were notpreviously engaged in cytolysis. These results indicate thatrather than exhausting NK cell function by interaction withinfected cells, a strong inhibition of NK cell activation occurs invivo during FMDV infection.

An array of surface receptors modulates the general func-tion of NK cells through a balance of the inhibitory and acti-vating receptors that recognize ligands on infected cells. Themeasurement of mRNA expression for some of these mole-cules shows little alteration in gene expression in cells frominfected animals, including NKG2D, NKp80, and granzyme B.On the other hand, the expression of another receptor, NKp30,was significantly induced, an expected result for cells originat-ing from an activated environment. However, these cells weredysfunctional.

In animal species where these molecules have been studiedin depth, the downregulation of activating receptors was asso-ciated with defective NK cell cytotoxicity (37). The upregula-tion of CCR5 has been reported in HIV infection that isassociated with assisting virus entry into CD4� T cells. Itsexpression on NK cells is not clearly understood, but possiblyit interacts with viral envelope protein to cause defects in theirfunction (31). Unfortunately, no information is available yetabout the precise function of the swine homologues of thesegenes expressed by NK cells. Therefore, it is difficult to spec-ulate how this pattern of expression relates to the observeddysfunctional nature of porcine NK cells during infection withFMDV. Moreover, the narrow selection of receptors analyzeddoes not allow the drawing of definitive conclusions on a pos-sible tipping of the balance toward a cell phenotype that isdysfunctional. Although cytokine receptor mRNAs were ap-preciably expressed, we could not detect appropriate cytokinesin the serum. This observation could argue in favor of NK celldysfunction, since no cytokine signals would be transduced,even though cytokine receptor expression was intact. However,due to the lack of reagents, we could not examine the NK cellsurface cytokine receptor expression.

Although the mechanisms of NK cell dysfunction duringFMDV infection remain unresolved, it is certain that FMDVinfection is associated with functional perturbations in NKcells, which include reduced cytotoxicity, the altered expressionof NK cell receptors, and a reduced ability to secrete cytokines.This dysfunction may result from the direct effect of FMDV onNK cells, although these cells are not productively infected.The nonproductive infection of these cells, below the detectionlevels of our assays, is a situation that cannot be ruled out.Alternatively, the virus may induce the deregulation of signalsfrom infected cells, resulting in the induction of an anergicstate in the NK cells. Potential mechanisms of such a situation

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presently are under investigation. In conclusion, these resultsillustrate another aspect of the evasion of host antiviral re-sponses by FMDV infection.

ACKNOWLEDGMENTS

This work was supported by CRIS 1940-32000-052-00D (W.T.G.)and 1235-51000-051-00 (H.D.) from the Agricultural Research Service,USDA, and interagency agreement number 60-1940-8-037 betweenthe Department of Homeland Security, Science, and Technology Di-rectorate and the USDA (W.T.G.).

C. Nfon was the recipient of a Plum Island Animal Disease CenterResearch Participation Program fellowship, administered by the OakRidge Institute for Science and Education (ORISE) through an inter-agency agreement between the U.S. Department of Energy and theUSDA.

We thank Mary Kenney for her technical assistance, Kathy Apicellifor assistance with illustrations, and the animal care staff at the PlumIsland Animal Disease Center for their professional support and as-sistance. We thank Richard Miller of 3M Pharmaceuticals, Inc. (Min-neapolis, MN) for the generous gift of the TLR7/8 agonist compounds.Finally, we thank D. Mark Estes, University of Texas Medical Branchat Galveston, for his consultation and discussion of this work.

No competing financial interests exist.

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