1 Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK
2 Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK
Correspondence
Javier Castillo-Olivares
javier.castillo-olivares{at}aht.org.uk
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ABSTRACT |
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INTRODUCTION |
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Virus-neutralizing antibodies (VNAbs) in equids are believed to play an important role in immunity against EAV (Fukunaga et al., 1981; McCollum, 1969
). Their appearance in serum coincides with clinical recovery and reduction of virus excretion, and passive transfer of colostral antibodies from immune mares to foals was found to moderate or prevent equine viral arteritis (McCollum, 1976
). Furthermore, protection in animals immunized with inactivated whole virus vaccines (Fukunaga et al., 1990
) or a prototype subunit vaccine comprising the GL (ORF5) ectodomain, an immunodominant virion component (Chirnside et al., 1995
; Castillo-Olivares et al., 2001
), correlated with VNAb titres at the time of challenge. Currently, nothing is known about cell-mediated immunity to EAV, such as the role it plays in clearance of virus infection and whether it would be desirable to stimulate such immune responses by vaccination.
Studies of other arteriviruses have investigated cell-mediated immune responses. Virus-specific cytotoxic T lymphocytes (CTL) have been detected in mice infected with LDV (Even et al., 1995; van den Broek et al., 1997
). There are some indications that cellular immunity plays an important role in clearance of SHFV infections since persistently infected patas monkeys with low-titre VNAb can clear subsequent acute infections caused by a different strain (Gravell et al., 1986
). It has also been shown that PRRSV infection of pigs stimulates a strong cellular immune response (Lopez Fuertes et al., 1999
) and that there is an increase in cytolytic lymphocytes in the lungs during infection (Samsom et al., 2000
). Animals that recover from EAV infection develop a long-lasting immunity against the disease (Gerber et al., 1978
), although not always against re-infection (McCollum, 1969
). However, EAV replication in chronically infected stallions, which is restricted to cells of the accessory sex glands, persists for several months or years, despite high levels of circulating VNAbs. In addition, experimental infections with EAV can cause a cell-associated viraemia that lasts several weeks after serum VNAb becomes detectable (Neu et al., 1987
; J. Castillo-Olivares, unpublished observations). One step towards a better understanding of virushost interactions in equine viral arteritis would be to determine whether virus-specific cytotoxic cell-mediated immune responses are elicited during infection in the natural host.
Various methods have been developed to study CTL responses against equine herpesvirus type 1 (EHV-1) (Allen et al., 1995; O'Neill et al., 1999
), equine infectious anaemia virus (McGuire et al., 1994
, 1997
, 2000
; Hammond et al., 1998
; Zhang et al., 1999
; Lonning et al., 1999
) and equine influenza virus (Hannant & Mumford, 1989
). The aim of this work was to determine whether EAV induces cytotoxic cell-mediated immune responses and to examine some of the features of this response in the natural host.
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METHODS |
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Animals.
Five 2-year-old castrated male ponies (7378, 027a, 5d66, 5062 and 697b) infected intranasally with 106 TCID50 EAV LP3A+ strain were used to obtain peripheral blood mononuclear cells (PBMC) and equine dermal cells (EDC). Both types of cell were used in cytolytic assays to detect EAV-specific cell-mediated cytotoxic responses. The animals presented a moderate to severe acute EAV syndrome, characterized by pyrexia, anorexia, lethargy, weight loss, mild ataxia and conjunctivitis. Virus was isolated from nasal secretions for the first week of infection and from blood up to day 21 post-infection (p.i.). VNAbs were first detected in serum by day 6 p.i., rose to titres of 1 : 100 and remained stable for more than 1 year.
Establishment of primary EDC lines.
EDC lines were established for each pony used in the study. Skin punch biopsies were performed under aseptic conditions, the epidermal portion of the sample removed and the dermal plug immersed in MEM/20 % FBS [Eagle's minimum essential medium with Earle's salts (M2279; Sigma) supplemented with 100 IU penicillin ml-1, 10 µg streptomycin ml-1, non-essential amino acids, L-glutamine to a final concentration of 2 mM and 20 % heat-inactivated foetal bovine serum (FBS)] and transported to the laboratory for immediate processing. The dermal plug was sliced using a scalpel and each slice laid in a well of a six-well flat-bottomed plate. Fresh MEM/20 % FBS was added to each well and the plates incubated at 37 °C, 5 % CO2 for 5 days. Once islets of fibroblasts were observed, the inoculum was removed, fresh MEM/20 % FBS was added and incubation continued at 37 °C, 5 % CO2 until a confluent monolayer was formed. The cells were then passaged into a 25 cm2 tissue culture flask, expanded and finally resuspended in freezing medium (10 % DMSO in FCS) for cryopreservation in liquid nitrogen. In the experiments described in this study, the cells were used between passages 5 and 14.
Preparation of targets for 51Cr release cytolytic assay.
Twenty-four hours before the effectors were incubated with the targets, the EDC monolayers were washed twice with PBS and the cells trypsinized. Once detached, the cells were resuspended in MEM/10 % FBS and centrifuged for 5 min at 1400 r.p.m., the supernatant discarded and the cells resuspended in MEM/20 % FBS at a concentration of 4x105 cells ml-1. Half of the cells were inoculated with EAV LP3A+ at an m.o.i. of 0·3 TCID50; the other half was left uninfected. Each set of cells was then inoculated with 15 µCi Na251CrO4 (Amersham) ml-1, and 100 µl per well of each suspension was added to half of the wells of a 96-well flat-bottomed plate and incubated at 37 °C, 5 % CO2 for 24 h.
Secondary stimulation of in vivo-primed PBMC (effectors).
Induction of cytotoxic cells from PBMC was done as described for the detection of EHV-1-specific CTL by Allen et al. (1995) with minor modifications. Briefly, whole venous blood was collected at various times after infection into vacuum tubes containing 1 IU sodium heparin in PBS ml-1 whole blood and the mononuclear cell fraction was isolated by FicollHypaque density gradient centrifugation. The interface was harvested and the PBMC were washed three times in PBS to reduce the number of platelets and resuspended in either freezing medium or induction medium [1 : 1, v/v, mixture of AIM-V/RPMI 1640 supplemented with 2 mM L-glutamine, minimal essential medium non-essential amino acids (0·05 mM each), 0·5 mM sodium pyruvate, 2-mercaptoethanol (55 µM), gentamicin (50 µg ml-1) and equine serum (7 %, collected from the ponies before EAV experimental infection and inactivated at 56 °C for 40 min)]. The PBMC were incubated in induction medium for 7 days in upright 75 cm2 tissue culture flasks at 1·12·0x108 cells per flask in 40 ml in the presence or absence of 106·1 TCID50 EAV LP3A+.
Measurement of cytolytic activity of in vitro-stimulated PBMC.
EAV-induced and mock-induced PBMC cultures were centrifuged at 800 g at 20 °C for 10 min without the brake, the supernatant discarded and cell pellets resuspended in CTL medium (RPMI 1640 containing 10 % heat-inactivated equine serum). Both cultures were adjusted to contain the same concentration of viable cells (determined by trypan blue exclusion) and diluted appropriately in CTL medium to obtain different effector:target ratios when added to the overnight-grown fibroblasts. Target cells (24 h after addition of 51Cr, ±EAV infection) were washed three times with RPMI 1640 using 125 µl per well in each wash before the addition of either the effectors, a CTL medium control, or cell lysis solution (2 % Triton X-100 in PBS). The plates were incubated for 4 h at 37 °C, 5 % CO2, after which the supernatants were harvested (Supernatant Harvesting System) for quantification of 51Cr release by gamma counting. The lytic activity of each PBMC culture dilution was assessed against four to six replicates of EAV-infected and uninfected autologous or allogeneic radiolabelled targets. The percentage of specific 51Cr release was calculated according to the formula: [(e-sp)/(t-sp)]x100, where e is the experimental 51Cr release in the presence of effectors, sp is the spontaneous 51Cr release in the presence of CTL medium and t is the total 51Cr release from targets incubated with cell lysis solution.
Indirect immunofluorescence.
Equine dermal fibroblasts or cytospins of PBMC were fixed in either acetone or 4 % formaldehyde, 0·4 % Triton X-100 in PBS for 15 min at room temperature. After washing in PBS, an anti-EAV nucleocapsid (N)-specific rabbit polyclonal antiserum (de Vries et al., 1992) diluted 1 : 100 in 2 % BSA in PBS (PBSA) was applied to the cells and incubated for 1 h at 37 °C. After washing in PBS, the samples were incubated for 1 h at 37 °C with an anti-rabbit IgG FITC-conjugated antibody (Dako) diluted 1 : 40 in PBSA, washed again in PBS and observed using a fluorescence microscope.
Flow cytometry.
Equine dermal cells were pretreated before immunostaining procedures in suspension were carried out. Confluent monolayers of EDC were trypsinized, washed in MEM/20 % FBS, resuspended in MEM/20 % FBS and incubated at 37 °C, 5 % CO2 for 2 h. In vitro-induced effectors and pretreated targets were washed in PBSG (0·5 % normal goat serum, 0·01 % sodium azide in PBS) three times and 2x106 cells resuspended in PBSG containing monoclonal antibodies (mAbs) (2 µg ml-1) specific for equine lymphocyte antigens (Lunn et al., 1998). Cells were incubated with either mAb H58A, H42A, HT14A or HB61A (VMRD Inc.) specific for MHC I, MHC II, CD8 and CD4 antigens, respectively. Background staining was determined using an IgG1 isotype control mAb (Dako). After 1 h incubation on ice, the cells were washed with ice-cold PBSG and resuspended in goat anti-mouse IgG FITC-conjugated antibody (Dako) diluted 1 : 40 in PBSG and incubated for 1 h on ice. After a final wash in PBSG, the cells were fixed in 2 % formaldehyde and analysed on a FACScalibur flow cytometer (Beckton Dickinson). Dead cells were identified using propidium iodide staining at a concentration of 20 µg ml-1.
Separation of CD4+ and CD8+ T cells from induced PBMC cultures.
Biomagnetic separation of effectors was performed using MACS goat anti-mouse IgG microbeads and MS Separation Columns (Miltenyi Biotec), essentially following the manufacturer's recommendations. Briefly, after 7 days incubation, the PBMC cultures were centrifuged, the supernatant discarded and the cells washed twice in separation buffer (0·5 % BSA, 2 mM EDTA in PBS). The stimulated PBMC were then incubated for 1 h at 4 °C in separation buffer with or without 2 µg of either anti-equine CD4 mAb HB61A or anti-equine CD8 mAb HT14A ml-1. The cells were washed twice in separation buffer and incubated with the anti-mouse IgG-conjugated microbeads for 1 h at 4 °C before being washed again in separation buffer and applied to the separation columns. Both enriched and depleted cell fractions were collected, centrifuged and the cell pellets resuspended in CTL medium to obtain equivalent concentrations of effectors. The cells were then assayed for cytotoxic activity as described above.
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RESULTS |
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Fig. 5(a) confirmed specific cytolysis of EAV-infected autologous targets by the unfractionated, EAV-induced effectors. Strikingly, despite the relatively low effector:target ratio (6 : 1), the CD8+-enriched fraction showed a high level of lysis of EAV-infected targets (60 % specific lysis). In contrast, the CD8+-depleted and the CD4+-enriched subpopulations showed very low cytolytic activities. These results indicate that the EAV-specific cytotoxicity observed in vitro is mediated by CD8+ T lymphocytes.
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DISCUSSION |
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In the present study, generation of virus-specific CD8+ T lymphocytes was achieved by incubating the PBMC for a few days in the presence of live virus without adding exogenous cytokines such as IL-2. Although exogenous cytokines were likely to enhance the activation of lymphocytes during the in vitro induction process, previous studies in horses have shown that high concentrations of exogenous IL-2 stimulated cytotoxic effector cells of broad specificity, which killed in a genetically unrestricted manner (Hannant & Mumford, 1989). It is worth noting that, unlike EHV-1 CTL induction (O'Neill et al., 1999
), the cytotoxic activity against EAV-infected cells could not be induced using cryopreserved PBMC. This is probably due to damage of an EAV-permissive subpopulation of cells during the freezing and/or thawing processes. For EHV-1, it is known that T lymphocytes are the major population susceptible to EHV-1 infection and these cells survive the cryopreservation procedures well. In our studies, EAV N antigen expression was clearly detected in freshly collected PBMC but not in cryopreserved PBMC (24 h after infection with EAV) and infected cells had a monocyte-like morphology. Furthermore, we observed a significant reduction in the number of cells of monocyte morphology visible for EAV-induced compared with mock-induced PBMC (7 days p.i.), suggesting that monocyte-lineage cells may be the EAV permissive cell type acting as the principal antigen-presenting cell during the in vitro stimulation procedures. However, dual staining with an antibody specific for the human monocyte/granulocyte marker L1 (calprotectin) demonstrated that EAV-infected cells (24 h p.i. of PBMC) were L1 negative (data not shown). This may indicate that the EAV-infected cells are not monocyte/macrophage lineage, that EAV infection down-regulates expression of L1, or that only a subpopulation of differentiated monocytes/macrophages is susceptible to EAV infection, since it has been shown that a subset of blood monocytes do not express L1 and that expression is modulated during differentiation (Zwadlo et al., 1988
). Further studies are required to identify the prinicipal antigen-presenting cells required for in vitro induction of CTL against EAV.
Following EAV induction, we observed activation of both CD8+ and CD4+ cells (indicated by increased size and granularity and upregulation of CD8 and CD4 antigen expression), with an overall increase in the CD8+/CD4+ ratio. Significantly, subfractionation of effectors indicated that CD8+ cells were responsible for the lysis of EAV-infected target cells. We were not able to determine the equine leukocyte antigen phenotype of the ponies used in this study. However, our results suggested the cytolytic activity was genetically restricted. Although we have not definitively demonstrated MHC I-restricted killing, these results are consistent with stimulation of a classical, CD8+ CTL response.
We have demonstrated that EAV-specific CTL precursors persist for at least 1 year p.i. Clinical recovery from EAV and the reduction of virus excretion from nasal secretions coincides with the development of VNAb in serum (usually after the first week of infection), as observed in the ponies used in this study and described in other reports (Fukunaga et al., 1981; McCollum et al., 1969
). However, cell-associated viraemia persists for longer periods ranging from 2 or 3 weeks to around 3 months p.i. (Neu et al., 1987
; J. Castillo-Olivares, unpublished data). The mechanisms responsible for the eventual disappearance of the viraemia are not known, but the detection of CD8+ CTL precursors from EAV-convalescent ponies indicates that cell-mediated immunity might play an important role in the ultimate clearance of the infection. Following PRRSV infection of pigs, it appears that a combination of cell- and antibody-mediated immune mechanisms is necessary to clear infection. PRRSV induced an antibody response that was detectable by day 9 p.i. (Labarque et al., 2000
), but lymphoproliferative responses and VNAbs were not detectable until 4 weeks p.i. (Lopez Fuertes et al., 1999
), coinciding with clearance of virus from blood and lungs. Observations in mice infected with LDV, which exhibit a life-long persistent infection, indicate that CTL are stimulated but disappear within 30 days (Even et al., 1995
). It is believed that the mechanism of viral persistence in LDV infections involves a process of clonal deletion of new LDV-specific CD8+ cells in the thymus, leading to a state of tolerance to LDV CTL epitopes. It will be interesting to determine the importance of CTL in the control and ultimate clearance of EAV infection, in particular, whether there are differences between the CTL responses of stallions persistently infected with EAV compared with those that clear the infection.
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ACKNOWLEDGEMENTS |
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Received 5 February 2003;
accepted 24 June 2003.
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