Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells

Veterinary Immunology and Immunopathology 67 (1999) 17±32

Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells E.M. Siedeka, M. Whelan1,b, N. Edingtona, A. Hamblina,* a

Department of Pathology and Infectious Diseases, The Royal Veterinary College, Royal College Street, London, NW1 0TU, UK b Institute of Animal Health, Compton, Nr Newbury, Berks, RG20 7NN, UK Received 7 May 1998; accepted 25 August 1998

Abstract Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses. As with other herpesviruses, cell-mediated immunity is considered important for both recovery and protection. Although virus-specific T-cell proliferation and cytotoxicity can be detected following in vivo infection, little is known about the role of antigen presenting cells such as dendritic cells (DCs) in these processes. Peripheral blood DCs were shown to express the viral glycoprotein gB perinuclearly following exposure to EHV-1 in vitro, demonstrating EHV-1 replication within them. Co-culture of infected DCs or their supernatants with a susceptible cell line (RK13) demonstrated that EHV-1 infection was productive. In vitro-infected DCs showed cytopathic effects, including loss of viability and syncytial formation. However, they were superior to other antigen presenting cells in stimulating both peripheral blood T-cell proliferation and cytotoxicity. Although ponies which had been intranasally infected with EHV-1 exhibited T-cell proliferation to live virus presented on DCs, the responses began to decline as early as 15 weeks and cease at 22 weeks post-in vivo infection. Cytotoxic responses were not detected 35 weeks after the first intranasal infection but were seen again 7 weeks following a second infection. These findings show that equine DCs, which are infected with EHV-1 in vitro, can stimulate memory T-cell responses but appear unable to circumvent the short-lived memory response found following this infection in vivo. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; Equine herpesvirus type 1; Proliferation; CTL; Immunosuppression

* Corresponding author. Tel.: +44-171-468-5319; fax: +44-171-383-4670; e-mail: [email protected] 1 Present address: The Edward Jenner Institute for Vaccine Research, Compton, Nr Newbury, Berks RG20 7NN. 0165-2427/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 9 8 ) 0 0 2 0 3 - 7

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1. Introduction Equine herpesvirus type 1 is a common pathogen in horses associated with respiratory disease, abortion and myeloencephalopathy (Allen and Bryan, 1986). Protective immunity to re-infection is short-lived (4±6 months) and horses suffer repeated outbreaks throughout their life (Allen and Bryan, 1986). None of the current vaccines induces effective or durable immunity (Burki et al., 1990). Infection with EHV-1 stimulates the production of neutralising antibodies, but these antibody responses are not protective (Mumford et al., 1987). As has been found for other herpesviruses (Sethi et al., 1983), cell-mediated immunity thus appears important for recovery from disease and for protection against re-infection. Cell-mediated immune responses are initiated by antigen presenting cells (APCs), of which dendritic cells (DCs) are the most potent both in vivo and in vitro (Banchereau and Steinman, 1998). DCs acquire antigens at epithelial surfaces and carry them via lymphatics to the draining lymph nodes where they present processed antigens to T-cells (Hamilton-Easton and Eichelberger, 1995). Additionally, DCs are able to secrete high levels of IL-12 which drives the development of Th1 responses (Koch et al., 1996) including anti-viral cytotoxic T lymphocytes (CTLs) (Macatonia et al., 1991; Burkly et al., 1995). DCs process viral antigens and present them in association with MHC Class I molecules to CTLs (Bhardwaj et al., 1994) which then clonally expand and develop into effector cells that detect and lyse virally-infected cells. CTL activity has been demonstrated in horses following in vivo experimental infection with EHV-1 (Allen et al., 1995) but it is not known whether DCs in equines are able to stimulate EHV-1specific CTLs. Infection of DCs by a number of viruses such as measles virus (Fugier-Vivier et al., 1997; Schnorr et al., 1997), human immunodeficiency virus, (HIV; Patterson and Knight, 1987), influenza virus (Bhardwaj et al., 1994) and LCMV (Borrow et al., 1995) has been reported and has been shown to interfere with their ability to stimulate T-cell responses (Macatonia et al., 1992; Fugier-Vivier et al., 1997). We have recently isolated cells from equine peripheral blood with the features of DCs (Siedek et al., 1997). Here we report that these equine DCs are susceptible to EHV-1 infection in vitro and that a cell population enriched for DCs and infected with EHV-1 can be used as APCs to stimulate both proliferative and CTL responses. We further show that in spite of their immunostimulatory capacity (Banchereau and Steinman, 1998) such DCs fail to maintain cell-mediated responses in animals which have been infected with EHV-1 in vivo. 2. Materials and methods 2.1. Animals Peripheral blood was collected for the infection studies from a total of 24 healthy abattoir horses after the animals had been killed. The breed, age, history and immunological status of these horses were unknown.

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Proliferation assays were performed on blood from two ponies 13±24 weeks after they had been intranasally infected with the EHV-1 isolate Ab4. Infection was confirmed in both ponies by virus isolation from nasal swabs and buffy coats (Patel and Edington, 1983). CTL assays were performed using blood from two tissue-typed Welsh Mountain ponies (Institute of Animal Health, Pirbright, UK) 7±35 weeks after intranasal infection with the EHV-1 isolate Ab4. 2.2. Virus stocks The EHV-1 isolate Ab4 from a gelding with paresis (Patel and Edington, 1983) was used throughout. For infection and proliferation studies the passaged virus had been further passaged six to eight times on RK13 cells and contained 4.4  106 pfu/ml. A stock of this EHV-1 was UV-inactivated by 3 kjrd for 60 min. Subsequent seeding onto RK13 cells confirmed that infectivity had been eliminated. For CTL assays plaquepurified EHV-1 (Ab4) had been passaged 49±53 times exclusively on RK13 cells and contained 2.5  107 pfu/ml. RK13 cells were grown in minimum essential medium with Earle's Salts (EMEM, Gibco Cat. no. 31095-029, UK), supplemented with 200 mM Lglutamine (Gibco), 100 mg/ml streptomycin (Gibco), 100 IU/ml benzylpenicillin (Gibco), 0.5 mg/ml Fungizone1 (Gibco) and 2±10% fetal calf serum (FCS; PAA, Austria) and referred to as sEMEM. Prior to use, virus supernatants were freeze-thawed once, sonicated for 2 min, clarified by centrifugation at 400g for 10 min and stored in aliquots at ÿ708C. 2.3. Cell populations PBMCs were separated according to the method of Bright et al. (1978). Heparinised (10±20 U/ml; Sigma, UK) peripheral blood from abattoir horses was left to stand for 0.5± 1 h to allow red blood cells to sediment. The white blood cell rich plasma was layered onto Nycoprep 1.077TM (Gibco) or Ficoll-paque (Pharmacia Biotech, UK) and spun for 25 min at 726g at room temperature without braking. Fresh blood from experimental ponies was diluted 1 : 1 with phosphate-buffered saline (PBS; Gibco) before gradient centrifugation. PBMCs at the interphase were harvested and washed twice in chilled (48C) PBS. Low density cells (LDCs) were isolated as described previously (Siedek et al., 1997). Briefly, PBMCs were resuspended at 5  106 cells/ml in RPMI 1640 Medium (Gibco Cat. no. 21875-034), supplemented with 200 mM L-glutamine, 100 mg/ml streptomycin, 100 IU/ml benzylpenicillin (sRPMI), and either 10% heat-inactivated FCS or heat inactivated autologous horse plasma (AHP). Cells were cultured overnight in tissue culture flasks (Gibco) at 378C in 5% CO2 in air and then non-adherent cells were layered onto 13.7% (w/v) MetrizamideTM (Nycomed, Gibco) in sRPMI/10% FCS or 10% AHP and centrifuged for 10 min at 726g at room temperature. The interphase, which contained the LDCs, and the pellet of high density cells (HDCs) were harvested and washed twice in chilled (48C) PBS. Adherent cells were detached from the tissue culture flasks with cell scrapers (Gibco) and washed. LDCs, prepared according to this protocol,

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were shown by immunocytochemistry to contain 62  6% Class II positive cells with DC morphology and were highly stimulatory cells in allogeneic MLRs (Siedek et al., 1997). Pokeweed mitogen (PWM) blasts were obtained by resuspending PBMCs at 3  107 cells/ml in AIM-SM with 2.5 mg/ml PWM (Sigma, UK). AIM-SM contained 50% (v/v) AIM-V (Gibco, Cat. no.12030-011), 5% (v/v) heat-inactivated autologous horse serum (AHS), 0.5% (v/v) MEM non-essential amino acids (100, Gibco), 55 mM 2mercaptoethanol (Sigma), 2 mM L-glutamine, 0.5 mM natrium pyruvate (Sigma), 100 mg/ml streptomycin, 100 IU/ml benzylpenicillin in RPMI 1640 medium. After incubation for 2 days in an upright tissue culture flask at 378C in 5% CO2 in air, the cells were passed over Ficoll-paque to remove dead cells and counted. 2.4. Infection of LDCs and immunocytochemistry Following centrifugation at 394g for 10 min, cells were resuspended in virus supernatant at 1±10 MOI and incubated for 1±2 h at 378C in 5% CO2 in air. Cells were washed three times in PBS and counted. Control cells were mock-infected by adding RK13 cell supernatants which had been incubated with mock-infected RK13 cells in sEMEM containing 2% FCS for 18 h. Prior to mock infection the supernatant was freezethawed and clarified in the same way as virus supernatant. Infected and mock-infected cells were cultured for up to 6 days in sRPMI/10% FCS. Samples were taken at 2±72 h and assessed for viability by trypan blue (0.1% w/v in PBS; BDH, UK) exclusion. Additional samples were cytocentrifuged onto slides and immunocytochemically stained (Siedek et al., 1997) with an anti-EHV-1 gB monoclonal antibody (mAb; Edington et al., 1987). To examine whether LDCs were particularly susceptible to the virus, cell populations enriched for lymphocytes (HDCs containing 76  3% T-cells and 11  2% B-cells, Siedek et al., 1997), adherent cells containing macrophages (63  7% negative for T- and B-cell markers, Siedek et al., 1997) and PWM-blasts (used as target cells in CTL assays, Allen et al., 1995) were also infected with EHV-1 (1 MOI) in four experiments. 2.5. Co-cultivation and titration of supernatants Co-cultivation was performed according to the method of Edington et al. (1994). Infected and mock-infected cells were counted and prepared as 10-fold dilutions. Equal volumes of the cell suspensions were cultured with confluent RK13 cells in quadruple flat-bottom wells of 24-well plates (Alpha Labs, UK) and checked daily for cytopathic effects (CPE). Cell-free supernatants of infected and mock-infected cell cultures were also titrated onto confluent RK13 cells as controls. To ensure that the CPE was EHV-1specific, RK13 cells from infected wells were collected, cytocentrifuged onto slides and immunocytochemically stained for EHV-1 with anti-gB mAb (Edington et al., 1987). Cell-free supernatants were collected at 2, 24 and 48 h post-infection and stored at ÿ208C. Virus titres were determined as tissue culture infective dose 50% (TCID 50) per ml by preparing half log dilutions of the stock and seeding 0.1 ml onto confluent RK13 cells in 96-well-plates (Alpha Labs). After 2 h incubation the virus supernatant was tipped off and the wells were filled with 0.2 ml sEMEM/5% FCS. Cultures were

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incubated for 5 days, when TCID 50 values were calculated with the Karber formula (Russell and Edington, 1985). 2.6. T-cell proliferation T-cells were positively selected by the VarioMACS (Miltenyi, UK) using anti-equine CD3 (Blanchard-Channell et al., 1994) mAb as the primary mAb and MACS magnetic microbeads conjugated to goat anti-mouse IgG (Miltenyi) as the secondary Ab. Briefly HDCs were stained for 15 min at 48C with the primary mAb followed by one wash in buffer containing 1% BSA in PBS with 5 mM EDTA (BDH) referred to as BPE. The cell pellet was resuspended in 80 ml BPE and 20 ml microbeads added for every 107 cells. After 15 min at 48C, cells were washed again, resuspended in 1±2 ml BPE and loaded onto a MACS column (Miltenyi). The column was washed three times with chilled BPE to remove unattached cells. Positively selected cells were eluted from the column following removal of the magnetic field by repeated washes with BPE. Cells were then washed once in sRPMI to remove BSA and purity assessed by flow cytometry (85±97% CD3 positive). Proliferation cultures in triplicate microtitre round-bottom wells (Alpha Labs) contained different numbers of LDCs or adherent cells and 105 T-cells/well in 0.2 ml sRPMI/10% AHP in). Proliferation was assessed by incorporation of 1 mCi/well 3 Hthymidine (specific activity 6.7 Ci/mmol, ICN Flow, UK) in an 18 h pulse from day 6 to 7. T-cells on their own and stimulator cells on their own served as controls. Cells were harvested with a TitertecTM cell harvester (Skatron, UK) and radioactivity measured on a b-scintillation counter (TriCarb11500, Canberra Packard). Results are expressed as mean CPM  SEM of triplicate cultures or as stimulation index (SI) which was calculated as follows: mean CPM with infected stimulator cells/mean CPM with mock-infected stimulator cells. 2.7. CTL assays CTL assays were performed following modification of the method of Allen et al. (1995). CTL induction cultures were set up with 1  108 PBMCs, 2.5  107 g-irradiated (5000 rad) PBMC feeder cells and infected or mock-infected stimulator cells (1  106 to 1  107 LDCs or PWM blasts), in 20 ml AIM-SM with 10 U/ml IL-2 (Boehringer, UK) and 10% TCS from ConA (Sigma) stimulated PBMCs. LDCs were prepared and infected at 10 MOI or mock-infected. Cells were washed and incubated in AIM-SM for 3 h to allow processing of viral immediate early and early proteins. LDCs were g-irradiated with 7500 rad and added to the induction cultures at different PBMCs to LDCs ratios. PWM blasts were infected at 10 MOI for 90 min, washed and incubated with 15 U/ml of IL-2 in AIM-SM for 18 h. Cells were g-irradiated with 7500 rad before adding to the induction cultures at different ratios. The complete induction cultures were incubated for 6 days upright in tissue culture flasks at 378C in 5% CO2 in air. On day 6 the cells were counted, resuspended at 2  107 cells/ml RPMI 1640 (Gibco, no. 21875-091) and diluted one in three. PWM blasts were used as target cells and were prepared and infected in the same way as for the induction culture PWM blasts.

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Blasts were washed and cultured at 3  106 cells/ml in AIM-SM with 15 U/ml IL2 (Boehringer) and 125 mCi 51 Cr (Dupont, France) for 18 h. Target cells were washed twice in PBS and once in RPMI 1640/10% FCS without additives to reduce background. Cells were counted and resuspended at 2  105 cells/ml in RPMI/10% FCS without additives. 1  104 target cells were mixed with different numbers of effector cells to give effector to target ratios of 100 : 1, 33 : 1, 11 : 1 and 3.7 : 1. The maximal and spontaneous 51 Cr release was determined by adding 50 ml of 1% NP 40 (Sigma) or 50 ml of the medium, respectively, to target cells. Cells were centrifuged at 150g, incubated at 378C for 4 h, centrifuged again and 25 ml supernatant was removed for counting. All cultures were performed as triplicates. Results are expressed as percent specific lysis calculated as follows: % specific lysis (SL) ˆ (mean test CPM minus mean spontaneous CPM)  100 / (mean maximal CPM minus mean spontaneous CPM). 2.8. Statistical analysis Virus titres, viability, proliferation and CTL data were statistically evaluated with SPSS for Windows Release 6.1, using the Mann±Whitney test and a 95% confidence interval, and presented using Microsoft Excel Version 5.0. 3. Results 3.1. EHV-1 infection of equine DCS Following in vitro infection of LDCs from 15 separate horses with EHV-1 (1 MOI) a proportion of the cells from all animals reacted with the anti-gB mAb by immunocytochemistry (Fig. 1(a)). Many infected LDCs were large, exhibited irregular nuclei and extended cytoplasmic processes characteristic of DCs (Fig. 1(b); Siedek et al., 1997). Staining with the anti-gB mAb was seen perinuclearly, previously reported to be the site for EHV-1 assembly (Roizman and Baines, 1991). Viral gB was first detected at 8 h post-infection, consistent with early/late gene products, like gB, being expressed 8± 12 h post infection (Caughman et al., 1985). EHV-1 infection also caused accumulation of cell debris, aggregation of LDCs and the formation of syncytia (Fig. 1(c)), which are characteristic of the cytopathic effects of herpesviruses (Neubauer et al., 1997). Since gB expression was predominant in syncytia the exact number of infected cells could not be determined, but estimation suggested that 5±25% of LDCs expressed gB. Infection with virus-free supernatants (RK13 TCS) or virus which was unable to replicate (UV-inactivated EHV-1) did not result in gB expression, cytopathic effects or syncytial formation (Fig. 1(d)). The appearance of cell debris in EHV-1-infected LDC preparations suggested that cells had been destroyed by the virus. Viability studies in 10 experiments undertaken by counting individual non-aggregated cells confirmed that the viability of infected LDCs decreased more rapidly particularly in the first 12 h than that of mock-infected LDCs (Fig. 2). The viability of mock-infected LDCs (>74%) was similar to that obtained for

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Fig. 1. EHV-1 replicates in equine DCs. LDCs stained immunocytochemically with anti-gB mAb ((a), (b)) 24 h post-in vitro infection with EHV-1 at 1 MOI ((a), (b), (c)) or mock-infection with RK cell TCS (d). Infected cells exhibited a DC morphology (b) and syncytia formation (c). Magnification: (a)10; (b)100; (c)40) (d040.

untreated LDCs cultured for up to 48 h (>69%). Thus the decline in viability was not due to factors released by RK13 cells, but to EHV-1 infection. The ability of EHV-1-infected LDCs to produce virus, was assessed by co-culturing confluent monolayers of RK13 cells with 102±104 infected (1 MOI) LDCs in four experiments. Virus-induced cytopathic effects were routinely seen in all wells 2±3 days after co-cultivation with 103±104 infected LDCs; in one experiment, the addition of only 102 infected LDCs, in which 77% of the cells were morphologically DCs, produced cytopathic effects in 50% of the replicate wells. RK13 cells showing the cytopathic effects stained with the anti-gB mAb, indicating infection with EHV-1. No changes were ever observed when mock-infected LDCs were co-cultivated with RK13 cells. Supernatants of infected (1 MOI) LDCs were collected in three experiments, and virus titres of the samples were determined by titration onto confluent RK13 cells. Virus titres increased 1±100-fold between 2 and 24 h post-LDC infection, indicating that the new

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Fig. 2. Viability of EHV-1-infected (1 MOI; *) and mock-infected (&) LDCs. Results are expressed as mean % viability SEM of 10 experiments. After 24 h the viability of infected differed significantly from that of mock-infected LDCs (p < 0.05).

virus was released into the supernatant. The increase was small but significant (p < 0.032) when compared to mock-infected controls and confirmed that some LDCs became productively infected with EHV-1. When different cell populations were compared, adherent cells were permissive to EHV-1 infection, to an extent comparable with LDCs as assessed by gB staining, and also showed loss of viability and syncytia formation. HDCs exhibited little gB staining (1%) and their viability remained similar to that of mock-infected cells. In contrast, PWM blasts contained 31  11% gB positive cells by immunocytochemistry, showing that activated as opposed to resting lymphocytes were susceptible to EHV-1 infection. 3.2. EHV-1-specific proliferation Infected LDCs (1MOI) but neither adherent cells nor mock-infected LDCs stimulated significant (p < 0.03) T-cell proliferation in two ponies (Fig. 3). Increasing the LDC : Tcell ratio but not the adherent cell : T-cell ratio increased the proliferative response. Thus, although adherent cells were as permissive as LDCs for EHV-1 infection, LDCs were superior in stimulating EHV-1-specific proliferation. When EHV-1-infected (1 MOI) LDCs were used to stimulate T-cell proliferation in two ponies at times up to 6 months post-experimental infection with EHV-1 (Fig. 4) the Tcells of one pony proliferated weakly at 13 weeks post-infection and then more strongly 15±24 weeks post-infection although the response declined during this time. In the second pony responses were significant 19 weeks post infection but below a stimulation index of three 22 weeks post infection. The findings are compatible with reports that immunity to EHV-1 lasts 4±6 months (Allen and Bryan, 1986). 3.3. EHV-1-specific CTL responses LDCs were more effective than PWM blasts in the in vitro induction of CTLs (at 50 : 1 p < 0.03; Fig. 5) although PWM blasts contained as many or more EHV-1-infected cells

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Fig. 3. T-cell proliferation to EHV-1-infected LDCs ( ), mock-infected LDCs( ), EHV-1- infected adherent cells ( ) and mock-infected adherent cells ( ) after 7 days in culture. Results on two ponies (a and b) are expressed as mean CPM  SEM of triplicate cultures.

than LDCs. Infected LDCs induced significantly more cytotoxicity than mock-infected LDCs (at 50 : 1 p < 0.01). CTL activity on MHC matched, EHV-1-infected targets was significantly greater than that on targets with a different MHC, and thus cytotoxicity was MHC-restricted (Fig. 5). NK activity was not detected since there was no significant difference between lysis of infected or mock-infected MHC-mismatched target cells. EHV-1-infected LDCs were used to stimulate virus-specific CTL responses on three occasions from a pony, that had previously been intranasally infected with 4  107 pfu

Fig. 4. Proliferation of T-cells (105) in the presence of EHV-1-infected LDCs (103) from two ponies (& and *) at various time points post infection. Results are expressed as SI of triplicate cultures.

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Fig. 5. CTL activity of PBMCs, which have been cultured with either LDCs or PWM blasts as stimulator cells, on EHV-1- (^,}) or mock-infected (*,*) MHC-matched (ÐÐÐ) or mismatched (- - -) target cells. Results are expressed as % specific lysis of mean CPM of triplicate cultures.

EHV-1. EHV-1-specific cytotoxicity was seen 30 weeks post-infection, but could not be detected 5 weeks later (Table 1). The pony was experimentally infected again 1 month later and 7 weeks following a second infection CTL activity was again detected (Table 1). 4. Discussion DCs have been found to be very susceptible to in vitro and in vivo infection with a variety viruses, such as retroviruses, paramyxoviruses and orthomyxoviruses (Patterson and Knight, 1987; Bhardwaj et al., 1994; Schnorr et al., 1997). Here we show for the first time that EHV-1 infection of equine LDCs resulted in the expression of the structural glycoprotein gB in large cells with extended cytoplasmic processes which are Table 1 CTL responses of PBMCs cultured with infected DCs Weeks post-infection

Effector to target cell ratio 4 : 1 (%)

11 : 1 (%)

Infection I 30 35

8a 0

4 0

Infection II 7

8%

19%

33 : 1 (%)

100 : 1 (%)

12 0

26 0

29%

52%

a % specific lysis of PBMCs cultured with EHV-1-infected DCs for 5 days. Cytotoxicity was assessed on autologous, EHV-1-infected PWM blasts.

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characteristic of DCs. Cells prepared as described here, also have other properties associated with DCs such as expression of MHC Classes I and II and LFA-1, lack of expression of T- and B-cell markers and stimulation of a vigorous mixed lymphocyte reaction (Siedek et al., 1997). Within the limitations of antibodies available to study equine DCs (see Siedek et al., 1997, p. 29 para 4). we are confident that our infected cells were DCs. Given the migratory life-style of DCs, the susceptibility to infection with EHV-1 may play an important role in transporting infectious virus, as well as viral antigens, from mucosal surfaces to other parts of the body including lymph nodes (Hamilton-Easton and Eichelberger, 1995). Upon reaching sites of immune activation infected DCs may both transmit the virus to other cells and initiate protective immune responses (Bhardwaj et al., 1994). In vivo EHV-1 has been reported to infect equal proportions of monocytes and lymphocytes or more lymphocytes than monocytes (Scott et al., 1983; Slater et al., 1994). In vitro infection with EHV-1 has only been studied previously using unseparated PBMCs (Dutta and Myrup, 1983). This is therefore, the first investigation into in vitro infection of peripheral blood isolates enriched for DCs (LDCs), lymphocytes (HDCs) and adherent cells (monocytes/macrophages). Infection of all three cell populations was achieved by exposure of the cells to virus for only 2 h indicating that all these cells can be the focus for primary infections. However, on the basis of gB staining, syncytia formation and loss of viability, LDCs enriched for DCs and adherent cells enriched for macrophages were much more susceptible to infection than resting lymphocytes. As previously reported activated lymphocytes were much more susceptible to EHV-1 infection in vitro than resting lymphocytes (Allen et al., 1995). Co-cultivation of infected LDCs or tissue culture supernatants from them showed that infection with EHV-1 was productive, although the increase of EHV-1 titres in tissue culture supernatants was small and compatible with only some LDCs being permissive. However, since LDC isolates contained a proportion of T-cells (15  3%, Siedek et al., 1997) it is possible that these may have become infected within DC clusters, and subsequently produced infectious EHV-1. Mitogen-activated equine T-cells, but not Bcells or monocytes, have been reported to produce infectious EHV-1 (Thomson and Mumford, 1977; Scott et al., 1983) and in vitro re-activation of latent virus in blood cells has been shown to be predominantly associated with T lymphocytes (Chester et al., 1997; Smith et al., in press). Activated T-cells are also the predominant producers of infectious HIV virions in vitro (Tsunetsugu-Yokota et al., 1995) with DCs rarely becoming productively infected (Patterson and Knight, 1987). Instead, they provide a reservoir for HIV and assist viral spread by transmitting infection to activated T-cells in DC-T-cell clusters (Cameron et al., 1994; Patterson et al., 1994; Pope et al., 1994; TsunetsuguYokota et al., 1995; Weissman et al., 1995). Taken together our findings are compatible with myeloid cells, such as DCs, being infected initially and subsequently infecting activated lymphocytes during DC clustering with T-cells. These T cells may then go on to be productively or latently infected. Our data suggest that only some of the equine DCs are susceptible to EHV-1 infection. The number of gB positive LDCs was estimated as <25% at 24 h, although the exact proportion of infected cells could not be determined because of cell lysis, cell aggregation and syncytia formation. Since most of the gB staining was contained in the syncytia they

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may represent a major site of viral replication, as has been reported for HIV infection (Canque et al., 1996). Most cell death occurred during the early period following infection, the rate of loss of viability thereafter being similar to the mock-infected controls. Again this suggests that not all LDCs are equally susceptible to infection. Maturity of DCs has been shown to be a major factor influencing their capacity to be infected with viruses. All immature DCs but only 40% of mature DCs can be infected with the influenza virus (Lanzavecchia, pers. comm.). A minority of blood DCs, prepared as here, became infected with HIV in vitro (Patterson and Knight, 1987) and DC susceptibility to HIV in vivo depends on their stage of development (Charbonnier et al., 1996). We have previously shown that our LDCs contain mostly mature DCs based on MHC Class II expression and allostimulatory potency (Siedek et al., 1997). It remains to be determined whether immature equine dendritic cells are more susceptible to infection with the virus. Proliferative and CTL responses to live EHV-1 have been demonstrated in vitro using PBMCs (Hannant et al., 1991; Allen et al., 1995; Ellis et al., 1995) but the APC stimulating EHV-1-specific T-cells have not been studied. Here we have shown that a population of cells enriched for equine DCs, when exposed to infectious virus, were able to stimulate EHV-1-specific T-cell responses in vitro. Thus in spite of the cytopathic effects of the virus on DCs, antigen presentation and T-cell activation could occur. Whether the infected cells, or other cells, processing viral antigens, are responsible remains to be determined. We did not test proliferative responses at an earlier stage after infection because activated serum TGF-b, which circulates in experimentally infected ponies for up to 8 weeks post-infection (Charan et al., 1997), has been shown to suppress proliferation to EHV-1. In one pony proliferation was small (SI ˆ 4) 13 weeks postinfection, peaked at 15 weeks and then declined. Due to lack of availability of blood samples, unfortunately, the second horse was not studied prior to 19 weeks. However, a decline in virus-specific response was seen in between 19 and 22 weeks post infection. These results suggest that LDCs can only stimulate memory T-cell responses with live virus for a limited time and are compatible with the observations that immunity is shortlived and wanes 4±6 months post-infection (Allen and Bryan, 1986). Although DCs have been reported to boost T-cell responses in animals that were unresponsive (Boog et al., 1985; Kast et al., 1988) the waning T-cell response seen here could not be circumvented by use of infected DCs as APCs. We were also able to show that DCs could support the generation of CTLs in an MHCrestricted fashion and that these were more effective on a cell-for-cell basis than PWM blasts. Although the PWM blasts contained more EHV-1-infected cells than the LDCs the latter were more efficient in activating MHC-restricted memory CTLs demonstrating that the APC-type was more important than the proportion of infected cells. It is likely that CTL activity was directed against immediate early and early viral proteins since only these proteins could have been expressed in the short time between infection and irradiation (Honess and Roizman, 1974). Immediate early gene products have been identified as important target antigens for CTLs in other herpesvirus infections (Nugent et al., 1995). Although only studied in one pony the CTL results were compatible with the proliferation studies and previous studies on CTL activity in in vivo-infected horses

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(Allen et al., 1995) in that CTL activity could not be induced 35 weeks after in vivo infection with EHV-1 and subsequent in vivo re-infection restored the CTL response. The failure of infected DCs to maintain high levels of immune responsiveness throughout the experiments described here is of interest since DCs have been reported to potently induce immune responses. CTLs from non-immune animals have been generated in vitro in other species with DCs (Hengel et al., 1987; Nair et al., 1993) and the activation of weak immune responses by DCs is an important component of current immunotherapeutic approaches (Banchereau and Steinman, 1998). It is unlikely that the separation method prevented T-cell activation, since mature DCs, similarly prepared (Knight et al., 1986), have been successfully used for the induction of antiviral CTLs and T-cell proliferation (Macatonia et al., 1989, 1991). Furthermore, the lack of CTL responses was not caused by the transmission of EHV-1 from LDCs to T-cells since the LDCs were irradiated and washed and thus infectious virions inactivated before being added to the T-cells. We conclude that at time points where T-cell responses were low or absent either the immunostimulatory capacity of DCs or the response of the memory Tcells was deficient. Impairment of DC function has been reported for HIV (Macatonia et al., 1990) and we have found EHV-1-induced downregulation of the cell surface expression of MHC products (data not shown). However, such downregulation would be predicted to affect antigen presentation at all times. Accordingly, these results suggest that the waning immunity of equines may be due to more impairment in the memory Tcells than in the antigen presenting capacity of the DCs. This study provides the first evidence that EHV-1 infects DCs in vitro. The results imply that EHV-1 could be disseminated by these cells in vivo. Furthermore, cells enriched for DCs stimulated virus-specific memory T-cells to proliferate to live virus and to lyse-infected targets, suggesting that DCs act as APCs to these T-cells in vivo. However, infection caused cell death, which may delay the activation of anti-viral T-cells in vivo and thus contribute to virus-induced immunosuppression (Hannant et al., 1991). The role of DCs in T-cell activation and in the establishment of memory T-cell responses in animals experiencing EHV-1 infection warrants further investigation. Acknowledgements E.S. was generously supported by the Wellcome Trust and M.W. by the Equine Virus Research Fund. We thank the RVC Biological Services Unit and the staff of the Animal Unit at Pirbright for expertly looking after the animals and the owners and staff at the abattoir for their patience and cooperation. References Allen, G., Yeargan, M., Costa, L., Cross, R., 1995. Class I MHC-restricted cytotoxic T lymphocyte responses in horses following infection with equine herpesvirus-1. J. Virol. 69, 606±612. Allen, G.P., Bryan, J.T., 1986. Molecular episootiology, pathogenesis and prophylaxis of equine herpesvirus-1 infection. Prog. Vet. Microbiol. Immunol. 2, 78±144. Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245±252.

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