Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs

Tiny G. M. de Bruin1, Eugene M. A. van Rooij1, Yolanda E. de Visser1, John J. M. Voermans1, Janneke N. Samsom1, Tjeerd G. Kimman1 and Andre T. J. Bianchi1

Department of Mammalian Virology, Institute for Animal Science and Health (ID-Lelystad), Postbus 65, 8200 AB Lelystad, The Netherlands1

Author for correspondence: Tiny de Bruin. Fax +31 320 238668. e-mail m.g.m.debruin{at}jo.wag-ur.nl


   Abstract
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Abstract
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Methods
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Discussion
References
 
We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6+ CD8bright+ CD4- (CTL phenotype), CD6+ CD8dull+ CD4+ (the fraction containing memory T helper cells), CD6+ CD8- CD4+ (T helper cell phenotype), CD6- CD8dull+ CD4- {gamma}{delta}-T+{gamma}{delta}-T cell phenotype), CD6- CD8dull+ CD4- {gamma}{delta}-T- (NK phenotype) and CD6- CD8- CD4- {gamma}{delta}-T- or  {gamma}{delta}-T+. Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6+ CD8+ and CD4+ CD8+ and fewer CD6- CD8+ and {gamma}{delta}-T+ CD8+ lymphocytes than IL-2 stimulation of naive PBMC (P<0·05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6+ CD8+ T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6- CD8+ T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Pseudorabies virus (PRV) is an alphaherpesvirus that causes Aujeszky’s disease in pigs. Both humoral and cellular immunity appear to be involved in the development of protective immunity to herpesviruses, which has been studied in detail for herpes simplex virus type 1 infection in humans. Previous reports have described the cellular lymphoproliferative immune response against PRV in pigs (Chinsakchai & Molitor, 1992 ; Kimman et al., 1995b ). Both CD4+ and CD8+ T lymphocytes proliferated (Kimman et al., 1995b ) after they were stimulated in vitro with PRV. Zuckermann & Husmann (1996) demonstrated the involvement of CD4+ CD8+ lymphocytes in proliferation, and this subset harbours putative memory cells. Zuckermann and co-workers also demonstrated that CD8+ T lymphocytes killed PRV-infected cells (Zuckermann et al., 1990 ). Saalmüller et al. (1994a ) demonstrated that the subset of CD5+ CD8+ lymphocytes might harbour classical CTL in pigs. Pauly et al. (1995 , 1996 ) demonstrated in the case of classical swine fever virus infection that CD6+ CD8+ lymphocytes killed virus-infected target cells in an MHC-I-restricted way, whereas the natural killer (NK) cells were CD6- CD8+.

Previously, we demonstrated killing of PRV-infected target cells by CD2+ CD4- and CD8- or CD8dull+ T lymphocytes (Kimman et al., 1996 ). Their ability to kill target cells appeared to be at least partially MHC-I-unrestricted. NK cells or lymphokine-activated killer (LAK) cells therefore appeared to be involved. However, the presence of MHC-I-restricted killing was not excluded. These results could be explained by assuming that PRV-stimulated T helper lymphocytes secrete IL-2 in vitro that may increase natural killing. Apparently, different cytolytic subsets are involved in the killing of PRV-infected target cells, but it is not clear which subset predominates under various circumstances.

The purpose of this study was, therefore, to investigate the significance of various cytolytic lymphocyte subsets in peripheral blood. We therefore stimulated PBMC from naive and immune pigs in vitro with PRV or IL-2 and separated the different subsets 6 days later by using anti- ({alpha}-)wCD6 monoclonal antibodies (MAbs). The phenotype and cytolytic function of these subsets were subsequently investigated.


   Methods
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Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus.
Virus stocks for inoculation and challenge of pigs were prepared on SK6 cells (Kasza et al., 1971 ) and secondary porcine kidney cells, respectively, as described by Kimman et al. (1995b ). Pigs were inoculated with a gE- PRV strain (M141 or 783) (Gielkens et al., 1989 ; De Wind et al., 1990 ). The pigs were then challenged with the wild-type PRV strain NIA-3 (McFerran & Dow, 1975 ). This strain was also used to infect target cells, as described by Kimman et al. (1995a ).

{blacksquare} Animals and experimental design.
Minnesota miniature pigs, which were inbred for swine leukocyte antigen complex (SLA) (haplotype d/d) (Sachs et al., 1976 ), were kept under specific-pathogen-free conditions in the breeding unit of our institute. The pigs were born from unvaccinated sows and had no antibodies against PRV. Pigs were inoculated intramuscularly with 105 p.f.u./ml 783 or M141 at 7 months of age and were subsequently challenged three times intranasally with 105 p.f.u./ml NIA-3 virus, first after 3 months and then at intervals of 6 months. Flow cytometry analyses was done randomly after one (singly inoculated pigs) or several (frequently inoculated pigs) challenge inoculations. Cytolytic experiments with immune PBMC were done randomly after several challenge inoculations (frequently inoculated pigs). Uninoculated pigs, aged 7 months at the start of the experiments, were used in parallel with the immune pigs as negative controls during the experimental period. Each experiment was repeated at least five times with blood taken from two pigs per group. The different groups contained four or five pigs (Table 1). The ethics committee for animal experiments of ID-Lelystad approved the experiments.


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Table 1. Experimental design

 
{blacksquare} Isolation and culture of PBMC.
PBMC were obtained from PRV-inoculated and naive pigs. Blood was collected from the superior vena cava in vacuum tubes containing heparin (Venoject, Terumo Europe). Blood samples were diluted at a ratio of 1:2 in PBS, pH 7·4, layered on an equal volume (5 ml) of Lymphoprep (Nycomed Pharma) and centrifuged for 20 min at 800 g at 20 °C. PBMC at the interface were collected and washed twice in PBS. Viable cells were counted by trypan blue exclusion. These PBMC were stimulated in vitro with NIA-3 virus or IL-2 and used in a cytolytic assay or used for flow cytometry analysis. Note that PBMC from naive pigs were not examined with PRV stimulation because previous studies showed that PBMC of naive pigs, cultured with PRV, did not show any stimulation (Table 1).

To stimulate the cells in vitro, they were adjusted to a final concentration of 5x106 viable cells, with 5x106 p.f.u. NIA-3 virus, per ml in RPMI 1640 Dutch modification medium (ICN Biomedicals) or with 500 IU/ml recombinant human IL-2 produced in Escherichia coli (Proleukin, Eurocetus). The medium contained 10% foetal calf serum (FCS), 200 U/ml penicillin, 0·2 mg/ml streptomycin, 100 U/ml mycostatin, 0·3 mg/ml L-glutamine and 5x10-5 M 2-mercaptoethanol (RPMI complete medium). These cells were incubated in 6-well plates (Greiner) for 6 days and killing was determined at various intervals after inoculation.

For flow cytometry analysis, stimulated cells were adjusted to a final concentration of 1x106 viable cells per ml in PBS with 0·5% FCS and 0·01% NaN3.

{blacksquare} Two-colour flow cytometry analysis.
PBMC were analysed on a FACScan flow cytometer (Becton Dickinson). Unfixed lymphocyte suspensions were first incubated with a mixture of two MAbs directed against the molecules of interest and then incubated with a mixture of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG2b or IgG1 (dependent on monoclonal isotype) and phycoerythrin (PE)-conjugated goat anti-mouse IgG2a (ITK Diagnostics). PBMC were incubated on ice for 30 min with saturating amounts of MAb. After each incubation, cells were washed three times in PBS containing 0·5% FCS and 0·01% NaN3. A total of 5000 cells was examined.

PBMC were stained for the combinations {gamma}{delta}-T/CD8, CD4/CD8 and CD6/CD8 and the frequency of various lymphocyte subsets was determined. The following MAbs were used to characterize the lymphocytes: MAb 74.12.4, directed against porcine CD4 (IgG2b) (Pescovitz et al., 1984 ); MAb SL2 (11/295/33), directed against porcine CD8 (IgG2a) (Jonjic & Koszinowski, 1984 ); MAb a38b2, directed against porcine wCD6 (IgG1) (Saalmüller et al., 1994b ); and MAb PPT16, directed against a component of the porcine {gamma}{delta}-T-cell receptor (TCR) (IgG2b) (Kirkham et al., 1996 ; Yang & Parkhouse, 1996 ). Cells staining positive for {gamma}{delta}-TCR are referred to in this article as {gamma}{delta}-T+ T lymphocytes. The separated populations were also stained with the combinations mentioned above. We could not investigate the populations after the PBMC were separated for CD8. Staining with conjugated anti-mouse antibodies directed against IgG2a+b cross-reacted with the bead-labelled MAbs on the CD8+-enriched populations. Note that the whole lymphocyte population was analysed, because after 6 days culture only a small number of lymphoblastoid cells were left (especially after IL-2 stimulation) and most lymphoblastoid cells had already matured into lymphocytes.

To illustrate how we analysed the flow cytometry results, flow cytometry plots of a representative experiment are depicted in Figs 1 and 2. We analysed the percentages of the different lymphocyte subsets by quadrant analysis (Figs 1 and 2). Student’s t-test was used for statistical analysis of the results.



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Fig. 1. Flow cytometry analysis of freshly isolated PBMC from frequently inoculated pigs (a) and PRV-stimulated PBMC isolated from singly inoculated (b) and frequently inoculated (c) pigs. Cells were double-stained with MAbs directed against porcine CD4/CD8, CD6/CD8 and {gamma}{delta}-T/CD8 as shown. Plots of a representative experiment are shown.

 


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Fig. 2. (a)–(c) Flow cytometry analysis of freshly isolated PBMC from a naive pig (a) and IL-2-stimulated PBMC isolated from naive (b) and immune (frequently inoculated) (c) pigs. Cells were double-stained with MAbs directed against porcine CD4/CD8, CD6/CD8 and {gamma}{delta}-T/CD8. (d)–(e) Flow cytometry analysis of the CD6- fraction of IL-2-stimulated PBMC isolated from naive pigs (d) and the same fraction from immune pigs (e). Cells were double-stained with MAbs directed against {gamma}{delta}-T/CD8. Plots of a representative experiment are shown.

 
{blacksquare} Immunomagnetic cell sorting.
Purified CD8+ and CD8- or CD6+ and CD6- effector lymphocyte populations were obtained by immunomagnetic cell sorting after 6 days of in vitro stimulation. PBMC were labelled with MAb 11/295/33 ({alpha}-CD8, isotype IgG2a) or MAb a38b2 ( {alpha}-wCD6, isotype IgG1). The PBMC were then incubated with magnetic microparticle-labelled goat anti-mouse IgG2a+b or IgG1 (dependent of the isotype of the MAb) (CLB) and subsequently incubated with FITC-conjugated F(ab’)2 fragment of rabbit anti-mouse immunoglobulins (Dako). PBMC were incubated on ice for 30 min with saturating amounts of antibody. After each incubation, cells were washed three times in PBS containing 0·5% BSA, 0·2 mM EDTA and 0·01% NaN3. The PBMC were sorted with a magnet-activated cell sorter (MACS, Miltenyi Biotec) as recommended by the manufacturer (CLB), which resulted in the isolation of highly purified antigen-positive and antigen-negative fractions (90–98%). The purity of all sorted fractions used in the experiments was controlled by flow cytometric analyses. To ensure that the labelling procedure did not influence the reactivity of CTL, MAb-labelled unfractionated cells were also included in all cytotoxicity assays.

{blacksquare} Target cells.
The following target cells were used in the cytolytic assays: PRV-infected and uninfected L14 cells (a retrovirus-immortalized B lymphoblastoid cell line of SLA haplotype d/d) (Kaeffer et al., 1990 ) and K562 cells (a cell line from a human erythroleukaemia) (ATCC). K562 cells were used because killing of these cells indicates that NK cells are involved (Pescovitz et al., 1988 ). Infected L14 cells were obtained by infecting the cells 24 h before the start of the cytolytic assay with NIA-3 virus at an m.o.i. of 10. The cells were labelled by incubating various numbers of cells in a volume of 50 µl serum-free medium containing 400 µCi 51Cr (Amersham, CJS4) for 2 h at 37 °C on a Rock ’n Roller (Labinco). After being labelled, the cells were washed three times in RPMI complete medium. Volumes of 50 µl medium containing 104 cells were added to the wells of 96-well V-bottomed microtitre plates (Nunc, Life Technologies).

{blacksquare} Cytolytic assay.
The cytolytic activity of the effector cells was measured by 51Cr release. Volumes of 50 µl medium containing effector cells and 50 µl medium containing 104 51Cr-labelled target cells were mixed with effector:target ratios of 50:1 to 6:1. The plates were then centrifuged for 5 min at 200 g. Maximal release of 51Cr was determined by adding 50 µl 20% Triton X-100. Spontaneous release was determined in wells that did not contain effector cells. Killing of the target cells was determined by measuring the release of 51Cr in the supernatant after an incubation period of 5 h. Volumes of 50 µl supernatant were mixed with 100 µl Optiphase supermix liquid scintillation fluid (EG&G Instruments). Radioactivity was then measured in a Wallac Microbetaplus 1450 scintillation counter (EG&G Instruments). The percentage of specific 51Cr release was calculated as 100x(c.p.m. experimental release-c.p.m. spontaneous release)/(c.p.m. maximal release-c.p.m. spontaneous release).


   Results
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Introduction
Methods
Results
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References
 
Comparison of phenotypes of PRV-stimulated PBMC from the singly and frequently inoculated groups
To examine the influence of subsequent PRV inoculations on the frequency of lymphocyte subsets, we compared the frequency of various lymphocyte subsets in immune PBMC from the singly inoculated group and the frequently inoculated group after stimulation in vitro with PRV. The percentages of CD6+ CD8+ (CTL phenotype, 60±8%) and CD4+ CD8+ (fraction containing memory T helper cells, 32±7%) lymphocytes were significantly higher in the frequently inoculated group than in the singly inoculated group (CTL phenotype 42±7%; fraction containing memory T helper cells 22±10 %). In contrast, the percentage of the {gamma}{delta}-T+ CD8+ lymphocyte subset (16±4%) was significantly lower in the frequently inoculated group than in the singly inoculated group (25±6%) (Fig. 3).



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Fig. 3. Frequency of various lymphocyte subsets from singly inoculated (shaded bars) and frequently inoculated pigs (filled bars; n=5) determined by flow cytometry analysis. PBMC were stimulated in vitro with PRV. Cells were double-stained with MAbs directed against porcine CD4/CD8, CD6/CD8 and {gamma}{delta}-T/CD8. The subset frequencies that differed significantly are indicated by * and {dagger}.

 
Phenotypes of PRV-stimulated and IL-2-stimulated PBMC
We subsequently examined the influence of IL-2 and PRV stimulation on the frequency of various lymphocyte subsets. When cells from immune pigs where stimulated in vitro with PRV, we observed the following lymphocyte subset frequencies: CD6+ CD8+, 49±11%; CD4+ CD8+, 26±10%; CD6- CD8+, 19±6%; and {gamma}{delta}-T+ CD8+, 21±7%. When cells from immune pigs where stimulated in vitro with IL-2, we observed the following lymphocyte subset frequencies: CD6+ CD8+, 44±13%; CD4+ CD8+, 21±6%; CD6- CD8+, 20±10%; and {gamma}{delta}-T+ CD8+, 22±4%. When cells from naive pigs where stimulated in vitro with IL-2, we observed the following lymphocyte subset frequencies: CD6+ CD8+, 34±5%; CD4+ CD8+, 16±5%; CD6- CD8+, 29±9%; and {gamma}{delta}-T+ CD8+, 34±7%.

The frequencies of various lymphocyte subsets were compared in the IL-2-stimulated PBMC of naive pigs, the IL-2-stimulated PBMC of immune pigs and the PRV-stimulated PBMC of immune pigs. We detected significantly more CD6+ CD8+ (CTL phenotype) and CD4+ CD8+ (fraction containing memory T helper cells) and fewer CD6- CD8+ (NK phenotype) and {gamma}{delta}-T+ CD8+{gamma}{delta}-T cell phenotype) lymphocytes when we compared the frequencies of various lymphocyte subsets after PRV stimulation of immune PBMC and after IL-2 stimulation of naive PBMC (Fig. 4) (P<0·05).



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Fig. 4. Frequency of various lymphocyte subsets from IL-2-stimulated PBMC isolated from naive pigs (shaded bars; n=5) and immune pigs (filled bars) and PRV-stimulated PBMC isolated from immune pigs (open bars). Cells were double-stained with MAbs directed against porcine CD4/CD8, CD6/CD8 and {gamma}{delta}-T/CD8. The subset frequencies that differed significantly are indicated by * and {dagger}.

 
Characterization of lymphoid subsets after cell separation with {alpha}-wCD6 MAb
CD6-negative selection.
In immune and naive PBMC (after PRV or IL-2 stimulation), CD6- lymphocytes appeared to be mainly CD4- (<8% CD4+). In immune PBMC (after PRV or IL-2 stimulation), the majority of lymphocytes were {gamma}{delta}-T+ CD8+ ({gamma}{delta}-T cell phenotype, 25%) or {gamma}{delta}-T- CD8+ (NK phenotype, 40–41%; in bold in Table 2), whereas in naive PBMC (after IL-2 stimulation), the majority of lymphocytes were {gamma}{delta}-T+ CD8+ ({gamma}{delta}-T cell phenotype, 67%; in bold in Table 2). Interestingly, the expression of the CD8 antigen was higher on the {gamma}{delta}-T- CD8+ (NK phenotype) lymphocytes than on the {gamma}{delta}-T+ CD8+ ({gamma}{delta}-T cell phenotype) lymphocytes (see the mean fluorescence values in Fig. 2d, e).


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Table 2. Percentage of lymphocytes positive after separation with {alpha}-wCD6 MAb

 
CD6-positive selection.
In immune and naive PBMC (after PRV or IL-2 stimulation), the CD6+ population contained CD4- {gamma}{delta}-T- CD8bright+ (CTL phenotype), CD4+ {gamma}{delta}-T- CD8- (T helper) and CD4+ {gamma}{delta}-T- CD8dull+ (fraction containing memory T helper cells) lymphocytes (Table 2). However, in PBMC from immune pigs after in vitro PRV stimulation, the percentage of CD4+ {gamma}{delta}-T- CD8dull+ (fraction containing memory T helper cells) cells was high (68%; in bold in Table 2), whereas when PBMC isolated from immune and naive pigs were stimulated with IL-2, the percentage of CD4+ {gamma}{delta}-T- CD8dull+ cells was low (20–30%) (Table 2).

Phenotypic and functional characterization of cytolytic subsets
In none of the experiments was killing inhibited by pre-incubation with the MAbs against porcine CD8 and wCD6. PBMC from immune pigs, stimulated in vitro with PRV, killed PRV-infected L14 cells more efficiently than uninfected L14 cells (Fig. 5a). In order to dissect the activity of various subsets, these stimulated PBMC were separated for CD8 and CD6. The CD8+ lymphocytes killed PRV-infected L14 cells more efficiently than uninfected L14 cells, indicating virus-specific killing (Fig. 5a). Both the total population and the enriched CD8+ lymphocyte subpopulation of PRV-stimulated PBMC isolated from immune pigs killed very few K562 cells.



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Fig. 5. Killing activity of CD8- and CD6-separated PBMC. PBMC that were stimulated in vitro with PRV or IL-2 were labelled with {alpha}-CD8 or {alpha}-wCD6 MAb and subsequently separated by MACS into CD8+ and CD8- or CD6+ and CD6- subpopulations, respectively. Killing of K562 cells ({blacktriangleup}), PRV-infected L14 target cells ({blacksquare}) and uninfected L14 target cells ({diamondsuit}) is shown. PBMC isolated from immune pigs were stimulated in vitro with PRV (a) or IL-2 (b). PBMC isolated from naive pigs were stimulated in vitro with IL-2 (c). One representative experiment of each is shown. E:T, Effector:target.

 
The CD6+ lymphocytes primarily killed the PRV-infected L14 cells (Fig. 5a), indicating virus-specific killing. The CD6- lymphocytes killed the PRV-infected, uninfected L14 and K562 target cells equally well, suggesting the activity of NK cells. We could not detect more efficient killing (i.e. percentage killing of PRV-infected targets) with the enriched populations compared with the total population. However, virus-specific killing (i.e. percentage killing of PRV-infected targets minus percentage killing of uninfected targets) was more efficient with the enriched populations, indicating enrichment of virus-specific CTL after separation.

When PBMC were isolated from immune as well as naive pigs and stimulated in vitro with IL-2, these PBMC killed the PRV-infected and uninfected L14 cells equally well. In addition, these cells also killed K562 cells, suggesting the activity of NK cells (Fig. 5b, c). The IL-2-stimulated PBMC were separated for CD8 and CD6. The CD8+ lymphocytes also killed PRV-infected and uninfected L14 cells and K562 cells equally well, indicating natural killing. In contrast, killing of target cells by the CD6+ lymphocytes was low after IL-2 stimulation (Fig. 5c). However, in two experiments with cells from immune pigs, the PRV-infected L14 cells were killed more efficiently than the uninfected L14 cells (26 versus 7% and 36 versus 14%), indicating that virus-specific killing can be induced after IL-2 stimulation of immune PBMC. In contrast, only natural killing was found when the cells were separated by using the anti-CD8 MAb.

The K562 cells were not killed by the IL-2-stimulated CD6+ lymphocytes. The CD6- lymphocytes killed PRV-infected and uninfected L14 cells equally well and K562 cells were also killed. Therefore, virus-specific killing was due to CD6+ lymphocytes and natural killing was due to CD6- lymphocytes.


   Discussion
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Discussion
References
 
We have demonstrated that we could discriminate various subsets of cytolytic cells. PRV stimulation in vitro of immune PBMC resulted mainly in CD6+ CD8+ lymphocytes (classical CTL phenotype and the fraction containing memory T-helper cells) and killing was mainly virus specific. In contrast, IL-2 stimulation of immune and naive PBMC resulted mainly in CD6- CD8+ lymphocytes (NK and {gamma}{delta}-T cell phenotypes) and killing was mainly non-virus specific. In addition, flow cytometry analysis demonstrated more CD6+ CD8+ and CD4+ CD8+ and fewer CD6- CD8+ and  {gamma}{delta}-T+ CD8+ lymphocytes after PRV stimulation of immune PBMC compared with IL-2 stimulation of naive PBMC. Therefore, the flow cytometry data are in line with the phenotype of cytolytic cells detected after PRV (more CD6+ cells) and IL-2 (more CD6- cells) stimulation.

PRV-stimulated immune PBMC killed PRV-infected target cells more efficiently than uninfected target cells and this killing was mediated by CD6+ CD8+ lymphocytes. These findings confirm the study of Pauly et al. (1996) , who reported that CD6+ CD8+ lymphocytes are responsible for virus-specific MHC-restricted killing. In contrast to Pauly et al. (1996) , we could not eliminate killing by the unseparated population by ‘blocking’ of the MHC-I molecule with a MAb (data not shown). However, both the CD6+ and CD8+ population showed decreased killing after ‘blocking’ of the MHC-I molecule, which indicated that classical CTL were present in PRV-stimulated immune PBMC. Flow cytometry analysis of the separated populations revealed that the PRV-stimulated CD6+ PBMC were CD4- {gamma}{delta}-T- CD8bright+ (CTL phenotype), CD4+ {gamma}{delta}-T- CD8dull+ (fraction containing memory T helper cells) or CD4+ {gamma}{delta}-T- CD8- (T-helper cell phenotype). The percentage of CD4+ CD8+ lymphocytes was very high after PRV stimulation (68%; Table 2) compared with that of freshly isolated PBMC (Fig. 1), which indicates that these lymphocytes may contribute to killing or are themselves directly involved in killing.

In addition to the killing by CD6+ lymphocytes, we also detected some killing by CD6- lymphocytes. These cells killed PRV-infected and uninfected L14 cells and K562 cells efficiently. Flow cytometry analysis of the PRV-stimulated CD6- PBMC revealed mainly {gamma}{delta}-T- CD8+ (NK phenotype) and {gamma}{delta}-T+ CD8+{gamma}{delta}-T cell phenotype) lymphocytes. Therefore, these results confirm our earlier observation (Kimman et al., 1996 ) that NK cells were induced in PRV-immune pigs.

In contrast to the predominant CD6+ CD8+ phenotype of cytolytic cells after PRV stimulation of immune PBMC, we demonstrated that the IL-2-stimulated cytolytic cells were mostly CD6- CD8dull+ lymphocytes. These CD6- CD8+ lymphocytes killed all the target cells investigated, indicating MHC-unrestricted or natural killing. Flow cytometry analysis revealed that the IL-2-stimulated CD6- PBMC were mainly {gamma}{delta}-T- CD8+ (NK phenotype) and {gamma}{delta}-T+ CD8+ ({gamma}{delta}-T cell phenotype). These findings indicate that the NK activity in the fraction containing CD6- lymphocytes is due to LAK cells or {gamma}{delta}-T lymphocytes with NK-like activity. The IL-2-stimulated PBMC isolated from naive pigs contained 67% {gamma}{delta}-T+ CD8+ lymphocytes. The high percentage of {gamma}{delta}-T lymphocytes, at least after IL-2 stimulation of PBMC of naive pigs, compared with the percentage in freshly isolated PBMC (Fig. 2; CD6- fraction) suggests a role in the NK activity. Killing by {gamma}{delta}-T+ CD8+ lymphocytes has also been reported after stimulation with an anti-CD3 MAb (Yang & Parkhouse, 1997 ) or with IL-2 (De Bruin et al., 1997 ).

In addition to the killing by the CD6- lymphocytes, we also detected some killing by CD6+ CD8+ lymphocytes after IL-2 stimulation. These lymphocytes killed the PRV-infected and uninfected L14 target cells equally well. We demonstrated previously (De Bruin et al., 1997 ) that IL-2-stimulated killing by CD5+ CD8+ lymphocytes (CTL phenotype) could be directed against infected and uninfected L14 target cells. Virus-specific killing was only detected after IL-2 stimulation of PBMC isolated from immune pigs, which indicates that PRV inoculation in vivo is needed to expand virus-specific CTL precursors. We did not detect killing by CD8- lymphocytes after either IL-2 or PRV stimulation, which confirms the results of Yang & Parkhouse (1997) . Therefore, killing by both classical CTL and NK activity is due to CD8+ lymphocytes, which confirms the results of Pescovitz et al. (1988) . However, the {alpha}-wCD6 MAb (Pauly et al., 1996 ) enabled us to discriminate between classical CTL and cells with NK activity (summarized in Table 3).


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Table 3. Results summary

 
In PBMC from immune pigs, the percentage of PRV-induced CD6+ CD8+ (CTL phenotype) lymphocytes was higher in frequently inoculated pigs compared with singly inoculated pigs (Fig. 3). This result agrees with the observed increase in virus-specific killing after frequent inoculations. Similarly, the percentage of CD4+ CD8+ (fraction containing memory T helper cells) lymphocytes was also higher in frequently inoculated pigs than in singly inoculated pigs. These lymphocytes contribute to the PRV-specific proliferative ‘helper’-like response of PBMC (Zuckermann & Husmann, 1996 ; Summerfield et al., 1996 ). Direct effector functions have not yet been described for CD4+ CD8+ lymphocytes; therefore a cytolytic effector function can not be excluded. Besides the high percentage of CD4+ CD8+ lymphocytes in the CD6+ population (mentioned above), the increase in CD4+ CD8+ lymphocytes after subsequent immunizations also indicates an important role for these lymphocytes after PRV inoculation in pigs. These findings may explain the observation that repeated immunizations with PRV vaccines are usually needed to provide a high level of protective immunity.

In conclusion, we have demonstrated that PRV stimulation induced predominantly classical CTL (CD6+ CD8+ lymphocytes) but also some NK cells, whereas IL-2 stimulation induced mainly LAK cells with NK activity (CD6- CD8+ lymphocytes). The possible involvement of CD4+ CD8+ lymphocytes (the fraction containing memory T helper cells) as cytolytic effector cells against PRV-infected cells after PRV stimulation and the NK-like activity of {gamma}{delta}-T+ CD8+ lymphocytes after IL-2 stimulation remain to be investigated.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Chinsakchai, S. & Molitor, T. W. (1992). Replication and immunosuppressive effects of pseudorabies virus on swine peripheral blood mononuclear cells.Veterinary Immunology and Immunopathology 30, 247-260.[Medline]

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De Wind, N., Zijderveld, A., Glazenburg, K., Gielkens, A. & Berns, A. (1990). Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus.Journal of Virology 64, 4691-4696.[Medline]

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Jonjic, S. & Koszinowski, U. H. (1984). Monoclonal antibodies reactive with swine lymphocytes. I. Antibodies to membrane structures that define the cytolytic T lymphocyte subset in the swine.Journal of Immunology 133, 647-652.[Abstract/Free Full Text]

Kaeffer, B., Bottreau, E., Phan Thanh, L., Olivier, M. & Salmon, H. (1990). Histocompatible miniature boar model: selection of transformed cell lines of B and T lineages producing retrovirus.International Journal of Cancer 46, 481-488.

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Received 29 November 1999; accepted 28 January 2000.