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
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Abstract |
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Introduction |
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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- (-)wCD6 monoclonal antibodies (MAbs). The phenotype and cytolytic function of these subsets were subsequently investigated.
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Methods |
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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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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.
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 -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
-T-cell receptor (TCR) (IgG2b) (Kirkham et al., 1996
; Yang & Parkhouse, 1996
). Cells staining positive for
-TCR are referred to in this article as
-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
). Students t-test was used for statistical analysis of the results.
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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).
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).
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Results |
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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 -T+ CD8+ (
-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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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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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.
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Discussion |
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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-
-T- CD8bright+ (CTL phenotype), CD4+
-T- CD8dull+ (fraction containing memory T helper cells) or CD4+
-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 -T- CD8+ (NK phenotype) and
-T+ CD8+ (
-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 -T- CD8+ (NK phenotype) and
-T+ CD8+ (
-T cell phenotype). These findings indicate that the NK activity in the fraction containing CD6- lymphocytes is due to LAK cells or
-T lymphocytes with NK-like activity. The IL-2-stimulated PBMC isolated from naive pigs contained 67%
-T+ CD8+ lymphocytes. The high percentage of
-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
-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
-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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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 -T+ CD8+ lymphocytes after IL-2 stimulation remain to be investigated.
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Received 29 November 1999;
accepted 28 January 2000.