Optimization of murine CD8+ cytotoxic T-lymphocyte responses to pseudorabies virus

Journal of Immunological Methods 203 Ž1997. 77–88

Optimization of murine CD8q cytotoxic T-lymphocyte responses to pseudorabies virus C. Depierreux a b

b,)

, I. Graff a , V. Lancelot a , M. Denis b, A. Vanderplasschen b, E. Hanon b, P.P. Pastoret b, C. Swysen a

Laboratoire d’Immunologie Appliquee, ´ SOLVAY s.a., rue de Ransbeek 310, B-1120 Bruxelles, Belgium SerÕice d’Immunologie de la Faculte´ de Medecine Veterinaire de l’UniÕersite´ de Liege, ´ ´´ ` Liege, ` Belgium Received 30 May 1996; revised 8 October 1996; accepted 21 January 1997

Abstract The aim of this work was to optimize the procedures used to elicit a cellular immune response to pseudorabies virus ŽPrV. in mice, using various immunization schedules and routes. An Eu-labeling-based cytotoxic T-lymphocyte ŽCTL. test was developed to measure the response. This necessitated optimization of numerous steps. In suspension, Eu labeling required high concentrations of dextran-sulfate ŽDXS. and Eu with a 30-min labeling time at room temperature. For anchored cells, the labeling time was 1 to 48 h, and the labeling efficiency depended strongly on the Eu concentration, but only marginally on the DXS concentration. In vivo and in vitro stimulation protocols were also optimized for the CTL test. For in vitro stimulation, spleen cells were cultured in T-25 flasks at a multiplicity of infection Žm.o.i.. of 2. The CTL test was validated by specific depletion of CD8q CTL, FACS analysis, and by comparing Eu and 51 Cr labeling. Then groups of mice were vaccinated once or twice by various routes Žintraperitoneal Ži.p.., intravenous Ži.v.., subcutaneous Žs.c.. and in the rear footpads ŽFP.. and according to different time schedules. CTL activity was detected only in boosted animals immunized FP, i.p. or i.v. That the cellular immune response contributes to protection was further suggested by the observation that i.p. immunization conferred better protection against challenge than s.c. immunization. Keywords: Europium; Pseudorabies; Immunization route; Cytotoxic T-lymphocyte induction

1. Introduction

Abbreviations: CMI, cell-mediated immunity; CTL, cytotoxic T-lymphocytes; DTPA, diethylenetriaminopentaacetate; DXS, dextran-sulfate; Eu, Europium; FP, rear footpads; m.o.i., multiplicity of infection; PrV, pseudorabies virus; TK, thymidine kinase ) Corresponding author. Universite´ de Liege, ` Faculte´ de Mede´ cine Veterinaire, Service d’Histologie-Embryologie, Boulevard de ´´ Colonster 20 Žbat. ˆ B43., B-4000 Liege, ` Belgium. Tel.: q32 Ž4. 3664081; Fax: q32 Ž4. 3664097.

Aujeszky’s disease, which affects various mammals, is caused by an a-herpes virus Žpseudorabies virus, PrV.. The pig, as the natural host, is most sensitive at birth. Infected newborns display high mortality; adults show few clinical signs, but the frequent abortions represent a serious threat to the pig industry ŽPensaert and Kluge, 1989..

0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 7 . 0 0 0 1 5 - X

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C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

Various modified live vaccines have been developed and used as attenuated marker strains in vaccine control programmes ŽGielkens et al., 1989; Visser and Lutticken, 1989; Kimman, 1992; Pensaert ¨ et al., 1992.. Most carry deletions or mutations in the thymidine kinase ŽTK. gene andror other non-essential genes ŽgE, gI. ŽMettenleiter, 1994.. Vaccination against PrV is routinely used and is known to effectively control the disease. Since no clear-cut correlation has been found between protection and the level of circulating antibodies in immunized pigs ŽChinsakchai and Molitor, 1994., it is generally accepted that CMI could play a major role in the protection mechanism. The high susceptibility of mice to the disease and the availability of cell markers and inbred lines make it possible to use the mouse model to study the immune mechanisms involved. In both mice and pigs, Zuckermann et al. Ž1990. have demonstrated PrV-specific cytotoxic CD8q T-cell activity, mostly directed against the gC ŽgIII. glycoprotein. The purpose of this work was to optimize procedures used to generate cytolytic T-cell activity in spleen cell populations, using various immunization schedules and routes. The research was based on Zuckermann’s PrV mouse cytotoxicity study. To assess the best vaccination route, we compared the footpad ŽFP., subcutaneous Žs.c.., intravenous Ži.v.., and intraperitoneal Ži.p.. routes. Eu labeling was used to measure the cytotoxic T-lymphocyte response. After optimization, this procedure was validated by comparison with the standard 51 Cr release test. We used specific depletion and flow cytometric analysis to demonstrate the contribution of CD8q T-lymphocytes to cytotoxic lysis.

2. Materials and methods 2.1. Mice and cell lines Female BALBrc ŽH-2 d . mice, 8–12 weeks old, were purchased from Charles River, Germany. The pooled spleen cells of 5–10 mice were used for each protocol tested. The histocompatible mouse fibroblast cell line 3T3 provided the target cells. These cells were grown in DMEM ŽDulbecco’s modified Eagle’s medium.

containing 10% fetal calf serum ŽFCS, Gibco Brl, Life Technologies, Merelbeke, Belgium. and antibiotics Ž100 Urml penicillin and 100 mgrml streptomycin.. Vero cells were used to grow PrV. They were cultured in F12rM199 medium ŽVrV. ŽGibco Brl, Life Technologies, Merelbeke, Belgium. supplemented with 5% FCS, 5% lactalbumin hydrolysate, 2 mM L-glutamine, and antibiotics. PD5 cells, used for the seroneutralization test, were cultured in the same medium as 3T3 cells. 2.2. PrV strains The avirulent strain NIA3M207 was used for both in vivo and in vitro stimulation and for target cell infection. This strain Žprovided by Dr. A. Bianchi, ID-DLO, Lelystad, the Netherlands., propagated on Vero cells, is a thymidine kinase-deficient strain obtained by double crossing-over recombination. Bartha strain K61 ŽBartha, 1961., used in the seroneutralization assay, and the virulent parent strain NIA3, used in challenge experiments, were obtained from Solvay Duphar. 2.3. Immunization routes Mice were injected with 100 ml Žper mouse. of culture medium containing PrV at 10 7 PFU or less. The routes tested were the i.p., i.v. and s.c. and injection into both rear footpads Žthe FP route. ŽColigan et al., 1992.. 2.4. Generation of cytotoxic T-cells CTL effectors were generated ŽZuckermann et al., 1990. from the spleen cells of animals immunized according to different protocols. Splenocytes were cultured for 5 days in the absence or presence of virus at a multiplicity of infection Žm.o.i.. of 2 in effector medium ŽDMEM ŽGibco Brl, Life Technologies, Merelbeke, Belgium. supplemented with 10% FCS, antibiotics Ž100 Urml penicillin and 100 mgrml streptomycin., 2 mM L-glutamine, 2 = 10y5 M 2-mercaptoethanol, 10 mM HEPES ŽpH 7.4., and 2 mM sodium pyruvate.. The cells Ž25 = 10 6 in 5 ml. were distributed into flasks Ž25 cm2 . and incubated upright at 378C, 95% humidity, and 3% CO 2 .

C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

2.5.

51

Cr labeling of the target cells (3T3)

51

Cr labeling was performed as described by Heymer and Leibbold Ž1993.. Briefly, 3T3 cells grown for 1 day to near-confluence in a 6-well plate ŽNunc. were labeled by removing the culture medium and incubating the cells for 2.5 h at 378C with a Ž mixture of 200 ml Na51 2 CrO4 250 mCi, Amersham, Bruxelles, Belgium. and 500 ml culture medium. The labeled cells were washed with culture medium Ž3 times. and with PBS-EDTA Žonce. before trypsinization and suspension in effector medium Ž5 = 10 4 cellsrml.. The labeling efficiency was assessed by incubating 5000 cells in 100 ml effector medium Žspontaneous release. or in 100 ml Triton X-100 at 1% vrv Žmaximal Cr release.. After centrifugation at 50 = g for 4 min, the supernatants Ž100 ml. were harvested and the radioactivity counted in a g-counter ŽPharmacia, LKB, Uppsala, Sweden.. 2.6. Eu labeling of the target cells (3T3) The Eu labeling procedure was adapted from Granberg et al. Ž1988.. Cells Ž3T3. at confluent growth were first trypsinized and washed once in HEPES buffer Ž50 mM HEPES, 93 mM NaCl, 5 mM KCl and 2 mM MgCl 2 6H 2 O at pH 7.4.. Then to 6 = 10 6 cells in 1700 ml HEPES buffer were added 200 ml Eu-DTPA solution ŽDTPA: diethylenetriaminopentaacetate.. This solution was prepared with 1.52 ml Eu Standard solution Ž1000 mgrml in 1% nitric acid; Aldrich, Bornem, Belgium., 8 ml HEPES buffer, and 0.5 ml DTPA solution Ž3.93 g DTPA in 10 ml HEPES buffer, pH adjusted to 7.4.. Two minutes later, 100 ml dextran-sulfate ŽDXS. Ž50 mg DXS in 10 ml HEPES buffer. was added to the cells. A 30-min incubation was needed for labeling at room temperature with gentle shaking every 10 min. After the addition of 7 ml repair buffer Ž0.588 g CaCl 2 2H 2 O and 1.8 g glucose in 1 l HEPES buffer. and 3 ml effector medium, 0.1% Žvrv. DNase solution Ž17 000 Urml in saline, Boehringer Mannheim. was added to the cell suspension. After 8 min, the cells were washed once with effector medium, suspended in 5 ml effector medium, and layered onto 5 ml Ficoll-Paque ŽPharmacia, Uppsala, Sweden.. The tube was centrifuged for 15 min at 800 = g and 208C. Living target cells were harvested from the

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upper phase including the interphase, then washed once in effector medium. The extent of labeling was estimated as for Cr labeling Žsee above., but in this case, 50 ml supernatant was added to 200 ml enhancement solution ŽDelfia, Wallac, Finland. at least 1 h before fluorescence was measured ŽTime Resolved Fluorometer, 1234 Delfia Research, Wallac, Finland.. Spontaneous release was estimated by incubating target cells with 100 ml effector medium only and maximum release was assessed in the presence of 100 ml Triton X-100 Ž1%, vrv, Merck, Overijse, Belgium.. Cells nearing confluence could also be labeled in flasks Ž175 cm2 . as follows. Part of the culture medium was removed, leaving 15 ml to which 1 ml of Eu-DTPA was added. After incubation for 24 h at 378C Ž3% CO 2 and ) 90% humidity., the labeling liquid was replaced with 20 ml repair buffer and the flask incubated for 10 more minutes under the same conditions. After two washes Ž1 = effector medium and 1 = PBS., the cells were trypsinized with 5 ml of trypsin-EDTA, whereupon effector medium Ž7 ml. was added. Further steps ŽFicoll-Paque, DNase treatments. were as described above. 2.7. Cytotoxicity assay Cytotoxic effector cells were collected 5 days after in vitro stimulation, washed twice in effector medium, and serially diluted into U-bottomed 96-well microdilution plates Žin triplicate., starting with 5 = 10 5 cells in 100 ml. Target cells Ž3T3 fibroblasts cultured in DMEM and 10% of FCS. were incubated for 90 min at 378C in the presence or absence of PrV at an m.o.i. of 10. The cells were then labeled with Eu or 51 Cr. The time course of infection was followed by immunofluorescence Žindirect labeling. and by microscopic observation of cell lysis. Surface immunofluorescence appeared 4–8 h postinfection Žcf. recommendations by Dr. Bianchi.. 5–7 h after infection with PrV, 5000 labeled target cells Žin 50 ml. were added to the serially diluted effector cells Žeffector : target ratios were 100:1, 50:1, 25:1, 12:1, 6:1, 3:1.. The plates were briefly centrifuged at 50 g and incubated for 4 h at 378C. After incubation followed by centrifugation at 50 = g for 4 min, the labeled cells were treated as follows under Ža. or Žb..

C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

80

Ža. Eu-labeled target cells: 20 ml supernatant from each well was transferred to the corresponding well of a flat-bottomed plate containing 200 ml enhancement solution ŽDelfia, Wallac, Finland., then stored in the dark at room temperature. Fluorescence ŽHemmila et al., 1984. was measured at least 1 h later Žusually 16 h later. ŽTime Resolved Fluorometer, 1234 Delfia Research, Wallac, Finland.. Žb. Cr-labeled target cells: 100 ml of supernatant from each well was transferred for counting to a g-counter ŽPharmacia, LKB, Uppsala, Sweden.. Percent specific Eu or 51 Cr release was calculated as: % specific lysis experimental release y spontaneous release s maximum release y spontaneous release = 100 2.8. Immunofluorescence staining and FACS (flow cytometric) analysis To quantify the proportion of various cell types in an effector cell population, we detected expression of specific surface markers by direct labeling with fluorescein-coupled monoclonal antibodies. Anti-mouseCD4 Žanti LCT4rCD4 fluorescein., anti-mouse-CD8 Žanti LY-2 fluorescein ŽCD8a.., and anti-mousemacrophage ŽMAC-1 fluorescein. antibodies ŽBoehringer Mannheim, Bruxelles, Belgium. were used. Spleen cells were isolated as described above. After in vitro stimulation in the absence or presence of PrV virus, effector cells were collected, centrifuged at 48C Ž220 = g, 4 min., washed once in PBS, and suspended at a concentration of 5 = 10 6 cellsrml. In a U-bottomed 96-well plate, 4 = 200 ml of effector-cell suspension were distributed into 4 wells Žone for calculating the background and 3 for incubation with the different antibodies.. To each well was added 100 ml of the appropriate MAb Žanti-CD4 or anti-CD8 MAb diluted 1r100 in PBS or anti-MAC1 MAb diluted 1r20.. The samples were then incubated in the dark for 30 min at 48C, whereupon 100 ml of PBS-FCS Ž5%. was added to minimize non-specific labeling. The cells were then kept in the dark at 48C. To distinguish live cells from dead ones, we added propidium iodate Ž5 mgrml. Žvrv. 5 min before analysis.

Flow cytometric analysis was performed with a Becton-Dickinson fluorescence-activated cell sorter ŽFacstar Plus. equipped with an argon laser ŽILT air cooled with a 100-mW excitation line at 488 nm.. Debris were excluded from the analysis by the standard scatter-gating method. The emission signal was collected via an appropriate filter at 530 nm Žband pass 30 nm.. In most cases, 10 000 events per sample were collected in list-mode fashion, stored, and analyzed by a Consort 32 system ŽBecton-Dickinson.. The fluorescence intensity threshold for a positive result was arbitrarily set on the basis of the negative control sample. 2.9. Depletion of (cytotoxic) CD8 q T-lymphocytes For CD8 depletion, magnetic beads ŽM-450 Dynabeads coupled to sheep anti-mouse antibody ŽDynal.. were used in a two-step procedure ŽVartdal et al., 1987. involving formation of a complex between the magnetic beads and the anti-CD8 antibody, followed by depletion itself. The magnetic beads Ž4 = 10 8 beadsrml. were washed twice in Tris buffer Ž0.1 M; pH 7.4.. A magnetic device ŽDynal MPC-1. was used to collect them. The beads were resuspended in PBS-BSA Ž0.1%.. Anti-CD8 antibodies Ž1 mg antibodyrmg Dynabeads. were then added and incubated for 30 min at 48C under gentle shaking. Finally, the beads were resuspended in 6 ml PBS-BSA Ž0.1%. supplemented with 1% FCS and incubated for 10 min at 48C with gentle shaking. They were then ready for the use in the depletion step. In vitro stimulated spleen cells Ž5 = 10 6 . were resuspended in 5 ml effector medium and 500 ml of magnetic bead suspension were added Ž10 beads per cell.. The samples were then incubated for 10 min at 48C with gentle shaking. The beads were then removed and the supernatant collected and depleted again for maximal efficacy. 2.10. Challenge of mice Groups of 10 mice were immunized i.p. or s.c. with PrV strain NIA3M207 at doses ranging from 10 7 to 3 = 10 4 PFUrmouse. Control mice received PBS Ži.p. or s.c... Four weeks later, the mice of all groups were inoculated with the virulent virus strain

C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

NIA3 Ž7000 PFUrmouses 100 = LD50 .. Mortality was monitored for 15 days postchallenge.

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each sample calculated as the reciprocal of the dilution corresponding to the 50% end-point.

2.11. PrV neutralization test 3. Results Sera were collected by retro-orbital puncture. Serial two-fold dilutions were made with assay medium Ž340 ml minimal essential Eagle’s medium ŽICN Biomedicals NV-SA, Asse, Belgium., 100 ml lactalbumin-hydrolysate 2.5%, 5–10 ml NaHCO 3 Ž5.6%., and 50 ml FCS. in 96-well plates. Bartha strain K61 w9x was diluted to 100 TCID50 in 50 ml assay medium, added to each serum dilution, and incubated for 24 h at 378C. PD5 cells Ž2 = 10 4 cells in 50 ml. were then added to each mixture and incubated for 5 days at 378C. Cytopathic effects were observed and the titer of

3.1. Optimization of Eu labeling 3.1.1. Labeling of 3T3 cells in suspension The parameters optimized were the Eu-DTPA and DXS concentrations, the number of cells, and the labeling time. Best results were obtained under conditions Žsee Table 1. which differed somewhat from those used by Granberg et al. Ž1988.: a lower cell concentration Ž3–6 = 10 6 cellsrml., incubation with Eu-DTPA for 2 min before addition of DXS, and labeling at room temperature for 30 min. The temperature and incubation time were adapted to the

Table 1 Optimization of Eu labeling of cells in suspension showing experimental design, labeling results without Ficoll treatment and labeling results after such treatment Experimental conditions tested

3T3 cell line 1

Cell volume Žml. 0.5 Ž6 = 10 6 cellsrml. HEPES buffer Žml. 375 Eu-DTPA Žml. 100 Incubation for 2 min DXS Žml. 25 Labeling time Žmin. 20 Labeling results without Ficoll treatment b Ža. Background 10 138 " 760 fluorescence Žcps. a Žb. Maximal 31 287 " 2 359 fluorescence Žcps. a Žc. Spontaneous release 32.4 " 4.9% Labeling results after Ficoll treatment b Ža. Background 7 216 " 404 fluorescence Žcps. a Žb. Maximal 40 669 " 5698 fluorescence Žcps. a Žc. Spontaneous release 17.7 " 3.5%

2

3

4

5

6

0.5

0.5

0.5

0.5

1

375 100

375 100

375 100

375 100

700 200

50 20

50 30

100 20

100 30

100 30

15 127 " 374

35 127 " 3 109

20 122 " 1 347

53 132 " 2 859

ND

81 063 " 6 230

166 719 " 8 384

105 545 " 12 861

328 927 " 10 995

ND

18.7 " 1.9%

21.2 " 2.9%

19.1 " 3.6%

16.2 " 1.4%

ND

10 199 " 387

16 342 " 3 263

7 545 " 333

28 436 " 2 107

11 694 " 740

99 254 " 4725

212 084 " 876

56 902 " 6 466

299 742 " 1 8728

214 094 " 7 443

10.0 " 0.9%

7.7 " 1.6%

13.3 " 2.1%

9.5 " 1.3%

3.7 " 0.3%

c

To 5000 labeled target cells in 50 ml effector medium, Ža. 100 ml effector medium was added for measuring spontaneous fluorescence and Žb. 100 ml Triton X-100 Ž1%. was added for measuring maximal fluorescence. Fluorescence Žin cps. was measured as described Section 2: Materials and methods. Žc. Background fluorescence= 100rmaximal fluorescence. a Means" SD of 3–4 repeats. b After the repair treatment Žsee Eu labeling method. and washing with effector medium, the cells were divided into two parts: one batch only was subjected to Ficoll treatment, followed by washing in effector medium for both batches. c Not determined.

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target cell line Ždata not shown.. A DNase digestion step followed by Ficoll-Paque treatment was included in order to remove dead cells from the target cell preparation. The Eu and DXS concentrations used in experiment 6 gave the best results and were adopted in further experiments. Ficoll-Paque treatment was also systematically included, because it decreased the spontaneous release without affecting labeling. A final addition to this protocol was the DNase treatment, applied during the 8-min of repairing incubation. This resulted in a better cell yield after the washing steps, avoiding cell entrapment in a loose DNA-containing pellet.

3.2. Optimization of in ÕiÕo and in Õitro stimulation 3.2.1. In ÕiÕo stimulation Various immunization protocols were compared. To estimate the efficacy of a booster injection, we first immunized two groups of mice intravenously with strain NIA3M207 Ž10 7 PFUrmouse.. A control group received an injection of PBS ŽGibco Brl, Life Technologies, Merelbeke, Belgium.. Four weeks later, one of the two immunized groups received a booster injection. After 6 more days, spleen cells were harvested from all groups for in vitro stimulation. The CTL effector cells were cultured for 5 days in the presence of PrV at an m.o.i. of 5. As shown in Fig. 1, CTL activity was detected only in boosted animals. No specific cytolysis of uninfected target cells was detected and effector cells from control mice failed to trigger lysis of infected target cells. The immunization route and the interval between injections both proved critical to induction of a cellular immune response. This was shown in experiments using two shots Žadministered either 1 or 4 weeks apart. and 4 different immunization routes. When the interval between injections was only 1 week ŽFig. 2., vaccination via the i.v. and s.c. routes yielded no detectable cytolytic activity; the FP and i.p. routes gave rise to low, but significant, lysis. In a repeat experiment, 40% cytolysis was observed after FP immunization. When the booster was administered 4 weeks after priming ŽFig. 3., increased cytolytic activity was obtained with mice immunized

3.1.2. Labeling of anchored 3T3 cells The parameters tested were the Eu-DTPA and DXS concentrations and the labeling time Žranging from 1 h at 378C in a water bath shaker to 24 and 48 h under static conditions, in an incubator at 378C, 3% CO 2 .. Labeling results are summarized in Table 2. When the labeling time was short Ž1 h., the labeling efficiency was highest when the Eu-DTPA concentration was high; increasing the DXS concentration had a lesser effect. In a preliminary 24-h trial, the DXS concentration was found to have but marginal impact. This was confirmed, as shown in Table 2. Omission of DXS during prolonged incubation Ž24 h. had little effect on cell viability. Under all conditions tested, the cells remained anchored, but became rounded in shape.

Table 2 Optimization of Eu labeling of anchored cells Flasks Ž175 cm

2.

1

Culture medium Žml. 8 Eu-DTPA Žml. 4 DXS Žml. 1.6

a

2

3

4

5

6

7

10 4 4.8

12 9 1.6

15 3 0.01

15 1 0.01

15 1 0.01

15 1 0

1h

1h

24 h

48 h

24 h

24 h

Labeling time 1h

Background 2145 " 107 9115 " 821 14 944 " 1197 6802 " 227 2437 " 155 7580 " 607 5099 " 140 fluorescence Žcps. Maximal 61 331 " 8859 84 586 " 2849 121 752 " 1772 265 710 " 20 554 199 384 " 6430 122 856 " 5042 99 991 " 6820 fluorescence Žcps. Spontaneous release 3.5 " 0.7% 10.8 " 1.3% 12.3 " 1.2% 2.6 " 0.3% 1.2 " 0.1% 6.2 " 0.8% 5.1 " 0.5% a

Calculation parameters are as defined in Table 1. Cells were subjected to Ficoll treatment.

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Fig. 1. Ž A. The time schedules show the intervals between priming Ži.v., with NIA3M207, 10 7 PFUrmouse. and the following steps: boosting Žif applicable., spleen cell isolation for in vitro restimulation, and the CTL assay. The same protocols were used as described in Section 2.4: Generation of cytotoxic T-cells, except that 5 = 10 6 spleen cells in 2 ml culture medium were cultured in 24-well plates. Ž B . Percent specific Eu release from target cells Ž3T3. at different effector : target ratios, in experiments using splenic effectors from boosted animals ŽB., primed animals receiving no booster injection Ž`., and control animals Ž-=-.. Effectors from control animals displayed no cytotoxic activity towards infected targets Ž-=-., nor did effectors from immunized animals display any cytotoxic activity towards uninfected targets Ž'.. Spleen cells of each group were pooled from 10 mice. ) Cut off: cytolytic activity is considered positive if exceeding the average lysis in negative controls by more than 3 standard deviations.

i.p. or i.v., but animals vaccinated via the s.c. route still displayed no cytolytic activity. In another experiment, mice received two FP injections 3.5 months apart. Spleen cells from these mice triggered 27% specific lysis. 3.2.2. In Õitro stimulation Proliferation of inguinal or popliteal lymph node cells and spleen cells was compared at the same

m.o.i. in 24-well plates Ž5 = 10 6 cells in 2 ml culture medium.. Spleen cells were preferred to lymph node cells because they were easier to use, even though the cell proliferation ratios were about equal. In vitro stimulation of spleen cells was also compared in 24-well plates Ž5 = 10 6 cells in 2 ml culture medium. and T-25 flasks ŽFalcon, 25 = 10 6 spleen cells in 5 ml culture medium. at different m.o.i. values Ž1, 5, and 10 for 24-well plates and 0.5, 2, and 5 for T-25

Table 3 Cell proliferation and cytolytic activity after in vitro stimulation at various m.o.i. m.o.i.

0.5 2 5

Number of cells used a

Number of viable cells after 6 days of culture

Cell yield Ž%.

150 = 10 6 125 = 10 6 150 = 10 6 125 = 10 6 50 = 10 6

9.95 = 10 6 6.60 = 10 6 8.10 = 10 6 4.60 = 10 6 None viable

7 5 5 4 0

b

Cytotoxic activity against infected cells c Ž% specific lysis. 11.9 " 7.1% 33.5 " 15.0% ND

a Splenic effector cells from mice immunized twice Ži.p., 4 weeks between priming and boosting. with 10 7 PFUrmouse of NIA3M207 virus. Six days after boosting, spleen cells were harvested, stimulated, and cultured at 25 = 10 6 cellsr5 ml in flasks Ž25 cm2 . in the presence of NIA3M207 virus at an m.o.i. ranging from 0.5 to 5. b Number of viable cells after 5 days in vitro = 100rnumber of cells engaged. c CTL test involving Eu-labeled target cells infected with the NIA3M207 strain at an m.o.i. of 10 for 6 h, effector : targets 100:1.

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Fig. 2. Ž A. The time schedule indicates the intervals between priming and the following steps: boosting ŽFP, i.p., i.v., s.c. with NIA3M207, 10 7 PFUrmouse., in vitro restimulation, and the CTL assay. The same protocols were used as described in Section 2.4: Generation of cytotoxic T-cells. Ž B . Percent specific Eu release from target cells Ž3T3. at different effector : target ratios, in experiments using splenic effectors from boosted animals against infected ŽB. or uninfected targets Ž'., for the 4 routes of immunization. Spleen cells of each group were pooled from 10 mice. ) Cut off: cytolytic activity is considered positive if higher by more than 3 standard deviations than the average lysis in negative controls.

flasks.. In 24-well plates, an m.o.i. of 5 proved optimal for cell viability ŽCTL lysis was not measured.. The optimal m.o.i. for stimulating cells in T-25 flasks was 2 ŽTable 3.. In further experiments, we adopted spleen cell stimulation with PrV at an m.o.i. of 2 in T-25 flasks, because these conditions yielded high cytolytic activity. 3.3. Validation of the CTL test 3.3.1. Effector cell analysis and depletion Spleen cell populations were analyzed by flow cytometry and by specific depletion of T-CD8q lymphocytes. When spleen cells from twice-immunized and naive mice were identically stimulated

with PrV in vitro Žm.o.i.s 2., flow cytometry revealed a higher proportion of both CD4q and CD8q cells among the former than among the latter ŽTable 4.. Stimulation in the absence of virus yielded a lower percentage of both CD4q and CD8q cells in the total spleen cell population. A specific depletion test was performed to identify the effectors of the cellular immune response. In this test, mice were immunized twice ŽFP with strain NIA3M207, 10 7 PFUrmouse.; spleen cells were restimulated in vitro with PrV at an m.o.i. of 2. Half of this population was then depleted of T-CD8q lymphocytes as described under Section 2: Materials and methods. FACS analysis of the population confirmed the elimination of T-CD8q lymphocytes Žde-

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85

Fig. 3. Ž A. The time schedule indicates the intervals between priming and the following steps: boosting Ži.p. and i.v. with NIA3M207, 10 7 PFUrmouse., in vitro restimulation, and the CTL test. The same protocols were used as described under Section 2.4: Generation of cytotoxic T-cells. Ž B . Percentages of specific Eu release from target cells Ž3T3. at different effector : target ratios, using splenic effectors from boosted animals against infected targets ŽB. or against uninfected targets Ž'. for the two routes of immunization. Spleen cells of each group were pooled from 10 mice. ) Cut off: cytolytic activity is considered positive if higher than the average lysis of negative controls increased by 3 standard deviations.

creasing from 24.63 to 0.64%.. Specific cytolytic activity was then measured in the depleted and undepleted populations. The significant activity observed in the undepleted population Ž18.5 " 3.6% specific lysis at an effector : target ratio of 100:1. was abolished in the CD8-depleted sample Ž4.7 " 1.3% at 100:1..

ceived PBS by the same route. Six days after the booster injection, spleen cells were isolated and restimulated in vitro Žm.o.i.s 2. with the same PrV strain. Five days later, target cells were infected, labeled with Eu or 51 Cr as described under Section 2: Materials and methods, and used in the CTL test. All control samples were negative and target cells labeled by the two methods displayed very similar maximum levels of specific lysis Ždata not shown..

3.3.2. Comparison of 51Cr and Eu labeling Eu labeling of target cells in suspension was compared with the standard 51 Cr method. Ten mice were twice immunized ŽFP, with a 1-week interval between injections. with strain NIA3M207 Ž10 7 PFUrmouse.. A control group re-

3.4. Protection against Õirulent challenge We also performed a mouse protection test to assess the contribution of the cellular and humoral

Table 4 Expression Ž%. of various cell surface markers after stimulation by different routes

Naive mice Immunized mice a b c d

d

Controls

a

No virus

b

ND 0.2%

Anti-mouse CD4 m.o.i. 2 0.2% 0.1%

c

No virus ND 28.2%

Fluorescein-coupled monoclonal antibodies. Spleen cells cultivated in vitro in the absence of virus. Spleen cells cultured in vitro at an m.o.i. of 2. Spleen cells from mice immunized twice with PrV.

b

a

m.o.i.s 2 22.2% 41.1%

Anti-mouse CD8 c

No virus ND 7.8%

b

a

m.o.i.s 2 11.5% 21.9%

Anti-mouse Mf c

No virus ND 2.7%

b

a

m.o.i.s 2 2.3% 7.2%

c

86

C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

Table 5 Results of the challenge test after i.p. or s.c. priming at various doses Virus inoculated for immunization ŽPFUrmouse. IP immunization Survivors a Seroneutralization titer prechallenge b Seroneutralization titer postchallenge c SC immunization Survivors a Seroneutralization titer prechallenge b Seroneutralization titer postchallenge c a b c d

10 7

3 = 10 6

10 6

3 = 10 5

10 5

3 = 10 4

PBS control

PI 50

d

10r10 8 90

9r10 6 8

9r10 ND ND

7r10 ND ND

9r10 ND ND

4r10 ND ND

0r10 y1.4 No survivors

10 4.99

9r10 6 128

10r10 y1.4 16

8r10 ND ND

4r10 ND ND

1r10 ND ND

0r10 ND No survivors

0r10 y1.4 No survivors

10 5.99

Number of surviving mice 2 weeks postchallengernumber of animals tested. Sera from each group were collected and pooled 3.5 weeks postimmunization. Sera from each group were collected and pooled 6 weeks postimmunization. PI, protective index.

immune responses to protection. BALBrc mice Žgroups of 10 mice. were immunized i.p. and s.c. with differing amounts of modified live virus NIA3M207 Ž10 7, 3 = 10 6 , 10 6 , 3 = 10 5, 10 5 and 3 = 10 4 PFUrmouse.. After 4 weeks, the mice were challenged with virulent NIA3 at 7000 PFUrmouse. About 50% of the challenged animals survived when immunized i.p. with 3 = 10 4 PFU or s.c. with 3 = 10 5 PFU. The i.p. route thus offered better protection. Seroneutralizing antibodies were detected in both cases prechallenge, but higher levels were observed in mice immunized by the i.p. route ŽTable 5..

4. Discussion Our goal here was to assess CTL activity in the mouse model after PrV immunization. This entailed, as an essential part of the work, optimizing not only the immunization protocol, but also various steps of the test used to measure CTL activity, notably labeling with the fluorescent marker Eu. Various markers of cytotoxic release have been proposed instead of 51 Cr, the aim being mainly to avoid work with radioactive material and cumbersome waste disposal problems. Colored substrates Že.g., MTT., specific cell enzyme release, and DNA fragmentation markers have been used, but the specificity andror sensitivity of these methods have often proved insufficient. A preliminary trial in our labora-

tory with LDH release was abandoned because serum was found to interfere with enzyme activity Ždata not shown.. Here we have studied Eu labeling of 3T3 target cells, both in suspension Žafter trypsinization. and anchored. In suspension, best results were obtained with high Eu-DTPA and DXS concentrations and a long incubation Ž30 min. at room temperature, as indicated by the high fluorescence observed in the maximum release test. The quality of a CTL test, however, also depends on the level of spontaneous release. Removal of dead cells by Ficoll-Paque treatment, combined with DNase treatment to avoid cell entrapment in a viscous pellet, yields high maximum fluorescence combined with low spontaneous release Žtypically below 15%.. When Eu labeling was applied to cells in suspension, the viable cell yield was low, routinely between 6 and 20%. This is the only case where Eu labeling compared unfavorably to 51 Cr labeling. Low cell yields are perfectly acceptable when working with murine cell lines, since on the average 2.6 = 10 7 cellsrflask Ž175 cm2 . are harvested. CTL assays in humans or non-laboratory animals, such as pigs, require gentler methods using as few target cells as possible. To avoid this loss of viable cells, we sought to label anchored cells. This ensures high survival, although no further growth was noted. Under these conditions, the Eu-DTPA concentration and treatment duration were critical. When the labeling time

C. Depierreux et al.r Journal of Immunological Methods 203 (1997) 77–88

was short Ž1 h., a high DXS concentration was needed to allow the Eu marker to penetrate the cells, while no DXS was needed for the long procedure Ž24 h.. The mortality observed after a 48-h labeling period was indicative of some Eu cytotoxicity. Because Eu shows extremely high fluorescence once in contact with enhancement solution, care must be taken not to contaminate equipment and laboratory surroundings while handling the labeling solutions. Contamination with Eu-containing aerosol is avoided by routinely decontaminating the pipetting tools with enhancement solution. Our conditions for labeling cells in suspension Ž30 min at room temperature. differ from those used by Granberg et al. Ž1988. and Blomberg and Ulfstedt Ž1993. on non-adherent cells Ž48C, 5 min.. Optimal conditions, however, depend strongly on the cell line used and must be adapted accordingly. Pacifici et al. Ž1993. have reported the labeling of an adherent cell line during growth, using lower Eu concentrations Ž1 mM instead of the 63 mM used here., but incubating for 4 days instead of one. We also find that DXS can be totally omitted when enough Eu is present. Onehour labeling in flasks in the presence of DXS resulted in the highest cell yields of all methods tested Ž37–85%.. For in vitro stimulation, spleen cell proliferation was compared at various m.o.i. in 24-well plates and in flasks Ž25 cm2 .. Flasks with a m.o.i. of 2 were selected. Cell survival, however, was rather low Žaccording to Bianchi’s own observations, personal communication., generally below 20% of the total spleen cell population stimulated in vitro. Viral cytopathic effects account for much of the loss. On the other hand, cells cultured in vitro for 5 days without virus showed a higher survival, but never induced any cytotoxic activity. The proportion of CD4q and CD8q cells in in vitro-stimulated spleen cell populations, as determined by flow cytometry, appeared higher when the spleen cells were isolated from twice-immunized mice than when they were obtained from naive mice. The exact nature of the effector cells was confirmed by loss of CMI activity after specific depletion of the T-CD8q lymphocyte population. The magnitude of the CTL-type response observed in mice after vaccination with modified live PrV depends strongly on the immunization protocol.

87

High doses and a long interval Ž1 month. between priming and boosting consistently induced high cytotoxic activity, as compared to low virus doses, a short interval Ž1 week. between injections, or no booster. In mice vaccinated via the i.p. route, we could reduce both the initial dose and the booster dose to 10 4 PFUrmouse Žinstead of 10 7 PFUrmouse. and still measure a cytotoxic response, albeit not consistently Ždata not shown.. The immunization route proved an important factor in determining whether a CTL-type response was obtained. While vaccination via the i.p., i.v., or FP always induced a strong CTL response, no CTL activity was detected upon s.c. immunization. Subcutaneous immunization can, however, induce infection and an immune response, mainly of the humoral type as indicated by the generation of seroneutralizing antibodies. Intraperitoneal and s.c. immunization yielded similar titers of seroneutralizing antibodies for a range of priming virus doses, but in a challenge protection test comparing the s.c. and i.p. routes at various priming doses, i.p. immunization provided better protection at low doses capable of eliciting a measurable cytotoxic response. We conclude from these observations that CMI contributes to the higher challenge protection level afforded by i.p. immunization.

Acknowledgements We wish to thank Dr. A. Bianchi, ID-DLO, Lelystad, for technical advice and Dr. H. Geerligs, SOLVAY DUPHAR, Weesp, for performing the seroneutralization assays and giving technical support. Purchase of the flow cytometer was supported in part by a grant from the Loterie Nationale Ž9.4505.92, Belgium.. A.V. is ‘Charge´ de Recherche au Fonds National de la Recherche Scientifique’ ŽF.N.R.S.. and E.H. is ‘Aspirant F.N.R.S.’.

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