Immunology 1978 34 291

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus III.

IN VIVO PROTECTIVE ACTIVITY OF EFFECTOR CELLS GENERATED IN VITRO

M. B. C. DUNLOP John Curtin School of Medical Research, Australian National University, Canberra, Australia

Received 18 April 1977; accepted for publication 25 April 1977

Summary. Secondary cytotoxic T cells were generated in vitro by culturing WE3-LCM virus-specific memory CBA/H spleen cells with WE3-LCM virusinfected syngeneic peritoneal cells at 370 for 5 days, and were found to be highly potent in reducing virus titres in the visceral organs of syngeneic recipients when transferred 24 h before intravenous virus challenge (e.g. 3-1 x 106 cells transferred gave approximately 3 logs reduction of virus titre). Protection was measured by titrating spleens for virus in a plaque assay, usually 2 days post viruschallenge. Intravenously administered secondary effectors did not, however, elicit a reduction in brain virus titres by 3 days after intracerebral inoculation of virus. Cells mediating protection were sensitive to treatment with anti-O ascitic fluid plus complement. Protection occurred only when donors of secondary cells, infected stimulator cells and recipients shared H-2K or H-2D, and there was a similar genetic restriction for these effectors to efficiently lyse virus-infected targets in vitro. Spleen cells from ectromelia virus-memory mice were cultured with ectromelia virus-infected syngeneic peritoneal 'stimulator' cells and generated secondary effector cells which protected syngeneic recipients from subsequent challenge with ectromelia but not LCM virus. However, secondary effector Correspondence: Dr M. B. C. Dunlop, Department of Microbiology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra 2602, A.C.T. Australia.

291

cells derived from in vitro stimulation of spleen cells from WE3-LCM virus-memory mice with WE3LCM virus-infected syngeneic stimulator cells caused a significant reduction in spleen virus titres in syngeneic recipients challenged witheither ectromelia or LCM virus. If WE3-LCM virus-infected stimulator cells were fixed and responder spleen cells were either from very old (6-12 months postpriming) WE3-LCM virus-memory mice, or from ARM-LCM virus-memory mice, this unidirectional cross-protection of ectromelia virus-challenged mice was less pronounced. One explanation for the unidirectional cross-protection was that the transferred secondary effectors contained and released infectious LCM virus (or defective virions) which adsorbed to recipients' spleen cells; these latter cells displayed both LCM and ectromelia virus-specific antigenic patterns after ectromelia virus challenge, rendering them susceptible to specific T-cell mediated lysis. Clear specificity of effector cells was demonstrated at the effector: target level in vitro between LCM virus and ectromelia virus. (Experiments to compare the in vivo activity of secondary effectors with primary effector T cells raised in vivo or in vitro were hampered because of the large amounts of infectious virus in the latter two populations.) Secondary effectors reduced spleen, lung and liver virus titres when transferred 24 h after virus challenge, but were less efficient in protecting recipients than when given before virus.

292

M. B. C. Dunlop INTRODUCTION

LCM virus-immune T cells have been shown to mediate LCM virus-induced immunopathology in vivo (Cole, Nathanson & Prendergast, 1972; Johnson & Cole, 1975; Doherty & Zinkernagel, 1974). Cytotoxic T cells appear to be incriminated, since the capacity to adoptively transfer fatal LCM disease to immunosuppressed, intracerebrally infected recipients with lymphocytes from LCM virus-primed mice requires that donor and recipient share at least one allele in H-2K or H-2D (Doherty, Dunlop, Parish & Zinkernagel, 1976). Transferred cells may, however, also be protective since Johnson & Cole (1975) showed that primary effector T cells can protect normal mice from developing lethal meningitis when given 18 to 24 h prior to intracerebral virus challenge. As well, reduction in virus titres was demonstrated in spleen, liver and lung when spleen T cells from immunized mice were transfused into infected recipients (Mims & Blanden, 1972). More recently, Zinkernagel & Welsh (1976) have mapped this latter activity to require sharing of H-2K or H-2D, but not I region, between donors and recipients. T-cell deficiencies in children demonstrate the paramount importance of cell-mediated immunity in determining recovery from certain virus infections in humans (W.H.O. Tech. Rep. Ser., 1973). Treatments with specific transfers have not however to date been attempted in these patients or, for example, in patients receiving immunosuppressants (who are at risk from various virus infections). An experimental model has thus been sought in which virusspecific T cells could be used to protect mice from virus challenge in a protocol potentially applicable to a therapeutic situation. In this paper, secondary virus-specific T cells generated in vitro (Dunlop & Blanden, 1976; Dunlop, Doherty, Zinkernagel & Blanden, 1976) were found to provide highly potent protection (as assessed by reduction in organ virus titres) under particular assay conditions. The properties in vivo of these secondary effectors induced in vitro

are

characterized here.

MATERIALS AND METHODS

Mice and virus stocks CBA/H, C57BL/6, BALB/c, SJL/J and A.TL recombinant inbred strains and the WEHI outbred

strain were bred in the animal house of the John Curtin School. Inbred recipients (usually female) were used at 5-6 weeks of age. Except where stated otherwise, mice were primed with 0-2 ml of a 10-s dilution of guinea-pig lung or spleen stock of the WE3 (viscerotrophic) strain of LCM virus (WE3LCM virus) (titrated at approximately 4000 intracerebral LD5o or 2500 PFU), given intravenously. Priming dose of the Armstrong (attenuated neurotrophic) strain of LCM virus (ARM-LCM virus) was 0-2 ml of a 10-3 dilution of suckling mouse brain stock (approx. 13,000 intracerebral LD50 or 8000 PFU). Priming dose of ectromelia virus was 2-0 x 104 PFU of the Hampstead Egg strain. Virusmemory mice were primed 6-8 weeks before, except where stated otherwise. Infectious ARM-LCM virus could not be detected in spleens longer than 1 month following primary challenge (M. B. C. Dunlop & G. A. Cole, unpublished observations). Ectromelia virus similarly appears not to persist in spleen cells more than 1 month post-challenge, since these spleen cells do not generate secondary ectromelia virus-specific T cells when cultured at 370 or 390 with uninfected stimulator cells (R. V. Blanden, personal communication; Gardner & Blanden, 1976). Cell cultures Continuous line mouse fibroblasts (L929) were grown at 370 in a modified Eagle's minimal essential medium (Autopow brand, Flow Laboratories, Md.) supplemented with 10% bovine serum and antibiotics. Plaque assay The plaquing procedure closely resembled that used by Blanden (1970) to titrate ectromelia virus. A similar plaquing assay for LCM virus has recently been independently described by Popescu & Lehmann-Grube (1976). 1 0x 106 L929 fibroblasts were seeded into each well of 35 mm, 6 well plastic dishes (Falcon Plastics, Los Angeles, Calif.) in 3 ml of Autopow containing 10% bovine serum. Cells were incubated at 370 overnight to permit adherence and spreading. Monolayers were infected with serial 10-fold dilutions of organ homogenates in ice-cold 0 5 % gelatine saline (Blanden, 1970), adding 0-1 ml samples per well. Plates were then incubated at 370 for 1 h to permit virus adsorption, following which the plates were overlaid with 2 ml Autopow thickened with the addition of 2% carboxymethyl-

Secondary effector cells protect against LCAM cellulose (Gardner, Bowern & Blanden, 1974) at 350 in an atmosphere of 1-2% CO2 in air for 6 or 7 days. Overlays were then tipped off and plates were soaked for 30 min in a solution of crystal violet containing approximately 0-25 % w/v fresh glutaraldehyde, gently washed, dried and plaques were counted. Permanent plaque records were obtained by this relatively easy method. There was a predominant turbid plaque variant and a minor clear plaque variant discernible using WE3-LCM virus guinea-pig spleen or lung stock. (For an investigation of the properties of these LCM virus plaque variants, see Popescu & Lehmann-Grube, 1976.) 'Streaming' of plaques due to convection currents was common. There was occasionally a prozone phenomenon with fewer plaques at the highest virus concentrations. Spleen homogenates from carrier mice gave generally turbid or hazy plaques. Assay of virus in spleen, lung and liver Organs were removed aseptically from individual mice and homogenized in ice-cold 0 5% gelatine saline with a motorized teflon pestle. Gall bladders were removed from the livers before grinding. Two ml portions of each homogenate were dispersed by 7 s ultrasound (Branson Sonifier, Connecticut) and centrifuged to remove debris. Supernates were appropriately diluted in ice-cold gelatine saline and assayed separately. Results were expressed as mean log10 PFU (plaque-forming units)/organ ± standard error of the mean for groups of four mice. Protection was defined as (log1o virus titre post-transfer of no cells or of normal cells) - (log,0 virus titre posttransfer of secondary effectors). A preliminary experiment showed no loss in infectivity when homogenates were subjected to 7 s ultrasound. Indeed, virus titres were lower if homogenates were disrupted by freeze-thawing rather than sonication. Moreover, a titration of the centrifuged organ pellets by intracerebral inoculation of C57BL/6 mice showed no excess loss of virus to the pellets.

Techniques of generation of primary and secondary responses to LCM virus-infected cells in vitro These techniques have been described in detail previously (Dunlop & Blanden, 1976; Dunlop, Doherty, Zinkernagel & Blanden, 1976; Dunlop & Blanden, 1977). In essence, in vitro primary responses were generated by culturing mixtures of unprimed, syngeneic spleen cells and systemic (para-aortic and inguinal) lymph node cells (in the H

293

proportion of 7 parts spleen cells to 1 part lymph node cells), in ten-fold excess with syngeneic, LCM virus-infected peritoneal cells for 6 days. Secondary responses were generated in vitro by culturing spleen cells from LCM virus-memory mice in 10-fold excess with syngeneic, LCM virus-infected peritoneal cells for 5 days. Usually 2 5 x 107 'responder' cells and 2 5x 106 infected 'stimulator' cells were cultured in F-15 medium (Gibco, New York) supplemented with 10% heat-activated foetal calf serum, antibiotics and 1-0 M 2-mercaptoethanol in small (surface area 25 cm2) Falcon plastic flasks; or three times this number of responder and stimulator cells were cultured in 30-40 ml medium in large (75 cm2) Falcon flasks. The references mentioned above also give details of formaldehyde fixation of infected peritoneal 'stimulator' cells, anti-l treatment and the 51Crrelease assay. Secondary effectors were generated with the use of unfixed infected peritoneal 'stimulator' cells in all tables and figures, except where indicated in Tables 1, 6 and 7 where fixed, infected stimulator cells were used. Results were expressed as percent specific lysis= (percentage 51Cr release in presence of effectors - percentage medium release) x 100/percentage water lysis. Data presented are the mean of 4 replicates. Standard errors of the mean were generally never greater than ±2 % and were omitted for clarity. Statistical significance was determined using Student's t-test. Induction of ectromelia virus-specific secondary

effectors and technique of ectromelia virus plaque assay are also described elsewhere (Gardner & Blanden, 1976; Blanden, 1970). The ectromelia half of the virus specificity experiments was performed by Miss U. Kees.

RESULTS Virus content of effector populations under test Intraperitoneal or subcutaneous immunization with LCM virus is known to sensitize normal recipient mice to a subsequent intracerebral virus challenge, and brain virus titres post-challenge may be lower if the interval between pre-immunization and challenge is 5 days or greater (Rowe, 1954; Hotchin, 1962; Nathanson, Monjan, Panitch, Johnson, Petursson & Cole, 1975). Therefore it seemed possible that in assays designed to show optimal

M. B. C. Dunlop

294

Table 1. Virus content of different effector populations

6-0r-

5-0k

Log1o virus titre Effector population*

Primary in vivo Primary in vitro Conventional secondary in vitro Secondary in vitro, very old memory responders, fixed infected stimulators

(PFU/106 cells) 3-37 3-73 0-57 (no plaques)

Primary in vivo effectors were spleen cells from mice 8 days after virus challenge; primary in vitro effectors were a mixture of 7 parts spleea cells; 1 part lymph node cells from unprimed mice cultured in ten-fold excess with syngeneic infected peritoneal cells for 6 days; conventional secondary in vitro effectors were spleen cells from mice approximately 7 weeks postinfection cultured with infected peritoneal cells for 5 days. Secondary in vitro effectors using very old memory responders were spleen cells from mice approximately 1 year post-infection cultured with formaldehyde-fixed, infected peritoneal cells for 5 days. For further details on preparation of different effector populations, see references cited in Materials and Methods. CBA/H mice and WE3 (viscerotrophic) strain of LCM virus were used in this experiment. *

protection where cells were given first, carriage of large amounts of virus by cells from cultures might modify subsequent virus titres in tissues after virus challenge. Accordingly, various CBA/H effector populations-in vivo primary (spleen cells, at the time of peak cytotoxic T cell activity, 8 days postchallenge), in vitro primary and in vitro secondary, were assayed for virus content by plaquing (Table 1). Secondary effectors which were prepared in the conventional manner using unfixed, infected peritoneal 'stimulator' cells contained approximately 3 logs virus/106 cells less than primary effectors which were generated in vivo or in vitro. Since a protective dose of 2 0-x 106 secondary effectors (Fig. 1) would have transferred less than 0 3 % of the virus challenge dose of 2500 PFU given 24 h later, carriage of virus or virus-infected cells by secondary effector populations under these circumstances was probably not significantly affecting final virus titres in recipient organs. Moreover, meaningful comparisons of protective activity could not be made with primary effectors when cells were transferred before virus administration because of the much greater virus content of primary effectors, relative to the challenge dose of

0

c 40

.o

a

~~z~ I~~~~~

2 30 Q.

0 -J

2-0

1-0 i

5-6

I

59

.

6-2 6-5 6-8 Log,, cells transferred

7-1

74

Figure 1. Plot of log1o protection vs log1o cells transferred when CBA/H secondary effectors were given on day -1, virus challenge (2500 PFU intravenously) was on day 0, and spleens were harvested on day +2. Spleen titre with transfer of 1.0 x 108 normal spleen cells was 5 9 ±0-3. Since a titre of 1-3 is the limit of detection of the assay, the maximal protection that can be shown is 4-6 log10 units. Vertical bars enclose one standard deviation.

virus. No plaques were detectable on titrating secondary effector cells generated using fixed, infected peritoneal stimulator cells and responder spleen cells from LCM virus-memory mice primed more than 1 year previously. Secondary effector cells generated using fixed, infected stimulator cells and very old memory responder cells were used in some experiments to define virus specificity in Tables 6 and 7. Time-course of protection by secondary effectors when given 24 h before virus challenge 2 5x 107 CBA/H secondary effectors generated in vitro or spleen cells from unprimed mice were transferred intravenously to groups of 4 CBA/H recipients. Recipients were challenged 24 h later with either 2500 PFU intravenously or 400 PFU intracerebrally. Spleens or brains respectively were individually assayed for virus 1, 2 or 3 days post-challenge (Table 2). Titrations of spleens for the upper half of Table 2 were performed by intracerebral inoculation of outbred mice before the LCM virus-plaquing assay was developed, and titres, expressed as loglo LD5, would be expected to be about 1 log higher than titres using the plaquing assay. Titrations of brains for the lower half of Table 2 and titrations

Secondary effector cells protect against LCM

295

Table 2. Time-course of protective activity of secondary effectors in spleens and brains following challenge with virus 24 h after cell transfer*

Organ assayed

Day of assay post virus challenge

Log10 virus titret Normal cells

Spleen

1 2 3

5 8 ±0-2 6-8 ±0-1 6-5 ±0-2

Secondary effectors

Log1o protection:

(LD50) 4 5

(PFU)

Brain

1 2 3

1-5±0-81

1-4±0-7 3-7 ±0-1

4 0 ±0-1¶

4-7±03

50±01M

0 0 0

* Groups of four CBA/H mice were transfused with 2-5 x 107 CBA/H cells I day before challenge with LCM virus. Mice were challenged with 2500 PFU intravenously and spleens were subsequently assayed; or challenge was 400 PFU intracerebrally and brains were assayed. Organs were individually titrated for virus. t Spleens were titrated intracerebrally in outbred WEHI mice and results were expressed as log1o LD5o. Brains were titrated on L929 cells and results were expressed as log1o PFU. Results are the mean ± s.e.m. for groups of four mice. All titrations in subsequent tables and figures were performed on L929 cells and were expressed as log1o PFU. $Log to protection = (Logio virus titre post-transfer of normal cells) - (log1o virus titre post-transfer of secondary effectors). § Significantly lower (P < < 0-001) than virus titre following transfer of normal cells (limit of titration was log1o 2-0). ¶I Not significantly different from virus titre following transfer of normal cells.

in all subsequent tables and figures were performed on L929 cells. There was a marked reduction in spleen virus titres when secondary effectors were transferred, compared with normal spleen cells. In contrast, secondary effectors did not reduce brain virus titres by 3 days after intracerebral inoculation. The optimal interval chosen to assay spleens, was 2 days after virus challenge. Dose-response of protection

CBA/H recipients were given different doses of syngeneic secondary effectors intravenously, were challenged 24 h later with 2500 PFU LCM virus and spleens were titrated a further 2 days later (Fig. 1). Secondary effectors gave a linear relationship between log cell dose transferred and log protection, and protection was marked, e.g. 3n1 x 106 secondary effectors caused approximately a 3 logo reduction in spleen virus titres. In the same assay primary effectors generated in vivo also protected well, 1 25 x 107 cells conferring 5 log1 protection; normal spleen cells or unstimulated spleen cells from LCM virus memory mice did not protect in doses up to 1 0 x 108 cells.

A second, similar experiment including CBA/H primary effectors generated in vitro was performed; these cells reduced spleen levels only slightly for the doses tested, that is, up to 5-0 x 107 cells. A third, similar experiment showed that 2 5 x 107 C57BL/6 primary effectors generated in vitro did not reduce spleen virus titres in C57BL/6 recipients. The simplest explanation for this apparent lack of protective activity was transfer of large doses of infectious virus with the cells (Table 1).

Anti-I susceptibility Because secondary effectors generated in vitro appeared to be less susceptible to lysis by anti-@ ascitic fluid plus complement than primary effectors generated in vivo (Dunlop et al., 1976), CBA/H secondary effectors were twice treated with anti-@ ascitic fluid plus complement, normal AKR ascitic fluid plus complement, or complement only, before transfer in doses of 2-0 x 106 cells to syngeneic recipients. Recipients were challenged 1 day later and spleens titrated after a further 2 days (Table 3). Treatment of effectors with anti-@ ascitic fluid

M. B. C. Dunlop

296

Table 3. Anti-@ susceptibility of CBA/H secondary effectors mediating protection following challenge with virus 24 h after cell transfer*

Treatment plus complementt

Log10 spleen virus titre (PFU)

Logi0 protection

4-7 ± 0-4t

0.1

2-4+±01 2-6 ±0h2

2-4 2-2

Anti-@ ascitic fluid Normal AKR ascitic fluid Nil (In vitro normal, untreated spleen control)

4-8 ±0-1

* 2-0 x 106 CBA/H secondary effectors transferred to syngeneic recipients on day -1; virus challenge was 2500 PFU on day 0; spleens were titrated on day + 2.

t Treated populations were given double treatments with sera plus complement. t Significantly higher (P < 0 001) than virus titre following transfer of other treated effectors.

plus complement abolished protective activity indicating that protecting cells were T cells.

infected peritoneal stimulator cells for 5 days, then transferred in doses of 2-0 106 cells to groups of 4 CBA/H, BALB/c and SJL/J recipients 1 day before routine

were co-cultivated with syngeneic, x

Protection involves genes mapping in K or D regions

virus challenge. In the

of the H-2 complex A.TL or CBA/H memory responder spleen cells

effectors

were

assayed

same

on

experiment, secondary

LCM virus-infected

uninfected CBA/H, BALB/c and SJL/J macrophage

Table 4. Genetic mapping of H-2 restriction of protective activity of secondary effectors following challenge with virus 24 h after cell transfer*

Secondary effectors transferred

Log1o spleen Recipients

virus titre (PFU) 55

A.TL

(skkkkd) CBA/H

CBA/H

±0-2t

1-9 ±0-2t

Log10 protection 0 3-3

(kkkkkk) Nil

5 2 ±0-2

A.TL CBA/H

3-4 ±0lit 5 2 ±O+3T

BALB/c (dddddd)

A.TL

CBA/H Nil

1.9

0-1

5.3 ±0.3

Nil

SJL/J (ssssss)

or

3-5 ±O-lt 4-6 ± 0-2t

1.5 04

5 0 ±0-1

* 2-0 x 106 A.TL or CBA/H secondary effectors were transferred to recipients on day -1; virus challenge was 2500 PFU on day 0; spleens were titrated on day + 2. H-2 maps are for K, IA, IB, IC, S and D regions of the gene complex. t Significantly less (P < 0-001) than control. $ Not significantly different from control.

297

Secondary effector cells protect against LCM Table 5. Genetic mapping of H-2 restriction of cytotoxic activity of secondary effectors*

Macrophage targets (difference)t Responder spleen cells

E: T ratio

A.TL (skkkkd) CBA/H (kkkkkk)

1 :1 0-5: 1 1 :1 0-5: 1

SJL/J

(kkkkkk)

BALB/c (dddddd)

1-4 0

22-4:

34-41 22-3t

69.5$ 53-9$

2-8 3-8

0 0-4

CBA/H

51-7:

(ssssss)

* A.TL or CBA/H virus-memory spleen cells were co-cultivated in 10-fold excess with LCM virus-infected, syngeneic peritoneal cells for 5 days, then assayed. t Difference = ( % specific lysis on infected targets) - ( % specific lysis on uninfected targets). $ Significantly higher (P 40 001) than lysis in those effector: target combinations where K or D regions are not shared.

targets (Tables 4 and 5). Protection in vivo and efficient lysis of infected macrophage targets occurred only where donors of infected peritoneal stimulator cells and respectively recipients or targets shared either H-2K or H-2D. In particular, no protection was conferred when the whole I region was shared (A.TL-CBA/H combinations). These results, in conjunction with the anti-O treatment experiment, suggest that the cells protecting in vivo also lyse virus-infected targets in vitro.

Specificity of protection In the first experiment, spleen cells from ectromelia virus-memory mice were cultured with ectromelia virus-infected, syngeneic peritoneal cells, and generated secondary effector cells which protected syngeneic recipients from challenge one day later with ectromelia but not LCM virus (spleens were assayed 2 days after virus challenge-experiment 1, Table 6). However, secondary effector cells derived from in vitro stimulation of spleen cells from WE3-LCM virus-memory mice with WE3-LCM virus-infected syngeneic stimulator cells caused a significant reduction in spleen virus titres on transfer at a dose of 2 0x 106 cells into syngeneic recipients challenged with either ectromelia or LCM virus (experiment 1, Table 6). A possible mechanism for this unilateral cross-protection would be that the infectious LCM virus transferred with effectors resulted in infection of many cells of the recipients' reticulo-endothelial system. These phagocytic cells could be dually infected after ectromelia challenge, be recognized because they expressed LCM virus-

specific surface antigenic patterns, and be lysed by the LCM secondary effectors, thus indirectly limiting spread of ectromelia infection. To obviate transfer of infectious virus, the experiment was repeated essentially unchanged except for the use of fixed, LCM virus-infected stimulator cells together with responder spleen cells from very old (6-12 months postpriming) WE3-LCM virus-memory mice (experiments 2 and 3, Table 6), or responder spleen cells from ARM-LCM virus-memory mice (experiment 4, Table 6). In these latter three experiments, unidirectional cross-protection was less marked compared with the reduction in ectromelia virus titres in recipients given secondary effectors from ectromelia virus memory cultures. Cell suspensions from LCM virus-memory cultures from experiments 2, 3 and 4 of Table 6 were disrupted by sonication and titrated for free LCM virus by plaquing in vitro and by intracerebral inoculation into C57BL/6 mice: no plaques were found and challenged recipients did not develop viral meningitis, confirming absence of free infectious virus. Effector cell populations from experiments 2, 3, and 4 of Table 6 were tested for control specificity in vitro at the effector: target level by assay on LCM or ectromelia virus-infected or uninfected H-2 compatible L929 targets; only when stimulator cells and target cells were infected with the same virus, did significant specific lysis occur, indicating clear specificity in vitro. (Representative results using effector cells from experiment 2 of Table 6 are given in Table 7.) It thus appeared that secondary effectors from LCM virus-memory cultures gave less protection to ectromelia virus-challenged recipients when pro-

M. B. C. Dunlop

298

Table 6. Capacity of CBA/H LCM or ectromelia virus-reactive secondary effectors to protect recipients following challenge with LCM or ectromelia viruses 24 h after cell transfer* LCM virus challenge Expt. no.

Cells transferred in vivo or assayed in vitrott §

Cell dose transferred

Ectromelia virus challenge

Logi0 spleen

Log10 spleen virus titre

Loglo

(PFU)

protection

virus titre (PFU)

protection

1 6

4 ±0-6¶

25

Log10

LCM virus 1

secondaryt

2-0 x 106

5

Ectromelia virussecondary Nil

2-0 x 106

6-2 ±0It: 65±01

03

5 0 ±O1¶T 6-5±03

1 5

secondaryt

2-0 x 106

4-5 ±0 5¶

1-6

4-3 ±0K3T

2-2

Ectromelia virussecondary

20x106

6-10-1tt

01

15±Ol¶T

50

2-0 x 106 10 x 106 0-5 x 106 4-0 x 106 2-0 x 106 1-0x106

6-2 +03 5-9 ±0-2¶ 6-1 ±0-3O 6-6 +03** 7-8 ±0-2$$ 7-6 ±0-1$$ 7-7 0±-1$ 7-6 ±0-1

1-7 1-5 10 0 0 0

6-5 ±0-2 6-1 ±0 3tt 6-3 ±0-2** 6-6+0 1$$ 3-8 ±061¶ 5.9 ±0-2** 6.5±0-1** 6-9 ±01

09 07 04 3-2 1-0 0-4

2-0 x 106 1.0x106 2-0 x 106 1.0 x 106

3-8 ±0-31¶ 5.2+0-2** 6-0 ±0l-tt 6-2 ±0lftt 6-3 ±0 1

25 11 03 0-1

±0-2¶

LCM virus 2

Nil

LCM virus

secondaryt 3

Ectromelia virus secondary Nil

4

LCM virus secondary§ Ectromelia virus secondary Nil

6-7 ±0

1lt

6-6±01$$ 5.2 ±0.3** 6-2 ±0-1**

0-2 03 17 07

6-9 ±0 1

* CBA/H LCM or ectromelia virus-reactive secondary effectors were transferred at various doses into syngeneic recipients on day -1; virus challenge was 2500 PFU WE3 LCM virus or 2 5 x 106 PFU Moscow strain (ectromelia) virus on day 0; spleens were titrated on day +2. Secondary effectors showed clear bidirectional specificity of lysis of H-2 compatible, LCM or ectromelia virus-infected L929 cells in vitro in the three assays performed (experiments 2, 3, 4-see Table 7). t LCM virus secondary effectors were generated using memory spleen cells from WE3 LCM virus-primed mice and unfixed, infected peritoneal 'stimulator' cells. t LCM virus secondary effectors were generated using very old (over 6-12 months) memory spleen cells from WE3 LCM virus-primed mice and fixed, infected peritoneal stimulator cells in experiments 2 and 3. § LCM virus secondary effectors were generated using memory spleen cells from ARM LCM virus-primed mice and fixed, infected peritoneal stimulator cells in experiment 4. If Significantly lower (P