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The cell-mediated immune response to ectromelia virus infection
CELLULAR
IMMlJNOLOGY
22,
The Cell-Mediated 1. Kinetics
(1976)
271-282
Immune
and Characteristics
Response to Ectromelia of the Primary
R. V. BLANDEN Department
Effector
Virus
T Cell Response in Vivo
AND I. D. GARDNER
of Microbiology, The John Curt&z School of Medical Australian National University, Canberra, 2601. A.C.T. Received
Infection
November
Research,
lo,1975
The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 X 1@ up to 5 X 10’ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a “Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4-22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5-6 days postinfection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-8 and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days l-4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.
INTRODUCTION Ectromelia virus is a poxvirus similar to vaccinia and variola viruses (l), which produces a disease, mousepox, with many features resembling human smallpox (1). Recovery from primary infection depends upon cell-mediated immunity (CMI) in which the virus-specific effector cells are thymus-derived lymphocytes (T cells) (2-5). Antibodies appear too late and in insufficient amounts to play a significant role in recovery, (2, 3) but appear to be an important component in resistance to re-infection (6). The role of CM1 in secondary infection (T cell memory) has not been elucidated and will be the subject of future studies (7). Previous evidence suggested that a number of different mechanisms could operate in concert to eliminate virus from foci of infection in the major, visceral target organs, the liver and spleen. These mechanisms depend upon localization of effector 271
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rights
o
1976 by Academic Press, reproduction in any form
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T cel1.sin foci, followed by the attraction of monocytes (4, 8) and perhaps their differentiation to activated macrophages (9). Macrophages ingest the virus, and even if infected, produce so little infectious progeny virus that the ultimate outcome is elimination of infection (4, 10). Activation of the macrophages and local interferon production (perhaps by T cells) may augment the efficiency of virus clearance (4, 11). Recent studies indicate that ectromelia-infected target cells can be lysed efficiently by specific cytotoxic T cells in vitro (12, 13). Rigorous criteria support the identification of the killer cells as T cells, i.e. they are sensitive to anti-8 and complement and do not bear surface immunoglobulin (5, 13). The cytotoxic process does not require exogenous complement, macrophages, or secretion of soluble factors, proceeds in a linear manner with time, and appears to require one hit (12, 13). Target cells acquire antigenic changes within a few hr after infection in the absence of viral DNA synthesis (14) and are thus vulnerable to lysis before the assembly of progeny virus particles. Therefore, T cell-mediated cytotoxicity could well contribute to retardation of virus growth rate and spread within target organs in tivo. One aspect of T cell recognition of virus-infected cells both in vitro (15, 16) and in tivo (5, 16) is that host cell genes, which map in either the K or D regions of the H-Z gene complex (17) appear to dictate an essential component of the antigenic moiety recognized (18, 19). The advent of a well-defined in vitro assay for T cell-mediated cytotoxicity (12, 13) has provided a second test for effector T cell function, in addition to cell transfers to infected recipient mice (3)) with obvious logistic and quantitative advantages. Both of these tests have been used to examine questions about the nature and heterogeneity of T cells which perform various functions in the response to ectromeIia infection. In this paper we examine in detail the response of individual mice to different doses of virus, the kinetics of virus growth in spleen in relation to the kinetics of production of effector T cells, and the nature of other T cell subsets in spleen which are apparently unrelated to the specific response to infection. MATERIALS
AND
METHODS
General. CBA/H and C57BL/6 mice, stocks of attenuated (Hampslead egg) and virulent (Moscow) strains of ectromelia virus (Z), the bacterium Listeria nzonocytogenes (3), methods of titration of infectious agents (2, 3), immunization of donor mice (5), preparation of spleen cell suspensions (5)) culture of target cells (12-15) and anti-8 and complement treatment (13) have all been described previously. Antiviral activity of i++zYtmune spleen cells. The techniques of cell transfer and the titration of virus in recipient tissues have been described in detail elsewhere (3, 5). Briefly, donor mice were immunized intravenously (iv) with 5 X lo4 plaque-forming units (PFU) of attenuated virus at varying times before harvest of immune spleen cells. Recipient mice were inoculated iv with 4 X lo4 PFU of virulent virus 24 hr before cell transfer and spleens removed for virus titration 24 hr after cell transfer. Cytotoxic assay. The 51Cr release method used for H-l sarcoma cells, mouse embryo cells and macrophages was basically as described in detail previously (12), utilizing 7 mm assay wells (15). Macrophages were seeded at lo5 cells per well
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and other target cells at 2 x 104. The procedure used for L929, P-815 and EL-4 cells was modified slightly as follows. Cells were first labelled with Yr in suspension. Infection with ectromelia virus was also performed in cell suspensions at a density of S-10 X 10Gcells per ml with 4-10 PFU per cell. Target cells were then dispensed into 7 mm wells (2 ~10” per well) in a volume of 100 ~1 with an automatic pipette. Tests showed variation in total counts per well to be no more than 1% either side of the mean. Killer cells were also dispensed in 100 ~1, giving all wells identical 200 ~1 volumes. After 16 hr of killer-target interaction at 37”C, the whole culture tray was gently centrifuged to deposit any cells in suspension, and 100 ~1 of supernatant from each well was removed and counted. The total counts per well were obtained from 100 ~1 aliquots of target cells to which had been added 0.9 ml of distilled water. These tubes were counted, then centrifuged and the supernatant and pellet counted to calculate % 51Cr releasable by water, (usually 85-90s of the total). Lysis in test wells (done in quadruplicate) was expressed as a percentage of the lysis achieved by water, and was derived from the formula : Counts in 100 jJ supernatant from test wells X 100 Mean counts in 100 ~1 supernatant in water lysis tubes RESULTS variation. Large doses of lymphocytic choriomeningitis (LCM) virus, which is not cytopathic, have a suppressive effect upon the production of virus-specific cytotoxic T cells (20). This effect was not seen with ectromelia virus, possibily because the dose level required to achieve it would be lethal, even with an attenuated strain of virus. With CBA/H mice, doses much above 5 x lo4 PFU given iv produce extensive necrosis in spleen and liver, and death in occasional animals. Thus groups of four mice 8 weeks old were immunized iv with different doses of virus (5 X 104, 104, 2 X lo3 and 4 X 10” PFU/mouse). Individual spleens were harvested 6 days later, known to be at about the time of peak effector T cell response (3, 12). Even at a virus dose of 4 X 10’ PFU, spleen cells from each of the four mice gave substantial cytotoxicity (Table 1). The two highest doses, 5 X lo4 and lo4 PFU, caused very consistent responses in individual mice, although the lower dose gave less cytotoxicity. The four mean percentages of 5*Cr release from individual mice within each of these two groups were averaged to give a mean value * SEM. In both cases, this SEM was similar to the SEMs from quadruplicate assay wells of the individual mice, showing that within this dose range, variations between individual mice were negligible (Table 1). In all subsequent experiments, virus doses of between 2 x lo4 and 5 X 10” PFU and spleen cells pooled from four mice were used, although the results suggested that within this dose range, one mouse would be representative of the group. Age dependence. Groups of four CBA/H mice of varying ages from 4 weeks to 22 weeks were immunized iv with 5 x lo4 PFU attenuated ectromelia virus per mouse, and their spleen cells assayed for cytotoxicity 6 days later. The results (not presented here) showed that mice aged from 4 to 12 weeks exhibited similar levels of cytotoxicity against infected target cells. Mice at 22 weeks gave a slightly, though significantly, lower level. Mice were routinely used at 7-8 weeks of age. Ejject
of virus
dose
and
individual
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TABLE Effects
Virus dose
of Virus Dose and Individual Against Virus-Infected CBA/H mouse
Target Ectromelia-infected
5 x 10’
104
2 x 103
4 x 102
Spontaneous
1
Variation on the Cytotoxic T Cell Response H-Z-Compatible Target Cells
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 release
cells
L929
Uninfected
80.2 76.0 75.6 78.0 62.7 63.2 61.1
f 1 1” f 0.8 f 1.4 f 1.1 f 0.6 f 1.7 f 1.0
62.1 50.1 36.2 56.9
f f f f
0.5 0.3 0.8 1.4
65.6 f 64.9 f 56.4 f
1.3 1.7 1.4
57.2 f 58.4 f
1.3 1.8
16.3 f 16.5 f
0.3 0.4
14.0 f
0.1
20.5 i
0.3
77.5 f
1.1*
62.3 f
0.5
19.0 21.1 18.2 14.6 13.2 13.0 13.7
6.2
16.4 16.1 17.1 14.4
1.9
16.5 i 0.1 17.0 f 0.3 15.5 f 0.5
52.2 f
59.2 f
i f * f f f f f f f f
0.6’ 0.5 0.2 0.4 0.3 0.3 0.4 0.5 0.3 0.3 0.2
L929
18.2 f
1.4*
14.1 f 0.8
16.0 f
0.6
16.3 f
0.3
0 Mean y0 Wr released iSEM from groups of four wells, assayed at a spleen cell: target cell ratio of 100: 1. Immune spleen cells obtained 6 days after immunization iv. * Mean fSEM, of the means of the four individuals receiving the same virus dose iv.
Growth of virus in the spleen. Over 99% of an intravenous inoculum of Moscow strain ectromelia virus is cleared from the blood within 5-7 min; most of this virus goes into the liver, with l-5% entering the spleen(4). Five groups of four CBA/H mice were infected iv with 2 x lo4 PFU/mouse of attenuated ectromelia virus. Thus < lo3 PFU would be expected to localize in the spleen. On each of the next 5 days, a group of mice was sacrificed and their spleens removed for virus titration. Figure 1 shows the behaviour of the virus in spleen. Logarithmic multiplication occurred for 2 days, then titers declined slightly on day 3 and dropped sharply on day 4. No virus was detectable on day 5 (less than 2 X lo2 PFU) . Kinetics of appearance of cytotoxic T cells in the spleen. Five groups of four CBA/H mice were immunized iv at 2 day intervals with a single injection of 5 x lo4 PFU of attenuated ectromelia virus per mouse. Two days after the last group was immunized the spleen cells of all groups were assayed against ectromeliainfected and uninfected L929 target cells (Fig. 1). CBA/H mice and L929 cells share H-2 haplotypes ( H-2k). Cytotoxicity against infected L929 cells increased to a peak at day 6 and had declined to a low level by day 10. (Fig. 1). The activity of immune spleen cells against uninfected L929 cells showed quite different kinetics. It was prominent on days 2 and 4, but declined almost to background by day 8. In a similar experiment, uninfected P-815 (H-2d) target cells were also used (Fig. 2). Activity of immune spleen cells against them was maximal on day 4. Thus, use of three
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6
I
2
3
4
5
6
7
8
9
10
DAYS AFTER INFECTION
FIG. 1. Growth of ectromelia virus in spleen (--¤-) after infection iv with 5 X 10’ PFU of attenuated virus, expressed as mean loglo PFU per spleen in groups of four CBA/H mice per point. Cytotoxicity generated in spleen cells was measured with virus-infected (---A-) or uninfected (- - n - -) L929 target cells at a killer target ratio of 100 :l. Each point is the mean of quadruplicates. Background “Cr release from infected ( l ) or uninfected (0) targets in the presence of normal CBA/H spleen cells at 100: 1 is shoddy by the single points below the curves. All vertical bars enclosed 2 SDS.
different target cell types revealed cytotoxic activity with three different kinetic patterns appearing in spleen as a consequence of infection. Rigorous criteria have been met to establish that the spleen cells responsible for killing infected H-Zcompatible target cells in this system are T cells (5. 13), that they are virusspecific (13, ZO), and that H-2K or H-2D homology is required between donors of T cells and infected target cells (17). Detailed studies of the kinetics of their appearance in spleen are described later in this paper. However, the possibility that
DAYS
AFTER INFECTION
FIG. 2. Cytotoxicity of spleen cells harvested from CBA/H mice at various times after iv infection with 2 X 10’ PFU of attenuated ectromelia virus against infected L929 targets at 50: 1 (-A--) or 1O:l (--A-) or against uninfected p-815 targets at 50: 1 (- - fJ - -). “Cr release in the presence of normal CBA/H spleen cells at each ratio has been subtracted. Each point represents the mean of four wells using pools of spleen cells from four mice, vertical bars enclose 2 SDS.
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TABLE Cytotoxicity”
2
of Early Ectromelia-Immune Spleen Cells from CBA/H H-2-Compatible (L929) or Syngeneic (H-l, Macrophage)
Expt.
1
2
Target
cells
Days after immunization
L929 H-l L929 CBA/H Macrophages
a Mean ye 6rCr released from cell ratios were 100: 1 for L929 presence of normal spleen cells highly significant (P < O.OOl), (P < 0.05). b N.D.. not determined.
Mice against Target Cells
Uninfected
iv
1
2
3
4
20.20 0
20.6 0.4
18.3 1.4
21.8 0
25.8
32.2
N.D.b
N.D.
0.8
3.7
N.D.
N.D.
three wells. SEMs were never larger than 3.4. Spleen cell: target and H-l cells and 30: 1 for macrophages. Background release (in at the same ratio) was subtracted. Release from L929 cells was release from H-l or macrophage target cells was not significant
activity of CBA/H immune spleen cells against H-Z-compatible, uninfected L929 cells reflected a transient break in self tolerance warranted further analysis. Therefore, CBA/H spleen cells harvested 1, 2, 3 and 4 days after immunization were tested against uninfected L929 cells, derived originally from C3H mice (21), and syngeneic target cells of two types (a) CBA/H peritoneal macrophages and (b) H-l sarcoma cells, from a methylcholanthrene-induced tumor of CBA/H mice (22). While spleen cells from days 1 through 4 killed uninfected L929 cells, they had no activity against the syngeneic cells (Table 2). Since both peritoneal macrophages and H-l sarcoma cells are readily lysed by T cells sensitized to H-2k (unpublished results), this data suggested that the cells which kill uninfected L TABLE
3
Effect of Anti-8 and Complement Treatment on Ability of Ectromelia-immune Spleen Cells from C57BL/6 Mice to Lyse” Uninfected Allogeneic Syngeneic (EL-4, MEC) Target Cells Immunizing infection
Ectromelia
Listeria
Treatment of spleen cells
Nil Anti-0 + complement Normal ascitic fluid + complement Nil Anti-B + complement Normal asciticfluid + complement
or L&&-immune (L929) or
Target
cells
L929
EL-4
MEC
44.3a 5.6b 39.7
15.2 Ob 14.5
46.8 22.5b 43.8
28.6 4.0b 31.2
25.8 10.P 29.3
35.1 12.lb 32.3
0 Mean $$ SrCr released from four wells. SEMs were never larger than 4.5. Spleens were harvested 4 days after immunization. Spleen cell: target cell ratios were 1OO:l. Spontaneous release subtracted. B Significantly less release than controls (P < 0.001).
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4
Competitive Inhibition of Cytotoxicity Exerted by 4-Day Ectromelia-immune CBA/H Spleen Cells against Uninfected H-2 Compatible (L929) or Allogeneic (P-815) Target Cells Unlabelled
competitors
@?051Crrelease from
Nil P-815 L929
(9:l)b (9:l)
L929
P-815
23.2a 16.1 0.6c
26.9 ll.lC 14.5
a Mean “ye Kr released from four wells. SEMs were never larger than 0.9. Spleen cells: target cell ratios were 100: 1. Control values (Wr release in wells containing normal spleen cells at the same ratio) are subtracted. b Numbers in parenthesis are ratios of unlabelled competitors to labelled target cells. c Significantly greater competitive effect than with the heterologous competitor (P < 0.001 for L929 labelled targets and P < 0.05 for P-815 labelled targets).
cells are not specific for H-2”, i.e., that self-tolerance to H-2 antigens is not broken. This phenomenon was investigated more widely. It was found that infection with a bacterium, Listeria wzonocytogenes, also induced apparent “anti-self” cytotoxicity. Various mouse strains were infected with ectromelia virus or Listeuia. Syngeneic target cells, either tumor cell lines, or embryo cell lines were killed by l-6 day immune spleen cells, e.g. C57BL/6 spleen cells killed C57BL/6 embryo cells (MEC) or EL4 lymphoma cells, and DBA/2 spleen cells killed P-815 mastocytoma cells (some representative data given in Table 3). Use of anti-0 serum and complement suggested that the cytotoxic process was largely, if not totally, T cell-dependent (Table 3). Killing appeared to depend, at least in part, upon specific recognition by cytotoxic cells bearing receptors for antigen of uniform specificity or a limited range of specificity, since it could be
A2
50
54 LOG,,
58
62
KILLERS
FIG. 3. The family of straight lines with 45” slope o%ained by plotting log, ectromeliainfected L929 target cells lysed against log,” immune spleen cells required to lyse them. The pools of immune spleen cells were harvested from groups of four CBA/H mice infected iv with 2 x lo4 PFU of attenuated ectromelia virus 3 (*), 4 (a), 5 (0), or 6 (0) days previously.
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TABLE Effector Days after infection
3 4 5 6
GARDNER
5
Unit Parameters’
Abscissaa intercept
Elector units6 per lo6 cells (103)
2.3 1.55 1.25 1.65
5 28 56 22
Lymphoid cells per spleen (X 108) 1.7 1.1 1.4 3.5
Effector units per spleen (X 105) 8.5 31 78 77
o Calculated from the data in Fig. 3. Experimental details in text. b Effector units bear a direct relationship to the actual number of cytotoxic T cells in the cell population. Since each T cell can kill more than one target cell, the actual number of T cells could theoretically be obtained by dividing the number of effector units by the mean number of target cells killed by each T cell. However, this number is not known in the present system.
partly inhibited by unlabelled competing target cells, and inhibition was more effective if they were the same type as the labelled targets (Table 4). This applied to both H-2 compatible and incompatible target cells, (Table 4) but the data were not clearcut in many experiments of this type and no firm conclusions can be drawn about the specificity of recognition involved in this phenomenon. These data suggested that subsets of cytotoxic cells (probably T cells) at least moderately specific for alloantigens, or non-H-2 antigens borne by some syngeneic, uninfected cells were briefly activated to a limited extent as a consequence of infection. Their specificity and kinetics, however, bore no obvious relationship to the major cytotoxic T cell response against ectromelia-induced antigenic changes on H-2-compatible infected cells. Quantitative kinetics of the generation of T cells specific for ectronzelia-induced antigenic changes. Groups of CBA/H mice were injected iv at daily intervals with 2 X lo4 PFU of attenuated ectromelia virus, and their spleen cells assayed for cytotoxic activity 3, 4, 5 and 6 days after immunization on infected L929 targets. Each immune cell pool was assayed at ratios of 40: 1, 20 : 1, 10 : 1 and 5 : 1, and the results plotted as loglo target cells lysed versus loglo number of splenic lymphocytes required to lyse them (Fig. 3). T cell killing systems give a straight line graph with a slope of 45” when specific 51Cr release is between 10% and 60% (K. J. Lafferty, personal communication) indicating a one-hit process (23). The day 3 population (Fig. 3) had three points which fell within these limits, and the line of best fit for these points had a 45” slope. The day 4 and day 6 data also had three points whose lines of best fit were 45” slopes. The line for the peak response (clay 5) was assumed also to have a 45” slope, since a 45” line could be drawn through the two points falling within the lo%--60% limits. Extrapolation of this family of lines to the abscissa gave the logarithms of the numbers of cells required to kill one target cell for each immune spleen cell population. The reciprocals of the antilogarithms of these abscissa intercept values represent the concentration of “effector units” in the spleen population (Table 5). Although the concentration of effector units was much higher on day 5 than day 6, the total number of effector units per spleen was no different. Thus the increase in mean cell numbers per spleen which occurred from days 5 to 6 was due to an influx or mu!tiplication of cells other than cytotoxic T cells.
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FIG. 4. Capacity of pools of immune spleen cells, from groups of four CBA/H mice infected iv with 2 X 10’ PFU of attenuated ectromelia virus at 2 day intervals, to lyse infected L929 target cells (-A-) at a ratio of 100: 1, or to reduce viral titers when transferred into preinfected CBA/H recipients (- - n - -) at 5 X 10’ cells per mouse. =Cr release in the presence of normal CBA/H spleen cells was subtracted and log protection values are the log,, reduction in spleen titer below the level in recipients of normal CBA/H spleen cells. Points represent the mean of four assay wells or four individual recipient mice; vertical bars, enclose 2 SDS.
Correlation of in vitro cytotoxicity with in viva effector activity. Five groups of five CBA/H mice were injected intravenously with 5 x lo4 PFU of attenuated ectromelia virus per mouse at 2 day intervals. Two days after the last group was injected, the spleens of all groups plus a group of six uninfected control mice were removed and the cells assayed for cytotoxicity against ectromelia-infected L929 cells, and for their ability to cause reductions in virus titers in spleens of preinfected mice (Fig. 4). The two effector activities showed similar kinetics, an observation consistent with, but not proof of, their being functions of the same T cel1 subset. DISCUSSION Since the CM1 response is crucial for recovery from primary ectromelia virus infections in mice (2-S)) we have examined the sequence of events in the virusinfected spleen in terms of effector T cell responses. This investigation has revealed much more complexity than that embodied in the conventional immunological view, based on Burnet’s “Clonal Selection Theory” (24), which would hold that the response should consist of the activation of T cell clones bearing receptors complementary to antigenic determinants specified by the viral genome. In fact, as discussed in detail elsewhere (5, 18, 19), there is thus far no clear evidence for the existence of these conventional T cells; the major T cell response seems to be directed against antigenic patterns dictated partly by the viral genome and partly by the H-2 gene complex of the host (5, 15, 16, lS, 19), i.e.,with CBA/H mice they are present on virus-infected H-2” target cells. Two other categories of T cells also appear to be activated by infection; these are discussed below. After iv injection of virus the chronological sequence is as follows: About l-5% of the inoculum localizes in the spleen within a few min, most of the remainder going to the liver (4). The immunofluorescence studies of Mims (25) and evidence reported in the following paper (26) suggest that virtually all classes of spleno-
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cytes are vulnerable to infection, including T cells which ultimately give rise to the immune response that triggers clearance of infection. Thus the full development of an immune response in the spleen depends on the dose of virus being such that the growing foci of infection do not become confluent before the first effector T cells begin to operate (25). By implication, this would happen between 2 and 3 days after infection, since virus titers in spleen increase for 2 days and then decline to undetectable levels by day 5. Effector T cell activity in immune spleen cells was greatest when the highest virus dose was used for immunization. This suggested that the number of infected cells, presumably the stimulus for cytotoxic T cell induction (26) was probably still a limiting factor up to the highest dose used (5 x lo4 PFU). Therefore, titers of virus in spleen on days 1 and 2 after infection, when specific immune induction is most likely to be taking place, may well be directly related to the number of effector T cells produced in the response. Effector T cells which kill virus-infected target cells in vitro and which trigger viral clearance in viva are readily detectable by day 4 and reach peak concentrations in the spleen cell population by days 5-6. These two different functional assays reveal very similar kinetics in the T cell response(s) measured. They could therefore reflect the activities of the same T cell subset, but absolute proof of this proposition is very difficult. Another similarity is that lysis of virus-infected cells in vitro and virus clearance in viva both require that immune T cell donors share a part of an H-2 haplotype with the infected target cells, or recipient mice, respectively. In both cases, the essential gene(s) map in either the K or D regions of the H-2 complex (17, Kees and Blanden, submitted). These findings are especially interesting in the light of results of Miller et al. (27)) that transfer of delayed hypersensitivity to fowl gamma globulin or dinitrofluorobenzene in mice requires I-A region homology between immune T cell donors and recipients. Experiments are in progress to investigate the presence and relative importance of such T cells during ectromelia infection. It is also noteworthy that the splenomegaly resulting from infection is not a direct reflection of virus-specific effector T cell production. In particular, the major increase in spleen size (three-fold) occurred from days 5 to 6 post-infection, but effector T cells did not increase in numbers in the spleen over this same interval. The increase in effector units from day 3 to day 4 was four-fold, suggesting that the cytotoxic cells may have undergone two divisions in this time. This conclusion may be invalid, however, as there would presumably be antigen available to induce new precursor T cells continuously over at least the first 3 days of infection, thus giving the impression of rapid cell cycles in the assumed proliferative response which followed. The increase in effector units from day 4 to day 5 was only about 2$-fold, perhaps reflecting waning numbers of precursors induced, a slower rate of division, or that significant numbers of cytotoxic T cells were leaving the spleen via the circulation. The lack of increase in total effector units from day 5 to day 6, suggested that egress of effector cells from the spleen kept pace with their production, and/or that some suppressive or regulatory effect is being exerted on division or activity or cytotoxic T cells. Therefore, these data suggest that studies of lymphoid cell proliferation during the immune response should be interpreted with caution, since the peak of the proliferative response may not necessarily reflect effector cell production.
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Furthermore, it seems that two categories of cytotoxic activity other than that specific for virus-induced antigenic changes in “self” cell surfaces are also present in spleen cell populations during the early response to infection. The first category is directed against alloantigenic target cells, e.g., cells from CBA/H (H-2”) mice lysed uninfected P-815 (H-2d) target cells. This activity reached a peak on day 4 post-infection. The second category was directed against H-2-compatible, uninfected target cells, e.g. CBA/H (H-2”) immune spleen cells killed uninfected L929 (H-ak) target cells. The kinetics of this activity were strange in that cytotoxic activity was present at similar levels through the first 4 days after infection and then gradually disappeared. Both these categories of cytotoxic activity appeared to be T cell-dependent since they were abolished or significantly depressed by anti-0 treatment of immune spleen cells. They seem unlikely to be dependent on K cells, which operate in herpesvirus infection (28, 29) for example, but are not anti-8 sensitive and would require a source of specific antibody (28, 30)) or on macrophages, since activated macrophages do not appear in the spleen until S-10 days post-infection (9). Therefore, they seem likely to be due to direct cytotoxic action of T cells. There are several possible explanations for these phenomena. First, T blast cells produced early in infection may bind to and kill any target cell for non-immunological reasons. Second, there may be a degree of immunological cross-reaction between, for example, infected CBA/H cells and uninfected L929 or P-815 cells. Third, there may be a multi-clonal T cell response, with the killing of each uninfected target cell type being due to specific clones or subsets of T cells with receptors reasonably specific for the particular target cell. The third interpretation above is favoured by several pieces of evidence. For example, the kinetics of the activities against uninfected L929 and P-815 are different, implying that they are functions of different T cell subsets. Furthermore, no cross-reaction can be shown at the cytotoxic T cell level between P-815 cells and infected L929 cells (unpublished results). Finally, reduction of the cytotoxicity against one type of uninfected target cells, with less impairment of the activity against another type, has been achieved by competition with excess numbers of uninfected, unlabelled target cells of the first type, thus indicating a degree of immunological specificity in the interaction between killer cell and target cell. A number of subsets of alloantigen-reactive precursors of cytotoxic T cells may, therefore, be activated during infection by either viral, bacterial or cellular products in a manner analogous to mitogen activation of T cells in z&o. (31, Cole, G. A., Blanden, R. V., and Dunlop, &I. B. C., in preparation). However, the appearance of cells reactive to H-2-compatible, uninfected target cells is more intriguing. It seems unlikely that virus present in the early spleen cell populations is sufficient to infect enough uninfected target cells to explain the phenomenon by invoking the action of the virus-specific cytotoxic T cells. In any case, lysis of deliberately infected target cells by the early immune spleen cells is less than that of uninfected targets, and there is no peak of activity corresponding to virus titers in the spleen. There was no detectable activity against strictly syngeneic target cells such as macrophages. Killing of syngeneic tumor cells or embryo cells occurred, but these cells could be expressing vertically transmitted viruses or embryonic antigens and may not be strictly “self” with respect to normal adult animals. For these reasons, it seems unlikely that there is a transient break in cytotosic T cell tolerance to normal “self” antigens.
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GARDNER
Even if this did occur, effector cells would need to be present at high levels in order for their effects against labelled syngeneic target cells to be seen in the presence of the large excess of autochthonous spleen cells which would act as “cold” competitors. Furthermore, such killer cells might be expected to kill each other, or even themselves during the act of endocytosis. Whether the cells responsible are reactive to minor alloantigens, embryonic antigens, or perhaps antigenic patterns induced by vertically transmitted viral genomes (3.2) remains to be determined. Whatever the outcome, the phenomenon could well be relevant to the problem of autoimmune disease. ACKNOWLEDGMENT We are very grateful to Narelle Bowern for expert technical assistance.
REFERENCES 1. 2. 3. 4. 5.
Fenner, F., J. Immunol. 63, 341, 1949. Blanden, R. V., J. I?.@. Med. 13’2, 1035, 1970. Blanden, R. V., J. Exp. Med. 133, 1074, 1971. Blanden, R. V., J. Exp. Med. 133, 1090, 1971. Blanden, R. V., Bowern, N. A., Pang, T. E., Gardner, I. D., and Parish, C. R., Azlsf. J. Exp. Biol. Med. Sci. 53, 187, 1975. 6. Fenner, F., Aust. J. Exp. Biol. Med. Sci. 28, 1, 1949. 7. Gardner, I. D., and Blanden, R. V., Cell. Immrcnol. 22, 283, 1976. 8. Blanden, R. V., Transplant. Rev. 19, 56. 9. Blanden, R. V., and Mims, C. A., Artst. J. Exp. Biol. Med. Sci. 51, 393, 1973. 10. Roberts, J. A., J. Zmmztnol. 92, 837, 1964. 11. Glasgow, L. A., Arch. Intern. Med. 126, 125, 1970. 12. Gardner, I. D., Bowern, N. A., and Blanden, R. V., Eur. J. Immunol. 4, 63, 1974. 13. Gardner, I. D., Bowern, N. A., and Blanden, R. V., Eur. J. Immunol. 4, 68, 1974. 14. Ada, G. L., Jackson, D. C., Blanden, R. V., Tha Hla, R., and Bowern, N. A., Stand. J. Immunol., in press. 1.5. Gardner, I. D.. Bowern, N. A., and Blanden, R. V., EUY. J. ZIIZNIZH~O~. 5, 122, 1975. 16. Blanden, R. V. In “Progress in Immunology II Vol. 4” (L. Brent and J. Holborow, Eds.) pp. 117-125, North Holland, Amsterdam, 1974. 17. Blanden, R. V., Doherty, P. C., Dunlop, M. B. C., Gardner, I. D., Zinkernagel, R. M., and David, C. S., Nature 254, 269, 1975. 18. Blanden, R. V., Hapel, A. J., and Jackson, D. C., Ilrl11~ltlzoch~ll~istry,in press. 19. Doherty, P. C., Blanden, R. V., and Zinkernagel, R., Transplant. Rev., in press. 20. Doherty, P. C., Zinkernagel, R. M., and Ramshaw, I. A., J. Immunol. 112, 1548, 1974. 21. Sandford, K. K., Earle, W. R., and Likely, G. D., J. Nat. Cancer Inst. 9, 229, 1948. 22. Kearney, R., A., and Nelson, D. S., Znt. J. Cancer 15, 438, 1975. 23. Miller, R. G., and Dunkley, M., Cell. Zmmzmol. 14, 284, 1974. 24, Burnet, F. M. “The clonal selection theory of acquired immunity,” University Press, Cambridge, 1959. 25. Minis, C. A., Bactcriol Rev. 28, 30, 1964. 26. Gardner, I. D., and Blanden, R. V., Cell. Immwol. 22, 283, 1976. 27. Miller, J. F. A. P., Vadas, A., Whitelaw, A., and Gamble, J., PYOC. Natl. Acad. Sci. U.S.A., 72, 5095, 1975. 28. Rager-Zisman, B., and Bloom, B. R., Nature 251, 542, 1974. 29. Ramshaw, I. A., Infect. Immun. 11, 767, 1975. 30. Forman, J., and Moller, G., Transplant. Rev. 17, 108, 1973. 31. Bevan, M. J., and Cohn, M., in press. 32. Phillips, S. M. In “Immunology of Cancer. Progr. Exp. Tumor Res. Vol. 19.” pp. 37-44, Karger, Basel, 1974.
IMMlJNOLOGY
22,
The Cell-Mediated 1. Kinetics
(1976)
271-282
Immune
and Characteristics
Response to Ectromelia of the Primary
R. V. BLANDEN Department
Effector
Virus
T Cell Response in Vivo
AND I. D. GARDNER
of Microbiology, The John Curt&z School of Medical Australian National University, Canberra, 2601. A.C.T. Received
Infection
November
Research,
lo,1975
The cell-mediated immune (CMI) response to ectromelia virus infection in mice was studied. Virus doses from 4 X 1@ up to 5 X 10’ PFU of an attenuated strain inoculated intravenously (iv) all induced cytotoxic T cell responses in the spleen as measured in a “Cr release assay using virus-infected target cells. Higher virus doses gave larger responses. There was little variation between individual animals, and mice ranging in age from 4-22 weeks gave similar responses. Following iv infection, virus grew logarithmically in spleen for 2 days, then titers declined to undetectable levels by day 5. The peak of the virus-specific cytotoxic T cell response occurred at 5-6 days postinfection, as determined by calculation of effector units based on a linear log-log relationship between killer cells added and targets lysed. T cells responsible for virus clearance in vivo gave similar kinetics, suggesting the possibility that both functions are mediated by the same T cell subset. Two other categories of cytotoxic activity were also generated at low levels in the spleen during ectromelia infection or during infection with a bacterium, Listeria monocytogenes. These activities were significantly sensitive to anti-8 and complement treatment, suggesting T cell dependence, but participation of other mechanisms has not been rigorously excluded. One category lysed allogenic target cells and reached a peak at 4 days post-infection. The other lysed H-2-compatible cells, syngeneic embryo cells, and some syngeneic tumor cells but not syngeneic macrophages, and was present at similar low levels through days l-4. These different kinetics and evidence from “cold” target competition experiments suggested that the total cytotoxic activity of immune spleen cell populations was a composite of the activities of separate cellular subsets (probably mainly T cells), killing of any one target cell type being the responsibility of a subset with receptors at least partly specific for antigens on that target cell.
INTRODUCTION Ectromelia virus is a poxvirus similar to vaccinia and variola viruses (l), which produces a disease, mousepox, with many features resembling human smallpox (1). Recovery from primary infection depends upon cell-mediated immunity (CMI) in which the virus-specific effector cells are thymus-derived lymphocytes (T cells) (2-5). Antibodies appear too late and in insufficient amounts to play a significant role in recovery, (2, 3) but appear to be an important component in resistance to re-infection (6). The role of CM1 in secondary infection (T cell memory) has not been elucidated and will be the subject of future studies (7). Previous evidence suggested that a number of different mechanisms could operate in concert to eliminate virus from foci of infection in the major, visceral target organs, the liver and spleen. These mechanisms depend upon localization of effector 271
Copyright 4
All
rights
o
1976 by Academic Press, reproduction in any form
Inc. reserved.
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T cel1.sin foci, followed by the attraction of monocytes (4, 8) and perhaps their differentiation to activated macrophages (9). Macrophages ingest the virus, and even if infected, produce so little infectious progeny virus that the ultimate outcome is elimination of infection (4, 10). Activation of the macrophages and local interferon production (perhaps by T cells) may augment the efficiency of virus clearance (4, 11). Recent studies indicate that ectromelia-infected target cells can be lysed efficiently by specific cytotoxic T cells in vitro (12, 13). Rigorous criteria support the identification of the killer cells as T cells, i.e. they are sensitive to anti-8 and complement and do not bear surface immunoglobulin (5, 13). The cytotoxic process does not require exogenous complement, macrophages, or secretion of soluble factors, proceeds in a linear manner with time, and appears to require one hit (12, 13). Target cells acquire antigenic changes within a few hr after infection in the absence of viral DNA synthesis (14) and are thus vulnerable to lysis before the assembly of progeny virus particles. Therefore, T cell-mediated cytotoxicity could well contribute to retardation of virus growth rate and spread within target organs in tivo. One aspect of T cell recognition of virus-infected cells both in vitro (15, 16) and in tivo (5, 16) is that host cell genes, which map in either the K or D regions of the H-Z gene complex (17) appear to dictate an essential component of the antigenic moiety recognized (18, 19). The advent of a well-defined in vitro assay for T cell-mediated cytotoxicity (12, 13) has provided a second test for effector T cell function, in addition to cell transfers to infected recipient mice (3)) with obvious logistic and quantitative advantages. Both of these tests have been used to examine questions about the nature and heterogeneity of T cells which perform various functions in the response to ectromeIia infection. In this paper we examine in detail the response of individual mice to different doses of virus, the kinetics of virus growth in spleen in relation to the kinetics of production of effector T cells, and the nature of other T cell subsets in spleen which are apparently unrelated to the specific response to infection. MATERIALS
AND
METHODS
General. CBA/H and C57BL/6 mice, stocks of attenuated (Hampslead egg) and virulent (Moscow) strains of ectromelia virus (Z), the bacterium Listeria nzonocytogenes (3), methods of titration of infectious agents (2, 3), immunization of donor mice (5), preparation of spleen cell suspensions (5)) culture of target cells (12-15) and anti-8 and complement treatment (13) have all been described previously. Antiviral activity of i++zYtmune spleen cells. The techniques of cell transfer and the titration of virus in recipient tissues have been described in detail elsewhere (3, 5). Briefly, donor mice were immunized intravenously (iv) with 5 X lo4 plaque-forming units (PFU) of attenuated virus at varying times before harvest of immune spleen cells. Recipient mice were inoculated iv with 4 X lo4 PFU of virulent virus 24 hr before cell transfer and spleens removed for virus titration 24 hr after cell transfer. Cytotoxic assay. The 51Cr release method used for H-l sarcoma cells, mouse embryo cells and macrophages was basically as described in detail previously (12), utilizing 7 mm assay wells (15). Macrophages were seeded at lo5 cells per well
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and other target cells at 2 x 104. The procedure used for L929, P-815 and EL-4 cells was modified slightly as follows. Cells were first labelled with Yr in suspension. Infection with ectromelia virus was also performed in cell suspensions at a density of S-10 X 10Gcells per ml with 4-10 PFU per cell. Target cells were then dispensed into 7 mm wells (2 ~10” per well) in a volume of 100 ~1 with an automatic pipette. Tests showed variation in total counts per well to be no more than 1% either side of the mean. Killer cells were also dispensed in 100 ~1, giving all wells identical 200 ~1 volumes. After 16 hr of killer-target interaction at 37”C, the whole culture tray was gently centrifuged to deposit any cells in suspension, and 100 ~1 of supernatant from each well was removed and counted. The total counts per well were obtained from 100 ~1 aliquots of target cells to which had been added 0.9 ml of distilled water. These tubes were counted, then centrifuged and the supernatant and pellet counted to calculate % 51Cr releasable by water, (usually 85-90s of the total). Lysis in test wells (done in quadruplicate) was expressed as a percentage of the lysis achieved by water, and was derived from the formula : Counts in 100 jJ supernatant from test wells X 100 Mean counts in 100 ~1 supernatant in water lysis tubes RESULTS variation. Large doses of lymphocytic choriomeningitis (LCM) virus, which is not cytopathic, have a suppressive effect upon the production of virus-specific cytotoxic T cells (20). This effect was not seen with ectromelia virus, possibily because the dose level required to achieve it would be lethal, even with an attenuated strain of virus. With CBA/H mice, doses much above 5 x lo4 PFU given iv produce extensive necrosis in spleen and liver, and death in occasional animals. Thus groups of four mice 8 weeks old were immunized iv with different doses of virus (5 X 104, 104, 2 X lo3 and 4 X 10” PFU/mouse). Individual spleens were harvested 6 days later, known to be at about the time of peak effector T cell response (3, 12). Even at a virus dose of 4 X 10’ PFU, spleen cells from each of the four mice gave substantial cytotoxicity (Table 1). The two highest doses, 5 X lo4 and lo4 PFU, caused very consistent responses in individual mice, although the lower dose gave less cytotoxicity. The four mean percentages of 5*Cr release from individual mice within each of these two groups were averaged to give a mean value * SEM. In both cases, this SEM was similar to the SEMs from quadruplicate assay wells of the individual mice, showing that within this dose range, variations between individual mice were negligible (Table 1). In all subsequent experiments, virus doses of between 2 x lo4 and 5 X 10” PFU and spleen cells pooled from four mice were used, although the results suggested that within this dose range, one mouse would be representative of the group. Age dependence. Groups of four CBA/H mice of varying ages from 4 weeks to 22 weeks were immunized iv with 5 x lo4 PFU attenuated ectromelia virus per mouse, and their spleen cells assayed for cytotoxicity 6 days later. The results (not presented here) showed that mice aged from 4 to 12 weeks exhibited similar levels of cytotoxicity against infected target cells. Mice at 22 weeks gave a slightly, though significantly, lower level. Mice were routinely used at 7-8 weeks of age. Ejject
of virus
dose
and
individual
274
BLANDEN
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TABLE Effects
Virus dose
of Virus Dose and Individual Against Virus-Infected CBA/H mouse
Target Ectromelia-infected
5 x 10’
104
2 x 103
4 x 102
Spontaneous
1
Variation on the Cytotoxic T Cell Response H-Z-Compatible Target Cells
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 release
cells
L929
Uninfected
80.2 76.0 75.6 78.0 62.7 63.2 61.1
f 1 1” f 0.8 f 1.4 f 1.1 f 0.6 f 1.7 f 1.0
62.1 50.1 36.2 56.9
f f f f
0.5 0.3 0.8 1.4
65.6 f 64.9 f 56.4 f
1.3 1.7 1.4
57.2 f 58.4 f
1.3 1.8
16.3 f 16.5 f
0.3 0.4
14.0 f
0.1
20.5 i
0.3
77.5 f
1.1*
62.3 f
0.5
19.0 21.1 18.2 14.6 13.2 13.0 13.7
6.2
16.4 16.1 17.1 14.4
1.9
16.5 i 0.1 17.0 f 0.3 15.5 f 0.5
52.2 f
59.2 f
i f * f f f f f f f f
0.6’ 0.5 0.2 0.4 0.3 0.3 0.4 0.5 0.3 0.3 0.2
L929
18.2 f
1.4*
14.1 f 0.8
16.0 f
0.6
16.3 f
0.3
0 Mean y0 Wr released iSEM from groups of four wells, assayed at a spleen cell: target cell ratio of 100: 1. Immune spleen cells obtained 6 days after immunization iv. * Mean fSEM, of the means of the four individuals receiving the same virus dose iv.
Growth of virus in the spleen. Over 99% of an intravenous inoculum of Moscow strain ectromelia virus is cleared from the blood within 5-7 min; most of this virus goes into the liver, with l-5% entering the spleen(4). Five groups of four CBA/H mice were infected iv with 2 x lo4 PFU/mouse of attenuated ectromelia virus. Thus < lo3 PFU would be expected to localize in the spleen. On each of the next 5 days, a group of mice was sacrificed and their spleens removed for virus titration. Figure 1 shows the behaviour of the virus in spleen. Logarithmic multiplication occurred for 2 days, then titers declined slightly on day 3 and dropped sharply on day 4. No virus was detectable on day 5 (less than 2 X lo2 PFU) . Kinetics of appearance of cytotoxic T cells in the spleen. Five groups of four CBA/H mice were immunized iv at 2 day intervals with a single injection of 5 x lo4 PFU of attenuated ectromelia virus per mouse. Two days after the last group was immunized the spleen cells of all groups were assayed against ectromeliainfected and uninfected L929 target cells (Fig. 1). CBA/H mice and L929 cells share H-2 haplotypes ( H-2k). Cytotoxicity against infected L929 cells increased to a peak at day 6 and had declined to a low level by day 10. (Fig. 1). The activity of immune spleen cells against uninfected L929 cells showed quite different kinetics. It was prominent on days 2 and 4, but declined almost to background by day 8. In a similar experiment, uninfected P-815 (H-2d) target cells were also used (Fig. 2). Activity of immune spleen cells against them was maximal on day 4. Thus, use of three
PRIMARY
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275
6
I
2
3
4
5
6
7
8
9
10
DAYS AFTER INFECTION
FIG. 1. Growth of ectromelia virus in spleen (--¤-) after infection iv with 5 X 10’ PFU of attenuated virus, expressed as mean loglo PFU per spleen in groups of four CBA/H mice per point. Cytotoxicity generated in spleen cells was measured with virus-infected (---A-) or uninfected (- - n - -) L929 target cells at a killer target ratio of 100 :l. Each point is the mean of quadruplicates. Background “Cr release from infected ( l ) or uninfected (0) targets in the presence of normal CBA/H spleen cells at 100: 1 is shoddy by the single points below the curves. All vertical bars enclosed 2 SDS.
different target cell types revealed cytotoxic activity with three different kinetic patterns appearing in spleen as a consequence of infection. Rigorous criteria have been met to establish that the spleen cells responsible for killing infected H-Zcompatible target cells in this system are T cells (5. 13), that they are virusspecific (13, ZO), and that H-2K or H-2D homology is required between donors of T cells and infected target cells (17). Detailed studies of the kinetics of their appearance in spleen are described later in this paper. However, the possibility that
DAYS
AFTER INFECTION
FIG. 2. Cytotoxicity of spleen cells harvested from CBA/H mice at various times after iv infection with 2 X 10’ PFU of attenuated ectromelia virus against infected L929 targets at 50: 1 (-A--) or 1O:l (--A-) or against uninfected p-815 targets at 50: 1 (- - fJ - -). “Cr release in the presence of normal CBA/H spleen cells at each ratio has been subtracted. Each point represents the mean of four wells using pools of spleen cells from four mice, vertical bars enclose 2 SDS.
276
BLANDEN
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TABLE Cytotoxicity”
2
of Early Ectromelia-Immune Spleen Cells from CBA/H H-2-Compatible (L929) or Syngeneic (H-l, Macrophage)
Expt.
1
2
Target
cells
Days after immunization
L929 H-l L929 CBA/H Macrophages
a Mean ye 6rCr released from cell ratios were 100: 1 for L929 presence of normal spleen cells highly significant (P < O.OOl), (P < 0.05). b N.D.. not determined.
Mice against Target Cells
Uninfected
iv
1
2
3
4
20.20 0
20.6 0.4
18.3 1.4
21.8 0
25.8
32.2
N.D.b
N.D.
0.8
3.7
N.D.
N.D.
three wells. SEMs were never larger than 3.4. Spleen cell: target and H-l cells and 30: 1 for macrophages. Background release (in at the same ratio) was subtracted. Release from L929 cells was release from H-l or macrophage target cells was not significant
activity of CBA/H immune spleen cells against H-Z-compatible, uninfected L929 cells reflected a transient break in self tolerance warranted further analysis. Therefore, CBA/H spleen cells harvested 1, 2, 3 and 4 days after immunization were tested against uninfected L929 cells, derived originally from C3H mice (21), and syngeneic target cells of two types (a) CBA/H peritoneal macrophages and (b) H-l sarcoma cells, from a methylcholanthrene-induced tumor of CBA/H mice (22). While spleen cells from days 1 through 4 killed uninfected L929 cells, they had no activity against the syngeneic cells (Table 2). Since both peritoneal macrophages and H-l sarcoma cells are readily lysed by T cells sensitized to H-2k (unpublished results), this data suggested that the cells which kill uninfected L TABLE
3
Effect of Anti-8 and Complement Treatment on Ability of Ectromelia-immune Spleen Cells from C57BL/6 Mice to Lyse” Uninfected Allogeneic Syngeneic (EL-4, MEC) Target Cells Immunizing infection
Ectromelia
Listeria
Treatment of spleen cells
Nil Anti-0 + complement Normal ascitic fluid + complement Nil Anti-B + complement Normal asciticfluid + complement
or L&&-immune (L929) or
Target
cells
L929
EL-4
MEC
44.3a 5.6b 39.7
15.2 Ob 14.5
46.8 22.5b 43.8
28.6 4.0b 31.2
25.8 10.P 29.3
35.1 12.lb 32.3
0 Mean $$ SrCr released from four wells. SEMs were never larger than 4.5. Spleens were harvested 4 days after immunization. Spleen cell: target cell ratios were 1OO:l. Spontaneous release subtracted. B Significantly less release than controls (P < 0.001).
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277
4
Competitive Inhibition of Cytotoxicity Exerted by 4-Day Ectromelia-immune CBA/H Spleen Cells against Uninfected H-2 Compatible (L929) or Allogeneic (P-815) Target Cells Unlabelled
competitors
@?051Crrelease from
Nil P-815 L929
(9:l)b (9:l)
L929
P-815
23.2a 16.1 0.6c
26.9 ll.lC 14.5
a Mean “ye Kr released from four wells. SEMs were never larger than 0.9. Spleen cells: target cell ratios were 100: 1. Control values (Wr release in wells containing normal spleen cells at the same ratio) are subtracted. b Numbers in parenthesis are ratios of unlabelled competitors to labelled target cells. c Significantly greater competitive effect than with the heterologous competitor (P < 0.001 for L929 labelled targets and P < 0.05 for P-815 labelled targets).
cells are not specific for H-2”, i.e., that self-tolerance to H-2 antigens is not broken. This phenomenon was investigated more widely. It was found that infection with a bacterium, Listeria wzonocytogenes, also induced apparent “anti-self” cytotoxicity. Various mouse strains were infected with ectromelia virus or Listeuia. Syngeneic target cells, either tumor cell lines, or embryo cell lines were killed by l-6 day immune spleen cells, e.g. C57BL/6 spleen cells killed C57BL/6 embryo cells (MEC) or EL4 lymphoma cells, and DBA/2 spleen cells killed P-815 mastocytoma cells (some representative data given in Table 3). Use of anti-0 serum and complement suggested that the cytotoxic process was largely, if not totally, T cell-dependent (Table 3). Killing appeared to depend, at least in part, upon specific recognition by cytotoxic cells bearing receptors for antigen of uniform specificity or a limited range of specificity, since it could be
A2
50
54 LOG,,
58
62
KILLERS
FIG. 3. The family of straight lines with 45” slope o%ained by plotting log, ectromeliainfected L929 target cells lysed against log,” immune spleen cells required to lyse them. The pools of immune spleen cells were harvested from groups of four CBA/H mice infected iv with 2 x lo4 PFU of attenuated ectromelia virus 3 (*), 4 (a), 5 (0), or 6 (0) days previously.
278
BLANDEN
AND
TABLE Effector Days after infection
3 4 5 6
GARDNER
5
Unit Parameters’
Abscissaa intercept
Elector units6 per lo6 cells (103)
2.3 1.55 1.25 1.65
5 28 56 22
Lymphoid cells per spleen (X 108) 1.7 1.1 1.4 3.5
Effector units per spleen (X 105) 8.5 31 78 77
o Calculated from the data in Fig. 3. Experimental details in text. b Effector units bear a direct relationship to the actual number of cytotoxic T cells in the cell population. Since each T cell can kill more than one target cell, the actual number of T cells could theoretically be obtained by dividing the number of effector units by the mean number of target cells killed by each T cell. However, this number is not known in the present system.
partly inhibited by unlabelled competing target cells, and inhibition was more effective if they were the same type as the labelled targets (Table 4). This applied to both H-2 compatible and incompatible target cells, (Table 4) but the data were not clearcut in many experiments of this type and no firm conclusions can be drawn about the specificity of recognition involved in this phenomenon. These data suggested that subsets of cytotoxic cells (probably T cells) at least moderately specific for alloantigens, or non-H-2 antigens borne by some syngeneic, uninfected cells were briefly activated to a limited extent as a consequence of infection. Their specificity and kinetics, however, bore no obvious relationship to the major cytotoxic T cell response against ectromelia-induced antigenic changes on H-2-compatible infected cells. Quantitative kinetics of the generation of T cells specific for ectronzelia-induced antigenic changes. Groups of CBA/H mice were injected iv at daily intervals with 2 X lo4 PFU of attenuated ectromelia virus, and their spleen cells assayed for cytotoxic activity 3, 4, 5 and 6 days after immunization on infected L929 targets. Each immune cell pool was assayed at ratios of 40: 1, 20 : 1, 10 : 1 and 5 : 1, and the results plotted as loglo target cells lysed versus loglo number of splenic lymphocytes required to lyse them (Fig. 3). T cell killing systems give a straight line graph with a slope of 45” when specific 51Cr release is between 10% and 60% (K. J. Lafferty, personal communication) indicating a one-hit process (23). The day 3 population (Fig. 3) had three points which fell within these limits, and the line of best fit for these points had a 45” slope. The day 4 and day 6 data also had three points whose lines of best fit were 45” slopes. The line for the peak response (clay 5) was assumed also to have a 45” slope, since a 45” line could be drawn through the two points falling within the lo%--60% limits. Extrapolation of this family of lines to the abscissa gave the logarithms of the numbers of cells required to kill one target cell for each immune spleen cell population. The reciprocals of the antilogarithms of these abscissa intercept values represent the concentration of “effector units” in the spleen population (Table 5). Although the concentration of effector units was much higher on day 5 than day 6, the total number of effector units per spleen was no different. Thus the increase in mean cell numbers per spleen which occurred from days 5 to 6 was due to an influx or mu!tiplication of cells other than cytotoxic T cells.
PRIMARY
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2’19
FIG. 4. Capacity of pools of immune spleen cells, from groups of four CBA/H mice infected iv with 2 X 10’ PFU of attenuated ectromelia virus at 2 day intervals, to lyse infected L929 target cells (-A-) at a ratio of 100: 1, or to reduce viral titers when transferred into preinfected CBA/H recipients (- - n - -) at 5 X 10’ cells per mouse. =Cr release in the presence of normal CBA/H spleen cells was subtracted and log protection values are the log,, reduction in spleen titer below the level in recipients of normal CBA/H spleen cells. Points represent the mean of four assay wells or four individual recipient mice; vertical bars, enclose 2 SDS.
Correlation of in vitro cytotoxicity with in viva effector activity. Five groups of five CBA/H mice were injected intravenously with 5 x lo4 PFU of attenuated ectromelia virus per mouse at 2 day intervals. Two days after the last group was injected, the spleens of all groups plus a group of six uninfected control mice were removed and the cells assayed for cytotoxicity against ectromelia-infected L929 cells, and for their ability to cause reductions in virus titers in spleens of preinfected mice (Fig. 4). The two effector activities showed similar kinetics, an observation consistent with, but not proof of, their being functions of the same T cel1 subset. DISCUSSION Since the CM1 response is crucial for recovery from primary ectromelia virus infections in mice (2-S)) we have examined the sequence of events in the virusinfected spleen in terms of effector T cell responses. This investigation has revealed much more complexity than that embodied in the conventional immunological view, based on Burnet’s “Clonal Selection Theory” (24), which would hold that the response should consist of the activation of T cell clones bearing receptors complementary to antigenic determinants specified by the viral genome. In fact, as discussed in detail elsewhere (5, 18, 19), there is thus far no clear evidence for the existence of these conventional T cells; the major T cell response seems to be directed against antigenic patterns dictated partly by the viral genome and partly by the H-2 gene complex of the host (5, 15, 16, lS, 19), i.e.,with CBA/H mice they are present on virus-infected H-2” target cells. Two other categories of T cells also appear to be activated by infection; these are discussed below. After iv injection of virus the chronological sequence is as follows: About l-5% of the inoculum localizes in the spleen within a few min, most of the remainder going to the liver (4). The immunofluorescence studies of Mims (25) and evidence reported in the following paper (26) suggest that virtually all classes of spleno-
280
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cytes are vulnerable to infection, including T cells which ultimately give rise to the immune response that triggers clearance of infection. Thus the full development of an immune response in the spleen depends on the dose of virus being such that the growing foci of infection do not become confluent before the first effector T cells begin to operate (25). By implication, this would happen between 2 and 3 days after infection, since virus titers in spleen increase for 2 days and then decline to undetectable levels by day 5. Effector T cell activity in immune spleen cells was greatest when the highest virus dose was used for immunization. This suggested that the number of infected cells, presumably the stimulus for cytotoxic T cell induction (26) was probably still a limiting factor up to the highest dose used (5 x lo4 PFU). Therefore, titers of virus in spleen on days 1 and 2 after infection, when specific immune induction is most likely to be taking place, may well be directly related to the number of effector T cells produced in the response. Effector T cells which kill virus-infected target cells in vitro and which trigger viral clearance in viva are readily detectable by day 4 and reach peak concentrations in the spleen cell population by days 5-6. These two different functional assays reveal very similar kinetics in the T cell response(s) measured. They could therefore reflect the activities of the same T cell subset, but absolute proof of this proposition is very difficult. Another similarity is that lysis of virus-infected cells in vitro and virus clearance in viva both require that immune T cell donors share a part of an H-2 haplotype with the infected target cells, or recipient mice, respectively. In both cases, the essential gene(s) map in either the K or D regions of the H-2 complex (17, Kees and Blanden, submitted). These findings are especially interesting in the light of results of Miller et al. (27)) that transfer of delayed hypersensitivity to fowl gamma globulin or dinitrofluorobenzene in mice requires I-A region homology between immune T cell donors and recipients. Experiments are in progress to investigate the presence and relative importance of such T cells during ectromelia infection. It is also noteworthy that the splenomegaly resulting from infection is not a direct reflection of virus-specific effector T cell production. In particular, the major increase in spleen size (three-fold) occurred from days 5 to 6 post-infection, but effector T cells did not increase in numbers in the spleen over this same interval. The increase in effector units from day 3 to day 4 was four-fold, suggesting that the cytotoxic cells may have undergone two divisions in this time. This conclusion may be invalid, however, as there would presumably be antigen available to induce new precursor T cells continuously over at least the first 3 days of infection, thus giving the impression of rapid cell cycles in the assumed proliferative response which followed. The increase in effector units from day 4 to day 5 was only about 2$-fold, perhaps reflecting waning numbers of precursors induced, a slower rate of division, or that significant numbers of cytotoxic T cells were leaving the spleen via the circulation. The lack of increase in total effector units from day 5 to day 6, suggested that egress of effector cells from the spleen kept pace with their production, and/or that some suppressive or regulatory effect is being exerted on division or activity or cytotoxic T cells. Therefore, these data suggest that studies of lymphoid cell proliferation during the immune response should be interpreted with caution, since the peak of the proliferative response may not necessarily reflect effector cell production.
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Furthermore, it seems that two categories of cytotoxic activity other than that specific for virus-induced antigenic changes in “self” cell surfaces are also present in spleen cell populations during the early response to infection. The first category is directed against alloantigenic target cells, e.g., cells from CBA/H (H-2”) mice lysed uninfected P-815 (H-2d) target cells. This activity reached a peak on day 4 post-infection. The second category was directed against H-2-compatible, uninfected target cells, e.g. CBA/H (H-2”) immune spleen cells killed uninfected L929 (H-ak) target cells. The kinetics of this activity were strange in that cytotoxic activity was present at similar levels through the first 4 days after infection and then gradually disappeared. Both these categories of cytotoxic activity appeared to be T cell-dependent since they were abolished or significantly depressed by anti-0 treatment of immune spleen cells. They seem unlikely to be dependent on K cells, which operate in herpesvirus infection (28, 29) for example, but are not anti-8 sensitive and would require a source of specific antibody (28, 30)) or on macrophages, since activated macrophages do not appear in the spleen until S-10 days post-infection (9). Therefore, they seem likely to be due to direct cytotoxic action of T cells. There are several possible explanations for these phenomena. First, T blast cells produced early in infection may bind to and kill any target cell for non-immunological reasons. Second, there may be a degree of immunological cross-reaction between, for example, infected CBA/H cells and uninfected L929 or P-815 cells. Third, there may be a multi-clonal T cell response, with the killing of each uninfected target cell type being due to specific clones or subsets of T cells with receptors reasonably specific for the particular target cell. The third interpretation above is favoured by several pieces of evidence. For example, the kinetics of the activities against uninfected L929 and P-815 are different, implying that they are functions of different T cell subsets. Furthermore, no cross-reaction can be shown at the cytotoxic T cell level between P-815 cells and infected L929 cells (unpublished results). Finally, reduction of the cytotoxicity against one type of uninfected target cells, with less impairment of the activity against another type, has been achieved by competition with excess numbers of uninfected, unlabelled target cells of the first type, thus indicating a degree of immunological specificity in the interaction between killer cell and target cell. A number of subsets of alloantigen-reactive precursors of cytotoxic T cells may, therefore, be activated during infection by either viral, bacterial or cellular products in a manner analogous to mitogen activation of T cells in z&o. (31, Cole, G. A., Blanden, R. V., and Dunlop, &I. B. C., in preparation). However, the appearance of cells reactive to H-2-compatible, uninfected target cells is more intriguing. It seems unlikely that virus present in the early spleen cell populations is sufficient to infect enough uninfected target cells to explain the phenomenon by invoking the action of the virus-specific cytotoxic T cells. In any case, lysis of deliberately infected target cells by the early immune spleen cells is less than that of uninfected targets, and there is no peak of activity corresponding to virus titers in the spleen. There was no detectable activity against strictly syngeneic target cells such as macrophages. Killing of syngeneic tumor cells or embryo cells occurred, but these cells could be expressing vertically transmitted viruses or embryonic antigens and may not be strictly “self” with respect to normal adult animals. For these reasons, it seems unlikely that there is a transient break in cytotosic T cell tolerance to normal “self” antigens.
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Even if this did occur, effector cells would need to be present at high levels in order for their effects against labelled syngeneic target cells to be seen in the presence of the large excess of autochthonous spleen cells which would act as “cold” competitors. Furthermore, such killer cells might be expected to kill each other, or even themselves during the act of endocytosis. Whether the cells responsible are reactive to minor alloantigens, embryonic antigens, or perhaps antigenic patterns induced by vertically transmitted viral genomes (3.2) remains to be determined. Whatever the outcome, the phenomenon could well be relevant to the problem of autoimmune disease. ACKNOWLEDGMENT We are very grateful to Narelle Bowern for expert technical assistance.
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