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*
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia;
Institut für Immunologie der Universität Heidelberg, Heidelberg, Germany;
Department of Immunology and Medical Microbiology, University of Florida, Gainesville, FL; and
§
Max-Planck-Institut für Immunbiologie, Freiburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, two pathways of target cell killing by cytolytic leukocytes have been described. One is the granule exocytosis pathway mediated by perforin and serine proteases or granzymes (gzm) (8, 9). This is generally believed to be the dominant mechanism by which CTL eliminate virus-infected cells (10). The second mechanism, called Fas-mediated pathway, requires the interaction of the Fas receptor on the target cell with the Fas ligand on the killer cell (11) and is supposedly involved in immunoregulation (12).
Over twenty years ago, Gardner et al. (13) reported on poxvirus-mediated suppression of alloreactive CTL-mediated target cell lysis using mouse poxvirus, ECT. They observed severe inhibition of lysis of ECT-infected target cells by alloreactive CTL, while the same targets were highly susceptible to lysis by MHC class I-restricted ECT-immune CTL. It was proposed then that the inhibition of alloreactive CTL-mediated lysis was due to a decrease in normal MHC class I cell surface expression and a replacement by virus-modified MHC, a consequence of poxvirus-mediated host protein synthesis inhibition (14).
More recently, it was found that poxviruses encode proteins related to the serpin family of proteinase inhibitors, termed SPI-1, -2, and -3 (15, 16). SPI-2 (or cytokine response modifier (crmA)) was shown to inhibit both Asp-specific serine and cysteine proteases, including components involved in CTL-mediated cytotoxic and inflammatory responses, such as gzmB (17) and caspases 1 (IL-1 converting enzyme (ICE)) (18), 3 (CPP32) (19), and 8 (Fas-associated death domain-like ICE (FLICE)) (20), respectively. Although the rates of inhibition of the various proteases by crmA were shown to differ by several orders of magnitude, they seem fast enough to be of biological significance (17, 18). Serpin-like genes have been found in all poxviruses analyzed so far (15), and the high homology observed between the serpins of vaccinia virus Western reserve (VV-WR), cowpoxvirus (CPV), rabbitpoxvirus (RPV), Variola, and ECT (R.W. et al., unpublished observations), indicate conserved function and evolutionary benefit. The fact that crmA is able to prevent target cell apoptosis, mediated largely via the Ca2+-independent (Fas-mediated) pathway, by alloreactive CTL (21), suggested that serpins evolved as an immune escape mechanism to avoid immune destruction of infected cells before viral replication and viral-induced cytolysis. This was also emphasised by findings of Macen et al. (22) that showed that target cell lysis by an alloreactive CTL line, as measured by 51Cr release, was greatly reduced for both cytolytic pathways, upon infection with either CPV or RPV, but restored by infection with virus deletions of the serpin SPI-1 and SPI-2 genes.
These findings, together with the evidence that functionally active ECT-immune CTL, and in particular the granule protein perforin (23), are required for recovery from primary ECT virus infections, led us to study the role of poxvirus-encoded serpins in the inhibition of target cell lysis by alloreactive and MHC-restricted CTL in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (KbDb) (B6), CBA/H (KkDk) (CBA), BALB/c (KdDd) (B/c), C3H.H2o (KdDk) (OH), B10.HTG (KdDb) (HTG), B10.A(2R) (KkDb) (2R), B10.A(5R) (KbDd) (5R), and the perforin-deficient mutant (Perf-/-) (24) and the granzyme A and B deficient mutant (A x B-/-) (25) were bred under pathogen-free conditions at the Animal Breeding Facility of the John Curtin School of Medical Research (Canberra City, Australia). The Fas ligand mutant mice (gld) bred on B6 background were obtained from the Centenary Institute (Sydney, Australia). Only female animals >12 wk of age were used.
Viruses and synthetic peptides
The cowpoxviruses wild type (CPV), the mutants with a defect in serpin 1 (CSPI-1) and serpin 2 (CSPI-2) (22), and the vaccinia virus (VV) WR strain (VV) were grown on CV-1 cell monolayers. The ECT virus Moscow strain was grown in mouse spleen. All poxviruses were titrated as described (26). The influenza virus strains A/WSN (H1N1) and A/JAP (H2N2) were prepared and titrated as described (26).
The synthetic peptide derived from the nucleoprotein of influenza A virus specific for Kd, TYQRTRALV, and specific for Kk, SDYEGRLI (NPP) (27), was synthesized at the Biomolecular Resource Facility (Australian National University, Canberra, Australia). The synthetic peptide derived from the hemagglutinin of A/JAP (H2N2) virus specific for Kd (LYQNVGTYV) (HAP) was obtained from Chiron (Melbourne, Australia).
Target cells
The mouse cell lines L929 (H-2k), L929-Fas (kindly provided by P. Krammer, Heidelberg, Germany), MC57 (H-2b), RMA (H-2b), HTG (KdDb), 2R (KkDb), and 5R (KbDd) were grown in Eagle’s minimal essential medium supplemented with 10% FCS. The cells were infected with poxviruses at a multiplicity of infection (MOI) of 10–20 PFU per cell for 16 h before being labeled with 51Cr for 1 h and used for analysis. Target cells were peptide-treated with NPP at the same time as labeled with 51Cr as previously described (26). Modification of targets with HAP was as for NPP.
Immunization
Animals were immunized with 106 PFU of ECT into hind footpad, 107 PFU of VV-WR, 1 x 106 PFU CPV, or 104 hemagglutination units of A/WSN (H1N1) i.p. For ECT virus, mice were infected with 1 x 106 PFU ECT into the hind footpads.
FACS analysis
L929 (KkDk) or HTG (KdDb) cells were infected with 20 PFU of ECT, as described for target cells. At 16 h after infection, the cells were washed, resuspended in growth medium at 107 cells per ml, and labeled at 4°C for 45 min with mAb HB-160 (American Type Culture Collection (ATCC), Manassas, VA) specific for Kk, mAb 15-5-5S (a gift from F. Momburg, Heidelberg, Germany) specific for Dk for L929 cells, mAb HB-159 (ATCC) specific for Kd, or mAb HB-27 (ATCC) specific for Db, followed by FITC-conjugated sheep anti-mouse Ig (Silenus, Hawthorn, Australia) staining. Cells were examined with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
Generation of CTL
For primary poxvirus, immune CTL splenocytes of 6-day immunized animals were used ex vivo. For the generation of alloreactive CTL, 8 x 107 responder splenocytes were cocultured with 4 x 107 irradiated (2000 rad) allogeneic stimulator cells for 5 days in 40 ml Eagle’s minimal essential medium, 10% FCS plus 10-5 M 2-ME. The generation of secondary NPP-immune CTL has been described (26).
51Cr release cytotoxicity assay
The methods used for target cell lines have been described in detail elsewhere (26). The duration of the assays was 6 h. Percentage of specific lysis was calculated by the formula: % specific lysis = [(experimental release - medium release)/(maximum release - medium release)] x 100. Data given are the means of triplicate determinations. SEM values were always <5%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm the earlier results of Gardner et al. (13) and to
extend them to other poxviruses, we used primary ex vivo-derived
splenic effector cells immune to VV, CPV, or ECT and tested them on
target cells infected with either the homologous or heterologous
poxviruses. Fig. 1
shows two
representative experiments using two mouse strains, CBA (Fig. 1A) and B6 (Fig. 1B), as donors of
poxvirus-immune CTL that were tested for their cytolytic potential to
lyse H-2-matched target cells, L929 and MC57, respectively. The results
clearly demonstrate that poxvirus-immune CTL are broadly cross-reactive
within the poxvirus family, indicating conservation of immunodominant
peptides among different viruses.
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, A and B,
reached plateau levels of 60–70% at E:T ratios of 30–60:1. Such
levels of lysis are similar to those found for other primary ex
vivo-derived virus-immune CTL (28), thus indicating that inhibition by
poxvirus-encoded serpins, if it occurs at all, may only be marginal in
affecting poxvirus-infected cell lysis by poxvirus-immune CTL. Differential inhibition of lysis of poxvirus-infected target cells by alloreactive and MHC-restricted CTL
To investigate further the role of serpins in the down-regulation
of target lysis by MHC-restricted vs alloreactive responses, we made
use of peptide modification of target cells. It is obvious that lysis
of target cells by poxvirus-immune CTL could not be used to evaluate
possible interference of lysis by serpins. In Fig. 2A, L929 target cells were
infected with ECT for 16 h or left uninfected. Infection of target
cells for 1 or 3 h before assay did not affect alloreactive CTL
lysis, but did sensitize for poxvirus-immune CTL lysis (data not
shown). The cells were tested for lysis by two alloreactive CTL
populations (B6 anti-2R, anti-Kk; B/c anti-OH,
anti-Dk) and two MHC-restricted CTL populations, namely
primary ex vivo-derived ECT-immune CTL and secondary influenza
NPP-immune CTL. For the latter, target cells were incubated for 1
h before assay with 10-4 M NPP peptide with the motif for
Kk. Fig. 2A (first panel)
shows the lysis by ECT-immune effectors. Levels of lysis of the
ECT-infected targets reaches 70–80%, demonstrating successful target
cell infection. When the mock and ECT-infected target cells were
treated with NPP and tested for lysis by NPP-immune effector cells
(second panel), mock and ECT-infected NPP-treated
target cells were lysed to the same extent and significantly exceeded
that of untreated targets. In contrast, using alloreactive CTL, target
cell lysis was greatly suppressed by ECT infection compared with
mock-infected targets. This suppression was more pronounced with CTL
directed against the D end than K end (third and
fourth panels). The presence of NPP did not to any
significant amount affect lysis of alloreactive or poxvirus-immune CTL
(data not shown).
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B extend the findings using ECT and
an additional poxvirus CPV and a similar experimental design, but
different effector and target cells. The two alloreactive CTL
populations, B6 anti-HTG (anti-Kd) and 5R
anti-B6 (anti-Db) lysed HTG
(KdDb) mock-infected targets to a similar
extent. Infection with either ECT or CPV reduced the release of
51Cr 9- to 20-fold, with CPV being the stronger inhibitor
(second and third panel). On the other
hand, B/c secondary influenza HAP-immune CTL lysed HAP-modified HTG
target cells to a similar extent, irrespective of poxvirus infection
(first panel). Although small inhibition is
apparent, the slopes of the killing curves are the same with HAP-immune
CTL, inferring the same rate of killing, but are different with
alloreactive CTL. Gardner et al. (13) have shown that H-2k alloreactive CTL lysed 80% of mock-infected L929 target cells but only 7% of ECT-infected targets, which, however, were lysed to 100% by ECT-immune effectors. These experiments, together with the present data, clearly establish that inhibition of target cell lysis by poxvirus infection is predominantly observed with alloreactive but not MHC-restricted CTL.
Cell surface MHC class I expression after poxvirus infection
To test whether the results obtained in Fig. 2 with alloreactive
CTL can be explained by a decrease in MHC class I cell surface
expression as originally proposed by Gardner et al. (13), we undertook
FACS analysis of mock- and virus-infected target cells. Fig. 3 shows the expression of MHC class I
Kk and Dk on L929 target cells 16 h after
ECT infection. The same cells were used for the experiment shown in
Fig. 2A. Fluorescence intensity increased after infection in
regard to anti-Kk Abs and decreased when infected cells
were labeled with anti-Dk Ab. Note that there is no
correlation between the level of MHC class I expression and inhibition
of CTL lysis.
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B, HTG cells were
infected with ECT or left uninfected and examined to determine to what
extent poxvirus infection alters cell surface expression of
Kd and Db. Fig. 3 (lower
panels) shows that both class I molecules are marginally reduced
on the cell surface as compared with mock-infected cells. Poxvirus inhibition of target cell lysis is cell type-dependent and varies with different CTL effector populations
The variability between target cells in poxvirus inhibition was
further explored within one experiment using two alloreactive effector
populations, 5R anti-B6 (anti-Db) and 2R
anti-B6 (anti-Kb). These effector populations were
tested on various Kb- and/or Db-expressing
target cell lines, RMA (KbDb), MC57
(KbDb), 5R (KbDd), and
2R (KkDb) (Fig. 4). The targets were either mock-infected
or infected with ECT, CPV, or the selective CPV-derived serpin mutants
CSPI-1 or CSPI-2 (15, 22). First, the level of lysis of RMA target
cells by either of the two alloreactive CTL effectors was, if at all,
only marginally affected after infection with any of the four virus
preparations, although the targets were susceptible to poxvirus-immune
CTL (data not shown). Similar results were found with other target cell
populations of hematopoietic origin, such as El-4, L1210, or P815 (data
not shown). Lysis of the other three targets (all of which are of
connective tissue origin and fibroblast-like) were affected by poxvirus
infection, however, to different degrees. Anti-Kb
alloreactive CTL lysed mock-infected MC57 and 5R targets to a similar
extent. Infection with ECT, CPV, and CSPI-1 completely inhibited lysis
of 5R targets, but only partially inhibited lysis of MC57 targets. The
mutation in CSPI-2 partially relieved the suppression in both targets
but substantially more in MC57 than 5R targets. Anti-Db
effectors lysed mock-infected MC57 targets to a lesser extent than 2R.
Suppression by CPV was 30- and 5-fold for 2R and MC57, respectively,
and by ECT, 5- and 3-fold, respectively. The mutation in SPI-1 did not
affect suppression as compared with wild-type CPV in any of the assays.
CSPI-2 virus completely abrogated suppression on MC57 targets and only
partially released inhibition on 2R targets. The data are consistent
with the interpretation that SPI-1 is not involved in suppression.
SPI-2, on the other hand, either completely or partially reduces target
cell lysis, depending on the effector cells employed. Thus, additional
virus molecules, such as SPI-3 (15), may be responsible for some
inhibition of CTL effector function in particular circumstances.
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To evaluate the influence of poxvirus infection on Fas-mediated
cytolysis vs the granule exocytosis pathway, we made use of three
mutant mouse strains. One was deficient in perforin
(Perf-/-) and as presently understood, CTL derived from
such mice rely on the Fas pathway for cell cytolysis (24). The second
strain was gld mice, which are defective in Fas ligand expression (29)
and thus kill primarily via the exocytosis pathway (24). The third
strain was A x B-/- mice; the CTL of which exert
cytolytic but not nucleolytic activity via the Fas-independent pathway
(25). Splenocytes from these mutant and wild-type B6 mice stimulated in
vitro with Kk (2R) allogeneic splenocytes were tested on
L929 and the Fas-transfected variant L929.Fas for 51Cr
release. 51Cr release was measured after assay times of 6
and 12 h (Fig. 5). Effectors from B6
wild-type mice lysed ECT- and CPV-infected L929 target cells three to
times less efficiently than mock-infected cells, at both time points.
No differences in lysis were observed between mock and CSPI-2-infected
L929 cells. The differential in susceptibility of mock vs ECT- and
CPV-infected targets was even greater on L929.Fas targets. In contrast
to L929, L929.Fas target lysis was partially suppressed by CSPI-2.
Gld-derived effectors lysed ECT- and CPV-infected L929 and L929.Fas
target cells substantially less efficient than mock-infected targets.
However, infection of both targets with CSPI-2 did not effect their
lysability. Thus, in the absence of the Fas-pathway, inhibition of
lysis is due exclusively to SPI-2 (crmA). Perf-/--derived
CTL did not lyse L929 target cells in a 6-h assay, but lysed L929.Fas
mock-infected targets efficiently. Lysis of the latter targets was
completely abrogated upon infection with ECT or CPV. Infection with
CSPI-2 only partially restored lysability. In 12-h assays, the same
effectors did lyse mock-infected L929 targets to a significant extent
and only slightly less CSPI-2-infected targets. However, again, ECT- or
CPV-infected targets were not lysed to any significant extent. The
effects of CPV, ECT, and CSPI-2 on lysis of L929.Fas by
perf-/--derived CTL in the 12-h assay were similar to
those seen at 6 h. In this experiment, the lysis profiles with
A x B-/--derived alloreactive effector cells are
lower on all target cells independent on the state of infection, but in
essence similar to that obtained with B6-derived effectors. This argues
against the possibility that the inhibition of the exocytosis pathway
by ECT and CPV is due to inactivation of granzymes, in particular of
gzmB by SPI-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data described here and previous results (13) are incompatible with recent interpretations (21, 22) that inhibition of CTL-mediated target cell lysis by poxvirus-encoded serpins, SPI-1 and SPI-2, is a means for virus to escape from cytotoxic T cell-mediated cytolysis. This is emphasized by 1) the original finding that virus-immune CTL cells are critical for recovery from infection with ECT (1, 2, 30), 2) the fact that Perf-/- mice are highly susceptible to ectromelia infection as compared with wild-type B6 mice (Ref. 23; and G. Karupiah, unpublished observations), and 3) the present demonstration that inhibition of target cell lysis, as observed with CPV and ECT, is predominantly seen with biologically irrelevant alloreactive, but not with relevant MHC class I-restricted, CTL. Together with the finding that poxvirus mainly interferes, most probably via crmA (SPI-2), with Fas-mediated cytolytic processes (Ref. 21, and data shown here), these data clearly establish that perforin-mediated control of ECT infections, a cytopathic virus, is of primary importance. The converse was inferred from experiments using the nonmouse pathogen VV (31), an infection that was shown before to be controlled in the absence of CTL (32). Similarly, studies presented here and elsewhere (22), using nonrelated poxviruses, CPV and RPV, in a mouse model, may also be inadequate to uncover strategies of either host or virus, which are of evolutionary significance. This is, in fact, exemplified by a recent comparison of the course of infection in perforin knockout mice upon inoculation with either ECT or CPV (23).
The differential susceptibility of the same poxvirus-infected target cells to alloreactive and MHC class I-restricted CTL can be the result of a number of different mechanisms. One possibility may be that the three different effector CTL employed here, primary ex vivo-derived poxvirus immune, in vitro alloreactive, and in vitro secondary influenza immune, may greatly differ in their activation state, which may influence their cytolytic potential in the presence of serpins. Another explanation may be fundamental differences in the mechanisms by which the two effector populations are engaged to deliver their lethal hit by yet unknown molecular basis. The possibility that the two CTL populations express distinctly different cytotoxic potentials is doubtful in light of their inherent capacity to specifically lyse their respective target cells and their overlapping repertoire. It is more likely that they possess TCRs with different affinities, and, consequently, require differing numbers of receptor/ligand interactions to achieve an avidity sufficient for triggering effector function. Quantitative consideration in TCR engagement to achieve triggering of Tc-mediated cytotoxic processes, such as exocytosis or Fas ligand-Fas ligation, may well be reflected in qualitatively different signals received by target cells (33).
Two distinct possibilities by which poxvirus infection may affect
alloreactive, but not MHC restricted, CTL lysis can be envisaged.
First, alloreactive CTL may induce a qualitatively different death
pathway than MHC-restricted CTL due to their requirement for multiple
receptor/ligand interactions. Such possibilities have been proposed
recently (34). Second, poxviruses may be able to alter the target cell
ligands recognized by CTL. This may occur in two ways; first, by a
possible down-regulation of MHC class I, as has originally been
proposed by Gardner et al. (13) due to poxvirus inhibition of host
protein synthesis (14). However, the data shown in Fig. 3 indicate that
the changes in cell surface expression of MHC class I after poxvirus
infection do not correlate with the CTL lysis inhibition results, as
lysis to both Kk and Dk is reduced but only
Dk cell surface expression is lower than that on
mock-infected targets. The more pronounced inhibition seen in the case
of Dk- vs Kk-reactive CTL (Fig. 2) may be due
to the decrease of Dk cell surface expression.
Alternatively, one could postulate that poxvirus infection affects MHC
class I cell surface motility, preventing aggregation of class I
molecules and TCRs bound to them, necessary for low-affinity
alloreactive CTL to kill, but which would leave CTL with high-affinity
TCRs unaffected. The different strength of inhibition seen with
different targets and different MHC class I molecules on one and the
same target is consistent with this hypothesis and may reflect
differential MHC class I cell surface concentrations of K and D Ags and
varying cell membrane fluidity. It is known that poxvirus infection
alters the cytoskeletal structures within the cell (35), and such
alterations may be responsible for changes in MHC class I cell surface
motility. We are at present investigating this possibility.
In addition, the observation that fibroblast-like target cells infected with poxvirus were prevented from lysis but not, or only marginally, cells of hematopoietic origin may be for the same reason, namely differential fluidity in the cell membrane. Alternatively, these two groups of target cells derived from different tissues, though similarly infected as indicated by their susceptibility to lysis by poxvirus-immune CTL, may express the poxvirus encoded "suppresser" molecules in different quantities.
Another possible explanation for the ability of CPV and ECT to inhibit
target cell lysis by alloreactive CTL has been proposed by Macen et al.
(22) and others (21, 17, 18, 19). Poxvirus-encoded serpins may interfere
with cytolysis and/or nucleolysis by inhibiting proteases involved in
the death pathways, such as caspases 1, 3, and 8, as well as gzmB. In
favor of this interpretation is the fact that CPV with a mutation in
serpin-2 restores, at least partially, target cell lysis by
alloreactive CTL (Figs. 4 and 5). In light of previous findings that
peptide caspase inhibitors blocked both nucleolysis and cytolysis by
the CTL-mediated Fas pathway, but only nucleolysis and not cytolysis
induced via granule exocytosis (36), the present data indicate that
target cell lysis elicited by virus-immune CTL is exclusively mediated
by perforin. They also suggest that the reduced capacity of
alloreactive CTL to exert their full cytolytic potential on
poxvirus-infected target cells is due to incomplete granule exocytosis
as a consequence of suboptimal TCR engagement.
As to the effect of poxvirus infection on the proteolytic activity on gzm, it was found that the cowpox serpin inhibitor SPI-2 was able to inhibit gzmB in vitro (17). Although the rate of inhibition is fast enough to be of physiological significance, its implication for the survival from natural poxvirus infection is unclear. We are currently investigating the role of gzmB in poxvirus infection and should be able to provide definitive evidence if serpin/gzmB interactions are biologically significant in determining the survival from natural poxvirus infections. What is clear is that, in the absence of gzmA and gzmB, lack of SPI-2 restores lysability to target cells in extended assays. As this is also true with effectors from Fas ligand-defective mice (gld), it suggests that SPI-2 interferes with yet an undefined pathway leading to 51Cr release. Inhibition of gzmA by poxvirus is rather unlikely because normal B6 mice are able to control infection, whereas gzmA-/- mice are highly susceptible under similar conditions with increased mortality and morbidity (37). Since alloreactive and MHC class I-restricted CTL from gzmA knockout mice express normal cytolytic potential, this was attributed to a direct effect of gzmA on virus replication rather than interference with CTL cytotoxicity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arno Müllbacher, Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. E-mail address:
3 Abbreviations used in this paper: ECT, ectromelia; VV, vaccinia virus; WR, Western reserve; CPV, cowpox virus; RPV, rabbitpox virus; SPI, serine proteinase inhibitor (serpin); crmA, cytokine response modifier; gzm, granzyme; ICE, IL-1 converting enzyme; Perf, perforin; NPP, nuclear protein peptide from influenza virus; HAP, hemagglutinin protein from influenza virus.
Received for publication November 30, 1998. Accepted for publication March 22, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or infected with VV (
), ECT (•),
or CPV (
) for 16 h. Cytotoxic assay time was 6 h. Each
point constitutes the mean of percent specific lysis of three separate
wells. Spontaneous release was always <20%.
) for 16 h were tested for lysis
by 2R anti B6 (anti-Kb) (top panels) and
5R anti B6 (anti-Db) alloreactive CTL. Cytotoxic assay
time was 6 h. Each point constitutes the mean of percent specific
lysis of three separate wells. Spontaneous release was always
<20%.