By
From the * Max-Planck-Institut für Immunbiology, Stübeweg 51, D-79108 Freiburg, Germany; Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland; and § Division
of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National
University, Canberra City, A.C.T. 2601, Australia
Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains
unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmA×B/
) with those from single knockout mice deficient in
gzmA (
/
), gzmB (
/
), or perforin (
/
) to induce nuclear damage and lysis in target cells.
With the exception of perforin
/
, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear
damage in target cells. In gzmA×B
/
mice, Tc/NK-mediated target cell DNA fragmentation
was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term
(4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely
to perforin and independent of both proteases.
Cytotoxic T (Tc) cells mediate target cell lysis by two
independent pathways, one involving exocytosis of
preformed granules, the other requiring ligation of Fas
ligand on the effector cell with the Fas receptor on target
cells (1). Both processes lead to target cell apoptosis and
lysis (7) by intracellular mechanisms which are still poorly
understood. Perforin, gzmA, and gzmB have been implicated as main contributors to membrane and/or nuclear disintegration of target cells by the secretory pathway (7). Membrane damage of target cells, as measured by 51Cr-release, is
mainly accounted for by perforin, which itself is capable of
causing target cell cytolysis (7). However, gzmA and B
have been shown to cause DNA fragmentation and apoptotic morphology of target cells, but only in the presence of perforin (10).
Controversy still remains as to the distinct contribution
of each of the two proteases to Fas-independent Tc/NK cell-
mediated DNA fragmentation of target cells and to what
extent they affect perforin-mediated target cell lysis. gzmA
and B seem to be able to cross the target cell membrane (7,
13) via a still undefined process, but their nucleolytic activities seem to follow distinct pathways. Whereas gzmB induces early DNA fragmentation by cleaving multiple cysteine proteases of the caspase cascade, including FLICE (14)
and CPP32 (15), the nucleolytic activity of gzmA seems to be delayed and independent of caspases (12, 18,
19). Although indirect evidence suggested that Tc cell-mediated target cell lysis correlates with the expression of gzmA
and/or B (11, 20, 21), no reduction of Fas-independent cytolytic activity was observed with Tc cell populations from
gzmA (22, 23) or gzmB knockout (ko) mice (18). Here we
use double ko mice deficient in both gzmA and B to investigate their role in cytotoxicity further.
Mouse Strains and Genetic Analysis.
gzmA×gzmB
ko mice
(gzmA×B
/
) were generated by crossing gzmA
/
(22) with
gzmB
/
(18) mice and by subsequent intercrossing of heterozygous F1 animals. C57BL/6 (B6), BALB/c (B/c), gzmA
/
,
gzmB
/
, and two strains of perforin deficient mice, perforin
/
(B6 background; reference 2) and perforin
/
(129 × B6 background; reference 24), were maintained at the Max-Planck-Institut
and the John Curtin School of Medical Research under pathogen-free conditions. No differences were found in any of the experiments between the two perforin
/
strains. Only mice of the
same sex were used in individual experiments at 12-20 wk of age.
/
: 5
-AGG AGC AAT ATA TAC CAA
TGG-3
and 5
-AGG TAG GTG AAG GAT AGC CAC-3
;
neo-primer: 5
-CGG AGA ACC TGC GTG CAA TC-3
.
gzmB
/
: 5
-CTG CTA CTG CTG ACC TTG TCT-3
and
5
-TGA GGA CAG CAA TTC CAT CTA-3
; neo-primer: 5
-TTC CTC GTG CTT TAC GGT ATC-3
. Perforin
/
: 5
-CCA
CTC CAC CTT GAC TTC AAA AAG GCG-3
and 5
-TGG
GCA GCA GTC CTG GTT GGT GAC CTT-3
; neo-primer:
5
-CGG AGA ACC TGC GTG CAA TC-3
. The genomic
DNA was subjected to amplification by PCR and analyzed as
previously described (22).
Fig. 1.
Analysis of wild-type (C57BL/
6) and the mutant mice gzmA/
, gzmB
/
,
gzmA×B
/
, and perforin
/
mice by
PCR. Tail DNA from individual mice was
analyzed by PCR amplification using the
gzmA-, gzmB-, and perforin-specific primer pairs, as indicated in Materials and Methods.
[View Larger Version of this Image (29K GIF file)]
Target Cells. P815 (H-2d), EL4.F15 (EL4), (H-2b), L1210.3 (H-2d), and L1210.Fas (EL4 transfected with Fas cDNA, H-2d; reference 1), TA-3 (H-2d), A1.1 (H-2d), and YAC-1 (H-2a) were maintained in culture as has been previously described (22, 25).
For detection of influenza virus-immune Tc cells, EL4 cells were pretreated with 10Generation of Tc and NK Cells In Vitro and In Vivo.
To generate alloreactive Tc cells in vitro, H-2b responder splenocytes from
mutant and B6 mice (106/ml) were cultivated together with irradiated (3,000 rad) stimulator splenocytes from B/c mice (106/ml)
at a ratio of 2:1 for 5-6 d. Restimulation for secondary MLC was
performed by incubating in vitro-derived Tc cells (5 × 104/ml)
with stimulator (2.5 × 106/ml) cells in medium supplemented
with supernatant (10% final solution) from rat spleen cells desensitized with 5 µg/ml Con A in medium for 24 h (Con A-SN, supplemented with 20 mg/ml -methyl-D-mannoside; Sigma
Chemical Co., Munich, Germany).
Cytotoxicity Assays. All cytotoxicity assays were performed in cell culture medium, in which FCS was replaced by BSA (2 mg/ml). The 51Cr-release assay was performed for 2-10 h, as previously described (22). The 125I-DNA-release assay was performed essentially as previously described (31), with some modifications (22). Target cells (2 × 106) were labeled with 10 µCi 125I-deoxyuridine (125I-UDR; Amersham Corp., Arlington Heights, IL) in 400 µl in polystyrol tubes for 3 h. Effector cells were mixed with 2 × 103-2 × 104 labeled target cells in triplicates at the indicated effector/target ratio (e/t) in 200 µl IMDM, complemented with 12 mM Hepes and 2 mg/ml BSA. The plates were centrifuged at 500 rpm for 5 min and incubated for 1 h. After centrifugation (1,200 rpm, 10 min) 100 µl of supernatant was removed and 100 µl of lysis buffer (10 mM Tris/HCl, pH 7.5, 1 mM EDTA, 0.4% Triton- X-100) was added. After 10 min of incubation at room temperature, the plates were centrifuged (1,200 rpm, 10 min). 100 µl of supernatant was collected and released radioactivity was measured. In some experiments, 3 mM EGTA plus 4.5 mM Mg2+ (EGTA-Mg2+) was added to the assay to exclusively assess Fas-based cytotoxicity (32).
In vitro-derived H-2d-reactive Tc cells from gzmA/
,
gzmB
/
, gzmA×B
/
, perforin
/
, and B6 mice were
tested for their ability to induce 51Cr-release in various target cell populations in 2-10-h cytotoxicity assays. Representative experiments are shown in Fig. 2, A-E. Tc cells
from all gzm ko mice, including gzmA×B
/
, expressed
cytolytic activities with kinetics and levels comparable to
those seen with B6 Tc, when tested on either L1210.3,
L1210.Fas, A1.1, or TA3 lymphoma target cells (Fig. 2, A-D,
top panels) and P815 mastocytoma targets (data not shown). In
addition, Tc cells from gzmA
/
, gzmB
/
, gzmA×B
/
,
and B6 also lysed L1210.Fas target cells in the presence of EGTA-Mg2+ to the same extent, when tested at 10 h, indicating the involvement of the Fas pathway (1). Tc cells
from perforin
/
mice did not express any cytolytic activity
on the respective target cells with the exception that 51Cr-release was observed with L1210.Fas target cells in a 10-h assay, which was reduced in the presence of EGTA-Mg2+.
As slight variations in lytic activity of gzmA×B
/
T cells
occurred between individual sets of experiments, we tested the cytolytic activity of alloreactive Tc cell populations
from eight (four male, four female) individual gzmA×B
/
mice and compared them with B6 alloreactive Tc cells in
a primary and secondary MLC (Fig. 2 E, only shown for
male mice). No differences in lytic activity could be discerned. Similar results were obtained with in vivo-derived
H-2d reactive splenocytes from two individual mice of B6,
gzmA
/
, gzmB
/
, gzmA×B
/
, and perforin
/
strains
(Fig. 3). This was true for both target cells, L1210.3 and
L1210.Fas, with the exception of one gzmA×B
/
mouse,
which showed reduced cytolytic activity on both target cells.
However, in vivo-generated alloreactive Tc cells from perforin
/
mice lysed L1210.Fas (Fig. 3) and P815 (data not
shown) targets at levels comparable to those from B6 and
gzm
/
mice. Lysis in the presence of EGTA-Mg2+ was
only marginally reduced. The phenotype of the in vitro-
and ex vivo-derived cytolytic cells from all mutant and B6
mice was CD4
CD8+ and no cytolytic activity was observed on H-2-matched EL4 target cells (data not shown).
The differences observed in the cytolytic activity of in vitro-
versus ex vivo-derived alloreactive Tc cells from perforin
/
mice may reflect differential regulation of Fas ligand and Fas expression during ex vivo induction and in vitro culture.
To determine if ex vivo-derived virus-specific Tc cells
possess cytolytic phenotypes similar to those of in vitro- or
ex vivo-derived alloreactive Tc cells, we tested ex vivo-
derived influenza virus A/WSN-immune Tc cell for 51Cr-release of NPP-modified EL4 target cells (Fig. 4). Effector populations of all three infected gzm mutant mouse strains,
but not perforin/
mice, showed specific cytolytic activities in 2- and 4-h assays, at levels and with kinetics comparable to those of B6 mice. No 51Cr-release was seen with
any of the Tc populations on untreated EL4 cells (data not
shown).
To assess the cytolytic activities of ex vivo-derived NK
cell populations of mutant and B6 mice, recipients were
treated with either poly I:C (29) or SFV (30). Splenocytes
were tested for lytic activity on 51Cr-labeled YAC-1 target
cells. Similar levels of cytolytic activity were observed with
effector cell populations from gzmA/
, gzmB
/
, gzmA×
B
/
, and B6 mice, when assayed between 2 and 4 h (Fig. 5,
top). The occasional reduction in the activity of NK cells
from gzm single or double ko mice as compared to B6
mice to induce 51Cr-release was neither significant nor reproducible in three additional experiments (data not shown).
As shown in previous studies (2), marginal or no NK cell-
mediated lytic activity was obtained with splenocytes from
perforin
/
mice.
Role of gzmA, gzmB, and Perforin in Target Cell DNA Fragmentation.
In vitro-derived H-2d-reactive Tc cells
from gzmA/
and B6 mice were indistinguishable in their
ability to induce DNA fragmentation in L1210.3, L1210.Fas,
A1.1, and TA3 target cells when tested between 2 and 8 h
of incubation (Fig. 2, bottom panels). In contrast, Tc cells
from gzmB
/
mice were defective in their nucleolytic potential on the same targets in short term assays, i.e., between 2 and 4 h. However, after incubation of 6-8 h, this
defect was either partially (L1210.3, 6 h) or completely
(L1210.Fas, 6 h; A1.1, 6 h; TA3, 8 h) abrogated. Most importantly, the absence of nucleolytic activity was more pronounced in Tc cells from gzmA×B
/
as compared to
gzmB
/
mice and only marginal nuclear damage was seen
in long-term assays with gzmA×B
/
Tc cells. Although
gzmA×B
/
Tc cells occasionally induced some DNA fragmentation in L1210.Fas and P815 target cells (data not
shown), the level of nucleolytic activity was always lower
than that seen with gzmB
/
Tc cells. Marginal or no nucleolytic activity was seen with Tc cells from perforin
/
mice. No 125I-release in H-2-matched control EL4 cells
was seen with any of the five Tc cell populations (data not
shown).
The analysis of the specific nucleolytic potential of ex
vivo-derived influenza virus-immune Tc cell populations
revealed that in a 2-h assay, perforin/
, and gzmA×B
/
mice were defective, whereas Tc cell populations from
gzmA
/
, gzmB
/
, and B6 mice each induced significant
and similar amounts of 125I-release in NPP-modified EL4
target cells (Fig. 4, bottom) at high e/t ratio. Interestingly, in
4-h assays similar levels of DNA fragmentation were obtained on NPP-modified EL4 target cells with Tc cells
from all mice tested, including gzmA×B
/
and perforin
/
.
Since EL4 cells express Fas (data not shown), Fas-dependent nucleolytic activities of ex vivo-derived influenza virus immune Tc cells may contribute to DNA fragmentation of target cells at later stages of the assay.
When tested for their nucleolytic activity on YAC-1 target cells in 2- and 4-h assays, ex vivo-derived NK cells
from perforin/
and gzmA×B
/
mice were defective
and caused, if any, only marginal DNA fragmentation. In
contrast, NK cells from gzmA
/
and gzmB
/
mice expressed nucleolytic activities which were already apparent in 2-h assays and increased in 4-h assays, comparable with
those of B6 mice.
The results of this study show that perforin-mediated target cell lysis by in vitro- and ex vivo-derived Tc and NK cells is independent of gzmA and B, but that DNA fragmentation induced by this pathway may involve both enzymes. gzmB appears to be the main effector molecule acting early during leukocyte-mediated apoptosis, whereas gzmA nucleolytic activity acts with delayed kinetics that may complement or substitute for a gzmB deficiency.
The data obtained with gzmA/
and gzmB
/
single ko
and gzmA×B
/
double ko mice clarify previous conflicting
results regarding the role of the two enzymes in Tc/NK
cell-induced 51Cr-release from target cells. The fact that a
noncytotoxic rat basophilic leukemia mast cell tumor line,
transfected with perforin cDNA, only conferred optimal
cytolytic activity upon coexpression of gzmA and B, was
taken as evidence for a synergistic role of the two enzymes
in perforin-mediated target cell death (11). On the other
hand, the recent findings that Tc/NK cell-induced 51Cr-release was not altered in gzmA
/
or gzmB
/
single ko
mice (18, 22, 23) suggested that neither of the two proteases
alone is critical for perforin-induced target cell membrane
damage. The present demonstration that in vitro- and ex vivo-
derived Tc and NK cell populations from gzmA×B
/
mice induced lysis in various target cells, at levels and with kinetics similar to those of single mutant or B6 mice, unequivocally demonstrates that the cytolytic pathway leading
to 51Cr-release is independent of gzmA and gzmB. However, the participation of other, as yet undefined, proteases
in perforin-mediated lysis cannot be ruled out. At present,
there is no obvious explanation for the discrepancy between the results presented here and a previous report
demonstrating a role for gzmB in NK- but not Tc- or LAK cell-mediated 51Cr-release (33). It is particularly confounding that comparable ex vivo-derived NK cell populations
(poly I:C) and target cells (YAC-1) were used in both studies.
Several recent reports have already provided evidence for
the involvement of gzms in perforin-initiated nuclear damage (10), the absence of early DNA fragmentation in
leukocyte-mediated killing of gzmB/
mice being the
most definitive (18). However, no such involvement of gzmA
in these processes could be established using gzmA
/
mice
(22, 23). The evidence suggests that the biological functions of gzmA and B are not merely redundant. This was
deduced from the distinct substrate specificities of gzmA
and B (34, 35) and because gzmA is not critical during early
events of DNA fragmentation (22, 23). However, an involvement of gzmA in later stages of this pathway could not
be excluded. This is also supported by the recent demonstration that inhibitors of caspases, in particular CPP32,
prevent the expression of early gzmB-based but not late
gzmA-based nucleolytic activity by Tc cells (19). This study
provides definitive evidence for such an interpretation and
presents additional information in that in vitro- and ex
vivo-derived gzmA×B
/
Tc/NK cells are distinct from
those of gzmB
/
mice in their perforin-based nucleolytic
activity. Impairment of nucleolytic activity of gzmA×B
/
Tc/NK cells was shown in short-term and, in most cases,
also in long-term assays.
In contrast to cytolytic and nucleolytic activities observed with in vitro-generated Tc cells from B6 and mutant mice, data obtained with ex vivo-derived influenza immune Tc cells were variable. This may be due to the
distinct genotypes of gzmA/
(B6; reference 22) versus
gzmB
/
(129 × B6; reference 18) and gzmA×B
/
(129 × B6) mice. It is well established that genes outside the MHC
play a crucial role in the recovery from ectromelia, a natural viral pathogen, and from influenza infection (reference 36
and Simon, M.M., and A. Müllbacher, unpublished data).
Thus, the various responses to the pathogens by non-MHC
genes may influence the kinetics of NK/Tc cell development
in gzmA
/
and gzmB
/
mice, thereby masking the actual
role of gzms in cytolysis/nucleolysis. To alleviate this problem we are currently breeding the gzmB
/
gene locus
onto the B6 background. This will provide us with the
ability to reveal the biological roles of gzmA, gzmB, and perforin not only in target cell cytolysis/nucleolysis, but
also in immunopathology and in the control of infections.
Address correspondence to Dr. Markus M. Simon, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany. Phone: 49-761-5108-533; FAX: 49-761-5108-529; E-mail: simon{at}immunbiol.mpg.de Klaus Ebnet's present address is Institut für Zellbiologie, ZMBE, D-48149 Münster, Germany.
Received for publication 13 August 1997 and in revised form 22 September 1997.
We would like to thank Ian Haidl for reading the manuscript and for valuable suggestions. We also thank the laboratory of R. Zinkernagel (Zürich, Switzerland) for the generous gift of perforin (B6) ko mice and T. Ley (St. Louis, MO) for providing the gzmB ko mouse.
This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft, Si 214/7-1.
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