1. Art and Diseño

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RAPID COMMUNICATION
The Role of Granzyme B in Murine Models of Acute Graft-Versus-Host
Disease and Graft Rejection
By Timothy A. Graubert, John H. Russell, and Timothy J. Ley
A complete molecular description the
of syndromes of graftversus-host disease (GVHD) and graft rejection could have
a significantimpact on clinical bone
marrow transplantation.
Recent in vitro experiments (Heusel et al. Cell 76977, 1994
and Shresta et al. Proc Natl Acad Sci USA 92:5679, 1995)
have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes(CTL) and natural killer (NK)
cells,
respectively,
initiate program
a
cell
of
death
(apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm
B). In the present study,we have analyzedthe phenotype of
gzm B-deficient mice using experimental transplant models
designed to isolate their CD8' CTL, CD4+ CTL, and NK com-
partments. We found a significantimpairment in class I-dependent GVHD mediated by gzm B -/- CD8' CTL, whereas
class Il-dependent GVHD was not altered using gzm B -/CD4' effectors. In ahybridresistancemodel, gzm B -/hosts rejected haplo-identical marrow grafts as efficiently
as did their wild-type littermates. This result is surprising
in
light of a severe defectin the ability of gzm B -/- NK cells
to induce apoptosis in susceptible targets in vitro. These in
vivo data define a significant role for gzm B in cytotoxicity
mediated by CD8' CTL, but not by C54+ CTL. Furthermore,
these resultsdo not support amodel of hybrid resistancein
which NK cells play a pivotal role.
0 1996 by The American Society of Hematology.
G
In vitro analysis of immune effector cells from mice bearing a null mutation in the gzm B locus has shown profound
functional defects. In standard lytic assays, gzm B-deficient
CD8' CTL and lymphokine-activated killer (LAK) cells exhibit a severe defect in theirability
to rapidly induce
apoptosis in allogeneic target cells, but late cytotoxicity recovers
In contrast, gzm
B-deficient NK cells cannot induce apoptosis in target cells despite high effector:target ratios or prolonged incubation times.'
The Chbdiak-Higashi Syndrome, an inherited disorder of
humans resulting in increased susceptibility to infections,
provides independent confirmation of the critical importance
of the granule exocytosis pathway for CTL- and NK-mediated cytotoxicity. CTL, NK cells, and neutrophils from these
patients share a common defect in granulopoiesis. The primary and secondary granules fuse aberrantly; consequently,
the lytic contents cannot be delivered to a target cell.' AIthough Chkdiak-Higashi CTL still exhibit some cytotoxicity,
Chbdiak-Higashi NK cells are virtually devoid of cytotoxicity.' Mice homozygous for the beige mutation have a similar
phenotype." These observations support the conclusion
drawnfromthe
analysis of perforin and gzm B-deficient
mice that the granule exocytosis pathway is important for
CTL cytotoxicity and essential for NK cytotoxicity.
Murine transplant models developed inthe 1980s dissected the roles of CD4' and CD8' CTL in the pathogenesis
of GVHD.I"l3 Congenic strains of C57BW6 mice expressing
either a mutant class I (B6.C-H-2b'"l) or class I1 (B6.C-H2b""2)allele served as hosts for these experiments. These
mice were reconstituted after lethal irradiation with syngeneic marrow combined with purified T-cell subsets from the
partially mismatched parental strain (BL/6, H-2b).After alloattack by T cells on the marrow graft, the hosts succumbed
as a result of marrow aplasia at day 10 to 14 (in the case of
a class I mismatch) or day 20 to 24 (class 11 mismatch)
post-BMT. These results indicated that single antigen H-2
mismatches that provoke either class I- or class 11-restricted
responses canleadto100%mortality
from acute GVHD
when as few as IO5 donor-derived T cells are infused.
The cellular basis of the host-versus-graft (ie, rejection)
response has not beenas well characterized. The prototypical
RAFT-VERSUS-HOST disease (GVHD) and graft rejection remain significant obstacles for successful allogeneic bone marrowtransplantation (BMT). The molecular
programs used by the immune effector cells responsible for
these clinical syndromes, cytotoxic lymphocytes (CTL) and
natural killer (NK) cells, are a subject of intense investigation. One manner in which these cells mediate tissue destruction is by direct contact-dependent cytotoxicity, resulting in
apoptotic death of the target cell. The granule exocytosis
pathway is probably the most important apoptotic mechanism used by CD8'
CTL and NK cells. In this model, the
recognition and tight binding of a susceptible target cell by a
CTL or NK cell causes vectorial alignment of their secretory
apparatus, promoting delivery of electron dense cytoplasmic
granules to the target cell membrane at areas of close intercellular contact.' One granule constituent is perforin, a proteinthat undergoes calcium-dependent polymerization on
the target cell membrane, forming a complement-like lesion
through whichother granule constituents, including the granzymes, probably enter the target cell cytoplasm.' The delivery of the granule component proteins, including granzymes
A and B, is required for induction of apopto~is.~.~
Granzyme
B (gzm B), a neutral serine protease that cleaves substrates
with aspartic acid at the P1 site,' is essential for the rapid
induction of this apoptotic pathway.
From the Division of Bone Marrow Transplantation and Stem
Cell Biology, the Departments of Internal Medicine and Genetics,
and the Department of Molecular Biology and Pharmacology.
Washington University Medical School, St Louis, MO.
Submitted November 6, 1995; accepted November 27, 1995.
Supported by National Institutes of Health Grants No. CA49712,
DK49786, und DK38682 and the Washington University-Monsanto
agreement.
Address reprint requests to Timothy J. Ley, MD, Washington University Medical School, Campus Box 8007, 660 S Euclid Ave, St
Louis. MO 63110.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore behereby marked
"advertisement" in accordance with I8 U.S.C. section 1734 solely 10
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8704-0049$3.00/0
1232
Blood, Vol 87,No 4 (February 15). 1996:pp 1232-1237
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1233
GRANZYME B IN GVHDAND GRAFT REJECTION
host-versus-graft reaction is rejection of parental marrow by
the F1 progeny of two inbred strains (a -+ a X b), a phenomenon knownas hybrid resistance.I4.l5Recent transgenic experiment~'~." support the notion that
the effector cell mediating
hybrid resistance lyses target cells that do not display the
full complement of self histocompatibility determinants (the
missingselfhypothesis).18Circumstantialevidencefavors
the NK cell as the likely mediator of hybrid resistan~e,'~-~'
although definitive proof of this is not yet available.
We performed the present study to analyze the contribution of gzm B to cytotoxicity mediated by
CTL and NK
cells in vivo using these established murine models of acute
GVHD and hybrid resistance. Our results suggest that gzm
B plays a significant role in acute GVHD mediated by CD8+,
but not CD4+ CTL. In contrast, gzm B does not appear to
be essential for hybrid resistance. In view ofthe critical role
of gzm B for in vitro NK cytotoxicity, this finding suggests
that NK cells are not required for hybrid resistance.
MATERIALS AND METHODS
Mice. gzm B-deficient mice were derived in a C57BL/6 X 129/
Sv background, as previously described! Heterozygote crosses
yielded littermates genotypically -I- or +I+ at the gzm B locus,
determined by Southern blot analysis of tail DNA.'B6.C-H-2b"",
B6.C-H-2bm'Z,Balbk, CBA/J, and 129/SvJ mice were obtained from
the Jackson Laboratory (Bar Harbor, ME).
Acute GVHD model. Lymphocytes were isolated from the mesenteric lymph nodes of 8- to 12-week-old gzm B +I+ or -I- sexmatched littermate donors by passage through a stainless steel mesh.
T-cell subsets were then prepared, as previously described." In brief,
antibody-mediated complement lysis was performed by adsorbing
antibodies to B cells (monoclonal antibody [MoAb] J1 Id) and CD4'
cells (MoAb RL.172) or CD8' cells (MoAb 3.155) at 4°C for 30
minutes at a concentration of lo7 IymphocyteslmL in RPM1 1640
(Sigma, St Louis, MO) supplemented with 5% (vol/vol) heat-inactivated fetal calf serum, 1 mmol/L sodium pyruvate, 15 mmol/L
HEPES, 2 mmol/L I-glutamine, nonessential amino acids at 0.1 moll
L each, 25 U/mL penicillin, 25 pg/mL streptomycin, and 75 pmoV
L /3-mercaptoethanol. The lymphocytes were then incubated at 37°C
for 1 hour in the presence of complement (rabbit Low-Tox-M; Cedarlane Laboratories, Hornby, Ontario, Canada). T cells were recovered by centrifugation through a 1.119 g/mL Histopaque gradient
(Sigma), washed twice in complete media before counting (>95%
viable by Trypan blue exclusion), and combined at a dose of 1.0 X
lo6 cells with the marrow graft before infusion. Unpooled T cells
derived from single donors were used in all transplants, except for
the fully allogeneic experiments.
Bone marrow grafts were prepared from the hosts by flushing
both femora with 1 mL of 1 X Hebs (20 mmol/L HEPES, pH 7.05,
137 mmol/L NaCI, 5 mmol/L KCI, 0.7 mmol/L Na,HPO,; 6 mmoV
L dextrose) through a 22-G needle. After settling debris, cells were
washed once in phosphate-buffered saline (PBS) and counted. A
dose of 2 X IO6 viable nucleated marrow cells was combined with
the T-cell graft in a volume of 450 pL of PBS and infused via a
lateral tail vein.
Hosts were conditioned with gamma irradiation from a "'CS
source at a rate of 95.2 cGylmin 24 hours before transplantation.
Balb/c hosts received 700 cGy, whereas the bml x 129 and bm12
X 129 hybrids received 1,OOO cGy. Animals were housed in a pathogen-free barrier facility in a manner approved by the Animal Investigation Committee at Washington University. Mice were observed
daily and killed if moribund or unable to take food.
Hybrid resistance model. gzrn B -1- or +I- (H-2bk)F2 hybrid
hosts (identified by Southern blot analysis of tail DNA and immunophenotyping of peripheral blood) were conditioned with 800 cGy 24
hours before intravenous (IV) infusion of 6 X 10' nucleated marrow
cells prepared from BL/6 X 129 (H-2b) donors. Engraftment was
measured at day +5 by the method of Bennett et al.', In brief,
each animal received a single injection of 10" m o w 5-flouro-2'deoxyuridine (to inhibit endogenous thymidylate synthetase) intraperitoneally (IP) followed 1 hour later by0.5 pCi '''IUdR (ICN
Biomedicals, Costa Mesa, CA) in 0.03 m o m citrate. Eighteen hours
later, the animals were killed and splenic incorporation of '%JdR
was measured in a gamma counter. Splenic uptake of the label,
an index of engraftment, was calculated by the following formula:
[(Spleek,,, - Mock Transplant Spleen,,,)/O.5 pCi dosecpm]X 100.
In a second series of experiments, gzm B -/- or +I- F2 hybrid
hosts received a single neutralizing dose (250 pg IP) of monoclonal
) ~antimouse
~
y interantimouse tumor necrosis factor a ( T N F ~or
feron (yIFNZ5;both provided byDr R Schreiber, Department of
Pathology, Washington University, St Louis, MO) 24 hours before
transplantation. The remainder ofthe protocol was performed as
discussed above.
H-2 immunophenotyping. One hundred microliters of whole
blood was obtained from the retro-orbital sinus of an anesthetized
animal. After 10 minutes of incubation at 4°Cin redblood cell
(RBC) lysis buffer (150 mmol/L NH,Cl, 10 mmol/L KHCO,, 0.1
mmol/L Na2EDTA, pH 7.1-7.4), the nucleated cells were washed
twice in PBS and resuspended in serum-free media. Cells (IO') were
stained with fluorescein isothiocyanate (FITC) antimouse H-2Kband
phycoerythrin (PE) antimouse H-2K' (Pharmingen, San Diego, CA)
and analyzed on a FACScan (Becton Dickinson Immunocytometry
Systems, San Jose, CA).
Statistics. Survival after BMT in the GVHD model was analyzed
by the Kaplan-Meier method.26 The groups were compared using
Log-Rank analysis. The Mann-Whitney test was used to compare
engraftment among the experimental groups in the hybrid resistance
model.
RESULTS
Acute GVHD model: CD8+ CTL. A single antigen class
I mismatch acute GVHD model was created using (bml X
129) F1 miceas hosts. These F1 hybrids share all
minor
histocompatibility loci with the gzrn B (BW6 X 129) T-cell
donor, ensuring thattheonlyallogeneicstimulusderives
from the mutant H-2b"'1 (class I) allele. Balb/c (H-zd) mice,
mismatched at all major and minor loci relative to the gzm
B mice, served as hosts in the fully allogeneic acute GVHD
model. The experimental design involved conditioning the
hosts with lethal irradiation and then rescuing them with an
infusion of syngeneic
marrow from a sex-matched littermate.
gzm B +/+ or -/-, CDCdepleted, CD8+ T cells from littermate donors were coinfused with the marrow graft into
sex-matched littermate hosts. With an intact T-cell effector
pathway, the target marrow cells are killed, leading to death
of the host from pancytopenia. If the gzm
B null mutation
disarms the T cells, the syngeneic marrow engrafts and the
animals survive. Mock-transplanted(PBSonly)negative
control hosts uniformlydiedofmarrowaplasiawithin
2
weeks. Onehundredpercentsurvivalofpositivecontrol
hosts (receiving marrow grafts without
T cells) confirmed
that the conditioning regimen did not put theanimals at risk
for death from nonhematologic causes.
CD8+ CTL directedagainst the singleantigen class Imismatched bml X 129 hosts caused significantly less death
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1234
GRAUBERT, RUSSELL, AND LEY
L
I
l
e
n
0.2
4
0.4
c
L
: i ;......, &m
B 4-CDB' T cells (n=25)
...__......._._...
1
I
0.0
G m B +/+ CDB' T cells (n.25)
mock ( n 4 )
0.0
I
I
I
I
l
l
I
l
0
10
20
30
40
50
M)
70
Days post BMT
Fig 1. Probabilityof survivalafter transplant of syngeneic marrow
combined with class l-mismatched CD8' T cells from gzm B + I + or
-I- donors. Survival is prolonged in recipients of gzm B - I - T cells
(P= .00657). Data are from eight separate experiments. All animals
receiving marrow grafts without T cells survived. All mock-transplanted animals died of pancytopenia at day 10 to 12.
from acute GVHD (Fig 1) when the lymphocytes were derived from gzm B -/- donors compared with gzm B +/+
donors (36% v 12% at 40 days; P = .00657). gzm B +/+
and +/- T cell donors were indistinguishable in this analysis
and were used interchangeably in these and subsequent experiments. Complete blood counts at day 10 post-BMT documented severe pancytopenia only in those animals that subsequently died of acute GVHD (data not shown).
Similar results were obtained in the fully allogeneic model
(Fig 2). Significantly fewer Balb/c hosts survived when gzm
B +/+, CDCdepleted, CD8+ T-cell donors were used compared with gzm B -/- donors (40% v 6% at 40 days; P <
.00622). These transplants into hosts mismatched at all H-2
and non-H-2 loci confirm that the data obtained in the bml
system are accounted for by the single class I mismatch and
i
0.2
L
..
G m B 4- CDB' T cells (n=15)
"
"
"
"
"
. .. . ..
0.0
0
T-
0
:
G m B +I+ CDB' T cells ( ~ 1 % )
..................................
I
I
I
l
l
I
1
10
20
30
40
50
60
70
Days post BMT
Fig 2. Probabilityof survival after transplant of syngeneic marrow
combined with fully mismatched CD8' T cells from gzm B +I+
or - I - donors. Survival is prolonged in recipients of gzm B - I - T
cells (P= .00622). Data are from six separate experiments.
2
4
6
8
10
1216
14
i
,
18
j
20
22
Days post BMT
Fig 3. Probabilityof survivalafter transplant of syngeneicmarrow
combined with class Il-mismatchedCD4+ T cells from gzm B + I + or
-I- donors. Survival is not significantly different between the
groups (P= .31254). Data are from four separate experiments.
are not anartifact of minor histocompatibility loci introduced
by the BL6 X 129 strain differences.
Of note, mortality in the gzm B +/+ cohort did not reach
loo%, as seen by Sprent et al.'3 This may be due to strain
differences in our model or to a reduced susceptibility to
death from infection in contemporary barrier facilities.
Acute GVHD model: CD4+ CTL. A single antigen class
I1 mismatch acute GVHD model was created using (bm12
X 129) F1 mice as hosts. In this case, gzm B +/+ or -/-,
CD8-depleted, CD4' T cells from littermate donors were
infused into sex-matched littermate bm12 X 129 hosts,
which were histocompatible at all major and minor loci except for the single mutant class I1 (H-2bm12)
allele. In contrast
to the class I model, there was no difference in survival of
the bm12 X 129 hosts when gzm B +/+ or -/- CD4+ CTL
were infused (Fig 3). A lower T-cell dose would be unlikely
to bring out a phenotypic difference between the two experimental groups, because mortality in the wild-type group is
reduced when fewer than lo6 cells are used.13 These findings
support the hypothesis that the perforin-granzyme pathway
is notessential for CD4+ CTL-mediated cytotoxicity in vivo.
Hybrid resistance model. Hosts for the hybrid resistance
model were created by crossing CBA/J (H-2k) and gzm B
-/- (H-2b) mice. The F1 progeny were then backcrossed to
the gzm B -/- parent. F2 hybrids bearing both haplotypes
(H-2b")and either gzm B genotype (+/- or -I-) were
the experimental hosts. Engraftment was measured after the
infusion of wild-type BL6 X 129 (H-2') marrow into a lethally irradiated host. The missing self hypothesis predicts
that a radioresistant cell within the F2 hybrid hosts will
recognize and kill the parental marrow stem cells because
those cells do not express the H-2k haplotype. Homozygotes
(H-2b)within theF2 litters provided concurrent positive controls because they provide no barrier to engraftment of the
parental BW6 X 129 marrow. Mock-transplanted animals
(PBS only) were used as negative controls.
All syngeneic control animals engrafted, with splenic
Iz5IUdRincorporation (mean, 2.2%) in good agreement with
previously published report^.'^ As expected, gzm B +/- H2b/khosts rejected bone marrow grafts from the H-2' parental
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1235
GRANZYME B IN GVHD AND GRAFTREJECTION
Q
3
h
syngeneic
mock
GUnB+/-
OUnBJ-
Fig 4. Engraftment(as measured by splenic '261UdR incorporation)
after transplantation of 6 x 10' parental bone marrow cells (H-Zb)
into lethally irradiated ( 8 0 0 cGy) F2 hybridhosts IH-2b'k)that are
homozygous or heterozygous for the gzm B null mutation. Hybrid
resistance was not significantly alteredthe
in gzm B -I- hosts compared with the gzm B +/- hosts ( P = 5672). Positive control hosts
eresyngeneic wlth themerrowdonor,whereasnegativecontrol
hosts are mock-transplantedwith PBS only. Results are the mean
2
SD of three separate experiments.
strain, as reflected by very low splenic Iz5IUdRuptake (mean,
0.19%; Fig 4). Surprisingly, gzm B -/- H-2m hosts also
rejected the mismatched marrow completely (mean
uptake, 0.08%). Pretreatment of the gzm B +/- or 4hosts with antimouse TNFa (n = 9), yIFN (n = 2), or the
combination of both antibodies (n = 2) did not significantly
change the outcome, because persistent complete rejection
of haplo-identical marrow was observed (data not shown).
'%JdR
DISCUSSION
The models used in the present study provide tools for
the in vivo dissection of the role of gzm B in each of the
lymphocyte compartments. To summarize the findings, we
note a moderate, but significant reduction in acute GVHD
mediated by gzm B 4- CD8+ CTL as compared with wildtype CD8+ CTL. In contrast, CD4+ CTL-mediated cytotoxicity and hybrid resistance were not affected by the loss of
gzm B activity in these models.
The molecular basis of cellular cytotoxicity is being elucidated using complementary experimental systems. Evidence
accumulated thus far implicates two major pathways used
by CTL. The importance of the Ca'+-dependent granule exocytosis pathway has been shown by the targeted disruption
of the key effector molecules, perforin and gzm B. CTL and
LAK cells from perforin- or gzm B-deficient mice have a
severe defect in their ability to rapidly induce apoptosis in
susceptible target ~ e l l s . ~ NK
, ' ~ cells from these mice are
completely unable to induce apoptosis in susceptible target
cell^.^.'^
The second mechanism of cellular cytotoxicity involves
the Ca'+-independent interaction of Fas, a ubiquitously expressed 45-kDtransmembrane protein in the TNF receptor
family, with its ligand present on the surface of CTL.** Al-
though Fas-induced cell death may serve primarily an immunoregulatory role,29*30Fas-dependent cytotoxicity can be
shown against nonlymphoid targets as well.31CTL from mice
with naturally occurring loss of function mutations of Fas
or its ligand have compromised cytotoxicity, particularly in
the CD4+ ~ompartment.~'
Although CTL may also kill target cells through secretion
of soluble mediators such as TNFa, experiments with mice
genetically deficient in both perforin and Fas suggest that
these two pathways account for nearly all T-cell cytotoxicity
in standard in vitro lytic assay^.^^.^
CD8+ CTL in the experiments described here were able
to kill mismatched bone marrow cells and cause aplastic
death in over half of the animals analyzed, despite the lack
of gzm B. This implies that, just as the in vitro assays have
shown, gzm B-independent apoptotic mechanisms are operative in CD8+ CTL. It further follows from the in vitro experiments that the Fas-Fas ligand interaction probably accounts
for much of this residual cytotoxicity in gzm B-deficient
mice. CTL deficient in both gzm B and Fas ligand have
a killing defect intermediate between gzm B-deficient and
perforin/Fas ligand-deficient CTL (S. Shresta and T.J. Ley,
unpublished observations). Because perforin is required for
delivery of gzm A and B to the target cell cytoplasm, but is
unable to induce apoptosis itself,35gzm A may account for
the difference between the gzm B/Fas ligand-deficient and
the perforin/Fas ligand-deficient CTL in these assays. Therefore, we would predict that the gzm B-independent cytotoxicity in this GVHD model is mediated primarily by the Fas
pathway with a smaller contribution from gzm A.
Our finding that gzm B is important, butnot essential
for CD8+ CTL-mediated attack on allogeneic hematopoietic
grafts is consistent with the recent report that perforin-deficient mice have impaired, but not fully disabled resistance
to cardiac allograft^.^^ In addition, Selvaggi et a137 have
shown that bone marrow grafts from perforin-deficient mice
will survive and engraft in fully incompatible hosts with no
mortality from GVHD unless the T-cell inoculum is increased substantially. Although this model differs from ours,
it further supports the conclusion that the perforin/granzyme
pathway is critical for in vivo cytotoxicity in the CD8+ compartment, but that important redundancy (eg, Fas) remains.
CD4+ CTL-mediated cytotoxicity, in contrast, appears to
be completely gzm B-independent in the class 11-restricted
GVHD model. This is consistent with the in vitro data that
suggets that Fas is the sole mediator in this compartment.
This possibility is being formally tested using Fas liganddefective CTL in similar transplant experiments.
It is harder to reconcile the failure of gzm B deficiency
to impact on hybrid resistance with the strong in vitro phenotype of gzm B -/- NK cells. One possible explanation is
that the pool ofNK cells is heterogenous, ie, armed with
different effector molecules. Similarly, individual NK cells
may have redundant pathways (eg. Fas or other granzymes)
that are used in vivo, but escape detection in an in vitro
assay using homogenous targets such as YAC-1 cells. However, Fas is unlikely to account for gzm B-independent NK
cytotoxicity, because Fas ligand-deficient NK cells have normal activity in lytic assays against NK susceptible target
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1236
GRAUBERT, RUSSELL, A N D LEY
cells (S. Shresta and T.J. Ley, unpublished observations).
Furthermore, allogeneic resistance was recently shown to be
unaffected by null mutations of perforin and Fas.3x This
experiment provides strong independent confirmation of our
observation that mutations that fully disarm NK cells have
no effect on hybrid resistance. Finally, the secretion o f the
cytotoxic cytokines TNFa or yIFN do not seen to account for
the gzm B-independent hybrid resistance we have observed,
although the in vivo administration of antibodies does not
conclusively rule out the possibility that some biologically
active cytokine may remain. In summary, it appears that
hybrid resistance remains intact when NK cells are fully
disarmed.
Alternatively, the paradigm that NK cells mediate hybrid
resistance maybe incorrect. Complementary experiments
from Aguila and Weissmad8”support this conclusion. These
investigators found that a sorted population of NK1.1+ cells
from an H-2 hybrid or allogeneic background were unable
to lyse hematopoietic stem cells invitro.38a Furthermore,
transgenic animals overexpressing a “gzm A-driven’’ diphtheria toxin gene39had no alteration in hybrid resistance,
although these animals contain little, if any, NK activity.3xa
This provides strong evidence that NK cells are not essential
for hybrid resistance. In this case, another radioresistant immune effector cell (such as the macrophage) may account
for hybrid and/or allogeneic resistance. Although it is clear
from our workand the work of Aguila et al that hybrid
resistance isnot altered whenNK cells are disarmed or
deleted, it remains a formal possibility that NK cells are the
primary mediators of hybrid resistance in the intact animal
and thatan alternative effector cell with allogeneic restriction
is able to fully compensate in the absence of an active NK
compartment.
Genetic models such as these mayultimately lead to a
complete understanding of the molecular basis of immune
cell effector function. Hopefully, this will provide new therapeutic strategies for the clinical problems of GVHD and
graft rejection.
ACKNOWLEDGMENT
We thank Robin Wesselschmidt and Pam Goda for expert technical assistance. We thank H.L. Aguila, I.L. Weissman, and E.R. Podack for sharing their results before publication.
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1996 87: 1232-1237
The role of granzyme B in murine models of acute graft-versus-host
disease and graft rejection
TA Graubert, JH Russell and TJ Ley
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