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TRANSPLANTATION
Protection from lethal murine graft-versus-host disease without compromise
of alloengraftment using transgenic donor T cells expressing
a thymidine kinase suicide gene
William R. Drobyski, Herbert C. Morse III, William H. Burns, James T. Casper, and Gordon Sandford
Donor T cells play a pivotal role in facilitating alloengraftment but also cause graftversus-host disease (GVHD). Ex vivo Tcell depletion (TCD) of donor marrow is
the most effective strategy for reducing
GVHD but can compromise engraftment.
This study examined an approach
whereby donor T cells are selectively
eliminated in vivo after transplantation
using transgenic mice in which a thymidine kinase (TK) suicide gene is targeted
to the T cell using a CD3 promoter/
enhancer construct. Lethally irradiated
B10.BR mice transplanted with major histocompatibility complex (MHC)–incompatible
TCD C57BL/6 (B6) bone marrow (BM) plus
TKⴙ T cells were protected from GVHD
after treatment with ganciclovir (GCV) in a
schedule-dependent fashion. To examine
the effect of GCV treatment on alloengraftment, sublethally irradiated AKR mice
underwent transplantation with TCD B6
BM plus limiting numbers (5 ⴛ 105) of B6
TKⴙ T cells. Animals treated with GCV
had comparable donor engraftment but
significantly reduced GVHD when compared with untreated mice. These mice
also had a significantly increased number
of donor splenic T cells when assessed 4
weeks after bone marrow transplantation.
Thus, the administration of GCV did not
render recipients T-cell deficient, but
rather enhanced lymphocyte recovery.
Adoptive transfer of spleen cells from
GCV-treated chimeric mice into secondary AKR recipients failed to cause GVHD
indicating that donor T cells were tolerant
of recipient alloantigens. These studies
demonstrate that administration of TK
gene–modified donor T cells can be used
as an approach to mitigate GVHD without
compromising alloengraftment. (Blood.
2001;97:2506-2513)
© 2001 by The American Society of Hematology
Introduction
The engraftment of allogeneic bone marrow (BM) is thought to be
dependent on the ability of transplanted donor immune effector cell
populations to eradicate or inactivate host cells capable of mediating rejection of donor hematopoietic stem cells.1-3 A significant
body of evidence indicates that donor T cells have a major role in
this process. Clinical trials have shown that T-cell depletion (TCD)
of donor marrow almost invariably leads to higher rates of graft
rejection.4-6 Moreover, this premise is supported by murine models
of transplantation where the addition of graded doses of donor T
cells to the marrow inoculum has been able to overcome graft
resistance in major histocompatibility complex (MHC)–incompatible
donor/recipient strain combinations.3
Although T cells are able to promote engraftment, they also play
a critical role in the induction of graft-versus-host disease (GVHD),
which is the major complication of allogeneic bone marrow
transplantation (BMT). T-cell recognition of alloantigens in the
context of MHC molecules is the initiating stimulus for graft-versushost (GVH) reactivity.7-11 Thereafter, T cells are thought to mediate
tissue destruction either directly or indirectly by recruiting secondary immune effector populations (macrophages, natural killer cells,
etc), which results in an amplification of immune reactivity and
dysregulated cytokine production.12-15 From a clinical perspective,
the pivotal role that T cells play in the pathogenesis of GVHD
results in the fact that durable engraftment in many patients is
achieved at the expense of increased toxicity from GVHD,
particularly in mismatched related and unrelated BMT.16 Furthermore, because morbidity and mortality from GVHD are inversely
proportional to the degree of donor/recipient histocompatibility,16
the effective application and expansion of allogeneic BMT into
broader clinical settings has been hindered.
The most effective strategy to reduce GVHD has been ex vivo
TCD whereby donor T cells are removed from the donor marrow
graft before transplantation.17,18 Although this approach has effectively reduced both the incidence and severity of GVHD, there has
been a corresponding increase in the risk of graft rejection in some
studies. Efforts to overcome graft resistance by increasing the
intensity of the conditioning regimen have reduced the incidence of
rejection but inadvertently increased regimen-related mortality.19
An alternative strategy is to eliminate host-reactive donor T cells in
vivo rather than ex vivo. With this approach, donor T cells are
given the opportunity to facilitate alloengraftment, but are eliminated before they can initiate a lethal GVH reaction. This strategy
is based on 2 assumptions. The first is that the persistence of mature
donor T cells is not required for long-term viability of the marrow
graft. The second is that durable engraftment can be achieved
before the GVH process progresses to the point where it becomes
unresponsive to selective in vivo T-cell elimination (ie, T-cell
independent). Whether these assumptions are valid is unknown. To
From the Departments of Medicine and Pediatrics, and the Bone Marrow
Transplant Program, Medical College of Wisconsin, Milwaukee, Wisconsin,
and the Laboratory of Immunopathology, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Reprints: William R. Drobyski, Bone Marrow Transplant Program, Froedtert
Memorial Lutheran Hospital, 9200 W Wisconsin Ave, Milwaukee, WI 53226;
e-mail: [email protected].
Submitted May 30, 2000; accepted November 22, 2000.
Supported by grants from the American Cancer Society (RPG 9808401), the
Charles E. Culpeper Foundation, and the Firefighter’s Fund (Milwaukee, WI).
2506
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
test this hypothesis, we have constructed transgenic mice that have
the thymidine kinase (TK) gene targeted to T cells. Transplantation of
mature transgenic T cells into MHC-mismatched recipients enables
alloreactive donor T cells to be selectively eliminated after exposure to
ganciclovir (GCV) allowing these assumptions to be tested.20,21 Prior
studies have shown that this strategy can be used to control GVHD in
both humans and in murine models.22-24 The effect of this approach on
alloengraftment, however, has not been fully studied. The purpose of
this study was to assess whether this strategy would allow for preservation of alloengraftment but mitigation of GVHD.
Materials and methods
Mice
C57BL/6 (B6) (H-2b, Thy1.2⫹) mice were obtained from either The
Jackson Laboratories (Bar Harbor, ME) or the National Institutes of Health
(Bethesda, MD). AKR/J (H-2k), B10.BR (H-2k), and B6.PL (H-2b, Thy1.1⫹)
mice were purchased from The Jackson Laboratories. All animals were
housed in the American Association for Laboratory Animal Care (AALAC)–
accredited Animal Resource Center of the Medical College of Wisconsin.
Mice received regular mouse chow and acidified tap water ad libitum.
Reagents
Ganciclovir (Cytovene; Roche Laboratories, Nutley, NJ) was dissolved in
phosphate-buffered saline (PBS) and stored in aliquots at ⫺20°C. GCV was
thawed prior to use and administered to animals intraperitoneally in PBS at
a dose of 50 mg/kg per day.
REGULATION OF GVHD AND ENGRAFTMENT BY TK⫹ T CELLS
2507
with a scalpel, admixed with Trizol reagent (Gibco, Gaithersburg, MD), and
homogenized using a 20-gauge needle and 1-cc syringe. After centrifugation, the supernatant was removed and transferred to a new microfuge tube.
RNA was isolated after chloroform extraction followed by isopropyl
alcohol precipitation. One microgram of total RNA was subjected to
first-strand complementary DNA (cDNA) synthesis in a 15-␮L reaction for
60 minutes at 37°C according to the manufacturer’s instructions (Pharmacia Biotech, Piscataway, NJ). After completion of first-strand cDNA
synthesis, 1.5 ␮L was used for each PCR reaction using the same protocol
noted above.
To confirm TK protein expression, Western blot analysis of representative tissues was performed. Briefly, tissues were suspended in RIPA buffer
(50 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, and 0.1% sodium
dodecyl sulfate [SDS]) and phenylmethylsulfonyl fluoride and underwent 3
cycles of freeze-thawing in a dry ice/ethanol bath. The suspension was
centrifuged and the supernatant quantitated for protein content using a BCA
protein assay kit (Pierce, Rockford, IL). Fifty to 100 ␮g protein lysate from
each tissue was then run on a 12.5% SDS-polyacrylamide resolving gel.
Protein was transferred onto nylon membranes using a Biorad Trans Blot
apparatus (Hercules, CA) (45 minutes at 2 mA/cm2). Detection was
performed using the enhanced chemoluminescence (ECL) detection system
according to the manufacturer’s instructions (Amersham Life Sciences,
Buckinghamshire, England). The primary antibody was a rabbit polyclonal
anti-TK antibody (obtained from Dr William Summers, Yale University,
New Haven, CT) and was used at a dilution of 1:1000. The negative control
for these experiments was the HU-143B human fibroblast cell line. This cell
line is TK⫺ and was generously provided by Dr R. Orentas (Medical
College of Wisconsin, Milwaukee, WI). The positive control was the AKIL
murine leukemia cell line26 that was transfected with the TK cDNA by
electroporation.
GCV killing assay
Generation of TK transgenic mice
Plasmid TGE-CD3 containing the murine T cell–specific ␦ enhancer and
promoter25 was obtained from Dr Ronald Evans (Salk Institute, San Diego,
CA). The enhancer region was isolated from the plasmid following
restriction digestion with BamHI and cloned into the BamHI site of the
pBluescript SK vector (Stratagene, La Jolla, CA). The murine CD3
promoter was cut with Pst I and blunted with T4 DNA polymerase. HindIII
linkers were ligated to the fragment and, following HindIII digestion,
cloned into the HindIII site downstream of the enhancer region. The herpes
simplex virus (HSV) type 1 thymidine kinase (TK) gene was excised from
the plasmid pHSV-106 (BRL Laboratories, Gaithersburg, MD) with restriction enzymes BglII and PvuII, blunted with DNA polymerase I, large
(Klenow) fragment, and cloned into the blunted Cla I site just downstream
of the CD3 promoter in the enhancer/promoter SK construct. Proper
orientation of all components was confirmed by sequence analysis (Sequenase, United States Biochemical, Cleveland, OH). The transgene was then
separated from plasmid sequences following restriction enzyme digestion,
agarose gel electrophoresis, and electroelution. Linearized DNA containing
the CD3-TK construct was microinjected into C57BL/6 murine blastocysts.
Blastocysts were then implanted into pseudopregnant C57BL/6 female mice.
Transgenic mice were identified by polymerase chain reaction (PCR)
amplification of tail DNA using TK-specific primers. PCR reactions
contained 25 mM of each dNTP, 1.5 mM MgCl2, 10 mM Tris-HCL, pH 8.3;
50 mM KCl, 1 U Taq polymerase (PerkinElmer, Norwalk, CT), and 50 nM
TK-specific primers. Reactions consisted of 30 cycles of denaturation at
94°C for 1 minute, annealing at 59°C for 1 minute, and polymerization at
72°C for 1 minute in a programmable heat block (PerkinElmer). The primer
sequences used for the amplification of the TK gene were 5⬘-CAATCGCGAACATCTACACCACA-3⬘ (sense strand) and 5⬘-CCGAAACAGGGTAAATAACGTGTC-3⬘ (antisense strand) and yielded a 593-base pair product.
TK transgene expression
To confirm messenger RNA (mRNA) expression of the TK gene, reverse
transcription–PCR (RT-PCR) was performed. Tissue samples were minced
Spleen cell suspensions from representative mice were obtained by pressing
spleens through wire mesh screens. Erythrocytes were removed from cell
suspensions by hypotonic lysis with sterile distilled water. Cells were then
resuspended in complete Dulbecco modified essential medium (CDMEM)
and 5% fetal calf serum (FCS) and cultured in flasks along with 5 ␮g/mL
concanavalin A (Con A). After 3 days, cell cultures were evenly split into
2 new flasks containing media and interleukin 2 (IL-2; Proleukin, Chiron Therapeutics, Emeryville, CA) at a concentration of 40 IU/mL. GCV
(10 ␮g/mL) was added to one of the 2 flasks. Viable cells as assessed by
trypan blue dye exclusion were counted daily for 5 days to determine the
degree of cell expansion.
BMT
Bone marrow was flushed from donor femurs and tibias with CDMEM and
passed through sterile mesh filters to obtain single-cell suspensions. BM
was T cell depleted in vitro with either anti-Thy 1.1 or anti-Thy 1.2
monoclonal antibodies (mAbs) plus low-toxicity rabbit complement (C6
Diagnostics, Mequon, WI). The hybridomas for HO-22-1 (anti-Thy1.1,
mouse IgM) and 30-H12 (anti-Thy 1.2, rat IgG2b) antibodies were
purchased from the American Tissue Culture Collection (Rockville, MD).
BM cells were washed and resuspended in DMEM before injection. Naive
donor T cells were obtained by passing erythrocyte-depleted spleen cells
once or twice through nylon wool columns to remove non-T cells. Host
mice were conditioned with total body irradiation (TBI) administered as a
single exposure at a dose rate of 75 cGy using a Shepherd Mark I Cesium
Irradiator (J. L. Shepherd and Associates, San Fernando, CA). Irradiated
recipients received a single intravenous injection of TCD BM (107 cells)
with or without added T cells within 24 hours. For studies of GVHD in a
fully MHC-mismatched system, B10.BR recipients (H-2k, Thy1.2) were
lethally irradiated (900 cGy) and transplanted with TCD B6 BM (H-2b, Thy
1.2) alone or admixed with graded doses of naive B6 TK⫹ T cells. For
studies of engraftment in a fully MHC-mismatched system, AKR recipients
(H-2k, Thy1.1) were sublethally irradiated (750 or 850 cGy) and transplanted with TCD B6.PL BM (H-2b, Thy1.1) alone or admixed with graded
doses of B6 T cells from TK⫹ mice.
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2508
DROBYSKI et al
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Flow cytometric analysis and assessment of chimerism
The mAbs conjugated to either fluorescein isothiocyanate (FITC) or
phycoerythrin (PE) were used to assess chimerism in BMT recipients.
FITC–anti-CD8 (clone 53-6.7, rat IgG2a) was purchased from Collaborative Biomedical Products (Bedford, MA). FITC–anti-CD45R (B220, rat
IgG2a) and PE–anti-CD8 (clone CT-CD8a, rat IgG2a) were obtained from
Caltag (San Francisco, CA). PE–anti-T-cell receptor-␣␤ (TCR-␣␤; clone
H57-597, hamster IgG), FITC–anti-Thy1.2 (clone 30-H12, rat IgG2b),
PE–anti-CD3 (clone 145-2C11, hamster IgG), PE–anti-Thy1.1 (clone
OX-7, mouse IgG1), FITC–anti-CD4 (clone RM4-4, rat IgG2b), PE–antiMac-1 (clone M1/70, rat IgG2b), and FITC–anti-H-2Kb (clone AF6-88.5,
mouse IgG2a) were all purchased from Pharmingen (San Diego, CA).
Peripheral blood was obtained from animals by tail vein bleeds. Red blood
cells were removed by Ficoll-Hypaque density gradient centrifugation.
Donor T cells and granulocytes were determined by constructing bit maps
and staining with either H-2Kb and CD3 or H-2Kb and Mac-1, respectively.
Spleen and thymus cells were obtained from chimeras at defined intervals
after transplantation, processed into single-cell suspensions, and stained for
2-color analysis. Red cells were removed when necessary by hypotonic
lysis. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The absolute number of splenic B and donor
T cells was determined by analyzing cells within gates that included the
entire spleen cell population after exclusion of red cells and nonviable cells
by forward- and side-scatter settings. Donor T-cell chimerism was determined by analyzing cells within the lymphocyte gate. Thymocytes were
analyzed within gates that included the entire thymus cell population. At
least 10 000 cells were analyzed for each determination whenever possible.
Statistics
Group comparisons of parameters of donor T-cell engraftment and thymic
and splenic reconstitution were performed using the Mann-Whitney U test.
A P value ⬍ .01 was deemed to be significant when pair-wise comparisons
were made between 3 groups. Otherwise a P value of ⬍ .05 was deemed as
significant in 2-group comparisons. Survival curves were constructed using
the Kaplan-Meier product limit estimator and compared using the log-rank
rest. Mice that were killed for secondary transfer experiments or engraftment studies were censored from the survival analysis at the time of death.
A P value ⬍ .05 was deemed to be significant in survival experiments.
Results
Derivation and functional characterization
of TK transgenic mice
A total of 47 pups were derived from impregnated female animals.
Genomic DNA from these offspring was screened using PCR to
detect the presence of the TK gene. Seventeen of 47 offspring were
positive for the transgene. TK⫹ mice were mated to normal
Figure 1. The TK transgene is expressed only in the spleen and thymus.
Western blot analysis of protein lysates derived from specified TK⫹ founder line
tissues were probed with a rabbit polyclonal anti-TK antibody. N signifies the negative
control (HU-143B human fibroblast cell line that is TK⫺). P denotes the positive
control (AKR murine leukemia cell line transfected with the TK cDNA).
Figure 2. Anti-CD3 antibody-activated TKⴙ T cells are killed by GCV in vitro.
Spleen cells from B6 TK⫺ and TK⫹ mice were activated in vitro with immobilized
anti-CD3 antibody and IL-2 for 3 days. Cells were then transferred into fresh flasks
and grown in IL-2 ⫾ GCV (10 ␮g/mL). The total number of viable cells was monitored
over the ensuing 5 days and is depicted for each of the groups.
C57BL/6 animals. Analysis of genomic DNA by PCR revealed
second-generation transmission of the transgene in only 6 of 17
lines. All 6 lines were then analyzed by RT-PCR to determine
whether the transgene was expressed. RNA from lung, liver,
spleen, BM, muscle, thymus, and kidney was obtained and
subjected to RT-PCR using TK-specific primers. One line was
identified that had expression of the transgene restricted to the
spleen and thymus.
To verify that there was appropriate protein expression of the
transgene, Western blot analysis was performed on lysates derived
from the same tissues of this founder. These data, which are shown
in Figure 1, demonstrate that TK protein was detectable only in the
spleen and thymus of transgenic animals, but not in the liver, lung,
muscle, kidney, or BM. To confirm the functionality of the
transgene in T cells, spleen cells from TK⫹ and TK⫺ mice were
activated in vitro with immobilized anti-CD3 antibody and IL-2 for
3 days. Cells were then transferred into fresh flasks and grown in
IL-2 ⫾ GCV (10 ␮g/mL). Cell expansion after addition of GCV
was assessed (Figure 2). Spleen cells from TK⫹ mice rapidly died
in vitro, indicating that the transgene was functional in these cells.
Normal cells were unaffected by GCV. Activated TK⫹ T cells
cultured in the absence of GCV had growth characteristics similar
to TK⫺ cells.
Treatment of recipients of HSV-TKⴙ T cells with GCV mitigates
lethal GVHD in a schedule-dependent fashion
Experiments were performed to confirm that T cells from transgenic mice were capable of causing GVHD and to determine
whether the in vivo administration of GCV could ameliorate GVH
reactivity in lethally irradiated recipients. B10.BR recipients were
transplanted with TCD B6 BM alone (control animals) or together
with 3 ⫻ 106 B6 TK⫹ T cells. Untreated mice transplanted with
naı̈ve TK⫹ T cells all died of GVHD within 45 days after BMT,
indicating that TK⫹ T cells were fully capable of causing lethal
GVHD (Figure 3). To assess the ability of GCV treatment to reduce
the severity of GVHD, similarly transplanted cohorts of mice were
administered GCV (50 mg/kg) on one of 3 schedules (ie, days 0-9,
3-16, or 7-20 after transplantation). Treatment with GCV resulted
in significantly prolonged survival in all 3 groups when compared
to untreated GVHD control mice. Administration of GCV on days 0
to 9 or 7 to 20, however, was less effective than treatment on days 3
to 16 (P ⬍ .0001 versus control). The administration of GCV on
days 3 to 16 to mice transplanted with normal B6 T cells had no
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
REGULATION OF GVHD AND ENGRAFTMENT BY TK⫹ T CELLS
2509
Selective elimination of donor TKⴙ T cells after transplantation
does not compromise early engraftment
Nontolerant donor T cells are thought to promote engraftment
by eliminating host immune cells capable of rejecting marrow
cells.3 Donor T cells recognize recipient alloantigens, become
activated, undergo clonal expansion, and differentiate into
competent cytotoxic T cells. We reasoned that if GCV were
given to recipients early after BMT to ameliorate GVHD, it was
conceivable that donor T cells might be eliminated before
engraftment had occurred. To test this question, we examined
the extent of donor engraftment immediately before and after
GCV treatment of recipients transplanted with mature TK⫹
T cells.
Cohorts of sublethally irradiated AKR mice underwent
transplantation with TCD B6.PL BM plus 5 ⫻ 105 B6 TK⫹ T
cells. This congenic model was used for 2 reasons. The first was
so that transgenic T cells could be identified in the recipients,
and the second was to determine if there was a correlation
between protection from GVHD and elimination of TK⫹ T cells
from the recipient. One group was given GCV on days 4 to 8
after BMT, whereas the other group was left untreated. The
GCV schedule was derived from the optimal schedule in GVHD
studies with the modification that the duration of administration
was shorter due to the fact that GVHD is less severe in this
nonmyeloablative (sublethal irradiation) model. Representative
animals from each group were killed on day 4 immediately
before GCV treatment and on day 9 after the completion of
GCV. On day 4, there was only minimal donor T-cell engraftment in the spleen or thymus of either group (Table 1). By day
9, donor T cells comprised 97% of all splenic T cells in mice
not treated with GCV. The majority of these donor T cells were
Thy 1.2⫹ TK⫹. Transgenic T cells also composed the majority of
T cells in the thymus of these animals. Mice treated with GCV
had an equivalent degree of donor T-cell chimerism in the
spleen; however, the percentage of T cells that were TK⫹ was
significantly reduced (14% versus 77%). The preponderance of
donor T cells in the spleens of GCV-treated mice were BMderived Thy 1.1⫹ T cells. These data indicate that GCV given on
days 4 to 8 did not reduce the percentage of donor T-cell
chimerism.
Table 1. Kinetics of donor T-cell engraftment early after transplantation in
GCV-treated mice
Spleen
Figure 3. GCV treatment mitigates lethal GVHD in mice transplanted with
HSV-TKⴙ T cells. Lethally irradiated B10.BR mice were transplanted with TCD B6
BM (107) alone (E, n ⫽ 6/group) or together with 3 ⫻ 106 B6 T cells. Animals
transplanted with T cells were then either left untreated (f, n ⫽ 9-10/group) or
received the specified schedule of GCV therapy (䡺, n ⫽ 10/group) (panel A, days
0-9; panel B, days 3-16; and panel C, days 7-20). Actual survival is depicted. Data are
cumulative results from 2 independent experiments per schedule.
Days
postBMT
4
9
GCV
Percent
TK⫹ CD3⫹
T cells
Percent
donor
Thy1.1
CD4
No
4
1
NE
4
4
Yes
3
1
NE
5
3
Percent donor
CD8
No
97
77
10
57
76
Yes
91
14
78
7
6
(Thy1.1⫹)
protective effect on GVHD, indicating that GCV itself could not
ameliorate GVH reactivity (data not shown). Collectively, these
data provided proof of the principle that GCV treatment was
capable of protecting animals transplanted with TK⫹ T cells from
GVHD and indicated that protection was, in part, schedule
dependent.
Thymus
Percent
donor T
cells
Irradiated (850 cGy) AKR mice
were transplanted with TCD B6.PL
(Thy1.1⫹) BM (107 cells) plus 5 ⫻ 105 B6 (Thy 1.2⫹) TK⫹ T cells. Cohorts of mice were
either treated with GCV (50 mg/kg) for 5 days beginning on day 4 after transplantation
or left untreated. Representative mice from each group (N ⫽ 3/group) were killed on
day 4 and day 9 after transplantation. Spleen and thymus cells from individual
animals in each group were pooled and analyzed for chimerism. Donor Thy1.1⫹ cells
in the spleen were defined as the percentage of donor Thy 1 cells that were Thy1.1⫹
and H-2b⫹. Donor CD4⫹ and CD8⫹ T cells in the thymus were defined as
Thy1.2⫹CD4⫹ and Thy1.2⫹CD8⫹, respectively.
NE indicates not evaluable due to insufficient donor T-cell engraftment at day 4.
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2510
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
DROBYSKI et al
Figure 4. The selective elimination of alloreactive donor T cells mitigates GVHD
without compromising engraftment. Irradiated AKR mice (850 cGy) were transplanted with TCD B6 BM alone (䡺, n ⫽ 9) or TCD BM plus 5 ⫻ 105 B6 TK⫹ T cells
with (f, n ⫽ 16) or without (E, n ⫽ 13) GCV administered after transplantation. GCV
was given to animals for either 5 (days 4-8, 2 experiments) or 10 (days 4-13, 1
experiment) days after transplantation and the results were combined. The mean
weights of animals are depicted over the first 4 weeks.
Elimination of HSV-TKⴙ T cells by GCV reduces GVHD severity
without compromising donor engraftment
Studies were then performed to determine whether early donor
engraftment was durable and whether GVHD was reduced by
elimination of alloactivated donor T cells. Cohorts of sublethally
irradiated AKR mice were transplanted with TCD B6.PL BM plus
5 ⫻ 105 B6 TK⫹ T cells. Control mice received TCD BM only.
Mice in all groups were killed 3 to 5 weeks after BMT. Animals
reconstituted with TK⫹ T cells but not treated with GCV had lost
about 30% of their pretransplant body weight due to GVHD at day
30 (Figure 4). Conversely, mice treated with GCV had weight loss
that paralleled non-GCV–treated mice for the first 2 weeks, but
thereafter regained weight similar to non-GVHD control animals.
Mice that did not receive GCV treatment had complete donor T-cell
chimerism but significantly reduced splenic and thymic cellularity
compared to TCD BM controls, consistent with ongoing GVHD
(Table 2). In contrast, although GCV-treated animals had splenic
donor T-cell engraftment that was comparable to untreated mice,
total spleen size (P ⬍ .001) and the number of splenic B cells
(P ⬍ .001) were significantly increased relative to GVHD controls.
The mean percentage of donor cells in GCV-treated versus
nontreated mice was also comparable (86% versus 88%), and
significantly higher than TCD BM controls (33%). GCV-treated
mice also had a striking increase in the percentage of doublepositive thymocytes (P ⬍ .0001) and overall thymic cellularity
(P ⬍ .0001), respectively, when compared with untreated animals.
Thymic reconstitution was comparable to that observed in nonGVHD control mice. Mice treated with GCV had an increased
absolute number of donor T cells in the spleen (Table 2), which was
directly attributable to the increase in the number of BM-derived
donor T cells (data not shown).
At a dose of 850 cGy, mice given TCD BM only still had a low
level of donor T-cell chimerism. To determine if transplantation of
TK gene-modified cells could facilitate engraftment under conditions where TCD BM was completely rejected, we performed
identical experiments at a TBI exposure of 750 cGy. At this TBI
dose, mice transplanted with TCD BM only rejected their grafts as
assessed by both T-cell and myeloid engraftment (Table 3).
Animals transplanted with 5 ⫻ 105 B6 TK⫹ T and treated with
GCV on days 4 to 8, however, had a higher incidence of graft
rejection compared to untreated mice (4 of 6 versus 1 of 6). When
the administration of GCV was delayed until days 8 to 12 after
transplantation, donor T-cell and myeloid engraftment was equivalent in both groups. Mice transplanted with TK⫹ T cells but not
treated with GCV lost 16% of their body weight by day 35.
Conversely, GCV-treated mice had a 3% gain in body weight that
was significantly different at 5 weeks after BMT (P ⫽ .004; data
not shown). Collectively, these data indicate that GVHD can be
substantially reduced without compromising alloengraftment under
conditions where engraftment is dependent on the presence of
mature donor T cells in the marrow graft.
Reconstituting BM-derived donor T cells are tolerant
of host alloantigens
The majority of donor T cells in mice after GCV treatment were
derived from BM. In the absence of GVHD, these cells undergo
thymic selection whereby self-reacting T cells are eliminated
before they egress into the periphery. During GVHD this
process can become deranged and “autoreactive” BM-derived
donor T cells can emerge into the periphery as a consequence of
impaired thymic selection.27 These cells have been shown to
have antihost reactivity in vitro27 and have been postulated to
cause GVHD. Because mice in our studies were allowed to have
a limited GVH reaction before GCV was administered, we
reasoned that some degree of thymic damage might have
Table 2. Treatment with GCV early after transplantation preserves alloengraftment and reduces GVHD
Spleen
Group
Donor T
cells added
GCV
Percent
donor T
cells*
Percent
CD3⫹ TK⫹
T cells†
Size‡
(⫻ 10⫺6)
B cells§
(⫻ 10⫺6)
Donor
T cells㛳
(⫻ 10⫺6)
Thymus
size¶
(⫻ 10⫺6)
Percent
CD4⫹8⫹
thymocytes#
I
None
No
26 ⫾ 8
0
40 ⫾ 4
9⫾2
ND
66 ⫾ 12
78 ⫾ 4
II
5 ⫻ 105 TK⫹
No
98 ⫾ 2
31 ⫾ 6
11 ⫾ 3
4⫾2
3⫾1
24 ⫾ 12
31 ⫾ 8
III
5 ⫻ 105 TK⫹
Yes
86 ⫾ 6
10 ⫾ 2
34 ⫾ 5
20 ⫾ 4
6⫾1
85 ⫾ 12
82 ⫾ 2
Irradiated (850 cGy) AKR mice (Thy1.1⫹) were transplanted with either TCD B6.PL (Thy1.1⫹) BM alone (107 cells) (group I) (N ⫽ 12/group) or together with 5 ⫻ 105 B6 (Thy
1.2⫹) TK⫹ T cells (groups II and III) (n ⫽ 13-19/group). Group III mice were treated with GCV (50 mg/kg) for 5 days beginning on day 4 after transplantation. Animals were
sacrificed 26-33 days after BMT and analyzed for splenic and thymic reconstitution. Donor T cells were defined as H-2b⫹/CD3⫹ and transgenic TK⫹ T cells as Thy1.2⫹/CD3⫹.
Data are presented as the mean ⫾ SE.
*Group I v III, P ⬍ .0002; II v III, P ⫽ .0018.
†Group II v III, P ⬍ .001.
‡Group I v III, P ⫽ .43; II v III, P ⫽ .003.
§Group I v II, P ⫽ .016; I v III, P ⫽ .04; II v III, P ⬍ .001.
㛳Group II v III, P ⫽ .02.
¶Group I v III, P ⫽ .34; II v III, P ⫽ .001.
#Group I v III, P ⫽ .67; II v III, P ⬍ .001.
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
REGULATION OF GVHD AND ENGRAFTMENT BY TK⫹ T CELLS
Table 3. T-cell and myeloid engraftment at TBI exposures of 750 cGy
Experiment
1
Donor T
cells
added
Percent donor
GCV
administration
T cells
None
No
1*, 1, 1, 53, 1
22*, 4, 6, 98, 1
5 ⫻ 105
No
99, 54, 2, 99,
95, 95, 65, 98,
99, 100
5 ⫻ 105
98, 99
d 4-8
2, 2, 3, 79, 98,
None
No
2, 2, 1, 1
6, 92, 5, 19
5 ⫻ 105
No
100, 99, 3, 98,
97, 99, 17, 98,
7
2
Granulocytes
99, 100
5 ⫻ 105
d 8-12
97, 1, 99, 99,
80, 89
12, 2, 12, 96,
96, 10
97, 97
99, 6, 94, 96,
97, 92
Irradiated (750 cGy) AKR mice were transplanted with either TCD B6.PL BM
alone (107 cells) or together with 5 ⫻ 105 B6 TK⫹ T cells. A cohort of mice in each
experiment transplanted with TK⫹ T cells was treated with GCV (50 mg/kg per day)
for 5 days after transplantation on the specified scheduled noted above. Animals
were bled on day 28 in experiment 1 and day 29 in experiment 2.
*One mouse in group 1 of experiment 1 was bled on day 18 immediately prior to
demise.
occurred before the reaction was abrogated. Thymic phenotyping studies (Table 2) did not reveal any abnormalities consistent
with GVHD in GCV-treated mice. As a functional test for
tolerance, we performed adoptive transfer experiments into
secondary AKR hosts. Splenic T cells from these chimeras
(0.5 ⫻ 106/mouse) were cotransplanted with normal TCD B6
BM into lethally irradiated secondary AKR recipients. Phenotypic analysis of the chimeric T-cell population indicated that
98% of T cells were donor in origin. Thy1.2⫹ TK⫹ T cells
represented 5% of all donor T cells, whereas the remaining cells
were Thy1.1⫹ (ie, BM derived). GVHD control mice consisted
of lethally irradiated AKR mice transplanted with an equivalent
number (0.5 ⫻ 106/mouse) of normal B6 Thy 1.2⫹ T cells.
GVHD control animals all died, whereas mice transplanted with
chimeric T cells survived without any clinical signs of GVHD
(Figure 5). These data provided evidence that reconstituting
BM-derived donor T cells underwent appropriate thymic selection and that the composite donor T-cell population was tolerant
of recipient alloantigens.
2511
these 2 events appeared to be based solely on the temporal
manipulation of T-cell survival by GCV administration resulting
in a limited GVH reaction and not on any inherent property of
transgenic T cells to differentially promote engraftment at the
expense of GVH reactivity.
The results of this study provide insight into the tempo of
donor engraftment early after BMT. Analysis of chimerism 4
days after transplantation demonstrated no evidence of donor
T-cell engraftment in either cohort of mice. Host T cells
comprised nearly all of the T-cell population. By day 9,
however, complete donor T-cell chimerism had been established
in the spleens of untreated mice and donor T cells constituted the
primary T-cell population in the thymus. These data are
consistent with the interpretation that donor T cells are able to
eliminate host immune cells capable of rejecting the graft within
the first week after transplantation. Notably, GCV treatment on
days 4 to 8 did not reduce the level of donor T-cell engraftment
in the spleen even though there was a substantial reduction in the
percentage of transgenic T cells in these animals. Thus, these
cells were able to eradicate host T cells before the majority of
TK⫹ T cells were themselves eliminated by GCV. Although we
cannot exclude that the small percentage of residual donor T
cells remaining after GCV treatment may have contributed to
engraftment, the inability of purified Thy 1.2⫹ TK⫹ T cells to
cause GVHD in adoptive transfer experiments (unpublished
Discussion
Donor T cells play a critical role in preventing graft rejection
after allogeneic BMT. A number of studies have shown that
T cells promote engraftment by eradicating or inactivating host
T cells that reject the marrow graft.1-3,6 CD8⫹ T cells are
responsible for preventing graft rejection in mice, primarily by
the generation of cytotoxic T cells that eliminate host immune
cells,28 although CD8 cells may also mediate veto activity.29-31
In animal models, the number of donor T cells can be adjusted to
facilitate engraftment without inducing significant GVHD.3 This
goal has been more elusive in clinical BMT where a similar
strategy has resulted in a prohibitively high risk of GVHD.32 In
this study, we used an alternative approach to address the
divergent role of T cells in engraftment and GVHD. We
demonstrated that GVHD could be significantly reduced without
compromising engraftment of the donor marrow by the selective
elimination of alloreactive TK gene–modified donor T cells.
These data also indicated that the ability of donor T cells to
facilitate alloengraftment could be dissociated from the ability
of these same cells to cause lethal GVHD. The dissociation of
Figure 5. Donor T cells from GCV-treated mice are tolerant of host
alloantigens. Irradiated AKR mice (850 cGy) were transplanted with TCD B6 BM
plus 5 ⫻ 105 B6 TK⫹ T cells. Mice were then treated with GCV from days 4 to 13 after
transplantation. Four weeks after BMT, mice were killed and pooled spleen cells from
these animals were cotransplanted with normal TCD B6 BM into lethally irradiated
(1100 cGy) AKR hosts (f, n ⫽ 6). Control groups consisted of similarly conditioned
mice transplanted with TCD B6 BM alone (䡺, n ⫽ 4) or TCD B6 BM plus 5 ⫻ 105 B6
naive T cells (F, n ⫽ 5). Survival is depicted in panel A and serial weights in panel B.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2512
DROBYSKI et al
observations, January 2000) suggests that the graft facilitating
capability of these cells is negligible.
Despite the administration of GCV and subsequent elimination
of alloactivated donor T cells, GCV-treated animals had an
increased number of splenic T cells 4 weeks after BMT (Table 2).
Thus, the administration of GCV did not render recipients T-cell
deficient. Rather, these animals had increased donor T-cell repopulation, presumably due to the reduction in GVHD,33,34 suggesting
that this approach may not only mitigate GVHD but also enhance
T-cell reconstitution. An important observation is the fact that the
majority of donor T cells in GCV-treated mice were derived from
BM (Table 1). This contrasted with what was observed in untreated
animals where splenic-derived T cells comprised the majority of T
cells. BM-derived T cells typically undergo selection in the thymus
where they are tolerized to host alloantigens, unless GVHDinduced thymic damage precludes normal deletional mechanisms.27 In these studies, adoptive transfer experiments showed
that donor T cells remaining after GCV treatment did not cause
clinically significant GVHD (Figure 5). Thus, donor T cells, of
which the majority were BM derived, were tolerant of host
alloantigens. Whether these T cells are competent to mediate
third-party responses against alloantigens and viral infections is a
significant question and one that is currently under investigation.
It should be emphasized that TK⫹ T cells persisted in mice after
GCV treatment. There are several possible explanations for this
observation. One is that an effective GCV concentration was not
maintained at all times that transgenic T cells were proliferating.
Hence some alloreactive T cells were able to escape elimination
over the 5-day treatment course. An alternative explanation is that
residual TK⫹ T cells did not recognize recipient alloantigens and
therefore were nonproliferative. In that regard, we would note that
the absolute number of TK⫹ T cells was approximately the same in
both GCV-treated and non-GCV–treated chimeras (Table 2). The
fact that the former cohort of mice had significantly reduced
GVHD suggests that these residual TK⫹ T cells were in fact not
reactive against recipient alloantigens. Adoptive transfer studies
also support the premise that these cells were tolerant of host
alloantigens. However, these data are not conclusive. An alternative explanation is that GCV treatment resulted in the more rapid
emergence of BM-derived donor T cells, some of which were able
to function as regulatory T cells capable of down-regulating GVH
reactivity.35 Ongoing studies using purified TK⫹ T cells in adoptive
transfer experiments (see above) are needed to definitively address
this question.
A fundamental aspect of this approach is that a GVH reaction is
allowed to begin but is abrogated before substantial tissue damage
has occurred in the host. The administration of GCV to TKtransplanted animals targets dividing T cells, the majority of which
are presumably responding to host alloantigens.36 Several studies
have substantiated that this strategy is feasible, although there has
been variability in the degree of GVHD protection afforded. In the
present study, we observed that administration of GCV over many
days to weeks was required for effective GVHD protection. This is
in contrast to studies by Lake-Bullock and colleagues23 where the
administration of as few as 2 doses of GCV was sufficient for
abrogation of GVHD. Cohen and coworkers24 reported more
variability in the degree of GVHD protection in their studies. They
postulated that this variability stemmed from the use of 2 different
transgenic lines of mice that differentially expressed the TK
transgene. This was supported by studies showing a difference in
GCV-mediated killing in vitro between activated T cells from the
respective lines. The level of transgene expression may therefore
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
be one explanation for the variable duration of GCV administration
required for effective prevention in different studies. An alternative
explanation may lie in differences in the murine transplantation
models used to evaluate GVHD. For example, in our studies, a
short course of GCV treatment was highly effective in preventing
GVHD in sublethally irradiated AKR recipients transplanted with a
low dose of donor B6 T cells. Conversely, a more protracted course
of GCV gave less protection in lethally irradiated B10.BR recipients transplanted with a 6-fold higher dose of B6 T cells. The
implication for clinical BMT is that the administration schedule of
GCV may be an important variable in modulating the intensity of
the GVH reaction and may vary depending on the intensity of the
conditioning regimen and the degree of MHC incompatibility
between donor and recipient.
In this study, the addition of mature donor T cells was
necessary to overcome graft resistance. Prior studies have
shown an inverse correlation between the intensity of the
conditioning regimen and the dose of T cells necessary to
facilitate alloengraftment.37-39 Thus, less intensively conditioned recipients require larger doses of donor T cells for
equivalent engraftment. Our study identifies the GCV administration schedule as another variable that also affects donor
engraftment when mice are transplanted with TK⫹ T cells. At
TBI exposures of 850 cGy, a GCV schedule of days 4 to 8
resulted in engraftment in all recipients (Table 2). In contrast,
the same schedule resulted in graft rejection in the majority of
mice conditioned with 750 cGy (Table 3). The latter group of
mice required that GCV be given later after transplantation (ie,
days 8-12) for engraftment to be achieved. We infer that this was
due to a higher percentage of host T cells surviving the less
intense conditioning regimen necessitating that donor T cells
persist in the host for a longer period of time. These findings
may have implications for human transplantation. In clinical
marrow transplantation, there is growing interest in the use of
nonmyeloablative conditioning regimens to reduce regimenrelated toxicity, particularly in older recipients or those with
underlying vital organ dysfunction.40,41 The infusion of large
numbers of donor T cells in the stem cell graft, however, has
been necessary to overcome graft resistance in patients given
nonmyeloablative conditioning and has been shown to cause
GVHD. Our data suggest that an alternative approach for these
patients may be to infuse larger numbers of donor T cells that
have been modified with insertion of the TK gene. However, the
current study would argue that the successful application of this
approach will be contingent on the careful modulation of
conditioning regimen intensity, donor T-cell dose, and GCV
administration schedule.
In summary, these results show that the ability of donor T cells
to facilitate engraftment and cause GVHD can be dissociated by
manipulating survival of alloactivated T cells through gene targeting of
the donor T cell. These data also demonstrate that the persistence of
host-reactive donor T cells after BMT is not a prerequisite for durable
engraftment. The extent to which this strategy may be beneficial in
patients with leukemia will depend on whether there is commensurate
retention of graft-versus-leukemia reactivity. In nonmalignant conditions (eg, marrow failure syndromes or immunodeficiency disorders),
however, where there is no risk of relapse, the ability of TK gene–
modified donor T cells to facilitate donor engraftment without causing
clinically significant GVHD may expand the spectrum of patients who
can benefit from allogeneic BMT.
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
REGULATION OF GVHD AND ENGRAFTMENT BY TK⫹ T CELLS
Acknowledgments
We would like to thank Tina Agostini and Sanja Jankovic-Vodanovic for
their technical assistance in performing the murine transplantation
2513
studies. The authors also acknowledge Rob Payne from the NIAID
transgenic facility who provided invaluable assistance in helping to
establish the founder line. Dr William Summers is acknowledged for
providing the thymidine kinase monoclonal antibody. We thank Robert
Truitt for critically reviewing the manuscript.
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2001 97: 2506-2513
doi:10.1182/blood.V97.8.2506
Protection from lethal murine graft-versus-host disease without compromise
of alloengraftment using transgenic donor T cells expressing a thymidine
kinase suicide gene
William R. Drobyski, Herbert C. Morse III, William H. Burns, James T. Casper and Gordon Sandford
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