Prolonged allograft survival through conditional and specific ablation of alloreactive T cells expressing a suicide gene.

Abstract

Control of antidonor activated T cells involved in allograft rejection while preserving immunocompetence is a challenging goal in transplantation. Engineered T cells expressing a viral thymidine kinase (TK) suicide gene metabolize the nontoxic prodrug ganciclovir (GCV) into a metabolite toxic only to dividing cells. We evaluated this suicide gene strategy for inducing transplantation tolerance in mice.
Transgenic mice expressing TK in mature T cells were analyzed for (i) specific T-cell depletion under GCV treatment upon various stimulations; (ii) outcome of allogeneic nonvascularized skin or heart allografts under a short 14-day GCV treatment initiated at the time of transplantation; and (iii) the capacities of T cells from such allotransplanted mice to proliferate in mixed lymphocyte reactions and to induce graft-versus-host disease in irradiated recipients with the genetic background of the donor allograft.
Upon in vitro or in vivo GCV treatment, only activated dividing TK T cells but not B cells were efficiently depleted. Acute rejection of allogeneic grafts was prevented and a significant prolongation of graft survival was obtained, although associated with signs of chronic rejection. Prolonged skin graft survival correlated with decreased in vitro and in vivo T-cell reactivities against donor alloantigens, whereas overall immunocompetence was preserved.
Efficient and specific depletion of alloreactive TK T cells can be achieved by administrating GCV. These results open new perspectives for the control of allogeneic graft rejection using suicide gene therapy.

Full text

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0041-1337/00/6910-2154/0
TRANSPLANTATION
Copyright © 2000 by Lippincott Williams & Wilkins, Inc.
Vol. 69, 2154–2161, No. 10, May 27, 2000
Printed in U.S.A.
PROLONGED ALLOGRAFT SURVIVAL THROUGH CONDITIONAL
AND SPECIFIC ABLATION OF ALLOREACTIVE T CELLS
EXPRESSING A SUICIDE GENE1
VÉRONIQUE THOMAS-VASLIN,2,3BERTRAND BELLIER,3JOSÉ L. COHEN,4OLIVIER BOYER,3
NATHALIE RAYNAL-RASCHILAS,5DENIS GLOTZ,5AND DAVID KLATZMANN3,6
Biologie et The ´rapeutique des Pathologies Immunitaires, UPMC/CNRS ESA 7087, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re,
75651 Paris Cedex 13; Ge ´nopoı ¨e ´tic, 75013 Paris; and Immunopathologie, INSERM U430, Ho ˆpital Broussais,
75674 Paris Cedex 14, France
Background. Control of antidonor activated T cells
involved in allograft rejection while preserving immu-
nocompetence is a challenging goal in transplanta-
tion. Engineered T cells expressing a viral thymidine
kinase (TK) suicide gene metabolize the nontoxic pro-
drug ganciclovir (GCV) into a metabolite toxic only to
dividing cells. We evaluated this suicide gene strategy
for inducing transplantation tolerance in mice.
Methods. Transgenic mice expressing TK in mature
T cells were analyzed for (i) specific T-cell depletion
under GCV treatment upon various stimulations; (ii)
outcome of allogeneic nonvascularized skin or heart
allografts under a short 14-day GCV treatment initi-
ated at the time of transplantation; and (iii) the capac-
ities of T cells from such allotransplanted mice to pro-
liferate in mixed lymphocyte reactions and to induce
graft-versus-host disease in irradiated recipients with
the genetic background of the donor allograft.
Results. Upon in vitro or in vivo GCV treatment, only
activated dividing TK T cells but not B cells were
efficiently depleted. Acute rejection of allogeneic
grafts was prevented and a significant prolongation of
graft survival was obtained, although associated with
signs of chronic rejection. Prolonged skin graft sur-
vival correlated with decreased in vitro and in vivo
T-cell reactivities against donor alloantigens, whereas
overall immunocompetence was preserved.
Conclusions. Efficient and specific depletion of allo-
reactive TK T cells can be achieved by administrating
GCV. These results open new perspectives for the con-
trol of allogeneic graft rejection using suicide gene
therapy.
Control of T-cell reactivity is a key issue in several situa-
tions such as organ transplantation, graft-versus-host dis-
ease (GVHD), and autoimmunity. Classical strategies for
treating or preventing T cell-mediated disorders are based on
the administration of chemotherapeutic immunosuppressive
drugs. Such treatments are usually not specific and can lead
to generalized immunodeficiency. In the long term, emer-
gence of tumors and increased susceptibility to opportunist
infections are frequent (1–3). Thus, it is important to develop
new strategies to control pathological T-cell responses (4).
Gene transfer offers new perspectives for achieving this
goal, and several experimental gene therapy approaches are
currently under investigation for immunomodulation in
transplantation (5, 6). One such approach is based on the
time-controlled selective destruction of reactive cycling T
cells, while sparing quiescent cells. It involves the expression
in T cells of a suicide gene whose product metabolizes an
inactive prodrug into a compound toxic only to cells that
actively synthesize DNA, such as proliferating T cells. The
thymidine kinase (TK) of the type 1 Herpes simplex virus is
the most frequently used suicide gene in experimental mod-
els (7-11) as well as in human therapeutic protocols (12). The
gene product phosphorylates the inactive nucleoside analog
ganciclovir (GCV) into toxic triphosphorylated-GCV, which is
then incorporated into growing DNA strands, blocking elon-
gation and ultimately leading to apoptosis of the dividing
cells.
Because the T cells involved in immunopathologies are
most often activated and dividing, this system is perfectly
suitable for developing specific genetic immunosuppression.
In addition, only TK-expressing T cells that are dividing at
the time of GCV administration will be killed. Consequently,
such a treatment should result in selective cell death of
reactive T cells involved in the pathology.
It has been shown that genetic immunosuppression of al-
loreactive T cells can control GVHD after allogeneic hemato-
poietic stem cell transplantation in mice and humans (13–
17). In the present study, we investigated whether this
approach could similarly control tissue or solid organ graft
rejection. Using transgenic mice expressing TK under the
control of specific regulatory sequences leading to TK expres-
sion in both CD4 and CD8 T cells (18), we demonstrated a
significant prolongation of skin and nonvascularized heart
allograft survival when mice were given a short course of
GCV at the time of the graft.
MATERIALS AND METHODS
Mice. FVB/N (H2q), DBA/2 and Balb/c (H2d), C57BL/6 (B6, H2b)
mice were obtained from Iffa Credo (L’Arbresle, France). DBA/1
2154
1This work was supported by Universite ´ Pierre et Marie Curie,
Centre National de la Recherche Scientique, Association pour la
Recherche sur les De ´ficits Immunitaires Viro-Induits, Assistance-
Publique Ho ˆpitaux de Paris, by a grant from Agence Nationale de
Recherche sur le SIDA and by Association Française contre les
Myopathies.
2Recipient of an allocation de recherche from ANRS.
3Biologie et The ´rapeutique des Pathologies Immunitaires, UPMC/
CNRS ESA 7087, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re.
4Ge ´nopoı ¨e ´tic.
5Immunopathologie, INSERM U430, Ho ˆpital Broussais.
6Address correspondence to: David Klatzmann, Biologie et The ´ra-
peutique des Pathologies Immunitaires, UPMC/CNRS ESA 7087,
Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, 83 Bd de l’Ho ˆpital, 75651 Paris
Cedex France. E-mail: veronique.thomas-vaslin@psl.ap-hop-paris.fr.

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(H2q) mice were from Harlan France SARL (Gannat, France). The
HSV1-TK transgenic FVB mice (EpTK line 40) have been previously
described and were bred in our specific-pathogen free animal facility
(19). They were obtained by microinjection of an HSV1-TK transgene
placed under the control of the human CD4 promoter (20) and the
murine CD4 minimal enhancer (21). Because of the absence of the
CD4 silencer (22), expression is obtained in both CD4 and CD8 T
cells (18, 23). In HSV1-TK mice, RNAse protection assays showed TK
transcripts in splenocytes and thymic CD4?, CD8?, and CD4?CD8?
cells. Mice were manipulated according to the European Economic
Community guidelines.
GCV administration. GCV (Cymevan; Roche, Neuilly-sur-seine,
France) was prepared by dilution in pyrogen-free H2O, filtered (0.22
?m), and then filled in mini-osmotic pumps (Alzet 2001 and 2002;
Alza corporation, Palo Alto, CA) that delivered GCV at a dose of 50
mg/kg/day for 7 or 14 days, respectively. The pump was implanted
subcutaneously under tribromo-ethanol anesthesia. GCV plasma
concentrations were determined by high-performance liquid chroma-
tography.
In vitro sensitivity to GCV. Cells were cultured in triplicate for 48
hr at 5?105/ml (200 ?l/well) with 3 ?g/ml concanavalin A (Con A) or
30 ?g/ml lipopolysaccharide (LPS; Sigma, St. Louis, MO) and in-
creasing concentrations of GCV. Cells were then pulsed for 16 hr
with [3H]thymidine (1 ?Ci/well) to measure cell proliferation.
Limiting dilution assay. Spleen cells were plated in limiting di-
lution (48 replicates/dilution, 5 dilutions) in flat-bottomed microwell
plates containing 4?105irradiated (25 Gy) syngeneic FVB spleen
filler cells in 0.2 ml of culture medium supplemented with murine
recombinant interleukin (IL)-2 (R&D Systems, Minneapolis, MN) (10
ng/ml) and Con A (5 ?g/ml) and in the presence or absence of GCV
(10 ?M). After a 7-day incubation period (37°C, 5% CO2), the number
of positive wells was scored by a proliferation assay using [3H]thy-
midine incorporation. For each cell concentration the culture wells
are considered as positive when the counts per minute (cpm) are
greater than the mean ? 3 SD cpm from irradiated filler alone. The
results were conformed to the first term of Poisson’s distribution, and
the frequencies were derived from semi-log plots to indicate the
number of cells per culture at which 37% of the cultures failed to
proliferate.
Mixed lymphocyte reaction (MLR). Spleen cells (2?105/well) were
cultured at 37°C 5% CO2in RPMI 1640 medium supplemented with
10% fetal calf serum, 2 mM glutamine, 5?10?5M ?-mercaptoetha-
nol, and penicillin/streptomycin/neomycin (all from Gibco, BRL,
Gaithersburg, MD) in triplicate in 96-well, U-bottomed microplates
in the presence of 20 Gy-irradiated stimulators (4?105/well in a total
volume of 200 ?l) for 4 days and pulsed with [3H]thymidine 1 ?Ci/
well for 16 hr.
Staphylococcal enterotoxin B (SEB) stimulation assay. SEB (Sig-
ma) was administered by intraperitoneal (i.p.) injection at a single
dose of 50 ?g. Forty hours later, the percentages of V?6 and V?7
were determined among CD4 and CD8 T lymphocytes by flow cytom-
etry.
Injection of allogeneic cells. B6 spleen cell suspensions were pre-
pared in phosphate-buffered saline (PBS) and irradiated with 20 Gy.
A total of 5?106viable cells were injected i.p. in 0.2 ml.
Immunostaining and flow cytometry. Direct or indirect immuno-
staining was performed by incubation of 106cells with optimal dilu-
tions of labeled monoclonal antibodies diluted in PBS 3% calf serum,
0.01% sodium azide for 20 min at 4°C followed by washing in PBS at
each step. Stainings were done with CD4-phycoerythrin, CD8-fluo-
rescein isothiocyanate, B220-Tricolor (Caltag Laboratories, San
Francisco, CA), V?6-biotin, V?7-biotin (PharMingen, San Diego,
CA), and Streptavidin-Tricolor (Caltag). A FACSCalibur (Becton
Dickinson, San Jose, CA) was used for the acquisition of 10,000
events, and analysis was performed after gating on viable lympho-
cytes on the basis of forward and side scatter parameters.
Skin and heart grafts. Skin grafts were performed under tri-
bromo-ethanol anesthesia using 0.8 cm2of donor tail skin, sutured
on the back of the mouse as previously described (24). Graft appear-
ance was monitored at least three times a week and every day
around the time of rejection, and the graft was considered rejected
when the skin was necrotic or had contracted more than 90%.
Heart transplants were removed from day 1 newborn mice and
grafted between the ear sheets (24). Graft acceptance or rejection
was evaluated by clinical examination under a stereomicroscope.
GVHD. GVHD was induced by intravenous (i.v.) injection of 107T
cells (as determined by the percentage of CD4 and CD8 T cells in
pooled spleen and lymph node lymphoid cells) from TK transgenic
L40 FVB mice together with 107bone marrow cells from FVB non-
transgenic mice into B6 mice subjected to lethal whole body irradi-
ation (11 Gy delivered by an x-ray upper and lower source; Saturne
I, CGR Mev, Buc sur Yvette, France) 24 hr before grafting.
RESULTS
In vitro selectivity of GCV toxicity for activated T cells but
not B cells from TK transgenic mice. We first compared GCV
toxicity to T and B cells from TK transgenic mice and control
nontransgenic littermates after in vitro T or B lymphocyte
polyclonal stimulation with Con A or LPS, respectively (Fig.
1). Cell proliferation was evaluated in cultures containing
increasing GCV concentrations, and the dose of GCV allow-
ing for 50% inhibition of proliferation (ID50) was defined. At
ID50, Con A-activated cells from TK transgenic mice were 100
times more sensitive to GCV than cells from control mice.
Furthermore, at concentrations close to the in vivo GCV
plasma concentrations obtained in mice under our experi-
mental conditions (7.4 ?M?4.5, n?4, day 3 of GCV treat-
ment; Fig. 1), Con A-induced T-cell proliferation is inhibited
by 80–90% in TK?cultured cells, whereas LPS-induced pro-
liferation is not (P?0.06 at 10 ?M GCV, Mann-Whitney U
test).
Using limiting dilution cultures, we determined that the
frequencies of spleen cells from TK?mice that proliferate to
Con A is 1/26 without GCV (upper and lower 95% confidence
limit 1/17–1/38) and 1/1562 in the presence of 10 ?M GCV
(upper and lower 95% confidence limit 1/1099–1/2272). The
ratio of these frequencies indicates that over 98% of Con
A-reactive T cells from TK?mice are sensitive to GCV treat-
ment.
We also compared the GCV sensitivity of T cells from TK?
and TK?transgenic mice upon syngeneic and allogeneic
stimulation. T-cell proliferation was inhibited by more than
90% at 10 ?M GCV (Fig. 1b).
Specific depletion in vivo of activated T cells in TK trans-
genic mice treated with GCV. The ability to specifically kill
activated dividing TK?T cells but not resting TK?T cells
was analyzed in vivo. In nontransgenic FVB mice, the popu-
lation of T cells expressing the V?7 T cell receptor can be
specifically activated and divides upon stimulation with the
SEB superantigen, whereas the V?6 T-cell population does
not. We thus analyzed these T cell subsets after SEB injec-
tion and concomitant GCV treatment in TK transgenic and
control mice (Fig. 2). In nontransgenic mice, SEB activation
led to the expansion of V?7?T cells. In contrast, in TK
transgenic mice under GCV treatment, the percentages of
V?7?T cells slightly decreased. The V?6 T-cell percentages
mirrored those of V?7?T cells (i.e., decreased in TK?mice
and slightly increased in TK?mice), indicating that they
were not affected by the treatment. Similar observations
were made with either CD4?or CD8?T cells. These results
show that specific T-cell depletion of activated dividing T
THOMAS-VASLIN ET AL.
May 27, 2000
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cells but not resting T cells occurs in GCV-treated TK trans-
genic mice.
Effects of GCV treatment on CD4 and CD8 T cell numbers
and function after injection of allogeneic cells in TK trans-
genic mice. T-cell depletion was evaluated in spleens of TK
transgenic versus nontransgenic mice stimulated by an i.p.
injection of allogeneic irradiated B6 spleen cells and treated
responds by mean to 37,800 and 35,000 cpm, respectively, in
TK?and TK?cells) or LPS (100% proliferation corresponds
by mean to 17,700 and 24,400 cpm, respectively, in TK?and
TK?cells), and represent the mean ? SD of three indepen-
dent experiments. The shaded zone represents the range of
GCV plasma concentrations obtained after 3 days of contin-
uous delivery of GCV (50 mg/kg/day) by osmotic pump in vivo.
(b) Reactivities of spleen cells from TK?and TK?mice were
tested individually in MLR against syngeneic FVB, allogeneic
B6, and Balb/c stimulators, with or without 10 mM GCV. On day
3 of culture, proliferation was assayed by [3H]thymidine incor-
poration. Results are expressed as mean percentage and SD
(n?4) of the response obtained without GCV (8,300, 42,100, and
25,600 cpm for FVB, B6, and Balb/c stimulators, respectively).
FIGURE 1. In vitro toxicity of GCV to activated T cells but not
B cells from TK transgenic mice. (a) GCV toxicity was tested
by dose response proliferation of peripheral lymphocytes
from TK?transgenic or TK?littermates mice under Con A or
LPS stimulation. Cells were cultured for 48 hr under optimal
Con A (upper panel) or LPS stimulation (lower panel) and
with various concentrations of GCV, and proliferation was
then assayed by [3H]thymidine incorporation. Results are
expressed as the percentage of thymidine uptake in cultures
in the absence of GCV, under Con A (100% proliferation cor-
FIGURE 2. In vivo toxicity of GCV to activated but not resting
T cells from TK transgenic mice upon superantigen stimula-
tion. Transgenic TK?(f) and TK?littermates (?) were
treated with GCV by osmotic pump for 7 days from day ?1. On
day 0, they received an i.p. injection of SEB (50 ?g), and 40 hr
later (day 2) the percentages of V?6 and V?7 were estimated
among the CD4 and CD8 T lymphocyte populations of the
inguinal lymph node and compared with values obtained
from the same mouse in the contralateral inguinal lymph
node before SEB treatment (day 0). Results represent the
mean ? SD of four mice and show significant depletion of V?7
but not V?6 cells on day 2 in TK transgenic mice (P value,
Mann-Whitney).
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for 7 days with 50 mg/kg/day of GCV administered by osmotic
pump. The absolute numbers of spleen CD4?or CD8?T cells,
as well as B220?B cells, were determined on day 7. CD4?T
cell number in TK transgenic mice was 41% lower than in
nontransgenic mice (P?0.03 Student’s t test) (Fig. 3, top).
Although statistically nonsignificant (P?0.07), CD8?T cell
number also decreased by 21%, whereas B cell counts re-
mained stable (P?0.93). These results indicate that activated
cycling CD4?and CD8?T cells can be depleted in vivo, and
in accordance with in vitro findings (Fig. 1), without toxic
effects on B cells. It is however important to note that the
T-cell depletion observed here is not the exclusive effect of
allostimulation. Indeed similar rates of T-cell depletion were
also observed in naive TK transgenic mice after 7 days of
GCV treatment (not shown). This is explained by the high
physiological T-cell turnover in mice (7, 25) that probably
hinders the observation of the specific alloreactive T-cell
depletion. For this reason, the specificity of the T cells that
persist after in vivo B6 allogeneic stimulation was assayed by
their capacity to proliferate in response to allogeneic stimu-
lators in MLR (Fig. 3, bottom). The in vitro proliferation of T
cells from B6 sensitized mice to B6 stimulators was de-
creased in TK transgenic as compared with nontransgenic
mice, whereas the primary response to a third-party stimu-
lator was equivalent in both groups. This indicates that T-cell
depletion was specific for B6 donor alloantigens to which the
mice were exposed and that T cells with other specificities
retained their capacity to proliferate.
Outcome of skin allografts in TK transgenic mice after GCV
treatment. In a first set of experiments, we analyzed fully
mismatched allograft survival in transgenic TK and non-
transgenic control littermates of the FVB albino background,
treated with GCV for 14 days starting from the day of the
graft. Mice were grafted with fully allogeneic B6 (Fig. 4, top)
or semiallogeneic F1 (B6?FVB) (Fig. 4, middle) pigmented
skin. In both cases GCV treatment led to a significant pro-
longation of allograft survival. The mean survival time
(MST) of the disparate B6 skin graft was 18.6?0.8 days in
TK?mice (n?5) and 39.1?6.8 days in TK?mice (n?17)
(P?0.01, log-rank). The MST of the semiallogeneic F1 skin
graft was 15.7?0.8 days in TK?mice (n?4) and 28.8?2.5
days in TK?mice (n?12) (P?0.01, log-rank). There was no
significant differences for survival of B6 or F1 skin grafts in
TK?mice (P?0.7, log-rank). This suggests that natural killer
(NK) cells are not the main mediator of the rejection process
because FVB class I expression on the donor graft did not
increase graft survival. In control mice, allograft rejection
was acute, with signs of necrosis appearing from day 8 fol-
lowed by complete and rapid elimination of the graft. In
contrast, in TK transgenic mice the first signs of rejection
were delayed and when it occurred, the rejection process was
chronic, with macroscopic inflammation but persistence of
the donor tissue. By day 25, 50% of the allografts were
completely rejected. Grafts persisting more than 25 days
generally lost hair and pigmentation, and atrophy occurred
followed by graft elimination. In mice kept alive, such grafts
persisted until at least day 140. One such graft with absence
of hair was removed on day 41 after graft and transplanted
again in a naive nontransgenic FVB mouse. The graft was
apparently still immunogenic because it was rejected by day
11 (not shown). FVB syngeneic skin grafts were always per-
manently accepted, with hair growing from day 12, in both
TK transgenic and control mice.
Since skin grafts with multiple major and minor histocom-
patibility differences are not permanently tolerated, a second
set of experiments was performed using skin allografts from
mice matched for MHC but differing for multiple minor an-
tigens. Skin from DBA/1 mice was grafted on FVB TK trans-
genic and nontransgenic mice treated for 14 days with GCV.
In nontransgenic mice, acute rejection of DBA/1 skin was
observed (MST 15.2?1.5 days, n?5). In TK transgenic mice,
DBA/1 skin graft survival was on average doubled (MST
29.6?9.8, n?5).
Thus, H2 matching in this particular combination does not
increase the length of skin graft survival as compared with a
FIGURE 3. Cell depletion and reactivities in allostimulated
and GCV-treated TK transgenic mice. Transgenic (f) and
nontransgenic (?) mice were stimulated by i.p injection of
allogeneic 20 Gy-irradiated B6 spleen cells, after a 7-day GCV
treatment. (Top) On day 7, the phenotype of cells was deter-
mined in spleen by immunofluorocytometry. Results repre-
sent the mean ? SD (n?3) of absolute numbers of CD4, CD8,
and B220 cells present in the spleen. (Bottom) On day 7,
reactivities of spleen cells from two mice were tested individ-
ually in MLR against B6 donor (peak response on day 3) and
DBA/2 third-party stimulators (peak response on day 4). Cpm
are corrected by deducing syngeneic reactivity values. P val-
ues comparing proliferative responses of TK?and TK?mice
by Student t test are indicated. Similar results were obtained
in another independent experiment.
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fully allogeneic graft, because both combinations led to a
doubling of skin graft survival time.
Outcome of nonvascularized heart allografts in TK trans-
genic mice after GCV treatment. We next evaluated the out-
come of newborn heart graft transplanted in a heterotopic
position in the ear (24). FVB and B6 nonvascularized new-
born heart grafts were performed in the right and left ear in
TK transgenic and nontransgenic mice treated with GCV for
14 days (Fig. 4, bottom). The survival of the B6 heart was
significantlyincreased in
MST?32.2?4.6 days, TK?MST?16.8?0.7 days, P?0.05 log-
rank), with 50% of the B6 hearts rejected on day 32.2 in TK?
versus day 16.0 in TK?mice. Syngeneic FVB heart grafts
were indefinitely accepted in control and transgenic mice. In
half the cases, microscopically observed beating of the trans-
plant started on day 9–15 and continued for at least 150
TK transgenicmice (TK?
days. In the other half, where no beating was detectable,
spontaneous vascularization of the heart (red appearance)
typical of graft acceptance was nevertheless observed and
further confirmed histologically. In contrast, allogeneic heart
grafts in control mice present signs of rejection such as swell-
ing and necrosis (dark red appearance) that led in a few days
to complete rejection (yellow/white appearance).
In vitro immunocompetence of T lymphocytes from GCV-
treated allografted mice. We next evaluated the reactivity of
T cells obtained from allografted mice. The proliferation of
splenic T cells from the TK transgenic and nontransgenic
mice on day 26 after grafting of DBA/1 tail skin and a 14-day
course of GCV was assayed. Irradiated stimulator cells were
obtained from syngeneic (FVB), donor (DBA/1), and third-
party (B6) mice. At this time point, some mice still bore the
allograft, whereas others had rejected it (Fig. 5).
Reactivities against B6 third-party stimulators were high
in all mice, attesting their immunocompetence. As expected,
there was no primary response against minor disparate
DBA/1 stimulators in control naive FVB mice. By contrast,
both TK?and TK?mice that had rejected the DBA/1 skin
graft early (on days 13 or 16) presented high cell prolifera-
tion. In mice that were still tolerant at the time of assay, the
longer the graft was accepted, the lower was the prolifera-
tion. There was a linear correlation between the ratio of
proliferation against C57BL/6 and DBA/1 and the day of
donor graft rejection (R2?0.91).
In vivo immunocompetence of T lymphocytes from GCV-
treated allografted mice evaluated by their capacity to induce
FIGURE 4. Prolonged allograft survival in GCV-treated TK
transgenic mice. TK transgenic (f) and TK?littermate mice
(?) were grafted with allogeneic B6 skin (top), F1(B6?FVB)
skin (middle), or B6 heart (bottom) and syngeneic FVB grafts
(thick line in all groups) and continuously treated with GCV
for 14 days from the day of grafting. Results show the Kaplan-
Meier cumulative survival plot, number of allogeneic grafts
in TK?and TK?with P values (log-rank).
FIGURE 5. Increased allograft survival correlates with de-
creased donor MLR responses. TK?transgenic and TK?lit-
termates mice were grafted with allogeneic DBA/1 tail skin
and syngeneic FVB skin and continuously treated with GCV
for 14 days from the day of grafting. On day 26, after partial
splenectomy, spleen cells were cultured in MLR for 3 days in
the presence of irradiated (FVB syngeneic, DBA/1 donor, and
B6 third-party) stimulator cells and then pulsed with [3H]thy-
midine. TK?and TK?mice were either still tolerant to DBA/1
skin on day 26 (these mice remained tolerant >110 days or
rejected on day 53) or had already rejected the graft (rejec-
tion on days 11, 13, or 16). Bars represent corrected cpm
values against allogeneic stimulators (by deducing syngeneic
reactivity values), and the numbers indicate the ratio of pro-
liferation between C57BL/6 and DBA/1.
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GVHD. We next investigated in vivo the functional reactiv-
ity of T lymphocytes from FVB TK transgenic and nontrans-
genic mice grafted with allogeneic skin and treated with GCV
by their capacity to induce GVHD in B6-irradiated mice.
T cells were removed from TK transgenic or nontransgenic
mice bearing a healthy B6 skin graft or having rejected it at
various time points after graft, and injected together with
FVB bone marrow cells in groups of five lethally irradiated
B6 recipients. As shown in Figure 6, recipient mice died of
acute GVHD when they received T cells from nontolerant
nontransgenic donors or from TK transgenic donors that had
rapidly rejected the B6 skin graft (on day 16). In contrast,
mice that received T cells from donors with delayed skin graft
rejection (on day 24) or still bearing the B6 skin graft at the
time of T cell harvesting, had a significantly longer survival
as compared with mice hosting T cells from allogeneic skin-
grafted, nontransgenic donors (P?0.05 and P?0.005, respec-
tively, log-rank).
DISCUSSION
We investigated the possible use of genetic immunosup-
pression for inducing transplantation tolerance, in a model of
transgenic mice expressing the TK suicide gene in both CD4
and CD8 T cells and submitted to a short course of GCV
treatment.
In TK transgenic mice, the GCV-mediated depletion of
TK?T cells should be limited to activated proliferating T
cells, sparing quiescent T cells and B cells. This was indeed
demonstrated in vitro and in vivo in various experiments: (i)
in vitro mitogen-activated T cells but not B cells were killed
at clinically relevant GCV concentrations; (ii) TK?T-cell
proliferation was reduced by 95% after in vitro alloactivation
in the presence of GCV; (iii) superantigen-activated CD4 and
CD8 T cells were specifically eliminated by GCV treatment in
vivo, sparing nonactivated T cells; and (iv) in vivo allostimu-
lation together with GCV administration resulted in a de-
crease in absolute CD8 and CD4 T cell numbers, stability of
B cell numbers, and a specific reduction of in vitro prolifer-
ation with the same allostimulators. These findings support
the use of transgenic mice expressing TK in T cells for ex-
perimental investigations requiring conditional and specific
ablation of activated dividing T cells, in immunopathological
situations such as graft rejection in tissue transplantation or
control of GVHD after allogeneic hematopoietic stem cell
transplantation as previously described (14).
Indeed, a significant prolongation of survival of nonvascu-
larized allogeneic skin and heart grafts was observed in TK
transgenic mice subjected to a short course of GCV. On av-
erage, the survival time was doubled for combinations of
either fully mismatched or MHC-matched donor and recipi-
ent. Allogeneic grafts were not indefinitely accepted, and the
grafted skin often displayed signs of chronic rejection such as
loss of pigmentation and hair, and shrinking. Nonetheless,
such chronically rejected grafts retained antigenic capacities
as demonstrated by their rejection after regrafting in naive
allogeneic hosts.
It should be noted that the survival of allogeneic skin
grafts was correlated with control of the primary T-cell re-
sponse by GCV treatment that prevents the induction of
T-cell memory against the donor allograft. Indeed, when T
cells from mice still bearing allogeneic skin grafts were sub-
mitted to a secondary stimulation either in vitro (Fig. 5) or in
vivo (Fig. 6), the response to the donor haplotype resembles
that of naive mice in both MLR and GVHD experiments (14).
So far, permanent survival of fully mismatched skin allo-
grafts can be achieved only with a limited number of immune
manipulations before skin allografting: (i) immune restora-
tion of the athymic host after donor-type thymic epithelium
graft (24, 26); (ii) injection of donor-type allogeneic T cells in
the portal vein followed by bone marrow cell infusion (27);
(iii) combined blockade of CD28 and CD40 T-cell activation
pathways (28); and (iv) antilymphocyte antibodies, either
depleting or not (29–31); but some of these treatments are
efficient only in certain specific donor-host combinations.
Thus, new approaches enabling the specific depletion/inacti-
vation of donor-reactive T cells with preservation of immu-
nocompetence are of vital interest for transplantation toler-
ance. The pharmacogenetic control of donor-reactive T cells
by suicide gene therapy, as demonstrated in the present
study, fulfills such a role. Nevertheless, although this system
is extremely efficient to control acute rejection, the absence of
complete tolerance and the delayed and chronic graft rejec-
tion may have several explanations.
First, incomplete depletion of alloreactive T cells may re-
sult from (i) insufficient local GCV concentrations or TK
expression in some cells; (ii) renewal of alloreactive cells from
recent thymic emigrants after cessation of GCV; or (iii) high
levels of CD4 T-cell depletion that could preclude CD8 acti-
vation/division and thus depletion.
Second, in addition to T cells, other cell types such as
macrophages, NK cells, and alloantibody-secreting B cells,
FIGURE 6. Increased allograft survival in GCV-treated trans-
genic mice correlates with decreased capacity to induce
GVHD. The in vivo functionality of T cells obtained from TK
transgenic and TK?littermate mice grafted with allogeneic
B6 skin and treated for 14 days with GCV as in Figure 4, was
tested by the capacity of their T cells to induce GVHD upon
transfer to B6-irradiated mice (five mice per group). The B6
recipient mice were either left untreated (?) or injected 24 hr
after irradiation with 107bone marrow cells from naive FVB
donor (O) or with 107bone marrow cells plus 107T cells, from
various mice previously grafted with B6 skin. These were
TK?mice that had rejected B6 skin on day 16 (E), TK?that
had rejected skin, respectively, on days 16 (Œ) and 24 (F) and
were killed on day 38, or a TK?mouse (f) still bearing a
healthy and hairy B6 skin graft on day 22 after grafting and
killed on this day. Results are expressed as the percent of
mice that survived GVHD after T-cell transfer.
THOMAS-VASLIN ET AL.
May 27, 2000
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Page 7

which are not depleted by GCV, may be involved in chronic
graft rejection. In this model, NK cells are unlikely to play a
significant role because the survival of semiallogeneic and
allogeneic grafts is similar, and NK may play a limited role in
allogeneic skin graft rejection (32).
Third, the nature of the tissue graft itself may influence
graft survival. Skin may indeed be more immunogenic than
solid organs due to the display of several cell types with
minor and major antigens and a high frequency of passenger
leukocytes (33-35). Heterogeneity in the survival of various
tissue grafts was noted by several groups. Anti-CD4 treat-
ment induced tolerance to vascularized adult but not fetal
heart and to adult but not fetal pancreas, and not to skin or
lung (29). In other studies involving treatment with anti-CD3
(36) or anti-V? antibodies (37), control of T-cell activation
after blocking CD40 or CD28 pathways (28), or intrathymic
injection of donor bone marrow cells (38), skin graft survival
was transiently prolonged whereas vascularized heart allo-
graft survival was more prolonged or definitively accepted.
Such differences might also be related to the vasculariza-
tion of the graft (36, 37, 39). Differences in blood supply may
lead to different ways of presentation of the donor antigens
and differences in tolerance or immunization. In nonvascu-
larized grafts, the allogeneic immune response is initiated in
the draining lymph nodes and leads to local sensitization by
donor antigen-presenting cells and graft rejection. In vascu-
larized grafts, the immune response could be initiated in the
spleen as proposed by others (40, 41). The same may occur
after infusion of donor lymphocytes intravenously (42) or in
the portal vein (27). Such priming seems to induce peripheral
tolerance by triggering suppressor cells or by anergy, induced
by direct recognition of donor cells in the absence of costimu-
latory molecules (43). Moreover, the persistence of the anti-
gen in the form of vascularized graft (44) or of long-lived
lymphoid donor cells (45) seems to be required to maintain
tolerance.
In the present model, T-cell depletion is the primary mech-
anism for induction of tolerance, but subsequent anergy
and/or suppression of T cells may also occur, particularly in
the case of vascularized grafts, as observed in other models of
transplantation tolerance induced by antilymphocyte anti-
body treatments (29, 46).
Finally, it should be emphasized that this system is versa-
tile and other treatment modalities might lead to improved
results. For example, depletion of alloreactive T cells can be
performed before transplantation by injection of donor cells
together with GCV treatment (Fig. 3).
Clinical application of genetic immunosuppression will re-
quire transducing T cells with TK gene and reinjecting them
into patients, as already performed in clinical trials for
GVHD treatment (16). However, numerous difficulties will
have to be overcome before such therapy can be used in
patients. Although transduction can now efficiently be per-
formed with available methods in hemopoietic progenitors or
in mature T cells, optimal conditioning of the recipient will
have to be developed to permit efficient gene modified T-cell
engraftment. In addition, it remains to be shown that a
functional and diverse immune system can be reconstituted
with such genetically modified cells.
Further studies, notably using vascularized allografts, are
required to define the potential role of genetic immunosup-
pression in transplantation medicine.
Acknowledgments. We thank C. Durieu for technical help.
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Received 27 May 1999.
Accepted 3 November 1999.
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