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T Cells Indirectly Regulate Murine Graft-Versus-Host Reactivity Following Donor Leukocyte Infusion Therapy in Mice1
Departments of
*
Medicine and
Biostatistics and
Bone Marrow Transplant Program, Medical College of Wisconsin, Milwaukee, WI 53226
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
were able to regulate graft-vs-host (GVH) reactivity mediated by 
ß
T cells in murine recipients transplanted with MHC-mismatched marrow
grafts. Studies were conducted using ex vivo-activated T cells
because this was a more clinically relevant strategy, and these cells
have been shown to be capable of facilitating alloengraftment without
causing GVH disease (GVHD). Coadministration of activated T
cells and naive ß T cells at the time of bone marrow
transplantation (BMT) significantly exacerbated GVHD when compared with
naive ß T cells alone. In contrast, when the administration of
naive ß T cells was delayed for 2 wk post-BMT, survival was
significantly enhanced in mice transplanted with BM plus activated
T cells vs those given marrow cells alone. Mitigation of GVHD by
activated T cells occurred only at high doses (150 x
106) and was a unique property of T cells, as
activated ß T cells were incapable of ameliorating the subsequent
development of GVHD. Protection from GVHD was not due to the direct
inhibition of naive ß T cells by T cells. Rather, T
cells mediated this effect indirectly through donor BM-derived ß T
cells that acted as the proximate regulatory population responsible for
the decrease in GVH reactivity. Collectively, these data demonstrate
that activated T cells are capable of modulating the ability of
MHC-incompatible nontolerant ß T cells to cause GVHD after
allogeneic BMT. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß or TCR heterodimer. Cells that express the
TCR-ß are thought to be the primary T cell subpopulation
responsible for mediating GVH/GVL reactivity and facilitating
alloengraftment (4, 5, 6). The role of T cells after
marrow transplantation is less well defined.
Murine BMT studies have provided conflicting data as to whether naive
T cells play a role in the pathophysiology of GVHD. Although
several studies have shown that T cells can contribute to GVH
reactivity (7, 8, 9), others have demonstrated that T
cells can inhibit GVHD that is induced by ß T cells
(10). A role for T cells in the facilitation of
alloengraftment in mice has also been proposed (11),
suggesting that these cells can effect some of the same immune
responses in allogeneic BMT as ß T cells. Because T
cells comprise only a small percentage of total T cells, direct
evaluation of the role of these cells in murine allogeneic marrow
transplantation has been difficult. Moreover, the paucity of these
cells in human peripheral blood makes the clinical translation of these
results potentially problematic. For that reason, we opted to evaluate
transplantation of ex vivo-activated and expanded T cells as a
more clinically relevant strategy. These studies showed
that large doses of ex vivo-activated and expanded T cells were
capable of facilitating alloengraftment across the MHC barrier
(12). Graft facilitating doses of these cells also failed
to cause GVHD in MHC-mismatched donor/recipient strain combinations
(13), demonstrating that transplantation of T cells
may be an approach to promote allogeneic engraftment without
causing GVHD.
T cells have been shown to have important immunological
functions in mice. Studies have demonstrated a role for these cells in
host defense against pathogens (14, 15). Additionally, a
number of reports support a primary role for T cells in the
induction of tolerance to both inhaled and ingested Ags
(16, 17, 18). In some, but not all, experimental settings,
these cells also are capable of contributing to the resolution of
inflammatory lesions (19, 20). One mechanism by which
T cells have been shown to induce tolerance and resolve
inflammatory lesions is by down-regulating immune responses mediated by
ß T cells (17, 21, 22). These reports prompted us to
explore whether T cells were capable of regulating immune
responses mediated by ß T cells after allogeneic marrow
transplantation. Because GVHD is the major complication of allogeneic
BMT and is mediated primarily by ß T cells, we elected to examine
how these two cell populations interacted in the setting of a GVH
reaction. To simulate human marrow transplantation, we assessed the
effect of T cells on the development of GVHD under two different
scenarios: that occurring from the infusion of naive ß T cells at
the time of BMT, and that resulting from a delayed infusion of naive
ß T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (H-2b), TCR
ß-/ß- (ß T
cell-deficient, C57BL/6 background, H-2b), AKR/J
(H-2k), and B10.BR (H-2k)
mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
C.B-17 scid mice (BALB/c background,
H-2d) were obtained from Taconic Farms
(Germantown, NY). All animals were housed in the American Association
for Lab Animal Care (AALAC)-accredited Animal Resource Center of the
Medical College of Wisconsin. Mice received regular mouse chow and
acidified tap water ad libitum. C.B-17 mice were housed in
microisolator cages, while all other animals were housed in
conventional cages.
Ex vivo activation and expansion of murine T cells
To expand T cells, spleen cells were obtained from TCR
ß-/ß- donor animals
and passed through nylon wool columns to remove B cells. The resulting
population was typically comprised of 
50% cells expressing the
TCR. Cells were then resuspended in complete DMEM plus 5–10%
FBS and cultured in flasks precoated with an immobilized T
cell-specific mAb (GL4, hamster IgG; PharMingen, San Diego, CA) at a
concentration of 5–10 µg/ml. Twenty-four hours after the initiation
of culture, human IL-2 (Proleukin, Chiron, Emeryville, CA) was added at
a concentration of 200 IU/ml. All cultures were split into fresh flasks
as needed to maintain a cell concentration of 0.5–1.5 x
106 cells/ml. Cells were exposed to immobilized
mAb for the first 3–4 days of culture and thereafter grown only in
medium plus 100 U/ml IL-2 to allow for reexpression of the TCR.
After a total of 7–8 days in culture, cells were counted, and the
percentage of T cells was analyzed by flow cytometry. Routinely,
a total of 5–10 x 108 cells was obtained
after expansion, with 95–99% of cells expressing the TCR.
Typically, 15–25% of activated T cells coexpressed CD8. CD4
expression was not detected on these cells. A similar approach was
employed for the activation and expansion of ß T cells with the
exception that spleen cells were obtained from C57BL/6 mice and
cultured in flasks precoated with an immobilized anti-CD3 Ab (clone
145-2C11) (kindly provided by J. Bluestone, University of Chicago,
Chicago, IL). Routinely, >95% of viable cells had reexpressed the
CD3/TCR complex at the time that these cells were transplanted into
recipients.
BM transplantation
BM was flushed from donor femurs and tibias with complete DMEM
and passed through sterile mesh filters to obtain single cell
suspensions. BM was T cell depleted (TCD) in vitro with anti-Thy1.2
mAb plus low toxicity rabbit complement (C6 Diagnostics, Mequon, WI).
The hybridoma for 30-H12 (anti-Thy1.2, rat IgG2b) Ab was obtained
from the American Tissue Culture Collection (Manassas, VA) and grown in
complete DMEM plus 5% FBS. The culture supernatants was then
harvested, precipitated in ammonium sulfate, and dialyzed against PBS
before use in in vitro depletion experiments. BM cells were then washed
and resuspended in DMEM before injection. To recover naive T cells,
spleen cell suspensions were obtained by pressing spleens through wire
mesh screens. Erythrocytes were removed from cell suspensions by
hypotonic lysis with sterile distilled water. Naive T cells for
admixture with TCD BM before transplantation were then obtained by
passing spleen cells once or twice through nylon wool columns (Robbins
Scientific, Sunnyvale, CA) to remove B cells. The percentage of
ß+ T cells from B6 donors was quantified by
flow cytometry and defined as Thy1.2+
TCR-ß+.
B10.BR or AKR/J recipient mice were given varying doses of lethal total
body irradiation (900 or 1100 cGy, respectively) as a single exposure
at a dose rate of 70 cGy using a Shepherd Mark I Cesium Irradiator
(J. L. Shepherd and Associates, San Fernando, CA). Irradiated
recipients then received a single i.v. injection of TCD BM (10 x
106) with or without ex vivo-activated T cells.
When a dose of 150 x 106 activated T
cells was administered to recipients, the total dose was split in half
or in fourths and given over a period of 24 h. This was done to
reduce immediate toxicity from the infusion of a large number of
activated T cells.
Flow cytometric analysis
mAbs conjugated to either FITC or PE were used to assess
chimerism in marrow transplant recipients. FITC-anti-Thy1.2 (clone
30-H12, rat IgG2b) was purchased from Collaborative Biomedical Products
(Bedford, MA). FITC-anti-Ly5 (B220, rat IgG2a) and PE anti-CD8
(clone CT-CD8a, rat IgG2a) were obtained from Caltag (San Francisco,
CA). PE anti-TCR ß (clone H57-597, hamster IgG), PE
anti-TCR (clone GL3, hamster IgG), PE anti-CD3 (clone
145-2C11, hamster IgG), PE anti-CD4 (clone GK 1.5, rat IgG2b),
FITC-anti-H-2Kb (clone AF6-885, mouse IgG2a)
were all purchased from PharMingen. Spleen and thymus cells were
obtained from chimeras at defined intervals posttransplant and stained
for two-color analysis. Red cells were removed by lysis in distilled
water. Cells were analyzed on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA). Red cells and nonviable cells were
excluded using forward and side scatter settings before analysis of
spleen cell populations. Splenic T cell chimerism was assessed within a
lymphocyte gated population, while thymic chimerism was evaluated using
open gates. Ten thousand cells were analyzed for each determination
whenever possible.
Statistics
Group comparisons of T cell chimerism in the spleen and thymus,
and thymic size were performed using the unpaired Student t
test. Data are presented as the mean ± SE. Survival curves were
constructed using the Kaplan-Meier product limit estimator and compared
using the log-rank rest. For experiments evaluating the effect of
delaying the administration of naive ß T cells to mice
transplanted with and without T cells, a Cox regression model
was used to compare survival between the respective groups. In this
model, no significant survival difference was found between groups of
mice transplanted with TCD BM followed by naive T cells on either day
0, 7, or 14 post-BMT. For that reason, a Cox model was fit pooling the
data from these three groups. Groups of mice transplanted with TCD BM
and activated T cells, and then given naive T cells on day 0, 7,
or 14 post-BMT were compared with these pooled data to evaluate
survival differences. In the T cell dose titration experiments,
survival was compared using the Mantel-Haenszel test. A two-tailed
p value 
0.05 was deemed to be significant in all
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells results in protection
from GVHD induced by a delayed infusion of naive donor T cells
Prior studies by others indicate that, in selected experimental
settings, T cells are able to down-regulate the ability of
ß T cells to mediate immune responses (17, 21). These
data raised the question as to whether T cells might be capable
of mitigating or preventing GVHD induced by ß T cells. To evaluate
the immunoregulatory role of T cells, we performed studies using
a previously described donor leukocyte infusion (DLI) model (23, 24) which allowed us to vary the time at which mice received
naive T cells posttransplant. Cohorts of lethally irradiated B10.BR
mice were transplanted with TCD B6 BM alone or TCD BM plus 150 x
106 activated T cells. Naive B6 T cells
(comprised of >97% ß T cells) were administered at the time of
BMT, or at 7 or 14 days after transplant. For GVH control mice
transplanted with TCD BM alone, the day on which naive ß T cells
were administered (day 0 vs 7 vs 14) did not significantly affect
survival between any of the three groups (p =
0.096). In contrast, the timing of naive ß T cell administration
significantly affected survival in mice transplanted with TCD BM and
activated T cells. When mice were transplanted with activated
T cells and naive T cells at day 0, animals had significantly
reduced survival relative to mice transplanted with naive T cells only
(Fig. 1
A)
(p = 0.019), indicating that activated T
cells exacerbated GVHD induced by naive ß T cells. Delaying the
administration of naive T cells for 7 days, however, resulted in
equivalent survival for both groups (Fig. 1B)
(p = 0.75). Conversely, when the infusion of
naive T cells was delayed to day 14 posttransplant, mice transplanted
with activated T cells were protected from lethal GVHD and had
an enhanced survival rate (p = 0.019) (Fig. 1C). These data indicated that T cells were able to
contribute to GVH reactivity when administered contemporaneously with
naive ß T cells. When the administration of naive T cells was
delayed for 2 wk, however, transplantation with T cells
protected mice from GVHD and prolonged survival.
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T cells
were not strain dependent, we repeated these experiments with a
different MHC-incompatible (B6
AKR) murine model. Administration of
DLI 2 wk after BMT to AKR mice transplanted with activated T
cells again resulted in a significantly higher survival rate as
compared with mice that received TCD BM only (p
= 0.01) (Fig. 2). Thus the effect of
T cells was not strain-specific.
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T cells is dose dependent
Experiments were performed to determine whether protection from
GVHD by activated T cells was dose dependent. AKR recipient mice
were transplanted with TCD B6 BM and either 5 x
106, 25 x 106,
150 x 106, or no activated T cells.
Two weeks later, animals were given 2 x 106
naive B6 ß T cells. No difference in survival was observed between
mice that received no T cells (20% survival at day 100,
n = 56) and those that received either 5 x
106 (16%, n = 55) or 25 x
106 (25%, n = 53) activated
T cells at the time of BMT. In contrast, transplantation with
150 x 106 activated T cells (36%,
n = 47) mitigated the development of lethal GVHD and
significantly prolonged survival when compared with the GVHD control
population (p = 0.023 by Mantel-Haenzel
test).
Protection from GVHD by activated T cells is a property unique to
T cells
We have shown previously that anti-CD3 Ab-activated ß T
cells cause significantly less GVHD than do an equivalent number of
naive T cells when administered at the time of transplant
(25). We therefore considered that protection from GVHD
after a delayed infusion of ß T cells might be a general property
of activated T cells and not a unique characteristic of T cells.
To address this question, AKR mice were transplanted with TCD B6 BM
alone or together with either a low dose (2 x
106) or a high dose (100 x
106) of anti-CD3 activated B6 ß T cells.
The mice were infused with 2 x 106 naive B6
T cells 2 wk later. The two doses of activated ß T cells were
selected to encompass a wide cell dose range and thereby account for
any effect that large doses of activated T cells per se might have on
GVHD protection. There was no difference in survival rates after DLI
between mice transplanted with 2 x 106
activated T cells vs mice given TCD BM only (p
= 0.14) (Fig. 3A). In
contrast, mice transplanted with 100 x 106
activated T cells had significantly worse survival after DLI than mice
transplanted with TCD BM followed by DLI (p =
0.016) (Fig. 3B). Death in the group of mice transplanted
with 100 x 106 activated T cells was due to
accelerated GVHD. These data indicated that protection from GVHD by
activated T cells was due to a unique property of T cells and
not a property of T cells in general.
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T cells do not protect against GVHD in
scid mice
The protective effect of activated T cells was likely due
either to a direct effect of these cells or to an indirect effect that
required the presence of another cell population. To address the
question of whether another cell population was involved, we examined
whether T cells mediated protection against GVHD in a
scid mouse model. This model was chosen because
transplantation can be performed without the need for preconditioning
or reconstitution with donor BM. scid mice can therefore be
transplanted with activated T cells alone and then given a
delayed infusion of naive GVH reactive T cells. The validity of this
model, however, is dependent upon activated T cells not being
rejected by host immune cells (e.g., NK cells) in the scid
animals. To address this issue, scid mice (n
= 8) were transplanted with 150 x 106
activated T cells and then analyzed 2 wk posttransplant for
donor chimerism. The mean percentage of T cells in the spleen
was 16.7% (range, 5.8–39.6), indicating that T cells were not
completely rejected by host NK cells. The percentage of T cells
was within the range observed for irradiated mice transplanted with TCD
BM and the same dose of activated T cells (W. R. Drobyski,
unpublished observations). Thus, we concluded that the scid
model was a valid one with which to address the role of other T cells.
scid mice were transplanted with activated B6 T cells
and then administered naive B6 T cells (4 x
106) 2 wk later. GVHD control mice received the
same naive B6 T cells without the antecedent infusion of T
cells. Mice transplanted with T cells followed by naive T cells
had significantly lower survival rates than animals which only received
naive T cells (Fig. 4)
(p = 0.003). These data strongly suggested that
the mitigation of DLI-induced GVHD by T cells was not a direct
effect of these cells but was dependent upon the presence of another
cell population(s).
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ß T cells
Because the scid mouse model did not employ donor BM,
we considered that the additional cell population might be a BM-derived
cell. Support for this hypothesis comes from a report by Johnson et al.
(23) who demonstrated the presence of a BM-derived T cell
that was able to down-regulate GVH reactivity after DLI. To address
this question, lethally irradiated AKR recipients were transplanted
with TCD BM from either normal B6 or ß T cell deficient
(ß-/ß-) B6 donors.
Mice in both cohorts received 150 x 106
activated T cells with their marrow grafts and 2 wk later were
administered an equivalent dose of 2 x 106
naive ß T cells. Animals transplanted with BM from
ß-/ß- donors had
significantly higher mortality from GVHD as compared with mice given
normal B6 BM (25% vs 69% 100-day survival, p = 0.021)
(Fig. 5). These studies suggested that
the protective effect of activated T cells required that the
donor marrow be capable of generating ß T cells and that a
BM-derived ß T cell was the proximate cell responsible for
mitigation of GVHD.
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T cells promote engraftment of BM-derived donor
Thy1.2+ T cells in the thymus
BM-derived T cell progenitors undergo selection and maturation in
the thymus before emigrating into the periphery. Because these cells
were necessary for mitigation of GVHD, we examined the thymi of mice
transplanted with and without T cells to determine whether
T cells enhanced thymic engraftment with BM-derived donor T cell
precursors. Thymic engraftment was examined 2 wk after BMT which
corresponded to the time that T cells were the predominant donor
T cell population in the spleen and the time that transplanted mice
were given naive ß T cells in the experiments above. Animals given
activated T cells had an increased percentage of donor T cells
in the spleen (80 ± 2% vs 39 ± 4%) ascribable solely to
the presence of donor T cells. There was a commensurate
reduction in the percentage of host T cells in these mice as well
(3 ± 0% vs 7 ± 2%;
p = 0.021). As shown in Table I, the thymi of mice transplanted with
activated T cells contained a similar average percentage and
absolute number of double-positive thymocytes as TCD BM control
animals. This is consistent with a lack of GVHD in mice transplanted
with T cells. At 2 wk, animals transplanted with activated
T cells had a significantly greater percentage of donor
Thy1.2+ T cells in the thymus when compared with
control mice (Table I). Both donor-derived CD4+
and CD8+ single-positive T cells were increased
relative to mice transplanted with TCD BM alone. Because the average
thymic size was the same in both groups, the absolute number of
donor-derived CD4+ and CD8+
cells was increased. There was also an increased percentage of donor
CD4+CD8+ double-positive
cells in mice transplanted with activated T cells. This provided
evidence that T cells increased the number of thymic T cell
progenitors that were derived from the donor BM.
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T cells (ranges, 6–67% and
13–61%, respectively). Therefore we examined whether there was a
correlation between the percentage of splenic donor T cells and
the degree of thymic engraftment by donor cells. A comparison of the
percentage of splenic donor T cells and the percentage of donor
thymic Thy1.2+ CD4+ cells
in individual mice 14 days posttransplant demonstrated a statistically
significant correlation between these two variables (Fig. 6) (Pearson’s coefficient,
r = 0.79, p < 0.0001). A similar
correlation was observed for CD8 cells (data not shown). These data
indicated that mice with the highest percentage of T cells in
their spleens at day 14 exhibited the greatest degree of donor T cell
engraftment in the thymus, suggesting a direct role for T cells
in promoting the posttransplant engraftment of BM-derived donor ß
T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cells were capable of regulating the ability of ß T cells to cause
GVHD. Initial studies showed that when T cells and ß T
cells were co-administered to lethally irradiated recipients, there was
an exacerbation of GVHD when compared with mice that received ß T
cells alone. Because transplantation of activated T cells alone
does not cause significant GVHD (13), these results
indicated that T cells could contribute to GVH reactivity but
required the presence of ß T cells. This is consistent with data
by Tsuji et al. (8) who showed that T cells were
incapable of inducing GVHD by themselves but could be induced to
proliferate by donor ß T cells. A possible explanation for the
exacerbation of GVHD is the release of soluble factors from ß T
cells that can stimulate the proliferation of T cells (26, 27). Activated T cells also secrete Th1-type cytokines,
such as IL-2 and IFN- (28, 29) and may thereby
contribute to the amplification of the GVH reaction in a paracrine
fashion. When the administration of naive ß T cells was delayed
until 2 wk posttransplant we observed that animals given T cells
at the time of BMT had a significant decrease in GVHD and improvement
in survival. Even though T cells were still detectable in the
spleen of recipients at 2 wk posttransplant, the infusion of naive
donor T cells did not exacerbate GVHD (Figs. 1
C and 2). This
effect was dose dependent and required transplantation of large numbers
of activated T cells (i.e., 150 x
106). At this dose there was a consistent 2- to
3-fold improvement in the survival rate of these mice (Figs. 1C and 2). Protection from GVHD was not complete, however,
as some mice did develop GVHD and recent data suggest that this effect
can be overwhelmed by a high dose of naive T cells (i.e., 4 x
106) (our unpublished observations). Notably,
mitigation of GVHD was specific to activated T cells because
activated ß T cells had no inhibitory role when examined at a low
dose (2 x 106) (Fig. 3A) and
exacerbated GVHD at a high dose (100 x 106)
(Fig. 3B). Collectively, these data suggested that T
cells altered the immune environment of these mice rendering them less
susceptible to GVHD when exposed 2 wk later to alloreactive donor T
cells.
Studies were conducted to examine the mechanism by which
transplantation with activated T cells protected mice from a
subsequent challenge of ß T cells. To determine whether T
cells had a direct regulatory effect on the alloreactivity of ß T
cells infused into host mice, transplants were done in scid
recipients which obviated the need for preconditioning therapy and
infusion of donor BM. Animals previously transplanted with T
cells developed significantly more severe GVHD after a delayed infusion
of naive ß T cells than mice that received only naive T cells
(Fig. 4). These results were similar to those observed when both T cell
populations were coadministered at the time of BMT (Fig. 1) and
indicated that T cells did not directly prevent ß T cells
from causing GVHD. This suggested that the protective effects induced
by T cells were indirect (i.e., dependent upon interaction with
another regulatory cell population), although these results did not
exclude the possibility that irradiation played a contributory role.
Because the scid model did not employ donor BM, we reasoned
that a putative regulatory cell population might be derived from the
marrow. Subsequent studies utilizing ß T cell-deficient donor BM
in an irradiation model resulted in a loss in the protective effect
induced by T cells (Fig. 5), providing confirmatory evidence
that the regulatory cell was a donor BM-derived
ß+ T cell. These results are supportive of
studies by Johnson et al. (23) who demonstrated the
existence of a donor, BM-derived, TCR-ß+ T
cell that is normally present in reconstituting chimeras and capable of
down-regulating GVH reactivity after DLI. They noted that this cell was
present only in marrow recipients that possessed an intact thymus,
indicating that these regulatory T cells were derived from the
donor-reconstituted host thymus. Elimination of this regulatory
population posttransplant significantly exacerbated GVHD induced by a
delayed infusion of donor T cells, demonstrating a critical role for
these cells in establishing peripheral tolerance.
The key question of how transplantation with activated T cells
promoted the ability of BM-derived ß T cells to mitigate GVHD has
not been fully answered. We have shown previously that activated
T cells can facilitate alloengraftment, but that high doses are
required (i.e., 150 x 106)
(12). In the current study, a protective effect was
observed only when a similarly high dose of T cells was
utilized. At this dose, there was a significant reduction in the
percentage of residual host T cells in the spleen after conditioning
therapy, substantiating a role for these cells in alloengraftment.
Based on these collective data, our hypothesis is that transplantation
of activated T cells enhances the emergence of a regulatory
population of donor BM-derived ß T cells from the thymus
posttransplant. The strong correlation between the percentage of
T cells in the spleen and percentage of donor-derived thymic cells 2 wk
after BMT supports this premise and suggests a causal role for T
cells in thymic engraftment of donor BM-derived ß T cell
precursors. How these cells augment donor thymic engraftment is
unknown. One possibility is that the graft-promoting effects of donor
activated T cells are responsible for protection from
DLI-induced GVHD. That is to say, by contributing to the elimination or
inactivation of host T cells that mediate graft resistance, T
cells may accelerate the emergence of a BM-derived regulatory
population.
A number of studies have supported a role for T cells in the
induction of tolerance in the nontransplant setting. Studies by Ke et
al. (16) and Mengel et al. (30) have
demonstrated that T cells are the critical cell population
necessary for the induction of oral tolerance when animals are
administered an antigenic challenge with OVA. Moreover, T cells
can prevent the development of insulin-dependent diabetes when
adoptively transferred into unaffected recipients (31) and
prolong skin allograft survival (32). Collectively, these
studies have shown that T cells can induce tolerance in both
humoral and cell-mediated responses. In some (17, 21, 22)
but not all instances (33, 34), T cells have also
been shown to be able to down-regulate immune responses mediated by
ß T cells. Kaufmann et al. (21) have shown that
ß T cells from mice treated in vivo with a T cell-specific
Ab had increased proliferation, cytokine secretion, and cytotoxicity in
vitro, suggesting that these cells had an inhibitory effect on ß T
cells. Supportive data for this premise also have come from work
showing that T cells down-regulate ß T cell responses in
murine listeriosis (35), mycobacterial tuberculosis
(36), and experimental Trypanosoma cruzi
infection (37). A common finding in the majority of these
studies is that the tolerogenic effects of T cells are mediated
by a direct interaction between these cells and ß T cells, or that
T cells elaborate cytokines that down-regulate the immune
response (35). The current study suggests another
mechanism by showing that T cells can mediate a tolerogenic
effect indirectly through a secondary regulatory ß T cell
population. Additional studies will be required to determine whether
this is unique to the BMT setting.
From a clinical perspective, these data may prove relevant to the
emerging use of adoptive immunotherapy as a means to prevent relapse
after allogeneic BMT. Due to the increased incidence and severity of
GVHD when donor T cells are administered at the time of transplant,
recent approaches have focused on the delayed infusion of T cells at
defined time points post-BMT (38, 39, 40, 41, 42). Although the
administration of DLI appears to cause less GVHD than when a comparable
number of donor T cells are given at the time of marrow grafting, GVHD
remains a formidable problem, particularly in recipients of mismatched
or unrelated grafts. The ability of activated T cells to protect
recipients from GVHD induced by a subsequent DLI infusion is an
observation that may allow for this strategy to be more clinically
efficacious. This approach has the added advantage that activated
T cells appear to be able to prevent graft rejection
(12). Therefore, alloengraftment might not be compromised,
but actually enhanced with this strategy. A barrier to transplanting
naive T cells is the limited number of these cells in the
peripheral blood. However, ex vivo expansion technologies for the
large-scale production of T cells are currently being developed
to make this a clinically feasible approach (43, 44).
These studies demonstrate that large numbers of relatively pure
populations of activated T cells can be obtained from the
peripheral blood. The continued refinement of this approach offers the
potential to translate these preclinical studies into clinical marrow
transplantation settings.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William R. Drobyski, Bone Marrow Transplant Program, Froedtert Memorial Lutheran Hospital, 9200 West Wisconsin Avenue, Milwaukee, WI 53226.
3 Abbreviations used in this paper: BMT, bone marrow transplantation; GVH, graft-vs-host; GVHD, GVH disease; DLI, donor lymphocyte infusions; TCD, T cell depleted.
Received for publication February 17, 2000. Accepted for publication May 16, 2000.
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) or TCD B6 BM plus 150 x 106 B6
activated 
). In A, 2 x
106 naive B6 T cells were administered on the day of
transplant to both groups (DAY 0)(n = 8–12
mice/group). In B, the same dose of naive B6 T cells was
administered 7 days post-BMT (DAY 7)(n = 12–17
mice/group). In C, the same dose of B6 T cells was
administered 14 days after transplant (DAY 14)(n =
23–27 mice/group). A control group received TCD BM only at BMT and no
naive T cells post-BMT in each of the experiments (
). Actual
survival is depicted. Data are cumulative results from two to five
independent experiments.
