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Departments of
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Medicine,
Radiology, and
Pathology, 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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B10.BR (H-2k)) and engraftment
(C57BL/6 (H-2b)AKR/J (H-2k)).
Transplantation with anti-CD3-activated T cells significantly
reduced GVHD compared with that in animals transplanted with equivalent
numbers of naive T cells. Protection from GVHD was not T cell subset
dependent, as highly enriched populations of either activated
CD4+ or CD8+ T cells caused less lethal GVHD
than comparable numbers of purified naive CD4+ or
CD8+ T cells. Transplantation with activated T cells also
resulted in protection from LPS-mediated GVH lethality in unirradiated
F1 recipients. Analysis of immune recovery indicated that
animals transplanted with activated T cells had thymic and splenic B
cell reconstitution that compared favorably to that in non-GVHD control
mice. When engraftment was analyzed, equivalent degrees of donor cell
engraftment were observed when animals were transplanted with limiting
numbers (5 x 105) of naive vs activated B6 T cells.
Further studies indicated that activated CD8+ T cells were
exclusively responsible for enhancing engraftment and that facilitation
of engraftment was dependent upon the direct recognition of host MHC
alloantigens. Collectively, these data demonstrate that transplantation
with anti-CD3 Ab-activated T cells results in a reduction in GVHD,
but these cells retain their ability to facilitate alloengraftment. The
use of this approach in allogeneic marrow transplantation may represent
an alternative strategy to mitigate GVHD without compromising
engraftment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most therapeutic approaches designed to reduce GVHD have focused on the ex vivo removal of alloreactive donor T cell populations from the marrow graft (3, 4). While this has significantly reduced GVHD, there has been a corresponding increase in the rate of graft rejection (10, 11, 12) due to the fact that the T cells that are capable of causing GVHD and are removed by these depletion procedures are also necessary to facilitate alloengraftment. Conversely, when T cells are retained in the donor marrow graft, engraftment occurs in the vast majority of patients; however, the incidence and severity of GVHD are significantly increased despite the use of pharmacologic agents designed to suppress GVH reactivity. This is particularly problematic in the case of mismatched related and unrelated BMT, where the greater degree of HLA disparity significantly increases toxicity from GVHD (13, 14, 15, 16).
T cell activation after encounter of alloantigen presented in the context of MHC molecules is generally accepted to be the proximate event in GVHD. Once activated, T cells undergo cellular proliferation and are able to secrete a variety of cytokines that contribute to the pathogenesis of GVHD (17). Activation of the T cell, however, also serves to open a cell death pathway that can be triggered by either withdrawal of growth factor or religation of the TCR (18, 19, 20, 21, 22, 23). This process, termed activation-induced cell death, results in apoptosis and is thought to be an important mechanism by which immune responses are regulated and peripheral tolerance is maintained in vivo (24, 25). Prior studies have also shown that signaling via the TCR/CD3 complex with anti-CD3 Abs or other mitogenic stimuli in vitro can serve as a priming stimulus for T cells to undergo subsequent apoptosis (19, 20, 21).
Whether T cells that have been ex vivo activated before transplantation into recipients behave in a biologically similar manner as naive T cells with respect to GVH pathophysiology has not been studied. This question is of emerging clinical relevance given that activated T cells are now being transplanted into allogeneic marrow transplant recipients, most notably in gene therapy protocols designed to modulate GVH and graft-vs-leukemia reactivity (26, 27). We reasoned that the propensity of ex vivo activated T cells to undergo apoptosis after either cytokine withdrawal or religation of the TCR could be advantageous in allogeneic marrow transplantation, since this might translate into a shortened life span and therefore a reduced ability to cause GVHD. However, since activated T cells possess cytotoxic capability and produce cytokines implicated in the pathophysiology of GVHD (28), it is possible that these cells could conversely exacerbate GVH reactivity. Moreover, if GVHD were reduced, the ability of these cells to promote engraftment might be compromised, since the presence of donor T cells is necessary to eradicate or inactivate host immune cells capable of causing graft rejection (29, 30). The purpose of this study was to examine these questions by comparing the relative effects of naive and activated T cells in mediating GVHD and facilitating alloengraftment in murine recipients transplanted with MHC-mismatched marrow grafts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) (H-2b), B10.BR (H-2k), B6.PL (H-2b), AKR/J (H-2k), and (C57BL/6 x A/J)F1 (B6AF1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). (C57BL/6 x AKR/J)F1 mice were bred at the Medical College of Wisconsin (Milwaukee, WI). All animals were housed in the American Association for Lab Animal Care-accredited Animal Resource Center of the Medical College of Wisconsin. Mice received regular mouse chow and acidified tap water ad libitum.
Ex vivo activation and expansion of T cells
Spleen cells were obtained from either C57BL/6 or B6.PL mice and
passed through nylon wool columns (Robbins Scientific, Sunnyvale, CA)
to remove B cells. Cells were then resuspended in complete DMEM (CDMEM)
plus 5% FCS and cultured in flasks precoated with an immobilized T
cell-specific mAb (either anti-CD3, clone 145-2C11 (provided by J.
Bluestone, University of Chicago, Chicago, IL), or anti-TCR-
ß,
clone H57-597 (American Type Culture Collection, Rockville, MD)) at a
concentration of 5 to 10 µg/ml. Twenty-four hours after the
initiation of culture, human IL-2 (Cetus, Norwalk, CT) was added at a
concentration of 20 to 40 U/ml (Cetus units). All cultures were split
into fresh flasks as needed to maintain a cell concentration of 0.5 to
1.5 x 106 cells/ml. Cells were exposed to immobilized
mAb for the first 3 days of culture and thereafter grown only in medium
plus IL-2 in fresh flasks for an additional 3 to 5 days to allow for
re-expression of the TCR. The continuous presence of IL-2 was necessary
for optimal cell proliferation. After a total of 6 to 8 days in
culture, cells were counted, and the percentage of T cells was analyzed
by flow cytometry. Routinely, 90 to 95% of viable cells had
reexpressed the CD3/TCR complex at the time that these cells were
transplanted into recipients. Approximately 75% of T cells were
CD8+, and 5 to 10% of cells were CD4+ after ex
vivo expansion. The remaining cells were either double positive
(CD4+CD8+) or expressed neither Ag. The
absolute number of activated T cells was quantified by multiplying the
percentage of Thy1.2+ ß+ T cells as
determined by flow cytometry by the total number of cells. For the
purpose of clarity, T cells activated with anti-CD3 Ab are
henceforth referred to as anti-CD3-activated T cells.
CD4+ and CD8+ T cell subset enrichment and activation
To obtain highly enriched populations of activated
CD4+ T cells, B6 or B6.PL spleen cells were passed through
nylon wool columns, and then CD4+ T cells were positively
selected using the MACS magnetic cell separation system (Miltenyi
Biotech, Auburn, CA). The resulting population was then ex vivo
expanded in immobilized anti-CD3 Ab-coated flasks and grown in
medium plus 20 to 100 U/ml of IL-2 for 6 to 8 days as described above.
Since ex vivo anti-CD3 activation under these conditions results in
the preferential outgrowth of CD8+ T cells, the resulting
population was first depleted of CD8+ T cells and then
positively selected for CD4+ T cells before performing the
transplant experiments. After the enrichment procedure,
CD4+ T cells represented 
90 to 95% of the entire cell
population, and CD8+ T cells represented 
5% of the total
cell population. Activated CD8+ T cells were obtained by
culturing nylon wool-purified T cells for 7 to 8 days, depleting the
cell population of CD4+ T cells, and then positively
selecting for CD8+ T cells. After these procedures were
performed, CD8+ T cells represented >95% and
CD4+ T cells represented <0.5% of the entire cell
population. Highly enriched populations of naive CD4+ or
CD8+ T cells were obtained by negative selection followed
by positive selection of nylon wool-purified spleen cells and resulted
in >95% purity of the respective T cell subsets.
Bone marrow transplantation
BM 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 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 Type Culture Collection (Manassas, VA) and grown in CDMEM plus 5% FCS. 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.
Naive T cells were obtained by pressing spleens through wire mesh
screens. Erythrocytes were removed from spleen cell suspensions by
hypotonic lysis with sterile distilled water. T cells for admixture
with TCD BM before transplantation were then obtained by passing spleen
cells once or twice through nylon wool columns to remove B cells. The
number of naive T cells was quantified by multiplying the percentage of
Thy1.2+ ß+ T cells, as determined by flow
cytometry, by the total number of cells. The average number of naive
ß T cells in the spleen cell suspensions after nylon wool
depletion was 70 to 85%. BM and T cells were always >90% viable as
assessed by trypan blue dye exclusion. Total body irradiation was
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 then received a single i.v.
injection of TCD BM (10–20 x 106) with or without
added naive or ex vivo activated T cells.
For studies of GVHD in a fully allogeneic system, B10.BR recipients
were lethally irradiated (900 cGy) and transplanted with 10 to 20
x 106 TCD B6 BM alone or admixed with 2 to 25 x
106 naive or activated T cells from B6 background donors.
In some experiments, congenic B6.PL donors were employed to allow for
the determination of the fate of spleen-derived and BM-derived naive or
activated T cells. In other experiments, GVHD was also assessed in a
parentF1 model as described by Nestel and colleagues
(9). Unirradiated B6AF1 mice were transplanted with
equivalent numbers of naive or ex vivo activated B6 T cells. Donors of
naive T cells were first in vivo depleted of NK cells with mAb PK136
(anti-NK 1.1; American Type Culture Collection) as previously
described (31) before T cells were harvested for the transplant
experiments. This was done so that recipients in both the naive and
activated experimental groups received only T cells, since NK cells
have been implicated in the pathophysiology of GVHD in this model (9)
and might therefore have been a confounding variable. Syngeneic control
mice were transplanted with an identical number of naive
B6AF1 T cells. Seven days after injection, all recipients
were administered 10 µg of LPS i.v. and assessed for mortality within
48 h. For studies of engraftment in a fully allogeneic system, AKR
recipients were sublethally irradiated (850 cGy) and transplanted with
10 x 106 TCD B6 BM alone or admixed with graded doses
of naive or activated B6 T cells. In the engraftment model, donor T
cells were ex vivo activated with immobilized H57-597 Ab as opposed to
145-2C11 Ab. In both models, donor and recipient were mismatched at
both class I and II loci to simulate unrelated and mismatched related
human transplantation where HLA class I and II disparities are often
present, and the risk of GVHD and graft rejection is highest (14, 16).
Flow cytometric analysis
mAb conjugated to either 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-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),
FITC-anti-Thy1.2 (clone 30-H12, rat IgG2b), PE-hamster IgG (isotype
control), 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), and FITC-anti-H-2Kb (clone AF6-88.5,
mouse IgG2a) were all purchased from PharMingen (San Diego, CA). Spleen
and thymus cells were obtained from chimeras at defined intervals
posttransplant processed into single cell suspensions and stained for
two-color analysis. Red cells were removed when necessary by hypotonic
lysis. Cells were analyzed on a FACS analyzer (Becton Dickinson,
Mountain View, CA) with Consort 32 computer support and LYSIS II
software. Donor T cell chimerism was determined by analyzing cells
within the lymphocyte gate. Donor T cells were defined as
H-2b+/CD3+. The percentages of donor cells and
B cells were 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 cells were
defined as H-2b+, and B cells were defined as
Ly5+. Thymocytes were analyzed within gates that included
the entire thymus cell population. At least 10,000 cells were analyzed
for each determination whenever possible.
Isolation and quantification of intrahepatic lymphocytes
Livers were perfused with PBS to wash out red cells and pressed through wire mesh screens to obtain single cell suspensions. Liver cell suspensions were then incubated in collagenase VIII (Sigma, St. Louis, MO) at a concentration of 50 µg/ml for 30 min at 37°C. Cells were then washed, passed through a nylon wool column to remove debris, and centrifuged on a 44 to 67% Percoll gradient for 20 min. Lymphocytes were collected at the 44 to 67% interface, washed, counted, and then analyzed by flow cytometry. Forward and side scatter characteristics were used to gate on the lymphocyte population and exclude liver parenchymal cells.
Radioactive labeling of T cells with 111indium oxine and in vivo detection of radioactive T cells
Naive and activated T cells were individually labeled in vitro with 111indium oxine by incubating 125 x 106 cells with 50 µCi of 1.85 MBq of 111indium oxine. Animals injected with 5 µCi of radiolabeled T cells were sacrificed 18 h posttransplant, and tissue samples from the spleen, liver, lung, kidneys, and thymus were removed. Tissue samples and whole organs were individually weighed, and the amount of incorporated radioactivity was determined in a sodium iodide scintillation counter using the 150 to 500 keV window. The biodistribution of radiolabeled T cells was calculated as the normalized uptake of radioactivity in the whole organ and expressed as the percentage of total injected dose per organ.
Histologic studies
Liver tissue was obtained from control and experimental animals, fixed in 10% neutral buffered formalin, and processed into paraffin blocks. Four-micron-thick sections were prepared from each block and were cut at two levels to optimize sampling. Routine tissue hematoxylin and eosin sections were prepared. For the evaluation of GVHD, tissue sections were screened with the examiner blinded to the treatment received by each animal. Tissues were scored positive for GVHD if there was evidence of bile duct necrosis with or without infiltration in the liver.
Statistics
Group comparisons of donor T cell chimerism, overall donor cell
chimerism in the spleen and thymus, splenic B cell content, and thymic
T cell subsets were performed using unpaired Student’s t
test. Survival curves were constructed using the Kaplan-Meier product
limit and were compared using the log rank test. A two-tailed
p 0.05 was deemed significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments were performed to assess the potential of
naive and anti-CD3-activated T cells to induce GVHD. Lethally
irradiated B10.BR recipients were transplanted with TCD B6 BM and
equivalent numbers of either naive or activated T cells. Mice
transplanted with 2 x 106 naive T cells quickly
developed GVHD, and only 10% of the animals survived to day 80 (Fig. 1
). In contrast, animals transplanted
with an equivalent number of activated T cells had significantly
enhanced survival (p = 0.0001), with all mice
surviving until the conclusion of the experiments. When weight loss,
which is a sensitive indicator of GVHD in this model, was assessed, we
observed only a slight reduction in weight in these mice compared with
that in TCD BM control animals. To determine whether this observation
was T cell dose dependent, the dose of T cells administered to
recipients in each cohort was increased. As expected, mice transplanted
with 5 x 106 naive T cells all died within 35 days of
GVHD. Transplantation with 5 x 106 activated T cells
significantly prolonged survival (p < 0.0001),
but weight loss in these mice was more pronounced than that at a dose
of 2 x 106 cells, indicating that GVHD was not
completely abrogated.
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ß T cells (either 2 x 106 or
5 x 106) had a similar outcome, as assessed by
survival and weight curves, compared with mice transplanted with
145-2C11-activated T cells (data not shown). These data indicated that
the observed results were not Ab dependent. Moreover, these data
collectively demonstrated that anti-CD3 Ab-activated T cells had a
reduced ability to mediate GVH reactivity compared with naive T cells,
which translated into significantly prolonged survival. Activation of T cells with anti-CD3 Ab qualitatively alters the ability of both activated CD4+ and CD8+ cells to mediate GVHD
Ex vivo activation and expansion with anti-CD3 Ab under
the conditions used in this study resulted in the preferential
expansion of CD8+ T cells (see Materials and
Methods). Therefore, the percentage of CD4+ and
CD8+ T cells administered to animals was not the same in
mice transplanted with activated vs naive T cells (i.e., mice
transplanted with activated T cells received more CD8+ T
cells and fewer CD4+ T cells than mice reconstituted with
naive T cells). To correct for this potential bias in assessing GVHD
mortality, we performed a series of experiments to evaluate the ability
of activated CD4+ and CD8+ T cells both
individually and collectively to cause GVHD compared with that of
equivalent numbers of naive CD4+ and/or CD8+ T
cells. In initial studies, animals were transplanted with the same
absolute number of a mixture of either activated or naive
CD4+ and CD8+ T cells. This was achieved by
mixing purified activated CD4+ and CD8+ T cells
in the same CD4:CD8 ratio as that obtained after nylon wool
purification of naive T cells. Mice transplanted with 2 x
106 activated T cells had significantly enhanced survival
compared with animals transplanted with an equivalent number of naive T
cells (67 vs 10% survival on day 60; p = 0.0005; Fig. 2A). Weight curves, however,
again demonstrated that surviving mice transplanted with activated T
cells were not free from GVHD, as these mice had less weight gain than
control animals (Fig. 2B).
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). Mortality in TCD BM control mice was
somewhat higher than that in previous experiments and was accompanied
by antemortem weight loss in deceased animals, which was ascribed to
graft failure. When similar experiments were performed using highly
enriched naive vs activated CD8+ T cells, there was a trend
toward improved survival at 60 days in mice transplanted with activated
T cells (93%; n = 15) vs naive T cells (67%;
n = 15; p = 0.067). These data
substantiated that both CD4+ and CD8+ T cells
activated with anti-CD3 Ab were qualitatively altered in their
ability to mediate GVH reactivity in an MHC-incompatible
donor/recipient strain combination.
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Murine studies of GVHD have generally involved the use of
irradiation in the conditioning regimen or have alternatively employed
parentF1 models in which no conditioning regimen is
used. An advantage of the latter is that the predominant effects of
GVHD are immunologic, and these can be examined in the absence of
irradiation-induced inflammatory changes that contribute to GVH
pathophysiology in lethal irradiation models. Using this approach,
Nestel and colleagues (9) have described a model in which GVH
reactivity due to alloreactive donor T cells results in macrophage
priming that renders recipients susceptible to LPS-mediated lethality
secondary to elevated TNF- production. As an alternative way to more
precisely assess the ability of activated T cells to cause GVHD in the
absence of radiation, we performed experiments using this model in
which F1 recipients were transplanted with equivalent
numbers of naive or activated T cells. The total number of administered
CD4+ and CD8+ T cells was again equivalent in
each group to control for a subset bias in the induction of GVHD.
Recipients of syngeneic naive T cells (F1F1)
had no mortality after LPS challenge (Table I). Conversely, animals transplanted with
25 x 106 naive T cells had 82% mortality within
48 h after LPS injection. Mice transplanted with activated T cells
were similar to TCD BM control mice, as all survived challenge with LPS
(p = 0.0002, by Fisher’s exact test, vs naive
T cell group). These data indicated that in the absence of a
conditioning regimen, activated T cells were inherently less capable of
causing GVHD than equivalent numbers of naive T cells.
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As another index to assess the severity of GVHD in animals
transplanted with activated T cells, long term immune reconstitution
was examined in chimeras 2 to 3 mo posttransplant. Specifically, thymic
reconstitution and splenic B cell repopulation were evaluated, since
the thymus has been shown to be a target tissue during the GVH reaction
(33, 34, 35), and we have also observed that B cell reconstitution is a
sensitive indicator of GVHD in this model (unpublished observations).
Surviving mice from Figure 2
transplanted with 2 x
106 activated T cells and those transplanted with TCD BM
only were comparatively analyzed 60 to 75 days after BMT (Table II). Analysis of T cell chimerism in the
spleen revealed that mice transplanted with activated T cells had
significantly enhanced donor T cell engraftment than control animals
(p = 0.03; Table II). Specifically, 10 of 12
mice in this group had >95% donor T cell chimerism in the spleen,
while the remaining two animals had predominant host T cell
reconstitution with <50% donor T cells. The presence of a small
percentage (5%) of Thy1.1+ (congenic B6.PL) T cells in the
spleen of these mice indicated that some activated T cells were able to
persist in these chimeras. Neither thymic size nor the percentage of
double-positive thymocytes was different between control mice and mice
transplanted with activated T cells. With respect to thymic size in the
latter group, we did observe some heterogeneity, as 3 of 12 animals had
nonmeasurable thymi, while the remaining mice reconstituted with
50 x 106 thymocytes. B cell repopulation, however,
was inferior to that observed in TCD BM animals
(p = 0.056), consistent with some ongoing GVHD,
which was also substantiated by serial weight curves (Fig. 2B).
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T cell activation has been shown to affect the regulation of
adhesion molecules as well as lymphocyte homing in vivo compared with
those in unactivated T cells (36, 37). We therefore considered that one
possibile cause of the reduction in GVHD mediated by activated T cells
might be the fact that these cells had a different trafficking pattern
in vivo compared with that of naive T cells. Experiments were therefore
performed to determine whether anti-CD3-activated T cells had
altered trafficking. To address this question, naive and
anti-CD3-activated B6 T cells were specifically labeled with
111indium in vitro and subsequently transplanted with
unlabeled TCD B6 BM into cohorts of lethally irradiated B10.BR
recipients. Animals were then analyzed 1 day after transplant to assess
the trafficking pattern of lymphocytes to representative organs (Table III). Since the binding of this
radioisotope to lymphocytes is stable, the amount of detectable
radioactivity in each of the respective organs was indicative of
specific lymphocyte trafficking. Approximately 50% of the administered
radioactivity was detected in both liver and spleen in each of the two
groups. However, while naive T cells preferentially localized in the
spleen (31.7%, vs 13.5% in the liver; Table III), transplantation
with anti-CD3-activated T cells resulted in the majority of
isotopic labeling appearing in the liver (36%, vs 16.4% in the
spleen). There was also increased radioactivity in the lungs of these
mice, while comparable amounts of radioactivity were detected in kidney
and thymus of these animals. These data indicated that ex vivo
activated T cells had an altered migratory pattern early posttransplant
relative to that in naive T cells.
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). There was also an early increase in
the percentage of lymphocytes that were Thy1.1+ in animals
transplanted with activated T cells, although by day 3 the relative
percentages in each of the two groups was equivalent. However, since
there was a much greater absolute number of intrahepatic lymphocytes in
the former group, the absolute number of Thy1.1+
lymphocytes in these mice was higher. By day 4 posttransplant,
approximately 50% of the total number of transplanted
Thy1.1+-activated T cells were detectable in the livers of
these chimeras, implicating the liver as a major site of anti-CD3
activated T cell accumulation posttransplant. Histologic analysis of
livers from representative animals from each cohort 4 days
posttransplant (n = 3/group) revealed no significant
tissue damage or incipient evidence of GVHD (data not shown). Thus,
despite the three- to sixfold greater number of lymphocytes in animals
transplanted with anti-CD3-activated T cells, there was no early
evidence of tissue destruction.
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The fact that activated T cells had a reduced ability to induce
GVHD raised the possibility that these cells might also be compromised
in their ability to facilitate alloengraftment. We therefore examined
the relative abilities of naive and activated T cells to promote
engraftment in a model (B6AKR) where TBI exposures of 850 cGy
result in rejection of TCD marrow grafts and where donor engraftment is
dependent upon the presence of mature T cells in the marrow graft (38).
In this model, transplantation with at least 1 to 5 x
105 naive ß T cells is required to significantly
enhance donor T cell engraftment. Dose titration studies with activated
T cells demonstrated that equivalent numbers of activated and naive
ß+ T cells (5 x 105) were required
to significantly enhance donor T cell and overall donor engraftment
relative to those in control animals (Table IV). Although the percentage of donor T
cells in the spleen was increased in mice transplanted with 5 x
105 naive vs activated T cells, overall donor engraftment
was equivalent between these two groups (p =
0.18), indicating that activated T cells were comparable to naive T
cells on a cell-to-cell basis in their ability to promote donor
engraftment. There was also a trend toward increased B cell
reconstitution in these chimeras (p = 0.06),
which was further evidence that equivalent doses of naive T cells were
more likely to cause GVHD and impair immune reconstitution than
activated T cells even in this sublethal irradiation model.
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Since activated CD4+ and CD8+ T cells were
both less likely to cause lethal GVHD compared with comparable numbers
of naive CD4+ and CD8+ T cells, we examined the
relative ability of each of these T cell subsets to prevent graft
rejection in the same engraftment model. Highly enriched populations of
activated CD4+ T cells were ineffective in facilitating
donor T cell (p = 0.36) or donor cell
(p = 0.84) engraftment when compared with TCD
BM alone (Table V). The number of splenic
B cells in these chimeras, which is another indicator of donor cell
engraftment (38), was also not significantly different from that in
control animals. In contrast, mice transplanted with equivalent numbers
of activated CD8+ T cells had significantly enhanced donor
T cell (p < 0.0002) and donor cell
(p < 0.0004) engraftment relative to control
animals. The degree of donor T cell and donor cell engraftment was
comparable to that in mice transplanted with equivalent numbers of
naive T cells that were comprised of both CD4+ and
CD8+ T cells. These data indicated that activated
CD8+ T cells were primarily responsible for facilitating
engraftment and that activated CD4+ T cells were
ineffectual in this model.
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Experimental data support direct MHC-restricted recognition of
residual host T cells by naive donor T cells as one mechanism by which
T cells facilitate engraftment (39). Whether activated T cells
facilitate engraftment in a similar fashion is unknown. We therefore
performed experiments using an F1parent model designed
to assess whether activated T cells that were tolerant of the recipient
and therefore unable to recognize host alloantigens could enhance donor
T cell engraftment. Analysis of the experimental control groups
revealed significantly enhanced donor T cell engraftment by naive T
cells and a minimal, but statistically significant, improvement in
engraftment by naive F1 T cells relative to that in TCD BM
animals. When mice transplanted with activated T cells were analyzed,
activated nontolerant T cells were again observed to enhance
engraftment relative to that in control mice (group I vs III;
p < 0.001; Table VI),
but activated T cells from F1 donors failed to improve
donor engraftment (p = 0.11). Notably, there
was a trend toward improved donor T cell engraftment after
transplantation with 5 x 105 naive nontolerant B6 T
cells compared with 5 x 105 nontolerant activated T
cells (p = 0.06), similar to what we had
previously observed (Table IV). These results indicated that
facilitation of donor engraftment under these conditions required that
activated donor T cells be capable of recognizing recipient
alloantigens.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of this study demonstrated the T cells activated with anti-CD3/TCR Abs had a markedly reduced ability to mediate GVHD in an MHC-incompatible donor/recipient strain combination. Similar results were observed when an alternative anti-CD3/TCR Ab was employed for ex vivo activation, indicating that the protective effect was not due to a unique property of one specific Ab. The reduction in GVHD was manifested not only as significantly improved survival and weight gain relative to those in GVHD control mice, but also by a relative preservation of B cell and thymic reconstitution, which are both sensitive immunologic parameters of GVHD (40, 41). Protection from GVHD, however, was not complete, as evidenced by the fact that some irradiated animals transplanted with activated T cells did succumb to GVHD or experienced weight loss despite prolonged survival. Transplantation with either highly enriched activated CD4+ or CD8+ T cells both resulted in a reduction in GVHD, indicating that ex vivo activation and expansion with anti-CD3 Abs plus IL-2 qualitatively altered the ability of both T cell subsets to mediate GVH reactivity. This effect therefore was not T cell subset dependent.
We reasoned that the relative ability of activated vs naive T cells to
cause GVHD might be confounded somewhat by the fact that mice in these
experiments were preconditioned with irradiation, which is known to
contribute to the pathophysiology of GVHD (42). To circumvent this
potential bias, we examined these cells using a parentF1
model in which transplantation with alloreactive donor T cells results
in subsequent susceptibility to LPS-mediated death in unirradiated
recipients (9). Animals transplanted with activated T cells had no
mortality, while nearly all mice transplanted with naive T cells died
of GVHD. Within the context of this model, these results indicated that
transplantation with activated T cells resulted in significantly less
priming of macrophages, such that the latter were not poised to release
TNF after LPS challenge. Transplantation with activated T cells
therefore appeared to result in less recruitment of secondary effector
cell populations that could contribute to the pathophysiology of GVHD.
These studies also underscored the fact that these cells had an
inherently reduced ability to cause GVHD, when examined in the absence
of a conditioning regimen.
There are several possible explanations for why GVHD was abrogated in mice transplanted with activated T cells. One is that activated T cells had an altered trafficking pattern and were less likely to migrate to lymphoid-rich organs where they would encounter APCs and MHC alloantigens that could trigger GVHD. Prior studies have shown that activated T cells have altered migratory properties in vivo compared with unactivated T cells (43, 44). Our studies demonstrated that activated T cells did indeed preferentially traffick to the lungs and liver in contrast to naive T cells, which migrated primarily to the spleen early after transplant. However, the facts that activated T cells were able to facilitate engraftment and that engraftment in this model was dependent upon recognition of recipient MHC alloantigens (38) were evidence that a significant portion of these cells had to directly interact with and eliminate host immune cells capable of rejecting the graft. Thus, an altered trafficking pattern does not appear to be a sufficient explanation for why activated T cells caused less GVHD, since the finding that activated donor T cells had to interact with host cells to promote engraftment should have been a similarly sufficient stimulus to induce clinically significant GVHD.
An alternative mechanism is that ex vivo activated T cells were more
susceptible to apoptosis than naive T cells. Either cytokine withdrawal
occurring after activated T cells were removed from culture and
transplanted into recipients or religation of the TCR after T cell
encounter with host alloantigens in vivo could have served as stimuli
for activated T cells to undergo apoptosis. Apoptosis secondary to
growth factor withdrawal has been shown to be mediated in part by a
deficiency of cytokines that signal through the IL-2 
-chain, such as
IL-2, IL-4, and IL-7 (18). Removal of IL-2 from culture after T cell
activation results in a rapid decline in Bcl-XL protein
levels (45). Since Bcl-XL inhibits apoptosis (46),
activated T cells are predisposed to undergo cell death. Alternatively,
the interaction between donor T cell TCR and host APCs could have
triggered activation-induced cell death. Apoptosis in activated T cells
occurring as a sequela of TCR religation has been shown to be mediated
primarily by way of Fas/Fas ligand interactions (47, 48), although
other pathways also appear to play a role (49). This latter process
could explain how GVH reactivity could be mitigated without
compromising engraftment, since a population of activated donor T cells
could have itself also undergone cell death after elimination of host T
cells. Concurrent cell death of both responder and target cells has
been previously described to occur in vitro in cytotoxic activated
T cells (50). The facts that a minority of activated T cells was
still detectable in the spleens of recipients 2 mo posttransplant
(Table II) and that these cells were still capable of inducing GVHD
(Figs. 2 and 3), however, indicated that not all activated T cells
would have had shortened survival.
The accumulation of anti-CD3-activated T cells in the liver is not inconsistent with a mechanism of accelerated in vivo T cell death. While it is possible that this observation could have been merely a trafficking event, an alternative explanation is that at least some of these cells specifically migrated to the liver because they were undergoing apoptosis. Huang and colleagues (51) have shown that previously activated murine T cells undergoing apoptosis in vivo are eliminated in the liver, implying that the liver is a site of peripheral T cell deletion. The daily incremental increase in activated T cells that we observed early posttransplant in the liver therefore could have been due to the fact that these cells localized in the liver because they were undergoing apoptosis. The observation that a significant portion of the activated T cells transplanted into recipients resided in the liver 4 days after transplant indicated that this organ was the major site of lymphocyte accumulation. While our attempts to document apoptosis in these cells by flow cytometry were unsuccessful (data not shown), the rapidity with which apoptotic cells are cleared by macrophages may have precluded us from detecting this event (52, 53). Studies are currently underway to define whether accelerated cell death of anti-CD3-activated donor T cells in vivo is indeed the mechanism that mitigates GVH reactivity in this setting.
Despite causing less GVHD, activated T cells were capable of
facilitating donor engraftment and were comparable to unseparated naive
T cells on a cell-to-cell basis. Thus, these cells retained functional
competency such that they could prevent graft rejection. Activated
CD8+ T cells were responsible for promoting engraftment, as
CD4+ T cells had no graft facilitory capability in this
model. These results are consistent with those of prior studies (29, 54), which have demonstrated that nontolerant CD8+ T cells
are far more effective in facilitating engraftment of MHC-disparate
marrow than CD4+ T cells. This is presumably due to the
fact that host T cells capable of rejecting the marrow graft do not
express class II molecules and therefore cannot be recognized by donor
CD4+ T cells. Both CD4+ and CD8+ T
cells, which are tolerant of the recipient, however, have been shown to
be capable of enhancing engraftment, presumably by a veto mechanism
(30, 39), although donor engraftment is generally less complete than
when nontolerant T cells are employed (39). To determine whether a
similar mechanism might be operable in activated T cells, we examined
whether tolerant activated T cells could promote engraftment. Using an
F1parent model in which donor T cells were incapable of
recognizing recipient alloantigens, these studies clearly indicated
that activated T cells had to be able to recognize host alloantigens
for engraftment to occur under these experimental conditions.
Consequently, if activated T cells did have a shortened life span in
vivo, they were still able to persist long enough in recipients to
eliminate host immune cells capable of causing rejection.
In summary, this study demonstrates that ex vivo activation of donor T cells before transplantation into recipients resulted in a significant reduction in GVHD. This study was limited to evaluating T cell activation with anti-CD3/TCR Abs, although we have observed similar protection from lethal GVHD with mitogen-stimulated (Con A) T cells as well (unpublished observations). While the mechanism(s) for GVHD protection has not been completely defined, altered T cell trafficking and/or shortened T cell survival are two possibilities that require further examination. In particular, if the latter mechanism proves to be primarily responsible for the reduction in GVHD, then therapeutic strategies that are able to more completely and selectively eliminate T cells capable of mediating GVHD may represent an effective clinical strategy. The fact that activated T cells, in particular CD8+ T cells, were able to facilitate alloengraftment indicated that these cells retained functional competency. The more complete selective elimination of these cells at a critically defined time point might therefore allow for the preservation of engraftment and the mitigation of GVHD. This might be accomplished by the use of agents that are capable of inducing apoptosis in preactivated CD8+ T cells or by incorporating suicide genes into T cells (55, 56), allowing them to be selectively eliminated at defined points posttransplant when engraftment will have occurred but before GVHD is clinically significant. This approach would be of particular therapeutic advantage in nonmalignant disorders where there is no risk of relapse. In this setting, donor T cells that subsequently reconstituted recipients would be BM derived and tolerant of the host after undergoing clonal selection in the thymus. This strategy might therefore allow for immune reconstitution to occur in a more ordered fashion in the absence of both the immune dysregulation induced by GVHD and the continued need for immunosuppressive agents to treat GVH reactivity.
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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 East Hospital, 9200 West Wisconsin Ave., Milwaukee, WI 53226-3596.
3 Abbreviations used in this paper: GVHD, graft-vs-host disease; GVH, graft-vs-host; BM, bone marrow; BMT, bone marrow transplantation; CDMEM, complete DMEM; TCD, T cell depleted; PE, phycoerythrin.
Received for publication February 10, 1998. Accepted for publication April 23, 1998.
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; n = 8) or TCD B6 BM plus
2 x 106 naive (
; n = 10),
5 x 106 naive (
; n = 10),
2 x 106 anti-CD3 activated (•;
n = 10), or 5 x 106 anti-CD3
activated B6 T cells (
; n = 10). Survival is
shown in A, and the mean weights of the same animals are
shown in B. Data are derived from two similarly
performed experiments.
; n = 21) from B6 or B6.PL donors.
Survival is shown in A, and the mean weights of the same
animals are shown in B. Data are cumulative results from
three similarly performed experiments. In one experiment, animals in
each of the two groups received a dose of 3 x 106 T
cells rather than 2 x 106 T cells.
