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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 2999-3006
Allo-Major Histocompatibility Complex-Restricted
Cytotoxic T Lymphocytes Engraft in Bone Marrow Transplant Recipients
Without Causing Graft-Versus-Host Disease
By
Liquan Gao,
Tian-Hui Yang,
Sophie Tourdot,
Elena Sadovnikova,
Robert Hasserjian, and
Hans J. Stauss
From the Departments of Immunology and of Histopathology, Imperial
College School of Science Technology and Medicine, Hammersmith
Hospital, London, UK.
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ABSTRACT |
Previous experiments in humans and mice have shown that allogeneic
donors can serve as a source of cytotoxic T lymphocytes (CTL) specific
for proteins, such as cyclin-D1 and mdm-2, expressed at elevated levels
in tumor cells. In vitro, allo-major histocompatibility complex
(MHC)-restricted CTL against these proteins selectively killed allogeneic tumor cells, including lymphoma, but not normal control cells. This suggested that these CTL may be useful for adoptive
tumor immunotherapy, provided that they (1) survive in MHC-disparate
hosts, (2) maintain their killing specificity, and (3) do not attack
normal host tissues. Here, we used cloned allo-restricted CTL isolated
from BALB/c mice (H-2d) that killed
H-2b-derived tumor cells expressing elevated levels of the
mdm-2 target protein. When these CTL were injected into bone marrow
transplanted (BMT) C57BL/6 (H-2b) recipients, they
consistently engrafted and were detectable in lymphoid tissues and in
the bone marrow (BM). Long-term survival was most efficient in spleen
and lymph nodes, where CTL were found up to 14 weeks after injection.
The administration of CTL did not cause graft-versus-host disease
(GVHD) normally associated with injection of allogeneic T cells. These
data show that allo-restricted CTL clones are promising reagents for
antigen-specific immunotherapy in BMT hosts, because they engraft and
retain their specific killing activity without causing GVHD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TWO WELL-DOCUMENTED immune reactions
mediated by donor lymphocytes play an important role in bone marrow
transplanted (BMT) leukemia patients. The graft-versus-host reaction is
mediated by donor T lymphocytes recognizing antigenic determinants
expressed by host cells. This can lead to destruction of normal host
tissues, resulting in severe graft-versus-host disease
(GVHD).1 The graft-versus-leukemia (GVL) reaction was first
descibed in murine models,2-4 and it was subsequently found
in humans that donor T lymphocytes can recognize antigenic determinants
expressed by recipient leukemic cells.5 It has been shown
that the GVL reaction can significantly improve the prognosis of
patients suffering from leukemia, particularly chronic myeloid
leukemia.6
The molecular nature of the antigenic determinants triggering GVHD and
GVL has not yet been fully identified. However, it is clear that host
HLA molecules presenting immunogenic peptide epitopes to donor T
lymphocytes are critically important. In the case of minor
histocompatibility antigen (mHA) mismatches, the peptide epitopes are
derived from cellular proteins that differ between donor and
host.7 To date, the peptide epitopes of 3 human8-10 and 7 murine11-17 mHAs have been
identified, but it is unclear whether expression of any of these
epitopes is restricted to hematopoietic cells and leukemias. This would
provide a rationale for immunotherapy in BMT leukemia patients using
donor-derived T cells specific for mHA expressed in recipient
hematopoietic cells.
In the case of major histocompatibility mismatches, serologically
undetectable polymorphism mapping to the peptide binding groove of HLA
molecules is likely to change the nature and conformation of HLA-bound
peptides.18,19 Consequently, donor T lymphocytes are
stimulated by a variety of novel peptide epitopes bound to the groove
of serologically matched host HLA molecules. In some cases, the peptide
epitopes recognized by human allo-reactive T cells have been
identified,20-22 but there is no evidence that these
epitopes might be preferentially expressed on leukemic cells compared
with normal host tissues. Thus, conventional allo-reactive T cells are
likely to attack both leukemic cells and normal tissues.
The identification of T-cell-stimulating peptide epitopes selectively
expressed in transformed cells is a prerequisite for dissociating GVL
from GVHD. We have recently developed a strategy of directing cytotoxic
T lymphocytes (CTL) of major histocompatibility complex
(MHC)-mismatched donors against selected peptide epitopes presented by
host MHC class I molecules.23,24 The selected peptide
epitopes were derived from proteins, such as cyclin-D1 and mdm-2, known
to be expressed at elevated levels in various human tumors, including
leukemia. Because these proteins are physiologically expressed at low
levels in normal tissues, the autologous immune system was found to be
tolerant and unable to mount efficient CTL responses.25 In
contrast, human and murine allo-MHC-restricted CTL against cyclin-D1
and mdm-2 were found to kill tumor cells expressing these proteins but
not normal control cells in vitro.
The in vitro specificity of allo-restricted CTL suggested that they
might be suitable for antigen-specific immunotherapy in BMT leukemia
patients, resulting in a GVL reaction without triggering GVHD. However,
there is currently no information on whether allo-restricted CTL can
survive in MHC-mismatched hosts and whether they retain their
peptide-specific killing activity without causing damage to normal host
tissues. To address these issues we have designed experiments to study
the fate of BALB/c-derived allo-restricted CTL clones injected into BMT
C57BL/6 recipients. The results show that the CTL engraft, that they
retain their killing specificity, and that they do not cause GVHD.
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MATERIALS AND METHODS |
Mouse strains.
C57BL/6 (MHC haplotype Kb, IAb,
Db), B10.A(4R) (MHC haplotype Kk,
IAk, IEk, Db), and BALB/c mice (MHC
haplotype Kd, IAd, IEd,
Dd, Ld) were purchased from Harlan,
UK Ltd (Bicester, Oxfordshire, UK), and housed in the
animal facility of Imperial College School of Medicine, Hammersmith
site (London, UK). Animals were used for experiments at an
age of 6 to 8 weeks.
CTL clones and cell lines.
In a previous study we described the isolation of allo-MHC-restricted
CTL clones specific for a peptide epitope, mdm100 (YAMIYRNL), derived
from the mdm-2 protein.24 The CTL were of BALB/c origin and
recognized the mdm100 peptide presented by H-2Kb class I
molecules. For this study, the CTL clone 6A5D was chosen. In vitro,
this CTL clone showed good killing of RMA lymphoma cells (H-2b) expressing mdm-2 endogenously, but not
Con-A-activated normal lymphoid cells from C57BL/6 mice. RMA is a
lymphoma cell line isolated from C57BL/6 mice, and RMA-S cells were
derived from RMA by in vitro mutagenesis and selection with anti-MHC
class I antibodies.26 RMA-S cells have a mutation in the
TAP-2 gene,27 impairing peptide transport from the cytosol
into the ER, which results in inefficient loading of Kb and
Db class I molecules with endogenous peptides. However, the
class I molecules of RMA-S cells can be efficiently loaded with
exogenously provided peptides.
BMT.
C57BL/6 mice received total body irradiation of 1,000 Rad, and the
following day they were transplanted with T-cell-depleted BM from
B10.A(4R) donors. T-cell depletion was achieved by mixing BM cells with
DYNAL beads (DYNAL UK Ltd, Bromborough, Merseyside, UK) coated with
anti-Thy-1 antibodies followed by magnetic removal of Thy-1-positive
T cells. All BM samples were treated twice with DYNAL beads and
successful removal of T cells was confirmed by fluorescence-activated
cell sorting (FACS) analysis using antibodies specific for
CD4 and CD8. In a typical depletion experiment, the percentage of T
cells in the BM was reduced from 0.57% to 0.05% (data not shown).
T-cell-depleted BM cells were injected intravenously (IV) at a dose of
2 × 107 per recipient. For the following 3 weeks,
recipient mice were observed daily for signs of weight loss and other
signs of poor health.
Antibodies.
Directly labeled anti-CD4-phycoerythrin (PE) and
anti-CD8-PE antibodies were purchased from Sigma (Poole, Dorset, UK).
To identify cells of C57BL/6, B10.A(4R), and BALB/c origin, antibodies against H-2Kb (AF6.88), H-2Kk (11-4.1), and
H-2Ld (HB31), respectively, were used. These antibodies
were used in combination with fluorescein isothiocyanate (FITC)-labeled
antimouse Ig antibodies as a second layer. AF6.88 was a gift from Dr D. Gray (University of Edinburgh, Edinburgh, UK), 11-4.1 was a gift from
Dr H. Reiser (I.C.S.M., Dept. of Immunology, London, UK), and HB31 was purchased from the American Tissue Culture Collection (Rockville, MD).
Limiting dilution assay (LDA) and bulk CTL cultures.
This assay was used to detect mdm100-specific CTL in the spleen, lymph
nodes, thymus, and BM of C57BL/6 recipient mice. To validate this
assay, pilot experiments were performed by mixing mdm100-specific CTL
and C57BL/6 splenocytes in vitro at a ratio of 1 in 300. Decreasing
numbers of cells taken from this mixture were stimulated under limiting
dilution conditions in 96-well plates with irradiated peptide-loaded
RMA-S stimulator cells (104 per well) and irradiated
C57BL/6 splenocytes (2 × 105 per well) in 200 µL
microcultures containing 10 U/mL recombinant interleukin-2 (IL-2).
After 10 days, microcultures were restimulated using the same number of
stimulator cells and feeder cells, and after 5 days, each well was
tested in a 51chromium-release killing assay against RMA-S
target cells coated with the mdm100 peptide or a Kb-binding
control peptide.25 Microcultures that killed mdm100-coated targets at least 10% more efficiently than control targets were counted as positive. Using these conditions, the calculated frequency in the mixture containing 1 CTL/300 splenocytes was 1/273
( 2 = 1.4; P = .682). This indicated that the
assay was suitable to accurately measure the frequency of
mdm100-specific CTL in cell mixtures isolated from BMT mice.
Similar conditions were used for measuring mdm100-specific CTL in bulk
cultures. Cultures were set up in 24-well plates, with each well
containing 2 × 106 irradiated C57BL/6 splenocytes as
feeders and 105 irradiated mdm100 peptide-loaded RMA-S
cells in 2 mL to which 106 cells isolated from spleen,
lymph nodes, thymus, or BM of transplanted mice were added. After one
restimulation using the same number of stimulator cells and feeder
cells, CTL activity was tested against RMA-S targets coated with the
mdm100 peptide or a Kb-binding control peptide.
Histology of tissues from recipient mice.
Samples of skin, stomach, liver, and colon of 4 BMT mice injected with
CTL and 4 control mice without CTL were fixed in 10% neutral buffered
formalin, processed routinely, and stained with hematoxylin and eosin
(H&E).
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RESULTS |
Experimental design.
We have previously generated allo-MHC-restricted CTL clones from
BALB/c mice against a Kb-presented peptide, mdm100, derived
from the mdm-2 protein. High avidity CTL clones were shown to kill
H-2b-derived tumor cells, including lymphomas, but not
normal cells of H-2b origin.24 For the in vivo
study described here, we selected a CTL clone of intermediate avidity
that displayed efficient killing of RMA lymphoma cells (eg, see Fig
4C). To test these CTL in vivo, they were injected into C57BL/6 mice
(H-2b) transplanted the previous day with BM from B10.A(4R)
donors (Fig 1). This experimental set up
was chosen to mimic a planned immunotherapy protocol for leukemia
patients.28 In the human immunotherapy, allo-restricted CTL
lines are generated from HLA-A2-negative donors and directed against
tumor-associated peptide epitopes presented by HLA-A2 class I
molecules.23 Once a leukemia-reactive CTL clone is
established, it would be ideal to use it for treatment of all
HLA-A2-positive leukemic patients, avoiding generation of new CTL
clones for each individual patient. Consequently, in some patients,
there may be a complete MHC-mismatch between injected CTL and host. To
mimic such a complete mismatch, BALB/c-derived CTL were injected into
C57BL/6 hosts in the murine model used in this study. B10.A(4R) BM
donors were chosen to mimic a partially mismatched transplant situation
that sometimes occurs intentionally or unintentionally in humans. The
H-2K locus mismatch is functionally important, because it renders
B10.A(4R) donor cells expressing the Kk allele
unrecognizable by the injected CTL that are specific for the mdm100
peptide presented by Kb molecules (Fig 1).
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| Fig 1.
Murine BMT model. Experimental model used in this study
involves a partial MHC-mismatch between recipient mice and BM donors
and a complete MHC-mismatch between recipients and CTL donors. CTL are
allo-MHC-restricted and recognize peptides presented by recipient
H-2Kb class I molecules. BM donor cells expressing
H-2Kk are not recognized by CTL.
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Analysis of BMT mice.
C57BL/6 mice underwent BMT, and after 4 to 14 weeks, mice were analyzed
by double-staining of lymphocytes with CD8 antibodies and antibodies
specific for Kb and Kk to identify cells of
C57BL/6 host and B10.A(4R) donor origin, respectively. FACS analysis
showed that the majority of the lymphoid cells were of B10.A(4R) origin
(Fig 2A and B). However, it was consistently observed that a proportion of lymphocytes, including CD8 T
cells, stained with anti-Kb antibodies, indicating that
they were of C57BL/6 host origin (Fig 2A). It is likely that these
cells represent mature T lymphocytes that escaped total body
irradiation, raising the possibility that these residual host T cells
might prevent engraftment of BALB/c-derived CTL clones.
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| Fig 2.
FACS analysis to monitor BM engraftment and to search for
injected CTL. Shown is the analysis of splenocytes isolated from
C57BL/6 mice that either received BM from B10.A(4R) donors (BMT group)
or BM and 1.5 × 107 cloned allo-restricted CTL of BALB/c
origin (BMT+CTL group). Splenocytes were stained with PE-labeled CD8
antibodies and with antibodies specific for the MHC class I alleles
Kb, Kk, and Ld. The cells displayed
in all panels were acquired through a lymphocyte gate set according to
the size (FSC) and granularity (SSC) of lymphocytes. Shown is a
representative analysis performed 6 weeks after BMT. Similar data were
obtained with mice analyzed between 4 and 14 weeks after BMT.
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Engraftment of allo-restricted CTL in BMT recipients.
C57BL/6 mice were transplanted with B10.A(4R) BM, and the following
day, one group of mice was injected IV with 4 × 106
BALB/c-derived CTL specific for the mdm100 peptide presented by
H-2Kb class I molecules. A control group did not receive
any CTL. Injection of an additional dose of 5 × 106
CTL was repeated after 9 days in the CTL group. Three weeks after the
second CTL injection, mice in each group were killed and lymphocytes isolated from spleen, thymus, lymph nodes, and BM were stimulated in
vitro with RMA-S cells presenting the mdm100 peptide in the context of
Kb class I molecules. Figure 3
shows that strong peptide-specific CTL activity was detected in all
tissues from BMT mice that had been injected with CTL. In contrast, BMT
mice that were not injected with CTL failed to generated any
peptide-specific CTL activity (Fig 3E). We further investigated whether
the observed CTL activity was caused by BALB/c-derived CTL. In an
independent experiment, the CD8 cells in cultures displaying
peptide-specific killing activity were stained with antibodies specific
for Ld, Kb, and Kk to identify
cells of BALB/c, C57BL/6, and B10.A(4R) origin, respectively. Nearly
all of the CD8 cells expressed the Ld molecule
(Fig 4A), suggesting that BALB/c-derived
CTL accounted for the observed killing activity.
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| Fig 3.
Reisolation of mdm100-specific CTL activity from BMT
recipients. C57BL/6 mice received a BMT from B10.A(4R) donors followed
by injection of 4 × 106 and 5 × 106
mdm100-specific CTL 1 and 10 days after the BMT (BMT+CTL group).
Control mice received a BMT but no CTL (BMT group). Three weeks after
the second CTL injection, mice were killed and cells isolated from the
spleen (A), thymus (B), lymph nodes (C), and BM (D) were stimulated in
vitro with irradiated RMA-S cells loaded with mdm100 peptides as
described in Materials and Methods. Peptide-specific CTL activity was
discovered in all tissues from BMT+CTL mice (A through D), and
similar results were obtained in independent experiments. In contrast,
no peptide-specific CTL activity was observed in tissues from BMT mice.
([E] shows a representative result obtained with BM cells. Similar
results were obtained when spleen, thymus, and lymph nodes were
analyzed.)
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| Fig 4.
MHC phenotype and killing specificity of CTL reisolated
from BMT recipients. C57BL/6 mice were transplanted with BM from
B10.A(4R) mice and injected the following day with mdm100-specific CTL
isolated from BALB/c mice. (A) After 4 weeks, mice were killed and
cells from lymph nodes were stimulated in bulk cultures as described in
Fig 3, resulting in mdm100-specific CTL activity similar to that seen
in Fig 3C. (B) After 4 weeks, mice were killed and cells from lymphoid
tissues were analyzed in LDA assays as described in Fig 5. The CTL bulk
cultures (A) or the CTL expanded from positive wells of LDA assays (B)
were then double-stained with PE-labeled CD8 antibodies and antibodies
specific for Kb, Kk, and Ld.
Staining with the anti-class I antibodies was detected with
FITC-labeled secondary antibodies (see Materials and Methods). Shown is
the staining of gated CD8 cells with the indicated anti-class I
antibodies. Similar results were obtained in independent experiments.
(C and D) CTL killing of RMA-S target cells coated with mdm100 and of
RMA lymphoma cells expressing naturally processed mdm100. (C) Analysis
of CTL used for injection into BMT mice and (D) analysis of CTL rescued
from mice and expanded from LDA plates as in (B).
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How many CTL are present in different tissues?
To determine the frequency of injected CTL, single-cell suspensions
prepared from various tissues were double-stained with antibodies to
CD8 and Ld, Kb, or Kk. The
proportion of CD8 T cells staining with anti-Kb and
anti-Kk antibodies was similar in BMT mice and in mice that
were additionally injected with BALB/c-derived CTL (Fig 2A, B, D, and
E). Staining with anti-Ld antibodies showed no difference
between the 2 groups of mice, indicating that FACS analysis was not
sufficiently sensitive to detect the injected CTL (Fig 2C and F).
Therefore, a more sensitive assay was used. Single-cell suspensions
from spleen, thymus, lymph nodes, and BM were analyzed by LDA to
measure the frequency of CTL specific for the mdm100 peptide. A high
frequency of CTL (up to 1/481) was detected in lymphoid tissues of mice
3 weeks after CTL injection (Fig 5A and B).
Weekly analysis showed good long-term survival of CTL in spleen and
lymph nodes: at week 10, the frequency in spleen and lymph nodes was
only 2- to 5-fold lower than at week 3 (Fig 5A). The longest follow-up
in our experiments was 14 weeks postinjection, at which time CTL were
still present in spleen and lymph nodes (Fig 5B). Most importantly, in
all recipient mice, CTL were consistently found in the BM, a site that
is likely to contain residual leukemic cells in BMT patients. After 3 weeks, the CTL frequency in BM was similar to that seen in lymphoid
tissues, followed by a more rapid decrease at weeks 6 and 7 in the BM
(Fig 5A and B). Analysis of (C57BL/ 6 × BALB/c) F1 mice
transplanted with BM from littermates showed that the frequency of
engrafted CTL and their decrease in the BM was similar to that observed in C57BL/6 recipients (Fig 5A). Thus, there is no evidence that engraftment of the BALB/c-derived, mdm100-specific CTL was impaired in
MHC-mismatched C57BL/6 hosts.
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| Fig 5.
Frequency of injected CTL in tissues of BMT recipients.
C57BL/6 mice were transplanted with 2 × 107
T-cell-depleted B10.A(4R) BM cells. Animals then received a single
dose (1.5 × 107) of mdm100-specific CTL at day 1 post-BMT
(A) or 2 doses (4 × 106 and 5 × 106) of CTL
at days 1 and 10 post-BMT (B). Mice in (B) were divided into a group
that did receive 2 doses of 50,000 U IL-2 at the time of CTL injection
and into a group that received CTL without IL-2. At the indicated weeks
post-CTL injection, mice were killed (1 or 2 mice per time point) and
single-cell suspensions were prepared from spleen ( ), thymus ( ),
lymph nodes ( ), and BM ( ). These cell suspensions were used in
LDA experiments as described in Materials and Methods to determine the
frequency of mdm100-specific CTL. Each data point in (A) and (B) shows
the calculated frequency of 1 LDA experiment. The solid symbols in (A)
show the frequency data of (C57BL/6 × BALB/c) F1 mice transplanted
with BM from littermates and injected with a dose of 1.5 × 107 mdm100-specific CTL ( , F1 mice:
spleen;  , F1 mice: lymph nodes). At weeks 3 and 5, the frequency was
analyzed only in the spleen, and at weeks 12 and 13, the frequency was
analyzed in the spleen, lymph nodes, thymus, and BM. Injected
mdm100-specific CTL were undetectable in thymus and BM at weeks 12 and
13. The frequency was calculated using zero-linear regression analysis,
and all data had statistically acceptable 2 less than 11 and P values greater than 0.1, except for 2 experiments (spleen
at 8 weeks [A] had a 2 of 17.5, and the lymph nodes at
3 weeks+IL-2 [B] had a 2 of 22.5). No data points
are shown for experiments in which the frequency was less than the
detection limit of the LDA assay (eg, thymus and BM at 7 and 14 weeks
in [B]).
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The possiblity that antigenic stimulation might prevent CTL decrease in
the BM will be addressed in future experiments. The observed homing of
CTL to the thymus raised the interesting possibility that they might
tolerize recipient mice against the allogeneic MHC molecules expressed
by the injected CTL.
To confirm that the mdm100-specific killing activity observed in the
LDAs was due to BALB/c-derived CTL, cells from positive wells from some
LDA plates were expanded to obtain sufficient cells for double-staining
with antibodies against CD8 and Ld, Kb, or
Kk. FACS analysis indicated that the CD8 T cells expanded
from LDA cultures were of BALB/c origin (Fig 4B).
Finally, we explored the possibility that low-level mdm-2 expression in
normal tissues in vivo might downmodulate the ability of injected CTL
to recognize naturally processed mdm100 antigen. Thus, CTL rescued from
BMT mice were tested for their ability to recognize RMA tumor cells
presenting naturally processed mdm100.24 The efficiency of
RMA killing by rescued CTL was similar to the killing by CTL before
injection into BMT hosts (Fig 4C and D). Thus, injected CTL retained
the ability to lyse mdm-2-expressing lymphoma cells.
Does IL-2 improve CTL engraftment?
This question was addressed in BMT mice that were injected twice with
CTL. The first injection was performed 1 day after BMT, followed by a
booster injection 10 days later. In one group of mice, both IV CTL
injections were performed in combination with subcutaneous injections of 50,000 U of recombinant IL-2 in an oil
emulsion to achieve gradual IL-2 release. The control group was treated
identically, except that IL-2 was omitted. Analysis 3 and 7 weeks after
the second CTL injection showed that the frequency of mdm100-specific
CTL in different tissues was similar in IL-2-treated mice compared
with the control group (Fig 5B). Thus, IL-2 did not detectably increase
the frequency of injected CTL, nor was it required for long-term CTL survival.
Allo-restricted CTL engraft without causing GVHD.
Previous in vitro studies showed that mdm100-specific, allo-restricted
CTL killed H-2b tumor cells, including lymphoma cells, but
not normal cells.24 However, only a limited selection of
normal cells such as dendritic cells and Con-A-activated lymphocytes
was available for in vitro tests. Thus, it remained unclear whether
some normal tissues in vivo express sufficient levels of mdm-2 to
trigger killing by the injected CTL. Several lines of evidence
suggested that injected CTL did not attack normal host tissues.
Firstly, none of the mice showed any acute side effects after CTL
injection. Secondly, the recovery from BMT and the general health
status were indistinguishable in mice that received CTL compared with
control mice. Thirdly, histological analysis of liver, gut, and skin
from 4 CTL treated and 4 untreated mice showed that these tissues,
which are normally affected by GVHD, showed similar histology in mice
with or without CTL administration
(Fig 6). This indicated
that CTL injection did not cause the tissue damage that is frequently
seen in BMT individuals after infusion of allogeneic T lymphocytes.
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| Fig 6.
Histology of BMT mice. (A through D) The histology of
C57BL/6 mice transplanted with B10.A(4R) BM. One group of mice was
injected with allo-restricted CTL while the control group did not
receive CTL. (A) Liver from a CTL-treated mouse 3 weeks posttransplant,
with no significant inflammation or necrosis. (B) Liver from a control
mouse 3 weeks posttransplant, with mild periportal inflammation. Skin
from a CTL-treated mouse (C) and a control mouse (D) 3 weeks
posttransplant, with no significant inflammation or keratinocyte
necrosis. Tissues were also examined at 4, 5, and 6 weeks
posttransplant and showed no evidence of GVHD in either CTL-treated or
control mice (not shown). (E-H) show the histology of (C57BL/6 × BALB/c) F1 mice, transplanted with BM from littermates. One group of
mice was injected with allo-restricted CTL and a control group did not
receive CTL. (E) Liver from a CTL-treated mouse 4 weeks posttransplant,
with no significant inflammation or necrosis. (F) Liver from control
mouse 4 weeks posttransplant, with no significant inflammation and
necrosis. Skin from a CTL-treated mouse (G) and a control mouse (H) 4 weeks posttransplant, with no significant inflammation or keratinocyte
necrosis. Tissues were also examined at 3 and 5 weeks posttransplant
and showed no evidence of GVHD in either CTL-treated or control mice
(not shown). (All H&E-stained sections, original magnification × 20.)
Sections from colon and stomach also showed no significant inflammation
(not shown).
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We explored whether immune responses of BMT C57BL/6 hosts against
BALB/c-derived CTL prevented the induction of GVHD by injected CTL. If
this were the case, we predicted that CTL injection into (C57BL/6 × BALB/c) F1 hosts would result in GVHD. Thus, (C57BL/6 × BALB/c) F1 mice were transplanted with BM from littermates using the
same conditioning protocol that was used for all experiments in this
study. One group of transplanted mice was injected with 1.5 × 107 allo-restricted CTL and a control group did not
receive any CTL. Histology of skin, liver, stomach, and gut
performed after 3, 4, and 5 weeks showed that injected CTL did not
cause GVHD in these F1 hosts (Fig 6E through H). Thus, lack of GVHD was
not dependent on host immune responses against injected CTL.
| |
DISCUSSION |
Allo-MHC-restricted CTL are promising reagents for treatment of BMT
leukemia patients. Currently, infusion of lymphocytes from BM donors is
used to successfully treat leukemic relapse in these
patients.6,29-40 However, in many cases, the donor
lymphocytes cause not only GVL but also GVHD. Because the antigen
specificity of infused donor lymphocytes is unknown, it is difficult to
selectively direct them against leukemic cells. In contrast,
allo-restricted CTL clones can be raised against peptide epitopes that
are preferentially expressed in leukemia.
Immunotherapy with allo-restricted CTL will always involve at least one
MHC-class I locus mismatch between injected CTL and recipient host. To
avoid generation of CTL clones for individual patients, it would be
desirable to use one established clone specific for a tumor-associated
peptide epitope presented by a common MHC class I allele, such as
HLA-A0201. This clone could then be used in the treatment of all
A0201-positive patients. However, in some cases, this may involve a
complete MHC-mismatch between host and injected CTL. For this reason,
we have investigated in the described murine model the fate of
BALB/c-derived CTL clones injected into MHC-mismatched, BMT C57BL/6 hosts.
Our data showed that CTL consistently engrafted and were detectable in
spleen, lymph nodes, thymus, and BM. Even in the absence of antigenic
stimulation, a high frequency of CTL was detected in lymphoid tissues 3 weeks postinjection, followed by a gradual decrease. This is similar to
the kinetics of antigen-specific immune responses in which, after
initial clonal expansion and death of effector CTL, the frequency of
memory CTL gradually decreases over time. Like memory CTL, injected
allo-restricted CTL responded readily to antigenic stimulation in vitro
and displayed peptide-specific killing activity. These results are
promising; further experiments will assess whether the injected CTL
expand and develop cytotoxic effector function when they encounter
their peptide antigen in vivo.
Although the frequency of CTL was lower in BM than in lymphoid tissues,
it was probably higher than the number of leukemic cells present in the
BM of patients undergoing BMT after myeloablative therapy. In the
experiments described here, the frequency of CTL in the BM 3 weeks
postinjection was 1/1,821 to 1/12,550. For comparison, reverse-transcriptase-polymerase chain reaction (RT-PCR)
used for detection of BCR/ABL-positive cells in the BM of CML patients can detect 1/105 to 1/106 leukemic
cells.41 Thus, in patients with minimal residual disease as
defined by RT-PCR, a high ratio of CTL over leukemic cells may result
in efficient elimination of malignant cells and prevent relapse.
Histological examination indicated that injection of allo-restricted
CTL against peptide epitopes expressed at elevated levels in
transformed cells did not trigger GVHD. It is currently unclear whether
the expected low level of peptide display in normal tissues can deliver
weak stimulatory signals to enhance the survival of injected CTL
without triggering effector function.42 Alternatively, low-level peptide display may be ignored, or it may cause
downmodulation of the TCR and/or CD8 molecules.43 Future
studies will investigate these alternate possibilities.
Because the allo-restricted T-cell strategy is not limited by
immunological tolerance, it provides an opportunity to target almost
any protein that is expressed in leukemic cells. These include
hematopoietic transcription factors, such as WT-1 and gata-1, and
differentiation antigens, such as myeloperoxidase and CD45.
Allo-restricted CTL clones against these proteins would be expected to
function as proficient GVL effectors and attack leukemic cells. The CTL
may also attack normal hematopoietic cells expressing the relevant
protein, but not nonhematopoietic tissues, which are usually the target
of GVHD. The patients' hematopoietic system could be protected from
CTL attack by selecting allogeneic BM donors who do not express the HLA
class I allele that is required for antigen recognition by the injected
CTL clones.28
| |
FOOTNOTES |
Submitted November 2, 1998; accepted June 24, 1999.
Supported by grants to H.J.S. from the Leukaemia Research Fund and the
Cancer Research Campaign.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hans J. Stauss, MD, ICSTM,
Hammersmith Hospital, Department of Immunology, Du Cane Road, London
W12 0NN, UK; e-mail: hstauss{at}rpms.ac.uk.
| |
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ABSTRACT
INTRODUCTION