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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2426-2433
TRANSPLANTATION
Immunization of allogeneic bone marrow transplant recipients with
tumor cell vaccines enhances graft-versus-tumor activity without
exacerbating graft-versus-host disease
Larry D. Anderson Jr.,
Cherylyn A. Savary, and
Craig A. Mullen
From the Departments of Experimental Pediatrics, Immunology, and
Surgical Oncology, The University of Texas M.D. Anderson Cancer Center,
Houston, TX.
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Abstract |
Allogeneic bone marrow transplantation (BMT) induces 2 closely
associated immune responses: graft-versus-tumor (GVT) activity and graft-versus-host disease (GVHD). We have previously shown that pretransplant immunization of allogeneic BMT donors with a
recipient-derived tumor cell vaccine increases both GVT activity and lethal GVHD because of the priming of donor T cells against putative minor histocompatibility antigens (mHAgs) on the tumor vaccine cells. The work reported here tested the hypothesis that tumor
cell vaccination after BMT would produce an increase in GVT activity
without exacerbating GVHD. C3H.SW donor bone marrow and splenocytes
were transplanted into major histocompatibility complex-matched,
mHAg-mismatched C57BL/6 recipients. One month after BMT, recipients
were immunized against either a C57BL/6 myeloid leukemia (C1498) or
fibrosarcoma (205). Immunized recipients had a significant increase in
survival and protection against tumor growth in both tumor models, and
significant tumor protection was seen even in recipients with
preexisting micrometastatic cancer before immunization. Alloreactivity
appeared to contribute to the in vitro anti-tumor cytolytic activity,
but in vivo immunity was tumor specific, and no exacerbation of GVHD
was observed. Although the immunodominant mHAg B6dom1 was
shown to be expressed by all B6 tumors tested and was largely responsible for the alloreactivity resulting from tumor immunization of
donors, the in vitro alloreactivity of immune recipients was more
restricted and was not mediated by recognition of B6dom1.
In conclusion, post-transplant tumor immunization of allogeneic BMT
recipients against either a leukemia or a solid tumor can increase GVT
activity and survival without exacerbating GVHD.
(Blood. 2000;95:2426-2433)
© 2000 by The American Society of Hematology.
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Introduction |
Allogeneic bone marrow transplantation (BMT) is
associated with an unequivocal graft-versus-tumor (GVT) immune response
mediated by donor T cells, but the benefit of GVT activity is often
offset by graft-versus-host disease (GVHD), a potentially fatal immune response caused primarily by mature donor T cells attacking normal cells in the recipient.1 Although it is commonly believed
that the target antigens of GVHD in MHC-matched BMT are minor
histocompatibility antigens (mHAgs), only a few have been identified
because of their polymorphic and heterogeneous
nature.2,3 Similarly the target antigens for GVT
activity are unknown. In theory they could include some or all of the
following categories: (i) ubiquitously expressed immunodominant host
mHAgs that are the targets of GVHD, (ii) ubiquitously expressed
nonimmunodominant host mHAgs, (iii) tissue-restricted host mHAgs not
expressed in physiologically critical GVHD target organs, (iv)
differentiation stage- and tissue-restricted host mHAgs, or (v)
tumor-specific antigens. The simplest hypothesis that would explain
"classical" graft versus tumor activity that occurs without
specific immune manipulation of either donor or host is that the
targets are ubiquitous, immunodominant mHAgs. However, at present it is
unclear to what extent the donor T-cell populations mediating GVT
activity and GVHD overlap or are identical in terms of either antigen
specificity or effector mechanism 2,3
Previous work in our laboratory has shown that recipient-derived tumor
cell vaccines given to donors before BMT cause not only
increased GVT activity but also lethal GVHD,4 which we postulated was caused by the recognition of immunodominant mHAgs expressed by tumor cells and target organs of GVHD. Nevertheless, both
clinical and experimental evidence suggests that some of the effector
cells mediating GVT activity may be distinct from those mediating
GVHD.5-9
Unlike solid organ transplantation, BMT does not require life-long
immunosuppression. This is most likely because of the development of
unresponsiveness or tolerance to immunodominant mHAgs after BMT. If the
spectrum of antigens on GVHD target tissues and tumor cells are not
identical, it is conceivable that in this setting tumor cell vaccines
could expand the donor-derived T-cell populations recognizing antigens
on tumor cells (eg, theoretical antigen categories ii to v), but not
those unresponsive or absent populations that recognize immunodominant
mHAgs present on most normal cells. Therefore, the experiments
described in this study tested the hypothesis that post-transplant
immunization of BMT recipients with a tumor cell vaccine would
substantially increase "nonclassical" donor GVT activity and
extend the survival of BMT recipients without exacerbating GVHD.
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Materials and methods |
Animals
Female C57BL/6 mice were purchased from the National Cancer
Institute (Frederick, MD), and female C3H.SW-H2b/SnJ (C3H.SW) mice were
purchased from the Jackson Laboratory (Bar Harbor, ME). They were used
for experiments at 6 to 12 weeks of age. Mice were housed in
conventional rooms with food and water ad libitum. From 2 or 3 days
before BMT until day 14, water was acidified (pH 2.5) and supplemented
with 2 g/L neomycin sulfate (Sigma, St Louis, MO).
Cell lines
The cell line 205 is a weakly immunogenic methylcholanthrene-induced
C57BL/6 fibrosarcoma cell line.10 This tumor is not spontaneously metastatic but reproducibly forms multiple lung nodules
when at least 1 × 104 cells are injected
intravenously into C57BL/6 mice. 205IL-2/TK is a 205 cell line modified
to express both the interleukin-2 (IL-2) gene and the herpes simplex
virus thymidine kinase (TK) suicide gene using the LXSN and pBabePuro
retroviral vectors, respectively. The pBabePuro vector has a puromycin
resistance gene,11 and the LXSN vector contains a neomycin
resistance gene.12 Transduced cells were selected in 2.5 µg/mL puromycin, 1 mg/mL G418, or both. C1498 is a C57BL/6
myelomonocytic leukemia cell line of spontaneous origin (ATCC,
Rockville, MD). When injected intravenously,
1 × 104 cells is a near-uniformly lethal dose of
cells; animals die with gross evidence of leukemia, including
hepatomegaly and splenomegaly. Both 205 and C1498 express major
histocompatibility complex (MHC) class I but not MHC class II. B16F10
is a spontaneous, weakly immunogenic C57BL/6 melanoma cell line (a gift
from Dr I. J. Fidler, M.D. Anderson Cancer Center). EL4 is a C57BL/6
lymphoma cell line (ATCC). P815 is a lymphokine activated killer
(LAK)-sensitive DBA/2 mastocytoma cell line (ATCC). Yac-1 is a natural
killer (NK)-sensitive A/Sn lymphoma cell line (ATCC). Cells were grown in tissue culture using RPMI-1640 (RPMI) supplemented with 5% heat-inactivated fetal bovine serum (Biowhittaker, Walkersville, MD),
and 2 mmol/L L-glutamine.
BMT recipient immunization
205IL-2/TK + ganciclovir immunization.
We have shown that ganciclovir-mediated ablation of live 205IL-2/TK
cells induces systemic immunity to unmodified 205 in C57BL/6 mice13 and in C3H.SW donors.4 In the initial
experiments recipients were injected subcutaneously in the flank with 3 to 5 × 106 live 205IL-2/TK cells in 0.2 mL
Hank's balanced salt solution (HBSS) 1 month after BMT and then
received 1 mg ganciclovir intraperitoneally in 0.2 mL
phosphate-buffered saline daily for 1 week starting 3 days after the
cell injection. Subsequent vaccines were given as above at 2-week intervals.
Irradiated 205IL-2/TK or 205 immunization.
Some recipients (and control C3H.SW donors) were injected
subcutaneously in the flank with 3 to 5 × 106
irradiated 205IL-2/TK or 205 cells in 0.2 mL HBSS 1 month after BMT.
Vaccines were given at 1-week intervals.
C1498 tumor immunization.
Mice were injected subcutaneously in the flank with
10 × 106 irradiated C1498 cells in 0.2 mL HBSS
starting 1 month after BMT. Vaccines were given at a 1-week interval
unless the vaccine was given at the time of intravenous leukemia
challenge, which was 10 days after the previous vaccine.
In vivo tumor inoculation
Micrometastatic lung tumors were established by injecting C57BL/6
mice with 1 × 105 205 tumor cells intravenously in
0.2 mL HBSS. C1498 leukemia was established by injection of 1 to
1.5 × 104 cells intravenously. The timing of tumor
challenge in relation to the vaccine schedule is specified in the text
and legends.
Bone marrow transplantation
BMT recipients received 850 cGy total body irradiation using a
60Co source 1 day before BMT. On the day of BMT, 2 to
4 × 106 bone marrow cells and 5 to
10 × 106 spleen cells were injected intravenously
together in a total volume of 0.2 mL HBSS. Bone marrow was isolated
from donors by flushing each femur and tibia with RPMI. Spleen cells
were isolated by macerating the spleens between 2 frosted glass slides,
followed by lysis of erythrocytes. Fewer than 5% of recipients were
excluded from these studies before the initiation of immunization
because of signs of severe GVHD.
Enumeration of pulmonary tumor nodules
After death, lungs from mice injected with 205 tumor were stained
black by suffusion with India ink instilled through the trachea. Lungs
were fixed, and white tumor nodules on the black lung surface were
counted without magnification.
Evaluation of GVHD
Recipients were weighed weekly and observed daily for signs of GVHD
(weight loss, alopecia, dermatitis, hunched posture, and death). In
some experiments histologic examination of livers for GVHD was
performed. Liver sections stained with hematoxylin and eosin were
examined for characteristic mononuclear cell infiltrates in portal triads.
Cytotoxicity assays
For use as effector cells, spleen cells were cultured in 6-well
plates at 1 × 106 cells/mL and 10 mL/well in RPMI
supplemented with 10% FBS (Summit Biotech, Fort Collins, CO), 100 U/mL
penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, 100 mmol/L
sodium pyruvate, 0.1 mmol/L nonessential amino acids, and 50 µmol/L
2-mercaptoethanol (complete medium). In assays for alloreactivity, 30 Gy-irradiated C57BL/6 spleen cells or B6dom1 peptide-pulsed
C3H.SW spleen cells were used as stimulators at a concentration of
1 × 106 cells/mL or 1.25 × 105
cells/mL, respectively. Peptide-pulsed stimulators were incubated for 1 hour at 37°C with 35 µg/mL B6dom1 peptide (AAPDNRETF,
synthesized by the Peptide Synthesis Core Laboratory of M.D. Anderson
Cancer Center). In assays for combined tumor-reactivity and
alloreactivity, 250 Gy-irradiated 205 tumor cells or 100 Gy-irradiated
C1498 leukemia cells were used as stimulators at a concentration of
5 × 103 cells/mL or 5 × 104
cells/mL, respectively. After 4 to 5 days in culture with the appropriate stimulator cells, effector cells were harvested and plated
in triplicate with 5 × 103 51Cr-labeled
target cells per well at effector:target ratios ranging from 200:1 to
12.5:1. Target cells were labeled by combining
5 × 106 cells in 0.1 mL complete medium with 20 µL FBS (Summit) and 0.1 mL (approximately 100µCi) sterile isotonic
Na2 51CrO (Amersham, Arlington
Heights, IL) for 60 minutes at 37°C. Concanavalin A (ConA)
lymphoblast (CAB) targets were generated by stimulating C57BL/6 or
C3H.SW spleen cells for 2 days with 2 µg/mL. ConA at
2 × 106 cells/mL in complete medium. They were
labeled with 51Cr as above for 45 minutes. Some C3H.SW CAB
were loaded with B6dom1 peptide by incubation for 90 minutes with 40 µg peptide (45 minutes before adding 51Cr
plus 45 minutes after adding 51Cr). Labeled targets were
washed 3 times before plating with effectors in a total volume of 0.2 mL/well in 96-well round-bottom plates. Plated cells were incubated for
4 hours at 37°C, after which 0.1 mL supernatant was counted in a
gamma counter (Wallac, San Francisco, CA). Percentage lysis was
calculated as 100 × (experimental
cpm  spontaneous cpm)/(maximum cpm spontaneous
cpm)]. Spontaneous release was usually less than 20% and always less
than 30% of the maximum release.
Statistical analysis
Prism 3.0 software (GraphPad Software for Scientists, Sorrento, CA)
was used for statistical evaluation of data. When more than 2 groups of
lung nodules were compared, a nonparametric 1-way analysis of variance
(Kruskal-Wallis test) was performed. If P < .05 overall,
then the groups were compared using a Dunn's multiple comparison test.
When only 2 groups were compared, a Mann-Whitney U test was
used. To compare numbers of mice with no lung nodules, the Fisher exact
test was used. To compare Kaplan-Meier survival curves, the log-rank
test was used.
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Results |
Tumor immunization of allogeneic BMT recipients increases GVT
activity
Experiments were conducted to test the hypothesis
that tumor immunization of allogeneic BMT recipients could protect
against the growth of metastatic cancer in BMT recipients. We
first assessed the kinetics of immune function recovery and found
that recipients in this model could not mount a detectable cytolytic
immune response at 2 weeks after BMT but began responding to tumor cell
antigens by 1 month after BMT and began responding to influenza
nucleoprotein antigen by 5 weeks after BMT, as assessed by in vitro
cytotoxicity assays (data not shown). Therefore, in the experiments
described in this study, we initiated vaccination at least 1 month
after BMT. C3H.SW C57BL/6 recipients in the initial
experiments were immunized with 205IL-2/TK cells plus ganciclovir. As
seen in Figure 1, recipients immunized only
a single time were not protected against tumor growth, but
those immunized again at the time of tumor challenge had complete
protection against growth of pulmonary tumor nodules
(P < .05) and significantly enhanced survival
(P = .0040) compared to nonimmunized recipients. All deaths
among recipients immunized twice resulted from growth of a
nonpulmonary metastasis. Nonpulmonary metastases usually
consisted of tumors in the connective tissue of the
sacral/pelvic/paraspinal region or ovary. Nonpulmonary tumors also
grew in some nonimmunized recipients.
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| Fig 1.
Immunization of BMT recipients increases survival and GVT
activity.
One month after BMT, SWB6 recipients were immunized with 205IL-2/TK
cells and ganciclovir. Two weeks after the first vaccine,
micrometastases were established by intravenous injection of
1 × 105 205 tumor cells, at which time 1 group of
recipients received a second vaccine. Lung nodules were counted at the
time of death or after day 100 for each recipient. The deaths that
occurred in the recipient group immunized twice resulted from the
growth of a nonpulmonary metastasis (sacral/pelvic mass) that
necessitated sacrifice. Groups and sizes were: no vaccine, n = 4; 1 vaccine, n = 5; and 2 vaccines, n = 5. *P < .05 for
lung nodules compared to 1 or no vaccine controls. **P = .004
compared to nonimmunized recipient survival.
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Because of evidence of greater efficacy with multiple vaccine
treatments, recipients in subsequent experiments were immunized 4 times. Recipients immunized 4 times with irradiated 205-IL-2/TK cells
had markedly extended survival (P < .0001; Figure
2A) and often had complete prevention of
lung nodule growth (P = .0011; Figure 2B). Furthermore, 92%
(11 of 12) survived more than 100 days without signs of tumor or GVHD.
Recipients immunized 4 times with irradiated, unmodified 205 cells also
had enhanced survival (P = .0016; Figure
3A) and significant reduction of lung
nodules (P = .0022; Figure 3B). Fifty percent (3 of 6)
survived more than 100 days, and 83% (5 of 6) were completely
protected against lung nodules.
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| Fig 2.
Recipient immunization with an irradiated tumor cell
vaccine increases GVT activity and prevents death from tumor.
One month after C3H.SWC57BL/6 BMT, recipients (205-immune, n = 12)
were immunized with 50 Gy-irradiated 205IL-2/TK cells. Control
recipients (nonimmune, n = 8) were not immunized. Ten days after the
first vaccine, micrometastases were established in all recipients by
intravenous injection of 1 × 105 205 tumor cells,
and 205-immune recipients received 3 more vaccines at weekly intervals.
Lung nodules were counted after death or after day 100 for each
recipient; pulmonary nodule counts were not available for 1 mouse in
the nonimmune group because the carcass was severely cannibalized
before autopsy. 205-immune recipients had significantly enhanced
survival (A, P < .0001) and a significant reduction of lung
nodules (B, P = .0011) compared to nonimmune recipients.
Results were pooled from 2 independent experiments.
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| Fig 3.
Recipient immunization with irradiated unmodified 205 cells increases GVT activity and prevents death from tumor.
One month after C3H.SW C57BL/6 BMT, recipients (205-immune, n = 6)
were immunized with 50 Gy-irradiated unmodified 205 cells. Control
recipients (nonimmune, n = 6) were not immunized. One week after the
first vaccine, micrometastases were established in all recipients by
intravenous injection of 1 × 105 205 tumor cells,
and 205-immune recipients received 3 more vaccines at weekly intervals.
Lung nodules were counted after death or after day 100 for each
recipient. 205-immune recipients had significantly enhanced survival
(A, P = .0016) and significant reduction of lung nodules (B,
P = .0022) compared to nonimmune recipients.
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We next tested the influence of post-BMT vaccines in a second
tumor model, the C57BL/6 C1498 model of acute myelogenous leukemia. Using the immunization method shown by Boyer et al14 to
induce a T-cell-mediated immune response in syngeneic C57BL/6 mice,
C3H.SW C57BL/6 BMT recipients were immunized 4 times with
irradiated C1498 cells. Immunization significantly extended survival
(P < .0001; Figure 4) compared
to nonimmunized recipients, and 71% (5 of 7) of recipients achieved
long-term survival for more than 100 days without signs of leukemia or
GVHD. In all recipients that died, there was gross evidence of
leukemia, including hepatomegaly, but not GVHD.
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| Fig 4.
Tumor immunization of BMT recipients increases GVT
activity against leukemia.
One month after BMT, SW B6 recipients (C1498-immune, n = 8) were
immunized twice with irradiated C1498 leukemia cells at a 1-week
interval. Control recipients (nonimmune, n = 8) were not immunized.
10 days after the 2nd vaccine, 1 × 104 C1498 cells
were injected intravenously to simulate relapse after BMT, and
C1498-immune recipients were immunized twice more at a 1-week interval.
Immunization significantly enhanced survival (P < .0001)
compared to nonimmune control recipients.
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GVT activity is induced by tumor cell vaccines in BMT recipients
with preexisting micrometastatic cancer
Both 205 and C1498 tumors progress rapidly in vivo, often leading to
death within 1 month. Because the induction of primary T-cell responses
usually does not occur until 7 to 10 days after the
administration of vaccines, we predicted that vaccine
efficacy would be reduced in mice with established, progressively
growing tumors. Indeed, in mice inoculated with C1498 leukemia cells 1 week before immunization, survival was not prolonged by tumor vaccination (Figure 5). However, in
recipients with C1498 leukemia established 3 days before the initiation
of vaccination, survival was significantly increased compared to
nonimmunized recipients (P = .0351; Figure 5). In the 205 tumor model, recipients that began vaccine treatment 3 days after the
establishment of 205 metastases had a significant reduction in
pulmonary tumor nodules (P = .0140, Figure
6). In a separate experiment in which
long-term survival was the endpoint, the observed increase in median
survival after 205 tumor challenge (from 31 to 41 days) was not
statistically significant (data not shown). Death in these
vaccine-treated animals resulted from growth of either nonpulmonary
tumors or progressive growth of a small number of pulmonary nodules.
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| Fig 5.
Tumor immunization of BMT recipients increases GVT
activity against preexisting leukemia.
One month after BMT, SW B6 recipients were challenged intravenously
with 1.5 × 104 C1498 cells to induce preexisting
leukemia. Recipients were then either not immunized (no vaccine; solid
line) or immunized 4 times with 10 × 106 irradiated
C1498 leukemia cells at a 1-week interval starting 3 days (day 3 vaccine; thick dashed line) or 7 days (day 7 vaccine; thin dashed line)
after tumor challenge. Although immunization beginning on day 7 did not
improve survival, immunization beginning on day 3 significantly
enhanced survival (P = .0351) compared to the nonimmune
controls.
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| Fig 6.
Recipient immunization with a tumor cell vaccine
increases GVT activity against preexisting 205 tumor.
One month after SW B6 BMT, micrometastatic fibrosarcoma was
established by intravenous injection of 1 × 105 205 tumor cells. Recipients were either not immunized (nonimmune) or
immunized 4 times with 50 Gy-irradiated 205IL-2/TK cells (205-immune)
at a weekly interval starting on day 3 after challenge. Lung nodules
were counted 1 month after tumor challenge. The day 3 205-immune group
had a significant reduction in lung nodules (P = .0140)
compared to the nonimmune recipients.
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Tumor immunization of allogeneic BMT recipients does not
exacerbate GVHD
Recipients surviving tumor challenge in the above experiments did
not develop signs of acute GVHD (eg, weight loss, fur loss, diarrhea,
dermatitis, or histologic evidence of GVHD in the liver). This
suggested that tumor cell vaccines administered after BMT did not
increase GVHD. To test this hypothesis directly, we also conducted 3 experiments in which recipients were immunized 4 times against 205 but
were not challenged with tumor. All immunized recipients in both
experiments (n = 16) survived more than 100 days without signs of
GVHD, including weight loss.
GVT activity induced by tumor immunization of recipients is
tumor-specific in vivo
We have previously shown that the GVT activity induced by tumor
immunization of allogeneic BMT donors is mediated by
alloreactive T cells that cross-react broadly with recipient tumors and
nonmalignant tissues.4 Therefore, experiments were
performed to determine whether recipient immunization would
induce alloreactivity. If so, tumor vaccination should induce
cross-protection against another immunologically unrelated recipient
tumor. Recipients were either not immunized (nonimmune) or immunized
against 205 (205-immune) or C1498 (C1498-immune) and then challenged
with either 205 or C1498. As seen in Figure
7, the GVT activity of 205-immune
recipients was tumor specific. The 205 vaccine again induced protection
against 205 (P = .0006; Figure 7B) but did not protect
against C1498 challenge compared to nonimmune controls (Figure 7A).
Similarly, vaccination against C1498 increased survival after challenge
with C1498 immunization (Figure 7A) but not after challenge with 205 (Figure 7B). Reduction in 205 pulmonary metastases was seen with the
use of 205 vaccines but not with the use of C1498 vaccine (Figure 7C).
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| Fig 7.
GVT activity after tumor immunization of BMT recipients
is tumor-specific in vivo.
One month after BMT, SW B6 recipients were immunized twice with
irradiated C1498 leukemia cells (C1498-immune) or were irradiated
205IL-2/TK cells (205-immune) at a 1-week interval. Control recipients
(nonimmune) were not immunized. Ten days after the 2nd vaccine,
1.5 × 104 C1498 cells (A) or
1 × 105 205 cells (B, C) were injected
intravenously to simulate relapse after BMT, and immune recipients were
immunized twice more at 1-week intervals. Lung nodules were counted in
the 205-challenged mice at the time of death or after 100 days.
Although tumor immunization against either 205 or C1498 induced
protection (P < .05) against the immunizing cell type, it
did not induce cross-protection against the other tumor
(P > .05 for both survival tests and lung nodules). Results
were pooled from 2 independent experiments.
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Tumor-immunized allogeneic BMT recipients exhibit in vitro cytolytic
antitumor activity but only limited alloreactivity
To evaluate further the specificity of the antitumor response, in
vitro cytotoxicity assays were performed. Splenocytes from immunized
BMT recipients or control immunized donors (C3H.SW) were stimulated in
vitro with tumor cells and tested for their ability to kill various
target cells (Figure 8). Immunization of
C3H.SW donors with either 205 or C1498 vaccines generated broadly alloreactive CTL activity, lysing all C57BL/6 targets. In contrast, BMT
recipients immunized with 205 or C1498 generated cytotoxic cells with a
different target specificity profile. In 3 independent experiments, BMT
recipients generated cytotoxic cells that killed the immunizing tumor,
but cross-reactivity was only seen against B16F10 in the case of
205-immune recipients and against 205 for C1498-immune recipients.
Neither immune recipients nor control immunized C3H.SW donor
splenocytes killed Yac-1 or P815, indicating that the cytolytic
response was not caused by nonspecific NK or LAK activity.
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| Fig 8.
Tumor immunization of BMT recipients increases antitumor
cytolytic activity and induces limited alloreactivity compared to
immune C3H.SW controls.
Three months (A) or 1 month (B, C) after BMT, C3H.SW B6 recipients
were immunized twice with irradiated 205IL-2/TK cells (205-immune) or
irradiated C1498 leukemia cells (C1498-immune) at a 1-week interval.
Control C3H.SW donors were also immunized in an identical manner. Ten
days after the 2nd vaccine, spleens were harvested, and spleen cells
were stimulated for 4 days in vitro with irradiated 205 (A, B) or C1498
(C). A 51Cr-release assay was performed using targets
specified in the legends. B6 CAB are C57BL/6 ConA lymphoblasts. A, B,
and C represent independent experiments with different panels of
targets; neither P815 nor Yac cells were used as targets in experiment
C because other experiments had demonstrated little or no LAK or NK
activity. Data shown are at an E:T ratio of 200:1, and each
effector:target condition was performed in triplicate using splenocytes
pooled from 2 or 3 mice.
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Unresponsiveness against the immunodominant recipient minor
histocompatibility antigen B6dom1 is not reversed by
post-transplant tumor immunization of recipients
Perreault et al15-17 have identified an immunodominant
peptide mHAg (B6dom1), which is presented by
H-2Db on normal C57BL/6 cells and is at least partially
responsible for GVHD in the C3H.SW C57BL/6 BMT model. Therefore, we
wanted to determine whether tumor cell vaccines induce a response to B6dom1 in C3H.SW mice, whether C57BL/6 tumor cell lines
express B6dom1, and whether BMT recipients fail to respond
to B6dom1, which would help explain their limited
alloreactivity compared to immunized donors. First, C3H.SW mice were
immunized with either C57BL/6 spleen cells (B6-immune) or irradiated
205IL-2/TK tumor cells (205-immune). Control mice were not immunized
(naïve SW). After stimulation of immune splenocytes with
B6dom1 peptide in vitro, we tested their ability to
specifically kill C3H.SW ConA lymphoblasts loaded with
B6dom1 peptide (B6dom1/SW CAB) versus
control unpulsed SW CAB. As seen in Figure
9A, both 205-immune and control B6-immune
donors developed a specific and almost identical response to
B6dom1, demonstrating that 205 tumor cells do express
B6dom1 as an immunodominant antigen. We next tested the
ability of B6-immune, B6dom1-stimulated C3H.SW cells to
kill various C57BL/6 tumor cell lines (Figure 9B). All lines tested
(205, C1498, B16F10, and EL4) were killed efficiently and therefore
express B6dom1, explaining why 205-immune C3H.SW
splenocytes were broadly cross-reactive against all of them as seen in
Figure 8.
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| Fig 9.
Tumor immunization of
C3H.SW BMT donors induces a
response to the mHAg
B6dom1 found on all
C57BL/6 tumor targets tested. C3H.SW (donor
strain) mice were immunized twice with 5 × 106
irradiated 205IL-2/TK cells (205-immune) or
20 × 106 C57BL/6 spleen cells (B6-immune) at a
weekly interval, or they were not immunized (naïve). Ten days
after the 2nd vaccine, their splenocytes were stimulated
for 5 days in vitro with C3H.SW spleen cells loaded exogenously with
B6dom1 peptide. A 51Cr-release assay was
performed using the targets specified in the graphs.
B6dom1/SW CAB indicates C3H.SW ConA lymphoblast targets
that were loaded exogenously with B6dom1 peptide, whereas
SW CAB were not loaded with peptide. Data shown are at an E:T ratio of
100:1, and each effector:target condition was performed in triplicate
using splenocytes pooled from 2 or 3 mice. Figures 9A and 9B represent
data from the same experiment.
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Because tumor-immunized recipients do not have broad cross-reactivity
against all C57BL/6-derived targets (unlike tumor-immunized C3H.SW), it
is possible that they do not mount a substantial response to
B6dom1. To test this hypothesis we examined the relative
ability of 205-immune BMT recipient splenocytes to kill
B6dom1-loaded SW CAB and regular SW CAB (Figure
10). Although 205-immune C3H.SW controls
again mounted a strong response to B6dom1, 205-immune BMT
recipients had virtually no response to B6dom1.
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| Fig 10.
Unresponsiveness to the recipient mHAg
B6dom1 is not reversed by post-transplant tumor
immunization of recipients.
One month after BMT, allogeneic BMT recipients or control C3H.SW donors
were immunized twice with irradiated 205IL-2/TK cells at a 1-week
interval. Ten days after the second vaccine, their splenocytes were
stimulated for 5 days in vitro with B6dom1-loaded C3H.SW
spleen cells. A 51Cr-release assay was then performed using
C3H.SW ConA lymphoblast targets that were either loaded with
B6dom1 peptide (B6dom1/SW CAB) or not loaded
with peptide (SW CAB). Data shown are at an E:T ratio of 50:1, and each
effector:target condition was performed in triplicate using splenocytes
pooled from 2 or 3 mice.
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Unresponsiveness against recipient mHAgs responsible for GVHD is not
reversed by secondary adoptive transfer of lymphocytes from
tumor-immunized BMT chimeras into new irradiated C57BL/6 hosts
Although use of tumor vaccines after BMT did not increase severe
acute GVHD (as measured by death, weight loss, dermatitis, and
histology), it is possible that tumor vaccines may have produced subclinical exacerbation of GVHD. To test this possibility, we transferred tumor-immunized BMT recipient splenocytes into newly irradiated C57BL/6 hosts. This is potentially a more sensitive bioassay
for the detection of GVHD because in it the secondary hosts may
generate the total body irradiation-induced cytokine storm, a critical
cofactor in the generation of severe GVHD.18-22 Long-term
survivors of C3H.SW C57BL/6 BMT and irradiated 205IL-2/TK immunization were reimmunized twice before transferring their bone
marrow and spleen cells to irradiated C57BL/6 recipients. Secondary
transfer of 205-immune BMT recipient (chimera) splenocytes did not
induce any signs of GVHD in any of the recipients tested (Table
1).
View this table:
[in this window]
[in a new window]
|
Table 1.
Secondary adoptive transfer of 205-immune BMT chimera
lymphocytes into irradiated recipients does not induce GVHD
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Discussion |
This work tested the hypothesis that immunization of MHC-matched
allogeneic BMT recipients with a tumor cell vaccine would substantially
increase GVT activity and prolong tumor-free survival without
exacerbating GVHD. Tumor vaccination of recipients against either a
fibrosarcoma or a myeloid leukemia produced a substantial increase in
GVT activity, which was capable of complete protection against tumor
growth and of preventing the growth of preexisting micrometastatic
cancer cells. Furthermore, BMT recipients did not develop signs of
acute GVHD after tumor cell vaccination, demonstrating that GVT
activity can occur independent of GVHD.
This study shows that tumor cells of diverse histology are capable of
both expression of a minor histocompatibility antigen (mHAg)
(B6dom1) and induction of an immune response against this
mHAg, which is an immunodominant GVHD target antigen in this BMT
model.15-17 This finding confirms at the molecular level
our previous findings in the allogeneic donor immunization
model4 and elucidates the mechanism whereby tumor-immunized
donor lymphocytes can kill various recipient targets and cause GVHD.
Specific restimulation of tumor-immune donor cells with the
H-2Db-restricted B6dom1 peptide in vitro caused
a similar level of tumor cell lysis compared to tumor cell
restimulation, indicating that much of the alloreactive cytolytic
T-cell activity seen after donor immunization with tumor cells
is caused by the recognition of B6dom1.
Although the same vaccines used in this study have been shown to
increase both GVT activity and lethal GVHD when used to treat donors
before BMT,4 they did not cause GVHD when administered after BMT. This is consistent with the hypothesis that after
transplantation, naive donor-derived T cells eventually become
unresponsive to the immunodominant mHAgs that are potential targets for
GVHD. Because the antitumor response of recipients after BMT does not exacerbate GVHD, it is likely that much of the immune response is
directed against other antigens, such as nonimmunodominant mHAgs, with
restricted tissue distribution or possibly even tumor-specific antigens. This is supported by the observation that in vivo tumor protection in this study was tumor specific; the fibrosarcoma and
leukemia vaccines induced immunity that did not cross-protect against
growth of the other tumor (Figure 7), despite the presence of common
mHAgs on both tumors (Figure 9).4 The hypothesis that
allogeneic BMT recipients become unresponsive to immunodominant mHAgs
that are targets of GVHD is also supported by the finding that although
donor immunization with recipient tumor cells induced a potent response
to B6dom1, immunization of recipients 1 month after BMT
failed to induce a significant response to B6dom1. Such
T-cell unresponsiveness to B6dom1 explains why immune
recipient cytolytic activity is not broadly cross-reactive against all
recipient targets. The mechanism for this unresponsiveness is unknown.
These data are consistent with either clonal deletion, anergy, or the
generation of suppressor cells. Further studies are being conducted to
help elucidate the mechanism of unresponsiveness to recipient mHAgs,
which will be helpful in learning how to better apply tumor vaccination
strategies to human BMT recipients.
The limited pattern of cytolytic activity seen in vitro was likely
caused by activation and expansion of some T-cell clones that
recognized tumor antigens and other T cells that recognized mHAgs other
than B6dom1. These other putative mHAgs appear to be
expressed on some but not all tumor cell lines and with limited or no
expression on normal lymphoblasts or targets of GVHD. For example,
205-immune recipient splenocytes recognized both 205 and B16F10 tumors
(Figure 8A). (205 and B16F10 do not induce cross-reactivity in normal, syngeneic C57BL/6 mice and therefore do not share tumor-specific antigens.13) That this cross-reactivity was attributable to the recognition of shared mHAgs is supported by the observation of
lysis of 205 and B16F10 after in vitro allostimulation of 205-immune recipient splenocytes with normal C57BL/6 splenocytes that do not
express tumor antigens (data not shown). Nevertheless, the mHAgs
recognized by post-transplant BMT recipients must be different than
those recognized by tumor-immunized donors because of the lack of broad
cross-reactivity against all recipient targets, which was seen after
donor immunization (Figure 8). The lack of expression of the
target antigens of alloreactivity by target cells of GVHD is
further supported by the observation that the alloreactive T
cells did not induce GVHD even when transferred to secondary
irradiated recipients (Table 1).
Although useful in many respects, the experimental BMT model used in
these studies does not exactly replicate the clinical situation in
which the BMT recipient is in clinical remission but harbors minimal
residual tumor at the time of BMT that may reappear clinically in
months or years. As was shown, the experimental murine tumors showed
rapid growth producing death in approximately 1 month. In these murine
transplants, as in human transplants, the capacity of the host to
respond to immunization to antigens in the first month is very poor.
Therefore, it was not practical within this model to test the efficacy
of the tumor vaccines in the setting of preexisting tumor. The results
must be viewed with this in mind. It is possible that the results of
post-BMT tumor vaccination would be different in such a setting.
One could hypothesize that minimal residual tumor at time of BMT could
make the donor-derived lymphocytes tolerant to antigens on
host tumor cells. We are developing BMT models to test this hypothesis.
Use of post-BMT tumor vaccines in human trials will necessarily require
careful scrutiny of the potential of such vaccines to exacerbate GVHD.
Because of the complex and uncharacterized expression of most mHAgs on
tumor cells, caution must be exercised about using whole tumor cells as
vaccines until methods are developed for identifying mHAgs, their
pattern of tissue distribution, and their relative ability to induce
GVT activity and GVHD. However, in this study we showed that the
induction of GVT activity can occur independent of GVHD if recipients
are immunized with cancer cells after the development of
unresponsiveness to immunodominant mHAgs. In some human malignancies,
such as melanoma, tumor-specific antigens and antigens with relatively
restricted tissue distribution have been identified.23,24
Perhaps the identification of such tumor antigens for other
malignancies may lead to the ability to immunize BMT donors or
recipients without the risk for exacerbating GVHD.
| |
Footnotes |
Submitted August 24, 1999; accepted December 6, 1999.
Supported in part by a Clinical Oncology Career Development
Award (CDA-96-61) from the American Cancer Society (C.A.M.), a Research
Project Grant (RPG-98-035-01-CIM) from the American Cancer Society
(C.A.M.), a grant from the Leukemia Research Foundation (C.A.M.), and a
Rosalie B. Hite Fellowship (L.D.A.). Support for Veterinary and
Peptide Synthesis core laboratory facilities was provided by NIH Cancer
Center Support Grant CA16672.
Reprints: Craig A. Mullen, Department of
Experimental Pediatrics, The University of Texas M. D. Anderson Cancer
Center, Box 88, Room B7.4518, 1515 Holcombe Boulevard, Houston, TX
77030; e-mail: mullen{at}mdacc.tmc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction