[Cancer Research 60, 5797-5802, October 15, 2000]
© 2000 American Association for Cancer Research
Pretransplant Tumor Antigen-specific Immunization of Allogeneic Bone Marrow Transplant Donors Enhances Graft-versus-Tumor Activity without Exacerbation of Graft-versus-Host Disease1
Larry D. Anderson, Jr.,
Shahram Mori,
Savita Mann,
Cherylyn A. Savary and
Craig A. Mullen2
Departments of Pediatrics [L. D. A., Sh. M., Sa. M., C. A. M.], Immunology [C. A. M.], and Surgical Oncology [C. A. S.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
 |
ABSTRACT
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Allogeneic bone marrow transplantation (BMT) causes a beneficial
graft-versus-tumor (GVT) immune response that is often
associated with graft-versus-host disease (GVHD). There
is substantial interest in developing therapeutic strategies that
augment GVT without GVHD. We have demonstrated recently that
immunization of BMT donors with cellular tumor vaccines leads to
curative GVT but induces unacceptable GVHD because of the presence of
recipient minor histocompatibility antigens (mHAgs) in whole-cell tumor
vaccines. This study tested the hypothesis that immunization of BMT
donors against a defined tumor-specific antigen with a vaccine not
containing recipient mHAgs would help to separate the two responses by
enhancing GVT activity without exacerbating GVHD, even when cellular
vaccines were used after BMT. Recipient strain C57BL/6 fibrosarcoma
cells engineered to express the well-characterized model tumor antigen,
influenza nucleoprotein (NP), were used in these studies. C3H.SW donors
were immunized against NP prior to BMT, and cytolytic T cells were
transferred along with bone marrow into irradiated H-2-matched,
mHAg-mismatched C57BL/6 recipients with established micrometastatic
205-NP tumors. Donor immunization led to a significant increase in GVT
activity, as measured by reduction in tumor growth and enhanced
survival. However, deaths in recipients of tumor antigen-specific
immune BMT ultimately occurred because of the growth of antigen-loss
variants; such tumor growth did not occur in animals receiving BMT from
donors treated with whole-cell vaccines. Donor immunization did not
lead to an exacerbation of GVHD, even when BMT recipients received
additional immunization after BMT with a 205-NP "whole" tumor cell
vaccine (which was shown to induce fatal GVHD when used for donor
immunization). In conclusion, immunization of allogeneic BMT donors
against a tumor-specific antigen significantly enhances GVT activity
without an associated exacerbation of GVHD.
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INTRODUCTION
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Tumor vaccine strategies require host immunocompetence for
effectiveness. However, cancer patients often have impaired immune
competence because of protracted chemotherapy and/or tumorinduced
immune suppression (1, 2, 3, 4, 5)
. In allogeneic
BMT,3
donor T lymphocytes have not been tolerized by tumor cell products or
chemotherapy and are transferred to patients with minimal residual
tumor burden. This setting may be more conducive to successful cancer
immunotherapy. Indeed, allogeneic BMT is associated with a GVT immune
response, but this response may be even more powerful if a tumor
vaccine is used to prime donor lymphocytes against tumor antigens prior
to BMT (6, 7, 8)
.
We have shown previously that allogeneic BMT donor immunization using
recipient-derived "whole" tumor cell vaccines produces
tumor-curative GVT activity but also produces unacceptable GVHD
(9)
. We have also demonstrated that this increase in GVHD
is attributable to the presence of immunodominant mHAgs on the
"whole" tumor cell vaccines (9
, 10)
. However, some
donor T cells mediating GVT activity have been shown to be tumor
specific and distinct from those mediating GVHD (6
, 9
, 11
, 12)
. In theory, if donors could be immunized against a
tumor-specific antigen without simultaneously being immunized against
mHAgs, it is conceivable that one could potentiate the GVT activity
without exacerbating GVHD.
Recently, there has been substantial progress in the molecular
identification of human tumor antigens (13, 14, 15)
. These
developments have created opportunities for selective immunization
using recombinant proteins, peptides, or even nucleic acid vaccines
without the use of "whole" cell vaccines, which induce responses to
mHAgs as well as tumor antigens (16
, 17)
.
Less is known about murine tumor antigens in well-characterized
allogeneic BMT systems. Therefore, to explore the biological effects of
immunizing allogeneic donors against molecularly defined tumor
antigens, we used an established model tumor antigen system, the NP
from the influenza A virus. The NP gene has been
cloned, and murine tumor cell lines have been modified to express this
gene (18
, 19) . Tumor cells expressing the NP
gene grow progressively without loss of antigen in immunocompetent
syngeneic mice (19)
. Furthermore, the immunodominant MHC
class I-associated NP peptide recognized by CD8+
T cells has been identified and can be used to study NP-specific
cytolytic T cell responses in vitro (20
, 21) .
It is possible that the relative potency of GVT activity could be
increased by using donor immunization to activate and expand donor T
cells capable of recognizing tumor antigens prior to BMT. An additional
possibility is that donor immunization to the defined antigen would
adversely affect GVHD because activated donor T cells secrete
proinflammatory cytokines (e.g., IFN-
), which may play a
role in the pathogenesis of GVHD. The experiments described in this
study tested the hypothesis that immunization of immunocompetent
MHC-matched donors against a model tumor-specific antigen would
increase GVT activity and extend survival of BMT recipients bearing
preexisting micrometastatic tumor without exacerbating GVHD.
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MATERIALS AND METHODS
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Animals
Female C57BL/6 (B6) mice were purchased from the National Cancer
Institute (Frederick, MD), and female C3H.SW-H2b/SnJ (C3H.SW or SW)
mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They
were used for experiments at 6–12 weeks of age. Mice were housed in
conventional rooms with food and water ad libitum and were
cared for by the Department of Veterinary Medicine and Surgery at
M. D. Anderson Cancer Center. From 2 or 3 days prior to BMT until day
14, water was acidified (pH 2.5) and supplemented with 2 g/l neomycin
sulfate (Sigma Chemical Co., St. Louis, MO).
Cell Lines
205 is a weakly immunogenic, methylcholanthrene-induced C57BL/6
fibrosarcoma cell line (22)
. This tumor is not
spontaneously metastatic but reproducibly forms multiple lung nodules
when at least 1 x 104 cells are
injected i.v. into C57BL/6 mice. 205-NP is a 205 cell line modified to
express the NP gene from the influenza A/PR/8/34 virus using
the LXSN retroviral vector (23)
. 205-B7-NP is a 205 cell
line transduced with the B7.1 immune costimulatory molecule (LXSN
vector) and also transfected with the NP gene (BP-NP-I-H
vector). The LXSN vector contains a neomycin resistance gene, and the
BP-NP-I-H vector carries a hygromycin resistance gene. Transduced cells
were selected in 300 µg/ml hygromycin and/or 1 mg/ml G418. EL4 is a
C57BL/6 lymphoma cell line (American Type Culture Collection,
Rockville, MD), and B16 is a spontaneous, weakly immunogenic C57BL/6
melanoma cell line (a gift from Dr. I. J. Fidler, M. D. Anderson
Cancer Center). Cells were grown in tissue culture using RPMI 1640
supplemented with 5% heat inactivated fetal bovine serum
(BioWhittaker, Walkersville, MD) and 2 mM
L-glutamine.
BMT Donor Immunization against NP Antigen
Influenza Vaccine.
Human influenza A virus (strain A/PR/8/34) was obtained from American
Type Culture Collection (Rockville, MD) and expanded by passage through
embryonated chicken eggs. C3H.SW BMT donors (Ag-Immune SW) were
immunized by i.p. injection of 5 x 106 influenza virus-infected C3H.SW splenocytes.
Splenocytes were infected by incubation for 1 h at 37°C with
live influenza virus on a rocker at a cell concentration of
20 x 106 cells/ml in RPMI 1640.
Donor immunization was repeated 2 weeks after the first injection.
Irradiated Tumor Cell Vaccine.
C3H.SW donors (Tumor-Immune SW) were injected s.c. in the flank with
5 x 106 50 Gy-irradiated C57BL/6
205-NP tumor cells in 0.2 ml of HBSS. The immunization was repeated 2
weeks after the first injection.
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BMT
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BMT recipients (C57BL/6) received 850 cGy TBI using a
60Co source 1 day before BMT. On the day of BMT,
2–4 x 106 bone marrow cells and
5–10 x 106 spleen cells were
injected i.v. together in a total volume of 0.2 ml of HBSS. Bone marrow
was isolated from donors (C3H.SW) by flushing each femur and tibia with
RPMI 1640. Spleen cells were isolated by macerating spleens between two
frosted glass slides, followed by lysis of erythrocytes. Mice that died
from sepsis during the first 10 days after BMT (<10%) were excluded
from these studies.
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BMT Recipient NP Antigen Immunization
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Some recipients were treated once by injection of 5 x 106 irradiated 205-NP cells on the day of
BMT. In other experiments, some recipients were injected with
5 x 106 irradiated 205-B7-NP
cells s.c. in 0.2 ml HBSS four times at weekly intervals starting the
day after BMT.
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Pulmonary Tumor Assays
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Micrometastatic lung tumors were established by injecting
C57BL/6 mice with 1 x 105 205-NP
tumor cells i.v. in 0.2 ml of HBSS 6 days prior to BMT. After death or
sacrifice, lungs 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.
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Analysis of Antigen Expression in Pulmonary Tumors
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After sacrifice, the thoracic cavity was opened in a sterile
manner to visualize the surface of the lungs. Individual lung nodules
were excised from the lung surface, macerated between two frosted glass
slides, and placed in tissue culture with complete medium. They were
stained for NP expression using the PT107 monoclonal antibody (mouse
IgG2b anti-NP; a kind gift of Dr. J. Schulman, Mount Sinai Medical
Center, New York, NY) after fixing and permeabilizing the cells with a
1:1 mixture of cold acetone and methanol. A FITC-labeled goat antimouse
secondary antibody was used for detection. Controls included unmodified
205 tumor cells labeled with both antibodies and cells incubated with
the secondary antibody only. Flow cytometric analysis was performed
using a FACScan and Lysis II software (Becton Dickinson, Moutainview,
CA).
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Evaluation of GVHD
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Recipients were weighed weekly and observed daily for signs of
GVHD (weight loss, alopecia, dermatitis, hunched posture, and death).
In some experiments, histological examination of livers for GVHD was
performed. Liver sections stained with H&E were examined for
mononuclear infiltrates in portal triads characteristic of GVHD.
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Cytotoxicity Assays
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For use as effector cells, spleen cells were cultured in
six-well plates at 1 x 106
cells/ml and 10 ml/well in RPMI 1640 supplemented with 10% FBS (Summit
Biotech, Ft. Collins, CO), 100 units/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 100
mM sodium pyruvate, 0.1 mM nonessential amino
acids, and 50 µM 2-mercaptoethanol (complete medium).
Stimulator cells were loaded with NP366 peptide (ASNENMETM) by
incubation for 1.5 h at 37°C with 35 µg/ml peptide
(synthesized by the Peptide Synthesis Core Lab of M. D. Anderson
Cancer Center). After 5 days in culture with stimulator cells, effector
cells were harvested and plated in triplicate with 5 x 103 51Cr-labeled target cells/well at E:T ratios
ranging from 200:1 to 12.5:1. 205-NP target cells were labeled by
combining 5 x 106 cells in 0.1 ml
of complete medium with 0.1 ml (
100 µCi) sterile isotonic
Na251CrO (Amersham, Arlington
Heights, IL) for 60 min at 37°C. Labeled targets were washed three
times before plating with effectors in a total volume of 0.2 ml/well in
96-well, round-bottomed plates. Plated cells were incubated for 4 h at 37°C, after which 0.1 ml of supernatant was counted in a gamma
counter (Wallac, San Francisco, CA). The percentage of lysis was
calculated as: 100 x [(experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous
cpm)]. Spontaneous release was usually <20% and always less than
30% of the maximum release.
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Statistical Analysis
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Prism 3.0 software (GraphPad Software for Scientists, Sorrento,
CA) was used for statistical evaluation of data. When more than two
groups were compared, a one-way ANOVA was performed. If
P < 0.05 overall, then the groups were
compared using a Tukey’s multiple comparison test. When only two
groups were compared, a Student’s t test was used. To
compare Kaplan-Meier survival curves, the log-rank test was used. For
regression analysis of NP+ cells, Statistica for Windows 5.1 (StatSoft,
Tulsa, OK) was used using the percentage of flow cytometry-positive
cells and the percentage of cytolysis as continuous variables.
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RESULTS
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Tumor Antigen-specific Immunity from BMT Donors Can Be Transferred
to Allogeneic BMT Recipients Treated with a Cellular Tumor Vaccine at
the Time of BMT.
Experiments were first performed to determine whether a cytolytic T
cell-mediated immune response to the model tumor antigen could be
efficiently transferred to allogeneic BMT recipients. Donors (Ag-Immune
C3H.SW) were selectively immunized against influenza virus to induce
immunity to the NP model tumor antigen, and both bone marrow and spleen
cells were transplanted into irradiated allogeneic C57BL/6 recipients.
Other experiments in a syngeneic BMT model (C57BL/6) had shown that
peritransplant recipient exposure to NP antigen was required for
efficient transfer of the cytolytic immune response (data not shown).
Some C57BL/6 BMT recipients were exposed to NP antigen on the day of
BMT by injection of 5 x 106
irradiated whole 205-NP tumor cells. Three weeks after BMT, CTL
activity against the immunodominant MHC class I-presented NP peptide
was assessed by in vitro NP peptide restimulation and
chromium release assay using NP+ tumor cells as targets. As seen in
Fig. 1
, potent CTL activity was present in recipients of immune donor cells
that had been exposed to antigen at the time of transplant. The
cytolytic activity was NP specific. C57BL/6 EL4 lymphoma cells were not
lysed unless they were preincubated with NP peptide (Fig. 1B)
. The cytolytic cells also failed to lyse B16, another
C57BL/6 tumor that does not express NP (data not shown). The cytolytic
activity in recipients of Ag-immune cells exposed to NP at the time of
BMT was not a primary immune response. In other experiments, we have
observed that recipients of cells from naive donors are incapable of
mounting a primary anti-NP cytolytic response until 5 weeks after BMT
(data not shown).
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Fig. 1. Anti-NP immunity can be efficiently transferred to
allogeneic BMT recipients treated with tumor vaccine at the time of
BMT. C3H. SW (SW) donors were either immunized against
NP with influenza virus-infected syngeneic spleen cells
(Ag-Immune SW) or not immunized (Non-Immune
SW). Two weeks later, their bone marrow cells (4 x 106) and spleen cells (10 x 106) were transplanted into C57BL/6 mice treated with 850
cGy TBI 1 day before BMT. Some Ag-Immune SW and Non-Immune SW were not
used for BMT and were not further manipulated until they were used as
controls for the cytotoxicity assay 3 weeks after BMT. One group of
recipients was exposed to NP antigen by s.c. injection of 5 x 106 irradiated 205-NP tumor cells on the day of
transplant (+ NP Ag Day 0), whereas the other group was
not exposed to NP antigen (No NP Ag). Three weeks after
BMT, splenocytes were isolated and stimulated for 5 days in
vitro with NP peptide-loaded C3H. SW splenocytes before testing
them for cytolytic activity against NP+ cells. In experiment
A, 205-NP tumor cells were used as the NP+ target.
Syngeneic B16 melanoma cells, which do not express NP, were the
negative control, and lysis of these targets was <10% (data not
shown). In experiment B, C57BL/6 EL4 cells pulsed with
MHC-binding NP peptide (NP-pulsed EL4) were the NP+
target, whereas EL4 cells not incubated with NP peptide were the
negative control. Each E:T condition was performed in triplicate using
splenocytes pooled from two or three mice/group. Bars,
SE.
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Transfer of Tumor Antigen-specific Immunity from Allogeneic BMT
Donors Decreases Tumor Burden in Recipients with Preexisting
Micrometastatic Cancer.
We have shown recently that immunization of donors with whole tumor
cell vaccines induces curative GVT activity against preexisting
micrometastatic disease (9
, 10)
. Although the experiments
described above indicate that tumor antigen-specific CTL cells could be
efficiently transplanted, this result did not prove that such
antigen-specific immunity without additional reactivity directed
against mHAgs could prove effective in vivo against
preexisting metastatic disease. To test this possibility, donors
(C3H.SW) were selectively immunized against the NP tumor antigen
(Ag-Immune) by exposure to syngeneic C3H.SW spleen cells infected with
influenza virus. Control donors were immunized with irradiated 205-NP
tumor cells (Tumor-Immune) that induce immunity to both tumor antigen
and allogeneic mHAgs. Donor bone marrow and spleen cells were
transplanted into irradiated C57BL/6 recipients with preexisting
micrometastatic 205-NP pulmonary tumors. Recipients of NP-immune
(Ag-Immune) donor BMT exhibited both prolonged survival
(P = 0.018) and a significant reduction in
pulmonary tumor nodules (P < 0.01) compared
with recipients of Non-Immune donor BMT (Fig. 2)
. None of these BMT recipients had GVHD. However, all recipients of
Ag-Immune BMT died from progressively growing lung tumors. This result
was in contrast to Tumor-Immune donor BMT, which completely eradicated
growth of pulmonary tumor nodules in most recipients but also induced
fatal GVHD in nearly all recipients.
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Fig. 2. Transfer of tumor antigen-specific immunity from
allogeneic BMT donors decreases tumor burden in recipients with
preexisting micrometastatic cancer. C3H.SW (SW) donors
were immunized with 5 x 106 influenza
virus-infected syngeneic spleen cells i.p. (Ag-Immune
SW) or 5 x 106 irradiated 205-NP
tumor cells s.c. (Tumor-Immune SW), and immunizations
were repeated 2 weeks later. Control donors were not immunized
(Non-Immune SW). Two weeks after the second vaccine,
donor bone marrow cells (4 x 106) and
spleen cells (10 x 106) were transplanted
into C57BL/6 mice with 205-NP lung micrometastases established by i.v.
injection of 1 x 105 205-NP cells 6 days
prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT.
**, recipients of either Ag-Immune or Tumor-Immune donor BMT
exhibited increased survival (P = 0.0180)
compared with recipients of Non-Immune donor BMT. ***, recipients
of Ag-Immune or Tumor-Immune donor BMT had a significant reduction in
pulmonary tumor nodules (P < 0.01)
compared with recipients of Non-Immune donor BMT. Recipients of
Ag-Immune BMT died from progressively growing lung tumors. Recipients
of Tumor-Immune BMT often had complete prevention of pulmonary tumor
nodule growth but developed fatal GVHD. This experiment is
representative of three independent experiments in which Ag-Immune BMT
caused significant but incomplete protection against 205-NP lung nodule
growth. In this experiment, each group had a sample size of 4. Bars, SE.
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Lung Metastases Growing in Recipients of Tumor Antigen-immune Donor
BMT Are Antigen-Loss Variants.
Because most but not all lung nodules were prevented from growing in
recipients of Ag-immune BMT, we hypothesized that the reason for
progressive tumor growth was loss or down-regulation of NP antigen
expression. Although Tumor-Immune donor lymphocytes from donors treated
with whole tumor cell vaccines can recognize both the NP antigen and
C57BL/6 minor histocompatibility antigens on 205-NP cells (9
, 10)
, the only antigen on 205-NP tumor cells recognized by
influenza virus-immune donor lymphocytes is the NP antigen. Therefore,
loss of NP expression could cause evasion from the immune attack. To
test this hypothesis, progressively growing lung nodules from
recipients of Ag-Immune BMT and control nodules from recipients of
NonImmune donor BMT were randomly selected and removed 1 month
after BMT. As seen in Fig. 3
, all tumors reisolated from Ag-Immune BMT recipients had complete or
nearly complete loss of expression of the NP antigen as assessed by
flow cytometry. In contrast, most of the lung nodules reisolated from
Non-Immune BMT recipients, where there was no transplanted selective
pressure for antigen loss, expressed NP at substantially higher levels.
(Several tumor reisolates from nonimmune recipients had reduced levels
of NP expression; this may have resulted from primary anti-NP immune
responses in the recovering immune system.) The observed reduction in
NP expression was not the result of short-term growth in
vitro in the absence of neomycin selection. 205-NP was grown in
continuous culture for 28 days in the absence of G418; at the end of
this time, 81% of the cells were positive by flow cytometry for NP
expression compared with 91% at the beginning of the month in culture
(data not shown). Cells that do not express NP as measured by flow
cytometry are not sensitive to NP-specific CTLs. In an independent
experiment, 205-NP tumors were reisolated from naive or influenza
immune C57BL/6 mice and analyzed for NP expression and sensitivity to
NP-specific CTLs. The positive control 205-NP tumor was 78% positive
for NP by flow, and the percentage of lysis was 36% at an E:T ratio of
130; the negative control 205 had undetectable antigen (2%) and 8%
lysis. NP expression in tumors from influenza immune hosts
(n = 2) had undetectable NP expression (1%),
and lysis ranged from 0–5%. In contrast, in 205-NP resiolates from
nonimmune hosts (n = 5), NP expression ranged
from 20 to 53%, and lysis ranged from 7 to 39%. Regression analysis
demonstrated a statistically significant relationship
(P < 0.016) between NP expression by flow
cytometry and sensitivity to anti-NP CTLs.
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Fig. 3. 205-NP Lung nodules growing in recipients of Ag-Immune
donor BMT exhibit loss of NP antigen expression. C3H.SW
(SW) donors were immunized with 5 x 106 influenza virus-infected syngeneic spleen cells i.p.
(Ag-Immune SW) or were not immunized (Non-Immune
SW), and immunizations were repeated 2 weeks later. Two weeks
after the second vaccine, donor bone marrow cells (4 x 106) and spleen cells (10 x 106) were transplanted into C57BL/6 mice with lung
micrometastases established by i.v. injection of 1 x 105 205-NP tumor cells (A)
6 days prior to BMT. Recipients underwent 850 cGy TBI 1 day before BMT.
One month after BMT, individual lung nodules were isolated from
recipients, macerated, and placed in tissue culture. Seven lung nodule
cultures were successfully grown from five recipients of Ag-Immune BMT,
and 10 cultures were grown from four recipients of Non-Immune BMT.
After culturing the cells for 2 weeks, they were stained with an
unlabeled NP-specific antibody and a FITC-conjugated secondary antibody
for analysis by flow cytometry. Background fluorescence was determined
by staining of unmodified 205 tumor cells and also by staining of lung
nodule cells with secondary antibody alone. Fluorescence histograms
representative of the lung nodule cell cultures from Non-Immune
recipients (B) and Ag-Immune recipients
(C) are shown. Using the markers shown, the
percentages of cells expressing NP were determined and plotted for each
culture (D).
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Transfer of Tumor Antigen-specific Immunity from Allogeneic
BMT Donors Does Not Exacerbate GVHD.
Recipients of Ag-Immune allogeneic donor BMT in these experiments
did not exhibit signs of GVHD. Instead, they died from progressive
tumor growth by day 40. However, because in this BMT model GVHD may not
become evident until after that time, early tumor growth may have
prevented detection of GVHD. Active immune responses after BMT
(e.g., to viral infections) have been associated with
exacerbations of GVHD (24)
; therefore, it remained a
possibility that transfer of an active T-cell response to
non-alloantigen could exacerbate GVHD. Additional experiments were
conducted to determine the effect of tumor-specific immunity on the
development of GVHD in recipients without the confounding factor
of progressively growing tumor. C3H.SW donors were immunized with
either influenza virus (Ag-Immune SW) or irradiated 205-NP tumor cells
(Tumor-Immune SW). Control donors were not immunized (Non-Immune SW).
Donor bone marrow and spleen cells were transplanted into lethally
irradiated C57BL/6 mice without tumors. Although Tumor-Immune
(allo-immune) control donor lymphocytes induced fatal GVHD, Ag-Immune
donor cells did not induce signs of GVHD in any of the recipients
(n = 10; Fig. 4A
). The capacity of NP Ag-Immune donor cells to cause GVHD
was further tested in a second experiment in which some recipients were
actively immunized with irradiated 205-B7-NP tumor cells that express
both NP and C57BL/6 mHAgs. (Tumor cells coexpressing NP, mHAgs, and the
B7.1 costimulatory molecule were chosen to help avoid the issue of a
deficiency of antigen-presenting cells in the peritransplant period.)
In this setting, it is conceivable that the anti-NP response could
induce cytokines that cross-prime a response to the mHAgs and worsen
GVHD. However, even when recipients were exposed to NP antigen on the
same cells with mHAgs after BMT, there was no increase in mortality
(Fig. 4B)
or other measures of GVHD (death, weight loss, fur
loss, or dermatitis) compared with recipients of nonimmune donor BMT.
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Fig. 4. Recipients of tumor antigen-immune BMT do not have an
increased incidence of GVHD, even when exposed to whole tumor cell
vaccines after BMT. C3H.SW (SW) donors were immunized
with 5 x 106 influenza virus-infected
syngeneic spleen cells i.p. (Ag-Immune SW) or
5 x 106 irradiated 205-NP tumor cells s.c.
(Tumor-Immune SW), and immunizations were repeated 2
weeks later. Control donors were not immunized (Non-Immune
SW). Two weeks after the second vaccine, donor bone marrow
cells (4 x 106) and spleen cells
(10 x 106) were transplanted into C57BL/6
mice given 850 cGy TBI 1 day before BMT. Recipients were not challenged
with live tumor cells. A, whereas Tumor-Immune
(allo-immune) control donor lymphocytes induced fatal GVHD in all
recipients, NP Ag-Immune donor cells did not
(n = 10). B, in an
independent experiment, some recipients were exposed to NP antigen by
s.c. injection of 5 x 106 irradiated
205-B7-NP tumor cells on days 0, 7, 14, and 21 after BMT to augment the
ongoing immune response. Even in this case, there was no increase in
the incidence of GVHD compared with recipients of Non-Immune donor BMT
(n = 5).
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DISCUSSION
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This work demonstrated that immunization of MHC-matched allogeneic
donors against a tumor-specific antigen with vaccines that do not
contain recipient mHAgs can enhance GVT activity and prolong survival
of BMT recipients with preexisting micrometastatic disease without
exacerbation of GVHD. This was also the case in experiments in which
BMT recipients received additional post-BMT vaccination with whole
tumor cell vaccines that contain recipient mHAgs. This is in contrast
to our observations published recently that immunization of BMT donors
with whole tumor cell vaccines, although highly effective in enhancing
GVT activity, leads to severe GVHD (9)
. Our results are
compatible with reports by others in which use of tumor
antigen-specific vaccines in conjunction with autologous BMT leads to
enhanced antitumor immune activity (25)
.
Although donor immunization produced a significant increase in GVT
activity that extended survival and reduced the number of pulmonary
tumor nodules growing in recipients (Fig. 2)
, the GVT activity was not
curative. Flow cytometry demonstrated that the lung metastases that
emerged in antigen-immune recipients had either down-regulated or lost
their expression of the NP gene (Fig. 3)
. Although BMT from
nonimmune donors did not eliminate NP expression, NP-immune donor cells
most likely caused a selective growth advantage for antigen-loss
variants. NP antigen expression in the tumor challenge population was
heterogeneous (Fig. 3A)
, reflecting the antigenic
heterogeneity of clinical tumors. Antigen-loss variants recovered
in vivo could be outgrowths of low- or nonexpressing cells
injected, or they could be antigen-loss variants generated in
vivo (26
, 27)
. The observation of antigen-loss
variants in this model indicates that effective vaccines will need to
be directed against more than one tumor-specific antigen.
Recipients of antigen-immune BMT did not develop GVHD, even when
exposed to NP antigen and allogeneic mHAgs multiple times by weekly
s.c. injection of irradiated 205-B7-NP tumor cell vaccines. In no
experiment was there an increase in the incidence or mortality of GVHD
compared with recipients of nonimmune donor cells (Fig. 4)
. Although
GVHD can be exacerbated in an antigen-nonspecific manner by the
cytokine release induced by BMT preparative regimens (28)
or viral infections (24)
, these experiments suggest that
the proinflammatory activity of the induced T cells did not contribute
in an antigen-nonspecific manner to GHVD.
Despite the problem of antigen-loss variant tumor growth using the
tumor antigen-specific vaccination strategy, these results suggest
potentially useful strategies for BMT patients. One approach would be
the simultaneous immunization of donors with several tumor antigens or
antigens with multiple epitopes to make variant selection in
vivo more difficult. Transfer of a polyclonal T-cell response to
tetanus toxoid from allogeneic donors boosted with tetanus vaccines
prior to cell harvest has been demonstrated in clinical transplantation
(29)
. For cancer, this approach will require
identification of multiple antigens in the same tumor. Although
tumor-specific antigens for many malignancies have not yet been
identified, several have been molecularly characterized for melanoma
and other cancers (16
, 17 , 30, 31, 32)
. In addition, tumor
specific idiotypes from myelomas can be molecularly characterized and
treated as tumor antigens; the clinical feasibility of using such
idiotypes as vaccines in conjunction with autologous transplantation
has been established (33, 34, 35)
. An alternate and possibly
more feasible approach is immunization of donors with one defined tumor
antigen, followed by post-BMT vaccination of recipients with a
"whole" cell tumor vaccine that may contain other target antigens
that have not been molecularly identified. Our recent work
(10)
and data here show that post-BMT immunization of
recipients with "whole" tumor cell vaccines does not exacerbate
GVHD; therefore, such a combined strategy would potentially allow
multiple tumor antigens to stimulate a more powerful GVT immune
response with reduced probability of provoking GVHD against mHAgs. Our
current work explores such strategies.
|
FOOTNOTES
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|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by Clinical
Oncology Career Development Award CDA-96-61 from the American Cancer
Society (to C. A. M.), Research Project Grant RPG-98-035-01-CIM from
the American Cancer Society (to C. A. M.), a grant from the Leukemia
Research Foundation (to C. A. M.), and a Rosalie B. Hite Fellowship
(to L. D. A.). Support for the Peptide Core Lab and veterinary
services was provided by NIH Cancer Center Core Grant CA 16672. 
2 To whom requests for reprints should be
addressed, at Department of Pediatrics, The University of Texas M. D.
Anderson Cancer Center, Box 88, Room B7.4518, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone: (713) 792-3314; Fax: (713) 794-4373;
E-mail: mullen{at}mdacc.tmc.edu
3 The abbreviations used are: BMT, bone marrow
transplantation; GVT, graft-versus-tumor; GVHD,
graft-versus-host disease; NP, influenza nucleoprotein;
mHAg, minor histocompatibility antigen; SW, C3H.SW; B6, C57BL/6; B7,
B7.1; TBI, total body irradiation.
Received 2/ 8/00.
Accepted 8/17/00.
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ABSTRACT
INTRODUCTION
