Active immunization of murine allogeneic bone marrow transplant

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1996 87: 3053-3060
Active immunization of murine allogeneic bone marrow transplant
donors with B-cell tumor-derived idiotype: a strategy for enhancing
the specific antitumor effect of marrow grafts
LW Kwak, R Pennington and DL Longo
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Active Immunization of Murine Allogeneic Bone Marrow Transplant Donors
With B-Cell Tumor-Derived Idiotype: A Strategy for Enhancing the Specific
Antitumor Effect of Marrow Grafts
By Larry W. Kwak, Robin Pennington, and Dan L. Longo
Persistence of the underlyingmalignancy remainsthe major
obstacle limiting thesuccess of high-dose chemoradiotherapy with allogeneic bone marrow transplantation(BMT) for
lymphomas and multiplemyeloma. We used the C3H 38C13
murine B-cell lymphoma, which expresses and secretes clonally derived Ig, the idiotype of whichcan serve as a tumorspecific antigen, t o test the principle of transfer of tumor
idiotype-specific immunity with BM. BALBlc marrow donors
were twice immunized with 38Cl3-derived lg, or with an
isotype-matched control Ig, conjugated t o keyhole limpet
hemocyanin. Lethally irradiated C3H recipients reconstituted with marrow from idiotypeimmune, but not nonspecifically immune, donors demonstrated protection against subsequent lethaltumor
challenge. The immunoprotective
effect of immune allogeneic marrow was abrogated by Tcell depletionof the marrow graft
before infusion. Low levels
of serumanti-idiotypic antibodyremained unalteredin recipients of T-cell-depleted immune marrow, consistent with a
primary role for T-cell immunity in the cellular mechanism
of thisphenomenon. A modest therapeutic effect
of immune
allogeneic marrow wasobserved against 10 day, 1cm established subcutaneous tumors, but only in combination with
a booster immunization of the recipient post-BMT. These
results provide therationale for a novel strategyfor enhancing the
specific antitumor effect of allogeneic marrow grafts.
This is a US government work. There are no restrictions on
its use.
H
immunity from syngeneic marrow donors, actively immunized with Ova,to lethallyirradiated
BMT recipients.”
These studies demonstrated theprotective and therapeutic
antitumor effect of immune syngeneic marrow. Based on the
success of this approach, we have nowexplored this strategy
with tumor idiotypein the model system of 38C13, a murine
B-cell lymphoma that both expresses and secretes idiotypic
Ig, and extended these studies to the immunization of fully
major histocompatibility complex (MHC)-mismatched donors.
IGH-DOSE chemoradiotherapy with autologous or allogeneicbonemarrow
transplantation (BMT)has
shown considerable promise as therapy for relapsed or refractory lymphomas and multiple m y e l ~ m a . ”The
~ immunologic concept underlying allogeneic BMT, in particular, is
attractive, because it provides an opportunity for exploiting
a potential antitumor effect of the marrow graft as adoptive
immunotherapy against a residual tumor burdennot completely eradicated by the conditioning regimen. For example,
there is growingevidence that a biologicallysignificant
graft-versus-leukemia (GVL) reaction,probablydirected
against minor histocompatibility determinants that differ between donor and recipient, may occur against some hematologic malignan~ies.’,~-~
Adoptive transfer of specific humoral, and to a lesser extent, cellular immunity to viral or other recall antigens, such
as hepatitis B surface antigen, keyhole limpet hemocyanin
(KLH), varicella zoster virus (VZV), and tuberculin purified
protein derivative (PPD), from immune donors to BMT recipients has been achieved previously in humans.’”‘ However, the transfer of tumor antigen-specific immunity from
BMT donor to recipient has not been studied in human patients, in large part because of the lack of well-defined, tumor-specific antigens on most human tumors.
The idiotype of the Ig expressed by malignancies of mature B cells, such as B-cell lymphomas and myelomas, can
serve as a unique tumor-specific antigen forvaccine development. Sirisinha and Eisen15 and Lynch et all‘ were the first
to observe that active immunization with tumor-derived Ig
induced anti-idiotypic immunity in syngeneic mice andproduced idiotype-specific resistance to malignantB-cell tumor
growth, an observation that has since beenreproducedin
several other B-cell tumor m0de1s.l”’~ Furthermore, a pilot
study by one of us in nine patients with B-cell lymphoma
showed that humoral and cellular idiotype-specific immunologic responses could be induced by active immunization
with autologous tumor-derived idiotypic Ig (Id), conjugated
to KLH, and administered with an immunologic adjuvant.”
We have previously used the model system of EG.7-Ova
to explore the strategy of transfer of tumor antigen-specific
Blood, Vol 87,No 7 (April l),
1996:
pp 3053-3060
MATERIALS AND METHODS
Mice and tumor. C3H/HeN MTV- and BALB/cANNCR female
mice 6 to 12 weeks of age were obtained from the AnimalProduction
Area (National Cancer Institute-Frederick Cancer Research and Development Center [NCI-FCRDC], Frederick, MD). The carcinogeninduced %C13 B-cell lymphoma has been previously described.”
The 38C13 tumor secretes and expresses IgM(K) on the cell surface.
Inoculation of 10’ tumor cells intraperitoneally (ip) into syngeneic
C3H/HeN mice results in progressive tumor growth andmedian
survival times of approximately 20 days. Tumor cells from a common frozen stock were passaged in vitro3 days before use. Injections
From the Biological Response Mod$ers Program, National Cancer Institute-Frederick Cancer Research and Development Center,
Frederick, MD; andBiologicalCorcinogenesis
and Development
Program, SAIC Frederick, Frederick, MD.
Submitted July 10, 1995; accepted November 20, 1995.
The content of this publication does not necessurily
reflect the
views or policies of the Department cf Health and Human Services,
nor does mentionof trade names, commercial products,or organization imply endorsement by the US government.
Address reprint requests to
L a r p W. Kwak, MD. PhD, Division
of Clinical Science. Rldg 567, Ronrn 205, NCI-FCRDC, Frederick,
MD 21702-1201.
The publication costs of this urticle were defrayedin part by page
churgepayment. This article must thereforebeherebymarked
“advertisement” in clccordance with 18 U.S.C. section 1734 solely to
indicate this fact.
This is a US government work. There are no restrictions on its use.
0006-4971/96/N707-0015$0.00/0
3053
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3054
KWAK, PENNINGTON, AND LONG0
for each experiment were made from the same suspension of tumor
37°C for 45 minutes. Cells were then washed two times before use.
cells.
Aliquots of BM cells were stained with fluorescein isothiocyanate
Vaccine preparation and administration. Idiotypic IgM (38C 13(FITC)-labeled anti-CD4 (YTS 191.1, rat IgG2b) and -CD8 (YTS
Id) was rescued by somatic cell hybridization as described," and
169.4. rdt IgGZb) monoclonal antibodies (MoAbs; both from Caltag.
38C13-Id and a control IgM(K) were purified from ascites and couSouthSanFrancisco,CA)aftertreatment
with C'alone or antipled to KLH using 0.1% glutaraldehyde. Donor
or recipient mice
Thyl.2 plusC'andanalyzed
by flow cytometry to monitorthe
were immunized subcutaneously (sc) with 50 pg 38C13-Id-KLH or
efficiency of TCD. These studies generally showed a
reduction of
control IgM-KLH emulsified in an adjuvant formulation consisting
T cells from approximately 5% to 10% of lymphocytes to less than
of 0.2% Tween80,2.5%PluronicL121,5%squalane,and
100
I%.
pglmL N-acetylglucosaminyl-(~1-4)-N-acetylmuramyl-L-ala1~yi-D- Analyses of lymphoid chimerism. Chimerism of splenocytes obisoglutamine (GMDP). GMDP was kindly provided by P. Cooper
tained post-BMT was analyzedby single-color flow cytometry using
(C-C Biotech Corp. Poway, CA) as a gift.
FITC-labeled anti-H-2d (SFI-l. 1, mouse IgG2a) or anti-H-2' (36-7Serum anti-idiofypic antibody. Mouse serum was serially diluted
5 , mouse IgG2a) specific MoAb (both from Pharmingen,San Diego,
over microtiter plates coated with 38C13-Id. Binding of antibodies
CA).Briefly,splenocytesweretreated
with red bloodcellACK
lysing buffer (Sigma, St Louis, MO), washed, and preincubated
with
intheserumto38C13-Idwasdetected
by goat-antimouseIgG2.4G2 antimouse Fcy receptor MoAb (rat IgG2b, American Type
horseradish peroxidase antibodies, which hadbeen absorbed against
CultureCollection[ATCC]HB197;ATCC,Rockville,
MD) to
38C13-Id. Serum anti-idiotypic antibody levels were quantitated by
of theprimaryantibodies.Cellswere
blocknonspecificbinding
comparing sera titration curves with a standard curve obtained with
incubatedwithprimaryantibodiesfor
IS to30minutesat
4°C.
a known concentration of a mixture of
purified monoclonal antiwashed. andfixed in I % paraformaldehyde, All results were ohtained
idiotypic antibodies. Antibody levels are expressed
in micrograms
using an EPICS Profile flow cytometer (Coulter Corp. Hialeah, FL).
SD of
per milliliter of serum for individual mice or as the mean
Forwardandsidescattersettings
were gatedtoexcludeany
refour to five individual mice.In each enzyme-linked immunosorbent
assay (ELISA), sera obtained either from mice immunized with con- maining redblood cells and debris. Ten thousand cells were analyzed
for each determination.
trol 1gM-KLH or from their lethally irradiated BM recipients were
Anu1y.si.s ofdura. Mice were checked daily to determine the date
included as negative controls. Such sera never showed any titratable
ofdeath.Tumor-bearingmice
with sc tumorswereinspected by
binding activity on 38C13-Id.
palpation,andtumordiametersweremeasuredonopposingaxes
Allogeneic BMT. Donor BALBk mice, which had been immuevery other day. Statistical comparisons of survival were performed
nized specifically with 38C13-Id-KLH, or nonspecifically with conInc. Los
trol IgM-KLH, in adjuvant were killed, and BM was collected from using BMDPILsoftware(BMDPStatisticalSoftware,
Angeles. CA) to generate nonparametric Mantel-Cox logrankP valboth left and right femurs and tibiae. The BM was filtered through
ues. Mice surviving greater than 90 days after tumor challenge were
a nylon mesh and washed three times before use. Viable cells were
killed and were reported as long-term survivors.
counted by trypan blue exclusion. Naive recipient C3H mice were
lethallyirradiatedwith 950 rad total body irradiation(TBI) in a
RESULTS
Mark I '"CS source irradiator (J.L. Shepherd and Associates, San
Fernando,CA).Irradiatedrecipientswereinjectedintravenously
Immune syngeneic BM dose response. Before studying
(W) with 20 X IOh BM cells in 0.5 mL RPM1 1640 medium in the
the
immunization
of MHC-mismatched donor-recipient
lateral tail vein.
pairs,
we
first
performed
several experiments in syngeneic
For experiments in tumor-bearing recipients, C3H mice were first
mice to determine the optimal dose of immune marrow for
injected with lo4 38C13 tumor cells sc in the right flank and monitransfer of tumor idiotype-specificimmunity.
C3H mice
tored for tumor growth by inspection, palpation, and measurement.
serving asmarrow
donors were immunized with either
Ten days after tumor inoculation,
all recipients received 950 rads
38C13-Id-KLH or control IgM-KLH in adjuvant, and then
TB1 andcyclophosphamide(CY)75
m g k g bodyweightip.One
daylater,conditionedrecipientswereinjected
IV with20 X 10"
again with the respective immunogen 2 weeks later. One to
BM cells.Sometumor-bearingrecipientsreceivedimmunizations
2 weeks after the booster immunization, graded numbers of
as described above on the day of marrow infusion.
BM cells pooled from these idiotype immuneor nonspecifiBM recipients were maintained in sterile microisolator cage units
cally immune donors wereused to reconstitute lethally irradiin a laminarflow hood for approximately3 weeks, duringwhich time
ated (950 rad TBI) normal C3H recipients. After 3 weeks'
they received sterile food and acidified water. Overall postoperative
recuperation, these recipients were challenged ip with a lemortality was approximately 10% (maximum 23%
in a single experithal dose of lo3 38C13 tumor cells and monitored for surment), and all surviving mice were clinically healthy without evivival. Recipients reconstituted with 20 X IO" immune BM
dence of graft-versus-host disease (GVHD; weight loss, alopecia,
diarrhea, hunching,or skin erythema). In addition, random mice were cells demonstrateda median survival time (MST) of 29 days,
compared withrecipientsreconstitutedwithan
equivalent
killed and submitted for histopathological examination performed
by
the PathologykIistotechnologyLaboratory, NCI-FCRDC. No histonumber of nonspecifically immune marrow cells (MST 25
logic evidence of GVHD was observed in any recipients. The abdays), as well as 24% long-term survival ( P = .OOOl). Howsence of lethal GVHD in the BALBlc "* C3H strain combination
ever, lower doses of immune marrow (5 X IOb and 10 X
has been observed by others.14
IO' cells) failed to confer any significant survival benefit on
Ex vivo T-celldepletion(TCD) of BM. TCD of BALBk BM
recipients, compared with nonspecifically immune marrow
cells was performed according to methods previously described by
(data not shown). Thus, for subsequent experiments, a marothers.'5,26 BM cells were adjusted to a concentration of
I O 7 cells/
row inoculum of 20 X 10' cells was routinely used.
mL in RPMI-1640 and incubated with 50 pg/mL anti-Thyl.2 for 30
Comparison of normal syngeneic and allogeneic BM and
minutes at 4°C (30-H-12, rat IgG2b; Hazelton Biotechnologies CO,
protective
effect o
f immune allogeneic marrow. We next
Vienna, VA). Cells were then pelleted, resuspended at lo7 cells/mL
performed experiments designed toevaluatethe effectof
in a 1 : 10 dilution of Low Tox-M rabbit complement (C'; Accurate
normal allogeneic, compared with syngeneic, BM on tumor
ChemicalandScientificCorp,Westbury,
NY). andincubatedat
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3055
DONOR VACCINATION WITH TUMOR IDIOTYPE
'oo[Th
A
5 x 10' challenge
80
60
b-0
n
2o
1
bp
p = 0.0001
W
2
B
>
CK
10' challenge
loo
6o
40
t
l
0
10
20
30
40
50
DAYS
Fig 1. Survival of lethally irradiated C3H recipients of normal syngeneic or allogeneic BALBlc BM after ip challenge with either (A) 5
x IO3or (B)10'38C13 tumor cells 3 weeks after BMT in two separate
experiments.
resistance in the strain combination selected. Lethally irradiated C3H recipients received marrow from BALBlc or syngeneic C3H donors at the marrow inoculum dose established
above. After 3 weeks' recuperation, both groups of mice
were challenged ip with either 5 X lo3 (Fig LA) or lo4 (Fig
1B) tumor cells and monitored for survival. A significant
resistance producing effect of BALBlc BM, compared with
syngeneic marrow, was observed at both tumor challenge
doses. Because no long-term survivors were observed in
recipients of BALBlc marrow at the higher tumor challenge
dose, the dose of lo4 was selected for the experiments of
primary interest testing immune allogeneic marrow.
BALBlc mice were twice immunized with either 38C13Id-KLH or control IgM-KLH in adjuvant as marrow donors.
Lethally irradiated C3H mice received marrow pooled from
these idiotype-immune or nonspecifically immune donors.
Three weeks later these recipients were challenged ip with
lo4 tumor cells and monitored for survival. The results of a
representative experiment are shown in Fig 2. This tumor
challenge dose was uniformally lethal in recipients of nonspecifically immune BALBlc marrow. However, recipients
of immune BALBlc marrow demonstrated significantly prolonged survival, with 50% of the mice surviving the tumor
challenge ( P = .016).
Recipient anti-idiotypic antibody and the effect of marrow
TCD. In an effort to explore the cellular mechanism of the
observed protective effect of immune allogeneic marrow, we
first determined whether the anti-idiotypic humoral response
elicited in donors was transferred to lethally irradiated recipients. Normal BALBk donor mice showed vigorous antiidiotypic antibody responses after two immunizations at the
time of marrow harvest (mean serum antibody level > 500
pglmL, data not shown). Serum samples were obtained from
the recipients of idiotype-immune or nonspecifically immune
marrowin the experiment inFig 2 atthetime
of tumor
challenge and assayed for anti-idiotypic antibody. Serum
anti-idiotypic antibody titers of five individual randomly selected recipients from each group and from five recipients
of immune BM from a second identical experiment are
shown in Fig 3A. In contrast to donor levels, extremely
lowbut detectable levels of anti-idiotypic antibody were
observed in each C3H recipient of immune marrow (mean
antibody levels 0.3 5 0.2 and 1.7 ? 3.0 pglmL, experiments
1 and 2, respectively). The consistent lack of binding to
38C13-Id by control sera from recipients of nonspecifically
immune marrow (complete lack of absorbance of undiluted
serum) showed the sensitivity of this ELISA.
We then tested the effect of in vitro marrow TCD on the
protective effect of immune marrow. Pooled BM from
BALBlc donors twice immunized with either 38C13-IdKLH or control IgM-KLH in adjuvant was subjected to treatment witheither anti-Thy 1.2 plus rabbit C' or with C' alone.
Lethally irradiated C3H recipients were then randomly allocated to receive one of the four resulting pools of marrow.
After 3 weeks, these recipients were challenged ip with 5 X
lo3 tumor cells and monitored for survival. The protective
effect of immune allogeneic marrow was again evident, as
demonstrated by comparison of the survival of recipients of
immune with recipients of nonimmune marrow treated with
C' alone ( P = .001, 83% long-term survival in recipients of
immune marrow at this tumor challenge dose). Treatment
100
lO4 challenge
t
p = 0.016
0
A
immune BM (nZl2)
Non-immune BM (n-6)
l
0
10
20
30
40
,
I
50
DAY S
Fig 2. Survival of lethally irradiated C3H recipients of immune or
nonspecifically immune BALB/c marrow after ip challenge with 10'
38C13 tumor cells three weeks after BMT. Immunization of donors
was performd as described in Materials and Methods. The experiment shown is representative of three such experiments.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
3056
KWAK,PENNINGTON, AND LONG0
A
7.7
I.
0
0.5
-
0
0.4
-
0
0.3
-
0
0.2 0.1
00
-
-
"
0.0
0
000
Immune BM
( e w . 1)
Immune BM
(exp.2)
2.9
4.1
L
v
Non-immune
BM
B
I.
0.5
-
0
0
0
0.4 -
0.3 0.2
-
0.1
-
00
0.0 -2
Non-Immune BM
c' alone
000
v
Immune BM
C' alone
Immune BM
anti-Thy
+ C'
Fig 3. (A) Individual serum anti-idiotypic antibody levels of five
randomly selected recipients of immune BALBlc marrow from t w o
separate experiments each. Lethally irradiated C3H recipients were
reconstituted with immune BALBlc marrow as described in Fig 2
and were bled 3 weeks after BMT at the time of tumor challenge.
Recipients of nonspecifically
immune BALBlc marrow demonstrated
no absorbanceto 38C13-Id coatedmicrotiter plates in the ELSA described in the Materials and Methods. (B1 Individual serum anti-idiotypic antibody levels of five randomly selected C3H recipients of
BALBlc BM from the experiment in Fig 4. Mice were bled 3 weeks
after BMT a t the time of tumor challenge.
recipients of otherwise identically treated normal BALB/c
BM. Spleens from two tothree individual recipients of antiThy 1.2 plus C' or C' alone-treated BM were stained with
anti-H-2"- or -H-2k-specific MoAb and analyzed by flow
cytometry at two timepoints. On day 21 post-BMT, the hematopoietic cells of recipients of BALB/c marrow treated
with C' alone werenearly all of donor origin with 96.7% and
3.0% H-2d- and H-2'-positive lymphocytes, respectively.
Similarly, at this timepoint the majority of splenic lymphocytes from recipients of anti-Thy 1.2 plus C' treated BALB/
c marrow were of donor H-2d phenotype (80.7%), although
a small fraction of H-2'-staining lymphocytes was still detectable (16.8%). At the latertimepoint(day 49). cellsof
recipients of anti-Thy l .2 plus C'-treated marrow were fully
of donor origin, with 94.7% and 5.5% of lymphocytes staining with H-2d and H-2k MoAb, respectively.
Serum samples from recipients of BALB/c marrow in the
experiment in Fig 4 were also obtained at the time of tumor
challenge andassayed for anti-idiotypic antibody. Consistent
with the prior results in Fig 3A, low but detectable antibody
levels were demonstratedin each of the individual recipients
of immune marrowtreated with C' alone(meanantibody
level 0.9 -t 1.1 pg/mL), compared with control recipients of
nonimmune marrow (Fig 3B). Of primary interest, low levels
of antibody of comparable magnitude were also shown in
recipients of TCD immune marrow (mean antibody level 0.9
f 1.6 pg/mL). Thus, the anti-idiotypic antibody alone is not
responsible for the antitumor effects seen after transfer of
idiotype-immune allogeneic marrow.
Therapeutic effect of allogeneic immune marrow against
established tumor. To more closely model the clinical status of a human patient bearing an established tumor burden,
we performed a series of
experiments designed to test the
potency of this approach against well-established tumors in
mice. The38C13lymphomagrows
rapidly, and l0 days
after the sc implantation of IO4 tumor cells in naive syngeneic mice, 1 0 0 % of mice develop macroscopic, l-cm tumor
100
5 x 10' choileogc
80
of immunemarrowwith anti-Thy 1.2 plusC' resultedin
abrogation of this immunoprotective effect (MST 27 days,
P < .OOl compared with C' alone-treated marrow; P = . l 8
compared with nonimmune C'-treated
marrow). A difference
in survival between anti-Thy 1.2 plus C' and C'alone-treated
nonimmune marrow recipients was also observed, presumably due to the depletionof mature alloreactive T-cells from
the BALB/c marrow graft (MST 23 and 29 days,
respectively; P < .001). A slight but significant difference in survival was also apparentbetween the two groupsof recipients
of TCD marrow ( P = ,002). Comparable results were observed in a second experiment after a tumor challenge dose
of lo4 cells (data not shown).
To establish that the loss of immunoprotection produced
by TCD of immune marrow was not due to the failure of
allogeneic marrow to engraft, weperformed separate analyses of chimerism in parallel in nontumor challenged C3H
A
x
v
1
BM. C' d o n s ("-12)
v Immune BM. C' alone ("-12)
v Non-immune EM. onti-Thy + C' (n-14)
8 Non-immune
60
Q
0 Immune BM, anti-Thy
2
2
+
C' ("-12)
40
3
m
20
v
"I
0.001
0
0
10
20
30
40
50
60
70
DAY S
Fig 4. Marrow TCD abrogates the protective effect of immune
marrow. Pooled marrow from specifically or nonspecifically immunized BALBlc donors was subjected to treatment with either antiThy 1.2plus C' or with C' alone beforeinfusion into Mhallyirradiated
C3H recipients. Three weeksafter BMT, all four groups of recipients
were challenged ip with 5 x 10' 38C13 tumor cells. The experiment
shown is representative of two such experiments.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
3057
DONOR VACCINATION WITH TUMOR IDIOTYPE
masses. Preliminary experiments in IO-day tumor-bearing
mice showed that 950 radTB1 was largely ineffective at
reducing tumor size or prolonging survival (data not shown).
The addition of CY ip at doses ranging from 25 to 75 mg/
kg to TB1 followed by rescue with normal BALBk marrow
was effective at reducing tumor masses and prolonging survival without effecting cures (data not shown). Based on
these results, a conditioning regimen of 950 rad TB1 plus
CY 75 mgkg was used for experiments in tumor-bearing
recipients.
Ten-day tumor-bearing C3H recipients were treated with
TB1 plus CY and received pooled marrow from BALB/c
donors that had been twice immunized with either 38C13Id-KLH or control IgM-KLH in adjuvant. Initial experiments
failed to demonstrate a survival advantage for recipients of
idiotype-immune, compared with nonspecifically immune
marrow. In an effort to more rapidly expand the adoptively
transferred T cells in their adoptive hosts, recipients from
each group were given a booster immunization with either
38C13-Id-KLH or control IgM-Id-KLH in adjuvant on the
day of marrow infusion. Data pooled from two such experiments are shown in Fig 5. Recipient mice injected with lo4
tumor cells S.C.in the right flank developed progressively
growing visible tumor masses, and by day 10 all of the mice
had l-cm tumors (100% tumor incidence). The administration of TB1 plus CY produced temporary remissions in all
recipients (1.2% tumor incidence, day 16), butwithin 10
days tumors started to recur. Ultimately, all but one of the
control mice that had received nonimmune BALBk marrow
and a nonspecific immunization post-BMT relapsed (95%
tumor incidence). The kinetics of tumor relapse were not
significantly different in recipients of nonimmune marrow
given a specific boost or in recipients of immune marrow
given a nonspecific boost, compared with this control group,
100 r
P
Table 1. Survival of Tumor-Bearing Recipients in Figure 5
No. Survivors1
Total No. Mice
Logrank
P Value’
1/22
-
0112
.66
+
1/22
.96
+
8/25
.02
Group
Treatment
1
Nonimmune BM +
control Id boost
Nonimmune BM +
38C13-Id boost
Immune BM
control Id boost
Immune BM
38C13-Id boost
2
3
4
* Compared with group 1.
with nearly all of the recipients eventually relapsing with
tumor. Relapsing tumors resulted in progressive tumor
growth and death of the hosts. However, a modest delay in
tumor relapse rate was observed for recipients of immune
marrow who had also received a specific booster immunization post-BMT. Although the majority of these recipients
also eventually relapsed, a significant proportion (32%) remained free of tumor during the period of observation and
were apparently cured (Table 1, P = .02 compared with
the control group). Thus, the therapeutic effect of immune
marrow transfer against established tumors required the addition of a specific booster immunization in the recipient postBMT.
All long-term survivors were found to contain hematopoietic cells dominantly of donor origin and were free of any
histologic evidence of GVHD (data not shown). Under the
experimental conditions used,no significant difference in
survival was observed between controls receiving nonimmune BALBlc marrowand other recipients reconstituted
with nonimmune syngeneic C3H marrow in parallel (MST
43 and 42 days, respectively; 0 of 12 survivors in the latter;
data not shown). Thus, allogeneic MHC recognition of host
tumor cells by donor lymphocytes was not sufficient to produce significant antitumor effects under these conditions.
DISCUSSION
L
W
0
0
Z
E
0
I
3
0 Non-immune
M
Non-immuna
lmmuna BY
0 Immuna BM
V
0
0
10
20
30
BM
+
control Id booat
BM + 3Bc-Id boost
+ control Id booat
+
38.2-Id
40
boost
50
DAYS
Fig 5. Therapeuticeffectof immune allogeneic marrow against
established tumors. C3H recipient mice were injected sc with I O 4
38C13 tumor cells and were left untreated for 10 days, after which
time all of the mice had developed macroscopic, l-cm tumor masses.
Recipients were treated with TB1 plus CY on day 10 and received
marrow from immune or nonspecifically immune BALBlc donors 1
day after this treatment. Recipients alsoreceivedan sc immunization
with either 38C13-Id-KLH or control IgM-KLH on the day of marrow
infusion and were monitored thereafter for reduction in tumor incidence and kinetics of subsequent relapse.
Exploiting the potential antileukemic effect of marrow
grafts remains an attractive concept. A GVL effect has been
inferred for most leukemias by the retrospective analyses of
higher rates of relapse after human syngeneic, compared
with allogeneic, BMT:,6 and a similar GVL effect may exist
for lymphomas’ and multiple myeloma. Efforts to manipulate the GVL effect have met with variable degrees of success, largely limited by the difficulty of dissociating GVL
reactions from GVHD.27-29
Perhaps the most definitive evidence of a GVL effect in humans has been provided by
the observations of cytogenetic remissions in patients with
chronic myelogenous leukemia (CML) receiving allogeneic
buffy coat mononuclear cells after relapse from allogeneic
marrow transplantati~n.’~”~~~
The experiments we have presented provide an alternative
strategy, designed to enhance the graft antitumor response
in a specific manner against a defined tumor antigen. C3H
recipient mice conditioned with 950 rad TBI, which had
been previously shown by others to be marrow ablative in
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
3058
this strain,’2 and infused with unfractionated BALB/c BM
were fully reconstituted with donor type cells 3 weeks after
BMT (seeResults) and were moreresistant to challengewith
38C13 lymphoma, compared withrecipients of syngeneic
marrow. The principal finding was that this basic effect of
allogeneic marrow on syngeneic tumor resistance could be
augmented significantly by active immunization of allogeneic marrow donors with Id-KLH. In addition, we have investigated the potential cellular mechanismof this phenomenon.
The mechanism of immunoprotection against the 38C13
lymphoma in normal syngeneic C3H mice immunized with
Id-KLH remains tobe fully elucidated but appears toinvolve
bothhumoral andcellulararms of immuneresponse. For
example, protection could betransferred with immune serum
containing anti-idiotypic antibodies in a Winn assay.19 However, protection is abrogated by in vivo depletion of CD4‘
or CD8’ T cells postimmunization,“ and in such mice antiidiotypicantibodylevelsremainunaltered
(L.W. Kwak,
manuscript in preparation). In the BCL-l lymphoma model,
protection after immunization with idiotypic Ig was primarily antibody mediated.Ix In contrast, Id-induced resistance to
MOPC-315 appeared to be predominantly a result of cellmediated
immunity,
as
postimmunization
thymectomy
abolished tumor resistance and had no effect on serum antiidiotypic antibody levels.?’ Id-induced resistance to MOPCI I could not be attributed to the predominance of either
antibody- or cell-mediated immunity.”In the current study,
TCD of immune allogeneic marrow before infusion clearly
abolished the transfer of the enhanced protective effect of
immune allogeneic marrow to syngeneic recipients (Fig 4).
Furthermore, no effect of marrow TCD was observed on
serum anti-idiotypic antibody levels in these recipients (Fig
3B). These observations, taken together, suggest a primary
role for antigen-specific T cells in the effector phase of this
phenomenon and that the transfer of humoral anti-idiotypic
immunity from donor to recipient at the low levels determined is not sufficient to mediate enhanced protection. This
finding was rather unexpected, given the fact that the percentage of mature T cells in murine BM is extremely low
(approximately 5%). This fact may also explain the requirement for arelativelylargemarrow
inoculum; specifically,
the protective effect of immune, comparedwith nonimmune,
marrow was not observed at doses less than 20 X 10‘ cells
(see Results). Nevertheless, the abrogation of protection by
as a slight but statistically srgnificant
TCD was not complete,
difference between immune and nonimmunemarrow recipients was still evident in the two anti-Thy 1.2 plus C’-treated
groups. It is tempting to speculatethat this residual degree of
protection may be caused by the low levels of anti-idiotypic
antibody demonstrated in the former.
The precise mechanism by which allogeneic BALB/c T
cells specific for idiotype mediate protection against the
38C I3 tumor is under further investigation. T-cell recognition of idiotypicpeptidepresented
by thetumor may be
occurring eitherin the contextof a self-MHC molecule crossreactive for an H-2k molecule expressed by the tumor target
or in direct association with an H-2‘ molecule (allo MHC).
It is noteworthy that T-cell responses specific for allogeneic
KWAK, PENNINGTON. AND LONG0
+
MHC antigenic peptide have been described previously.
Alternatively, indirectly activated effector mechanisms such
as non-antigen-specific effectors (eg, macrophages, natural
killer cells) recruited by cytokines secreted by antigen specific CD4’ T cells, are also being considered. For example,
tumor-derived idiotype may be taken up and processed by
antigen-presenting cells of donor haplotype and subsequently
presented to specific T cells. A precedent for suchan indirect
antitumor effect of T cells in vivo was provided recently by
the demonstration that CD4’, MHC class11-restricted T-cell
clones, which were specitic for myeloma idiotype, protected
againstthe MOPC-315 myeloma, which lacks MHC class
11 mo~ecules.”
The therapeutic effect ofimmune allogeneic marrow could
be demonstrated against rapidly growing, macroscopictumors, in which setting a slight delay in tumor relapse rate
and a modest cure rate were observed (Fig 5 and Table 1).
The full expression of thistherapeuticeffectrequiredthe
addition of a specific booster immunization against idiotype
in the recipient post-BMT, as neither immune marrow alone.
nor a specific recipient immunization
alone were sufficient
to produce any survival benefit compared with nonimmune
allogeneic marrow. Although the potency of immune marrow could be demonstrated, by comparison, it is noteworthy
that against these same established tumors no survival advantage was observed for recipients of nonimmune allogeneic,
compared with syngeneic, BM (see Results). Future studies
aimed atquantitatingthemagnitude
of thiseffect, ie. log
tumor cell elimination by immune marrow, may be worthwhile.
It is unclear why BALB/c marrow did not mediate signiticant GVHD,despiteMHC
differencesbetween donor
andrecipient.Evidently, the smallnumber of T cells that
were present in the marrow inoculum to transfer protection
were not sufficient to induce clinically or histologically evident GVHD (see Materials and Methods). Furthermore. as
the goal of the current study was to establish the principle
of transfer of protection with idiotype
immune allogeneic
marrow under controlled experimental conditions, no additional attempts were ~ n a d eto actively induce GVHD in marrow recipients. Inasmuch as the transfer of protection with
immune allogeneicmarrow is predominantly T-cell mediated, it may be worthwhile to explore the following: ( 1 1 the
addition of T cells from other, relatively enriched peripheral
sources (blood or splenocytes) from immune marrow donors
to increase potency, and (2) assessing the potential offsetting
GVHD onimmune transfer. We
effectofaccompanying
have recently transferred a human tumor idiotype-specific
T-cell response with unmanipulated marrow from an HLAmatched sibling donor, who had been immunized with myeloma idiotype protein, to a myeloma patient who experienced grade I1 GVHD.36 Thus, GVHD and its prophylaxis
may not be ineluctablebarriers to thetransfer of donorderived T-cell immunity.
Finally, it is acknowledged that the experimental setting
of fully MHC-mismatched differences between donor and
recipient is not reproducible in humans and that some findings may not be generally applicable. Furthermore, the requirement forisolation of idiotypes from individual patients’
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
DONORVACCINATION WITH TUMOR IDIOTYPE
lymphomas or myelomas for formulation into safe, refined
of this stratdonor vaccines may limit the routine application
egy in
Nevertheless, the clinical translation of
these results would probably best be performed using nonTCD allogeneic marrow (unless subsequent infusion of donor T cells was planned). The dose of BM infused may not
bealimitingfactor,
as human BM graftsaremost
often
contaminated with significant amounts of peripheralblood
containing T cells. Finally, the experiments in tumor-bearing
recipients suggest that recipient booster immunizations
postBMT may benecessary. A precedentforthisobservation
exists inhuman allogeneic BMT, in which thetransfer of
T-cell immunity to VZV or PPD and humoral immunity to
KLH required both donor and recipient to be
In principle, this therapeutic approach could be applied in
the future to other malignancies for which BMT has a role
in treatment andfor which definedtumor antigens have been
identified (eg, CML).
ACKNOWLEDGMENT
We thank William J. Murphy, PhD, for helpful discussions and
Laura Knott for preparation of the manuscript.
REFERENCES
1. Jones W, Ambinder RF, Piantadosi S, Santos GW: Evidence
of a graft-versus-lymphoma effect associated with allogeneic hone
marrow transplantation. Blood 77:649, 1991
2. Demirer T, Weaver CH, Buckner CD, Petersen FB, Bensinger
WI, Sanders J, Clift RA, Lilleby K, Anasetti C, Martin P, Storb
R, Chauncey T, Doney K, Sullivan K, Appelbaum FR: High-dose
cyclophosphamide, carmustine, and etoposide followed by allogeneic bone marrow transplantation in patients with lymphoid malignancies who had received prior dose-limiting radiation therapy. J
Clin Oncol 13:596, 1995
3. Gahrton G , Tura S, Ljungman P, Belanger C, Brandt L, Cavo
M, Facon T, Granena A, Gore M, Gratwohl A, Lowenberg B, Nikoskelainen J, Reiffers JJ, Samson D, Verdonck L, Volin L: Allogeneicbonemarrow transplantation in multiple myeloma. N Engl J
Med 325:1267, 1991
4. Dimopoulos MA, Alexanian R, Przepiorka D, Hester J, Andersson B, Giralt S, Mehra R, van Besien K, Delasalle KB, Reading
C, Deisseroth AB, Champlin RE: Thiotepa, busulfan, and cyclophosphamide: A new preparative regimen for autologous blood stem cell
transplantation in high-risk multiple myeloma. Blood 82:2324, 1993
5. Weiden PL, Sullivan K, Floumoy N, Storb R, Thomas ED,
The Seattle Marrow Transplant Team: Antileukemic effect of
chronic graft-versus-host disease. Contribution to improved survival
after allogeneic marrow transplantation. N Engl J Med 304: 1529,
1981
6. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J,
Kolb HJ, Rimm AA, Ringden 0, Rc-man C, Speck B, Truitt RL,
Zwaan FE, Bortin MM: Graft-versus-leukemia reactions after bone
marrow transplantation. Blood 75555, 1990
7. Kolb HJ, Mittermuller J, Clemm C. Holler E, Ledderose G,
Brehm G, Heim M, Wilmanns W: Donor leukocyte transfusions for
treatment of recurrent chronic myelogenous leukemia in marrow
transplant patients. Blood 76:2462, 1990
8. Kohler PC, Hank JA, Exten R, Minkoff DZ, Wilson DG,
Sondel PM: Clinical response of a patient with diffuse histiocytic
lymphoma to adoptive chemoimmunotherapy using cyclophosphamide and alloactivated haploidentical lymphocytes. A case report
and phase I trial. Cancer 55:552, 1985
3059
9. Starling KA, Falletta JM, Fernbach DJ: Immunologic chimerism as evidenceof bone marrow graft acceptance in an identical twin with acute lymphocytic leukemia. Exp Hematol 3:244,
1975
10. Lum LG, Seigneuret MC, Storb R: The transfer of antigenspecific humoral immunity from marrow donors to marrow recipients. J Clin Immunol 6:389, 1986
1 1. Wimperis JZ, Prentice HG, Karayiannis P, Brenner MK, Reittie JE, Griffiths PD, Hornrand AV: Transfer of a functioning humoral immune system in transplantation of T-lymphocyte-depleted
bone marrow. Lancet 1:339, 1986
12. Kat0 S, Yabe H, Yabe M, l m u r a M, Ito M, Tsuchida F,
Tsuji K, Takahashi M: Studies on transfer of varicella-zoster-virus
specific T-cell immunity from bone marrow donor to recipient. Blood
75306, 1990
13.Wimperis JZ,Gottlieb D,DuncombeAS,HeslopHE,
Prentice HG, Brenner MK: Requirements for the adoptive transfer of antibody responses to a priming antigen
in man. J Immunol
144:541, 1990
14. Rouleau M, Senik A, Leroy E, Vernant J-P: Long-term persistence of transferred PPD-reactive T cells after allogeneic bone marrow transplantation. Transplant 55:72, 1993
15. Sirisinha S, Eisen HN: Autoimmune-like antibodies tothe
ligand-binding sites of myeloma proteins. Proc Natl Acad Sci USA
68:3 130, 1971
16. Lynch RG, Graff RJ, Sirisinha S, Simms ES, Eisen HN:
Myeloma proteins as tumor-specific transplantation antigens. Proc
Natl Acad Sci USA 69:1540, 1972
17. Freedman PM, Autry JM, Tokuda S, Williams RC Jr: Tumor
immunity induced by preimmunization with BALBlc mouse myeloma protein. J Nat Cancer Inst 56:735, 1976
18. George AJT, Tutt AL. Stevenson FK: Anti-idiotypic mechanisms involved in suppression of a mouse B-cell lymphoma, BCLl .
J Immunol. 138:628, 1987
19. Campbell MJ, Esserman L, Byars NE, Allison AC, Levy R:
Idiotype vaccination against murine B-cell lymphoma. Humoral and
cellular requirements for the full expression of anti-tumor immunity.
J Immunol 145:1029, 1990
20. Kwak LW, Campbell MJ, Czerwinski D, Hart S, Miller RA,
Levy R: Induction of immune responses in patients with B-cell
lymphoma against the surface iriununoglobulin idiotype expressed
by their tumors. N Engl J Med 327:1209, 1992
21. Homung RL, Longo DL, Bowersox OC, Kwak LW: Tumor
antigen-specific immunization of bone marrow transplantation donors as adoptive therapy against established tumor. J Natl Cancer
Inst 87: 1289, 1995
22. Bergman Y, Haimovich J: Characterization of a carcinogen
induced murine B lymphocyte cell line of C3HleB origin. Eur J
Immunol 7:413, 1977
23. Eshhar Z , Blatt C, Bergman Y, Haimovich J: Induction of
secretion of IgM from cells of the B cell line 38C13 by somatic cell
hybridization. J Immunol 122:2430, 1979
24. Wang E, Conant JM, Li D, Visconti V, Chourmouzis E, Lau
C: Ex vivo spermine dialdehyde treatment prevents lethal GVHD in
a murine bone marrow transplantation model. Bone Marrow Transplant 6235, 1990
25. Siu H, Vitetta ES, May R D , Uhr JW: Regression of BCLl
tumor and induction of a dormant tumor state in mice chimeric at
the major histocompatibility complex. J Immunol 137:1376, 1986
26. Blazar BR, Taylor PA, Sehgal SN, Vallera DA: Rapamycin, a
potent inhibitor of T-cell function, prevents graft rejection in murine
recipients of allogeneic T-cell-depleted donor marrow. Blood
83:600, 1994
27. Goldman JM, Gale RP, Horowitz MM, Biggs JC, Champlin RE, Gluckman E, Hofman RG, Jacobsen SJ, Marmont AM.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
3060
McGlavePB,MessnerHA,RimmAA,RozmanC,SpeckB,
Tura S, Weiner RS, Bortin MM: Bone marrow transplantation
for chronic myelogenous leukemia in chronic phase: Increased
risk for relapse associated with T-cell depletion. Ann Intern Med
108:806, 1988
28. Marmont A, Horowitz MM, Gale RP, SohocinskiK, Ash RC.
van BekkumDW,ChamplinRE,DickeKA,GoldmanJM,Good
RA, Herzig RH, Hong R, Masaoka T, Rimm AA, Ringen 0, Speck
B, Weiner R, Bortin MM: T-cell depletion of HLA-identical transplants in leukemia. Blood 78:2120, 1991
29. Sullivan KM, Storh R, BucknerCD, Fefer A,Fisher L,
Weiden PL, Witherspoon RP, Appelhaum FR, Banaji M, Hansen J.
Martin P, Sanders JE, Singer J, Thomas ED: Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms. N Engl J Med 320:828, 1989
30. Drohyski WR, Keever CA, Roth MS, Koethe S, Hanson G,
McFadden P, Gottschall JL, Ash RC, van Tuinen P, Horowitz MM,
Flomenberg N: Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after
allogeneic hone marrow transplantation: Efficacy and toxicity of a
defined T-cell dose. Blood 82:2310, 1993
31. Porter DL, Roth MS, McGarigle C, Ferrara JLM, Antin JH:
KWAK,PENNINGTON,
ANDLONG0
Induction of graft-versus-host disease
as immunotherapy for relapsed
chronic myeloid leukemia. N Engl J Med 330:I00, 1994
32. Samlowski WE, Araneo BA, Butler MO. Fung MC, Johnson
HM: Peripheral lymph node helper T-cell recovery after syngeneic
hone marrow transplantation in mice prepared with either y-irradiation or husulfan. Blood 74:1436. 1989
33. Daley MJ,GehelHM,Lynch
RG: Idiotype-specifictransplantation resistance to MOPC-315: Abrogation by post-immuniztion thymectomy. I Immunol 120: 1620, I978
34. Heher-Katz E, Schwartz RH, Matis LA, Hannum C, Fairwell T, Appella E, Hanshurg D: Contribution of antigen-presenting cell major histocompatibility complex gene products to the
J ExpMed
specificity of antigen-induced T cellactivation.
155:1086, 1982
35. Lauritzsen GF, Bogen B: The role of idiotype-specific, CD4+
T cells in tumor resistance against major histocompatihility complex
negative
plasmacytoma
cells.
Cell
Immunol
class I1 molecule
148:177,1993
36. Kwak LW, Taub DD, Duffey PL, Bensinger WI. Bryant EM.
Reynolds CW, LongoDL:Transfer of myelomaidiotype-specitic
immunity from an activelyimmunizedmarrowdonor.
Lancet
345:1036, 1995