[CANCER RESEARCH 34, 1851 1856, August 1974]
Prolonged Survival in Long-Passage AKR Leukemia Using
Chemotherapy, Radiotherapy, and Adoptive Immunotherapy1
Mortimer M. Bortin, ' Alfred A. Rimm, Glenn E. Rodey, Roger H. Giller, ' and
Edward C. Saltzstein
The May andSigmund Winter Research Laboratory, Mount Sinai Medical Center [M. M.B.,A.A.R.,R.H.G.,E.
C. S.\; The Milwaukee Blood Center
[G. E. R.]: and the Departments of Medicine [M. M. B., G. E. R.], Biostalistics [A. A. R.}, and Surgery [E. C. S.], Medical College of Wisconsin,
Milwaukee, Wisconsin 53233
SUMMARY
A chemoradioimmunotherapeutic
model, with potential
for clinical applicability, was tested in AKR mice bearing a
long-passage lymphocytic leukemia. A temporary graftversuj-leukemia reaction was the key feature of the model.
In the most successful experimental groups, graft-versushost disease was circumvented and "cell cure" of a trans
planted acute lymphocytic leukemia was achieved in at least
88% of the mice. Late mortality occurred in 58% of these
mice and was due mainly to bone marrow and lymphoid
aplasia of unknown cause. Appearance of spontaneous
leukemia-lymphoma was significantly delayed or prevented
in those mice that survived the first 100 days, and 20% lived
beyond 21 months of age.
INTRODUCTION
Despite recent advances in the treatment of acute leu
kemia in man (13, 14, 16, 19), most patients die of recurrent
leukemia (10). Chemotherapy usually is much less effective
when used following relapse of leukemia after an initial
remission and, with only occasional exceptions, sustained
subsequent remissions do not occur (1). The experiments
described below were performed in order to test a strategy
that might have therapeutic applicability for the leukemic
patient in whom conventional therapy has failed.
In brief, AKR mice bearing advanced acute lymphocytic
leukemia were treated with a combination of chemotherapy,
irradiation therapy, and adoptive immunotherapy using a
transplant of bone marrow and lymph node cells from
1Presented in part at the Second Annual Conference of the Interna
tional Society for Experimental Hematology, Paris, France, June 25 to 27,
1973. Research supported by American Cancer Society Grant ET-55; a
grant from the Board of Trustees, Mount Sinai Medical Center; a
memorial to William Heller, Sr.; Grant HE 13-629 from the National
Heart and Lung Institute; and a grant from the Milwaukee Division of the
American Cancer Society. This is Paper 4 in a series titled "Grnf{-versusLeukemia."
2To whom reprint requests should be addressed, at the Winter Research
Laboratory, Mount Sinai Medical Center. 948 North 12th Street, Mil
waukee, Wis. 53233.
3Special Fellow, Winter Research Laboratory, Mount Sinai Medical
Center, 1972.
Received December 26, 1973; accepted April 22, 1974.
AUGUST
//-2-incompatible DBA/2 donors. After 6 days, the AKR
mice were "rescued" from potentially lethal GVH disease4
by killing the DBA/2 cells using DBA/2-specific antiserum
plus chemotherapy. Subsequently, bone marrow transplants
from //-2-compatible CBA donors were administered for
final hematopoietic reconstitution of the AKR mice. A
significant proportion of the treated mice were "cured" of
the transplanted leukemia and were long-term survivors.
The unique feature of the treatment plan consists of a
temporary transplant of immunocompetent cells from un
related donors for their adoptive immunotherapeutic effect.
This approach is conceptually similar to that originally
described by Boranic (4). Several modifications of Boranic's
treatment plan have been introduced in order to simulate
more closely problems encountered in clinical bone marrow
transplantation for leukemia. The most important modifi
cation is the use of cells from allogeneic histocompatible
donors for final hematopoietic reconstitution, rather than
transplants of cells from syngeneic donors.
MATERIALS
AND METHODS
Experimental Design. The model used is diagramed in
Chart 1. Normal young AKR mice were given i.v. injections
of IO5leukemic "blast" (large) cells obtained from spleens
of AKR mice that had received similar injections 1 week
previously (Chart 1, Step I). After a time interval of 4 days
during which leukemia cells multiplied (Chart 1, Step //),
groups of approximately 15 mice were given antileukemic
and immunosuppressive therapy (Chart 1, Step III) consist
ing of 400 R TBR and 5 mg (approximately 185 mg/kg) CY
(generously supplied by Dr. Paul M. Worrall, Mead
Johnson Research Laboratories, Evansville, Ind.). These
doses of TBR plus CY were insufficient to eliminate all
leukemia cells but were sufficiently immunosuppressive to
assure engraftment of allogeneic cells (7). Approximately 4
to 6 hr later, 2 x IO7bone marrow cells and IO7lymph node
cells from //-2-mismatched DBA/2 donors were adminis
tered i.v. (Chart 1, Step IV}. This transplant of immuno
competent cells from unprimed histoincompatible donors
4The abbreviations used are; GVH, graft-vfrsuj-host: TBR, total-body
X-irradiation; CY, cyclophosphamide: GVL, graft-vfrsui-leukemia; MST,
median survival time; MLC, mixed leukocyte culture.
1974
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1851
M. M. Bortin et al.
into immunosuppressed leukemic mice (adoptive immunotherapy, GVL reaction) was the key step of the model. The
transplanted DBA/2 cells were used to "seek and destroy"
all residual leukemia cells. A time interval of 6 days for the
GVL reaction (Chart 1, Step V} was selected because
bioassay studies (6, 23) showed that all viable clonogenic
leukemia cells (i.e., leukemia cells with sufficient proliferative potential to cause death of the animal) were eliminated
from the spleens of leukemic AKR mice following a 6-day
GVL reaction. Thus, after 6 days, treatments were initiated
to rescue the AKR mice from the potentially lethal
concomitant GVH disease induced by the DBA/2 cells
(Chart 1, Step VI). The mice were treated with i.p.
injections of 0.4 ml CBA anti-DBA/2 serum plus 2 mg
(approximately 75 mg/kg) CY. Later that day, the AKR
mice were given i.v. injections of 2 x IO7bone marrow cells
from allogeneic, but //-2-matched, CBA donors in order to
provide hematopoietic repopulation (Chart 1, Step VII).
After a 7-day delay, 2 groups of mice received a 2nd set of
rescue maneuvers consisting of a 2nd injection of 0.4 ml
CBA anti-DBA/2 serum and a 2nd transplant of CBA
bone marrow cells.
Mice. Inbred 10- to 12-week-old AKR/J (H-2") and 10to 14-week-old DBA/2J (H-2") and CBA/J (H-2") female
mice, obtained from The Jackson Laboratory, Bar Harbor,
Maine, were used.
Transplanted Lymphocytic Leukemia. A long-passage
lymphocytic leukemia (BW5147) was the source of leu
kemia cells. BW5147 leukemia originated spontaneously in
an AKR mouse at The Jackson Laboratory in 1954 (12) and
has been passaged s.c. there as a solid tumor through more
than 875 transplant generations. It had been maintained as
a lymphocytic leukemia by serial i.v. passage in our
laboratory through more than 100 transplant generations
when these experiments were initiated.
The estimated growth pattern of BW5147 cells (Chart 2)
was based upon serial dilution experiments done just prior
to this study. The hypotheses upon which Chart 2 is based
have been described (6, 20). Known numbers of blast cells in
the spleen cell suspensions (rather than total number of
spleen cells) were administered, because this procedure has
resulted in highly reproducible mortality patterns (20). The
concentration of blast cells in the spleen cell suspensions
n
z
Chart 1. Experimental model. Step I, i.v. inoculation of IO5BW5I47
blast cells to a young AKR mouse: Step II. time for multiplication of
leukemia cells: 4 days; Slep III. combined 400 R TBR and CY, 185mg/kg,
as antileukemic and immunosuppressive therapy: Slep IV, initiation of
GVL reaction: i.v. inoculation of 2 x 10' bone marrow and 10' lymph node
cells from //-¿-mismatched DBA/2 donor: Slep V. duration of GVL
reaction: 6 days: Slep VI. termination of GVL reaction: CY, 100 mg/kg.
and 0.4 ml CBA anti-DBA/2 serum: Step VII, i.v. inoculation of 2 x IO7
bone marrow cells from //-2-compatible CBA donor.
1852
10«
10«
103
MST. Ig«O*ts(V31
102
U5T ISO DAYS IX*
10"
024
6
B 10 12 14 16 18 20
TIME (days)
Chart 2. Theoretical cytokinetic construct for BW5I47 leukemia based
on serial dilution studies. Numbers in parentheses, number of mice tested.
ranged from 61 to 69%. During the period of growth from
IO2to IO6cells, the generation time was estimated to be 14.4
hr. We assumed, as did Skipper et al. (25), that the
generation time remained more or less constant during
growth from 1cell to IO8cells. The generation time was then
presumed to have decreased slightly until death, when the
mice held an estimated IO9leukemia cells (25). With doses
of IO3to 10" blast cells, no AKR hosts survived. When IO2
blast cells were administered, 16.7% (5 of 30) of the mice
survived. Therefore, assuming a Poisson distribution of the
clonogenic leukemia cells in the cell suspensions, approxi
mately 2% of the blast cells were clonogenic. By extrapola
tion, administration of 1 clonogenic leukemia cell would
have caused death with a MST of 18.9 days (Chart 2).
X-irradiation and Chemotherapy. The irradiation condi
tions have been described (5). Dosimetry was verified with a
Victoreen R-meter. CY was given i.p. and cells were
inoculated 4 to 6 hr following CY administration.
Cell Suspensions. Bone marrow was flushed from the
femora of donor mice with Hanks' balanced salt solution
and passed 5 times through stainless steel sieves with a pore
size of 70 urn. Lymph node cells were obtained from the
mesenteric lymph nodes of donor mice. The lymph nodes
were cleansed of fat and connective tissue and processed
into a single-cell suspension by methods similar to those
described for preparing fetal liver cell suspensions (8).
Leukemia cells were collected from the spleens of mice
bearing BW5147 for 7 days. Following excision of the
spleen, 1 ml of Hanks' solution was injected into the
parenchyma, and the spleen was tamped lightly. Cells in
Hanks' solution poured out of the splenic artery and vein
and the suspension was passed twice through a 27-gauge
needle. Cells were maintained at 4°while aliquots were
counted in a hemocytometer and tested for viability by
means of nigrosin dye exclusion.
CANCER RESEARCH VOL. 34
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GVL
Immunization of CBA Mice against DBA/2 Tissues. Pilot
studies were performed to determine effective treatment
regimens that could be used to rescue AKR mice after
initiation of a lethal GVH reaction (unpublished studies).
Best results were obtained when bone marrow transplants
from //-2-identical CBA donors were preceded by i.p.
injections of CY, 100 mg/kg, or 0.4 ml of an antiserum (a
gift of Dr. G. D. Snell, Bar Harbor, Maine) specific for the
H-2 antigens of the //-2-mismatched cells.
For preparation of antiserum for the present experiments,
CBA mice were hyperimmunized with cells from DBA/2
mice. The immunization schedule was similar to that used
by Snell (personal communication) in the preparation of his
antiserum. CBA mice were given 2 i.p. injections of the
equivalent of 1/25 thymus gland from DBA/2 donors with
an interval of 16 to 21 days between injections. Each mouse
was then given 3 to 7 i.p. injections of a brei containing the
equivalent of 1/10 thymus gland, 1/10 spleen, and 1/10
submaxillary gland at 4- to 10-day intervals. Blood was
collected from the retroorbital venous plexus at 3- to 7-day
intervals starting after the 5th immunization. The blood was
permitted to clot at room temperature and then centrifuged
at 4°;the serum was removed and stored at -20°. In vitro
cytotoxicity tests of pooled antiserum disclosed >95% kill
of DBA/2 spleen cells in a dilution of 1/256. Bone marrow
cells from CBA mice immunized to DBA/2 tissues were
used as donors of bone marrow cells in Chart 1,Step VII, in
certain experiments.
Neuraminidase Treatment of Cells. In other experiments,
DBA/2 bone marrow and lymph node cells were incubated
with neuraminidase (from Vibrio cholerae. General Biochemicals, Laboratory Park, Chagrin Falls, Ohio) and used
to immunize CBA mice or for adoptive immunotherapy.
Following incubation with neuraminidase for 15 min at 37°,
the DBA/2 cells were placed on ice for 5 to 10 min to stop
the enzyme action. Cells were centrifuged at 1000 x g at 4°
for 7 min, supernatants were discarded, and the cells were
resuspended in fresh Hanks' solution. Three concentrations
of neuraminidase were used during incubation: 7.5 units/
donor when the immunizing dose was 1/25 thymus gland;
20 units/donor when the immunizing dose was 1/10 thymus
gland, spleen, and submaxillary gland: and 2.5 units for
each IO7bone marrow or lymph node cells. Cell counts and
tests of viability were performed following washing and
resuspension of the neuraminidase-treated bone marrow
and lymph node cells.
Observation. Mice were observed daily for survival.
Following the 1st 100-day period of observation, approxi
mately one-third of surviving mice were killed for tests of
chimerism. Daily observation continued for the remaining
mice.
Tests of Chimerism. Reciprocal 1-way MLC tests (2)
were performed to test for chimerism using pooled spleen
cells obtained from 7 experimental mice following the 1st
100-day period of observation. Use of MLC for testing
chimerism in rats was reported originally by Elkins (11).
The technique for MLC in mice has been described (21).
Histology. Mice that died were autopsied. Histological
study of the thymus, spleen, liver, and bone marrow was
AUGUST
possible in almost all of the mice, although postmortem
autolysis precluded definitive histological diagnosis in a few
instances. The tissues were evaluated for histological evi
dence of leukemia, hematopoietic
aplasia, lymphoid
aplasia, and GVH disease. Death was ascribed to leukemia
when the mice had (a) massive splenomegaly; (b) replace
ment of normal splenic architecture with diffuse infiltrates
of monotonously uniform large lymphoid cells (probably
lymphoblasts) with numerous mitotic figures: and (c) large
perivascular infiltrates of similar cells in the liver. In those
mice with a diagnosis of leukemia, infiltrates of leukemic
cells almost always were found in the bone marrow and in
approximately one-half of the thymus glands. Death was
attributed to hematopoietic aplasia or hypoplasia when the
bone marrow and spleen were devoid, or nearly devoid, of
granulocytic, erythroid, and megakaryoctic cell lines.
Lymphoid aplasia or hypoplasia was diagnosed when the
spleen was devoid, or nearly devoid, of lymphocytes. A final
diagnosis of GVH disease was made in the absence of
leukemia and aplasia when the mice had dermatitis and/or
focal or diffuse periportal hepatic necrosis.
RESULTS
Overall results from the 1st 100 days of the experiments
are summarized in Chart 3 and Table 1. The MST for all
leukemia control mice (Table 1, Group I; Chart 3, Curve x )
was 8.1 days, and no mouse survived beyond Day 12.
Effect of Neuraminidase-treated DBA/2 cells on GVL
Reaction. Bone marrow and lymph node cells from DBA/2
donors were incubated with neuraminidase prior to their
administration to initiate the GVL reaction (Table 1, Group
2; Chart 3, Curve •¿).For rescue, all mice received
antiserum from CBA mice immunized with neuraminidasetreated DBA/2 cells, and 13 of these received bone marrow
cells from the immune CBA donors. The remaining 15 mice
20
•¿40 50
60
70
80
90
100
TIME (day»)
Chart 3. Survival of AKR mice bearing BW5I47 leukemia following
combined chemotherapy, radiotherapy, and adoptive immunolherapy. In
A, AKR (H-2*) mice were given IO5BW5147 leukemic blast cells on Day 0;
in B, the leukemic mice were treated with TBR. CY, and immunocompetent cells from DBA/2 (H-21) donors on Day 4; in C, administration of
therapy was directed against DBA/2 cells followed by a bone marrow
transplant from CBA (H-2*) donors (rescue procedure) on Day 10; in D, a
2nd rescue procedure was performed for Groups 4 and 5 on Day 17. For
definitions of symbols, see Table 1.
1974
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1853
M. M. Bortin et al.
Table I
AKR mice bearing a long-passage lymphocytic leukemia receiving chemoradiolherapy plus adoptive immunotherapy (GVL reaction): expérimental
design and observations through 1st 100 days
for[DBA/2
cells
(W-^)AKR
+host(//-.?*) bone marrow
node"]35
mice"
lymph
Group12345Effector
to "rescue"
diseaserCY,
mice from GVH
Anti-DBA/2mg/kg
100
serum0
(H-2")bone
marrowcells
MST0
0+
+'0
++
028
+'29
+28
+31
++*+
+Procedures
(%)SymbolsSurvival
++CBA
8r+«
42+
41++'
62+
+
of death
(%)at
lOOdays01132535Leukemia10082101123Aplasia014587959Other"0
70Cause
°AKR mice, 8 to 10 weeks old, received 10s leukemic blast cells on Day 0.
" On Day 4, the AKR mice received 400 R TBR and CY, 185 mg/kg, for leukemia cytoreduction and immunosuppression. This was followed by 2 x
IO7bone marrow and 10' lymph node cells from //-2-mismatched DBA/2 donors to initiate the GVL reaction.
'On Day 10, the AKR mice received CY and/or specific antiserum to inactivate the DBA/2 cells and received 2 x IO7bone marrow cells from
//-¿-matched CBA donors to provide hematopoietic restoration.
'' Includes deaths associated with GVH disease and unknown cause.
' No mouse survived beyond Day 12.
' Cells from DBA/2 donors were incubated with neuraminidase prior to administration to AKR host mice or prior to use for immunization of CBA
mice to produce specific antiserum.
'CBA bone marrow donors for 13 of 28 AKR host mice were immunized with neuraminidase-treated DBA/2 cells.
" + + , procedure performed on Day 10 and repeated on Day 17.
' CBA donors had been immunized with DBA/2 cells.
received bone marrow cells from normal CBA donors. clonogenic leukemia cell remained should have died well
There were no significant intragroup differences and the before Day 50. Of the 7 mice that died before Day 50, 4 had
results have been combined. Most (22 of 28) AKR mice died histological evidence of leukemia. Thus, apparent cell cure
of BW5147 leukemia. Neuraminidase treatment of the of the transplanted leukemia was accomplished in 88 (52 of
DBA/2 cells appeared to interfere with their GVL capabil
59) to 93% (55 of 59) of the mice in Groups 4 and 5, Table 1.
Between Days 50 and 83, an additional 34 mice died.
ity.
Rescue from GVH Disease Using Antisemiti without CY. Survival at 100 days for the 2 groups was 31% (18 of 59).
In Table 1, Group 3 (Chart 3, Curve O) the rescue Gross and histological examinations of the tissues of mice
treatment consisted of a single i.p. injection of 0.4 ml CBA that died between Days 50 and 83 disclosed that 30 mice
anti-DBA/2 serum followed by a bone marrow transplant
died with hematopoietic and lymphoid hypoplasia or
from CBA donors. CY was not administered as part of the aplasia, 1 mouse died with findings typical of GVH disease,
rescue procedure. Although the mortality patterns for and 3 mice died with leukemia. The peak incidence of
Groups 2 and 3, Table 1, were similar (cf. Chart 3, Curves •¿ aplasia was delayed significantly (p < 0.001) to between
and O), the cause of death was dissimilar. Leukemia was Days 55 and 70 when compared with Group 3, Table 1.
Use of MLC's to Test for Chimerism. Following the
not a significant factor in Group 3. Histological study
indicated that 58% of these mice died with aplasia, and at initial 100-day period of observation, 7 of the surviving 18
least 21% died with GVH disease. The peak incidence of mice in Groups 4 and 5, Table 1, were sacrificed and
aplasia occurred between Days 35 and 45. The results reciprocal 1-way MLC's were used to test for chimerism
suggested that a 2nd transplant of CBA bone marrow might (Table 2). Spleen cells from these mice were stimulated to
prevent aplasia and that more intensive treatment directed synthesize DNA when cultured with mitomycin C-treated
against the DBA/2 cells might reduce mortality from GVH cells from AKR or DBA/2 donors. Conversely, cells from
AKR or DBA/2 mice were stimulated when cultured with
disease.
Rescue from GVH Disease Using Antiserumwith CY. For mitomycin C-treated cells from the experimental mice.
rescue, mice in Groups 4 and 5, Table 1 (Chart 3, Curves A Reciprocal nonstimulation was found when cells from the
and A) were treated with CY, antiserum, and CBA cells on experimental mice were cultured with CBA cells. Thus,
Day 10. On Day 17 the mice were given a 2nd injection of AKR mice that had received sequential transplants from
antiserum and a 2nd transplant of bone marrow cells from DBA/2 donors and from CBA donors approximately 100
CBA mice. Treatment regimens differed in that CBA days earlier were chimeric, and the predominant cell
donors of bone marrow in Group 4, Table 1 (Chart 3, Curve population in their spleens appeared to be of CBA origin.
Long-Term Observations. Of the experimental mice avail
A) had been immunized against DBA/2 tissues. During the
1st 50 days, only 12% (7 of 59) of the mice in these 2 groups able for long-term observation (Table 3), overall survival at
died. Assuming that the various treatments caused no 1 year of age was 73% (II of 15); 53% (8 of 15) survived
significant perturbation in the cytokinetic characteristics of beyond 15 months of age, and 3 mice lived more than 21
this leukemia (Chart 2), mice in which even 1 residual months. AKR mice surviving more than 15 months repre-
1854
CANCER RESEARCH
VOL. 34
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GVL
Table 2
Results of reciprocal ¡-wayMLC'.s used to test chimerism in leukemic AKR mice that received
sequential bone marrow transplants from DBA /2 and CBA donors approximately 100days earlier
ofstudies3333AKRm"8,067'27,08421,73021,861s/tt5.52.23.4DBA/2m84,7184,96751,52760,554s/u10.55.39.4CBAm65.135
ResponderAKRDBA/2CBAXNo.
" Subscript m, mitomycin C treatment of cells; X, pooled spleen cells from 7 AKR experimental
mice that were tested for chimerism.
6Stimulation ratio expressed as X cpm stimulated (s)/X cpm unstimulated (u).
' cpm of thymidine-3H incorporation by responder cells. Numbers shown are mean values of
triplicate cultures from 3 separate experiments.
Table 3
AKR mice bearing a long-passage lymphocylic leukemia received
chemoradiotherapy plus adoptive immunotherapy (GVL) reaction:
long-term observation
No. of experimental
mice treated and
Alive at
Group"
Alive at 6
mo. of age"
Available for
observation1
12
mo.
15
mo.
18
mo.
21
mo.
24"
mo.
123450317II313811181115111210110000
°Group designations are the same as in Table I.
"These mice were the 100-day survivors from Table I.
' Seven mice were sacrificed in order to test for chimerism.
" None of the 5 mice that died between 18 and 24 months of age had
gross or histológica!evidence of leukemia, aplasia, or GVH disease.
sent great longevity (26). Of additional interest was the
finding that all 8 of the surviving mice available for
observation in Group 5, Table 3, survived beyond the age of
1 year.
DISCUSSION
Importance of GVL Reaction to Eliminate Transplanted
Leukemic Cells. The efficacy of adoptive immunotherapy to
treat BW5147 leukemia has been reported in bioassay
models (6, 7, 23). In those experiments, clonogenic leukemia
cells were eliminated from the spleens of leukemic mice by
means of a GVL reaction alone (6) or in combination with
chemoradiotherapy (7, 23). Bone marrow and lymph node
cells exposed to 3000 R did not produce effective GVL
reactions (23).
Im and Simmons (17) reported that the severity of GVH
disease was reduced if the effector cells were incubated with
neuraminidase prior to administration. In the present
experiments, we incubated the DBA/2 bone marrow and
lymph node cells with neuraminidase to determine whether
(a) pretreated DBA/2 cells were more vulnerable to immunoelimination (Chart 1, Step VI) because of unmasked
AUGUST
antigens (9); (b) GVL reactivity was unchanged; and (c)
GVH reactivity was reduced or otherwise altered. The effect
of neuraminidase on vulnerability of DBA/2 cells for
immunoelimination and on GVH disease could not be
evaluated because of the large number of mice that died of
leukemia. The results indicated that the GVL capability of
DBA/2 cells was markedly reduced (but not eliminated) as
a result of the treatment. Inasmuch as mice in this group
received all of the antileukemic chemoradiotherapeutic
procedures used in the full model, we consider them to
represent a control group that demonstrates the require
ment for an unimpaired GVL reaction in order to eliminate
residual leukemia cells.
Rescue of Successfully Treated Leukemic Mice from
GVH Disease. Termination of an established GVH reaction
is a formidable problem (4, 15, 18). In pilot studies
(unpublished studies) we found that CY or specific antiserum was most effective in eliminating the cells mediating
GVH disease. However, in the present study, specific
antiserum without CY was not consistently effective in
eliminating DBA/2 cells. Death from GVH disease was
reduced markedly when CY was combined with antiserum,
although a strict comparison of these rescue protocols is not
possible because of a change in the antiserum schedule.
Prior immunization of CBA bone marrow donors with
DBA/2 cells had no significant effect on the incidence of
fatal GVH disease.
Delayed Mortality Associated with Hematopoietic and
Lymphoid Aplasia. We observed aplasia in AKR (//-2*)
mice treated with TBR, CY, and DBA/2 (H-2") cells and
rescued with antiserum and CBA (H-2';) cells. A similar
finding was reported by Barnes and Mole (3), who treated
sublethally irradiated CBA (H-2*) mice with lymph node
cells from C3H (H-2*) donors. They found delayed aplasia
but did not observe clinical or histológica!evidence of GVH
disease. The cause for aplasia in their experimental mice is
not known (D. W. H. Barnes, personal communication). In
both our experiments and in the experiments of Barnes and
Mole, aplasia occurred when a transplant of lymph node
cells was performed within the H-2* genotype, although in
our experiments a transplant from DBA/2 (H-2d) donors
was interposed.
As a consequence of these observations, in subsequent
experiments a 2nd transplant of bone marrow from CBA
1974
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1855
M. M. Bonin el al.
Leukemia. Science, 179: 811 813, 1973.
donors was performed 1 week after the intial transplant.
7. Bortin, M. M., Rimm. A. A., Saltzstein, E. C., and Rodey, G. E. Graft
Many of these mice also developed aplasia and died.
versus Leukemia III. Apparent Independent Antihost and AnHowever, mortality associated with aplasia was delayed
tileukemic
Activity of Transplanted Immunocompetent Cells. Trans
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plantation, 16: 182 188, 1973.
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8. Bortin, M. M., and Saltzstein, E. C. A Modified Technique for
aplasia.
Collection and Processing of Mouse Fetal Hematopoietic Tissue for
1-Way MLC's to Test for Chimerism. A-tthe conclusion
Production of Radiation Chimeras. Exptl. Hematol., 10: 21 30, 1966.
of the 1st 100-day period of observation we wished to 9. Currie, G. A., Doorninck, W. V., and Bagshawe, K. D. Effect of
determine whether lymphoid cells of surviving experimental
Neuraminidase on the Immunogenicity of Early Mouse Trophoblast.
mice were of AKR, DBA/2, or CBA origin, or a mixture of
Nature, 219: 191 192, 1968.
742 743, 1971.
cell types. The data indicated that experimental mice were 10. Editorial. Long Remissions in Leukaemia. Lancet, /.•
11.
Elkins,
W.
L.
The
Sources
of
Immunogenic
Stimulation
of Lymphoid
chimeric and that their spleen cells had the in vitro
Cells
Mediating
a
Local
Graft-versui-Host
Reaction
in
Rat Kidney.
characteristics of the CBA donors. The results reported here
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confirmed the observation of Elkins (11) that MLC tests
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may prove useful as a test for chimerism.
Bull., 2: 61 62, 1955.
Significance of Very Long Survival in AKR Mice. Ap 13. Frei, E. Combination Chemotherapy of Acute Leukemia and Hodgkin's Disease. J. Am. Med. Assoc. 222: 1168, 1972.
proximately 90% of untreated virgin female AKR mice
develop and die of spontaneous leukemia-lymphoma before 14. Freireich, E. J. Research Contributions to Combination Chemother
they reach I year of age, and less than 1% survive to 15
apy. J. Am. Med. Assoc. 222: 1169, 1972.
months (22, 26, 27). Following the clinical appearance of 15. Glucksberg, H., and Fefer, A. Combination Chemotherapy for
Clinically Established Graft-ver.siw-Host Disease in Mice. Cancer
AKR spontaneous leukemia-lymphoma, mortality from the
Res., 33: 859 861, 1973.
disease can be delayed by intensive combination chemother
apy (24). However, chemotherapy does not appear to 16. Holland. J. S. Combination Therapy of Acute Lymphocytic Leukemia
of Children. J. Am. Med. Assoc. 222: 1169 1170. 1972.
eliminate the leukemogenic inducing agent because appar
17. Im, H. M., and Simmons. R. L. Modification of Graft-vervus-Host
ent cell cures are followed by reinduction of spontaneous
Disease by Neuraminidase Treatment of Donor Cells. Decreased
leukemia-lymphoma (26).
Tolerogenicity of Neuraminidase-treated Cells. Transplantation, 12:
In the present studies there were 15 experimental mice
472 478, 1971.
available for long-term observation. Following the 1st 18. Merritt, C. B., Darrow, C. C., II, Vail, L., and Rogentine, G. N., Jr.
100-day period of observation, the mice were approaching 6
Bone Marrow Transplantation in Rhesus Monkeys following Irradia
tion. Modification of Acule Graft-vprciis-Host Disease with Antilymmonths of age and were at risk of development of and death
from spontaneous leukemia-lymphoma. The fact that the
phocyte Serum. Transplantation. 14: 9 20, 1972.
majority survived beyond 1 year of age was of considerable 19. Pinkel, D. Total Therapy of Acute Lymphocytic Leukemia. J. Am.
Med. Assoc. 222: 1170, 1972.
interest. Although several explanations might be offered for
the observed long-term survival of these AKR mice [e.g., 20. Rimm, A. A., Bortin, M. M., and Saltzstein, E. C. Graft-versi«Leukemia: a Proposed Cytokinetic Basis for a Successful Adoptive
the treatment was; in effect, a medical thymectomy (28)],
Immunotherapeutic Model for Murine Leukemia. Federation Proc.,
we believe that our results may reflect an antiviral effect of
31: 768, 1972.
transplanted cells. That is, in addition to GVH and GVL 21. Rodey, G. E.. Good, R. A., and Yunis, E. Progressive Loss in Vitro of
reactions, there may have been a graft-versws-virus reaction.
Cellular Immunity with Ageing in Strains of Mice Susceptible to
ACKNOWLEDGMENTS
Expert technical assistance was provided by Donna M. Eastman,
Marcia R. Hilger, Anita F. Lapp, Jesse J. Miller, III. Marc Rasansky.
Evangeline R. Reynolds, James Seltzer, M. Refaat Shalaby, and John
Sprader.
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CANCER RESEARCH VOL. 34
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1974 American Association for Cancer Research.
Prolonged Survival in Long-Passage AKR Leukemia Using
Chemotherapy, Radiotherapy, and Adoptive Immunotherapy
Mortimer M. Bortin, Alfred A. Rimm, Glenn E. Rodey, et al.
Cancer Res 1974;34:1851-1856.
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