Download Inhibition of in Vitro Lymphocyte Transformation

[CANCER RESEARCH 27, 98-105, January 1967]
Inhibition of in Vitro Lymphocyte Transformation
Chemotherapy in Man1
during
EVAN M. HERSH- AND JOOST J. OPPENHEIM
Medicine Branch, National Cancer Institute,
NIH, Bethesda, Maryland
Summary
The in vitro transformation responses of lymphocytes to stim
ulation with phytohemagglutinin (PHA) and smallpox vaccine
(vaccinia) were studied in cells from 20 patients with ocular and
malignant diseases receiving chemotherapy. The transformation
of lymphocytes to lymphoblast-Iike cells was reduced from 71%
in the pretreatment PHA-stimulated cultures to 1.5% during
therapy. The response to vaccinia was reduced from 12% before
therapy to 0% during therapy. The mitotic indices fell from
1.5% (PHA) and 1.2% (vaccinia) to 0% for each during therapy.
Intensive combination therapy with parenteral 6-mercaptopurinc and methotrexate, with or without prednisolone com
pletely abolished transformation after 3 days of treatment. Sub
stantial recovery occurred within 3 days after the end of therapy.
Nontoxic therapy with methotrexate or 6-mercaptopurine which
did not induce leukopenia took 2-5 weeks to cause maximum
suppression.
The abnormality seemed due to intrinsic damage to the lym
phocytes and not to persistent antimetabolite in the plasma.
In vilro lymphocyte transformation is an easy and reproducible
way of evaluating the immune competence of an individual's
circulating lymphocytes.
Introduction
Impaired immunologie competence has been recognized in a
variety of human diseases (27), and suppression of immunologie
competence has been observed during certain types of therapy
(16). Clinically, immunosuppressive antimetabolite therapy is
used to maintain homografts (21) and in the treatment of certain
"auto-immune" disorders (33). In general, antimetabolite ther
apy can delay homograft rejection, prevent the development of
delayed hypersensitivity, inhibit the mononuclear cell phase of
the local inflammatory response, and block primary antibody
responses (16). Partial suppression of the secondary response
has also been achieved with antimetabolites (18, 30).
Immunologically competent small lymphocytes play a central
role in mammalian host defense mechanisms (8). The recently
developed technics of short-term in vitro lymphocyte culture
offer means of directly assessing the coni|>etence of an individ1Presented in part at the Annual Meeting of the American
Association for Cancer Research, held at Philadelphia,
Pa.,
April 9, 1965.
2 Present address: M. D. Anderson Hospital and Tumor Insti
tute, The University of Texas, Houston, Texas.
Received July 2, 1965; accepted August 18, 1966.
ual's peripheral blood lymphocytes. When PHA,3 or antigens
with which the subject has had prior contact, or homologous or
heterologous tissue antigens are added to cultured peripheral
blood leukocytes, a proportion of the lymphocytes transform
into lymphoblast-like cells and undergo mitosis (29). There is
no detectable response to primary antigens. The transformed
lymphocytes are morphologically similar to the pyroninophilic
lymph node cells which develop in nodes draining sites of recent
antigenic stimulation (1, 34).
Impaired in vitro lymphocyte transformation has been noted
in several disorders where there is impaired immunologie com
petence. These include chronic lymphocytic leukemia (25),
Hodgkin's disease (12), ataxia telangectasia (23), and Boeck's
sarcoid (15). The above mentioned observations suggest that
the in vitro lymphocyte transformation response reflects the
immunologie competence of an individual's circulating lympho
cytes.
In the current study in vitro leukocyte cultures with PHA
and smallpox vaccine (vaccinia) were used to evaluate the im
munologie competence of lymphocytes from patients receiving
chemotherapy. Suppression of in vitro lymphocyte transforma
tion was noted. This was felt to reflect the effect the therapy
would have on secondary immune responses and therefore its
potential effect on homograft rejection, "auto-immune" dis
orders, and resistance to infection.
Materials
and Methods
Lymphocyte Culture Technic
Fifty ml of venous blood were mixed with 500 units of heparin
(heparin sodium, Upjohn) and allowed to sediment at 37°Cfor
2 hr in 150- x 20-mm screw-cap tubes. The supernatant WBCrich plasma obtained had a count of 1000-4000 WBC/cu mm
with approximately 50% lymphocytes (range: 30-70%).
WBC-rich plasma was added to Eagle's minimal essential
medium in a ratio of 1:2 and divided into 6- or 12-ml aliquots
for culture. The medium was supplemented with 100 units of
penicillin, 100 units of streptomycin, and 50 /ig of glutamine
per ml (all from Flow Labs, Rockville, Md.).
Each set of cultures consisted of an unstimulated control
culture, a culture containing 0.2 ml of phytohemagglutinin-M
(Difco Labs, Detroit, Mich.), and 3 cultures containing 0.5 ml
of 1:10, 1:100, or 1:1000 dilution of vaccinia (Dryvax-Wyeth),
3 The following abbreviations
tinin; 6-MP, 6-mercaptopurine;
are used: PHA, phytohemagglu
and MTX, methotrexate.
CANCER
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Inhibition of Lymphocyte Transformation
TABLE 1
THE PATIENTS' DIAGNOSESANDTHERAPY RECEIVED
GROUP orPATIENTS7224311DIAGNOSISAcute
No.1234567No.
leukemiaAcute
leukemiaAcute
m7.5
mg/sq
m1000 mg/sq
m1000mg/sq
m15mg/sq
m1000mg/sq
days7
days5
m500
mg/sq
mg/sq
leukemiaUveitisCarcinomaHemolyticanemiaAcute m7.5
m25 mg/sq
m7.5
mg/sq
mg/kg0.75
mg/kg
7.5
mg/kg0.025
mg/kg2.0
m25-50mg/sq
days42
days5
days30
i.V.i.V.p.o.i.V.ScheduleDailyDailyDailyQ4dDailyQ2d
Q2dWeeklyDailyDailyDuration5
days12
mq/sq mRoutei.V.i.V.i.V.i.V.i.V.i.V.weeks
arab-inosideDose500
leukemiaTHERAPYDrug6-MP«MTXPrednisolone6-MPMTXPrednisolone6-MPMTXMTX5-FUMTXCyclophosphamideVin
0 6-MP, 6-mercaptopurine;
MTX, methotrexate;
5-FU, 5-fluorouracil;
arabinofuranosylcytosine;
Q4d, every 4th day; Q2d, every other day.
cytosine
arabinoside,
1-/3-D-
respectively. Cultures were incubated for 5 days at 37°Cin
cemide (Ciba Pharmaceutical, Summit, N. J.) were added to
each culture 4 hr before harvesting. The polystyrene particles
are usually phagocytized by macrophages and other phagocytic
Harvesting and Counting
cells, thus aiding in their differentiation from the lymphoblastPolystyrene particles (0.1 ml, 1:100 saline dilution, 1.3 11 like transformed cells (28). Colcemide arrests mitosis in metadiameter; Dow Chemical Co., Midland, Mich.) and 4 jig of col- phase and thus makes the mitotic index of the cultures easier to
count (22).
The cultured cells were collected by centrifugation at 1400
rpm for 7 min (International centrifuge, No. 240 head), fixed in
a 1:9 glacial acetic acid:ethyl alcohol mixture for 10 min, recentrifuged, resuspended in fixative, pipetted on slides, air dried,
and stained with Giemsa (Harleco, A. H. Thomas, Philadelphia
Pa.).
stationary stoppered bottles in air.
TABLE 2
THE EFFECTOF CHEMOTHERAPY
ON IN VITROLYMPHOCYTE
TRANSFORMATION"
(%)ControlBlasts73667072737281Mitoses3.01.02.62.04.03.05.0TherapyBlasts0.5074271046Mitoses0000002.5TUIEDays
TRANSFORMATION RESPONSE
GROUP
No.1234567PHA6
CHART 1. The effect of chemotherapy on in vitro lymphocyte
responses to phytohemagglutinin
and small pox vaccine. The % of
transformed lymphocytes (blasts) and % of mitoses in the leuko
cyte cultures of 20 patients before and during chemotherapy are
shown. PHA, phytohemagglutinin;
Pox, vaccinia. Therapy values
represent the maximum inhibition of the response observed.
JANUARY
to
to
maximum
recovery3255C8Cd
suppression323.53961435Days
" The groups are the same as in Table 1. Values given are
means.
6PHA, phytohemagglutinin.
r Discharged at termination of therapy.
d Started on another drug immediately
sine arabinoside.
after cessation of cyto
1967
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Evan M. Hersh and Joost J. Oppenheim
Five hundred cell differential counts and 1000 cell mitotic
indices were done on each side. Cells counted included trans
formed lymphocytes, small untransformed lymphocytes, macro
phages, dead cells, polymorphonuclear leukocytes, and mitoses.
Transformed lymphocytes were characterized by their large
size, a large oval nucleus, 1-4 prominent nucleoli, heterochromatic nucleoplasm and basophilic nongranular cytoplasm.
Small untransformed lymphocytes were small in size, had
clumped nuclear chromatin, no nucleoli, and scanty pale cyto
plasm. Macrophages were large cells, had intermediate-sized
nuclei, often with light staining nucleoli, and abundant granular
cytoplasm filled with phagocytized polystyrene particles. Their
cytoplasm ¡nucleusratio was estimated at about 2. Dead cells
were characterized by disruption of the nuclear membrane and/or
cytoplasmic membranes and often showed karyolysis, karyorrhexis, or pyknosis of the nucleus.
i.v. 6-MP and MTX without prednisolone. Intermittent i.v.
MTX therapy was given to 4 patients who were in good health
except for uveitis. Three patients with metastatic carcinoma re
ceived combination therapy with 5-fluorouracil, MTX, cyclophosphamide, and vincristine in 5-day courses. Finally, 1 patient
with hemolytic anemia receiving daily p.o. 6-MP and 1 with
acute leukemia receiving cytosine arabinoside were also studied.
Patients receiving intensive 5-day therapy (Groups 1, 3, and 5)
were tested only twice during and 2-4 times after each par
ticular course of therapy. Samples were obtained on different
days hi different patients so that values were available for each
day during and 5 days after therapy. Patients in Groups 2, 4, 6,
and 7 were tested weekly. Each patient served as his own control
while not on therapy. Control values were obtained before any
therapy except in the patients with acute leukemia, where control
values were obtained between courses.
Patients Studied and Therapy Administered
Results
Two hundred fifteen sets of peripheral blood leukocyte cul
tures were done before, during, and after chemotherapy in 20
patients, (Table 1). The patients with acute leukemia were all
in peripheral blood remission (no circulating leukemic cells)
when studied. Seven patients with acute leukemia were given
intensive combination chemotherapy with 5-day courses of i.v.
6-MP, MTX, and prednisolone. Two patients with acute leu
kemia received more intensive 7-day courses of the same drugs.
Two patients with acute leukemia received 5-day courses of
20
30
The Control Transformation
The control responses to PHA and vaccinia in the 20 patients
are seen in Chart 1. After 5 days of culture with PHA, 71% of
the cells in the cultures were transformed lymphocytes or lymphoblasts; 1.5% of these cells were in mitosis. With vaccinia, 12%
of the cells were transformed cells and 1.2% were hi mitosis.
The responses to PHA were identical with the responses of the
16 normal individuals studied and are similar to the observa-
40
DAYS
I
1.0
0
7.5
0
1000
I
Response
I
50
1.0
0
7.5
0
-1000
PREDNISONE,mg/sq m
MTX, mg/sq m
6-MP, mg/sq m
0
0
CHART2. The effect of intensive combination chemotherapy on lymphocyte transformation. The figure demonstrates the rapidity of
inhibition and recovery during and after chemotherapy in a patient with acute leukemia. All tests done in the patient are shown. The
response to vaccinia was more variable than to PHA. The rapidity of inhibition and recovery is noteworthy.
100
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Inhibition of Lymphocyte Transformation
tions of others (29). Only 1 patient had a poor control response.
He had finished a course of Group 1 therapy 5 days prior to the
test.
The Effects of Therapy
Chart 1 also shows the over-all effects of therapy. Each type
of therapy resulted in an inhibition of lymphocyte transforma
tion and a fall in the mitotic index from control levels. This
occurred to a similar degree and at a similar rate in both the
PHA- and the vaccinia-stimulated cultures. The maximally
inhibited cultures showed no transformed cells but only small
lymphocytes and dead cells. Even the response of the patient
with the low control value fell further during therapy (34-0%
in response to PHA). In the pretreatment cultures most of the
transformed cells were of fairly uniform size. In the cultures
done during therapy, when inhibition was incomplete, those
cells that did transform appeared smaller.
There were striking differences among the various types of
therapy with regard to the rate of inhibition of lymphocyte
transformation. The effects of the individual types of therapy
are seen in Table 2. Intensive combination therapy with 6-MP
and MTX (Groups 1-3) inhibited transformation rapidly and
completely (Chart 2). The effect was the same whether or not
prednisolone was used in conjunction with the other 2 drugs.
The effects of 4-drug therapy given to the patients with
metastatic carcinoma were similar (Group 5, Chart 3). The
lymphocytes recovered rapidly from the inhibition. Within a
median of 3 days after the end of combination 6-MP and MTX,
transformation with PHA was within normal limits. However,
Absolute
O
IO
20
•
•
M
30
B
M
50
•
MO
30
40
DAYS
II
II
||
CYTOXAN
MTX
I
VINCRISTINE
• 5-FU
CHART3. The effect of combination chemotherapy on lympho
cyte transformation. The effect on lymphocyte transformation of
4-drug combination therapy in a patient with metastatic carci
noma is shown. This patient never responded well to vaccinia.
Lymphocyte
40
20
10
~~ • •
Count /
60
70
80
90
PREDNISONE, mg/sqm
O
7.5 METHOTREXATE, mg/sq m
15
O
500 6-MERCAPTOPURINE, mg/sq m
1000
CHART4. The effect of intensive combination therapy on lymphocyte transformation. The prolonged inhibition of lymphocyte trans
formation induced by intensive combination therapy given for 7 days. Note that the absolute lymphocyte count did not correlate with
the transformation
response and that it had risen above pretreatment levels by Day 58 while normal lymphocyte transformation did
not return until Day 72. For absolute lymphocyte count each division equals 500 lymphocytes/cu mm.
JANUARY
1967
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101
Evan M. Hersh and Joost J. Oppenheim
lOOi
1
1
25
C/)
PHA
Q
LJ
5
(T
O
so
20
60
15
g 40 ^ BLAST/LYMPH
er
20X
RATIO
\
IO
5
VACCINIA
IO
20
30
40
50
DAYS
60
70
80
mg/sqm
CHART5. The effect of intermittent methotrexate therapy on lymphocyte transformation. The gradual decline in the % of trans
formed cells in the cultures of cells from a patient with uveitis on MTX therapy is noteworthy. The ratio of transformed to untransformed lymphocytes fell before significant reduction in the % of total cells transformed was observed.
if the drugs were given at double the usual dose for 7 rather
than 5 days (Group 2, Chart 4), recovery was prolonged to 25
days. In patients receiving only intermittent i.v. MTX there was
a gradual fall in the percentage transformation (Chart 5). The
decline did not occur until after 2-3 weeks of therapy and
reached a maximum only after 5 weeks. At that time the degree
of suppression was the same as for the more intensive therapy.
Recovery was not studied in this group or Group 6 because the
patients were discharged within a day or 2 after the end of
therapy. Daily p.o. 6-MP also produced gradual, rather than
rapid, suppression. Little effect was noted in the patient re
ceiving cytosine arabinoside in spite of the fact that the patient
was followed through two 6-week courses of therapy, both of
which produced significant lymphopenia.
There was a clear correlation between the degree of cell death
in the patients' unstimulated cultures while on therapy and
inhibition of transformation in the patients' PHA-stimulated
cultures (Table 3). Thus, in those sets where the unstimulated
culture contained more than 50% dead cells, there was signifi
cantly less transformation in the corresponding PHA-stimulated
cultures than when there were less than 50% dead cells (P <
0.02). This observation suggested that cell death alone might
account for the impaired transformation and that the surviving
lymphocytes could transform normally. To investigate this
possibility, the ratio of transformed cells to untransfonned
lymphocytes in the patients' PHA cultures was studied (Chart
6). The majority of morphologically intact lymphoid cells in
cultures done during therapy were untransformed, and there
fore these cultures showed a median ratio of 0. The majority of
the lymphoid cells in cultures done during the pretreatment
period or after recovery were transformed and showed a median
ratio of 9.5. This suggests intrinsic damage to the morphologi
cally intact lymphocytes.
Patients' cells collected during treatment on 19 occasions were
102
TABLE 3
THE RELATIONSHIPBETWEENCELL DEATH IN UNSTIMULATED
CULTURESANDTRANSFORMATION
IN THE CORRESPONDING
PHYTOHEMAGGLUTININ
(PHA)-sTiMULATEDCULTURES"
TRANSFORMATIONMedian676162515424312Range1-807-802-800-824-800-721-680-45%
NO. OF CULTURE
DEAD
CELLS0-1011-2021-3031-4041-5051-6061
SETS626424233221628%
" This tabulation includes sets of cultures done before, during,
and after therapy. Cultures done while the patients were off
therapy and responding normally to PHA always had less than
50% dead cells.
washed and resuspended in calf serum rather than in autologous
plasma. Cultures were then set up in the usual way. The lympho
cyte transformation response was not restored by removing
autologous plasma from the patients' cells. The patients' plasma
did not inhibit transformation of cells obtained from normal
people.
Lymphopenia developed during the more intensive types of
therapy. It did not develop, however, in the patients receiving
intermittent i.v. MTX. There was a correlation between the
depressed circulating lymphocyte count and the subsequent
degree of inhibition of in vitro transformation observed only at
levels below 500/cu mm. For circulating lymphocyte counts
ranging from 500 to over 3500/cu mm (Table 4), there was no
CANCER
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VOL. 27
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Inhibition
THE EFFECT OF CHEMOTHERAPY ON
IN VITRO LYMPHOCYTE TRANSFORMATION
of Lymphocyte
Transformation
TABLE 4
THE RELATIONSHIP
BETWEENCIRCULATING
LYMPHOCYTE
LEVELS
AND THE LYMPHOCYTETRANSFORMATIONRESPONSE
ix VITRO"
—30-Uli-_j^o
tu
1=^•,„*„.,Mediän•t•st1
lymphocyte
transformation
Xo. of cultures37544232132413Absolute
countmm)0-500500-10001000-15001500-20002000-25002500-3000over
(cells/cu
response to
PHA*
(%)3595454495871Range0-800-770-800-770-775-8010-
3000Median
20bjSo<nzLYMPHOCYTE
" Only the group with 0-500 lymphocytes/cu
mm in peripheral
blood was significantly different from the other groups (P < 0.01
by chi square test).
1 PHAjphytohemagglutmin.
Discussion
RATIO
II
Ov.
•
*1t
1••'
e1-ffl0-i
THERAPY
PATIENT
NORMAL
CONTROLS CONTROLS
CHART 6. Phytohemagglutinin-induced
lymphocyte
transfor
mation in antimetabolite
treated and untreated patients and in
normal controls. The similarity of patient controls and normal
controls with regard to the proportion of lymphocytes trans
formed is noteworthy.
correlation. The patients with lymphocyte counts below 500/cu
mm were all under intensive therapy when their counts reached
a low point. In some patients the transformation response
returned to normal prior to the resolution of the lymphopenia
or even while the lymphopenia was worsening. In others the
lymphocyte count returned to normal before the lymphocyte
transformation response recovered (Chart 4).
Cells were added to the cultures in final concentrations ranging
from 350 to 1300 WBC/cu mm. Within these limits the cell
concentration in the cultures did not correlate with the PHA
transformation response.
In vitro lymphocyte transformation is related to in vivo im
munologie responsiveness. The transformed lymphocytes are
similar morphologically to the pyroninophilic lymph node cells
which develop after antigenic stimulation in vivo and which
go on to form plasma cells (1, 34). In vitro transformation re
sponses of lymphocytes cultured with antigens represent sec
ondary responses of these lymphocytes. Thus, only subjects
with prior exposure to tuberculosis (as evidenced by a positive
tuberculin skin test) will show an in vitro lymphocyte transfor
mation response to purified protein derivative (26). The subject
must have had prior immunizing contact with the antigen before
his lymphocytes will respond to it in vitro (6, 17). In cultures of
mixed human peripheral blood lymphocytes the degree of trans
formation response is greater in unrelated than in related subjects
and is almost completely absent in mixes of cells from monozygotic twins (3). During skin graft rejection there is a marked in
crease of the in vitro lymphocyte transformation response of
recipient cells in mixed donor-recipient leukocyte cultures (24). A
number of human diseases where abnormalities of the lymphoid
tissue, impaired immunologie competence, and impaired in vitro
lymphocyte transformation coexist have already been mentioned.
The teehnic of lymphocyte culture with various mitogenic agents
can be used therefore to evaluate lymphocyte competence
during chemotherapy directed at inhibition of the immune
response. One such situation would be to follow the effects of
continuing chemotherapy on the lymphocytes of patients who
have received homografts (31).
The current study has shown that several tyjws of chemo
therapy can inhibit in vitro lymphocyte transformation in man.
The duration of therapy before maximum inhibition was di
rectly related to drug dose as was the time it took for normal
lymphocyte transformation to return after the end of treatment.
Single-drug non-leukopenia-producing therapy inhibited trans
formation to the same degree as intensive combination therapy
but only after a longer period of treatment. The rapid inhibition
of transformation and the rapid recovery after the end of therapy
are noteworthy. The degree of inhibition did not correlate with
JANUARY 1967
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10.3
Evan Al. Hcrsh and Joost J. Oppenheim
the patients' absolute lymphocyte count except at lymphocyte
levels below 500/eu mm.
Impaired in vitro transformation of lymphocytes from patients
receiving chemotherapy is probably due to inhibition of the
nucleic acid and protein synthesis which is a necessary part of
the transformation process (7, 9, 14, 20). Although steroids can
block lymphocyte transformation when added in vitro (5),
steroid therapy does not explain our observations since only
9/20 patients received prednisolone.
This study outlines another type of host responsiveness which
can be modified by chemotherapy in man. The suppression of
in vitro lymphocyte transformation appears at approximately
the same time after initiation of intensive therapy as the in
hibition of the inflammatory reaction and the primary antibody
response (11, 13, 32). There is complete inhibition of the primary
antibody response to antigens given 24 hr after the start of
intensive chemotherapy. There is a good antibody response,
however, to another antigen given 24 hr after the end of the
course of treatment (10). This is analogous to the rapid inhibi
tion and recovery of transformation demonstrated in this study
during and after short-term intensive therapy. Similarly, the
effect of chemotherapy on transformation of peripheral blood
lymphocytes can be compared to the inhibition by chemotherapy
of the appearance of pyroninophilie lymph node cells after
homografting or antigenic stimulation in vivo (2, 34). However,
these studies were done during primary stimulation, and the
type of therapy used did not block the lymph node response to
secondary stimulation (2). The secondary stimulus was prob
ably given too early in the course of therapy.
The results of the current study suggest that even therapy
which does not induce leukopenia (such as intermittent M TX)
would eventually suppress the secondary response. However,
the most rapid and complete inhibition of immune responses,
including the primary antibody response, the local inflammatory
response, and in vitro lymphocyte transformation, is achieved
with intensive combination 6-MP and M TX therapy (11, 13,
32). If continued immunosuppression is the objective of therapy,
it might be achieved with relatively nontoxic doses of either
drug.
It is noteworthy that patients who have acute leukemia but
who are in remission (or at least without any circulating leukemic cells) and off therapy have normal in vitro lymphocyte
transformation. Similarly, such patients have normal local
inflammatory (4) and primary antibody responses (19).
Acknowledgments
We would like to thank Drs. Emil J. Freireich and Emil Frei,
III, for their guidance and support in all aspects of this work.
We would also like to thank Mrs. Ligita Novikovs and Mr. Robert
Colligan for their capable technical assistance.
References
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The Morphologic Responses of the Lymphoid System to
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2. —•——.
The Morphologic Responses of the Lymphoid System
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104
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105
Inhibition of in Vitro Lymphocyte Transformation during
Chemotherapy in Man
Evan M. Hersh and Joost J. Oppenheim
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