Differential Interleukin 12 Responsiveness for Interferon y

Vol.
4, 2425-2432,
October
1998
Clinical
Differential
Interleukin
Production
in Advanced
Correlates
Kazuko
Uno,’
Atsuko
Kishi,
Hiromi
Louis
with
Setoguchi,
Makoto
Ogawa,
and
Center
Medical
Institute,
Man
Tsunataro
for Medical
of Cancer
School,
Osaka
Inc., Cambridge,
Research
cells
606,
Japan
Center,
[K. U.,
Osaka
cells
enhances
NK
(3)
growth
factor
antitumor
activity
mechanisms
has been
in murine
including
duction
by T cells
present
study
the
and
was
tumor
capacity
natural
to examine
shown
to exhibit
models
through
cancer
patients
stimulation
IFN-’y
cells.
aim
Samples
from
responsiveness.
patients
displayed
those
observed
exhibited
of IL-12
IV)
increased
these
phocytes
contained
When
in
peripheral
mor
necrosis
patients
crosis
showed
exhibited
after
factor
factor
LL-12
a
blood
responses.
a production
stimulation,
was
IL-12-stimulated
induced
IFN-”y
tions indicate
that a remarkable
reactivity
among
cancer
patients,
responsiveness
depends
largely
the
a critical
significant
effects
(18).
therapy
abrogates
the
The
status.
of lymthe
tumor
were
enhanced
observed
in some
elevation
of tumor
in
samples
These
tunethat
observa-
exists
in IL-12
differential
IL-12
status.
the
high
stimu-
levels
these
of an
reactivated
IFN-’y
T
that
plays
In tumor-bearing
not
mdi-
sufficient
Nevertheless,
IL-12-mediated
tumor-bearing
to
induce
this
initial
antitumor
monoclonal
antitumor
state
logical
condition
induced
and by which
in Ref.
19).
mains
in
Whereas
the United
effect
antibody
efficacy
be-
before
of IL-l2
responses
(1 1,
of
NK
tumor-induced
we
cancer
the
whole
blood.
exists
in IL-l2
ferential
tumors
nor
rather
with
a given
implication
by
the
correlates
number
of
the PS of either
patients
These
in
stage.
evaluating
in ongoing
PATIENTS
AND
Subjects.
the
clinical
and
healthy
The
abbreviations
peripheral
blood
SN, supernatant;
investigation,
subjects
used
dif-
type
of
blood
stages
an
responsiveness
of IL-12
The
whole
provide
in
but
or those
important
of
cancer
immunotherapy.
METHODS
In this
patients
IL-12
trials
with
difference
the
at all cancer
results
from
contained
patients.
in
present
itself
neither
present
advanced
blood
cancer
with
PBMCs
similarly
PBMCs
a remarkable
among
reactivity
re-
in various
by PBMCs
that
responsiveness
in
issue
In the
of
whole
production
show
started
can
patients
(20).
stimulating
results
just
IL-12
various
responsiveness
LFN-y
The
IL-l2
have
is
(reviewed
a fundamental
whether
from
IL-12
patients
and measuring
of IL-12
immunomodulation
assessed
production
are modulated
Japan,
cells
pathophysio-
cytokine
trials
and
regarding
and
rIL-l2
2
T-cell
to be resolved
various
at
abnormal
Europe,
T cells
is a representative
which
clinical
States,
stimulate
patients
Received 4/17/98; revised 7/20/98; accepted 7/24/98.
The costs
of publication
of this article
were
defrayed
in part by the
payment
of page charges.
This article
must therefore
be hereby
marked
advertisement
in accordance
with
18 U.S.C.
Section
1734 solely
to
indicate
this fact.
I To whom
requests
for reprints
should
be addressed,
at Louis Pasteur
Center
for Medical
Research,
103-5,
Tanaka
Monzen-cho,
Sakyo-Ku,
Kyoto
City, Kyoto
606-8225,
Japan.
Phone:
81-75-791-7726;
Fax: 8175-7 15-5850;
E-mail:
[email protected].
for
is
by
strikingly
produce
(17).
alone
com-
responses
12).
study,
on performance
therapeutic
is critical
and
in
recently
induces
of immune
to produce
regression
production
IL-l2
samples
from
comparable
to
states
difference
and that
in tumor
(16)
anti-IFN-’y
cells
production.
sites
and
observed
it has
15): IL-12
cells
with
nor
blood
role
NK
cyto8);
(1, 9).
been
(1 1, 12), and allows
tumor
IFN--1
viduals,
has
of IL-12
14 and
and
IFN-y
to infiltrate
iso-
also
T cells
cytokine,
cells
showing
null/marginal
was examined,
7 of 13
was
only
antitumor
in Refs.
treatment
number
Whereas
(reviewed
T-cell
(3,
subset
Moreover,
a series
and
T cells
T-cell
administration
on T
cytokine
of various
and
helper
through
cause
performance
mononuclear
systemic
IL-12
for reduced
the
regression
positive
of the samples
samples
that
tumor
NK
(10-13).
process
incidences
neither
blood
blood
samples
responsiveness
positive
the
shown
plete
lates
by
This
as a NK
secretion
of IL-12
models
IL-12.
the rest
with
with
the
after
activity
tumor
effects
2).
acts
the
IFN-y
of
1 and
activities;
of the Thl
been
effector
(4)
pleiotropic
Refs.
stimulates
therapeutic
the host
in
CTL
that
murine
to exhibit
pg/mI
and null IL-12 responsiveness
stages or at a given advanced
along
correlated
lated
from
patient
responses,
and their
samples
The
production
at all cancer
However,
types.
1000
showed
whereas
responses.
in induc-
induced
with
individuals
for controls,
of IFN-y
patients
(stage
production
samples
proof the
A comparison
was
served
as controls
Approximately
half
of the
levels of LFN-y
production
almost-null
capacity
in cancer
stage
blood
all healthy
The
the efficacy
for IFN-y
of whole
various
to stimulate
killer
ing IFN-y
secretion
in cancer
patients.
made
between
healthy
individuals
who
and
potent
and
(5-7);
maturation
The
12 (IL-12)
shown
(reviewed
particularly
promotes
ABSTRACT
been
NK
kines,
565, Japan
[M. 0., H. F.]; and
Massachusetts
02140
[H. S.]
has
and
various
Interleukin
y
Patients
IL-122
Saotome,
Kyoto
Interferon
INTRODUCTION
Tanigawa,
Kishida
Research,
for
2425
Research
Status
Hideo
J. S., M. T., A. K., T. K.l; Biomedical
University
Genetics
Stages
Performance
Junko
Fujiwara,
Pasteur
12 Responsiveness
Cancer
are:
were
IL, interleukin;
mononuclear
PS, performance
cell;
various
selected
rIL-l2,
status.
from
NK,
cancer-bearing
those
natural
recombinant
who
killer;
human
were
PBMC,
IL-I 2;
2426 Differential
IL- 12 Responsiveness
receiving
medical
polyclinic
of Louis
care
Blood
was
subjects
had
no acute
or
patients
with
tumors
(n
9), ovarian
other
types
after
tests.
hepatic
(n
24),
age,
Patients
who
the past
2 months
(n
Cytokines.
13) and
58. 1 ±
infec-
no abnormal
value
12.4
consisted
tumors
19),
included
years;
9),
=
tumors
(n
3),
50 men
and
range,
25-88
and
59
years).
or chemotherapy
within
excluded.
rIL-l2
was
supplied
by
Genetics
Institute,
0
Inc.
(Cambridge,
was
supplied
Japan)
MA).
by
and
ized
to NIH
tional
Units
tube
of Blood
was
tube
at 3000
were
suspended
mented
with
FCS,
rpm
to the
procedure
method
(21):
diluted
For
at -80#{176}C until
of IFN-y
was
measured
by
was
prepared
using
monoclonal
antibody
(Cambridge,
MA)j
IFN--y.
ANOVA
tistical
(Abacus
ELISA.
[201
rpm
of
were
20
and
h.
stored
SE.
purchased
activity,
(Genzyme
Corp.,
protected
were
Inc.,
IFN--y
Endogen
was
ELISA
X
pur-
as well
corresponded
kits (recom10”
Cambridge,
units/mg).
MA)
were
concentration.
are presented
was
significant
computed
Berkeley,
ELISA
from
system
analysis
least
IFN-y
(Bl33.5
6.7
of TNF-a
Results
concen-
in our laboratory)
specific
Statistical
IFN-y
anti-human
Bl33.5
in PharMingen
and Fishe’s
calculations
was
of
0. 1 unit/mI
Analysis.
Concepts,
Cultures
human
types
in our ELISA
kits
±
The
two
IU/ml
IFN--y;
Statistical
mean
Intern,
Concentration.
biotinylated
for the determination
metic
well
at 37#{176}C
for
at 3000
and biotinylated
One
TNF-a
used
2Gl
and
Endogen
to approximately
Human
PBMCs
NUNC
with
CA).
as the
performed
difference.
StatView
(pg/mi)
Assessing
IFN--y
Blood
after in Vitro
Venous
blood
PBMCs
were
was
drawn
isolated
from
of rIL- 1 2 for 20 h. Culture
and assayed
for IFN--y
blood and
to rIL-l2.
isolated
IFN-’y
dependent
concentrations.
Production
way
in amounts
from
five
PBMC
sample.
contained
The
in whole
be assessed
stimulating
results
blood
isolated
blood
10-1000
or
10000
on stimulation
sample
and one
of PBMCs
in response
blood
1, both
in response
IL-12
dose-
the capacity
IFN--y
whole
of IFN-’y
Healthy
Individuals
whether
the capacity
IFNcould
also
with
from
that
harvested
in Fig.
to IL-l2
directly
instead
of
PBMCs.
A Comparison
between
amined
produce
show
by stimulating
were
WN-y
in an
to produce
portion.
PBMCs
suswith different
produced
induced
ranging
healthy
blood
SNs
response
was observed
except
for one blood
by
Stimulation
each
As shown
PBMC
samples
production
was
pg/ml,
and the maximal
with 1000 pg/ml
IL-l2,
rIL-12.
by Whole
Blood
patients
was
of rIL-12
in parallel
(Fig. 2). The results
with
show
that
individuals
all blood
Whole
Production
and Cancer
Patients.
We cxof PBMCs
from cancer
patients
to
be assessed
by stimulating
whole
cultured
with different
concentrations
that from seven healthy
individuals
samples
blood
from
responses,
differed
in IL- 1 2 responsiveness.
displayed
served
levels
for
almost-null
positive
10000
whereas
of
healthy
or only
samples
pg/ml
IL-12,
from
healthy
positive
using
Sta-
for
in Whole
blood diluted
1 :4 with MEM
and isolated
in RPM!
1640 were stimulated
in vitro
arith-
software
and
concentrations
can
system
human
CO2)
Ltd.,
to each
of 1 ml/well.
(5%
centrifugation
tration
binant
(Nalge
con-
Co.,
isolated
distributed
plates
various
System
rLL-12.
Whole
pended
blood
use.
Measurement
from
of PBMCs,
and
Stim-
whole
Kizai
for stimulation
#{149},
Contained
individuals,
according
with
(Eiken
with
rIL-12.
MEM.
the
cultured
tubes
1640
by
1 :4 with
as
incubator
harvested
with
previously
at a volume
in a CO,
PBMCs
performed
culture
Denmark)
as human
diluted
was
RESULTS
An Assay
MEM.
supple-
antibiotics.
or PBMCs
2 used
after
with
1640
rIL- 12 was
stimulation
in RPMI
washed
of ilL-i
ic;
San
layers
in RPMI
ic;#{149}1
io2
0
#{149}
,
cell
Dickinson,
the upper
and
and
was
blood
collection
heparmnized
CPT
(Becton
Blood
with
NUNCLON
chased
of PBMCs.
from
HEPES,
described
Japan).
were
and Isolation
for 20 mm
blood
blood
suspended
Roskilde,
in Interna-
heparin
of rIL- 1 2 in F-spitz
conducted
expressed
PBMCs,
10.-
Fig. I
IFN--y production
in whole
blood
or by isolated
lymphocytes
from healthy
individuals
after stimulation
in vitro with IL-12.
Diluted
(1:4) whole
blood
or isolated
PBMCs
(106
cells/ml)
from five healthy
individuals
were cultured
with various
concentrations
of rIL- 12 for 20 h
in F-spitz
tubes or 48-well
culture
plates,
respectively.
Culture
SNs were
assayed
for IFN--y concentrations.
Each symbol
represents
whole
blood
and PBMCs
from
the same
subject
(#{149},
subject
1;
subject
2; A,
subject
3;
subject
4; V. subject
5).
standard-
blood
10
‘
Concentrations
(Hyogo,
and
at 10” cells/ml
venous
of whole
48-well
Ltd.
was
a VACUTAINNER
recovered
of Whole
ulation
SNs
sodium
5 mivi
Stimulation
Tokyo,
Co.,
10
form)
of IFN
To isolate
using
were
PBMCs
Heparinized
(natural
titer
an evacuated
heparmn.
with
centrifugation
were
using
drawn
PBMCs
centrations
Samples
drawn
sodium
blood
CA).
Research
The
Gg23-901-530
was
preparation
IFN--y
milliliter.
containing
Jose,
Chemicals
reference
per
blood
venous
leukocyte
as the standard.
Preparation
Venous
Human
Nihon
used
iymphocytes
of
(n
breast
tumors
radiotherapy
were
healthy
=
uterine
isolated
The
group
(n
at the
(Kyoto,
of chronic
stomach
tumors
=
had received
29),
3),
tests
consent.
patient
=
(n
(n
blood
Research
exhibited
cancer
lung
of tumors
(mean
informed
and
tumors
=
with
Medical
and no history
The
tumors
women
for
infection
disease
blood
Patients
check
Center
drawn
autoimmune
on routine
colon
or a health
Pasteur
Japan).
tious
in Cancer
samples
IFN--y
production
the
marginal
responses.
to those
cancer
again
the
on
of
exhibited
cancer
patients
six of nine
comparable
rest
obtained
similar
from
Whereas
controls,
were
nine
to those
the
samples
The
peak
stimulation
observed
samples
ob-
exhibited
levels
with
for samples
in six
1000-
from
Clinical
Fig.
2
of IFN--y
cancer
(1:4) whole
blood
healthy
individuals
patients
samand
were cultured
with
of rIL-12 for 20 h
various concentrations
in F-spitz
tubes,
and culture
assayed
for IFN--y concentrations.
SNs
were
0
10
10
i02
0
Concentrations
more
1
c
mately
..-
half
by
#{149}0
0
of IL-12
responsiveness
0
0
ferential
S
5
0
0
10
various
So
0
#{149}
Cancer
Healthy
Comparison
and
healthy
individuals.
responders
stimulation
IFN-’y
Diluted
individuals
and cancer patients
for 20 h in F-spitz tubes.
positive
were
Because
with
some
exhibited
10000
pg/ml
from
from
samples
The
with
1000
Patients
to be modulated
responses
to those
cancer
mean
stage
ity
IFN--y
made
patients
stimulation
IFN-y
IU/ml)
was
between
(n
due
values
in
significantly
1000
in the
lower
of IFN--y
part
to
cancer
of lympho-
of tumors.
of
stage
III and
on
ally
it
high
is
than
the
production
presence
30)
(n
IL-12
Cancer
whether
that
(Fig.
3).
patients
(3.2
±
seen
healthy
The control
group
and the difference
between
of
in the
healthy
the two
groups
individuals
also
In
more
positive
classified
IV may
that
more
(low
of the
all patients
half
difference
in stage
III. Thus,
between
exhibiting
null
at earlier
of the
stage
responses.
based
on
in Fig. 4 as one of three
cancer
stages,
different
to the description
results
patients
values
show
was
that
a reduced
observed
of IFN-)I
Committee
capacity
as their
production
on
of IFN-y
PS status
are as follows:
(Fig.
(22)
healthy
The
5).
production
progressed.
we
PS grades
Cancer
Joint
it
IV patients
(PS 0, PS 1 + 2, and PS 3 + 4) according
American
re-
stages,
by
the
± 0.7
capac-
III (P < 0.05).
difference
IV than
to considerable)
classification
2.7
to the exception-
patient
a
4.
III, and
a lower
a statistical
patients
than
and
at stage
actually
at stage
in Fig.
I + H, stage
be related
of one
is
Canblood
± 4.5,
and
although
frequent
obvious
Instead
III
Contre
IV showed
did those
there
fact,
10.5
at stage
than
stages
in whole
is summarized
of stage
± 0.8,
in stage
cancer
production
stages
responsiveness
obscure
showed
production
IL-l2
pg/mi
cancer
and
4.4
stage
different
Internationale
values
production
between
obtained
for IFN-’y
27)
=
with
production
controls
individuals
(7.9 ± 1.3 lU/mi;
P < 0.005).
showed
a wide variation
in IFN-y production,
in the mean
included
to the tumor-node-metas-
IFN-y
Patients
value
was
IL-l2-induced
We
dif-
number
type
according
at various
mean
A comparison
after
the
of three
of Union
were
respectively.
for
IV)
production
IV patients
IU/ml,
were
blood
patients.
for producing
variables
the
and
as one
patients
IFN--y
sponses
cancer-bearing
These
classification
stimulation
with 1000 pg/mI
IL-12, the following
analyses
done by stimulating
whole blood with 1,000 pg/mI IL-l2.
by whole
in cancer
is responsible
IL-12-stimulated
stages.
was
conditions
to IL-l2,
graded
A high
groups
both
lower
compared
between
blood
were cultured
samples
IL-12
production
whole
somewhat
(TNM)
cer (UICC).
cancer
be
responses.
in Cancer
of examination,
I + II, III, and
tasis
patients
IL-l2-stimulated
cancer
patients.
of
individuals
from healthy
pg/mI rIL-l2
may
approxi-
almost-null
is considered
variable(s)
as responders
patients
g
individuals
0.73
exhibited
whereas
0
(stages
various
IU/ml),
Responsiveness
and/or
which
PS at the time
cytes
000
000000g
after
were
Nevertheless,
individuals
showed
IL- 1 2 responsiveness.
stages,
S
o
S
0
factors
investigated
0
0
Fig. 3
healthy
1 .5
patients
PS.
production.
healthy
than
of the cancer
(pg/mi)
0
20
:
(more
IL-12
i02
IFN--y
30 of the
Correlation
with
C
0
high
all
responses
10
for stimulation
a particularly
importantly,
positive
3
10
of riL-1 2 used
exhibiting
.
2427
Research
IL-12 dose-dependent
stimulation
production
in the whole blood
system.
Diluted
pies from
seven
nine
Cancer
in the
The
mean
individuals,
2428
Differential
IL- 1 2 Responsiveness
in Cancer
Patients
P<0.05
A
-
S
40
E
E
..-
..-
.30
C
0
0
U
U
0 20
0.
C..
0
0.10
S
±
A
A
10
.::
:ss5
:.
±
#{149};:
t*
U-
A
AAAA
,$2.
t
-
S
-
-
S
Cancer
.
PSO
stage
PS1+2
PS3+4
Cancer patients
IL-12-stimulated
IFN--y production
with various
stages.
Diluted
whole
at various
stages
were cultured
with
tubes.
A, and
represent
the
patient
at cancer
stages
I + II, III,
#{149},
S
.:
w
Ill
I +11
Fig. 4
patients
patients
in F-spitz
by each
A
A
S
A
in whole
blood from cancer
blood samples
from cancer
1000 pg/mi rIL-l2
for 20 h
value of IFN--y production
#{149}
and IV, respectively.
IFN-y production
in whole
blood
PS groups. Diluted whole blood
patients
were cultured
with
1000 pg/mi
tubes. #{149},
PS 0 cancer patients; A, PS I +
Fig.
in
3
6
from
4 cancer
+
stage
samples
various
IV cancer
patients
from stage IV cancer
rIL-12
for
2 cancer
20
h in F F-spitz
patients;
#{149}
, PS
and
patients.
VU
IV patients
50
E
0
lu/mi
40
C
ck3
0
00
30
.
PS
3
+
4 patients,
indicate
#{163}
AA
oGO
Healthy
individuals
PSO
PS1+2
whether
in each
cant
IL-l2-stimuiated
IFN-y
production
in whole
blood from cancer
in various
PS groups.
Diluted
whole
blood
from healthy
mdiand cancer
patients
in various
PS groups
were
cultured
with
1000 pg/mi rIL-12 for 20 h in F-spitz tubes. 0,
A, and
represent
the value
of IFN--y
production
by healthy
individuals,
PS 0 cancer
patients,
PS 1 + 2 cancer patients,
and PS 3 + 4 cancer
patients,
respectively.
difference
4 patients
patients,
3.2
PS 0 patients,
± 0.6
lU/mi;
6.4
and
PS
lU/mI
(statistical
analysis:
healthy
tients,
P < 0.001
; healthy
individuals
P < 0.001
0.001
; PS 0 patients
0 patients
significant
0.8
IU/ml)
remarkable
responses
(34.
; healthy
1%)
individuals
versus
±
1.5 IU/ml;
3 + 4 patients,
individuals
1 + 2
1.0
± 0.8
PS 0 pa-
versus
PS 1 + 2 patients,
versus
PS 3 + 4 patients,
versus
PS 1 + 2 patients,
P < 0.05;
P <
and PS
PS 3 + 4 patients,
P < 0.01). There
was no
difference
in IFN-y
production
between
PS 1 (3.7 ±
versus
and
PS
that
the incidence
increases
to PS
3 +
2 (3.3
from
4 (85%).
±
0.9
IU/ml)
of patients
PS
with
0 (20.5%)
This
was
patients.
also
null
It is more
PS
the case
when
1 +
stage
PS grading,
even
also
tients
bearing
there
was
depend
the
types
significant
2
0
the
to the numwe
the
determined
of lympho-
mean
there
number
was
patients
and
decrease
in
of
no signifiPS
responsiveness
IFN--y-producing
of
tumors.
difference
patients.
decreases
PS
on
different
these
To
3
+
4
in PS 3 +
the
number
of
to IL-12.
compared
no
Although
of IL-l2
The
with
capacity
As
shown
in IL-l2
results
PS
show
grade
in
Fig.
8,
responsiveness
that
each
in pain
IL-12
respon-
categorized
tumor
group.
IL-12
Responsiveness
Samples
Showing
whether
PBMCs
marginal
responses
isolated
from
of plasma.
(PS
in
have
the capacity
1 + 2) exhibiting
in Fig.
(>2.0
isolated
zero
9, 7 of 13 PBMC
IU/ml).
These
blood
and stimulated
were
results
Isolated
Responses.
contained
the blood
PBMCs
of PBMCs
Null/Marginal
from
to only
of WN--y production
by whole
RPMI
1640 were stimulated
with
or marginal
through
7).
with
as responders
We
PS
not
of
of Tumors.
the number
(Fig.
the reduction
does
lymphocytes
siveness
lU/mi;
group
cancer
PS.
to the Number
blood,
and
null
these
with
is related
in whole
capacity
between
Thus,
among
2.4
IL- 12 responsiveness
of
among
correlates
and to the Type
contained
of
± 3.7
collectively,
Responsiveness
Blood
decreased
patients.
#{149}, #{149}
±
PS
lymphocytes
Fig. 5
patients
viduals
15.2
of IL-12
4.8
incidence
differs
responsiveness
the IFN-’y-producing
cytes
PS3#{247}4
Cancer patients
the
Taken
IL- 1 2 responsiveness
of lymphocytes
both
___
grading.
grades
were
in PS 1 + 2 patients,
(PS 0 patients
versus
and
0.05),
PS
differential
in the
determine
<
with
that
and
Lymphocytes
ber
P
increased
Relationship
0
0
to the three
production
in PS 0 patients,
2.7 ± 0.6 lU/nil
± 0.9 lU/mI in PS 3 + 4 patients
00
A
according
of IFN--y
0.3
patients,
0
classified
values
and
data
0
were
6); the mean
responses
0
‘,
only
PS (Fig.
from
Blood
We examined
samples
showing
nulL’
to respond
to IL- 12 when
with
IL-l2
in the absence
the blood
of 13 patients
marginal
levels
(< 1.0 IU/ml)
blood.
PBMCs
suspended
in
1000 pg/ml rIL-12.
As shown
samples
indicate
produced
that
positive
PBMCs
responses
in
approxi-
Clinical
Healthy
individuals
S
40#{149}
Cancer
Research
2429
S
S
55
30
.
S
20
,
S
E
#{149}S
S
S
10
S
S
S
55
j
S
C
.
0
S
I.’:,te,
#{149}
,#55,
4-’
55
.
PS3+4
PS1+2
C.,
1- 30
20
S
S
10
S
Whole
blood
.S
:.?Ii*
..
i’o
_o_
.
20
30
.
,
40
No. of lymphocytes
Fig.
7
Relationship
between
IFN--y
S&..,.S5
500
10
(xl O5cells
production
20
30
40
50
/ml)
and
the
number
of
lymphocytes
in whole blood from cancer patients in various PS groups.
Whole
blood samples from healthy individuals
and various PS groups of
cancer patients were examined for IL-12 (1000 pg/ml)-stimulated
IFN--y
production.
The number of lymphocytes
contained
in each of these
blood samples was also determined.
The point indicating
IFN-’y production and the lymphocyte
number
in each subject
were individually
plotted.
Fig. 9 IFN--y production
in whole
biood
or by isolated
lymphocytes
from patients
after stimulation
with IL- 12. Diluted
( 1 :4) whole
blood or
isolated
PBMCs
(106 cells/mi)
from 13 patients
were cultured
with 1000
pg/mi rIL-12
in a culture
system
similar
to that described
in the Fig. 1
legend.
Dashed
line, 2.0 lU/mi.
Production
TNF-a
lation.
TNF-a
We finally
(23).
Liver
#{149} Gastro-intestinal
40
30
4-a
U
d’
A
PSO
PS1+2
thL
#{149}#{149}A
PSO
PS3+4
10). Patterns
2 and
5 were
PS1+2
#{149}.:..
PS3+4
50
ulation,
most
enhanced
almost
no
of
of IL-l2
2:
Uterus
cate that TNF-a
IL-12
stimulation
30
S
10
S.5
5
e
44e
I’I
U
PSO
S
-
PS1+2
PSO
PS3+4
A
bearing
1000 pg/mi
half
responsiveness
expression
plasma.
healthy
in all
besamples,
of TNF-a
were
detected
After
IL-12
individuals
and
PS
In contrast,
of TNF-a
IFN--y
concentration
level
of PS grades.
of TNF-a.
in PS 3 + 4 patients,
to induce
IFN-y
in Figs.
as in each
0 patients
only
production
which
Thus,
pro-
marginal
was
correlated
production.
in
stim-
with
these
the failure
mdi-
results
production
is enhanced
in whole
in association
with the induction
or
observed,
blood
after
of IFN--y
DISCUSSION
AjA
The
#{149}.e.
PS1+2
PS3+4
Fig. 8 IFN-y production
in whole blood from patients bearing various
types of tumors in different
PS groups.
Diluted
whole blood from
mately
the
concentration
production.
20
patients
levels
irrespective
rIL-12
to those
as well
zero
Each
pg/ml
TNF-a
individuals
at the
Stimucytokine
is also
stimulation.
1000
similar
Whereas
almost
enhancement
especially
LI.
patients.
levels
Ovary
and
IL-12
antitumor
production
IL-l2
production
to considerable
duced
after
with
production
in healthy
was
blood
after
stimulated
of IFN--y
of cancer
Breast
Lung
IFN--y
stimulation
0. 40
2
blood
samples
observed
unstimulated
I
0
for
assayed
(Fig.
significant
S
10
C
0
was
fore
20
E
blood
Blood
representative
whether
TNF-a
in whole
of the whole
PS group
S
in Whole
is another
examined
induced/enhanced
,u
Isolated
lymphocyte
various
rIL-12
types
of tumors
for 20 h in F-spitz
of the blood
samples
(shown)
were
cultured
with
showing
null/marginal
IL-l2
to IL- 12, but
in the presence
the
of
results
to healthy
difference
duction
whole
tive
tubes.
have the capacity
to respond
of such a capacity
is inhibited
pared
obtained
in IL-12
in whole
blood
after
from
although
contrast,
approximately
showed
levels
of
present
cancer
necessarily
number
tumor
of
types;
was
subjects
IFN-y
production
correlate
with
by IFN-’y
IL-l2.
pro-
Namely,
produced
posi-
variation.
cancer
comparable
the
corn-
In
patients
to those
for
of the samples
exhibited
IL- 12 responsiveness
did
stages
contained
it correlated
from
that
a substantial
a considerable
half of the samples
lymphocytes
rather,
with
all healthy
there
show
exhibit
as measured
stimulation
healthy
samples,
whereas
the rest
almost-null
responses.
The reduced
not
study
patients
responsiveness
blood
samples
responses,
in the
individuals,
in
with
of
blood
the
cancers,
samples,
the
or
the PS of the patients.
the
2430 Differential
IL-12
Responsiveness
in Cancer
ia
;
Healthy
PS 0
PS 1 +2
detrimental
represent
PS 3+4
#{149}
45
E
Patients
considered
C
0
to
relation
with
remains
to
reflected
10
U
effects
of the tumor
the overall
condition
be
in
TNF-a
5
1000
3
/
E
#{149})
600
0.
C
0
400
0
0.
+
+
-
Stimulation
+
-
with
1000
+
The
antitumor
various
murine
administration
efficacy
of IL-12
models,
which
has been
( 1 8), IFN-y
production
of
in the
assessing
the
IFN-’y
in response
determine
by T cells
IL-12-induced
of
whether
results
demonstrate
IL-12
production
after
necessarily
correlate
with
be
the PS of patients.
stages
increases
of PS.
ness
was
Because
with
also
the grade
expressed
TNF-a
on stimulation
ment
with
of TNF-a
by
be expected.
blood
culture
system
but
most
does
rather
closely
remarkable
IL-12
production
mainly
coris
IFN--y
observed
in whole
evaluating
focusing
concept
of PS
the effects
on the
state
was
first
of the
tumor,
seems
described
as a criterion
takes
(26).
into
Instead
account
be
immune
IFN-y/
by the number
the mean
to produce
system
blood
IFN--y
found
control
group,
the major
that
there
of
but
the
responders
was
CD56
18.6
culture
to
no substantial
NK
cells
± 1.5 (healthy
the
system
capacity
blood
used
(21).
63.9 ± 1.8
4 patients)].
of
here
PBMCs
and
to a lack
of
We
production
was
originally
showed
that
in
in healthy
has some advantages:
permits
us to examine
by
in subjects
system
the
this
whole
individuals,
with
and
correctly
than
in
are
stimulated
with
11-12
positive
culture
medium
possible
such
instead
that
is modulated
IFN--y
by
the
IL-l2
more
the
assay
in
in the
growth
factor
which
present
in the
levels
31),
FCS-
However,
in the
contained
3 (30,
isolated
of
serum.
induced
factors
event
in this
presence
own
production
actual
be reflected
be the real in vivo event.
shown that abnormally
high
as transforming
the
(b)
may
of their
various
as little as 1 ml of
effect
of IL-12
on
(a)
PBMCs;
treated
PBMCs
the
production/secretion
cytokine,
IFN--y,
context,
we found
of
by T cells
that
and
when
blood,
of cytokines
IL-6
(32-34),
the cytokine-producing
or in the whole blood
factors
for
of
any
found
and
that
and
responses,
suggesting
mechanism
leading
Whereas
2 patients
determine
progression.
(22,
from
whole
PBMC
samples
are consistent
responses
culture
system
coexisting
from
samples
the existence
to the
these
cells
two
mdito
antitumor
32,
33).
blood
In this
samples
null/marginal
LL-12 responsiveness
with rIL-12
in culture
medium,
leukocytes
PBMCs
a representative
NK
PBMCs
of some patients
exhibiting
were isolated
and stimulated
selves.
of
patients.
contained
that,
and
individuals)
is not ascribed
in
study
cytokines,
induced
also
of
in the PS 1 + 2 and PS
cells
(1-3,
8), we analyzed
the
several
cancer
patients,
especially
previous
the
of
value
is a close
correlation
between
cytokine
production
in
blood
and that in isolated
PBMCs.
The whole
blood
serum
to be secondary
drugs
PS
assess
culture
venous
vivo
the enhance-
(23-25),
here
of chemotherapeutic
can
there
whole
that
blood.
depending
to IFN-’y production.
Consistent
with this, an almost
complete
correlation
was observed
between
the enhanced
production
of
TNF-a
and the PS of the patients.
The
system
blood
lower
cells
approximately
half of these
responses.
These observations
responsive-
by macrophages
may
express
Namely,
responsiveness
blood
in our
inhibit
not
feature
cor-
of PS
IL-lO (35, 36) are detected
in blood
from tumor-bearing
viduals.
Each
of these
cytokines
has been
demonstrated
by IFN--y
patients,
Differential
T cell-derived
production
to
some of the cancer
patients
incidence
of null responders
TNF-a
is produced
test
as measured
The
whole
developed
it is also
to produce
screening
its
IL- 12-stimulated
a healthy
T cells/NK
although
it may
It has been
patients
by
Because
We
IL-12
Thus,
in cancer
subjects,
and the
null
reaction.
first
PS
to
± 3.5% (PS 3 + 4 patients);
CD3CD56,
individuals)
and 52.6 ± 4.8% (PS 3 +
cascade
the
in
[CD56,
system
whole
IL- 12 stimulation
that in contrast
to healthy
produce
null responses,
or
it was
T cells
to the
can
in our
cancer
systemic
slightly
percentages
is central
responsiveness,
with
the
regression
was
the
NK cells
in cancer
effects
obtained
that
and
would
the IL-l2
investigated
tumors
(10-13).
Although
to induce
antitumor
effects
PBMCs
to IL-l2
that
complete
antitumor
capacity
well
revealed
of rIL- 12 results
substantial
inhibition
of s.c. growing
IFN--y production
is not sufficient
relates
in
The
Fig. 10
IL-12-stimulated
TNF-a
production
in whole
blood from cancer patients
in various
PS groups.
Diluted
whole
blood
samples
from
healthy
individuals
and cancer
patients
were cultured
in the presence
or
absence
of 1000 pg/mI rIL-12
for 20 h in F-spitz
tubes,
and culture
SNs
were assayed for IFN-y and TNF-a
concentrations.
Each symbol
represents
IFN--y production
and TNF-a
production
from the same sample.
that
suggests
patient
blood.
not significant.
IL-12-responsive
ilL-i 2
entity
was not determined
PS 3 + 4 patients.
and 20.8
(healthy
-
pg/mI
of
the
here
T cells
and NK
population
from
Therefore,
-
than
was
CD3CD56
I-
The
4 groups
difference
5-.
scenario
+
from
e 200
because
the
in whole
numbers
IL-l2
are
lymphocyte
z
factor
Nevertheless,
study
of
responsiveness
difference
U-
This
capacity
contained
lymphocyte
in
clarified.
as exemplified
PBMCs
U.
prognostic
(27-29).
host and may thereby
patient.
PS has been
production.
IL-12
±
a useful
the
responsiveness
0
0
0.
C.-
be
survival
on the
of the
dysfunction
mechanisms
produced
positive
with the possibility
of T cellsfNK
cells
could be modulated
by
in blood.
we
However,
still exhibited
almost-null
of an additional
suppressive
of responding
were
observed
cells
in
them-
in PS
in this study,
further
analysis
will be required
how the ratio of the two mechanisms
changes
with
1 +
to
PS
Clinical
Our
results
illustrate
IL-l2-stimulated
patients,
and
stimulated
that
IFN--y
such
IFN-’y
antitumor
efficacy
of
contribute
not
to the
only
step
is a substantial
by
a difference
production
first
there
production
correlates
Thus,
selection
of the
the
essential
the
of
IL-l2
among
with
is an initial
IL-12.
difference
PBMCs
PS.
exhibit
the
effect
but
also
opment
function
of approaches
to analyze
and correct
the
occurring
in patients
with reduced
IL-l2
induces
tumor
could
who
can
to the develimmunodysresponsive-
tive
immunity:
pressed
1135-1145,
13.
14.
for Medical
and
help
with
this work.
Mu,
J., Zou,
REFERENCES
senting
T-helper
4027,
a cytokine
cells with immunoreguiatory
cells type I and cytotoxic
produced
functions
lymphocytes.
by
M. J. Interleukin-12.
Kobayashi,
M.,
Fitz,
J. Leukoc.
in the generation
of
Blood, 84: 4008-
L.,
Ryan,
M.,
Bioi.,
55:
Hewick,
R.
280-288,
M.,
1994.
Clark,
S. C.,
Chan, S., Loudon,
R., Sherman,
F., Perussia,
B., and Trinchieri,
G.
Identification
and purification
of natural killer cell stimulatory
factor
(NKSF),
a cytokine
with multiple biologic effects on human lymphocytes. J. Exp. Med., 170: 827-845,
1989.
4. Stem, A. S., Podlaski, F. J., Huimes, J. D., Pan, Y. C., Quinn, P. M.,
Wolitzky,
A. G., Familletti,
P. C., Stremlo, D. L., Truitt, T., Chizzonite,
R., and Gately, M. K. Purification
to homogeneity
and partial characterization
of cytotoxic
lymphocyte
maturation
factor from human Blymphobiastoid
cells. Proc. Nati. Acad. Sci. USA, 87: 6808-6812,
1990.
J. P., Yamamoto,
N.,
to
H..
Zou,
of cellular
tumor
regression.
Res.
underlying
795: 294-309,
Ogawa,
M.,
Tsutsu,
Herrmann,
Enhanced
induction
sites
2216-2222,
T.,
S.,
following
K.,
8. Chan,
S. H., Perussia,
B., Gupta,
J. W.,
Kobayashi,
M.,
Santoii,
D.,
Pospisii,
M.,
Young, H. A., Wolf, S. F., Young, D., Clark, S. C., and Trinchieri,
G.
Induction
of interferon
-y production
by natural killer cell stimulatory
factor:
characterization
inducers.
J. Exp. Med.,
of the responder
173: 869-879,
cells
1991.
and
synergy
with
other
9. Manetti, R., Parronchi,
P., Giudizi, M. G., Piccinni, M. P., Maggi, E.,
Trinchieri,
G., and Romagnani,
S. Natural killer cell stimulatory
factor
(interleukin
12 [IL-12])
induces
T helper
responses
and inhibits the development
J. Exp. Med., 177: 1199-1204,
1993.
10.
Brunda,
M. J., Luistro,
B. R., Murphy,
Nastaia,
C.
L.,
1 (Thl)-specific
of IL-4-producing
R. R., Wright,
M., Wolf, S. F., and Gately,
activity
of interleukin
12 against
antimetastatic
Med., 178: 1223-1230,
11.
L., Warner,
type
immune
Th cells.
R. B., Hubbard,
M. K. Antitumor
and
murine tumors. J. Exp.
1993.
Edington,
D.,
McKinney,
T.
G.,
Tahara.
Koba-
H.,
Nalesnik,
M. A., Brunda, M. J., Gately, M. K., Wolf, S. F., Schreiber,
R. D., Storkus, W. J., and Lotze, M. T. Recombinant
IL-12 administra-
nodes.
Cancer
Res.,
55:
T. Cellular
tumor
J. P.,
T.,
and molecular
regression.
Mu,
J.,
Fujiwara,
Ann.
Wijesuriya,
N.
R.,
Y.
Yu,
H., and
Hamaoka,
T-ceil
migration
to
Res.,
57:
of IL-12.
Cancer
T.
1997.
lates
and its secondary
induction
62: 450-457,
1997.
18.
Brunda,
of anti-tumor
M. J., Luistro,
G., and Gately,
M. K. Role
efficacy
of interleukin-l2.
Immunol.,
17: 71-77,
1995.
19.
Fujiwara,
H.,
and
mor T cell responses
pathways.
L., Hendrzak,
Garotta,
antitumor
J. Leukoc.
Biol.,
J. A., Fountoulakis,
of interferon--y
J. Immunother.
M.,
in mediating
the
Emphas.
Tumor
Hamaoka,
T. Regulatory
mechanisms
in the tumor-bearing
state. Immunol.
of antituRes., 14:
1995.
Atkins, M. B., Robertson,
M. J., Gordon, M., Lotze, M. T., DcCoste, M., DuBois,
J. S., Ritz, J., Sandier,
A. B., Edington,
H. D.,
Garzone,
P. D., Mier, J. W., Canning,
C. M., Battiato, L., Tahara, H.,
and Sherman,
M. L. Phase I evaluation
of intravenous
recombinant
20.
12 in patients
with
advanced
malignancies.
Clin.
1997.
Uno, K., Nakano, K., Maruo, N., Onodera, H., Mata, H., Kurosu, I.,
Akatani, K., Ikegami, N., Kishi, A., Yasuda, Y., Tanaka, K., Setoguchi,
J., Kondo, M.. Muramtsu,
S., and Kishida, T. Determination
of inter21.
22.
cer,
in whole
blood
healthy
persons.
American
Joint Committee
on Cancer.
3rd ed. Philadelphia:
J. B. Lippincott
cultures
from patients
J. Interferon
Cytokine
Manual
for Staging
Co., 1988.
with
Res.,
of Can-
23. Yamamoto,
N., Zou, J. P., Li, X. F., Takenaka,
H., Noda, S., Fujii,
T., Ono, S., Kobayashi,
Y., Mukaida, N., Matsushima,
K., Fujiwara,
H.,
and Hamaoka,
T. Regulatory
mechanisms
for production
of IFN--y and
TNF by antitumor
T cells or macrophages
in the tumor-bearing
state.
J. Immunol.,
154: 2281-2290,
1995.
24. Tracey,
K. J., and Cerami, A. Tumor necrosis factor, other
kines and disease.
Annu.
Rev. Cell Biol.,
9: 317-343,
1993.
25. Collart,
M. A., Belin,
D.,
Vassalli,
P. -y Interferon
enhances
necrosis factor/cachectin,
controlled
by short-lived
1986.
26.
H.
X. G.,
W. G., Ogawa,
M., Mu, J., Umehara,
K., Tsujimura,
T.,
H., and Hamaoka,
T. IL- 12-induced
tumor regression
correwith in situ activity of IFN--y produced
by tumor-infiltrating
cells
feron-a-producing
capacity
various
diseases
and from
16: 91 1-918, 1996.
Tsuji,
Tai,
Yu,
Fujiwara,
17.
7.
A.,
lymph
administration
interleukin
D’Andrea,
T.,
of VLA-4/LFA-i-dependent
Res., 3: 409-417,
H.,
7:
S., and Hamaoka,
T. A seevents involved
in IL-12-induced
146: 638-644,
1995.
Zou,
Kubo,
Cancer
S.
Tsutsui,
IL- 12-induced
1996.
human
Chan,
of sup-
Herrmann,
Immunol.,
6. Gately, M. K., Desai, B. B., Wolitzky,
A. G., Quinn, P. M., Dwyer,
C. M., Podlaski, F. J., Familletti,
P. C., Sinigagiia,
F., Chizzonite,
R.,
Gubier, U., and Stem, A. S. Regulation
of human lymphocyte
proliferation by a heterodimeric
cytokine,
IL- 12 (cytotoxic
lymphocyte
maturation factor). J. Immunol.,
147: 874-882,
1991.
Pospisil,
M., Young, D., Wolf, S. F., and Trinchieri,
G. Natural killer
(NK) cell stimuiatory
factor or IL-12 has differential
effects on the
proliferation
of TCR-a3+,
TCR--y6+
T lymphocytes,
and NK cells.
J. Immunol.,
149: 3495-3502,
1992.
and
molecular
271-291,
B.,
reversal
mt. Immunol.,
T cells.
H., Clark, S. C., and Hamaoka,
W. G.,
tumor
lung
J. P.,
and
Robertson,
M. J., Soiffer, R. J., Wolf, S. F., Manley, T. J., Donahue,
C., Young, D., Herrmann, S. H., and Ritz, J. Response of human natural
killer (NK) cells to NK cell stimuiatory
factor (NKSF): cytolytic activity
and proliferation
of NK cells are differentially
regulated
by NKSF.
J. Exp. Med., 175: 779-788,
1992.
5.
Perussia,
a striking
1995.
Fujiwara,
quence
16.
antigen-pre-
1994.
2. Brunda,
3.
Interieukin-12:
with
by anti-tumor
spontaneously
mechanisms
Acad.
Sci.,
G.
is correlated
production
1995.
15. Fujiwara,
1. Trinchieri,
production.
1994.
yashi,
M., Herrmann,
S., Fujiwara,
H., and Hamaoka,
T. Administration
of recombinant
interleukin
1 2 prevents
outgrowth
of tumor cells metas-
ACKNOWLEDGMENTS
for assistance
response
IFN-gamma
4404-4408,
Center
IFN--y
with
12. Zou, J. P., Yamamoto,
N., Fujii, T., Takenaka,
H., Kobayashi,
M.,
Herrmann,
S. H., Wolf, S. F., Fujiwara,
H., and Hamaoka,
T. Systemic
administration
of rlL- 12 induces
complete tumor regression
and protec-
in the
tasizing
Pasteur
in association
IL-12-
ness.
We thank the members
of the Louis
Research
and the Kyoto
Red Cross Center
regression
153: 1697-1706,
2431
J. Immunol.,
study
patients
tion
Research
cancer
process
present
cancer
in
Cancer
Kamofsky,
D. A.,
Vassalli,
J.
macrophage
interleukin
repressors.
and
Burchenal,
191-205,
1949.
D., de Kossodo,
S., and
transcription
of the tumor
1 , and urokinase
J. Exp. Med.,
J. H. The
chemotherapeutic
agents in cancer. Symposium,
N. Y. Acad. Med., N. Y., 1948. New York:
cyto-
genes,
clinical
which
evaluation
Microbiology
Columbia
are
21 13-2118,
164:
of
Section,
Univ.
Press,
2432
Differential
IL-I 2 Responsiveness
in Cancer
27. Stanley,
K. E. Prognostic
inoperable
lung cancer.
J. Nati.
28.
Berry,
Prognostic
of the
W.
R.,
factors
prostate.
Laazlo,
Cancer
29. Maldazys,
J. D.,
static renal carcinoma.
30. Li,
Nagata,
growth
bearing
in
31.
liver
E.,
Walker,
and hormonally
(Phila.),
44:
and deKernion,
J. Urol.,
136:
susceptibility
Res.,
763-775,
A.,
patients
1980.
and
determination
munol., 145:
with
Paulson,
unresponsive
D.
factors
Shirai,
Y., Kawata,
patients
diseases.
selectively
(Baltimore),
in meta-
34.
T cell function.
S., Tamura,
S., Ito, N., Tsushima,
Y. Plasma
transforming
Jpn. J.
carcinoma.
73: 2275-2279,
Y., Tada,
production
Comparison
1994.
with
T., Ohzeki,
S., Fujiwara,
of IL-6 in tumor-bearing
factor-3
1
chronic
H.,
mice
and
and
J.
Y.,
I., Murachi,
of allograft
production.
M., Tai,
responses
J. Im-
X-G., Toyooka,
K., and
induced
by IL-6 which
IFN--y but not IL-2 production.
757-763,
Transplantation
1997.
Negrier,
S.,
prognosis
factor
in metastatic
3317-3322,
1992.
H., Takaishi,
growth
Blay,
modulates
64:
for its augmented
1990.
Combaret,
V.,
Attali,
S.,
Goillot,
E.,
Merrouche,
Y., Mercateilo,
A., Ravault, A., Tourani, J. M., Moskovtchenko, J. F., Philip, T., and Favrot, M. Serum level of interleukin
6 as a
1993.
with hepatoceiiular
Cancer
(Phila.),
32. Utsumi,
K., Takai,
Hamaoka,
T. Enhanced
CD4+
of cells responsible
397-403,
33. Tomura,
M., Nakatani,
Fujiwara,
H. Suppression
carcinoma
1979.
J. B. Prognostic
376-379,
1986.
of anti-tumor
84: 315-325,
K., Kiso, S., and Matsuzawa,
in
Cox,
for survival
in
Inst., 65: 25-32,
X. F., Takiuchi,
H., Zou, J. P., Katagiri,
T., Yamamoto,
N.,
T., Ono,
S., Fujiwara,
H., and Hamaoka,
T. Transforming
factor-3
(TGF-3)-mediated
immunosuppression
in the tumorstate: enhanced
production
of TGF-3
and a progressive
increase
TGF-3
Cancer
J.,
in metastatic
factors
Cancer
Patients
renal
cell
carcinoma.
Cancer
Res.,
52:
35. Fiorentino,
D. F., Zlotnik, A., Vieira, P., Mosmann,
T. R., Howard,
M., Moore, K. W., and O’Garra, A. IL-lO acts on the antigen-presenting
cell to inhibit
cytokine
3444-3451.
1991.
36.
Chang-you,
W.,
production
Warner,
R.
by
R.,
Thi
cells.
Wang,
X.,
Gately, M. K. Regulation
of interleukin-12
receptor
and interleukin-l2
binding
by human peripheral
cells.
Eur.
J. Immunol.,
27:
147-154,
1997.
J. Immunol.,
Presky,
D.
146:
H.,
and
3i chain expression
blood mononuclear