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This information is current as leukocyte reaction.
cell activity induced in the syngeneic mixed
mice. II. The I region control of suppressor T
The syngeneic mixed leukocyte reaction in
U Yamashita, S Ono and H Nakamura
http://www.jimmunol.org/content/128/3/1010.citation
1982; 128:1010-1017; ;J Immunol
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Copyright © 1982 by American Association of Immunologists
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
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0022-1767/82/1283-1010$0200/0
Copyrlght
0
1982
by
The
American Associallon
of
Immunologists
THE
JOURNAL
OF
IMMUNOLOGY
VoI
128.
No.
3.
March 1982
Prrntedm
U.S.A
THE
SYNGENEIC MIXED LEUKOCYTE REACTION IN MICE
II.
The
I
Region Control
of
Suppressor
T
Cell Activity Induced in the Syngeneic Mixed
Leukocyte Reaction'
UKI YAMASHITA, SHlRO ON0,2
AND
HlROSHl NAKAMURA
From the Department
of
Immunology, Unrversity
of
Occupational and Environmental Health, School
of
Medicine,
1-1
lseigaoka, Yahatanishiku,
Kitakyushu
807,
Japan
In order to study the functional activity of T cells
responsible
for
the syngeneic MLR, the effect of T cells
stimulated
in
the syngeneic MLR on the induction
of
hapten-reactive cytotoxic
T
cells was analyzed. T cells
cultured
in
vitro with syngeneic spleen cells suppressed
the development of cytotoxic T cells. However; T cells
cultured with allogeneic spleen cells did not, suggesting
that only syngeneic MLR can induce suppressor cells.
The suppressor cells induced in the syngeneic MLR are
Ly-l+ T cells, because the induced suppressor activity
was sensitive
to
the anti-Thy-1 and anti-Lyt-1 serum plus
C
treatment, but was resistant to the anti-Lyt-2 and
anti-
Lyt-3 serum plus
C
treatment. The induction of suppres-
sor T cells required the stimulation by other cell types.
The stimulator cells for the induction of suppressor T
cells were macrophage-like adherent cells, because the
stimulation activity was depleted in a nylon and a Seph-
adex
G-10
column-passed cell population and enriched
in
a
plastic dish-adherent cell population. Suppressor
T
cells induced in the syngeneic MLR showed genetic re-
striction; namely, suppressor T cells induced
in
the syn-
geneic MLR worked on syngeneic cytotoxic
T
cells, but
not on allogeneic cytotoxic T cells. Furthermore, sup-
pressor
T
cells of F, mice stimulated
with
spleen cells of
one parent suppressed cytotoxic T cells of the same
parent, but not of the other parent, suggesting the exist-
ence
of
two distinct clones
in
F1
suppressor
T
cells that
can preferentially suppress cytotoxic T cells of one
or
the other parent. The activity of suppressor
T
cells in-
duced in the syngeneic MLR was regulated by the
I-A-
subregion of the MHC, because T cells cultured
in
vitro
of
BlO.BR,
A.TL, BlO.A,
BlO.R(4R)
mice suppressed the
development of cytotoxic T cells of C3H/He mice, but T
cells cultured
in
vitro of
B10,
BlO.A(5R),
and A.TH mice
did not.
All
these results are compatible
with
the results
obtained by the study of proliferative T cell response.
Thus, the
I
region products of the MHC on syngeneic
macrophages could induce a proliferative response of
T
cells, and these activated
T
cells could suppress the
development of cytotoxic T cells sharing the
I
region of
the MHC. The mechanism of suppression and the role of
suppressor T cells induced in the syngeneic MLR was
Received for publication May
22,
1981.
Accepted for publication November 16, 1981
The costs
of
publication of this article were defrayed in part by the payment
of
page charges. This article must therefore be hereby marked
advertsement
in
accordance with 18
U.S.C.
Section 1734 solely to indicate this fact.
'
Thls work was supported by a Grant-in Aid for Scientiflc Research from the
Japanese Government (Grants
501046
and 557129).
Institute for Cancer Research, Osaka Universlty Medical schook.
1-1-50.
Fukushma. Fukushimaku. Osaka
553,
Japan.
discussed from the standpoint of cellular interaction and
self regulation.
Although a phenomenon of autologous or syngeneic mixed
leukocyte reaction (MLR) has been reported in many labora-
tories
(1
-6),
the functional activity
of
the syngeneic MLR in an
immune response remains to be analyzed.
In
the accompanying
paper
(7)
we have established an experimental system
of
the
syngeneic MLR in mice and reported a cellular and genetic
mechanism of the syngeneic MLR. Our results suggested that
the responder cells were
T
cells, the stimulator cells were
adherent macrophages, and the induction of the syngeneic
MLR was controlled by the
I
region of the major histocompati-
bility complex (MHCh3
In order to further analyze the functional activity of
T
cells
responsible for the syngeneic MLR, we have studied the effect
of
T
cells stimulated
in
the syngeneic MLR on some immune
responses. In this report we will present evidence that the
T
cells stimulated in the syngeneic MLR have suppressor activity
on the induction
of
hapten-reactive cytotoxic
T
cells
in
vitro
and that the activity of suppressor
T
cells is controlled by the
I
region
of
the MHC.
MATERIALS AND METHODS
The strains of mice and the procedures of cell fractionation are the same
as reported in the accompanying paper (7).
Tumors. MOPC104E plasmacytoma cells (MOPC) from BALB/c mice
and X5563 plasmacytoma cells (X55631 from C3H/He mice maintained by
serial i.p. passages into syngeneic mice in ascitic form were utilized as
target cells for the assay of cytotoxic T cell activity.
column-purified
T
cells
(5
x
lo6) with mitomycin C (Kyowahakko
Co..
In vitro selection culture. Unpurified spleen cells
(8
x
lo6)
or nylon
Tokyo, Japan) treated spleen cells
(3
x
1
06)
were cultured
in
vitro in 2 ml
of Peck-Click culture medium
(8)
containing 10% fetal calf serum (FCS;
Grand Island Biological Co.. Grand Island.
NY)
in
a well of 24-well Linbro
culture plates (Flow Laboratories, Rockville, MD) at 37°C for 7 days in
5%
C02 and
95%
air. In order
to
study a functional activity of
T
cells stimulated
in
the syngeneic MLR, the recovered cells from these cultures were added
to
the induction phase of trinitrophenyl- (TNP) reactive cytotoxic
T
cells
at
the concentration of 0.4
to
10
x
1
05/well.
stimulator spleen cells were incubated at 37°C for 1 hr in
RPMI
1640
lnduction
of
hapten-reactive cytotoxic T cells
in
vitro.
10
X
106/ml
culture medium (Grand Island Biological Co.) containing
50
bg/ml
Of
mito-
treated spleen cells were then treated with
10
mM trinitrobenzene sulfonate
mycin C, then washed four times
to
remove mitomycin C. The mitomycin C-
for TNP coupling at 37°C for 10 min according
to
Shearer
(9).
2
X
1
O6
TNP-modified or unmodified spleen cells were cultured with
5
X
lo6
responder spleen cells in a well
of
24-well Linbro culture plates in
2
ml
of
Peck-Click culture medium containing
10%
FCS. Cultures were maintained
for
5
days
at
37OC in
5%
CO,
and
95%
air.
patibility complex:
MOPC.
MOPC104E plasmacytoma cells; NMS, normal mouse
Abbreviations used in this paper:
HS.
human serum:
MHC.
major histocom-
serum: X5563. X5563 plasmacytoma cells.
1010
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
19821
SUPPRESSOR
T
CELLS IN SYNGENEIC MLR
101
1
Assay system
of
cytotoxic
T
cell activity. The 5'Cr release assay was
performed according to Cerottini et a/.
(1
0).
MOPC or
X5563
tumor cells
were radiolabeled by incubation
of
3
x
10' cells in
1
ml of RPMl 1640
medium containing
10%
FCS and 100 pCi of 5'Cr sodium chromate (New
England Nuclear, Boston, MA) in Falcon No. 2057 tubes (Falcon, Oxnard,
treated with 2 mM trinitrobenzene sulfonate for TNP-coupling at 37°C for
CA) at 37°C for 60 min. After washings, radiolabeled tumor cells were
10
min. After extensive washings,
2
X
1
O4
radiolabeled target tumor cells
were mixed with the graded numbers
(50
to
100
X
1
04)
of
in
vitro-sensitized
spleen cells in 0.2 ml of RPMl 1640 medium containing
10%
FCS in a well
of round-bottom microtiter culture plates (Nunc, Roskilde. Denmark). After
the effector-to-target cell interaction was performed at 37°C for
5
hr in
5%
COP and
95%
air, the culture supernatant in each well was harvested by the
aid of a Titertek supernatant collection system (Flow Laboratories, Rockville,
MD), and the radioactivity collected in the filter was counted by a gamma
counter. The percentage of specific lysis was calculated as
Experimental release
-
control release
Maximal release
-
control release
%
Specific lysis
=
x
100
where the maximal release was obtained by incubating 51Cr-labeled target
cells in the presence of
1
% Nonidet P-40 (Sigma Chemical Co.,
St.
Louis,
MO). and the control release was given by incubating target cells with
unsensitized spleen cells. The results were expressed as the mean and
standard error of the percentage
of
specific lysis of triplicate cultures.
RESULTS
Suppressor activity
of
T
cells stimulated
in
the syngeneic
MLR
on the induction
of
hapten-reactive cytotoxic
T
cells
in
vitro. Nylon column-purified T cells of C3H/He mice were first
cultured
in
vitro with mitomycin C-treated syngeneic C3H/He
or allogeneic BALB/c spleen cells for
7
days. The recovered
cells from these selection cultures were added to the induction
phase of hapten-reactive cytotoxic T cells at a concentration
of
0.4
to
10
x
1
05/well.
As
shown in Table
I,
the culture of
5
X
lo6
responder spleen cells with
2
X
lo6
TNP-modified
syngeneic spleen cells developed a cytotoxic activity that lysed
TNP-coupled target cells. This cytotoxic activity is mediated by
T cells and is restricted to self MHC as reported previously
(1 1).
When the stimulated T cells
(1
0
X
1
05)
in the syngeneic
MLR were added to the induction phase of cytotoxic
T
cells,
the development
of
TNP-reactive cytotoxic T cells was mark-
edly suppressed. This suppression was not due to the change
of the kinetics in the development of cytotoxic T cells, because
the suppression was always observed when assayed on days
3,
5,
or
7
of the culture. The suppressor activity depended on
the added cell number. When the small number of stimulated
T cells was added, a less suppressive effect was observed.
Culture supernatant of the syngeneic MLR had no suppressor
activity on the induction of cytotoxic T cells. Furthermore, the
stimulated T cells in the syngeneic MLR had no suppressive
effect when added to the effector phase of cytotoxic T cells
culturing with TNP-coupled target cells (data not shown). On
the contrary in the syngeneic MLR, C3H/He T cells stimulated
with allogeneic BALB/c spleen cells had no suppressor activity
on the induction of cytotoxic T cells of C3H/He mice. These
results suggest that the syngeneic MLR activates suppressor
cells that suppress the induction of cytotoxic T cells
in
vitro.
Table
II
shows kinetics in the development of suppressor
cells in the syngeneic MLR. C3H/He spleen cells were first
cultured
in
vitro for several days, and the recovered cells
(5
x
1
05)
from each culture were added to the induction phase of
TNP-reactive cytotoxic T cells. Two-day cultured cells had no
suppressor activity. Four-day cultured cells suppressed the
development of cytotoxic T cells about
50%.
If spleen cells
were cultured more than
6
days, the stimulated cells completely
suppressed the induction of TNP-reactive cytotoxic T cells.
These results suggest that the suppressor T cell activity in the
TABLE
I
Evidence
for
suppressor activity
of
cells induced in the
syngeneic
MLR
on the mduction
of
hapten-reactive cytotoxic
T
cells in vitro"
Suppressor Cell Source'
%
Speclfic Lysis on Target Cells'
Responder Cells Stimulator Cells Cell
No.
(x
1
05) Responder cells Stlmulator cells
TNP-X5563 X5563
50-1
25.1 50-1
~~~~ ~~
C3H/He TNP-C3H/He 44.0
f
1.3 34.4
f
1.2
0.0
f
3.3
10
2 C3H/He C3H/He 0.9
f
1.0 -0.1
f
1.3 -3.3
f
0.9
C3H/He
0.4 C3H/He C3H/He 9.6
f
5.7 4.6
f
1.0 3.6
f
1.9
C3H/He 25.6
f
2.5 13.5
f
1.9
-5.6
f
2.9
10 C3H/He BALB/c 29.5
f
5.9 8.9
f
3.3
2 42.6
f
6.3
C3H/He BALB/c 45.5
f
3.0
0.4 C3H/He BALB/c 43.8
f
1.5
31.1
f
2.8
28.8
f
1.2 9.4
f
0.8
9.4
?
3.4
(2
x
10') at 37°C for
5
days.
*
For the induction of TNP-reactive cytotoxic T cells, C3H/He spleen cells (5
x
1
0')
were cultured in vmo with TNP-modified or unmodified C3H/He spleen cells
BALB/c spleen cells (3
x
10') at 37°C for 7 days. Cells recovered from these cultures were added at the induction phase of hapten-reactive cytotoxic T cells at the
For the induction of suppressor cells, nylon column-purified C3H/He spleen cells
(5
X
10') were cultured with syngeneic C3H/He spleen cells or allogeneic
number indicated.
Cytotoxic assay was performed by incubating sensitized cells with 5'Cr-labeled TNP-modified or unmodified X5563 cells at an effector/target cell ratio of 50:l
or 25:l at 37°C for
5
hr. The values represent the mean and
SE
of triplicate determinations.
TABLE
II
Kinetics
of
the inducbon
of
suppressor cells by the syngeneic
MLR"
%
Specillc Lysis on Target Cells
Responder Cells
Stimu'ator
Cells Spleen Cells Cultured lor
Suppressor Cell Source:
TNP-X5563 X5563
C3H/He TNP-C3H/He
days
4
2
6
10
8
12
50:l
71.9
f
3.2
65.3
f
2.1
38.8
f
2.2
-4.1
f
1.8
-0.9
f
3.2
2.4
f
1.1
-3.0
f
2.3
25:
1
44.4
f
0.8
47.1
f
0.6
20.4
f
1.3
-3.6
f
2.2
-1.2
f
0.9
2.4
f
3.1
7.6
f
1.5
50
1
-2.1
f
1.9
3.1
f
3.3
4.1
f
0.9
-7.2
f
0.7
-7.6
f
1.0
N.D.b
N.D.
responder cells
(5
x
10') and TNP-stimulator cells (2
X
10') for the induction of TNP-reactwe cytotoxic T cells in vitro.
a
C3H/He spleen cells (5
X
lo6)
were precultured for several days
as
indicated. Cells (2
x
lo5)
recovered from these cultures were added to the mixture of
N.D.. not determined.
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
1012
UKI YAMASHITA. SHlRO ONO, AND HlROSHl NAKAMURA
[VOL.
128
TABLE
111
Suppressor cells are specific
for
spleen cells but not for heterologous serum added
%
Specific Lysis on Target Cells
Responder Sttmuiator Calls Serum Source' Spleen Cells TNP-X5563
Suppressor Cell
Cells
c,llti,rc.d
in
X5563
-
-
. .
-
.
- -
. . .
50:l 251
50:
1
C3H/He TNP-C3H/He FCS 67.4
f
3.2 68.4
f
0.4
FCS
HS
-19.2
f
1.2
-20.9
f
1.5
-1 1.5
f
2.0
-66.4
f
0.5
-34.1
f
2.8 -26.8
f
2.0
-23.1
f
2.2
were added to the mixture of responder cells
(5
X
1
0')
and TNP-stimulator cells (2
x
10') for the induction of TNP-reactive cytotoxic T cells in vitro.
*
C3H/He spleen cells (5
X
10') were cultured in 10% of fetal calf serum (FCS) or human serum
(HS)
for
7
days. Cells (5
x
lo5)
recovered from these cultures
TABLE
IV
Suppressor Cells lnduced in the Syngeneic
MLR
are
T
Cells
Expt. No. Stimulator Cells Suppressor Spleen Cells'
Treated with
I
C3H/He TNP-C3H/He NMS
+
C
Anti-Thy-1
+
C
II
C3H/He TNP-C3H/He Untreated
Nylon-passed
Nvlon-Dassed
+
sDleen
%
Specific Lysis on Target Cells
TNP-X5563 X5563
50'1
25:l 50:l
59.5
f
0.7
27.5
f
1.4 44.6
f
1.6
5.0
f
3.2
26.2
f
4.3 11
.o
f
0.9
53.2
f
1.4 43.4
f
0.3 3.9
f
3.2
60.2
f
0.8
14.8
f
0.5 38.0
f
0.8
5.4
f
1.0
48.8
f
1.5 35.6
f
2.3
19.5
f
2.6
5.0
f
2.0
-5.9
f
1.3
-2.4
f
2.2
-0.2
f
1.4
5.3
f
1
.o
__
~
a
In Expt.
I,
spleen cells (5
X
10')
were first cultured for 7 days. Cells recovered from these cultures were treated with normal mouse serum (NMS) or anti-Thy-1
induction of TNP-reactive cytotoxic T cells in vitro. In Expt.
II.
nylon column-purified spleen cells
(5
x
10') were cultured in the presence or absence of spleen cells
serum and complement (C). Then the viable cells (3
x
1
05)
were added to the mixture of responder cells
(5
X
10') and TNP-stimulator cells
(2
x
10') for the
(3
x
10') for 7 days. Cells recovered from these cultures
(5
X
1
05)
were added to the mixture of responder cells
(5
x
10') and TNP-stimulator cells (2
x
10') for the
~~ ~~
induction of TNP-reactive cytotoxic T cells in vitro.
syngeneic MLR develops gradually and becomes dominant
from
4
days after the initiation of culture.
Suppressor cells are not specific for heterologous serum
used for the
in
vitro culture. One possibility of the specificity of
suppressor cells induced in the syngeneic MLR is that the
suppressor cells may be specific for heterologous serum used
for the
in
vitro culture rather than for syngeneic spleen cells
themselves. In order to exclude this possibility, we changed
the serum source for the induction phase and the expression
phase of suppressor cells. As shown in Table
111,
C3H/He
spleen cells were first cultured in 10% FCS or human serum
(HS), and the recovered cells from the culture were added to
the induction phase of cytotoxic T cells in FCS. However, cells
cultured
in
vitro in both FCS and HS showed the same sup-
pressor activity on the induction
of
cytotoxic T cells in FCS.
FCS and HS are immunologically not cross-reactive when
assayed by the stimulation of proliferative T cells. Thus, sup-
pressor cells induced in the syngeneic MLR seem to be specific
for syngeneic spleen cells themselves, but not for heterologous
serum used for the
in
vitro culture.
Suppressor cells induced
in
the syngeneic MLR are Ly-1
+
T
cells. In order to study the nature of suppressor cells,
in
vitro
cultured spleen cells of C3H/He mice were treated with anti-
Thy-1 serum and rabbit complement (C), then added to the
induction phase of cytotoxic T cells.
As
shown in experiment
I
of
Table
IV,
the cultured cells treated with normal mouse serum
(NMS) and C had suppressor activity on the induction of
cytotoxic T cells. However, the treatment of anti-Thy-1.2 serum
and C abrogated this suppressor activity. In order to further
study the nature
of
suppressor cells, normal spleen cells were
fractionated using nylon column and cultured
in
vitro in the
presence or absence of mitomycin C-treated syngeneic spleen
cells. As shown in experiment
II
of Table
IV,
the culture of
unfractionated spleen cells induced suppressor activity. How-
ever, the culture of nylon column-purified T cells alone did not
induce suppressor activity. The culture of nylon column-puri-
fied spleen cells with mitomycin C-treated unfractionated
spleen cells induced suppressor activity. These results further
suggest that suppressor cells are T cells, but the induction
of
suppressor T cells requires stimulation by a nylon column-
adherent cell population. In the accompanying paper
(7),
we
have shown that the proliferative T cells reactive for syngeneic
spleen cells belong to Ly-l+ cell populations. Thus, it is impor-
tant to study the Ly phenotype of suppressor T cells induced
in the syngeneic MLR. T cells of (C3H/He
X
BALB/c)F, mice
cultured
in
vitro were treated with anti-Lyt-1
,
anti-Lyt-2, or anti-
Lyt-3 serum and c, then added to the induction phase of
cytotoxic T cells. As shown in Table
V,
the treatment of T cells
with anti-Lyt-1.2 serum and C abrogated their suppressor
activity. However, the treatment of the same cells with anti-Lyt-
2.2 and anti-Lyt-3.2 serum and C gave no effect on the sup-
pressor activity. These results suggest that suppressor T cells
induced in the syngeneic MLR also belong to Ly-l+ cell pop-
ulations.
Stimulator cells are adherent macrophages for the induction
of suppressor
T
cells
in
the syngeneic MLR. In order to study
the nature of stimulator cells for the induction of suppressor T
cells in the syngeneic MLR, the stimulator spleen cells were
fractionated using a Sephadex G-10 column and plastic dishes
and cultured with nylon column-purified T cells. As shown in
Table
VI,
the nylon column-purified T cells alone did not induce
suppressor activity. The addition of unfractionated spleen cells
to the nylon column-purified T cells clearly induced suppressor
activity. The addition of plastic dish-adherent spleen cells also
induced suppressor activity. However, the addition of Sepha-
dex G-1 0-passed spleen cells did not induce suppressor activ-
ity. These results suggest that the stimulator cells for the
induction of suppressor T cells in the syngeneic MLR are
adherent macrophages.
Genetic restriction of suppressor
T
cells induced
in
the
syngeneic MLR. In the accompanying paper
(71,
we have
presented evidence that the restimulation of T cells with syn-
geneic macrophages shows genetic restriction and that this
restriction is controlled by the
I
region of the MHC. Thus, it is
also important to study the genetic restriction of suppressor T
cells induced in the syngeneic MLR. Nylon column-purified T
cells were first cultured
in
vitro with syngeneic spleen cells for
7
days. The recovered cells from these cultures were added to
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
19821
SUPPRESSOR
T
CELLS IN SYNGENEIC
MLR
1013
TABLE
V
Suppressor cells induced
in
the syngeneic MLR are Ly-1
+
T
cells
B
SDeclflc
LYSIS
on
Taraet Cells
Expt No Responder Cells Stimulator Cells Suppressor Cells Treated
TNP.X5563
wlth"
-
X5563
50-1
50-1
I
(C3H/He
x
BALB/c)Fq TNP-(CBH/He
x
BALB/c)F, 13.3
f
0.5
-10.3
f
0.9
NMS
+
C
Anti-Lyt-1
+
C 3.4
f
1.4
11.6
f
2.2 2.5
f
2.2
5.2
f
3.8
Anti-Lyt-2
+
C 2.7
k
1.7 -1.3
f
3.0
II
(C3H/He
x
BALB/c)F, TNP-(C3H/He
X
BALB/c)F, NMS
+
C 46.5
f
0.7 -6.4
f
1.3
19.8
f
2.0
Anti-Lyt-1
+
C -8.6
f
2.4
Anti-Lyt-3
+
C 31
.O
f
1.9 -4.4
f
1.3
15.7
f
2.1 -8.3
f
1.4
days. Cells recovered from these cultures were treated with normal mouse serum (NMS). antl-Lyt-1.2, anti-Lyt-2.2, or anti-Lyt-3.2 serum and C. Then the vlable cells
a
Nylon Column-purified T cells
(5
x
1
06)
of (C3H/He
x
BALB/c)F, mice were cultured
in
vitro with mitomycin C-treated syngeneic spleen cells (3
x
1
06)
for
7
(5
x
1
05)
were added to the mixture of responder cells
(5
x
10') and TNP-stimulator cells (2
X
1
06)
for the induction of TNP-reactive cytotoxic T cells
m
vitro.
TABLE
VI
Stimulator cells are adherent macrophages
for
the induchon
of
suppressor
T
cells
In
the
syngeneic
MLR
YO
Speciflc LYSIS
on
Target Cells
Expt. No. Responder Suppressor Cells": Nylon Column-
Spleen Cells Treated with
Stimulator purified T
Cells
Cultured with TNP-X5563 X5563
50'1 25.1
25
1
I
C3H/He TNP-C3H/He 60.3
f
1.6
Not Added 45.9
f
0.7
67.2
k
7.7
Untreated 41.7
f
2.1
Plastic dish-adherent 22.1
f
1.2
24.5
f
0.1 12.1
f
1.9
1.3
f
2.5
Sephadex G-1 0-passed 57.7
f
0.7 26.2
f
1.9
-5.7
f
1.1
N.D.
N.D.
N.D.
N.D.
I1
C3H/He TNP-C3H/He 84.0
k
1.8
Not Added 77.3
f
1.6
Untreated 52.8
f
2.9
Plastic dish-adherent 36.3
k
2.2
Sephadex G-1 0-passed
-4.6
f
1.3
-6.7
k
0.9
-4.4
f
1.7
-2.8
f
0.6
73.2
k
1.8
-1.7
f
1.8
Nylon column-purified T cells
(5
x
lo6) were cultured
in
vitro for
7
days with spleen cells
(5
x
lo5)
which had been treated as indicated. Cells
(5
x
lo5)
recovered from these cultures were added
to
the mixture of responder cells
(5
X
10') and TNP-stimulator cells (2
X
10') for the Induction of TNP-reactive cytotoxic
T cells
in
vitro.
the induction phase of either syngeneic or allogeneic cytotoxic
T cells. As shown in Table
VII,
in vitro cultured BALB/c T cells
suppressed the development
of
cytotoxic T cells
of
syngeneic
BALB/c mice. However, the suppressor T cells
of
BALB/c
mice did not suppress that of allogeneic C3H/He mice. Simi-
larly, the suppressor T cells of C3H/He mice suppressed the
development of cytotoxic T cells
of
C3H/He mice but not of
BALB/c mice. Thus, suppressor T cells induced in the synge-
neic MLR work on only histocompatible cytotoxic T cells. This
genetic restriction of suppressor T cells was further confirmed
with suppressor T cells of
F,
animals induced by parental
spleen cells. Nylon column-purified Fl T cells were first cultured
in vitro with spleen cells
of
each parent for
-7
days. The
recovered cells from these cultures were added to the induction
phase of cytotoxic T cells of each parent. As shown in Table
VIII,
the suppressor T cells of (C3H/He
x
BALB/c)F, mice
stimulated with BALB/c spleen cells suppressed the develop-
ment
of
cytotoxic T cells of BALB/c mice but not of C3H/He
mice. Similarly, the suppressor T cells of
F,
mice stimulated
with C3H/He spleen cells suppressed the development of
cytotoxic
T
cells of C3H/He mice but not of BALB/c mice.
These results suggest that there are two distinct clones of
suppressor T cells in
F1
animals: One can preferentially sup-
press the induction of cytotoxic
T
cells of one parent but not of
the other parent.
Genetic mapping
of
the
MHC
genes controlling the activity
of
suppressor
T
cells induced in the syngeneic
MLR.
Finally
we studied which genetic regions control the activity of sup-
pressor T cells induced in the syngeneic MLR using A.TL,
A.TH, and C3H/He mice. As shown in Table
IX,
the T cells of
C3H/He mice cultured in vitro suppressed the development of
cytotoxic T cells of C3H/He mice. The T cells cultured in vitro
of
A.TL mice who share the I-region of the MHC with C3H/He
TABLE
VI1
Genetic restrlction
of
suppressor
T
cells Induced
In
the syngeneic MLR
on
the
induction
of
hapten-reactive cytotoxic
T
cells
in
vitro
Suppressor
Straid
%
Speclflc Lysis
on
Target Cells
Re~~~der
Stmulator Cells Cells TNP(+) TNP(-)
_.
50'1
25
1
50
1
BALB/c TNP-BALB/c 37.0
f
1.4 31.9
f
1.9 11.9
f
1
.O
C3H/He 35.7
f
0.6 30.7
f
5.2 16.7
f
1.0
C3H/He TNP-C3H/He 88.9
f
2.9 85.5
f
1.8 -3.1
f
1.1
C3H/He 16.1
f
3.3 17.2
f
1.6 13.3
f
0.4
BALB/c 75.2
f
3.4 56.5
f
0.7 15.9
f
2.1
BALB/c -7.0
f
2.5 -1.9
f
0.8 -9.6
f
2.8
syngeneic spleen cells (3
X
lo6) for 7 days. Cells
(5
x
lo5) recovered from
Nylon column-purified T cells
(5
x
1
0')
of each strain were cultured with
these cultures were added to the mixture of responder cells
(5
X
1
06)
and TNP-
stimulator cells
(2
X
1
06)
for the induction of TNP-reactive cytotoxic T cells
~n
vitro.
mice also suppressed the development of cytotoxic
T
cells of
C3H/He mice. However, the T cells cultured in vitro
of
A.TH
mice who do not share the
I
region of the MHC with C3H/He
mice did not suppress the development
of
cytotoxic T cells of
C3H/He mice. Thus, the activity
of
suppressor T cells induced
in the syngeneic MLR seems to be controlled by the
I
region of
the MHC. Genetic mapping
of
the MHC genes controlling the
activity
of
suppressor T cells induced in the syngeneic MLR
was further studied using the congenic resistant lines
of
a
61
0
series of mice. As shown in Table
X,
T
cells
cultured in vitro
of
B1 O.BR
(kkkkkkkkk),
61
0.A
(kkkkkdddd), and
61
O.A(4R)
(kkbbbbbbb) mice suppressed the development
of
cytotoxic T
cells of C3H/He (kkkkkkkkk) mice. However, T cells cultured
in vitro of
61
O.A(5R) (bbbkkdddd) mice had less suppressive
activity, and T cells cultured in vitro of
61
0
(bbbbbbbbb) mice
had no suppressive activity on the development
of
cytotoxic T
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
1014
UKI YAMASHITA, SHlRO
ONO,
AND HlROSHl NAKAMURA
[VOL.
128
TABLE
Vlll
Selection
of
(C3H/He
x
BALB/c)F, suDpressor
J
cells by parental spleen cells
in
fhe svnaeneic MLR
Expt No. Responder Cells Stirnulator Cells
X
BALB/c)F, T Cells Selected with
Suppressor Cell Source": (C3H/He
Spleen Cells Straln
I
BALB/c TNP-BALB/c BALB/c
C3H/He
C3H/He TNP-C3H/He BALB/c
C3H/He
I1
C3H/He TNP-C3H/He BALB/c
C3H/He
YO
Specific Lysis
on
Target Cells
TNP(+) TNP(-)
..
50
1
25:l
50:
1
45.8
f
4.5 13.3
f
3.1 -13.3
f
2.2
17.1
f
3.8 -1.8
f
1.8
58.9
f
1.6 27.2
f
3.4 -9.8
f
3.8
6
6
f
3.1
84.0
f
0.9
75.4
f
1.5 76.0
f
1.5 4.2
f
1.8
61.6
f
1.4 14.6
f
0.3
41.7
f
3.9 24.3
f
3.8 8.1
f
0.4
79.6
f
2.6
59.5
f
1.1 70.1
f
0.7
-1.6
f
0.7
41.7
f
1.9
24.7
f
0.3 15.3
f
0.4 10.8
f
2.5
1.3
f
1.8
recovered from these cultures were added
to
the mixture of responder cells
(5
X
10') and TNP-stimulator cells (2
X
10') for the induction of TNP-reactive cytotoxic
e
Nylon column-purified (C3H/He
X
BALB/c)F, T cells
(5
X
10') were cultured with C3H/He or BALB/c spleen cells (3
x
10') for 7 days. Cells
(5
x
1
05)
T cells
In
vitro.
TABLE
IX
Activity
of
suppressor
J
cells Induced
in
the
syngeneic
MLR are controlled by the /-region
of
the MHC
o/,
Specific LYSIS
on
Target Cells
Expt
No
Responder Cells Stlmulator Cells Suppressor Cells Straln" TNP-X5563 X5563
50-1
25:l
25.1
I
C3H/He TNP-C3H/He C3H/He 78.4
f
2.1
11.1
f
1.8 72.7
f
0.4
-6.1
f
1.9
A.TL -10.4
f
0.3
A.TH -9.3
f
1.7
78.1
f
2.1 57.7
f
1.0
8.8
f
1.7
2.6
f
1.0
14.3
f
3.5
1.2
f
1.2
II
C3H/He TNP-C3H/He 84.0
f
1.8
C3H/He 36.3
f
2.2
A.TL
A.TH
-4.6
f
1.3
-2.8
f
0.6
26.8
f
1.5 3.6
f
1.5
68.1
f
0.7 -7.2
f
0.6
x
10') and TNP-stimulator cells (2
X
10') for the induction of TNP-reactive cytotoxic T cells
in
vitro.
a
Spleen cells
(5
x
1
0')
of each strain were cultured for 7 days. Cells
(5
x
1
05)
recovered from these cultures were added
to
the mixture of responder cells
(5
TABLE
X
Activity
of
suppressor
J
cells induced in the syngeneic MLR are controlled by the I-A-subregton
of
the
MHC
%
Speciflc Lysis
on
Target Cells
Expt. No Responder Cells Stimulator Cells Cells Regions of Identity'
Strain" TNP-X5563 X5563
50:l
251
50'1
I
C3H/He TNP-C3H/He 61.2
f
1.3
B1O.BR KABJECSGD 43.8
f
1.6 -3.4
f
0.8
BIO.A 21.4
f
1.4 8.0
f
0.3
KABJE 34.1
f
1.9 -5.4
*
0.6
BlO.A(4R) 17.1
f
2.0
KA -4.0
f
1.2
23.8
f
0.3 10.8
f
1.5 -4.0
f
1.4
BlO.A(SR) JE 39.5
f
0.4 23.3
f
0.8
E1
0
None 45.8
f
0.9 30.5
f
0.6 -2.5
f
1.3
-3.6
f
1.3
II
C3H/He TNP-C3H/He 63.8
f
1.4 44.9
f
2.6
B1O.BR -7.5
f
0.7
KABJECSGD 0.3
f
3.9 -2.5
f
0.0
B1 0.A KABJE 23.2
f
2.8 7.2
f
2.8 -2.0
f
0.4
-4.6
f
0.7
B1 O.A(4R)
BlO.A(BR) KA
JE 25.2
f
2.0
34.2
f
1.7 27.4
f
5.2
13.4
f
2.1 12.8
f
0.7
E10 8.3
f
0.3
None 61.7
f
1
.O
40.9
?
2.4 -0.7
*
1.3
a
Nylon-column-purified T cells
(5
x
1
0')
of each strain were cultured
in
vitro for 7 days with syngeneic spleen cells (3
X
1
0').
Cells (2.5
X
1
O5
in Expt.
I
and
5
x
1
O5
in Expt.
II)
recovered from these cultures were added to the mixture of responder cells
(5
x
10') and TNP-stimulator cells (2
x
10') for the induction of TNP-
reactive cytotoxic T cells
in
vitro.
Letters refer to regions or subregions of the MHC which are shared by the responder cells and the suppressor cells. A,
B.
J.
E.
and C represent the subregions
of
I
cells of C3H/He mice.
All
these results, taken collectively with
the results
of
the genetic control of the proliferative T cell
response presented in the accompanying paper
(7)
suggest
that the activity
of
suppressor T cells induced in the syngeneic
MLR is controlled by the I-A-subregion of the MHC.
DISCUSSION
In this report, we have presented evidence that T cells
stimulated in the syngeneic MLR have suppressor activity on
the induction
of
hapten-reactive cytotoxic T cells
in
vitro
and
that the activity of suppressor T cells induced in the syngeneic
MLR is controlled by the
I
region of the MHC. There are some
controversial reports on the functional activity of the syngeneic
MLR in an immune response. In the study of human lympho-
cytes, Hausman and Stobo (1
2)
and Chiorazzi et
a/.
(13)
reported that the
T
cells activated in the syngeneic MLR had
helper activity in the antibody response, whereas Smith and
Knowlton (14), lnnes
et
a/.
(1
5),
and Sakane and Green (1
6)
reported that T cells responsible for the syngeneic MLR had
suppressor activity. In the study of mouse lymphocytes, Burus
et
a/.
(1
7)
and Hodes and Hathcock
(1
8)
found that
in
vitro
culture of mouse spleen cells induced suppressor
T
cells that
could suppress the development of cytotoxic T cells and anti-
body response
in
vitro,
although they did not mention this
phenomenon as the syngeneic MLR. In order to further clarify
the functional activity of
T
cells responsible for the syngeneic
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
19821
SUPPRESSOR
T
CELLS IN SYNGENEIC MLR
1015
MLR, we have studied the effect of T cells stimulated in the
syngeneic MLR on the induction of hapten-reactive cytotoxic
T cells
in vitro.
When T cells cultured
in vitro
for
7
days with syngeneic
spleen cells were added to the induction phase of TNP-reactive
cytotoxic T cells, the development of cytotoxic T cells was
markedly suppressed (Table
I).
This suppressive effect was not
due to a nonspecific cytotoxic effect by stimulated T cells,
because the cell recovery from the induction culture of TNP-
reactive cytotoxic T cells was no different in both the control
group and the suppressed group. T cells cultured with alloge-
neic spleen cells had no suppressor activity in the development
of cytotoxic T cells. The suppression was observed when the
suppressor cells were added to the induction phase of cytotoxic
T cells, but not to the expression phase of cytotoxic T cells on
TNP-coupled target cells. Furthermore, these suppressor T
cells worked on only histocompatible cytotoxic T cells (Table
VII).
There are some reports that the allogeneic mixed leuko-
cyte reaction can induce suppressor T cells (1 9, 20). However,
looking at suppressor T cells induced in the syngeneic MLR,
we found that they were different in many aspects from the
suppressor T cells induced in the allogeneic MLR (21). From
the reports of Rich and Rich (1 9) and Hirano and Nordin (20),
the T cells cultured for 2 days with allogeneic spleen cells had
suppressor activity, whereas suppressor T cell activity in the
syngeneic MLR developed after
4
days of the culture and
lasted more than 2 wk (Table
11).
However,
T
cells cultured for
7
days with allogeneic spleen cells had no suppressor activity
as reported here. Suppressor T cells induced in the allogeneic
MLR were substituted by culture supernatant (19). whereas
suppressor T cells induced in the syngeneic MLR were not
substituted by culture supernatant (data not shown). Further-
more, the genetic control of the suppressor T cell activity
induced in the syngeneic MLR are different from that of the
suppressor T cell activity induced in the allogeneic MLR as
discussed later. Thus, the syngeneic MLR seems to induce
different clones of suppressor T cells from the allogeneic MLR.
The suppressor cells induced in the syngeneic MLR are Ly-
1
+
T cells, because the suppressor activity induced in the
in
vitro
culture were sensitive to the anti-Thy-1 and anti-Lyt-1
serum and C treatment, but were resistant to the anti-Lyt-2 and
anti-Lyt-3 serum and C treatment (Tables
IV
and
V).
These
results suggest that the suppressor T cells induced in the
syngeneic MLR belong to the same populations as proliferative
T cells. This notion is further confirmed by the finding of the
macrophage requirement for the induction of suppressor T
cells and the genetic control of suppressor T cell activity. For
the induction of suppressor T cell activity by the
in vitro
culture,
T cells should be stimulated by other cell populations (Table
IV).
The stimulator cells for the induction of suppressor T cells
were macrophage-like adherent cells, because the stimulation
activity for suppressor T cells was depleted by passing the
spleen cells through a nylon and a Sephadex G-10 column,
but it was retained in the plastic dish-adherent cell populations
(Table
VI).
Thus, the suppressor
T
cells can be activated by
recognizing syngeneic macrophages.
Another important finding concerning the suppressor T cells
induced in the syngeneic MLR are genetic control of suppres-
sor T cell activity. The suppressor
T
cells induced in the
syngeneic MLR worked on syngeneic cytotoxic T cells but not
on allogeneic Cytotoxic T cells (Table
VII).
Furthermore, F1 T
cells stimulated with spleen cells of one parent showed a
suppressive effect on cytotoxic T cells of the same parent but
not on cytotoxic T cells of the other parent (Table
VIII).
These
results suggest that there are two distinct clones in Fl sup-
pressor T cells that can preferentially suppress cytotoxic T
cells of one or the other parent. The activity of suppressor T
cells induced in the syngeneic MLR are controlled by the I-A-
subregion of the MHC, because the cultured T cells of A.TL
mice suppressed the development of cytotoxic T cells of C3H/
He mice, but the cultured T cells of A.TH mice did not (Table
IX).
Furthermore, the cultured T cells of BlO.BR, BlO.A, and
BlO.A(4R) mice suppressed the development of cytotoxic T
cells of C3H/He mice, but the cultured T cells of BlO.A(5R)
and B10 mice did not (Table
X).
All
these results-the nature
of suppressor T cells, the requirement of macrophages, and
the genetic control of suppressor T cell activity-are com-
pletely consistent with the results obtained from the study of
the proliferative T cell response reported in the accompanying
paper
(7)
and suggest that the suppressor T cells and the
proliferative T cells belong to the same T cell populations.
The target determinants recognized by suppressor T cells
induced in the syngenic MLR are not completely defined yet,
but we do not think these suppressor T cells are specific for
the heterologous serum used for the
in vitro
culture, because
even when the serum source, FCS and HS, was changed for
the induction and the expression phase of suppressor T cells,
a similar suppressor activity was observed (Table
IV).
We rather
consider that these suppressor T cells recognize the I-region
products of self MHC as discussed in the accompanying paper
(7).
From this standpoint, these suppressor T cells are not
antigen specific, but specific for self MHC. Thus, the prolifer-
ative response
of
T
cells is induced by recognizing the I-region
products of the MHC on syngeneic macrophages, and these
stimulated T cells work suppressively on the development of
cytotoxic T cells having the same haplotype of the
I
region of
the MHC.
Then what mechanism works for the suppression of the
development of cytotoxic T cells in these systems? One expla-
nation is that during primary culture of T cells in the syngeneic
MLR, cytotoxic T cells specific for syngeneic spleen cells may
develop, and these cytotoxic T cells directly eliminate re-
sponder
T
cells and/or TNP-coupled stimulator cells during
the induction phase of cytotoxic T cells. In fact, Altman and
Katz (22) reported that the culture of spleen cells in the
allogeneic effect factor induced cytotoxic T cells specific for
self MHC. However, we do not think that this is the case in our
system, because T cells cultures
in vitro
had no cytotoxic T
cell activity when assayed with "Cr-labeled tumor cells or
normal spleen cells. The cell recovery from the induction of
TNP-reactive cytotoxic T cells was the same in both the control
group and the suppressed group. These suppressor T cells do
not work on the effector phase of cytotoxic
T
cells on TNP-
coupled target cells. Furthermore, the activity of suppressor T
cells is mediated by the Ly-l+ T cell population and is con-
trolled by the
I
region of the MHC, whereas cytotoxic T cells
reported by Altman and Katz (22) belong to Ly-23+ T cell
populations and are specific for the H-2K and
D
end of the
MHC. Thus, the syngeneic MLR will induce different T cell
populations from those induced by an allogeneic effect factor.
We rather consider that the T cells induced in the syngeneic
MLR work as regulator cells in immune responses. The sup-
pressor T cell activity develops after
4
days of the initiation of
the
in vitro
culture and becomes very dominant after
6
days
(Table
Ill.
At that time, antigen-specific immune responses
in
vitro
have usually declined. The suppressor T cell activity
reported here is also induced during the course of antigen-
specific immune responses
in vitro
(data not shown). Thus, the
by Shiro Ono on January 18, 2016http://www.jimmunol.org/Downloaded from
1016
UKI YAMASHITA. SHlRO ONO. AND HlROSHl NAKAMURA
[VOL.
128
development of suppressor T cells in the syngeneic MLR may
terminate antigen-specific immune response
in vitro.
However,
the nature of suppressor T cells reported here is different in
many aspects from the nature of suppressor
T
cells reported
by some investigators. Antigen-specific and mitogen-induced
suppressor T cells belong to Ly-1- 23' cell populations and
they have I-J products on their surface (23-26). However, the
suppressor T cells reported here belong to Ly-l+ 23- cell
populations.
So
far, we have not found
I-J
products
on
these
suppressor
T
cells (unpublished observation). Concerning the
MHC determinant recognized by suppressor T cells, there are
some reports. Miller
eta/.
(27) and Jandinski
et
a/.
(28) reported
that suppressor T cells induced by the injection
of
hapten-
coupled spleen cells, which suppressed delayed hypersensitiv-
ity reaction
in vitro
or antibody response
in vitro,
recognized
the H-2K or H-2D end
of
the MHC. Kim
et
a/.
(29) reported that
suppressor
T
cells were induced by the I-C,
S,
and
G
region
differences in the allogeneic MLR. Furthermore, Rich and Rich
(30) reported that the suppressor factors produced in the
allogeneic MLR worked on the I-C subregion-compatible target
cells. However, the suppressor T cells induced in the syngeneic
MLR seem to recognize the
I-A
subregion products of the MHC.
From these observations, we consider that the suppressor
T
cells induced in the syngeneic MLR are in the helper/amplifier
T cell lineages and they exert their suppressive effect by some
different mechanism.
On the mechanism of the suppression of the development of
cytotoxic
T
cells by suppressor
T
cells induced in the syngeneic
MLR, some possibilities can be considered. Because the de-
velopment of cytotoxic T cells requires the participation of Ly-
1
+
helper
T
cells and la-positive macrophages (31 -33,
1
l),
one explanation is that the suppressor T cells induced in the
syngeneic MLR, which are specific for la antigens, may com-
pete with helper T cells on la-positive macrophages and may
inhibit the generation
of
effective helper function.
A
second explanation is that these T cells may be helper T
cells, and too much help from these T cells and antigen-specific
helper T cells may result in the suppression of cytotoxic
T
cell
development.
The most intriguing explanation of the regulation by these
suppressor T cells is an immunoregulatory circuit mechanism
proposed by Eardley, Cantor et
a/.
(34, 35), who reported that
Ly-1
+
T cells are inducer cells and they can induce a variety
of
effector cells, including
B
cells and cytotoxic
T
cells, and
delayed hypersensitivity, and at the same time they can also
induce suppressor
T
cells, which suppress the immune re-
sponse. Thus, in our system, Ly-1'
T
cells are not direct
suppressor
T
cells, but they may work on Ly-1 23+ cells to
induce Ly-2
3'
suppressor
T
cells that are contained in the
responder cell populations
of
cytotoxic T cells, and these Ly-2
3'
suppressor T cells may directly suppress the development
of
cytotoxic
T
cells. This possibility is now under investigation.
There are some observations to suggest that these suppres-
sor T cells work not only
in vitro
but also
in vivo
on immune
responses; namely, the syngeneic MLR is deficient in the
patients of systemic lupus erythematosus or lymphocytic leu-
kemia (36, 37) and in autoimmune mice
(38,
39). Furthermore,
the feedback inhibition by Ly-l+
T
cells is also deficient in
autoimmune mice (40). Thus, the dysfunction of suppressor T
cells inducible in the syngeneic MLR may result in a generation
of autoimmune diseases.
Although many questions remain unresolved regarding the
syngeneic MLR, the evidence presented here will suggest the
presence of T cell clones that recognize self MHC and regulate
immune responses. Furthermore, this evidence will give a fur-
ther explanation for the pathogenesis
of
autoimmune diseases.
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