Eur. J. Immunol. 1991.

21: 335-341 IL2-dependent induction of functional high-affinity IL2R 335

Jacques J. ProustoA,
Nancy L. Shaper+,
Meredith A. BuchholzO and
Albert A. NordinO
Clinical Immunology Sectiono,
Gerontology Research Center,
National Institute of Aging, NIH
and Oncology Center+, The Johns
Hopkins University School of
Medicine, Baltimore
T cell activation in the absence of interleukin 2
(IL2) results in the induction of high-affinity IL2
receptor unable to transmit a proliferative signal
Although interleukin 2 (IL2) clearly up-regulates the expression of the p55 chain
of the IL2 receptor (IL2R) little is known about its role in the induction of the
high-affinity IL 2R. RestingT lymphocytes were induced to express IL2R under
experimental conditions in which IL2 production was not induced or was
prevented. Under these conditions high- and low-affinity IL2R were easily
demonstrated by Scatchard analysis. Northern blot analysis confirmed the
accumulation of p55 specific mRNA and the absence of the IL2 transcript.
High-affinity IL2R induced in the complete absence of IL2 were unable to
transmit a proliferative response unless exposed to extremely high concentrations
of IL2.The addition of picomolar amounts of recombinant IL2 or the initiation of
endogenous IL 2 production during the induction period restored the function-
ality of high-affinity IL2R. Also, T cells induced to generate IL2 displayed
functional high-affinity IL 2R even in the presence of monoclonal antibodies
blocking extracellular IL 2 and IL 2R. These results indicate that the presence of
IL2 during the early phase of T cell activation is an absolute requirement for the
induction of fully operational high-affinity IL2R and that low amounts of
intracellular IL 2 are sufficient to confer functional properties to these receptors.
The data also suggest that an intracellular as well as an extracellular high-affinity
structure, expressed as a consequence of cell activation, is responsible for
conferring competence to the high-affinity IL 2R involved in IL 2-dependent
proliferation.

1 Introduction Interestingly, IL2 has been shown to up-regulate the

Recent studies have indicated that at least two IL2 binding
proteins termed p55 and p75 participate in the structure of
the IL2R [l-51. Each subunit can exist as a single
polypeptide or as preformed heterodimers [6] correspond-
ing to the three forms of the IL2R that have been identified
by ligand binding studies. Although there are no stoichio-
metric studies available, it is believed that the high-affinity
-
IL 2R (Kd 10-11 M) results from an association of p75
with p55 whereas the low (Kd - M) and the interme-
diate ( K d - M) affinity IL2R correspond to isolated
p55 and p75, respectively. p75 appears to be the transducing
structure involved in the generation of the proliferative
signal [7] whereas p55, although of prime importance in the
formation of the high-affinity heterodimer, seems to be
devoid of signaling capacity [8,9]. It has been suggested
that the p75 IL 2 binding protein is present constitutively on
the surface of human restingT lymphocytes whereas p55 is
the inducible subunit of the IL2R [lo]. Full expression of
the functional high-affinity p55/p75 heterodimer can be
induced by exposure of T lymphocytes to ligands that
cross-link antigen-specific receptors on the cell surface
[ll-131 which also results in the generation of IL2.
expression of the low-affinity IL2 binding peptide, p55
[lo, 14-17]. Although there is general agreement concern-
ing the up-regulation of p55 expression by IL2, the role
played by IL2 in the modulation of the high-affinity IL2R
is still controversial. It has been shown that rIL2 alone can
induce increased levels of high-affinity IL 2R in an antigen-
specific Th cell clone [18] whereas in other systems, the
interaction of IL2 with its high-affinity receptor results in
diminished expression of such receptors while considerably
augmenting the levels of Tac antigen [19]. The present
studies investigate the role of IL2 in the induction of
high-affinity IL 2R. Using various experimental ap-
proaches, we show that purified resting T lymphocytes,
activated in the complete absence of exogenous or endo-
genous IL2, express detectable amounts of high- and
low-affinity IL2R. IL2R induced under these conditions
are functionally aberrant in that they are unable to transmit
a proliferative signal when exposed to IL 2 concentrations
in the range of the Kd of the high-affinity site.The addition
of exogenous IL2 or the initiation of endogenous IL2
synthesis during the induction period restores the function-
al capacity of these IL2R.

2 Materials and methods

[I 87201 2.1 Animals

~ ~~~ ~~

A Recipient of a research fellowship from Hoffmann-La Roche.
Correspondence: Albert A. Nordin, Gerontology Research Cen-
ter, National Institute of Aging, NIH, Baltimore, MD 21224,
USA
Abbreviations: Bmax:Binding capacity CsA: Cyclosporin A
Four-month-old C57BL/6 male mice (The Jackson Labo-
ratories, Bar Harbor, ME) maintained at the Gerontology
Research Center animal facility, were used for these
experiments (the Gerontology Research Center is fully
accredited by the American Association for Accreditation
of Laboratory Animal Care).

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 OO14-2980/91/0202-0335$3S O + .25/0

336 J. J. Proust. N. L. Shaper, M. A. Buchholz and A. A. Nordin Eur. J. Immunol. 1991. 21: 335-341

2.2 Chemicals, antibodies, reagents and medium over FCS, centrifuged and resuspended in complete
medium devoid of agonists. Alternatively, the T lympho-
PBu2, ionomycin, forskolin and pronase were purchased cytes were treated with pronase [31], washed and resus-
from Calbiochem (La Jolla, CA). CsA was a gift from the pended in complete medium.
Sandoz Research Institute (East Hanover, NJ). The hybri-
doma PC61, described by Lowenthal et al. [20], was
provided by Dr. F. Fitch (University of Chicago, Chicago, 2.5 U51-rlL2binding assay
IL).The hybridoma 145-2C11specific for the E chain of the
CD3 complex [21] was obtained from Dr. S. Sharkis (Johns Before determining the level of binding, the activated
Hopkins University, Baltimore, MD). The hybridomas T lymphocytes were incubated twice for 1 h, at 37°C in
DMS-1 (anti-human IL2) [22], GK1.5 (anti-L3T4) [23] and complete medium. After each incubation the cells were
the cytotoxicTcel1 line CTLL-16 were provided by Dr. K. layered over and centrifuged through FCS to remove
Smith (Dartmouth Medical School, Hanover, NH). The unbound IL2. Because of the uneven distribution of IL2R
mAb corresponding to these hybridomas were isolated in theT cell preparations, the total amount of rIL2 bound
from ascites fluid using a protein A antibody purification to 2.5 x loh cells was expressed as binding capacity (Bmax).
kit obtained from Beckman (Fullerton, CA). The affinity- The radiolabeled rIL 2 binding assay was performed as
purified rabbit anti-rat IgG and rabbit anti-hamster IgG described [32].
antibodies used in cell preparations were from Zymed (San
Francisco, CA) and Jackson Immunoresearch (West
Grove, PA), respectively. A highly purified human rIL2 2.6 IL 2-dependent proliferative response of activated
(18 x loh U/mg) [24, 251 was generously supplied by Cetus lymphocytes
Corporation (Emeryville, CA). The 1251-rIL2 (33 to
42 pCi/pg) and ["IdThd (2 Ci/mmol) were purchasedfrom After IL2R induction, activated T lymphocytes were
New England Nuclear (Boston, MA). Deoxycytidine 5 ' - cultured at 10h/ml, for 48 h, in 96-well plates, in the
(a"P)triphosphate (3000 Ci/mmol) was from Amersham presence of rIL2 concentrations ranging from 1 PM to
(Arlington Heights, IL). The plasmid pcEXV-mIL 2-R8 100 nM. Proliferation was measured by the incorporation of
containing the coding region of the murine IL2R [26] was a 1 pCi of [3H]dThd/well during the last 4 h of culture.
gift from Dr. D. Pardoll (Johns Hopkins University, Balti-
more, MD). The plasmid pMIL2-20 [27] containing the
murine IL2cDNA was obtained from Dr. N. Holbrook 2.7 IL2 bioassay
(National Institute on Aging, Baltimore, MD).The culture
medium used throughout these experiments was IL2 biological activity was determined by measuring the
RPMI 1640 (Hazelton, Lenexa, KS) containing 10% FCS IL 2 concentration-dependent proliferation of the cloned
(Hyclone, Logan, UT), 20 mM Hepes, 5 x M 2-ME murine cytotoxic T cell line CTLL-16 [33], which is suffi-
and 100 pg/ml gentamycin, and is referred to as complete ciently sensitive to detect as low as 5.0 pM rIL2.
medium.
2.8 Northern blot analysis
2.3 Cell preparation
Total RNA was extracted and isolated from 7 X lo7cells by
The preparation of restingT lymphocytes was as previously solubilization in guanidine thiocyanate according to Chirg-
described [28] except for the use of Tcell recovery column win et al. [34]. Aliquots of 10 pg of RNA were electropho-
(Sci-Can Diagnostics, Alberta, Canada) in place of nylon resed on formaldehyde-agarose gels and transferred to
wool columns. The CD8+ subset was isolated by negative nitrocellulose membranes. The filters were hybridized
panning. Briefly, purified resting T lymphocytes were overnight at 42°C with the murine IL2 and IL2R cDNA
incubated on ice for 30 min, at 107/ml,in HBSS containing probes radiolabeled with a-32P-dCTPby nick translation.
2% FCS and 10 pg/ml anti-L3T4 mAb. After centrifuga- Conditions for nick translation, hybridization and washing
tion through FCS, 4 x lo7 cells were resuspended in 5 ml of were as described [35].
cold PBS containing 5% FCS and poured onto a petri dish
(Falcon 1029) previously coated with affinity-purified rab-
bit anti-rat IgG as described [29]. Of the nonadherent cell 3 Results
population 90% to 95% recovered was CD8+ with essen-
tially no contamination by CD4+ cells. 3.1 High-affinity IL 2R induced by PBuz are not
functional unless IL 2 is present during the induction
period
2.4 Induction of IL2R
Whereas mitogenic activation of T cells induces both the
Purified resting T lymphocytes, or the CD8+ subset, were secretion of IL 2 and the expression of IL 2R [36], activation
placed in culture in complete medium, at 37 "C, at a density of purified T lymphocytes by PBu2 does not induce the
of 106/ml. for 10 h , in the presence of the appropriate generation or the secretion of IL2 [37, 381. Twenty-hours
agonists at the indicated concentrations.When activated by after exposure to 50 nM PBu2, high-affinity (Kd = 12 pM,
immobilized anti-CD3 mAb, 3 x lo7 cells were incubated B,,, = 3 PM) and low-affinity IL2R ( K d = 9500 pM.
for 20 h, in 10 ml of complete medium, in a petri dish B,,, = 260 PM) could easily be demonstrated by Scatchard
previously coated with anti-CD3 as described [30]. At the analysis of rIL2 binding to the surface of activated
end of the induction period, the cell suspension was layered T lymphocytes (Fig. lA).The addition of 50 pM rIL2 to the
Eur. J. Immunol. 1991. 21: 335-341
c
0 Although these preparations of T lymphocytes activated in
x
- 0
0 3 6 9 1 2 1 5
- 0
r112 (M)
0 10 20 Jo 40 50
PI PM
Figure 1. Effect of exogenous and endogenous IL2 on the induc-
tion of IL2R in PBu2-activated T lymphocytes. Purified resting
T lymphocytes were incubated for 20 h with 50 n M PBu2 in the
absence (A)and in the presence of 5OpM rIL2 ( 0 )or 0.3 pM
ionomycin (A). The activated cells were then processed as
described in Sect. 2 to evaluate the rIL2 binding characteristics (A
and B). The functionality of the IL2R expressed under these
conditions was assessed by measuring the ability to transmit a
proliferative signal in response to various rIL2 concentrations (C).
The SE determined on triplicate samples were < 5% of the mean
values shown on t h e graph.
cell culture during the 20-h induction period resulted in an
approximately 3-fold increase in the quantity of high-
affinity IL2R (Kd = 18 pM, B, = 10 pM) and a 2-fold
increase in the quantity of low-affinity IL2R
(Kd = 8200 pM, B,,,450 pM) (Fig. 1B). Similar results were
obtained when endogenous IL2 production was initiated
by the addition of ionomycin in place of exogenous rIL2.
When compared to the induction of IL2R by PBu2 alone,
activation of resting T cells by 50 nM PBu2 and 0.3 pM
- 0
0 5 10 15 20 r112 (M)
P I PM
Figure 2. Effect of CsA suppressions of endogenous IL2 on the
expression of IL2R by Con A-activated T cells. RestingT lympho-
cytes were activated for 20 h by exposure to 5 p g h l Con A in the
absence ( 0 )and in the presence (A)of 100 nM CsA. As a control,
Con A-activated cells were also exposed to CsA in the presence of
1nM rIL 2 (0).The radiolabeled rIL 2 binding properties (A and
B) of the activated cells and the IL 2 concentration-dependent
proliferation (C) were evaluated as in Fig. 1.
IL2-dependent induction of functional high-affinity IL2R 337
ionomycin resulted in a 2-fold increase in the expression of
high-affinity (Kd-10 pM, B,, = 6 PM) and low-affinity
(Kd-6000 PM, Bmax = 530 PM) IL2R (Fig. 1B).
the absence or in the presence of IL2 all expressed a
measurable amount of both high- and low-affinity IL2R,
they differed dramatically in their requirement for the
amount of rIL2 needed to signal for proliferation. Tcells
activated by PBu2 alone were unable to proliferate in
response to rIL2 concentrations in the range of the Kd of
the high-affinity IL 2R. A significant proliferation was
nevertheless observed between M and lo-' M rIL2,
indicating that the proliferative mechanism was intact but
that the proliferative signal was not being transmitted
through a fully functional high-affinity IL 2R. However,
when exogenous or endogenous IL2 was present during the
induction phase the activated T lymphocytes expressed
fully functional high-affinity IL 2R as indicated by their
ability to proliferate in response to picomolar concentra-
tions of rIL2 (Fig. 1C).
3.2 Other models of T lymphocyte activation achieved
in the complete absence of IL2 also result in the
generation of high-affinity IL2R unable to signal for
proliferation unless exposed to extremely high
concentrations of IL 2
Because phorbol diester appears to induce IL 2R through
direct activation of PKC [39,40], we sought an alternative
experimental approach in which T lymphocytes could be
triggered to express IL 2R via a transmembrane signaling
pathway. RestingT lymphocytes activated by Con A in the
presence of CsA, a known inhibitor of IL 2 gene expression
[41], display a measurable amount of high-affinity IL2R
(Kd = 14 pM, B,, = 0.4 pM) (Fig. 2A). The addition of
rIL2 toTcells activated by Con A in the presence of CsA
induced a 3-fold increase in the quantity of high-affinity
IL2R (Kd = 26 pM, B,,,- 1.2 pM) (Fig. 2B). Allowing
endogenous IL2 synthesis by omitting CsA during the
induction period also increased the quantity of high-affinity
IL2R (Kd-25pM, Bma,-2.5pM) (Fig.2B) to a level
comparable t o that induced by PBu2 alone. As with
PBu2-activated lymphocytes, the high-affinity IL2R
induced by the combination Con A/CsA appeared to be
refractory to IL2 in that they were unable to transmit a
proliferative signal unless excessively high concentrations
of rIL2 were available (Fig. 2C). It should be noted that
cells induced by PBu2 alone expressed 8 times more
high-affinity IL2R thanT lymphocytes activated by Con A
in the presence of CsA and yet showed an identical
proliferation pattern, indicating again that the signal was
not transmitted through a fully functional high-affinity
IL 2R. In spite of the presence of CsA, however, exogenous
rIL2 during the induction period was able to fully restore
the functional properties of these receptors as indicated by
the fact that picomolar concentrations of rIL2 were
sufficient to signal for proliferation (Fig. 2C). Interestingly,
T lymphocytes activated by the combination Con A/CsA in
the presence of rIL2 express approximately half the
quantity of high-affinity IL 2R displayed by cells activated
by PBu2 alone and yet proliferate in response to picomolar
amounts of rIL2 while nanomolar concentrations of rIL2
are required for the proliferation of PBu2-activated cells.
 338 J. J. Proust. N. L. Shaper, M. A. Buchholz and A. A. Nordin
rlL2 (M)
- 0
0 5 10 I5 20 25 30
PI PM
Figure 3. Effect of exogenous rlL 2 o n the generation of functional
1L2R in CD8+ T lymphocytes activated by the soluble anti-CD3
mAb 2C11. Purified resting CDX+ T lymphocytes were induced to
express IL2R by a 20-h incubation with 50 yg/ml soluble anti-CD3
mAb in the absence (A)and in the prcsence ( 0 )of 1 nM rIL2.The
activated CD8' Tcells were then processed for 1251-rlL2binding
assays ( A and B) and proliferation (C) as described.
Taken together, these results strongly suggest that the
defective transmission of the proliferative signal is not
linked to a problem of receptor density. Comparable results
were obtained when T lymphocytes were triggered by
immobilized anti-CD3 in the presence of CsA or forskolin
(data not shown). Because the action of forskolin or CsA is
most likely not limited to the inhibition of IL2 gene
expression [42-441. an alternative was sought where cells
could be triggered to express IL2R without concomitant
IL2 synthesis. It has been shown that T cell antigen
receptor cross-linking is required for IL2 production and
release but not for IL2R expression [13]. Purified resting
CD8+ T lymphocytes activated for 20 h by 50 pg/ml of
1 2 3 4 5 6 1 2 3 4
._
Figurr4. RNA blot analysis of 1L2R and IL2 mRNA. Resting
T lymphocytes were activated for 20 h in the complete absence (A
and C) o r in the presence o f endogenous or exogenous IL2 (B and
D). Ten micrograms of total RNA was electrophoresed on a
formaldehyde-agarose gel. After transfer to nitrocellulose, the blot
was hybridized with the '?P-labelcd IL2R (A and B) and IL2 (C
and D) probes. (A) and (C): 1, control: 2. Con A CsA: 3, PBu2;
4,soluble anti-CD3; 5 . immobilized anti-CD3 + CsA; 6, immobil-
ized antLCD3 + forskolin. (B) and (D): 1. IL2: 2, Con A; 3.
Con A + CsA + IL2; 4. PBu? + IL2: 5. PBu2 + ionomycin; 6.
soluble anti-CD3 + IL2: 7. immobilized anti-CD3.
Eur. J. Immunol. 1991. 21: 335-341
soluble anti-CD3, E-chain-specific mAb expressed a detect-
able amount of high-affinity IL2R ( K d - 13 pM.
B,,,-0.6 pM) (Fig. 3A). As in the other experimental
systems, the addition of rIL2 during the induction period
resulted in the generation of functional high-affinity IL2R
(Kd = 12 pM, B,, = 24 pM) (Fig. 3B) capable of signaling
for proliferation when exposed to picomolar quantities of
rIL2 while those induced in the absence of rIL2 required
approximately 2 orders of magnitude more rIL2 to induce
an equivalent level of proliferation (Fig. 3C).
In the experimental systems where IL2 production was
either not induced or prevented, the absence of endoge-
nous IL2 during the induction phase of the IL2R was
ascertained by CTLL bioassay. N o biologically active
protein was detected in the culture supernatant generated
during the various conditions of activation. The accumula-
tion of p55-specific mRNA and the absence of IL2
transcript was further confirmed by Northern blot analysis
(Fig. 4A and C).When endogenous IL2 production was
allowed, the simultaneous presence of IL 2 and IL 2R
transcripts was observed (Fig. 4B and D, lanes 2 , 5 and 7).
When exogenous rIL2 was provided only the accumulation
of p55-specific mRNA could be seen (Fig. 4B and D.
lanes 1, 3, 4 and 6).
3.3 Exposure of resting T lymphocytes to rlL2 alone
results in the expression of low-affinity IL2R only
Because the presence of IL2 during the induction period
appeared to be the determining factor that conferred
functional properties to the high-affinity IL 2R induced as a
consequence of cell activation by either phorbol esters,
mitogens or anti-antigen receptor mAb, it was essential to
evaluate the effect of IL2 on unstimulated lymphocytes.
Resting lymphocytes, which did not express detectable
levels of either high- or low-affinity IL2R as evaluated by
Scatchard analysis, acquired a measurable amount of
low-affinity IL2R (Kd = 3000 PM, B,,, = 40 pM). when
5 6 7
exposed for 20 h to 1 nM rIL2 (Fig. 5A). The analysis of
RNA blots confirmed the presence of p55-specific mRNA
- (B) (Fig. 4B, lane 1).Irrespective of whether the restingT lym-
- 36kb phocytes were incubated with medium alone, with 50 pM
rIL2 or with 1 n M rIL2, the subsequent proliferation was
- 2.5 kb dependent on the presence of rIL2 concentrations in the
order of M (Fig. 5B).
- 0.9 kb
olE
O 00 2 4
101"1
6
&=e&g?-i=+
,ou 10" 1030
.a -:I
10.
IrlLPl IMI
Figurc.5. rIL2 induction of IL2R in resting T lymphocytes.
-:
108 16
+ Resting T lymphocytes were exposed for 20 h to medium alone
(0),50 pM rIL2 (A)or 1 nM rIL 1 ( 0 ) The. physical characteri-
zation of the 1L2R induced under these conditions was determined
by Scatchard analysis (A) and the functional properties were
assessed by the proliferative assay (B).
Eur. J. Immunol. 1991. 21: 335-341 IL2-dependent induction of functional high-affinity IL2R 339

3.4 T lymphocytes induced to synthesize IL2 express

fully functional high-affinity IL2R even in the
presence of mAb anti-IL2 and anti-lL2R during the
induction period

In order to assess the roles of intra- and extracellular IL 2 in

the induction of high-affinity IL2R, restingT lymphocytes
were activated for 20 h under conditions in which the
extracellular IL 2 (either exogenous or endogenously pro-
duced) was not available because of the presence of mAb
anti-IL2 (DMS-1) and anti-murine p55 (PC61). T lympho-
cytes were activated to 50 nM PBu2 for 20 h in the presence
of 100 PM rIL2 and 20 pg/ml PC61. These PBuz-activated
cells, which do not synthesize IL2, were unable to prolifer-
ate unless exposed to a nanomolar concentration of rIL2,
demonstrating that the presence of PC61 during the
incubation period prevented exogenous rIL 2 from gaining
access to the external structure responsible for the induc-
10" io'o iod
tion of functional high-affinity IL 2R. Omitting PC61 10'2 10.9 107

lrlul (M)
during the incubation period restored the normal efficiency
of the high-affinity IL2R induced by PBu2 in the presence Figure 7. Effect of extracellular I L 2 blockade on the induction of
of picomolar concentrations of rIL 2 (Fig. 6) demonstrating IL2R in IL 2-producing Con A-activated T lymphocytes. Resting
the high-affinity binding property of this structure. Con A- T lymphocytes were activated by a 20-h exposure to 5 pglml Con A
activated T lymphocytes which synthesize IL2 were also in the absence ( 0 )and in the presence (A)of 20 pglml anti-p55
induced to express IL2R for 20 h in the presence of mAb PC61 and 500 vglml anti-IL2 mAb DMS-1.After pronase
500 vg/ml DMS-1 and 20 pg/ml PC61. IL2 was undetect- treatment, Fig. 6.
the cells were prepared for the proliferative assay as in
able by CTLL bioassay in the culture supernatant of the
mitogen-activated T cells indicating that the DMS-1 and
PC61 used were at concentrations sufficient to effectively
block the activity of extracellular IL2. To ensure complete generated by Con A alone (Fig. 7).These data suggest that
removal of Con A and the mAb anti-IL2R, the mitogen- the binding of endogenous IL2 to some internal receptor is
activated cells were treated with pronase before being sufficient to induce the expression of functional high-
exposed to rIL2.The high-affinity IL 2R induced by Con A affinity IL2R on the plasma membrane of activated
activation in the presence of mAb blocking extracellular lymphocytes.
IL2 and p55 were fully functional and transmitted a
proliferative signal as efficiently as the high-affinity IL 2R
4 Discussion

The observation that high concentrations of IL2 are

capable of inducing the proliferation of human resting
T cells in the apparent absence of other activation signals
r' [45] has led to the following plausible model of T cell

!I
Q
I
150
activation: IL 2 induces proliferative competence by bind-
ing to the p75 protein shown to be constitutively expressed
on the surface of resting Tcells [10].This interaction results

E 1wc I
i
I
in the rapid induction of various genes involved in T cell
activation, including the gene coding for p55. The expres-
sion of p55, in turn, permits the assembly of the high-
affinity p55/p75 heterodimer capable of transmitting a
proliferative signal delivered by extremely low concentra-
tions of IL2. Our assessment of the action of rIL2 on
murine T lymphocytes confirms the fact that resting Tcells
I I can be induced to proliferate only when exposured to rIL2
concentrations in the range of the K d of isolated p75,
suggesting that such proteins are present on their surface
although they are not detectable by Scatchard analysis.
Although a 20-h preincubation with 1 n M rIL2 induces the
ClUl (M) expression of p55 as judged by Scatchard and Northern blot
analysis, this does not result in the formation of functional
Figure 6. Effect of extracellular IL2 blockade on the induction of high-affinity heterodimers as indicated by the proliferative
IL2R in PBu2-activated T cells. Resting T lymphocytes were
induced to express IL 2R by a 20-h incubation with 50 nM PBu;! and
data as well as by the Scatchard analysis. Therefore, even
100 PM rIL2 in the absence ( 0 )and in the presence (A)of 20 pglml though IL 2 alone increases p55 expression, full prolifera-
of the anti-p55 mAb PC61.The activated T cells were then treated tive competence is not confered to resting T lymphocytes.
with Pronase and processed for the IL2 concentraiton-dependent These results are in partial disagreement with those
proliferation assay. reported by Siege1 et al. [46] in which resting human
340 J. J. Proust, N. L. Shaper, M. A. Buchholz and A. A. Nordin
lymphocytes, incubated for 16 h with 1 nM rIL2, acquired
functional high-affinity IL 2R.
In our experimental model, high-affinity IL2 binding sites
can be induced by a variety of ligands and/or PKC
activators. However, if these stimuli fail to induce the
synthesis of IL2, or if IL2 production is prevented, the
resulting high-affinity IL 2R is unable to signal for prolifer-
ation when exposued to IL2 concentrations consistent with
the Kd of the binding site. The presence of picomolar
amounts of rIL2 during the induction period is both
necessary and sufficient to confer functionality to these
high-affinity IL 2R. These results suggest that ligand-
induced cell activation leads to the expression of a structure
that binds IL2 with a high affinity and is responsible for
confering competence to the high-affinity IL 2R involved in
IL 2-dependent proliferation. Because the presence of a
sufficient concentration of anti-p55 mAb can totally sup-
press the inductive activity of picomolar levels of rIL2 in
PBu2-activated cells, p5S most likely participates in the
formation of that structure. Moreover, in a ConA-
activated IL 2-generating system, the presence of mAb
blocking both IL 2 and IL2R does not prevent the induction
of fully competent high-affinity IL2R. This suggests that
this structure need not be displayed at the surface of the cell
and that endogenous intracellular IL 2 is sufficient to confer
functionality to the IL2R. It is unclear whether this
structure is the classic pS5/p75 heterodimer, implying that
the same receptor is responsible for the transduction of
both the inductive and the proliferative signals or repre-
sents a different type of receptor involved only in the
tansmission of an inductive signal. Interestingly, the struc-
ture of the murine IL2R appears to be more complex than
initially thought. Recently, Herrmann and Diamantstein
[47] presented evidence that a third molecule, termed
y-chain, was exclusively associated with the high-affinity
IL2R. Saragovi and Malek [6, 481 confirmed that observa-
tion and provided additional data suggesting that the
high-affinity murine IL 2R may be at least a tri-molecular
strucure consisting of p55, p75 and a putative 30-kDa
protein linked by disulfide bridges to p75. Whether this
30-kDa protein can be expressed independently and has a
signaling function on its own is presently unknown. It is not
unreasonable to propose that this multichain complex may
be able to transduce discrete signals provided by the same
ligand binding under different affinities.
It is still unclear how the presence of picomolar amounts of
IL 2 during the induction period can convert a nonfunction-
al high-affinity IL 2R into a fully operational receptor.
However, a novel signaling mechanism recently reported
for the IL6R [49] may provide a clue for other cytokine-
receptor systems. IL6 was shown to trigger the association
of the 80-kDa IL6R with another nonligand-binding
glycoprotein involved in signal transduction, thus trans-
forming an inactive IL6 binding polypepitde chain into a
fully functional IL 6R. Interestingly, the intracytoplasmic
portion of the IL2R, like that of the IL6R, has no kinase
domain [ 5 0 ] , implying that transduction of the growth
factor signal is mediated by another molecule associated
with the receptor.
Because IL 2 up-regulates the expression of p55 and most
likely also the expression of p75 in activated T cells, a mere
increase in the density of high-affinity IL 2R/cell beyond a
Eur. J. Immunol. 1991. 21: 335-341
critical threshold may elicit a proliferative signal. Since the
actual size of the population of T cells responsive to the
various activators is unknown, any attempt to quantify the
number of IL2R/cell can be misleading. However, T
lymphocytes activated by soluble anti-CD3 or a combina-
tion of Con A and CsA in the presence of exogenous rIL2
express fewer high-affinity IL 2R than do cells activated by
PBu2 alone, and still proliferate in response to picomolar
concentrations of IL2 while PBu2-activated cells do not.
This indicates that the faulty signal transmission is not
strictly related to a problem of receptor density. Indeed, the
inability of a sufficient density of high-affinity IL2R to
transmit a proliferative signal has also been observed with
human CD4+ lymphocytes [37] and non-cytotoxic suppres-
sor CD8+ T cells [51]. Moreover, under certain experimen-
tal conditions, antigen-specific murine cytotoxicT lympho-
cyte clones are unable to proliferate in response to IL2 in
spite of high levels of high-affinity IL2R. However, engage-
ment of these high-affinity IL2R by IL 2 results in enhanced
expression of p55, clearly indicating that inductive and
proliferative signals can be dissociated [52].
Considering the possible regulatory steps of the cell cycle,
these data suggest that additional control points may exist
during the early stages of cell activation. In the IL2-
dependent proliferation of T lymphocytes, the established
restriction point that determines entry into S phase is
defined by the expression of functional high-affinity IL 2R.
However, IL2 regulation of Tcell proliferation appears to
be also involved at an earlier stage in the GIphase of the cell
cycle. Following ligand stimulation, reactive T cells leave
the Go phase but progression to the main restriction point is
dictated by the availability of IL2. In the absence of IL2
early in the GI phase, adequate levels of high-affinity IL 2R
are expressed but are unable to transmit the signal that
allows entry into the S phase. This putative IL2 sensitive
switch point, situated upstream of the main restriction
point, would appear as a new control mechanism and could
physiologically serve as an abortive pathway during the
process of cell activation.
Received July 16, 1990; in revised form September 14. 1990.
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