-
0022-1767/91/1478-2734$02.00/0 THE JOURNAL OF IMMUNOLOGY
Copyright 0 1991 by The American Assoclatlon of Immunologists
Vol. 147,2734-2739. No. 8. October 15. 1991 Prlnted Ln U. S.
A.
NUCLEAR TRANSCRIPTION FACTORS THAT BIND TO ELEMENTS OF THE IL-2
PROMOTER
Induction Requirements in Primary Human T Cells
ANGELA GRANELLI-PIPERNO AND PATRICIA NOLAN From the Laboratory
of Cellular Physiology and Immunology, The Rockefeller Uniuersfty,
New York, NY 10021
Prior studies have identified several elements that contribute
to the activity of the 1L-2 promoter in the stimulated T cell line,
Jurkat. The sites and their corresponding nuclear binding factors
include: NF- KB, AP-1, AP-3, OCT-1, and NF-AT. The latter nu- clear
factor for activated T cells likely contributes to the tissue
specificity of 1L-2 gene expression. Using electrophoretic mobility
shift assays, we have studied these transcription factors in
primary T cells from human blood to verify their presence in a
physiologic setting and to identify the signals that stimulate
factor activity. All factors are induced in the nuclei of T cells
upon activation with mitogens but not with exogenous IL-2 growth
factor. However, the signaling requirements and sensitivity to pro-
tein synthesis inhibitors differ considerably. Only the activities
for NF-AT and AP-1 sites require two signals for optimal induction,
i.e., PMA plus either lectin or antibody to the CD3 or CD28 surface
mol- ecules. Other factors are induced by lectin, anti- body,
and/or PMA alone. After appropriate stimu- lation, both NF-AT and
AP-1 are peculiarly sensitive to the protein synthesis inhibitor
anisomycin. Our data correlate the activity of NF-AT and AP-1 in
gel shift assays with the two signals requirements for IL-2 gene
expression.
The induction of lymphokine gene expression is a piv- otal step
in the function of T lymphocytes. Lymphokines and their mRNA are
undetectable in resting T cells but are rapidly induced by specific
Ag or polyclonal mitogens, the latter including certain lectins
such as PHA and Con A, as well as mAb to T cell surface molecules
such as CD2, CD3, and CD28 (1-7). A feature of all these stimuli is
that a second signal is required in addition to the Ag, lectin, or
mAb. This second stimulus is provided by PMA or by APC (8).
Given the two signal requirement for the stimulation of
lymphokine gene expression, it is perhaps not surpris- ing that the
regulation of transcription is proving to be complex. The principal
lymphokine that has been studied
Received for publication February 28, 1991 ..
The costs of publication of this article were defrayed in part
by the Accepted for publication July 29, 1991 ..
payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indi- cate this fact.
This work was supported by the National Institute of Health
Grant
Address correspondence and reprint requests to Dr. Angela
Granelli- GM-37643 to A.G.-P.
Plperno. The Rockefeller Unlversity, Box 280. New York. NY
10021.
is IL-2 or T cell growth factor. Several promoter elements are
necessary for full inducibility of the IL-2 gene upon transfection
into the human T cell line, Jurkat (9-12). Five positive regulatory
elements have been identified. An NF-KB binding site is found at
positions -188 to -208 and is present in other lymphokine promoters
such as TNF (1 3). granulocyte-macrophage-CSF (1 4), IL-6 (1 5, 16)
as well as the IL-2R p55-gene and the HIV-1 LTR (17- 191. Two PMA
responsive sites are evident. An AP-3 binding site at positions
-185 to -200 lies within the NF- KB site, whereas an AP-1 binding
site is present at posi- tions - 15 1 to - 145. OCT- 1 sites, which
are found in many promoters such as those for Ig and histone genes
(20- 22), are located at -63 to -93 (OCT-1 proximal) and -240 to
-250 (OCT-1 distal) (23).
Positions -285 to -265 of the IL-2 enhancer is a prin- cipal
candidate for regulating the tissue specificity of the IL-2 gene.
This site binds a factor NF-AT found to date only in activated
Jurkat cells (24) and perhaps some B lymphocytes (25). The
induction of NF-AT is known to be sensitive to the
immunosuppressive drug, CsA3 (26, 27). which primarily acts by
reducing the induction of lymphokine gene transcription (28-30). It
is proposed that NF-AT is the link between signaling at the T cell
surface and the induction of IL-2 gene expression (25).
Inasmuch as most of the studies involving the control of the
IL-2 gene have been done in the Jurkat T cell line, we believed it
important to assess the various transcrip- tion factors of the IL-2
promoter in primary T cells. Primary cells are of intrinsic
importance, particularly for studying the role of various stimuli
on IL-2 gene activity. We find that all of the above transcription
factors can be identified in nuclei from human blood T cells upon
stim- ulation. We then use primary T cells to study the signals for
inducing the activity of the factors in EMSA. Interest- ingly, both
NF-AT and AP- 1 require two signals for their induction and are
sensitive to agents that block IL-2 gene transcription, whereas
factors for the other sites can be induced by one signal such as
PHA, mAb to CD3, or PMA. Exogenous IL-2, which does not induce
expression of the IL-2 gene, does not induce any of the nuclear
factors that we studied.
MATERIALS AND METHODS
Cell cultures. Human blood mononuclear cells were isolated from
buffy coats (purchased from the New York Blood Center, New York.
NY) on Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ)
density gradients. The cells were collected, washed in PES. and
Abbrevlations used in this paper: CsA, cyclosporin A: EMSA.
electro- phoretic mobility shift assay: DTT. dlthiothreitol.
2734
-
INDUCTION OF NUCLEAR FACTORS FOR IL-2 PROMOTER 2735
rosetted with neuraminidase-treated (Vlbrfo cholera neuramini-
dase, Calbiochem-Boehringer Diagnostics. La Jolla. CA) sheep E and
separated over Ficoll-Hypaque. The E rosette-positive fraction was
further purified by passage over a nylon wool column and used a s a
source of T cells. By FACS analysis. these T cells were >96%
CD3' and el% CD14* and
-
2736 INDUCTION OF NUCLEAR FACTORS FOR IL-2 PROMOTER
mologous oligonucleotides but not by the oligonucleotide with
mutation within the appropriate consensus binding site. Moreover
50x molar excess of cold NF-AT oligo did not compete for the
binding of AP-1 to the AP-1 binding site and conversely cold AP-1
oligo did not compete for the binding of NF-AT to the NF-AT site
(not shown).
Distinct signaling requirements for NF-AT and AP-I relative to
other nuclear transcription factors. The in- duction of IL-2 gene
expression is known to require "two signals." One signal ligates
the TCR. or some other T cell surface molecule such as CD2 or CD28.
and can be provided by a lectin like PHA or a mAb. The second
signal is provided by accessory cells or PMA. PHA alone induced
NF-KB and AP-3 activities, and low level of AP- 1, whereas PMA
alone induced NF-KB. OCT- 1, AP-3, and low levels of AP-1 and
OCT-Id (Fig. 3). The combination of PHA and PMA was required for
the induction of NF-AT and greatly increased the activity of AP-1.
Therefore the two signal requirement for the induction of IL-2 gene
expres- sion is associated with a similar requirement for the
induction of NF-AT and AP- 1. It is noteworthy that pro- tein in
nuclear extracts of primary T cells bounded very poorly to the
OCT-ld site when compared to protein in nuclear extracts from
Jurkat cells (data not shown).
In initial reports, cycloheximide did not block the in- duction
of IL-2 gene expression (39-41). subsequent studies (24, 42)
including our own (data not shown) have indicated that the more
rapidly acting inhibitor, aniso- mycin. does block. After
stimulation with PHA and PMA. the induction of NF-KB. OCT-lp. and
AP-3 were each resistant to anisomycin (Fig. 4). However, the
induction of NF-AT and AP-1 was ablated by anisomycin (Fig. 4).
?f$:: . . . . f E $ Z Z ! g $ z Z f J $ Z $ J 4 $ % g bl$z? - .
- . - ,- - -.. . . - . . .
h w NF-AT AP-3 AP : NF-kE OCT-ip OCT i d
Figure 3. Signaling requirements for the induction of nuclear
factors that bind to sites of the IL-2 promoter. Human T cells were
left unstimu- lated or were stimulated with PHA ( 1 pg/ml) and/or
PMA (5 ng/ml). After 8 h. the cells were collected and the nuclear
extracts tested for binding to the Indicated sites of the IL-2
enhancer a s described in Figure 1 .
NF-AT AP-3 AP-1 OCT-lp NF-LE
Anisomycin (50 pM) was added to T cells 30 min before PHA/PMA
[P/P) Ffgure4. Effect of anisomycin on the inductlon of nuclear
factors.
stimulation. After 8 h nuclear extracts prepared and analyzed a
s In Figure 1 .
again correlating the activity of these factors with IL-2 gene
expression.
Localization of NF-AT to nucleus and sensitivity to CsA. T cells
were stimulated for 15 h with the combina- tion of PHA and PMA
(Fig. 5; P / P ) in the presence or absence of CsA, which is known
to block the induction of IL-2 and other lymphokine genes (29. 43).
When ex- tracts of nuclei or cytoplasm were tested separately, only
the nuclei contained NF-AT (Fig. 5). The induction was blocked by
CsA. but not by the inactive analogue CsH (not shown). Mixing
experiments (not shown) indicated that the cytoplasm did not
contain an inhibitor of nuclear NF-AT activity.
Use of a-CD3 and a-CD28 mAb to induce nuclear transcription
factors. Similar experiments were per- formed using mAb to CD3 and
to CD28, rather than PHA. as ligands for more restricted sites on
the T cell surface. It is known that a-CD3 and a-CD28 also must be
added together with PMA to induce high levels of IL-2 mRNA in
primary human T cells (8.44). The a-CD3 mAb OKT3 by itself induced
AP-3 and NF-KB, but both OKT3 and PMA were needed to induce NF-AT
and AP-1 (Fig. 6). Another polyclonal T cell stimulant, a-CD28 also
induces the group of nuclear transcription factors (Fig. 7).
Induction of nuclear transcription factors in T lym- phoblasts.
Because Jurkat is unresponsive to IL-2. it has not been possible to
test the effects of this T cell growth factor on nuclear
transcription factors. We prepared pri- mary lymphoblasts and
tested their responses to IL-2 (Fig. 8, [eft). and to PHA/PMA (Fig.
8. right). It has been shown that PHA/PMA is needed to induce IL-2
gene expression in these blasts, and that IL-2 induces the p55
una PIP Plplc N C N C N C -
.
stimulated T cells, and the induction is sensitive to CsA. T
cells were left Ffgure5. NF-AT activity is detected only in the
nuclear extracts of
unstimulated or were stimulated for 5 h with P/P (PHA at 1
&ml. PMA at 5 ng/ml). in the presence or absence of CsA
[P/P/C). A total of 10 pg of nuclear [N) and cytosolic (C) proteins
were incubated with p2 end-labeled NF-AT site.
NF-AT AP-3 AP-1 NF-kB
Figure 6. Induction of nuclear factors with 0-CD3 mAb. OKT3.
Human T cells were left unstimulated or were stimulated for 6 h
with OKT3 [ 100 ng/ml) with or without PMA (5 ng/ml). Nuclear
extracts were isolated and samples analyzed as above.
-
INDUCTION OF NUCLEAR FACTORS FOR IL-2 PROMOTER 2737
NF-AT AP-1 AP-3 OCT-lp N F I B
Ffgure 7. Induction of T cell nuclear factors after stlmulatlon
wlth a- CD28 mAb. Human T cells were left unstlmulated or were
stlmulated for 6 h wlth a-CD28 mAb and PMA. Nuclear extracts were
Isolated after 6 h of stlmulatlon and samples analyzed a s
above.
3 ' *
- . NF-AT AP-3 AP-1 NF-kB
NF-AT AP-3 AP-1 NF-kB OCT-lp 1 L i
F f g u r e 8 . Comparlson of IL-2 and PHA/PMA for the Induction
of nu- clear factors ln T lymphoblasts. T blasts were cultured for
5 h with 100 U/ml rIL2 ( le f ) or the blasts were restlmulated
wlth PHA/PMA (P/P). Nuclear extracts were prepared 6 h later and
assayed for binding to sites of the IL-2 promoter.
NF-un, A P - 3 NF-AT NF-un *.I. m - 1 AP-1 A P - 3
ments of the IL-2 promoter (see Dfscusslon). FLgure9. Dlagram of
the slgnallng requirements for dlfferent ele-
IL-2R gene but not itself (29. 44). Without further stim-
ulation. the lymphoblasts lacked NF-AT and the OCT- 1 p factors,
but contained detectable NF-KB. AP-3. and AP-1. IL-2 did not induce
any of the factors, but PHA/PMA upregulated both AP-1 and NF-AT in
T blasts (Fig. 9 ). Therefore again the induction of IL-2 gene
expression in T blasts correlates with the up-regulation of both
AP-1 and NF-AT.
DISCUSSION
During the past few years, a wide range of nuclear factors have
been shown to bind to the IL-2 enhancer. Studies of the factors
that bind to the IL-2 enhancer have been mostly carried out by
transfection studies with re- porter genes driven by wild type or
deletion mutant in the Jurkat T cell line. The binding sequences
defined by these functional assay were subsequentially used in mo-
bility shift experiments to identify and characterize nu-
clear proteins that recognize their cognates. Some of these
factors, Le.. NF-KB, OCT-1, AP-3, are
ubiquitosly expressed and are found to bind to many
promoter-enhancer genes; however. the NF-AT protein is one of the
tissue specific nuclear transcription factor that is expressed
predominantly in T cells (25, 45).
The results presented in this report suggest that all the
nuclear transcription factors that were previously de- fined to
participate in the transcription of the IL-2 gene, using as a model
system the Jurkat cell line. can be identified by EMSA in primary
population of T cells from the blood.
Studies using homologous oligonucleotides and mutant oligos
demonstrate that nuclear proteins isolated from T cell bind to the
selected sites of the IL-2 enhancer in a sequence-specific manner.
Although function is not yet attributed to any of these bindings in
this T cell system (one of the reason is the difficulty to
efficiently transfect primary T cell populations). the main impact
of our find- ings is that the signaling requirements for IL-2 gene
expression in primary T cells correlate with the induction of both
NF-AT and AP-1 activities. The known require- ments for IL-2 gene
expression in stimulated T cells are protein synthesis and the
addition of two signals, Le.. either PHA. a-CD3. or a-CD28 plus
PMA. The induction of both NF-AT and AP-1 was blocked by anisomycin
and also required two signals. In contrast, the other factors we
studied (NF-KB, AP-3, and OCT-1) all could be induced by one signal
and were resistant to anisomycin. However, some differences with
the Jurkat results were noted. One is that the factor for the OCT-1
proximal site behaves like other factors, Le., it is low in
activity in unstimulated cells (although variations were noted for
different donors) and rises after application of mitogens. Second,
that the factor that bind to the OCT-1 distal site is present in
very little amount in T cell compared with Jurkat cells. More-
over, in a study of the effects of various immunosuppres- sive
drugs (46) we find that the immunosuppressive drug CsA also blocks
the induction of AP-1 that is different from the report in the
Jurkat cell line (26. 27). but the induction of NF-AT is sensitive
to cyclosporin A as was reported in the Jurkat T cell line (26.
27). The require- ments for the induction of nuclear factors for
the IL-2 promoters are diagramed in Figure 9.
Prior work has stressed the need for NF-AT to explain the
two-signal requirement for IL-2 gene expression (25. 47). However,
even though the AP-1 factor is a PMA- responsive element, two
signals also seem required for induction of high levels of AP-1 in
primary cells. AP-1 is the equivalent of c-jun protooncogene and
can associate with c-fos protein and augment transcription in
several system (48-51). It is known that the induction of c-fos
mRNA in T cells can be mediated by PMA alone (29). Therefore the
two signal requirement for optimal AP-1 in T cells may be exerted
at the level of the c-jun gene expression, or at the level of the
activity of c-jun or c-fos proteins.
Inasmuch as Jurkat grows continuously and does not respond to
IL-2, the cell line is not useful for studying signaling with this
growth factor. T blasts are exquisitely responsive to exogenous
IL-2. We found that none of the nuclear transcription factors that
could be induced with PHA/PMA were sensitive to IL-2 (Fig. 8). This
implies that IL-2 must induce other factors to exert its effects
on
-
2738 INDUCTION O F NUCLEAR FACTORS FOR IL-2 PROMOTER
growth including the induction of gene expression such as the
mRNA for the low affinity, IL-2R p55 polypeptide (52). When T
blasts were restimulated with combination of mitogens, NF-AT
induction was apparent. This induc- tion correlates with de novo
expression of IL-2 transcripts (29), strengthening the involvement
of NF-AT in the reg- ulation of IL-2 gene.
Further studies, using characterized factors and func- tional
assays, both in vivo and in vitro should provide a clearer picture
of the exact role of the various binding sites present in the IL-2
promoter in gene transcription.
Acknowledgement. We thank Dr. R. M. Steinman for critical review
of the manuscript and helpful discussion and Williams Avery for
expert technical assistance.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
REFERENCES
Meuer, S. C.. R. E. Hussey. M. Fabbi, D. Fox, 0. Acuto. K. A.
Fitzgerald, J. C. Hodgdon, J. P. Protentis. S. F. Schlossman, and
E. L. Reinherz. 1984. An alternative pathway of T-cell activation:
A functional role for the 50 kd T11 sheep erythrocyte receptor
protein. Cell 36:897. Van Wauwe. J. P.. J. R. DeMey, and J. G .
Goossens. 1980. OKT3: a monoclonal anti-human T lymphocyte antibody
with potent mito-
Meuer. S. C., R. E. Hussey, D. A. Cantrell. J. C. Hodgdon, S. F.
genic properties. J. Immunol. 124~2708.
Schlossman, and E. L. Reinherz. 1984. Triggering of the
T3/Ti
through an interleukin-2 dependent autocrine pathway. Proc.
Natl. antigen receptor complex results in clonal T-cell
proliferation
Acad. Scl. USA 81 : 1509. Samelson. L. E.. R. N. Germain. and R.
H. Schwartz. 1983. Mono- clonal antibodies against the antigen
receptor on a cloned T-cell hybrid. Proc. Natl. Acad. Scl. USA
80x3972. Meuer. S. C.. K. A. Fitzgerald, R. E. Hussey. J. C.
Hodgdon. S . F. Schlossman. and E. L. Reinherz. 1983. Cionotypic
structures in- volved in antigen-specific human T cell function:
relationship to the
Martin, P. J., J. A. Ledbetter, Y. Morishita, C. H. June, P. G.
Beatty, T3 molecular complex. J. Exp. Med. 157: 705.
and J. A. Hansen. 1986. A 44 kilodalton cell surface homodimer
regulates interleukin 2 production by activated human T lympho-
cytes. J. lmmunol. 136~3282. Hara. T., S. M. Fu, and J. A. Hansen.
1985. Human T cell activation. 11. A new activation pathway used by
a major T cell population via a disulfide-bonded dimer of a 44
kilodalton polypeptide (9.3 antigen). J . Exp. Med. 161: 151 3.
Hara. T., and S. M. Fu. 1985. Human T cell activation. I. Monocyte-
independent activation and proliferation induced by anti-T3 mono-
clonal antibodies in the presence of tumor promoter 12-o-tetradeca-
noyl phorbol-13-acetate. J. Exp. Med. 161:641. Fujita, T., H.
Shibuya, T. Ohashi, K. Yamanishi, and T. Taniguchi.
sequences in the 5 flanking region for the gene expression in
acti- 1986. Regulation of human interleukin-2 region functional
DNA
vated T lymphocytes. Cell 46:401. Serfling, E., R. Barthelmas,
1. Pfeuffer, B. Schenk. S. Zarius, R. Swoboda, F. Mercurio. and M.
Karin. 1989. Ubiquitous and lympho-
interleukin 2 gene in T lymphocytes. EMBO J. 8:465.
cyte-specific factors are involved in the induction of the
mouse
Durand. D. B., M. R. Bush, J. G . Morgan, A. Weiss, and G. R.
Crabtree. 1987. A 275 base pair fragment at the 5 end of the
interleukin-2 gene enhances expression from a heterologous pro-
moter in response to signals from the T cell antigen receptor. J .
Exp. Med. I65:395. Williams. T. M., L. Eisenberg. J. E. Burlein, C.
A. Noms, S. Pancer, D. Yao, S . Burger, M. Kamoun, and J. A. Kant.
1988. Two regions within the human IL-2 gene promoter are important
for inducible IL- 2 expression. J. Immunol. 141:662. Collar. M. A.,
P. Baeuerle, and P. Vassalli. 1990. Regulation of tumor necrosis
factor alpha transcription in macrophages: involve- ment of four
KB-lik.2 motifs and of constitutive and inducible forms
Schreck, R., and P. A. Baeuerle. 1990. NF- bas inducible
transcrip- of NF-KB. Mol. Cell. Biol. 10: 1498.
tionai activator of the granulocyte-macrophage
colony-stimulating
Shimizu, H.. K. Mitomo, T. Watanabe, S. Okamoto, and K-I. Ya-
factor gene. Mol. Cell. Blol. IO: 1281.
mamoto. 1990. Involvement of an NF-KB-like transcription factor
in the activation of the interleukin-6 gene by inflammatory lympho-
kines. Mol. Cell. Blol. 10561. Libermann. T. A., and D. Baltimore.
1990. Activation of interleukin- 6 gene expression through the
NF-KB transcription factor. Mol. Cell.
Bohnlein, E., J. W. Lowenthal, M. Siekevitz. D. W. Ballard, B.
R. Blol. 10:2327.
Franza. and W. C. Greene. 1988. The same inducible nuclear pro-
teins regulates mitogen activation of both the interleukin-2
receptor-
18. Hoyos. B., D. W. Ballard, E. Bohnlein. M. Siekevitz, and W.
C. alpha gene and type 1 HIV. Cell 53:827. Greene. 1989. Kappa
B-specific DNA binding proteins: role in the regulation of human
interleukin-2 gene expression. Science 244:457.
1 9. Cross. S. L., N. F. Halden, M. J. Lenardo, and W. J.
Leonard. 1989. Functionally distinct NF-rB binding sites in the
immunoglobulin k and IL-2 receptor alpha chain genes. Sclence
244:466.
20. Fletcher, C., N. Heintz, and R. G. Roeder. 1987.
Purification and characterization of OTF-1, a transcription factor
regulating cell cycle
21. LaBella, F.. H. L. Sive, R. G. Roeder. and N. Heintz. 1988.
Cell-cycle expression of a human histone H2b gene. Cell 51:773.
regulation of a human histone H2b gene is mediated by the H2b
subtype-specific consensus element. Genes Dev. 232.
22. Poellinger, L., and R. G. Roeder. 1989. Octamer
transcription fac-
immunoglobulin heavy-chain promoter. Mol. Cell. Blol. 9:747.
tors 1 and 2 each bind to two different functional elements in
the
23. Kamps, M. P., L. Corcoran, J. H. LeBowitz. and D. Baltimore.
1990. The promoter of the human interleukin-2 gene contains two
octamer- binding sites and is partially activated by the expression
of Oct-2.
24. Shaw, J. P.. P. J. Utz, D. B. Durand, J. J. Twle, E. A.
Emmel. and Mol. Cell. Blol. 10:5464.
G. R. Crabtree. 1988. Identification of a putative regulator of
early T cell activation genes. Science 241:202.
25. Ullman. K. S., J. P. Northrop. C. L. Verweij, and 0. R.
Crabtree. 1990. Transmission of signals from the T lymphocyte
antigen recep- tor to the genes responsible for cell proliferation
and immune func-
26. Emmel, E. A., C. L. Verweij, D. B. Durand, K. M. Higgins, E.
Lacy, tion: The missing link. Annu. Rev. Immunol. 8:421.
and G. R. Crabtree. 1989. Cyclosporin A specifically inhibits
func-
246:1617. tion of nuclear proteins involved in T cell
activation. Sclence
27. Schmidt, A., L. Hennighausen, and U. Siebenlist. 1990.
Inducible nuclear factor binding to the KB elements of the human
immunode- ficiency virus enhancer in T cells can be blocked by
cyclosporin A in a signal-dependent manner. J. Virol. 64:4037.
28. Granelli-Pipemo, A., K. Inaba. and R. M. Steinman. 1984.
Stimu- lation of lymphokine release from T lymphoblasts:
requirement for mRNA synthesis and inhibition by Cyclosporin A. J.
Exp. Med. 160: 1 792.
29. Granelli-Piperno, A., L. Andrus, and R. M. Steinman. 1986.
Lym- phokine and nonlymphokine mRNA levels In sUmulated human T
cells: kinetics, mitogen requirements, and effects of cyclosporin
A. J. Exp. Med. 163:922.
30. Kronke, M.. W. J. Leonard, J. M. Depper, S. K. Arya, F.
Wong- Staal, R. C. Gallo, T. A. Waldman, and W. C. Greene. 1984.
Cyclo- sporin A inhibits T-cell growth factor gene expression at
the level of mRNA transcription. Proc. Natl. Acad. Sci. USA
815214.
31. Dignam, J. D.. R. M. Lebovitz, and R. G . Roeder. 1983.
Accurate transcription initiation by RNA polymerase I1 in a soluble
extract
32. Ohlsson. H., and T. Edlund. 1986. Sequence-specific
interactions of from isolated mammalian nuclei. Nuclelc Aclds Res.
11:1475.
33. Bradford, M. 1976. A rapid and sensitive method for the
quantitation nuclear factors with the insulin gene enhancer. Cell
4535.
of microgram quantities of protein utilizing the principle of
protein- dye binding. Anal. Blochem. 72:248.
34. Fried, M.. and D. M. Crothers. 1981. Equilibria and kinetics
of lac repressor-operator interactions by polyacrylamide gel
electrophore-
35. Garner, M. M.. and A. Revzin. 1981. A gel electrophoresis
method sis. Nuclelc Acids Res. 9:6505.
for quantifying the binding proteins to specific DNA regions:
appll-
tory system. Nucleic Acids Res. 9:3047. cation to components of
the Escherlchla coli lactose operon regula-
36. Maniatis. T., E. F. Fritsch, and J. Sambrook. 1982. In
Molecular Clontng: A Laboratory Manual. Cold Spring Harbor
Laboratories, Cold Spring Harbor, NY. p. 191.
37. Holbrook, N. J., M. Lieber, and G. R. Crabtree. 1984. DNA
sequence of the 5 flanking region of the human interleukin 2 gene:
homologies
38. Chiu, R.. M. Imagawa, R. J. Imbra. J. R. Bockoven. and M.
Karin. with adult T-cell leukemia virus. Nucleic Aclds Res.
12:5005.
tional response to phorbol esters. Nature 329:648. 1987.
Multiple cis- and trans-acting elements mediate the transcrip-
39. Kronke. M., W. J. Leonard, J. M. Depper, and W. C. Greene.
1985. Sequential expression of genes involved in human T
lymphocyte
40. Granelli-piperno, A.. and R. Steinman. 1986. Induction of
gene growth and differentiation. J. Exp. Med. 161: 1593.
Immune Regulation by Characterized Polypeptides. G. Goldstein.
expression in human T lymphoblasts: effect of cyclosporin A. In
41. Efrat. S. . and R. Kaempfer. 1984. Control of biologically
active J. F. Bach, and H. Wigzell. eds. p. 139.
interleukin 2 messenger RNA formation in induced human lympho-
cytes. Proc. Natl. Acad. Scl. USA 81:2601.
42. Weiss, A., R. Shields, M. Newtown. B. Manger. and J.
Imboden. 1987. Ligand-receptor interactions required for commitment
to the activation of the interleukin 2 gene. J. Immunol.
138:2169.
43. Granelli-Piperno, A., and M. Keane. 1988. Effects of
cyclosporine A
-
INDUCTION OF NUCLEAR FACTORS FOR IL-2 PROMOTER 2739
plant. Proc. 20: 136. on T lymphocytes and accessory cells from
human blood. Trans-
44. Granelli-Piperno, A., M. Keane. and R. M. Steinman. 1988.
Growth factor production and requirements during the proliferative
response of human T lymphocytes to anti-CD3 monoclonal antibody. J.
im- munol. 1424138.
45. Verweij. C. L., C. Guidos. and G. R. Crabtree. 1990. Cell
type
of activated T-cells) transcriptional activity determined by a
new specificity and activation requirements for NFAT-1 (Nuclear
factor
method using transgenic mice to assay transcriptional activity
of an
46. Granelli-Piperno, A., P. Nolan. K. Inaba, and R. M.
Steinman. 1990. Individual nuclear factor. J. BLol. Chem.
265:15788.
The effect of immunosuppressive agents on the induction of
nuclear factors that bind to sites on the interleukin 2 promoter.
J. Exp. Med. 1 72: 1869.
47. Crabtree. C. R. 1989. Contingent genetic regulatory events
in T
48. Chiu, R.. W. J. Boyle. J. Meek, T. Smeal, T. Hunter. and M.
Karin. lymphocyte activation. Science 243:355.
transcription of AP-1 responsive genes. Cell 54:541. 1988. The
c-Fos protein interacts with c-Jun/AP-1 to stimulate
49. Sassone-Corsi, P.. W. W. Lamph, M. Kamps, and I. M. Verma.
1988. fos-Associated cellular p39 is related to nuclear
transcription factor
50. Abate, C.. D. Luk, E. Gagne, R. G. Roeder. and T. Curran.
1990. AP-1. Cell 54:553.
Fos and Jun cooperate in transcriptional regulation via
heterologous activation domains. Mol. Cell. BLoZ. i0:5532.
51. Nakabeppu, Y., K. Ryder. and D. Nathans. 1988. DNA-binding
activities of three murine jun proteins: stimulation by Fos.
Cell
52. Toledano, M. B.. D. G. Roman, N. F. Halden. B. B. Lin. and
W. J. 55:907.
tant for activation of the interleukin 2 receptor alpha-chain
gene in Leonard. 1990. The same target sequences are differentially
impor-
two distinct T-cell lines. Proc. Natl. Acad. Scl. USA
87:1830.