Week 6 IL2 TF 2

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 nuclear 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 protein 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 molecules. Other factors are induced by lectin, antibody, and/or PMA alone. After appropriate stimulation, 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 positions - 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 principal 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 transcription 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 stimulation. 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. electrophoretic mobility shift assay: DTT. dlthiothreitol.

2734

INDUCTION OF NUCLEAR FACTORS FOR IL-2 PROMOTER 2735

rosetted with neuraminidase-treated (Vlbrfo cholera neuraminidase, 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 induction 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 expression is associated with a similar requirement for the induction of NF-AT and AP- 1. It is noteworthy that protein 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 induction 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, anisomycin. 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 unstimulated 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 combination 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 extracts 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 lymphoblasts. 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 primary 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.


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 nuclear 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 stimulation. 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 mobility 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 defined 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 requirements 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 immunosuppressive 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 requirements 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 induction correlates with de novo expression of IL-2 transcripts (29), strengthening the involvement of NF-AT in the regulation of IL-2 gene.

Further studies, using characterized factors and functional 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.

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Week 6 IL2 TF 2