Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy

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SPECIAL INVITED ASH1 PAPER David D. Eckels, Guest Editor

Differential Induction of the NF-AT Complex During Restimulation and the Induction of T-Cell Anergy David Wotton, Julie A. Higgins, Robyn E. O’Hehir, Jonathan R. Lamb, and Richard A. Lake ABSTRACT: Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to upregulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cellspecific expression of the IL-2 and the p chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells.

ABBREVIATIONS APC antigen-presenting cell intracellular calcium concentration fCa2+1, dT deoxythymidine HA hemagglutinin MHC major hiscocompatibility complex

The inducible factors NK-KB, p E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription. The failure to induce NF-AT completely may be a consequence of a diminished calcium flux, since the PKC pathway was apparently intact. It was found that the calcium ionophore ionomycin could either induce anergy or abrogate the induction of nonresponsiveness according to the dose, also suggestive of differences in calcium signaling. The pattern of expression of transcription factors during the induction of T-cell anergy is consistent with the inability of anergic cells to produce IL-2. These results demonstrate that there are differences in the early nuclear events characteristic of stimuli, the outcome of which leads to cells that are phenotypically similar, but are functionally different. Htuwn immunology 42, 95-102 (1995)

NF-AT PBMC PHA PKC TcR

nuclear factor activated T cells peripheral blood mononuclear cell phytohemagglutinin protein kinase C T-cell receptor

INTRODUCTION Immunologic tolerance results from deletion of some self-reactive T lymphocytes in the thymus; other selfreactive T cells that escape deletion can be rendered non-

From the Department of Immunology (J.A. H., R. E.O., J.R. L., R.A. L.), St. Mary’s Hospital Medical School, imperial ColIege of Science, Technology and Medicine; and the Laboratory of Lymphocyte Molecular Biology (D. W. ), imperial Cancer Research Fund, London, England. Address reprint requests to Dr. R. A. Luke, Department of Immunology, St. Mary’s Hospital Medical School, Norfolk Place, London W2 IPG, England. Received (U) December I, 1993; accepted December 6, 1993. Human Immunology 0 American

42, 95-102 (1995) Society for Histocompatibility

and Immunogenetics,

1995

responsive or anergic (reviewed by Kroemer and Martinez El]). The second process is of particular interest because the ability to render specific clones of T cells nonresponsive in vivo may contribute to attempts to control immunologically mediated disease processes. Toward this end, we have investigated the mechanisms of induction of anergy with cloned T cells in vitro. T-cell activation and interleukin-2 (IL-2) production are regulated by several signals, including those generated through the T-cell antigen receptor (TcR), protein kinase C (PKC), and changes in the intracellular free cal019%8859/95/$9.50 SSDI 0198-8859(94)00004-A

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cium concentration ([Ca’ + Ii). Additionally, other costimulatory signals can act synergistically to cause synthesis of IL-2 and proliferation of the cells. The bestdocumented costimulatory pathway is mediated through CD28 on the T cell and the activation antigen B7/BB-1 present on the antigen-presenting cell (APC) 12-41. Stimulation through the TcR without a costimulatory signal results in anergy 153, and under some circumstances a signal through CD28 can block the induction of anergy in murine T-cell clones [6]. For human CD4 + T cells, however, stimulation through the TcR with high doses of appropriate peptide, in either the presence or absence of APCs, will inhibit subsequent antigendependent proliferation 171. These experiments clearly show that the optimum dose of peptide for the induction of anergy is higher in the presence of APCs. Thus, it seems that the magnitude of the signal received through the TcR can override an otherwise immunogenic stimulus and induce anergy. Fully activated T cells can also be rendered nonresponsive [81. And these observations anticipate clinical intervention in immunologically mediated diseases, where the target T cells are more likely to be activated than in a resting state and the T cells will certainly be in the presence of APCs. The phenotype of T cells during the induction phase of anergy is similar to the phenotype of cells undergoing stimulation. Key changes at the cell surface include downmodulation of the TcR complex and upregulation of CD2 and CD25 receptors. Interestingly, signals through the TcR upregulate transcription of the p chain of the TcR gene {9}, whereas transcription of the CD2 gene is not inducible. However, a complex array of posttranscriptional mechanisms regulates TcR gene expression [lo], such that the overall effect of activating or anergizing signals is downmodulation of cell surface TcR. After an activating signal, T cells upregulate their expression of the CD28 antigen. In contrast, T cells that receive a high dose of specific peptide as a tolerizing signal do not 18, 111. A signal through CD28 is a pleiotropic event: it is costimulatory for T-cell proliferation and results in the upregulation of several T-cell-derived cytokines by stabilizing their mRNAs 112). Ligation of CD28 affects IL-2 synthesis by the generation of a nuclear complex (CD28RC) that increases transcription from the IL-2 gene promoter { 131. The induction of the CD28RC is, however, clearly not specific for signals through the CD28 receptor as the complex is induced by the mitogenic combination of PMA and anti-CD3 antibodies [ 141. In this report, we have analyzed the effect of activating and anergizing regimens on the induction of some selected transcription factors that regulate expression of the B chain of the TcR and the IL-2 gene. These factors are well characterized and both T-cell-specific and gen-

era1 transcription factors are represented. The IL-2 promoter contains binding sites for nuclear factors including NF-AT, Ott, NF-KB, and AP- 1, which are all potentially sensitive to activation of PKC. Members of a family that include the nuclear oncogenes Fos and Jm bind as a heterodimeric complex to an AP-1 DNA-binding site. Dimeritation occurs by the interaction of leucine zipper domains in the two proteins and is a prerequisite for DNA binding. The AP-1 site is the major target of transcriptional induction after PKC activation in the IL-2 gene promoter {15]. NF-AT is an inducible factor that is restricted to T cells and is also a major regulator of IL-2 gene transcription. NF-AT is formed when a preexisting cytoplasmic subunit translocates to the nucleus and combines with newly synthesized Fos and Jun proteins { 161. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit E171, suggesting that the translocation is a calcium-dependent event. In nonstimulated cells, NF-KB resides in the cytoplasm in an inactive complex with the inhibitor I-KB. Stimulation causes release of I-KB and allows NF-KB to enter the nucleus, bind to DNA control elements and, thereby, aid transcription. Activation is triggered by a variety of agents including IL-l, TNF, and phorbol esters. The TcR P-chain enhancer responds to PKC-mediated activation signals through a functional domain that includes the BE2 element 1181. Multimerized BE2 can act in isolation as a phorbol-esterresponsive element; it contains a consensus Ets-binding site and binds directly to the product of the c-ets-1 protooncogene. PE2 can also bind a second family of nuclear factors, the core-binding factors l’9]. TCF-1 is a noninducible T-cell-specific transcription factor: it is known to bind within the CD3 E enhancer and in the TcR (Y enhancer. It shares a region of homology with other transcription factors termed the high-mobility group 1 (HMG) box 1191.

MATERIALS AND METHODS Peptides. The influenza hemagglutinin peptide (residues 307-3 19; HA 307-3 19) was synthesized using standard solid-phase technology and further purified by reversephase HPLC. The constitution of the peptide was confirmed by amino acid analysis as previously described 1201. Antibodies. Flow-cytometric analysis was performed using fluorescein-conjugated murine monoclonal antibodies. Anti-Leu4 (CD3), anti-IL-2 receptor (CD25), and a mouse IgG, control were purchased from Becton Dickinson (Oxford, UK). The murine anti-CD3 antibody used for cell activation was a kind gift from H. Spits

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(The Netherlands dam).

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Amster-

Cloned T lymphocytes. The isolation of HA 1.7, a clone of T cells reactive with HA 307-319 has been reported in detail previously 171. Briefly, peripheral blood mononuclear leukocytes (PBMCs) were stimulated with purified HA 307-3 19 in RPMI-1640 (Gibco Laboratories, Uxbridge, UK) supplemented with 100 U/ml penicillin, 100 kg/ml streptomycin, 2 mM L-glutamine, and 5% human AB + serum. The activated T cells were cloned by limiting dilution in the presence of irradiated autologous PBMCs, IL-2 (10% Lymphocult T; Biotest Folex, Frankfurt; i.e. 10 U/ml) and antigen. Growing HA1.7 cells were expanded by stimulation with antigen and filler cells every 7 days. After stimulation, the cells were expanded with 10% Lymphocult T every 3-4 days. The cells were rested 7-8 days after the last addition of filler cells and antigen prior to their use in experiments. Resting cloned T cells (2 X 105/well) were activated by incubation with anti-CD3 antibody insolubilized on tissue culture plates (12 p.g/ml) together with 10% Lymphocult T. To induce anergy, T cells were incubated with HA 307-319 (50 p.,g/ml). In some experiments, the calcium ionophore, ionomycin (0.2-l pg/ml) was added. After 24 hours, the cells were pooled and washed three times. The majority were used to make nuclear extracts, whereas some were assessed for their ability (2 X 104/well) to respond to an immunogenic challenge: either by APCs (irradiated histocompatible IDRl+I PBMCs 2 X lO*/well) and antigen (HA 307-3 19, 0.3 pg/ml) or by incubation with anti-CD3 antibody insolubilized on tissue culture plates (12 kg/ml). T-cell proliferation assays. After a further 60 hours of incubation E3H)methyl thymidine (1 pCi/well; Amersham International, Amersham, UK) was added and the cultures harvested 8-16 hours later. Proliferation as correlated with 13H)methyl thymidine incorporation was measured by liquid scintillation spectroscopy. The results are expressed as mean counts per minute (cpm) for triplicate cultures. Flow cytometry. T cells (3 X 105) were stained directly by using saturating concentrations of fluorescein-conjugated murine monoclonal antibodies. Forward and side scatter was used to identify viable cells. Cell populations were analyzed by flow cytometry using an Epics-Profile II (Coulter, Luton, UK). Data are expressed as the channel number representing mean fluorescence intensity.

plied Biosystems), according to the manufacturers’ eral instructions. The sequences used were PE2: NF-AT: NF-KB: TCF-1: CD28RC: AP-1:

gen-

5'-GATCCACAACAGGATGTGGTTTGACATTTA-3' 5'-GATCTAAGGAGGAAAAACTGTTTCATCG-3' 5'-GATCCAGTGGGAAATTCCTCG-3' 5'-GATCCTGGGAGACTGAGAACAAAGCGCTCTCACACGGGA-3' 5'-GTCTGATGACTCTTTGGAATTTCTTT-3' 5'-GATCTATCTCTGAGTCAATCAGCG-3'

The reverse and complement of each sequence was also synthesized with a noncomplementary 5’ dG and without the 3’dC. Electrophoretic mobility-shift assay. Nuclear extracts were prepared by a rapid method as described by Dignam et al. 1211, with several modifications. Cells (2 X 106) were washed in ice cold PBS, pelleted and resuspended in lysis buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl,, and 10 mM KCl). The mixture was vortexed and left on ice for 5 minutes. Cell nuclei were pelleted by centrifugation and resuspended in glycerol buffer (25% vol/vol glycerol, 20 mM Hepes pH 7.9, 0.42 M NaCl, 1.5 mM MgCl,, and 0.2 mM EDTA). The nuclear lysate was left on ice for 1 hour with occasional vortexing. The nuclear debris was pelleted and the extract retained. Protein concentrations were estimated using the Biorad protein assay kit. All buffers contained 0.5 mM PMSF, 1.0 mM benzamidine, 5 kg/ml aprotinin, 5 kg/ml pepstatin, 30 pg/ml leupeptin, and 0.5 mM DTT. Oligonucleotides were radiolabeled with 32P EdCTP] by filling in a dG overhang using AMV-RT, and the probes were then purified by separation on 7.5% polyacrylamide gels. Binding reactions contained 20 p,g acetylated BSA, 2 kg poly(dI-dC) * poly(dI-dC), 0.5 kg salmon sperm DNA, 0.005% NP40, 5 mM MgCI,, 80 mM NaCl, 10 mM Hepes pH 7.9, 2.5 P_.gof nuclear extract, and 0.5 ng of probe with an activity of around 10,000 cpm. Oligonucleotide competitors were preincubated with the nuclear extract for 5 minutes before the addition of probe. Nonspecific oligonucleotides were used in the final analysis as an indication that complexes were not being retarded through a general capacity to stick to DNA. In each case, the nonspecific oligonucleotide was TCF- 1, except in the TCF-1 gel shift, where the AP-1 oligonucleotide was used. Reactions were incubated at room temperature for 20 minutes and then separated on 5% polyacrylamide gels. Gels were dried for autoradiography or were quantitated directly from the dried gels by using an Ambis scanner.

RESULTS Oligonucleotides. Oligonucleotides were synthesized using an Applied Biosystems DNA synthesizer and purified using OPC (oligonucleotide purification columns, Ap-

Phenotypic changes during activation and the induction of anergy. The phenotypic modulation of the HA 307-319reactive CD4+ T cells (HA1.7) after treatment with

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activating and tolerizing regimens has already been reported in detail 1221. T cells were rested for 7 days after the last cycle of expansion and then activated with antiCD3 antibody and 10% Lymphocult T, or exposed to a tolerizing concentration of HA 307-319. Cells were analysed by fluorescence flow cytometry to determine the level of surface CD3, CD25, and CD2 (Fig. 1). Consistent with our previous findings, both treatments resulted in cells that had markedly downregulated membrane CD3 expression. In contrast, IL-2 receptor (CD25) and CD2 expression were upregulated. Induction of T-cell anergy by treatment of cells with HA 307The T cells were tested for their ability to respond to specific peptide, 24 hours after receiving an activating or anergizing signal (Fig. 2). The data show that preactivation enhances the ability of cells to proliferate in response to an immunogenic challenge when compared with cells incubated in medium alone. This phenomenon 3 19.

FIGURE

1

Phenotypic

modulation

of T cells exposed to

activating and anergizing signals. Membrane expression of CD2 (solid bars), CD3 (woss-batchedbars), and CD25 (stippled baavs)of T cells exposed to different conditions were compared by flow cytometry at 24 hours after stimulation.

Medium

Anti-CD3 + IL-2

Pretreatment

HA (50pglml)

of T cells

FIGURE 2 The ability of specific peptide to induce anergy in activated T cells. Cloned T cells (HA1.7; 106/ml) were pretreated by culturing with insolubilized anti-CD3 antibody and IL-2, with HA 307-3 19 at 50 Fg/ml or in medium alone for 24 hours. After pretreatment, the T cells (lOs/ml) were assayed for their ability to respond to restimulation with HLADRl + APCs (irradiated PBMCs) alone (solid bars), HA 3073 19 (0.3 p,g/ml) + APCs (cross-hatched bars)j or IL-2 alone (stippledbars). Proliferation as correlated with 1 Hlmethyl thymidine incorporation was determined 72 hours later.

BOO

600

is probably due to the induction of CD25 and the manifest hyperresponsiveness of activated cells to IL-2 (Fig. 2). After incubation with HA 307-319 (50 p,g/ml) for 24 hours, the T cells failed to proliferate when restimulated with HA 307-3 19 presented in an immunogenic form (Fig. 2). The addition of exogenous IL-2 caused proliferation of the anergized cells, confirming functional inactivation was not the result of cytolysis. Drffwential expressionof the NF-AT

Medium

Anti-CD3 + IL-2

Pretreatment

HA (50pglml)

of T ceils

complexin activated and tohized T cells. Nuclear extracts were incubated with radiolabeled double-stranded DNA probes that contained known cis-acting sequences to determine which transcription-factor-binding activities were expressed after treatment of cells with activating or tolerizing stim-

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characteristics

divide

the

factors

four groups, three of which were chosen for study: T-cell-specific inducible factors include NFKB, NF-AT, PE2, and the CD28RC; AP- 1 is a general and inducible molecular complex, whereas TCF- 1 1197 is an example of a T-cell-specific noninducible factor. TCF- 1 and PE2 are involved in the regulation of transcription of the p chain of the TcR, whereas the others have a role in transcription of the IL-2 gene. As expected, the noninducible factor, TCF- 1, is the only factor clearly present in the nuclei of untreated cells and its concentration does not vary in activation and anergy (Fig. 3). All six probes formed complexes with extract from activated cells that were competed by specific, but not by nonspecific, oligonucleotide competitors. The lower band in the NF-KB shift is a nonspecific complex, whereas the upper is authentic NF-KB. Equal amounts of protein were used from each nuclear lysate, and this is substantiated by the observation that binding to the TCF- 1 site is the same in the nuclei of resting, activated, and anergized cells. Differences in the pattern of induction of specific transcription factors were quantified using an Ambis scanner; into

FIGURE 3 Electrophoretic mobility shift assay. Nuclear extracts were prepared from resting T cells incubated in medium alone (M), and from activated (A) or tolerized (T’) cells. Double-stranded DNA probes (as indicated) were incubated with the extracts in the presence of 100-fold excess of either a specific (S) or nonspecific (N) oligonucleotide competitor and separated on polyacrylamide gels.

radioactivity was measured directly from the dried gels. These results confirm a relative failure of anergic T cells to upregulate NF-AT (Table 1). The inducible factors NF-KB, PE2, CD28RC, and AP-1 are not expressed in resting cells, and are 1.6- to 2.2-fold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT than cells that received an activating signal. Ftlnctional inactivation in the presence of calcium iono@ore. The failure to establish good NF-AT expression in the induction phase of anergy suggested that these T cells may be missing some biochemical aspect of an activating signal. The observation that AP-1 and BE2 binding were relatively well induced in anergized T cells suggested that the pathway through PKC was intact. There is an approximate fourfold difference between the induction of AP-1 and NF-AT when activated cells are compared with anergized cells, and both of these treatments involve stimulation through the TcR. Since NFAT consists of AP- 1 plus a second component that translocates to the nucleus as a result of calcium-dependent

PE2

NF-AT Ekkract Caqzetitor

M -

4-&l -NSNS-

M

M

I-+& --NSNS-

--NSNS-

AP-1 Extract Ccqetitor

M -

G-l+ -NSNS-

NFKB

TCF-1

4-G-l

CD28RC

M

M

I-%& --NSNS-

G-IA --NSNS.

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TABLE

1

Ratio of transcription factor binding activities in activation and anergy

DISCUSSION

Ratio” activated:anergized

Factor

8.1:1 1.8:l 1.6:1 2.1:1 2.2:1 1.0:1

NF-AT PE2 NF-KB AP- 1 CD28RC TCF- 1

n Retarded complexes were quantified directly from the dried gel using an Ambis scanner.

phosphorylation, it seemed logical to investigate the Ca2+ pathway further. It is noteworthy in this regard that both core-binding factor (CBF) and the ets transcription factors bind to the PE2 element: ets is inducible whereas CBF is not f91; this explains the small shift of the PE2 element when using the extract from resting T cells (Fig. 3). PE2 activity is inducible principally through PKC and not the Ca2+ pathway 1231, thus providing further evidence that the PKC pathway is relatively intact during the induction of anergy. T cells were therefore treated with calcium ionophore at the time that they received an anergizing dose of peptide to establish whether an additional calcium flux could abrogate the induction of T-cell anergy. T cells were restimulated with either insolubilized anti-CD3 or HA 3073 19 presented on irradiated histocompatible (DR 1+) PBMCs. The results (Table 2) show that ionomycin itself can induce anergy in a dose-dependent manner, but that peptide-induced anergy is abrogated only by a low dose of ionomycin. This applies both to restimulation with peptide and APCs and with anti-CD3. TABLE

2

Proliferative capacity various pretreatments

of the human

T-cell

Several groups have developed systems to study T cell anergy in vitro. Each method includes a signal through the TcR, though this can be delivered specifically, for example with peptides, or nonspecifically, using antiCD3 antibodies or lectins. T-cell proliferation is augmented by costimulatory signals, and anergy can be induced in murine T cells by antigen receptor stimulation in the absence of costimulation. The costimulatory molecule B7/BB-1 is functionally inactivated by a chemical fixation process, and the use of such cells to presenr antigen results in anergy. This mechanism does not result in the phenotypic changes that characterize highdose peptide anergy of human T cells, but the cells have in common a reduced ability to release IL-2 on rechallenge. Go and Miller examined the expression of a series of transcription factors after stimulation of murine T-cell clones with normal and with chemically modified APCs [24]. The tolerogenic stimulus induced less NF-AT and lower amounts of one of the two NF-KB-binding factors. The AP-1 transcription factor was induced and was not obviously differentially regulated. These results are in very close accord with the results presented here, but differ markedly from a third independent study [25]. This group showed that anergic T cells, generated by exposing the cells to concanavalin A, did not make IL-2. However, this was associated with a specific downregulation of the AP-1 complex. These experiments showed no differences in the NF-AT complex, a surprising finding since NF-AT contains AP- 1. Collectively, these data indicate that the control of IL-2 gene expression during anergy induction and during normal stimulation of anergized cells is distinct, and suggest the presence of additional regulatory elements that control synthesis and release of IL-2.

clone HA 1.7 after

Restimulation” APCb

Pretreatment Medium HA50d HA50 + I” (1 pg/ml) HA50 + I’ (0.2 pg/ml) I’ (1 pglml) I’ (0.2 pglml) Anti-CD3

109 1041 616 7254 167 105 202

(22) (30) (31) (11) (34) (19) (58)

APC + peptide’ 17,959 8250 1770 18,344 1602 6033 16,896

(4) (34) (9) (10) (9) (12) (55)

IL-2 12,855 27,620 20,936 34,092 14,313 13,183 13,306

Anti-CD3 (7) (23) (12) (12) (12) (6) (8)

3422 1084 441 6774 2 12 1768 7270

(18) (30) (24) (21) (38) (IO) (24)

a Cells were restimulated 24 hours after pretreatment and proliferation as correlated with [3Hlmethyl poration was determined 72 hours later in duplicate. The numbers in parentheses refer to SEM. b APC, irradiated DRl ’ Peptide, HA 307-319 d HA50, HA 307-319

+ PBMC. (0.3 pg/ml). (50 pg/ml).

’ I, ionomycin (concentration

as shown).

Anti-CD3

+ IL-2

22,565 (5) 33,502 (4) 24,317 (3) 45,441 (7) 17,286 (10) 20,557 (10) 30,65 1 (14) thymidine

incor-

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In an interesting series of experiments designed to investigate the relationship between NF-AT and gene expression, the lacZ gene was placed under the control of tandem copies of the NF-AT-binding site in transfected T cells. Following exposure of the cloned stably transfected T cells to TcR-specific stimuli, a bimodal pattern of gene expression was noted. Increasing concentrations of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell 1261. The pattern of expression was not dependent upon cell-cycle position or heritable variation. And the results, therefore, suggest that the concentration of NF-AT must exceed a critical threshold before transcription of a linked gene is induced. Other transfected T cells, in which IacZ is controlled by NF-KB or the entire IL-2 promoter, also show bimodal expression patterns after stimulation 1271. It seems likely then that transcription factors have concentration thresholds below which they cannot initiate transcription of linked genes. A completely different phenotype might, therefore, occur with only a partial loss of an inducible factor. T-cell anergy is mediated, at least in part, by a loss of the costimulatory pathway, and the CD28 receptor is only one component of a complex system regulating T-cell activation. There are several surface molecules that transmit costimulatory signals, and these might substitute if the CD28 pathway were blocked. For example, superantigen-induced proliferation of human T cells is not dependent on costimulation through CD28, but is affected by interactions between the CD1 la-CD 18 complex and its counter receptors ICAMl-3 [28]. The results reported here show that the induction of T-cell anergy, when compared with T-cell stimulation, is characterized by a reduced expression of the NF-AT complex. NF-AT is induced in T cells stimulated through the TcR and is required for IL-2 gene induction. The induction of NF-AT probably requires two activation-dependent events: the translocation of a preexisting cytoplasmic component and the synthesis of a nuclear component. The newly synthesized nuclear component of NF-AT is the transcription factor AP- 1, and it contains the Fos and Jun proteins [l6], whereas the translocation depends on dephosphorylation by calcineurin [29]. Our results show no primary failure in the ability of tolerized cells to produce AP-1; it therefore seems likely that the difference lies in the ability of these cells to translocate the cytoplasmic component of the complex to the nucleus. Maximal expression of NF-AT in T cells requires at least two signals: triggering of the TcR in association with either PKC activation or a [Ca2’}i flux. Activation through the TcR has many downstream biochemical effects, and signals other than a calcium flux or PKC can regulate NF-AT expression in peripheral blood-derived T cells 130). The TcR can regulate multiple intracellular

signals in T cells, and the biochemical events following ligation of the receptor may depend upon ligand density as well as the nature of additional signals. We have shown that the combination of these signals, generated by the addition of ionomycin during the induction of anergy, leads to a different end state according to the concentration of ionomycin. And this suggests that the triggering of particular downstream events is sensitive to different thresholds of [Ca2’li. Molecules associated with the induction of anergy (anergens) are likely to be a diverse group that may affect processes in distinct cellular compartments. Induction of anergy is an active process that requires protein synthesis 1221. Therefore, some anergens will be newly synthesized molecules and others may be activated by phosphorylation or translocated to an active site. While this study has not directly identified any potential anergen, the observation that the amount of NF-AT available in the nucleus for transcription is different in activation and anergy shows that the cells have taken alternative developmental steps.

ACKNOWLEDGMENTS

This work was supported by (MRC) and Wellcome Trust. Clinical Research Fellow. The to Mark Larch6 and Alex Faith ing the manuscript.

the Medical Research Council R.E.O. is a Wellcome Senior authors express their gratitude for helpful discussions concern-

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