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Originally published In Press as doi:10.1074/jbc.M402194200 on May 5, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29130-29138, July 9, 2004
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Tumor Necrosis Factor-{alpha} and CD80 Modulate CD28 Expression through a Similar Mechanism of T-cell Receptor-independent Inhibition of Transcription*

Dorothy E. Lewis{ddagger}, Maria Merched-Sauvage{ddagger}, Jörg J. Goronzy§, Cornelia M. Weyand§, and Abbe N. Vallejo¶||

From the {ddagger}Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, §Lowance Center for Human Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, and Division of Rheumatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905

Received for publication, February 27, 2004 , and in revised form, April 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28null T cells during repeated stimulation in vitro as well as in chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28null cells from CD28+ precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28null {leftrightarrow} CD28hi phenotypic transitions. JT and Jtag cells were CD28null and CD28lo, respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-{alpha} reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The CD28 molecule is a membrane glycoprotein with nearly restricted expression to T lymphocytes. Its role as the major costimulatory receptor for the induction and maintenance of T-cell activation and differentiation of effector function is well documented (1). In humans, chronological aging is associated with the in vivo accumulation of T cells that are deficient in CD28 expression (2, 3). Although mechanisms leading to immunosenescence are complex, CD28null T cells are the most consistent biological indicators of aging in the immune system. Indeed, clinical studies have shown that a high frequency of the CD28null T cells is a key predictor of immunoincompetence in the elderly (4, 5). These unusual T cells have also been found among patients with various inflammatory syndromes (6–10) and chronic infections, including HIV1 (11–14). In these pathological conditions, CD28null T cells are postulated to represent prematurely senescent T cells as a result of persistent immune activation (15).

The notion that CD28null T cells have advanced senescent features comes from observations that they are highly oligoclonal (2, 5, 16) and have significantly shortened telomeres compared with their CD28+ counterparts (9, 17). Therefore, they have extremely limited proliferative potential (18, 19). However, they are long-lived cells because of their resistance to apoptosis (18, 20).

The CD4+ T-cell compartment of elderly persons and patients with inflammatory conditions or chronic infections may consist of up to 50% CD28null T cells (3, 7, 11, 14). CD4+CD28null T cells lack expression of CD154 and are therefore unable to provide help for B-cell proliferation and immunoglobulin production (21). They have large cytoplasmic stores of interferon-{gamma} (22) and express CD161 (23), giving them the potential to be inflammatory. They express a variety of receptors normally found on natural killer cells (24). They have also acquired perforin and granzymes (34), which render CD4+CD28null T cells highly cytotoxic (25).

CD28null T cells may occupy ≥90% of the CD8 compartment of elderly persons (4) and ≥40% of CD8+ T cells among patients with chronic inflammatory or infectious diseases (6, 9–14). In most cases, the loss of CD28 in the CD8 compartment is a terminal developmental stage because the majority of CD8+CD28null T cells have lost their proliferative capacity (18, 26). Many of these cells also lack expression of CD11b; like their CD4+ counterparts, CD8+CD28null T cells have also acquired a variety of natural killer cell receptors, including CD56 and Fc{gamma} receptor IIIA (27, 28). Although some have retained their cytotoxic function, a large proportion of CD8+CD28null T cells are suppressors that specifically inhibit T-cell effectors as well antigen-presenting cells (29).

Such gain and/or loss of function among the CD28null T cells are consistent with the idea that cellular senescence involves protection from apoptosis and the development of new phenotypes accompanying the limitation or cessation of proliferation (30). The novel phenotypes of these senescent lymphocytes may provide a basis for immunoincompetence during normal aging as well as increased severity of clinical manifestations of various chronic diseases that are correlated with the frequency of CD28null T cells (4, 5, 7, 31, 32).

We reported previously that the loss of CD28 expression in T cells is associated with the inactivation of the transcriptional initiator (INR) of the gene promoter (3, 33). The CD28 INR is a novel regulatory element that lacks homology with the consensus INR sequence (34). It consists of two contiguous sequence motifs that function as a unit (33). In vivo-derived CD28null T cells uniformly lack INR-specific transcription factors (19), which explains why a CD28null phenotype is generally irreversible (19, 24, 35). Modulation in the levels of cell surface expression of CD28 during the proliferative life span of T cells is also coupled to INR activity (19). Indeed, quantitative decreases in the levels of cell surface expression of CD28 and the ultimate generation of CD28null T cells in vitro by the continuous activation and passage of CD28+ precursors are accompanied by the repression of the INR (19, 36, 37). To validate that CD28 INR function is a determinant of phenotypic transformations of T cells, we used a T-cell system in which the dynamics of CD28 expression could be rapidly assessed under controlled conditions. Because existing in vitro models involve highly protracted procedures (19, 36, 37), a biological system that has an inducible but transient expression of CD28 is advantageous in dissecting molecular events controlling CD28+ to CD28null transition. Here, we show the usefulness of the T-cell lines JT and Jtag as models to track changes in CD28 expression. Studies were conducted to examine whether expression and/or loss of CD28 in a variety of situations are directly related to the functional competence of the CD28 INR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—JT cells were derived from Jtag (provided by Dr. David Spencer, Baylor College of Medicine, Houston, TX), a subline of Jurkat T cells that is stably transfected with SV40 large T-antigen (38). Jtag cells have very low levels of cell surface expression of CD28, but upregulate CD28 expression upon exposure to phorbol 12-myristate 13-acetate (PMA). This was in contrast to Jurkat cells, which expressed higher magnitudes of CD28 that were unaffected by PMA or other pharmacologic agents2 (19, 20). To produce CD28null cells, Jtag cells were subjected to at least two rounds of flow cytometry selection for the complete lack of CD28 expression. The absence of CD28 was also confirmed by the lack of specific transcripts in standard reverse transcription (RT)-PCR assays for all the known splice variants of CD28 (19). The sorted CD28null cells were subsequently tested for the induction of CD28 expression with PMA by flow cytometry, resulting in the establishment of the CD28-inducible JT cell line.

JT, Jtag, and Jurkat cells were cultured in RPMI 1640 medium (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) supplemented with 10% fetal calf serum (Summit Biotechnology, Fort Collins, CO), 2 mM L-glutamine, 50 units/ml penicillin, and 5 µg/ml streptomycin (Invitrogen). Cells were maintained at a density of 0.5–5.0 x 106 cells/ml in a humidified 5–7.5% CO2 tissue culture incubator.

The murine P815 mastocytoma cell line (American Type Culture Collection, Manassas, VA) was also cultured in complete RPMI medium but incubated in 5% CO2. Wild-type P815 cell lines or those stably transfected with human CD80 or CD86 (provided by Dr. Lewis Lanier, University of California San Francisco; Ref. 39) were used as indicated in specific experiments.

Flow Cytometry—CD28 and CD45 expression on JT, Jtag, and Jurkat cells were monitored by direct immunostaining with a fluorochrome-conjugated anti-human CD28 or anti-CD45 monoclonal antibody and analyzed by flow cytometry using a FACSCalibur cytometer (BD Biosciences) or a XL cytometer (Beckman Coulter).

The kinetics of PMA-induced expression of CD28 on JT cells was examined. JT cells were incubated in PMA (Sigma Aldrich Corp.) at the indicated concentrations and CD28 expression was examined by flow cytometry after overnight incubation. The stability of CD28 expression after induction with PMA was also examined. PMA-stimulated JT cells were washed extensively and cultured in fresh medium, and CD28 expression was monitored during a 15- to 20-day culture period. Over this time, the cells remained viable with no significant levels of apoptosis as determined by propidium iodide staining and flow cytometry (data not shown).

In other experiments, the influence of exogenous tumor necrosis factor (TNF)-{alpha} or CD28 ligation on the PMA-induced expression of CD28 on JT (or Jtag) cells was also examined. As indicated, 10 ng/ml recombinant human TNF-{alpha} (R&D Systems, Minneapolis, MN) was added to appropriate cultures during incubation with PMA or to PMA-stimulated cells cultured in fresh medium. In appropriate cultures of PMA-stimulated JT (or Jtag) cells, 2–5 µg/ml of anti-CD28 monoclonal antibody ANC28 (Calbiochem-EMD Biosciences), L293, or 28-2 (BD Biosciences Pharmingen), or an IgG isotype control was also added. In parallel experiments, PMA-treated JT (or Jtag) cells were cocultured with CD80+, CD86+, or control P815 cells at 5:1 JT/P815 cell ratio. After a 48 h-incubation, CD28 and CD45 expression were examined by flow cytometry.

The levels of TNF-{alpha} receptor I and II were measured by indirect immunofluorescence staining and flow cytometry. Cells were stained with goat anti-TNF-{alpha} receptor I (R&D Systems) followed by a fluorochrome-conjugated rabbit anti-goat immunoglobulin (BD Biosciences), washed, then incubated with mouse monoclonal anti-TNF-{alpha} receptor II (R&D Systems) followed by rat anti-mouse IgG conjugated with a different fluorochrome (BD Biosciences).

Reporter Gene Bioassays—The CD28 promoter-driven reporter plasmids p42 and p52 have been described previously (3). Two additional luciferase reporters, KPN and RV, were constructed from a genomic clone of the 5'-flanking region of human CD28 gene (40; provided by Dr. Kelvin Lee, University of Miami Medical School, Miami, FL). The original clone was inserted in the pGEM-3Z vector (Promega, Madison, WI) at the EcoRI site and contained 1.7 kb extending into the first intron. DNA was digested with EcoRI and StuI, yielding an 812 bp-fragment that was subcloned into the pGL3 basic vector (Promega) at the SmaI site. The first 460 bp, corresponding to an Alu sequence, were deleted at the KpnI restriction site in the vector and in the CD28 promoter sequence, yielding the KPN construct. The RV construct was generated by restriction enzyme digestion at the KpnI site of the vector and the EcoRV site in the CD28 promoter sequence. DNA fragments were repaired using T4 DNA polymerase to generate blunt ends and were ligated to the pGL3 vector. Recombinant plasmids were used to transform DH5{alpha} E. coli hosts (Invitrogen Life Technologies). Plasmids were isolated using commercial kits, and DNA sequencing was performed to authenticate the sequence of the cloned CD28 promoter.

The KPN reporter was a longer reporter construct than the RV, p42, and p52 reporters (Fig. 4B, diagram). It contained additional 5' sequences flanking the minimal CD28 promoter sequences in the p42 and p52 reporters (3). We had reported previously that the activity of the CD28 minimal promoter was regulated by the CD28 INR (33), which functioned independently of a downstream GR element (41). To further validate that the CD28 INR comprised the minimal promoter, the GR element was deleted in the RV construct, the shortest of the reporters. The KPN construct also lacked the GR element, whereas the p42 and p52 constructs have an intact GR element.



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  		FIG. 4.
PMA-induced expression of CD28 is antagonized by TNF-{alpha}. A, triplicate cultures of JT cells were incubated in medium alone (U), 125 ng/ml PMA (P), 10 ng/ml TNF-{alpha} (T), or a combination of PMA and TNF{alpha} (PT) for 24 h. Parallel cultures were also stimulated overnight with PMA, washed, and transferred into TNF-{alpha}-containing medium (P/T) for an additional 24 h. CD28 expression was measured by immunofluorescence staining and flow cytometry. B, Jtag cells were transfected with four different firefly luciferase reporter plasmids (KPN, RV, p42, and p52) that were under the control of CD28 promoter sequences. Transfectants were incubated with medium alone (U), 125 ng/ml PMA (P), or 10 ng/ml TNF-{alpha} (T) overnight. Parallel cultures of transfectants were also stimulated overnight with PMA, washed, and transferred into TNF-{alpha}-containing medium (P/T) for an additional 48 h. The luciferase reporters, as indicated, contained the CD28 INR (33) with (p42 and p52) or without (KPN and RV) the GR element and a5'-Alu element. Data shown are specific firefly luciferase activity from four transfection experiments for each of the indicated reporters and were normalized for transfection efficiency by cotransfection of a TK promoter-driven R. reniformis luciferase plasmid.

 
Reporter plasmids were prepared using the EndoFree plasmid kit (Qiagen, Valencia, CA) and were used in transient transfection experiments with the GenePorter kit (Gene Therapy Systems, San Diego, CA). For each transfection, 4.5 x 106 JT cells were resuspended in 0.45 ml of serum-free RPMI 1640. A mixture of 8 µg of the appropriate luciferase reporter plasmid DNA, 1 µg of control pRL TK-Renilla reniformis luciferase (Promega), and 64 µl of the GenePorter reagent was added, and the final volume was adjusted to 2.5 ml with serum-free RPMI 1640. After 4 h of incubation, 2.5 ml of RPMI 1640 supplemented with 20% fetal calf serum was added and the transfectants were aliquoted to two tissue culture plates. PMA was added to one plate at a final concentration of 125 ng/ml. After 24 h, the cells were washed, resuspended in complete medium, and aliquoted into six 1-ml fractions in a 24-well culture plate. To the appropriate wells, 10 ng/ml TNF-{alpha}, 2 µg/ml anti-CD28 (ANC28, L293, or 28-2), or 2 µg/ml IgG isotype control was added. After an additional 48 h of incubation, CD28 expression was measured by flow cytometry, and luciferase activity was determined using the dual luciferase kit (Promega). Specific reporter luciferase activity was normalized against the internal R. reniformis luciferase activity and total protein concentration of cell lysates using a kit (BCA; Bio-Rad).

Gel Shift and Transcription Assays—JT cells were exposed to PMA followed by the addition of 10 ng/ml TNF-{alpha}, 2–5 µg/ml anti-CD28 monoclonal antibody (ANC28, L293, or 28-2), 5 µg/ml IgG, or mouse P815 cells as indicated. Nuclear extracts were prepared and used in electrophoretic mobility shift assays (EMSA) using DNA-binding probes corresponding to the CD28 INR site {alpha} as described previously (3, 19, 33). As system controls, similar EMSAs were conducted using nuclear extracts from Jurkat T cells and the use of Sp1-binding oligonucleotides as probes.

The nuclear extracts were also used in transcription assays to assess their competence in supporting transcription of CD28 INR-regulated templates. Transcription assays were conducted as described previously (33, 42). In brief, CD28 {alpha}{beta}-INR sequences were cloned at the 3' flank of a canonical TATA box and upstream of a heterologous 180 bp G-less cassette. This template was incubated in a transcription reaction containing JT or Jurkat nuclear extracts and a ribonucleotide triphosphate mixture with [{alpha}-32P]UTP (Amersham Pharmacia Biotech). Transcription products were subjected to RNase T1 (Roche Molecular Biochemicals) digestion, extracted with phenol-chloroform, size fractionated on 8% polyacrylamide/6 M urea sequencing gels, and visualized by autoradiography. As system controls, similar transcription assays were conducted using Jurkat extracts as well as DNA templates that were under the control of the INR element of the terminal deoxyribonucleotidyl transferase (TdT) gene.

RNase Protection Assay and RT-PCR—Jtag cells were incubated overnight with or without 125 ng/ml PMA. Cells were washed and resuspended in fresh medium. To appropriate cultures, TNF-{alpha}, anti-CD28 monoclonal antibody (ANC28 or 28-2), IgG control, or P815 cells were added as indicated. After 48 h, the cells were washed and total RNA was extracted using the RNAwiz reagent (Ambion, Austin, TX) and subjected to RNase protection assay (RPA) using the Ambion RPAIII kit. CD28 expression was detected by hybridization to a biotinylated RNA probe corresponding to exon 4 of the CD28 gene (25). As an internal control, hybridization was carried out using a kit probe to human {beta}-actin. RNA-RNA hybrids were fractionated by electrophoresis on 5% polyacrylamide-7 M urea gels and transferred to BrightStar plus membranes, and hybridization signals were detected by the BrightStar BioDetect kit (Ambion).

Levels of CD28 transcription in stimulated and unstimulated Jtag cells were examined by RT-PCR using the Superscript one-step kit (Invitrogen). PCR reactions included amplification primers that were specific for CD45, which served as the internal control. The gene-specific primer sets were designed such that the PCR-amplified CD45 and CD28 transcripts were identified as 990 and 100-bp products, respectively. The primer sets used were as follows: 5'-AGGCTCCTGCACAGTGACTA-3' and 5'-GAGCGATAGGCTGCGAAGT-3' for the amplification of CD28 exon 4; and 5'-CTTCTGGAAGCGCTGTCATT-3' and 5'-CGCAATTCTTATGCGACTCA-3' for CD45 amplification. Total RNA samples were subjected to first strand cDNA synthesis at 55 °C followed by 2 min denaturation at 94 °C. PCR was carried out in 29 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 2 min, with a terminal 7 min extension period. PCR products were fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Patterns of CD28 Expression on Jurkat T Cells and Its Variants—Jurkat and its variants, Jtag and JT, have varying levels of CD28 expression. As depicted in Fig. 1A, Jurkat has high constitutive levels of CD28 expression consistent with previous reports (3, 19). In contrast, its derivative subline Jtag (38) expressed low to negligible levels of CD28 at immunostaining levels less than a half-magnitude over that of isotype controls. A sequential selection of Jtag cells that have no detectable expression of CD28 led to the establishment of the JT subline, which was characteristically CD28null. Both Jtag and JT cells had the capacity to re-express high levels of CD28 when exposed to PMA (Fig. 1, B and C). PMA induced the expression of CD28 in a dose-dependent fashion. However, PMA did not affect the parental Jurkat cells, presumably because CD28 expression was already maximal.



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  		FIG. 1.
Patterns of CD28 expression on Jurkat T cells and its variants. The constitutive levels of CD28 expression on Jurkat cells and its variants Jtag and JT were measured by direct immunofluorescence staining and flow cytometry (A). Likewise, modulation in the levels of CD28 expression on Jtag (B) and on JT cells (C) were measured after a 24-h exposure to PMA at the indicated concentrations. Data shown are representative of four experiments.

 
The induction of CD28 was directly associated with transcriptional activation through the functional reconstitution of the CD28 INR, a core promoter element that regulates CD28 expression in T cells (33). In EMSAs, nuclear extracts from unstimulated JT cells consistently showed complete absence of DNA-binding complexes specific to the CD28 INR, which were found at high levels in similar extracts from PMA-stimulated cells (Fig. 2A). Formation of CD28 INR-protein complexes was apparently a specific effect of PMA. The levels of DNA-binding activities of ubiquitous nuclear proteins such as Sp1 were unaltered by PMA (Fig. 2B).



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  		FIG. 2.
PMA-induced expression of CD28 involves reconstitution of CD28 INR function. JT cells were incubated with PMA at the indicated concentrations (0, 25, 125 ng/ml) for 24 h. Nuclear extracts were prepared and used in EMSAs with binding probes corresponding to the site {alpha} of the CD28 INR (INR{alpha})(A) with Sp1-binding sequences as controls (B), or in transcription assays with CD28 INR-driven DNA templates (C). Nuclear extracts from Jurkat T cells (lane k), a prototypical CD28hi cell line, were also prepared and used as positive controls. DNA templates containing the wild-type (wt) or mutated variant (mt) of the TdT INR were used as positive and negative controls, respectively, in the transcription assays (C). Data shown are representative of three experiments for each assay using different batches of nuclear extracts.

 
In transcription assays, nuclear extracts from PMA-stimulated, but not unstimulated, JT cells were highly capable of activating transcription of DNA templates driven by the CD28 INR (Fig. 2C). Parallel transcription assays with control DNA templates driven by the TdT INR, a well studied canonical INR element (42, 43), showed equivalent levels of transcriptional activation with nuclear extracts from PMA-stimulated and unstimulated JT cells. Templates containing mutated variants of the TdT INR were inactive regardless of the nuclear extracts used.

As expected, extracts from unstimulated Jurkat cells, which constitutively expressed high levels of CD28 (3, 19, 33), contained CD28 INR-specific transcriptional activators as detected in EMSAs and in transcription assays (Fig. 2, A and C).

It might be noted that the degree of PMA-induced transcriptional reconstitution of the CD28 INR in both JT and Jtag cells was equivalent. However, JT cells exhibited dose-dependent induction of transcription more strongly than Jtag cells because of negligible backgrounds in DNA-protein binding activities detected in EMSAs (Fig. 2B) and INR-directed transcription of DNA templates (Fig. 2C).

Decay of PMA-induced CD28 Expression—Stability of CD28 expression on JT cells was examined. As depicted in Fig. 3A, JT cells exposed to PMA expressed high levels of CD28 that decreased progressively upon re-culture of the cells in fresh media. CD28 was invariably lost 15 to 20 days after the initial exposure to PMA. The loss of CD28 was accompanied by the extensive decrease of CD28 INR-specific nuclear protein complexes (Fig. 3B). The reduction of these specific DNA-binding complexes was functionally correlated by the inactivation of CD28 INR-dependent transcription of DNA templates (Fig. 3C).



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  		FIG. 3.
Induction of CD28 expression by PMA is reversible. JT cells were incubated with 125 ng/ml PMA (P). After 24 h, the cells were washed and transferred to fresh culture medium without additional stimulation. A, CD28 expression during a subsequent 15-d culture period was measured by direct immunofluorescence staining and flow cytometry at the indicated days (d2, d5, d10, d15). U, unstimulated JT cells. From parallel cultures, nuclear extracts were prepared and used in EMSAs with binding probes corresponding to CD28 INR site {alpha} (INR{alpha}) (B) or in transcription assays with CD28 INR-driven DNA templates (C) as in Fig. 2. Transcription assays included extracts from CD28hi Jurkat T cells (lane k) as a positive control. DNA-binding and transcription reactions were carried out in duplicate. Data shown are representative of three independent experiments for each indicated assay.

 
Inhibition of CD28 Induction by TNF-{alpha}—Although PMA induced CD28 expression, TNF-{alpha} inhibited it. As shown in Fig. 4A, the addition of 10 ng/ml TNF-{alpha} significantly reduced the PMA-induced expression of CD28 in JT cells. TNF-{alpha}-mediated down-regulation of CD28 was observed whether TNF-{alpha} was added during or after incubation with PMA. Although the degree of TNF-{alpha}-induced down-regulation of CD28 in JT and Jtag cells was equivalent, there seemed to be quite a broad distribution of resulting CD28lo to CD28null cells. The reason for this phenomenon is unclear in that both cell lines had similar unimodal high levels of TNF-{alpha} receptors (data not shown). It should be noted that the concentration of TNF-{alpha} used in these experiments was empirically determined to be non-toxic and had been shown in previous work to effectively down-regulate and eventually block cell surface expression of CD28 in long-term cultures of primary CD28+ T cells (37).

The inhibitory effect of TNF-{alpha} on CD28 expression was much more evident at the level of transcription as demonstrated in reporter gene bioassays. For these experiments, we used four types of CD28 promoter-driven reporters (i.e. luciferase constructs with the minimal CD28 promoter containing the INR element only or those that also contained other putative cis-acting sequences (3, 19)). As shown in Fig. 4B, Jtag cells transiently transfected with these constructs showed remarkably high levels of induction of luciferase gene expression after exposure to PMA regardless of the presence or absence of other cis-acting elements surrounding the INR. Reporters under the control of the CD28 INR-containing minimal promoter (the RV construct) showed levels of luciferase activity equivalent with those containing longer CD28 promoter sequences (KPN, p42, and p52 constructs). Neither the presence of Alu (KPN and p52 constructs) nor the presence of GR elements (p42 and p52 constructs) significantly affected the activity of the CD28 minimal promoter containing the INR element. Subsequent incubation of these PMA-stimulated transfectants in TNF-{alpha} showed equivalent marked reduction of luciferase activities. As expected, TNF-{alpha} alone did not induce luciferase activity or cell surface expression of CD28 (Fig. 4, A and B). These data clearly indicated that the INR element was necessary for the transcriptional activity of the CD28 promoter, which functioned independently of other putative regulatory sequences, and was a specific target for inhibition by TNF-{alpha}.

Repression of CD28 Gene Transcription by TNF-{alpha}—To further assess whether TNF-{alpha}-dependent inhibition of CD28 occurred at the level of transcription, DNA-binding and transcription assays were conducted. As shown in Fig. 5A, nuclear extracts from JT cells exposed to PMA showed high levels of CD28 INR protein-binding activities compared with unstimulated cells as detected by EMSAs. However, similar extracts from cells incubated with TNF-{alpha} alone or those incubated with TNF-{alpha} during (or after) exposure to PMA did not show significant assembly of such specific DNA-protein complexes.



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  		FIG. 5.
TNF-{alpha}-mediated repression of CD28 expression is caused by transcriptional silencing. JT cells were cultured in medium alone (lane U), 125 ng/ml PMA (lane P), 10 ng/ml TNF-{alpha} (lane T), a combination of PMA and TNF-{alpha} (lane PT), or a regimen of PMA followed by TNF-{alpha} (lane P/T) as in Fig. 4A. Nuclear extracts were prepared and used in EMSAs with CD28 INR site {alpha} (A) or Sp1 (E) sequences as binding probes, or in transcription assays with INR-driven DNA templates (B) as in Fig. 2. Jurkat nuclear extracts (lane k) were used as positive controls. From parallel Jtag cultures, total RNA was extracted and used in RPA (C) and in RT-PCR (D) experiments to assess CD28 gene transcription. {beta}-Actin and CD45 transcripts were used as internal controls. Mr, RNA (or DNA) size ladder, relative molecular mass. Data shown are representative of at least three independent experiments for each assay.

 
In transcription assays (Fig. 5B), nuclear extracts from PMA-stimulated JT cells supported the transcription of CD28 INR-driven DNA templates, but similar extracts derived from unstimulated JT cells or those incubated in TNF-{alpha} alone or with TNF-{alpha} during (or after) PMA exposure showed little or no transcriptional activity. Parallel transcription assays with control TdT INR-driven templates showed equivalent transcriptional activities of nuclear extracts from JT cells regardless of the mode of stimulation. DNA templates containing TdT INR mutants were inactive regardless of the nuclear extract used.

As expected, nuclear extracts from CD28hi Jurkat cells showed CD28 INR-binding activity and supported CD28 INR-driven transcription (Fig. 5, A and B). Neither PMA nor TNF-{alpha} affected the activity of Sp1 (Fig. 5E).

TNF-{alpha} inhibited the transcription of CD28 itself. Whereas a small amount of CD28 transcripts (or none at all) were found in unstimulated Jtag cells as examined by RPAs (Fig. 5C) and by RT-PCR (Fig. 5D), large amounts of specific transcripts were found in PMA-stimulated Jtag cells. More significantly, addition of TNF-{alpha} during (or after) PMA incubation resulted in the marked reduction or the complete lack of CD28 transcripts. The levels of {beta}-actin and CD45 transcripts were unaffected by PMA and TNF-{alpha}.

Autoregulation of CD28 Expression—During a screening of anti-CD28 monoclonal antibodies, we observed that one of them down-regulated CD28 expression in PMA-stimulated JT or Jtag cells. The degree of CD28 down-regulation was equivalent between the two cell lines. As shown in Fig. 6A, cells incubated overnight with soluble ANC28 antibody showed significant reduction in the level of cell surface expression of CD28. Incubation of cells with other anti-CD28 antibodies, such as 28-2 and L293, as well as the IgG isotype control, did not alter the levels of PMA-induced CD28 expression. Although the specific epitopes of these anti-CD28 antibodies are not known, they stained CD28+ Jurkat T cells with equivalent intensity as measured by flow cytometry, and they can cross-compete specific binding to CD28. However, only ANC28 had the unusual property of down-regulating CD28 on PMA-stimulated JT and Jtag cells, which was also observed in varying degrees in Jurkat and in primary T cells incubated with the monoclonal antibody (data not shown).



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  		FIG. 6.
Specific ligation of CD28 is autoregulatory. A, triplicate cultures of JT or Jtag cells were incubated with 125 ng/ml PMA overnight, washed, transferred into complete medium containing 2 µg/ml anti-CD28 monoclonal antibodies (ANC28, 28-2, L293) or mouse IgG, and incubated for an additional 24 h. CD28 expression was measured by immunofluorescence staining and flow cytometry. B, parallel cultures of PMA-stimulated Jtag cells were transfected with CD28 promoter-driven firefly luciferase reporter plasmids (KPN, RV) and incubated in 2 µg/ml anti-CD28 (ANC28 or L293) or IgG for 24 h. Specific luciferase activities were measured as in Fig. 4B. Data shown are relative luciferase activities of three independent transfection experiments.

 
ANC28-mediated down-regulation of CD28 expression was also observed in reporter gene bioassays. As shown in Fig. 6B, PMA-stimulated Jtag cells transfected with CD28 promoter-driven luciferase constructs showed high levels of luciferase activity, which was significantly reduced when the transfectants were incubated with ANC28. In contrast, luciferase activity remained at high levels in transfectants incubated with the L293 anti-CD28 monoclonal antibody or with the IgG isotype control.

Repression of CD28 Gene Transcription by the Ligation of CD28 —The basis for ANC28-mediated inactivation of CD28 promoter-driven reporters was examined further. In EMSAs (Fig. 7A), CD28 INR protein-binding activities were found in nuclear extracts from PMA-stimulated JT cells incubated with anti-CD28 monoclonal antibodies L293 and 28-2 and the IgG isotype control. In contrast, cells incubated in the ANC28 antibody showed low levels, or complete absence, of CD28 INR-protein complexes.



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  		FIG. 7.
Autoregulation of CD28 occurs at the level of transcription. JT or Jtag cells were incubated in medium alone (lane U) or in 125 ng/ml PMA overnight, washed, and transferred to complete medium (lane M) with 2 µg/ml anti-CD28 monoclonal antibody L293 (lane 1), ANC28 (lane 2), 28-2 (lane 3), or IgG (lane 4), and incubated for an additional 24 h. Nuclear extracts were prepared and used in EMSAs with CD28 INR site {alpha} (INR{alpha})(A) or Sp1 (E) sequences as binding probes or in transcription assays with CD28 INR-driven DNA templates (B) as in Fig. 2. Extracts from Jurkat cells (lane k) were used as a positive control. From parallel Jtag cultures, total RNA was also isolated and used in RPA (C) and in RT-PCR (D) experiments to assess CD28 gene transcription. {beta}-Actin and CD45 transcripts were used as internal controls. Mr, RNA (or DNA) size ladder, relative molecular mass. Data shown are representative of four independent experiments.

 
In transcription assays (Fig. 7B), nuclear extracts from PMA-stimulated JT cells incubated with L293 or 28-2 monoclonal antibodies or with IgG supported the transcription of CD28 INR-driven DNA templates. However, extracts derived from cells incubated with ANC28 (lanes 2) showed little or no transcriptional activity with these DNA templates. Extracts from Jurkat cells incubated with ANC28 showed a similar pattern of transcriptional repression (data not shown).

Parallel transcription assays using control TdT INR-driven templates (Fig. 7B) showed equivalent transcriptional activity of nuclear extracts from JT cells (or Jurkat), regardless of the presence or absence of anti-CD28 antibodies. Templates containing the mutated TdT INR were inactive regardless of the nuclear extract used.

As expected, nuclear extracts from CD28hi Jurkat cells showed high levels of CD28 INR-binding activity and supported transcription of CD28 INR-driven templates (Fig. 7, A and B). Neither the anti-CD28 monoclonal antibodies nor the IgG control affected the activity of ubiquitous transcription factors such as Sp1 (Fig. 7E).

Ligation of CD28 by the ANC28 antibody inhibited the transcription of the CD28 gene itself. There was an abundance of CD28 transcripts in PMA-stimulated Jtag cells in the presence of L293 anti-CD28 monoclonal antibody or the IgG isotype control as examined by RPAs (Fig. 7C) and by RT-PCR (Fig. 7D). In contrast, there was a significantly lower level of CD28 transcripts in cells incubated in ANC28. The levels of {beta}-actin and CD45 transcripts were unaffected by any of the anti-CD28 antibodies or by IgG.

CD80-CD28 Interaction Down-regulates CD28 Expression—To validate the biological significance of ANC28-mediated down-regulation of CD28, experiments were conducted to examine whether the CD28 ligands CD80 and CD86 elicited similar biochemical events. As shown in Fig. 8A, coculture of PMA-stimulated JT or Jtag cells with human CD80-expressing murine P815 mastocytoma cells resulted in the down-regulation of CD28 expression. In contrast, PMA-stimulated JT or Jtag cells cultured with human CD86-expressing or wild-type P815 cells retained high levels of cell surface expression of CD28.



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  		FIG. 8.
CD80-CD28 interaction down-regulates CD28 expression. JT or Jtag cells were cultured overnight in 125 ng/ml PMA. Cells were washed and resuspended in fresh medium, and wild-type murine P815 cells (lane P5) or those expressing human CD80 (lane 80) or CD86 (lane 86) were added and incubated for an additional 24 h. CD28 expression was measured by immunofluorescence staining and flow cytometry (A), by RT-PCR (B), and by RPA (C). Quantification of CD28 expression by RPA (C) was measured by phosphorimaging and expressed as relative arbitrary units normalized to the amount internal {beta}-actin control. Nuclear extracts were also prepared and used in transcription assays with CD28 INR-driven DNA templates (D) as in Fig. 2. U, unstimulated cells; Mr, RNA (or DNA) size ladder. Data shown are representative of two (C) or three (A, B, D) independent experiments for each indicated assay.

 
At the mRNA level, RT-PCR assays (Fig. 8B) and RPAs (Fig. 8C) showed abundance of CD28 transcripts in PMA-stimulated Jtag cells cultured with CD86+ P815 cells. In contrast, there was a marked reduction in the amount of specific transcripts in similar Jtag cells cultured with CD80+ P815 cells. In transcription assays (Fig. 8D), nuclear extracts from PMA-stimulated JT cells cultured with wild-type P815 cells or those expressing human CD86 supported the transcription of CD28 INR-driven DNA templates. In contrast, extracts from PMA-stimulated JT cells cultured with CD80+ P815 cells showed little or no transcriptional activity. As expected, nuclear extracts from CD28null unstimulated JT cells were transcriptionally incompetent, whereas those from CD28hi Jurkat cells supported transcription of CD28 INR-driven DNA templates.

It should be noted that P815 cells used in the co-culture experiments had equivalent levels (≥2 orders of magnitude) of CD80 and CD86 expression as determined by flow cytometry. Similar results showing differential down-regulation of CD28 by CD80 were also found with CD28hi Jurkat cells incubated with CD80+ P815 cells (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Various in vitro models of T-cell replicative senescence (19, 36, 37, 44) rely on protracted procedures to generate CD28null T cells from CD28+ precursors. To facilitate elucidation of the regulatory pathway(s) leading to CD28 silencing, we established a biological system that allows for rapid assessment of CD28 expression under controlled conditions. In this study, we show the versatility of the T-cell lines JT and Jtag in tracking CD28+ {leftrightarrow} CD28null phenotypic transitions. JT cells lack CD28 but are capable of expressing it when exposed to PMA (Fig. 1). Induction of CD28 is fairly transient (Fig. 3), permitting the molecular dissection of the machinery regulating CD28 expression. The present data show that both the gain and the loss of CD28 are dependent on the functional competence of the CD28 INR, a core promoter element that regulates CD28 transcription (33). The induction of CD28 in JT (or Jtag) cells is directly related to the reconstitution of the CD28 INR (Fig. 2), reminiscent of that seen in the acquisition of CD28 in transformed B cells and plasma cells (19, 45) and in some in vivo-derived CD28null T cells (46). On the other hand, the progressive loss of CD28 on PMA-stimulated JT (or Jtag) cells is coupled to the inactivation of the CD28 INR (Fig. 3), reminiscent of the situation in normal T cells losing CD28 during repeated stimulation and continuous passage (19).

Induction of CD28 expression in JT (or Jtag) cells is antagonized by TNF-{alpha} (Fig. 4). TNF-{alpha} represses the CD28 INR by inhibiting the assembly of INR-specific transcription factor complexes, which effectively shuts down CD28 transcription (Fig. 5). In agreement with previous studies (37), these data suggest that prolonged exposure of activated T cells to TNF-{alpha} probably accelerates the loss of CD28. Although the TNF-{alpha} signaling cascade leading to CD28 INR inactivation remains to be examined, normal aging, inflammatory syndromes, and chronic infections are known to be associated with elevated levels of TNF-{alpha} (47–49). More significantly, inflammatory syndromes, such as rheumatoid arthritis and Wegener's granulomatosis, or chronic infections such as Chagas' disease and HIV, are also associated with the high frequency of oligoclonal CD28null T cells disproportionately with patient age, and the severity of disease manifestation is highly correlated with the frequency of CD28null T cells (7–11, 16, 31, 50). Because there is an underlying persistent immune activation in these pathologic states, CD28null T cells have been postulated to be indicators of premature senescence during chronic disease (15).

T-cell activation studies (51) and the existing models of T-cell replicative senescence (18, 19, 36, 44) have shown that down-modulation, and the ultimate loss, of CD28 results from repeated TCR triggering in concert with CD28 costimulation. It is curious that CD80-CD28 interaction in conjunction with TCR engagement elicits a greater degree of CD28 down-regulation than CD86-CD28 interaction (52). However, ligation of CD28 alone elicits no significant decrease in the cell surface levels of CD28 (19). In contrast, the present work clearly demonstrates that specialized ligation of CD28 by a particular monoclonal antibody, namely ANC28, can result in CD28 down-regulation (Fig. 6). Although all of the anti-CD28 monoclonal antibodies used in the present and in previous studies recognize CD28 with equivalent affinity and elicit a costimulatory signal (19, 53, 54), the agonistic effect of ANC28 includes the specific repression of CD28 INR activity that leads to the silencing of CD28 transcription (Fig. 7). Although the epitope on human CD28 recognized by ANC28 remains to be identified, so-called "super-agonistic" antibodies to both rat and human CD28 have recently been reported (55). Such super-agonists seem to elicit signals sufficient to activate nuclear factor-{kappa}B, the downstream target of CD28 signaling, without activating signaling molecules associated with the TCR. Whether or not nuclear factor-{kappa}B activation is linked to the autoregulation of human CD28 remains to be studied.

The finding that CD80, one of the natural ligands of CD28, also elicits CD28 down-regulation validates the antibody-mediated autoregulation of CD28. Like the ANC28 monoclonal antibody (Fig. 7), CD80 interaction with CD28, in the absence of TCR ligation, inhibits CD28 transcription through the direct inactivation of the CD28 INR promoter element. CD86, the other CD28 ligand, does not affect CD28 expression (Fig. 8). Although the CD80/CD28 signaling pathway that leads to CD28 INR inactivation is yet to be elucidated, our data are consistent with the idea that CD80 and CD86 can differentially trigger CD28 signaling and elicit different outcomes (56, 57). It is significant that the present data show that CD80/CD28 signaling can occur without TCR signaling, resulting in a distinctive biological outcome. The relevant in vivo situations in which CD80, but not C86, elicits CD28 down-regulation remain to be explored. However, it is noteworthy that CD86 is constitutively expressed and rapidly up-regulated early in the immune response, whereas CD80 is inducibly expressed at high levels much later during an ongoing immune response (58). Thus, it is possible that down-regulation of CD28 by CD80 could be a mechanism for dampening the immune response. Consistent with this suggestion is the observation that CD8+CD28dim T cells in HIV are highly susceptible to apoptosis (26, 59). In this case, development of CD8+CD28null T cells could be a way to escape death-inducing signals after TCR triggering. On the other hand, persistent immune activation, such as that seen in inflammatory states and in chronic infections, could be associated with differentially higher levels of expression of CD80 on antigen-presenting cells (59–62) such that sustained CD80-CD28 interactions could accelerate and maintain the development of CD28null T cells. The notion of the strength of CD80-CD28 interaction, which leads to the extinction of CD28 transcription and the development of CD28null T cells, is supported by the recent finding of a CD28-specific microdomain within the N-terminal V-region of CD80 that is not found in CD86 (63).

In summary, the present work shows unequivocally that CD28 phenotypic transitions of T cells are regulated at the level of transcription. Although a number of regulatory motifs are found in the CD28 gene promoter (3), the CD28 INR is sufficient for CD28 transcription. In previous work, mutations in the CD28 INR have been demonstrated to inactivate the transcriptional competence of the CD28 minimal promoter (3, 33). The present data show that CD28 INR functions independently of other regulatory motifs, such as the so-called GR element (Fig. 4; Ref. 41). The CD28 INR is a specific target for inhibition by TNF-{alpha} (Figs. 4 and 6) as well as by a signaling cascade emanating from the specific ligation of CD28 by CD80 (Figs. 7 and 8). Recent studies have shown that the CD28 INR is a site of assembly of a transcription factor complex consisting of nucleolin and the A-isoform of heterogeneous ribonucleoprotein D0, which regulate, at least in part, the expression of CD28 (64). Down-regulation and the eventual loss of CD28 expression during the proliferative life span of T cells is caused not by the presence, relative abundance, or absence of these two proteins but by the lack of a functional DNA-binding protein complex that trans-activates the CD28 INR. The basis for the formation of this INR-binding complex is not yet known. Whether or not TNF-{alpha} and CD80/CD28 signaling also involves similar cascades that inhibit assembly of these INR-specific transcription factor complexes will have to be examined. The JT and Jtag cell lines are valuable tools in the dissection of the molecular events underlying CD28 phenotypic transitions, and the silencing of CD28 INR is a key event in the replicative senescence of T cells.


   FOOTNOTES
 
* This work was supported by the Mayo Foundation and by National Institutes of Health research Grants R01-AG22379 (to A. N. V.), R01-AG15043 (to J. J. G.), and R37-AI36682 (to D. E. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Children's Hospital of Pittsburgh, 2122 Rangos Research Center, University of Pittsburgh School of Medicine, 3705 Fifth Ave., Pittsburgh, PA 15213. E-mail: abbe.vallejo{at}chp.edu.

1 The abbreviations used are: HIV, human immunodeficiency virus; INR, initiator; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcription; TNF, tumor necrosis factor; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay; TdT, terminal deoxyribonucleotidyl transferase; RPA, RNase protection assay; TdT, terminal deoxyribonucleotidyl transferase; TCR, T cell receptor. Back

2 A. N. Vallejo, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kelvin Lee (University of Miami Medical School) for providing the CD28 genomic clone used in the construction of reporter plasmids, Dr. David Spencer (Baylor College of Medicine) for the parental Jtag cell line, Dr. Lewis Lanier (University of California, San Francisco) for the P815 cell lines, Xiao-Ping Wang for assistance in the construction of KPN and RV reporter plasmids, and Cindy D. Johnson and Terry Saulsberry for secretarial support.



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