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Departments of Medicine and Immunology, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, we examined whether this proinflammatory cytokine influences
CD28 expression. Incubation of T cell lines and clones as well as
Jurkat cells with TNF- induced a reduction in the levels of cell
surface expression of CD28. This effect of TNF- was reversible;
however, continuous culture of CD4+CD28+ T cell
clones in TNF- resulted in the appearance of a CD28null
subset. In reporter gene bioassays, TNF- was found to inhibit the
activity of the CD28 minimal promoter. Inactivation of the promoter was
accompanied by a marked reduction in DNA-protein complex formation by
two DNA sequence motifs corresponding to the transcriptional initiator
of the CD28 gene. Indeed, in vitro transcription assays showed that
nuclear extracts from TNF--treated cells failed to activate
transcription of DNA templates under the control of a consensus TATA
box and the CD28 initiator sequences. In contrast, similar extracts
from unstimulated T cells supported transcription. These results
demonstrate that TNF- directly influences CD28 gene transcription.
We propose that the emergence of CD4+CD28null T
cells in vivo is facilitated by increased production of
TNF-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although CD28 is constitutively expressed on T cells, the levels
of its expression on the cell surface are constantly modulated. In
humans, CD28 is down-regulated following activation, and levels of
expression decline progressively with replicative senescence (1, 5). CD28 down-regulation has also been reported during acute and
chronic viral infections (6, 7). Interestingly,
CD4+ T cells that completely lack expression of
CD28 have been found in unusually high frequencies among patients with
various inflammatory conditions such as rheumatoid arthritis
(RA)4 (1, 8), Wegener’s granulomatosis (9), and unstable
coronary artery disease (10).
CD4+CD28null T cells also
emerge during normal aging (11), with frequencies up to
50% of the total CD4 compartment found among some
individuals older than 65 years. Recent studies demonstrate that such
modulation and/or loss of CD28 expression on T cells are attributed to
two regulatory sequences, sites and 
, in the gene promoter
(5, 11). Changes in the levels of cell surface
expression of CD28 are associated with the coordinate modulation of the
protein-binding activities of sites and (5).
Although the binding activities of and are independent of each
other (11), they constitute a functionally singular
transcriptional initiator (INR) element (12). Among
CD28null T cells, this -INR is inoperative
because of the lack of - and -binding complexes.
The central role of CD28 in the productive costimulation of T cell
activation (13) suggests that modulation or loss of its
expression could profoundly influence immune responses. Thus, we have
been interested in examining situations that influence CD28 expression.
Inasmuch as CD4+CD28null T
cells have been first described from patients with RA (14)
and that their high frequencies correlate with severe disease
(8), we tested the hypothesis that proinflammatory
cytokines, such as TNF-, may regulate CD28 expression. Because
TNF- is considered to be the major humoral modulator of the
pathogenic process in RA (15), it is reasonable that
chronic elevated levels of TNF- in vivo may directly impact T cell
phenotype and function. Indeed, previous studies have demonstrated that
TNF- induces nondeletional and reversible T cell hyporesponsiveness
in vitro and in vivo (16). Such T cell hyporesponsiveness
is associated with the uncoupling of the proximal TCR signaling pathway
(17). Although the impact of chronic exposure to TNF-
on the CD28 costimulatory pathway is not known, recent studies also
show that TNF--induced T cell hyporesponsiveness may be in part due
to the down-regulation of CD3
(18). Along these lines,
it is important to note that although the nonapoptotic
effects of TNF- largely involve induction of genes (19, 20), there is increasing evidence for TNF--specific
repression of gene expression (18, 21, 22, 23). In the present
work, we directly examined whether TNF- contributes to the loss of
CD28 expression, and consequently, the emergence of
CD4+CD28null T cells in a
chronic inflammatory disease like RA. A similar argument might be made
for the progressive increase in the frequencies of these unusual cells
with normal aging (11), which is also associated with
elevated TNF- production (24, 25).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T cell lines Jurkat and 6TCEM20 (American Type Culture Collection (ATCC), Manassas, VA) were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) containing 10% FCS (Summit Biotechnology, Fort Collins, CO), 2 mM L-glutamine, 50 U/ml penicillin, 5 µg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA). Cells were maintained at a density of 5 x 106 cells/ml in a humidified 5% CO2 tissue culture incubator.
Human primary T cell lines and clones were established from fresh PBMC
as described previously (5, 11, 26). Briefly, PBMC at
0.5–2 x 106 cells/ml were incubated on
plate-immobilized anti-CD3 (OKT3; ATCC) for 3 days.
CD4+ T cells were subsequently isolated by
positive selection using the VarioMACS system (Miltenyi Biotec, Auburn,
CA). Cells were passaged every 5–7 days in the RPMI 1640 medium
described above supplemented with 20 U/ml recombinant human IL-2
(Proleukin; Chiron, Emeryville, CA) and feeder cells consisting of
-irradiated neuraminidase-treated EBV-transformed B lymphoblastoid
cells. Cells were maintained in a humidified 7.5%
CO2 tissue culture incubator.
T cell clones were established by limited dilution cloning of freshly
isolated CD4+ T cells or from short-term cell
lines (5, 11, 26). Clonality was determined by standard
semi-nested RT-PCR for TCR -chain V and J elements,
size-fractionation, and sequencing. As with the primary cell lines, T
cell clones were maintained on feeder cells of EBV-transformed B
lymphoblastoid cells in the presence of 20 U/ml IL-2.
TNF- treatment of T cells and detection of CD28 transcripts
For each experiment described, triplicate cultures of 1 x
106 T cells were added to 24-well plates. Human
rTNF- (R&D Systems, Minneapolis, MN) was added to appropriate wells
and incubated for the indicated time periods. Based on initial
experiments (data not shown), a concentration of 10 ng/ml TNF- was
found to be the optimal concentration that induces CD28
down-regulation.
The relative amounts of CD28 transcripts before and after exposure to
TNF- were monitored by RT-PCR. Total RNA was isolated using TRIzol
reagent (Invitrogen Life Technologies) treated with DNase I (Roche
Molecular Biochemicals/Boehringer Mannheim, Indianapolis, IN), and
first-strand cDNA was synthesized by standard techniques. PCR
amplification of specific cDNA fragments was conducted using
gene-specific primers (5). Parallel PCR experiments were
also conducted for -actin as a system control (5, 12).
Flow cytometry
The expression of CD3, CD4, and CD28 on cell surfaces was examined by immunostaining and flow cytometry. Cells were incubated with PE-conjugated anti-CD28, FITC-conjugated anti-CD3, and PerCP-conjugated anti-CD4 (BD Biosciences, San Jose, CA) mAbs on ice for 15 min, washed, and fixed in 1% paraformaldehyde in PBS. A parallel sample of cells was also incubated with Ig isotypic controls (Simultest; BD Biosciences). All samples were immediately analyzed on a FACScan flow cytometer (BD Biosciences). Fluorescence data from at least 10,000 cells were collected from each sample and stored as list-modes. Off-line analyses of raw data were performed using WinMDI software (J. Trotter, Scripps Institute, La Jolla, CA).
Flow cytometric quantification of the number of CD28 molecules on the cell surface was conducted using the QuantiBrite System (BD Biosciences). Briefly, cells were immunostained with PE-conjugated anti-CD28 Ab by standard protocols. Cytometric data for cells and four QuantiBrite-PE bead standards of known levels of PE fluorescence were acquired using QuantiQuest (BD Biosciences) on the same flow cytometer at identical instrument settings. The FL2 axis was converted into the number of PE molecules bound per cell, and was calibrated by the QuantiBrite beads. Slope and intercept information from the regression analyses was automatically saved with the acquired data files. The actual number of CD28 molecules per cell was estimated using QuantiCALC software (BD Biosciences).
Apoptosis assay
The rates of apoptosis in the cultures were determined by double staining of cells with annexin V and propidium iodide (PI) using a kit (Annexin V Fluos Staining kit; Roche Molecular Biochemicals/Boehringer Mannheim). Cells were immediately analyzed by flow cytometry. Apoptosis was determined as the percentage of annexin V+ cells that excluded PI.
As system control, annexin V staining was also conducted with cells
incubated with the apoptosis-inducing drug campothecin
(27). Cells were incubated with 4 µg/ml camptothecin
(Acros-Fisher Scientific, Pittsburgh, PA) for 24 or 48 h and
stained for annexin V, and the rate of apoptosis was compared with
cells incubated with TNF-.
Proliferation assay
The proliferation rates of cultures were measured by two
methods, namely, standard [3H]thymidine
incorporation and staining with CFSE (Molecular Probes, Eugene, OR)
(28). For thymidine incorporation assays, triplicate
cultures of 1–5 x 104 T cells were added
to 96-well plates. TNF- was added at a final concentration of
10 ng/ml to the appropriate wells and cultured for 24 and 48
h. Approximately 1 µCi of [3H]thymidine (NEN,
Boston, MA) was added to the cells during the last 4 h of
incubation. Cells were harvested onto fiberglass filters (XtalScint;
Beckman Instruments, Palo Alto, CA), and radioactivity was measured by
scintillation spectrometry.
For CFSE assays, triplicate cultures of 2 x
106 cells were stained with 10 µM CSFE for 10
min, washed, and cultured in the presence or absence of 10 ng/ml
TNF-. At the indicated time periods, cells were harvested and
washed, and CSFE staining was analyzed by flow cytometry.
Reporter gene assay
The CD28 minimal promoter (11) was cloned into
reporter plasmids containing the soluble alkaline phosphatase (SEAP)
gene (BD CLONTECH Laboratories, Palo Alto, CA) and used to transform
Escherichia coli DH5 (Invitrogen Life Technologies).
Recombinant plasmids were prepared from randomly selected bacterial
colonies and screened for the presence of the CD28 promoter sequences
by restriction analysis and sequencing. Two bacterial clones were
selected and subjected to two rounds of cesium chloride density
centrifugation to isolate the plasmids.
Transient transfection of Jurkat cells was conducted as described
previously (11) with the following modifications.
Approximately 1 x 107 cells were
resuspended in 250 µl of drug-free medium and transferred to
electroporation cuvettes (4-mm gap Cuvette Plus; BTX, San Diego, CA).
CD28-SEAP reporter plasmid (28 µg) was added to the suspension to a
final volume of 300 µl, gently mixed, and incubated for 10 min on
ice. Samples were subjected to a single pulse of 300 V for 25 ms using
a square wave electroporator (T820 Electro Square Porator; BTX
Electroporation Systems) and then incubated on ice for 10 min. Cells
were gently resuspended in 1 ml of drug-free culture medium and
incubated at 37°C in a humidified atmosphere of 5%
CO2 for 4 h. TNF- was subsequently added
to appropriate cultures and incubated for another 20 h. SEAP
activity in the culture supernatant was measured by a specific
colorimetric assay using the Great Escape SEAP detection kit (BD
CLONTECH Laboratories). SEAP activity is heat stable unlike the
endogenous alkaline phosphatase activity, which is heat labile. Enzyme
activities were quantified using a luminometer (Lumat LB9501; Berthold
Analytical, Nashua, NH).
Similar transient transfection assays were conducted using SEAP reporters under the control of SV40 promoter and enhancer sequences (BD CLONTECH Laboratories), which served as positive system controls. As negative controls, promoterless SEAP plasmids were used.
To normalize for transfection efficiency, each sample was cotransfected
with 2 µg of CMV-luciferase reporter plasmid (29).
Luciferase activities of samples were detected from the cell pellets by
chemiluminescence using a kit (Promega, Madison, WI) and quantified
using a luminometer. The quotient of luciferase activities between
TNF--treated cells and unstimulated controls was determined. This
quantity was then used as a divisor for the corresponding SEAP
activity.
EMSA
Nuclear extracts were prepared as described previously (5, 11, 29). Briefly, cells were lysed in HEPES hypotonic buffer, and the nuclei were isolated by centrifugation. Nuclear proteins were extracted in a high-salt buffer, and protein concentration was determined using a protein assay kit (Bio-Rad, Richmond, CA). EMSA was conducted as described previously (5, 11). Briefly, 10 µg of nuclear extract were combined with 3 µg of poly(dI-dC) (ICN Biomedicals, Costa Mesa, CA) and 3 µg of a nonspecific oligonucleotide (5'-TCGAAGTACTCAATTGCTCGAGATCGATAGATCTGAATTCAGTACTCC-3') (29) in a binding buffer to a total reaction volume of 50 µl. Reactions were incubated on ice for 30 min, after which radiolabeled probes were added and incubated for another 30 min at room temperature. Protein-DNA complexes were resolved using 6% nondenaturing polyacrylamide gels and autoradiography.
Binding probes consisted of oligonucleotides corresponding to sites
and sequences of the CD28 minimal promoter (11). Sites
(5'-CGTTATATCCTGTGTGAAAGCTGCA-3') and
(5'-TGTGGTTTGAGTGCCTTGAT-3') sequences and their respective
complimentary strands were synthesized, end-labeled with
[32P]-ATP (NEN), and annealed by standard
procedures. As system controls, similar probes corresponding to SP1
(5'-GCTCCAGGCGGGGGCGGGGCCCGGGTTCGG-3') (29) were
synthesized and used in parallel EMSAs. Probes were stored at -20°C
and used within 7 days. All oligonucleotide probes were synthesized at
the Mayo Molecular Biology Core Facility and purified by column
chromatography as described previously (30).
In vitro transcription assay
Two nonoverlapping sequences, sites and , constitute the
CD28 INR element (12). This contiguous sequence
(5'-CGTTATATCCTGTGTGAAAGCTGCAGTCAGGATGCCTTGTGGTTTGAGTGCCTTGAT-3';
the underlined 5' and 3' sequences correspond to and ,
respectively) (11) was cloned into transcription plasmid
templates provided by Dr. J. Kaufmann (Chiron) (12). These
plasmids contained a consensus TATA box, the INR of TdT, and a 180-bp
G-less cassette (31, 32). The contiguous sequences
were introduced into these plasmids replacing the TdT-INR by the
gene-splicing-by-overlap-extension technique (30).
Constructs were amplified in E. coli DH5 (Invitrogen Life
Technologies) by standard transformation procedures. Randomly selected
bacterial colonies were screened for the presence of -INR
sequences by direct PCR. DNA sequencing of plasmids authenticated the
correct introduction of -INR. Plasmids were prepared using a
purification kit (EndoFree; Qiagen, Chatsworth, CA).
Conditions of transcription were as described previously
(12). Briefly, dialyzed nuclear extracts were incubated
with 300 ng of plasmid template at 30°C for 60 min. A mixture of
nucleotides (100 mM ATP, 100 mM CTP, d-CD3-GTP, 50 µM
[32P]-UTP (Amersham Pharmacia Biotech,
Piscataway, NJ)) was added, and the total reaction volume was adjusted
to 100 µl and incubated for 90 min at 30°C. Products were digested
with 60 IU RNase T1 (Roche Molecular Biochemicals/Boehringer Mannheim),
extracted with phenol-chloroform, and ethanol precipitated. Samples
were subjected to urea-polyacrylamide gel electrophoresis and
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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preferentially down-regulates CD28 expression on T cells
Because aging and RA are associated with elevated levels of
TNF- (15, 24, 25) that coincides with the emergence of
CD4+CD28null T cells
(8, 11), we examined whether there is a causal
relationship between these two phenomena. T cells were incubated in
TNF- and the levels of CD28 expression were examined. Depicted in
Fig. 1
is a representative flow
cytometric analysis of the immunostaining of CD28 on a
CD4+CD28+ T cell line.
After 24 h of incubation with 10 ng/ml TNF-, there was at least
a half-magnitude reduction of the cell surface expression of CD28. Such
a decrease in CD28 expression was specific because the level of CD4
expression was unaffected by TNF-. Similar results were seen with
several T cell lines and clones and transformed T cells such as Jurkat
and 6TCEM20 (data not shown). In all assays conducted, incubation of T
cells with TNF- consistently resulted in significant decreases in
the cell surface levels of CD28 expression without affecting the
expression levels of other T cell Ags including CD4 and CD3.
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leads to a CD28null
phenotype. CD4+CD28+ T cell
clones were isolated and maintained in standard culture conditions with
or without TNF-. As shown in Fig. 2, maintenance of T cells with exogenous TNF- resulted in the emergence
of a subset of CD28null cells within 8 wk of
culture. This subset comprised 
30% of the culture at the time of
analysis. The levels of CD4 and CD3 were unaffected by prolonged
exposure of the cells in TNF- (data not shown).
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induces a quantitative decreases in CD28 expression at the
protein and mRNA levels
Because of the consistent responses of all types of T cells
examined, Jurkat cells were subsequently used as a model to further
elucidate the mechanism(s) of TNF--induced down-regulation of CD28.
Fig. 3 demonstrates that incubation of
Jurkat cells with TNF- elicited a significant decrease in the actual
number of CD28 molecules on the cell surface. Such decreases were
elicited in a dose- and time-dependent fashion. Interestingly, these
TNF--induced decreases in the number of CD28 molecules were
reversible as indicated by the restoration of baseline levels of
expression when TNF- was removed from the cultures. Moreover, these
TNF--induced changes in the number of CD28 molecules correlated with
equivalent changes in the relative amounts of specific transcripts.
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has the capacity to induce cell death, regardless
of the receptor with which it interacts (33, 34), we also
examined whether apoptosis might be a confounding factor in the
observed TNF--induced down-regulation of CD28. For these studies, we
determined the levels of annexin V staining, which measures the rate of
apoptosis before and after exposure of cells to TNF-. As shown in
Fig. 4, there was no significant
difference in the level of annexin V staining between the unstimulated
controls and cells exposed to TNF-. At the two concentrations of
TNF- used in these studies (10 and 50 ng/ml), there were no apparent
changes in annexin V staining of cells. As expected, exposure of cells
to the apoptosis-inducing drug camptothecin (27) induced a
significant number of cells to undergo apoptosis.
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is also known to have a growth-promoting effect
(35). Thus, we examined whether the addition of exogenous
TNF- affected cell growth patterns. Cells were labeled with CSFE and
incubated with TNF-, and the rate of decay of CSFE fluorescence, as
a consequence of cell division, was followed. As shown in Fig. 5, the levels of CSFE staining were
virtually identical between the unstimulated controls and the cells
incubated with TNF-. Despite the fact that cells used in these
studies were unsynchronized, TNF- did not appear to affect the rate
of cell division during a 5-day culture period. Similar results were
obtained in proliferation assays using
[3H]thymidine incorporation (data not shown).
Triplicate cultures of cells incubated with or without TNF- showed
no significant differences in proliferation after 3 days of culture.
Clearly, neither increased apoptotic cell death nor changes in cell
turnover kinetics was related to the observed diminution of CD28
expression on T cells after TNF- exposure.
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represses the CD28 gene promoter
The observation that TNF- specifically induces a significant
reduction in the amount of CD28 mRNA (Fig. 3
) suggested that TNF-
directly affected transcription. Therefore, we examined whether TNF-
influences the activity of the CD28 promoter. In previous studies
(11), we described the minimal promoter consisting of the
5' proximal 400-bp sequence flanking the translation initiation codon
(36). Truncation and mutational studies showed that this
400-bp sequence contained the minimal regulatory elements required for
CD28 gene transcription. Such regulatory elements, subsequently
identified as sites and , were found to control the basal
expression of CD28, and were responsive to down-regulation by
activation through the TCR or by pharmacologic agents such as phorbol
esters (5, 11, 12). Therefore, we examined whether the
TNF--induced down-regulation of CD28 transcription (Fig. 3) might
also be attributed to the repression of the 400-bp minimal promoter
sequence. This sequence was used in reporter gene bioassays.
As shown in Fig. 6, transfection of
reporter plasmids containing the CD28 minimal promoter into
CD28+ Jurkat cells resulted in high levels of
expression of the reporter gene. Incubation of transfectants with
TNF- showed pronounced repression of reporter gene activities of two
independent reporter clones examined. In contrast, bioassays using
reporter plasmids containing SV40 promoter and enhancer sequences
showed high levels of reporter gene activity that were unaffected by
incubation of transfectants in TNF-. As expected, bioassays using
vector controls did not yield significant reporter gene activities.
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induces the down-regulation of site - and -binding
proteins
In previous studies, we demonstrated that two sequence motifs,
sites and , regulate the activity of the CD28 minimal promoter
(11). Structural analyses showed that these tandem
sequences are unique to the CD28 gene and do not resemble other known
regulatory elements (12). In EMSAs, these two sequences
exhibited nonoverlapping and noncompeting DNA-protein complexes,
formation of which was unaffected by mutated variants or by
structurally divergent sequences in competitive EMSAs
(11). The formation of such motif-specific DNA-protein
complexes were found only in CD28+ cells, and
were subject to modulation by TCR-derived signals or by pharmacologic
agents (5). Therefore, similar experiments were conducted
to examine whether the observed repression of the CD28 minimal promoter
after TNF- exposure (Fig. 6) might be due to the loss of
-/-bound complexes.
As shown in Fig. 7, DNA-protein complex
formation by these two sequences coordinately and progressively
decreased with incubation of cells in TNF-. After 48 h of
incubation, binding activities of both sequences were barely
detectable. However, the subsequent removal of TNF- from the cell
cultures restored the motif-binding activities.
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.
TNF- inhibits the transcriptional activity of the CD28
-INR
Although sites and have nonoverlapping and noncompeting
protein-binding activities (5, 11), they function as a
singular INR element (12). Thus, we examined whether
TNF--induced decreases in the levels of - and -binding
activities correlated with the loss of transcriptional activity. For
these studies, in vitro transcription assays on INR-driven DNA
templates were conducted using nuclear extracts from cells treated with
TNF- or unstimulated controls. Such templates contained either the
CD28 -INR (12) or the TdT-INR (31, 32).
As shown in Fig. 8, the transcriptional
activities of templates controlled by the CD28 -INR and the
TdT-INR were comparable in assays using nuclear extracts from
unstimulated cells. As expected, the mutated variant of the TDT-INR
consistently yielded low levels of activity. However, in assays using
extracts from TNF--treated cells, the CD28 -INR activities
significantly decreased. Such reductions in specific INR activities
correlated with the incubation time in TNF- but were restored to
high levels when TNF- was subsequently washed away from the
cultures. In contrast, the levels of transcriptional activities of
wild-type and mutant forms of INR-TdT were unaffected by TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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down-regulates the level of cell surface expression of CD28 on T cells.
The modulatory effect of TNF- on CD28 is at the level of
transcription (Fig. 3). Exposure of T cells to TNF- leads to the
repression of the transcriptional activity of the gene promoter
(Fig. 6) by inhibiting the binding of nuclear protein complexes
(Fig. 7) that specifically recognize two basal regulatory sequences,
sites and (5, 11). In recent work, we reported
that these sequences constitute a functionally singular INR element,
which is responsible for the initiation of transcription of the CD28
gene (12). Indeed, the present data show the inability of
nuclear extracts from T cells treated with TNF- to initiate
transcription of DNA templates under the control of the -INR
(Fig. 8). Although the composition of DNA-protein complexes recognizing
-INR is currently being examined, the present data are the first
indication that TNF- can have a strong repressive effect at the
level of transcriptional initiation. This is in marked contrast with
studies showing the repressive effects of TNF- principally through
the inactivation of auxiliary transcription factors and/or enhancers. A
typical example is the TNF--induced inhibition of type II collagen
synthesis (22, 37) that involves the inactivation of Sox9,
a member of the high mobility group of transcription factors
(38) that are generally not associated with the basal
transcription complex. Another example is the repression of IL-12
production that occurs through the inactivation of a yet unidentified
enhancer protein that may interact with NF-
B (21).
The specific inhibition of the CD28 -INR element by TNF-
leading to the down-regulation and eventual loss of the CD28 Ag on the
T cell surface (Fig. 2) further supports the hypothesis that the loss
or gain of INR function is an important determinant of cell phenotype
(12). Although INRs are core promoter elements (39, 40), they can direct the cell-specific expression of genes such
as the lymphocyte-restricted expression of TdT (41) and
the TCR V chain (42). This is because INR sequences may
define binding sites for gene-specific transcription factors
(43, 44, 45, 46) in lieu of the components of the basal
transcriptional initiation complex (47, 48, 49). In the
present work, there is an apparent specificity of TNF- in
inactivating the CD28 -INR. Other INR-regulated genes, such as
CD4 (50, 51), are unaffected by TNF- (Fig. 1). This may
be attributed to the fact that the CD28 -INR has no homology to
the consensus INR sequence (12, 52) as the CD4 INR does.
Inasmuch as DNA binding of transcription factors is largely motif
specific, sequence diversity is an important factor that influences
INR-dependent regulation of genes. We suspect that the protein
complexes bound by the CD28 -INR are different from those that
recognize CD4 INR, hence their differential susceptibility to
inactivation by TNF-.
Although TNF- exerts a strong inhibitory effect on the transcription
of the CD28 gene, it is unclear why there appears to be a more gradual
decrease in the levels of the cell surface expression of the CD28 Ag.
Neither a 48-h (Figs. 1 and 3) nor a 7-day exposure (data not shown) of
cells to TNF- results in the complete loss of CD28 on the cell
surface. One possible explanation is that the CD28 molecule has a long
half-life. It is also possible that preformed or previously
internalized CD28 Ags are being recycled to the cell surface to
compensate for the lack of de novo synthesis due to the inhibition of
transcription. Such recycling of CD28 has been previously reported in
murine mast cells (53).
A more likely possibility is that T cells become refractory to TNF-
because it down-regulates its own receptors. Previous studies have
shown that TNF- induces a rapid internalization of the p55 receptor
and the shedding of the p75 receptor (54). It is thought
that receptor shedding is a means to prevent harmful effects of
cytokine pleiotropy (55) such as apoptosis, which can be
one of the ill effects of TNF- on T cells (33, 34).
However, in the present work TNF-, at the concentrations used, did
not elicit cell death (Fig. 4) nor did it affect the rate of T cell
proliferation (Fig. 5).
Interestingly, our results show that CD28null T
cells can emerge from CD28+ precursors that are
continuously exposed to TNF- (Fig. 2). Because of TNF receptor
shedding and internalization, these data indicate that only by
sustained exposure of primary CD4+ T cells to a
TNF--rich environment can a CD28null subset
emerge. Presumably, a cycle of synthesis and depletion of TNF- may
not be sufficient enough to completely inhibit CD28 transcription. This
suggestion is supported by observations that withdrawal of TNF- from
T cell cultures restores the level of CD28 expression on the cell
surface (Fig. 3) as well as transcriptional activity of the gene
promoter (Figs. 7 and 8).
Whether CD28null T cells derived from
CD28+ T cells chronically exposed to TNF-
(Fig. 2) can re-express CD28 remains to be examined.
CD28null T cells generated in vitro with TNF-
are refractory to stimulation through the TCR, hence they are not
amenable to propagation (data not shown). This is consistent with
studies reporting that prolonged in vitro exposure of T cells to
TNF- also leads to the inhibition of CD3 phosphorylation
(18). This inhibitory effect of TNF- is only partially
reversed by the removal of TNF- from cultures,
suggesting that functional restoration of in vitro-generated
CD28null T cells may depend on a
culture system that completely alleviates the repressive effects of
prolonged TNF- exposure. Interestingly, we recently
found that some, but not all, in vivo-derived CD28null T
cell clones may re-express CD28 under certain culture conditions
(J. J. Goronzy, C. M. Weyand, and K. J. Warrington,
manuscript in preparation). Although a CD28null
phenotype is largely stable (8, 11, 12, 14), we have
preliminary data indicating that the formation of protein complexes
that bind the CD28-INR and are the specific targets of inhibition by
TNF- (Figs. 7 and 8), can be restored in certain cases, suggesting
that CD28 expression might be reversibly modulated in vivo.
The emergence of CD4+CD28null T cells in vivo
among patients with inflammatory diseases such as RA (8)
and during normal aging (11) seems to be more than
coincidental with elevated levels of TNF- (15, 24, 25).
Our data strongly indicate that among the consequences of chronic
exposure of T cells to TNF- is the repression of CD28 transcription
that may lead to CD28null phenotype. On the one
hand, it may be of interest to examine whether the reported efficacy of
anti-TNF- therapies in RA (56, 57, 58) involves a
resurgence of T cell populations that re-express CD28. On the other
hand, the emergence of
CD4+CD28null T cells among
healthy individuals such as those found among the elderly might also
involve other mechanisms independent of TNF-. Although our data
provide definitive proof that CD28null T cells
may emerge from CD28+ cells, the possibility that
in vivo these two cells may also come from separate lineages remains to
be examined.
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Acknowledgments |
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Footnotes |
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2 E.B. and A.N.V. contributed equally to this work and are regarded as co-first authors.
3 Address correspondence and reprint requests to Dr. Abbe Vallejo or Dr. Jörg J. Goronzy, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail addresses: vallejo.abbe@mayo.edu and goronzy.jorg{at}mayo.edu
4 Abbreviations used in this paper: RA, rheumatoid arthritis; INR, initiator; PI, propidium iodide; SEAP, soluble alkaline phosphatase.
Received for publication May 7, 2001. Accepted for publication July 16, 2001.
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