From the Department of Immunology and the Division of Rheumatology, Department of Medicine, Mayo Clinic Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Changes in T cell populations and concomitant
perturbation of T cell effector functions have been postulated to
account for many aging-related immune dysfunctions. Here, we report
that high frequencies of CD28null CD4+ T cells were
found in elderly individuals. Because deviations in the function of
these unusual CD4+ T cells might be directly related to CD28
deficiency, we examined the molecular basis for the loss of CD28
expression in CD4+ T cells. In reporter gene bioassays, the minimal
promoter of the CD28 gene was mapped to the proximal 400 base pairs (bp) of the 5' untranslated region. CD28 deficiency was
associated with the loss of two noncompeting binding activities within
a 67-bp segment of the minimal promoter. These binding activities were
not competed by consensus Ets, Elk, or
AP3 motifs that were found within the sequence stretch. The DNA-protein complexes were also not recognized by antibodies to Ets-related transcription factors. Furthermore, introduction of mutations into the 67-bp segment at positions corresponding to the two
DNA-protein interaction sites, i.e. nucleotides spanning 206 to
179 and
171 to
148, resulted in the loss of specific nuclear factor binding activities and the abrogation of promoter activity. These observations implicate at least two regulatory motifs
in the constitutive expression of CD28. The loss of binding activity of
trans-acting factors specific for these sequences may
contribute to the accumulation CD4+CD28null T cells during
aging.
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INTRODUCTION |
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Among the many consequences of aging is the progressive decline in protective immunity. Elderly individuals have increased susceptibility to infections and blunted responses to vaccines. The incidence of various forms of malignancies and the risk of developing autoimmune diseases also increases with age. Although the molecular processes leading to the aging of the immune system are not yet clear, there is compelling evidence that changes in T cell populations and concomitant changes in T cell effector functions may underlie many aging-related immune dysfunctions (1). Because of thymic involution from the onset of reproductive maturity, aging leads to the inability to generate new T cells. Thus, there is a progressive replacement of naive (CD45RA+) T cells with those having a memory (CD45RO+, CD29hi, and CD44hi) phenotype (2-5). Paradoxically, expansion of the memory T cell population is not associated with enhanced memory immune responses in the elderly. Although the reason for this phenomenon is not known, previous studies indicate that T cells undergo functional changes with aging. Defects in signal transduction and, consequently, decreased proliferation and interleukin (IL)1-2 production have been reported (6-11). Other reported aging-related changes in T cell function are the inability of T cells to support B cell proliferation and immunoglobulin production (1, 12). Thus, T cell-dependent B cell responses are impaired with aging as demonstrated by the marked reduction in the efficacy of vaccines in the elderly (13, 14).
In an attempt to identify mechanisms of impaired immune responses, we studied a cohort of elderly individuals. Phenotypic T cell profiles of these individuals indicate that a subgroup had accumulated CD4+ T cells deficient in the expression of CD28, a membrane glycoprotein typically found on CD4+ T cells that provides the requisite costimulatory signal for T cell activation (15, 16). In the elderly, these CD28null cells can comprise up to 45% of the total CD4+ T cell compartment. A similar CD4+ T cell subset is typically found among patients with rheumatoid arthritis. Analysis of the T cell receptor usage of these in vivo expanded CD4+CD28null T cells (17, 18) indicated that they are oligoclonal (19) and persist for many years (20).
We propose that the emergence of CD4+CD28null T cells may reflect an aging immune system. Consistent with this hypothesis are the features of these cells, namely, their in vivo clonality and longevity, memory phenotype, and unusual functional profiles, including defective B cell help activity (17-22). All of these characteristics are considered to be hallmarks of an aging immune system (1). Inasmuch as CD4+CD28null T cells appear to be a distinct subset of functional T cells in the elderly but not among younger individuals, further assessment of the immunobiology of these cells may provide insight into the cellular processes involved in immunosenescence.
Our interest in studying the biology of CD4+CD28null T cells in humans lies in the well documented role of CD28 in immune responses as the major costimulatory molecule required for T cell activation (15, 16). In the mouse, the CD28 molecule is expressed on virtually all CD4+ T cells and has been unequivocally shown to be required for the maintenance of T cell proliferation, IL-2 production, and the de novo synthesis of many T cell-specific genes. In the absence of CD28-mediated signals, T cell recognition of antigen results in anergy or the induction of apoptosis. Perhaps the most compelling evidence of the critical requirement for CD28 in T cell-mediated immunity is the CD28-deficient mouse, which exhibits various forms of immune dysfunctions (23-26).
Among the important issues related to the biology and emergence of the CD4+CD28null T cells is the molecular basis for the deficiency of CD28 expression. The genomic and cDNA structures of the gene have been reported previously (27, 28), but the structural and biochemical requirements for gene transcription remain to be elucidated. Therefore, we initiated studies to examine the structural requirements for the constitutive expression of CD28. It is important to note that although CD28 is constitutively expressed on CD4+ T cells, previous studies showed that the levels of its expression are not static. The level of CD28 expression has been shown to either transiently increase or decrease, depending on the stimulus (29, 30), implying that the levels of its expression profoundly influence its costimulatory function.
The observed deficiencies in cell surface expression of CD28 among CD4+ T cells is correlated with a lack of CD28 mRNA.2, 3 Similar observations have also been reported for transformed CD4+CD28null T cells without apparent genomic lesions of CD28 itself (27). Therefore, we evaluated the hypothesis that CD28 deficiency in human CD4+ T cells is associated with a transcriptional block. In the present paper, bioassays were carried out to assess the transcriptional activity of the CD28 gene promoter in CD28-deficient cells. Additionally, studies were conducted to identify sequence motifs that may be associated with the differential surface expression of CD28 in primary human CD4+ T cells. Given the important role of CD28 in the immune response, elucidation of the biochemical and molecular basis for the modulation of its expression will facilitate our understanding of the biology of CD4+CD28null T cells. Because these unusual T cells appear to be a feature of the elderly, studies of the pathways controlling CD28 expression will provide insight on molecular mechanisms associated with the emergence of CD28null cells in the aging CD4 T cell compartment.
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MATERIALS AND METHODS |
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Study Population-- A total of 60 Caucasians who did not have a personal or family history of autoimmune diseases were chosen. Mononuclear cells were isolated from heparinized peripheral blood by isopycnic centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech). Cells were subjected to standard immunofluorescence staining with monoclonal antibodies to CD3, CD4, and CD28 (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells were fixed with 1% paraformaldehyde in phosphate-buffered saline, and the lymphocyte population was analyzed by flow cytometry using either a FACScan or FACSVantage cytometer (Becton Dickinson Immunocytometry Systems). All analyses of the staining profiles of lymphocytes were carried out using PC-LYSIS II software (Becton Dickinson Immunocytometry Systems).
The frequency patterns of CD28+ and CD28null cells among CD3+CD4+ lymphocytes were analyzed as a function of age. Statistical analyses of the frequencies were performed using the Mann-Whitney test (SYSTAT for Windows, SYSTAT, Inc., Evanston, IL).Cell Culture--
The CD28null CD4+ and CD28+CD4+ T
cell clones used in this study have been described previously (17, 21).
They were propagated in RPMI 1640 medium (BioWhittaker, Walkersville,
MD) containing 10% fetal calf serum (Summit Biotech, Ft. Collins, CO),
2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin sulfate (Life Technologies, Inc.). To enhance
their expansion, cultures were also supplemented with recombinant human
IL-2 (Genzyme Diagnostics, Cambridge, MA) to a final concentration of
20 units/ml and cocultured with 1 × 105 cells/ml of
neuraminidase-treated -irradiated (10,000 rad) Epstein-Barr virus-transformed B lymphoblastoid cells every 7-10 days. Cells were
maintained in a humidified 37 °C/7.5% CO2 incubator.
Cells were periodically examined for their expression of CD3, CD4, and CD28 by flow cytometry.
Isolation of the CD28 5'-Untranslated Region (UTR)-- The genomic template for the cloning of the CD28 gene 5'-UTR was obtained by a combined strategy (see Fig. 2) of restriction enzyme digestion, Southern blotting, and by polymerase chain reaction (PCR). Genomic DNA isolated from peripheral blood lymphocytes of a healthy donor was prepared using standard techniques and digested with a mixture of restriction enzymes containing HindIII, BglII, and ScaI. Restriction fragments were fractionated by agarose gel electrophoresis and blotted onto nylon membranes. The appropriate fragment was identified by hybridization with a synthetic 41-bp oligonucleotide probe (see Fig. 2B, probe 41) corresponding to a segment within the CD28 5'-UTR described previously (28). This 41-bp segment of the 5'-UTR contains the putative Ets1 and AP3 binding motifs. This combination of the two motifs within the 41-bp segment was found to be unique for the CD28 gene. Ets and AP3 motifs were determined by structural analysis of the published sequence of the CD28 5'-UTR (28) using the Map program in GCG software (Genetics Computer Group, University of Wisconsin, Madison, WI) interfaced with the transcription factor data base (NCBI, National Institutes of Health, Bethesda, MD).
The appropriate restriction fragment was purified and used as a template for the isolation of the 5'-UTR by PCR with gene-specific primers using the Expand PCR System (Boehringer Mannheim). All PCR primers used in this study contained an engineered 5' restriction enzyme site to facilitate subsequent cloning into plasmid vectors. Synthesis and purification of oligonucleotide probes and primers and the PCR conditions have been described (31). The appropriate PCR amplification products were identified by the Southern hybridization procedure described above. These PCR products were digested with the appropriate restriction enzyme, purified by GeneClean (BIO 101, La Jolla, CA), and cloned into a cloning/expression plasmid (see below). Plasmids were introduced into Escherichia coli DH5Reporter Gene Bioassays-- For the present studies, a cloning/reporter plasmid was specifically created. The plasmid, pSVGFPAmpR, was a fusion of the appropriate BamHI-HindIII fragments from pGLPromoter (Promega, Madison, WI) and phGFP-S65T (CLONTECH). It contains the ampicillin resistance gene, f1ORI, a multiple cloning site, and the minimal SV40 promoter. The humanized green fluorescence protein (hGFP) cDNA sequence with the associated SV40 poly(A) site is located downstream of the minimal SV40 promoter. This plasmid was used as one of the control plasmids in transient transfection assays, as well as the cloning vector for the construction of a series of hGFP reporter plasmids containing fragments of the CD28 5'-UTR.
Truncation variants of the CD28 5'-UTR were generated by PCR and cloned into the BglII and HindIII sites of pSVGFPAmpR after excision of the minimal SV40 promoter. These plasmids were amplified in bacteria as described above. Sequencing authenticated all CD28 5' UTR reporters generated, and two independent clones for each truncation variant were chosen for subsequent bioassays. All plasmids used in reporter bioassays were purified by cesium chloride density centrifugation. The plasmid preparations were then dialyzed against 1× Tris-EDTA, desalted by column chromatography (NAP-10 columns, Amersham Pharmacia Biotech), and stored atElectrophoretic Mobility Shift Assays (EMSAs)-- In the present work, EMSAs were conducted using nuclear extracts from both CD28+ and CD28null CD4+ T cell lines and clones described above. Additional extracts from Jurkat, HUT78, other non-T cell ATCC lines (HeLa, K562, RD, and U937), and the Epstein-Barr virus-transformed B lymphoblastoid cell line HT10 were also prepared. Binding probes used in these assays were as indicated (see "Results" and Fig. 4). In competitive EMSAs, competitors were added to the binding reaction at 5-300-fold excess concentrations relative to the binding probe. The conditions for the preparation of nuclear extracts, radiolabeling of oligonucleotide probes, binding reactions, nondenaturing polyacrylamide electrophoresis, and visualization of gel shifts have been described (33).
Mutational Analysis--
Results of EMSAs indicated that there
are two sequence motifs in the CD28 promoter 5'-UTR that
bound nuclear factors from CD28+, but not CD28null, CD4+ T
cells (see "Results"). To further evaluate the role of these two
motifs in the promoter activity of the 5'-UTR, site-specific mutations
were introduced into reporter constructs containing the minimal
CD28 promoter (see "Results" and Fig. 3). Cluster of
overlapping mutations, as indicated, were introduced by the gene
splicing by overlap extension technique described previously (31). For
these studies, the mutants were cloned into the
BglII-HindIII sites of the vector, pGLPromoter
(Promega) after the excision of the minimal SV40 promoter. Two
independent clones for each mutant were selected. The plasmids were
purified by cesium chloride density centrifugation and used in
transient transfection assays with Jurkat cells as described above. For
each transfection experiment, the plasmid pCMVLuc (32) was used as
positive control, and the pGLPromoter plasmid served as vector control.
Transfection efficiency was normalized by the cotransfection of 2 µg
of the -galactosidase reporter, pCMV
gal (32). Luciferase and
-galactosidase activities were determined by bioluminescence using
the Dual-Light Assay kit (Promega), and photoemissions were measured by
a luminometer (Lumat LB9501).
Sequence Mapping of the CD28 5'-UTR-- The cloned 600-bp segment of the CD28 5'-UTR was subjected to transcription factor mapping using the Map program in the GCG software interfaced with the transcription factor data base. Mapping algorithms were set at high stringency in that the relevant nuclear factor binding sites were identified with exact matches or with a single allowable mismatch.
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RESULTS |
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Deficiency of CD28 Expression in Human CD4+ T Cells Is Correlated with Age-- Our previous studies indicated that CD4+CD28null T cells are extremely infrequent in the majority of healthy individuals; however, a subset of individuals showed increased frequencies (18). We examined an extended cohort of healthy Caucasian individuals to identify factors associated with the increased numbers of CD4+ CD28null cells. As shown in Fig. 1, the frequency of CD4+ CD28null T cells was significantly correlated with age. These cells were rarely found among individuals younger than 40 years. In contrast, many of the elderly have a marked increase in the frequency of CD4+CD28null T cells. In some elderly individuals, these cells comprised up to 45% of the total CD4+ T cells (Fig. 1, inset). It is important to note that there were also elderly individuals who had extremely low frequencies of these unusual cells (<1% of the total CD4+ T cells) and those who completely lacked them. Conversely, there were occasionally those younger than 40 years with high frequencies (>2%) of CD4+CD28null T cells. These results indicate that there are other age-independent factors influencing the presence of these cells in the peripheral blood.
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The Promoter Activity of the CD28 5'-UTR Is Localized to the
Proximal 400-bp Region--
The deficiency in the cell surface
expression of CD28 in CD4+ T cells was previously shown to be due to
the uniform lack of CD28 mRNA.2, 3 By
standard reverse transcription-PCR techniques, none of the splice
variants of CD28 mRNA (27, 28) were detectable. To test the
hypothesis that a transcriptional block might account for the
deficiency in CD28 expression, studies were initiated to isolate the
5'-UTR of the gene for functional assays. The genomic structure of the
human CD28 gene has been described (28). The restriction map
of the gene has also been reconstructed by the mapping of contiguous
phage clones. However, the sequence encompassing the entire gene domain
remains to be determined. The regions of the gene that have been
sequenced include 760 bp of the 5'-UTR, exons 1-4, and parts of
introns 1-3. Intronic sequences that have been determined are those
that immediately flank the exons. Using this information, we conducted
a limited restriction enzyme analysis to isolate the appropriate
genomic template for the cloning of the CD28 5'-UTR. For
these studies, HindIII, BglII, and
ScaI enzyme digestion was performed. As indicated previously
(28), ScaI and HindIII sites are located within
introns 1 and 2, respectively (Fig.
2A). Thus, a mixture of these
enzymes was predicted to yield restriction fragments containing the
CD28 5'-UTR. By Southern hybridization using a 41-bp
sequence (Fig. 2B), the 5' UTR was found to be localized
within a 2.5 kb HindIII fragment (Fig. 2A).
This fragment was within larger BglII and ScaI
fragments because digestion of DNA with the all three enzymes or a
combination of HindIII with either BglII or
ScaI yielded hybridizing fragments of the same size as the
HindIII digested DNA. These results indicated that
ScaI, BglII, and HindIII sites are
situated on either side of exon 1. Using the Map programs in the GCG
software, none of these restriction sites were found within exon 1 or
the 760-bp sequence of the 5'-UTR (data not shown).
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The Promoter Activity of the Proximal 400-bp Segment of the 5'-UTR Is Suppressed in CD28null T Cells-- As indicated earlier, the deficiency of CD28 expression among CD4+ T cells was associated with a uniform lack of CD28 mRNA.2, 3 This suggested that a transcriptional block may be responsible for the lack of cell surface expression. To test this hypothesis, reporter gene bioassays were conducted using the CD4+CD28null T cell line HUT78. As we have found among our T cell clones, the lack of CD28 expression in HUT78 also occurred at the mRNA level without any apparent lesions in the genomic sequence of the gene (27). Results of transient transfection experiments with hGFP reporters showed that the promoter activity of the 400-bp segment of the CD28 5'-UTR was suppressed in HUT78 cells (Fig. 3D). The hGFP activities of cells transfected with the CMV promoter-driven reporters were maintained as expected. The negligible hGFP activities of HUT78 cells transfected with the other reporters were not significantly different from similarly transfected Jurkat cells.
A Lack of DNA-Protein Complex Formation with a 67-bp Sequence of the 5'-UTR Is Associated with a CD28-deficient Phenotype-- The observation that the promoter activity of proximal 400 bp of the CD28 5'-UTR was suppressed in HUT78 suggested that deficiency in CD28 expression may be associated with the lack of a relevant trans-acting factor(s). To evaluate this hypothesis, experiments were conducted to determine whether segments of the 400-bp region exhibited differential in vitro binding activities for nuclear factors from untransformed CD28+ and CD28null CD4+ T cells. In initial EMSAs, a 41-bp sequence (referred to as probe 41) was used (Figs. 2B and 4). Results of binding assays showed differential binding activities of probe 41 (Fig. 5). Binding activities to the probe were consistently observed with extracts from untransformed CD4+CD28+ T cell lines or clones and from Jurkat cells. In contrast, extracts from CD4+CD28null T cells, either transformed or untransformed, showed negligible binding activities. This differential binding activity was shown to localize to the opposite ends of probe 41. EMSAs using segments of probe 41 (Fig. 4), namely, the 5' half (probe 41A) and the 3' half (probe 41B), also showed differential binding activities (factors A and B) that correlated with the CD28 phenotype (Fig. 5). In contrast, binding assays using a probe corresponding to the central segment (probe 41C) did not show any binding activity regardless of the extract used in the assay. Interestingly, the binding activities seen with the 3' half (probe 41B) showed DNA-protein complexes with gel mobilities that were very similar to those seen with the intact 41-bp sequence (probe 41, factor A). This is in marked contrast to the activities seen with the 5' half (probe 41, factor B) which showed faster gel mobilities. Binding assays involving the same nuclear extracts using probes for the transcription factor SP1 (33) showed little difference among the T cell lines and clones examined. Moreover, EMSAs conducted using extracts from non-T cell lines also showed variable binding profiles (data not shown) that were unlike those seen with T cells as depicted in Fig. 5. Among the non-T cells examined were HeLa (epithelioid carcinoma), K562 (erythroid cell line), U937 (promonocytic line), RD (rhabdomyosarcoma), and HT10 (Epstein-Barr virus-transformed B lymphoblastoid line). These observations indicated that the differential binding activities of the 41-bp sequence or its specific segments were clearly correlated with the CD28 phenotype of the cells.
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There Are Two Noncompeting DNA-Protein Interaction Sites within the 67-bp Segment of the 5'-UTR-- To further ascertain that the two binding activities within the 67-bp sequence defined by probes 41A and 41B were nonoverlapping, competitive EMSAs were conducted. The experiments showed that probe 41A activities were not reciprocally competed by the 41B activities (Fig. 6). The respective DNA-protein complexes seen with the 41A probe were very stable in that even a 300-fold excess of competitor probe 41B or the 41B-related probe 5152 (data not shown) did not abrogate binding activity. Conversely, 41B activities were not competed by high concentrations of probe 41A or the 41A-related probes 4441A and 44. As expected, competition assays involving related probes 41A, 4441A, and 44 effectively competed each other, just as the related probes 41B and 5152 were effective competitors (Fig. 6 and data not shown).
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The Two DNA-Protein Interaction Sites Are Situated Immediately
Downstream from the CAAT-TATA Boxes--
Mapping analysis of the
cloned 600-bp segment of the CD28 5'-UTR showed that the
67-bp segment containing the two nonoverlapping DNA-protein interaction
sites lies downstream from the putative CAAT and TATA boxes (Fig.
7). As indicated by the results of gel shift assays (Figs. 5 and 6), these two sites, herein referred to as
sites and
, lie on opposite sides of an Ets1-like
binding sequence. Site
was the 5' site defined by probes 41A and
4441A, whereas site
was the 3' site defined by probes 41B and 5152. As indicated, sites
and
overlap with Elk1- and
AP3-like sequences, respectively. As shown by the gel shift
assays, however, neither Ets1, Elk1, nor
AP3 appeared to influence the observed binding activities of
sites
and
(see above and Figs. 5 and 6).
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Mutations in the Two DNA-Protein Interaction Sites Abrogate Nuclear
Factor Binding and Ablate Reporter Activity of the Minimal CD28
Promoter--
To evaluate the significance of the nuclear factor
binding activities of sites and
, a panel of reporter constructs
containing mutations in these CD28 promoter motifs were
generated. To identify the critical subsites for both motifs, a cluster
of four nucleotide substitutions were introduced across the sequence
stretch, thereby generating a series of overlapping mutants. As
depicted in Fig. 8, the mutagenesis
yielded motif variants that ablated reporter gene activities in that
mutations in either site
or site
independently elicited the
inactivation of the minimal CD28 promoter. Mutations in site
that resulted in the loss of promoter activity were within a
cluster of seven centrally located nucleotide positions, i.e. defined by mutants M3 and M4 and spanning positions
194 to
186, whereas in site
, the inactivating mutations
clustered in four to six more distal positions, i.e. defined
by mutants M9, M10, and M11 and spanning positions
161 to
154.
Curiously, mutations located between the two sites (mutant M5) did not
ablate reporter activities.
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DISCUSSION |
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Studies presented here address molecular mechanism(s) underlying the deficiency in CD28 expression of CD4+ T cells. CD4+ CD28null T cells are uncommon in healthy young individuals but emerge in the elderly. In some cases, these cells comprise up to 45% of the CD4+ T cell compartment. Given the critical role of CD28 in the immune response (15, 16, 23, 24), defective CD28 expression may contribute to T cell dysfunction characteristic of immunosenescence. Here we show that the lack of CD28 expression correlates with the absence of two trans-acting factor binding activities within the minimal CD28 promoter.
Accumulation of CD4+CD28null T Cells in an Aging Immune System-- The occurrence of CD4+CD28null T cells at unusually high frequencies among the elderly (Fig. 1) raises the issue of the relevance of these cells in immunity of the elderly. As has been well documented in both mice and humans, CD28 is the critical costimulatory molecule required for T cell activation, proliferation, and effector function (16). Its absence or interruption of its interaction with its ligands results in T cell anergy or the induction of apoptosis (15, 23, 25). In mice, the targeted deletion of CD28 resulted in a severely immunocompromised animal (24, 26). Thus, the observed accumulation of CD4+CD28null T cells in vivo raises the hypothesis that CD28 deficiency leads to deviations in T cell effector function and contributes to age-related immune dysfunctions.
As demonstrated previously, CD4+CD28null T cells are functional and are not anergic as might be expected (21). T cell receptor cross-linking either by immobilized anti-CD3 or soluble anti-CD3 and monocytes has been found to elicit proliferation. Under these stimulation conditions, these cells also produce high levels of cytokines, among which are IL-2, IL-4, andDelineation of cis-Acting Elements and Identification of Sequence Motifs Associated with CD28 Deficiency-- Inasmuch as the regulatory pathways involved in the normal expression of CD28 are not yet known, the present work provides basic information on the minimal structural requirements for CD28 gene transcription. Here, we have cloned 600 bp of the 5'-UTR and provide evidence that the proximal 400 bp contain the minimal cis-acting elements necessary for gene expression as assessed by reporter gene bioassays. As the data show, only reporter plasmids containing the proximal 400 bp yielded promoter activity. Structural analysis of the 5'-UTR (Fig. 7) revealed that this proximal 400-bp segment contained the putative CAAT and TATA boxes and various constituent binding sites where the RNA polymerase complex presumably assembles (44, 45). Deletion of this region, as shown by transfection experiments with reporters containing only the most proximal 200-bp segment (Fig. 3B), did not yield any significant promoter activity. Conversely, deletion of the proximal 200 bp also did not yield significant promoter activity, indicating that the TATA-containing region requires downstream elements to function.
Flanking the 400-bp region is a 200-bp segment that appeared to be inhibitory to promoter activity. The reason for this inhibition is not clear. However, results of the structural analysis (Fig. 7) revealed the presence of sequences within this distal 200-bp segment that are homologous to repressor elements found in other genes. As shown previously, CD28 expression transiently increases upon T cell activation, and ligation of CD28 by specific antibody induces reduction in its surface expression (16, 29). These findings suggest that there are regulatory elements responsive to T cell activation signals that are involved in feedback control. Our data indicate that such elements may be contained within the proximal 400-bp minimal promoter. Cells transfected with reporter constructs under the control of this minimal 400-bp promoter showed significantly down-regulated reporter activity when exposed to ionomycin and PMA (Fig. 3C). An important finding of the present study was that the promoter activity of the proximal 400-bp segment of the 5'-UTR is suppressed in HUT78 cells (Fig. 3D). This CD4+ T cell lymphoma has been shown to lack CD28 at both the mRNA and protein levels without apparent genomic lesions in the CD28 gene itself (27). This observation, together with the fact that reverse transcription-PCR analysis on a variety of untransformed CD4+CD28null T cell lines and T cell clones showed the uniform absence of CD28 transcripts,2, 3 supports the notion that deficiency in CD28 expression may be due to the lack of relevant trans-acting factors. This hypothesis was evaluated by EMSAs with nuclear extracts from CD28+ and CD28null CD4+ T cells. The oligonucleotide probes used encompass a 67-bp segment containing putative binding sites for the Elk1, Ets1, and AP3 transcription factors (Figs. 4 and 7). As the data show, there are two noncompeting DNA binding activities within this sequence for extracts from CD28+ but not CD28null CD4+ T cells. These binding activities correspond to two nucleotide stretches that lie on opposite flanks of the Ets1 motif (Figs. 4-7). The 5' site, referred to as site ![]() |
ACKNOWLEDGEMENTS |
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We thank Brenda Goehring for technical assistance and James Fullbright for assistance in statistical analysis and manuscript preparation.
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FOOTNOTES |
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* This work was supported by the Mayo Foundation, the Deutscher Akademischer Austauschdienst (Germany), Grants RO1 AR41974 and RO1 AR42527 from the National Institutes of Health, and Grant AF16 from the National Arthritis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: School of Medicine,
Ruprecht-Karls-Universität 69120 Heidelberg, Germany.
§ Present address: Department of Medicine, University of Innsbruck, A-6020 Innsbruck, Austria.
¶ To whom correspondence should be addressed: Mayo Clinic Foundation, 200 1st St. S.W., Rochester, MN 55905. Tel: 507-284-1650; Fax: 507-284-5045.
1 The abbreviations used are: IL, interleukin; EMSA, electrophoretic mobility shift assay; hGFP, humanized green fluorescence protein; PCR, polymerase chain reaction; PMA, phorbol 12-myristyl 13-acetate; RA, rheumatoid arthritis; UTR, untranslated region; bp, base pair(s); GCG, Genetics Computer Group; CMV, cytomegalovirus.
2 J. J. Goronzy and C. M. Weyand, unpublished data.
3 T. Namekawa, C. M. Weyand, and J. J. Goronzy, submitted for publication.
4 A. N. Vallejo, J. C. Brandes, C. M. Weyand, and J. J. Guronzy, manuscript in preparation.
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REFERENCES |
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