Download CTLA-4 gene expression is influenced by promoter and

Genes and Immunity (2001) 2, 145–152
 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00
www.nature.com/gene
CTLA-4 gene expression is influenced by promoter
and exon 1 polymorphisms
A Ligers1,2, N Teleshova1, T Masterman1, W-X Huang1 and J Hillert1
1
Division of Neurology, NEUROTEC, Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden; 2Center of
Biotechnology, Department of Bioscience, NOVUM, Karolinska Institutet, Stockholm, Sweden
CTLA-4, expressed mainly on activated T cells, helps maintain, through its inhibitory function, immune-system
homeostasis. Polymorphisms in the CTLA-4 gene (CTLA4) are known to be important in several autoimmune diseases,
including multiple sclerosis (MS). Here, we have performed genotyping for CTLA4 polymorphisms, and investigated
expression by peripheral blood mononuclear cells of CTLA-4 mRNA and protein, in patients with MS and myasthenia
gravis and in healthy controls. Expression levels for mRNA and protein were similar in the patient and control groups;
however, there was a clear relationship between genotype and CTLA-4 expression. Specifically, individuals carrying
thymine at position −318 of the CTLA4 promoter (T−318) and homozygous for adenine at position 49 in exon 1 showed
significantly increased expression both of cell-surface CTLA-4 after cellular stimulation and of CTLA-4 mRNA in nonstimulated cells. The association was seen most clearly for unsorted CD3+ cells and was absent in the CD8+ subset. The
T−318 allele has been shown to be negatively associated with susceptibility to MS in an earlier study by our group. Thus,
we propose that the susceptibility-influencing role of CTLA4 in MS may be related to genotypically conditioned promoter
function, whereby high gene expression may decrease the risk of disease. Genes and Immunity (2001) 2, 145–152.
Keywords: CTLA-4; gene expression; multiple sclerosis; polymorphism
Introduction
Multiple sclerosis (MS) is believed to be an autoimmune
disease resulting from aberrant immune responses to
central nervous system (CNS) antigens, and T cells are
thought to be involved in its pathogenesis. In the CNS,
organ-specific T cells mediate tissue damage by activating effector immune cells and by regulating recruitment
of non-antigen-specific lymphocytes and monocytes to
MS lesions.1
Naı̈ve T cells require two signals to proliferate and differentiate. Signal one is antigen-specific and is generated
by interaction of the T-cell receptor (TCR) with an antigenic peptide presented on an MHC molecule. Signal
two, essential for a functional response in T cells, is transduced most commonly through interaction of CD28 on
the T cell with B7 on the antigen-presenting cell.
Expression of cytotoxic T lymphocyte-associated molecule4 (CTLA-4) is subsequently up-regulated on T cells activated in this manner. CTLA-4 also binds B7, but this
interaction results in down-regulation of the ongoing
immune response.2–4
Correspondence: Arturs Ligers, Division of Neurology, NEUROTEC,
Karolinska Institutet at Huddinge University Hospital R54, S-141 86
Stockholm, Sweden. E-mail: Arturs.Ligers얀neurotec.ki.se
This study was supported by grants from the Swedish Medical
Research Council (project numbers 11023 and 11220), the Society
for the Neurologically Disabled, the Sigurd and Elsa Goljes Minne
Foundation, Karolinska Institutet, the Magn. Bergvall Foundation,
the Åke Wiberg Foundation, the Bibbi and Nils Jensen Foundation
and the Marcus Borgström Foundation.
Received 19 December 2000; revised and accepted 1 March 2001
CTLA-4 was first identified through the screening of a
murine cytolytic T-cell cDNA library,5 and the gene encoding the molecule is in humans located on chromosome
2q33-q34, 25–150 kilobases from the CD28 gene, with
which it shares extensive sequence homology.6
Expression of CTLA-4 is mainly restricted to CD4+ and
CD8+ activated T-lymphocyte subsets,7 but has also been
reported on B cells.8
The human CTLA-4 gene (CTLA4) is known to contain
polymorphisms in three regions: a cytosine-thymine singlebase substitution in the promoter at position −318
(C−318/T−318), and adenine-guanine dimorphism in the
exon 1 leader sequence at position 49 (A49/G49), an a
multiallelic dinucleotide repeat in the 3′ untranslated
region (UTR) of exon 4 (Figure 1).
These three polymorphisms have been investigated for
linkage and association in a number of human autoimmune diseases. The G49 allele has been shown to be
associated with several organ-specific autoimmune diseases, including type I diabetes mellitus (association with
the G49 allele and microsatellite 102 bp allele),9,10 Graves’
disease (association with microsatellite 102 bp allele)11
and MS (association with T−318 allele (negative) and G49
allele)12,13; but results are less clear with regard to the
non-organ-specific disease systemic lupus erythematosus
(SLE) (lack of association with T−318/C−318 alleles)14 or
rheumatoid arthritis (RA) (association with G49 allele
after stratification for HLA class II alleles).15
Little is known about the functional involvement of
CTLA-4 in human diseases—partly due to the fact that
detection of the CTLA-4 protein on non-stimulated cells
is difficult due to low density of expression. Previous
studies have employed in situ expression, flow cytometry
CTLA4 polymorphisms and gene expression
A Ligers et al
146
Results
CTLA-4 promoter region, exon 1 and exon 4
polymorphisms
Five genotypes were identified for the promoter-exon 1
region and 12 alleles for the dinucleotide repeat in exon
4. Of the 72 individuals typed, none was homozygous for
T−318; nor did we observe any instance of T−318 occurring
on the same chromosome as G49. Precise genotypes
counts for MS patients and controls are shown in Table 1.
Figure 1 Schematic representation of known polymorphic sites
within the CTLA-4 gene (CTLA4): aT/C (thymine/cytosine) dimorphism of the CTLA4 promoter region, located at position –318 from
ATG start codon. bA49/G49 (adenine/guanine) dimorphism of the
leader sequence of exon 1, located at position 49 from the ATG
start codon. c(AT) dinucleotide-repeat sequence, located in the 3′
untranslated region of exon 4. Distance between exon 1 polymorphism and dinucleotide repeat at exon 4 is 5421 bp. The segments
represented are not to scale.
or reverse transcriptase (RT) PCR methods for detection
of CTLA-4 protein or mRNA levels.
Expression of CTLA-4 appears to be selectively
induced in certain pathological conditions in vivo,16 but
the mechanism underlying this induction is unknown.
SLE patients were found to have higher expression of
CTLA-4 on T cells compared with normal controls, but
after cell stimulation, expression levels became similar.17
In patients with RA, increased levels of CTLA-4-positive
T cells were observed in synovial fluids and synovial
membranes; addition of anti-CTLA-4 antibody led to
increased production of TNF-␣ and IL-1␤, suggesting
that CTLA-4 expression plays a down-regulatory role in
rheumatoid articular inflammation.18 Mena et al19 found
no difference in CTLA-4 expression between IFN-␤treated and untreated MS patients.
The complexity of CTLA-4’s role in cell activation was
recently underscored by the identification of a soluble
form of the molecule, generated by alternative splicing of
the primary transcript. In humans, the soluble form has
been shown to be produced by B cells and nonstimulated T cells of both the CD4+ and CD8+ phenotypes; it has been detected in mice and rats as well.20,21
Thus, it appears that the CTLA-4 molecule may be constitutively produced in a soluble form that regulates
immune homeostasis by blocking B7 family receptors
during both early and late stages of the immune
response.
Because CTLA-4 appears to be a negative regulator of
the normal immune response, it is of interest to investigate whether altered expression of CTLA-4 on T cells
may contribute to the impaired immune response seen
in MS.
The aims of this study were as follows: to measure and
compare CTLA-4 mRNA and protein levels in patients
with MS and myasthenia gravis (MG) and in healthy controls; to determine whether three well-characterized
CTLA4 polymorphisms modulate protein and mRNA levels in these groups; and to analyze CTLA-4 protein levels
in different cell populations.
Genes and Immunity
Flow cytometry analysis
Repeated analysis by flow cytometry on unstimulated
cells showed that expression of CTLA-4 protein was low
intracellularly and undetectable on the cell surface.
Therefore, cells used for flow-cytometry analysis were
later subjected to mitogen-induced stimulation with ConA (see Materials and methods). For CTLA-4 protein
expression, no differences were found between MS
patients receiving immunosuppressive therapy and
untreated patients (data not shown); treated and
untreated MS patient groups were therefore combined
for subsequent analysis.
In both patients and controls, the number of cells
expressing CTLA-4 intracellularly was higher than the
number expressing CTLA-4 on the cell surface. When MS
patients were compared to controls, no differences were
found in intracellular or cell-surface CTLA-4 protein
expression in unsorted peripheral blood mononuclear
cells (PBMCs) or in any T-cell subpopulation. In addition,
Table 1 Distribution of CTLA4 genotypes among MS patients and
controls. Allele-specific PCR with simultaneous amplification of
dimorphisms at positions −318 and 49 allowed identification of the
following promoter-exon 1 ‘haplotypes’: C-A (haplotype 1), C-G
(haplotype 2), T-A (haplotype 3); exon 4 dinucleotide-repeat alleles
are designated by their length in base-pairs. No significant differences in the distribution of alleles, ‘haplotypes’ or genotypes
were observed
Promoter-exon 1
1,1
1,2
1,2
1,2
1,2
1,2
1,2
1,2
2,2
2,2
2,2
2,2
2,2
2,2
2,2
2,2
2,2
1,3
1,3
1,3
2,3
2,3
2,3
2,3
Exon 4
(AT)n
Patients
(ntot = 38)
Controls
(ntot = 34)
84/84
84/84
84/102
84/104
120/120
104/124
102/106
102/120
102/102
110/120
84/84
84/102
100/104
102/114
84/110
110/110
102/118
118/118
84/84
84/94
84/84
102/118
102/102
130/130
3
2
8
3
1
1
1
0
5
2
1
2
1
0
0
0
0
1
3
1
1
1
1
0
2
5
7
0
0
0
0
1
6
0
0
0
0
1
1
1
1
1
6
0
0
0
1
1
CTLA4 polymorphisms and gene expression
A Ligers et al
Table 2 Median percentage (and interquartile range) of peripheral
blood mononuclear cells expressing CTLA-4 on the cell surface in
multiple sclerosis (MS) patients and controls stratified for genotype
at promoter and exon 1 dimorphisms and exon 4 dinucleotide
repeat
MS
(n = 38)
Controls
(n = 34)
MS and controls
(n = 72)
Promoter
T/C
C/C
3.5 (2.1 to 6.8)
2.2 (1.8 to 4.5)
4.0 (2.9 to 27.0)a
1.7 (1.4 to 2.5)
4.0 (2.4 to 8.0)e
2.0 (1.4 to 3.0)
Exon 1
A/A
A/G
G/G
3.1 (1.4 to 6.8)
2.3 (1.8 to 3.0)
2.6 (1.8 to 6.0)
5.2 (4.0 to 27.0)b,c
1.7 (1.5 to 3.0)
1.5 (1.1 to 2.0)
4.2 (2.0 to 8.0)f,g
2.1 (1.5 to 3.0)
2.0 (1.2 to 2.9)
Exon 4
84/84
84/X
X/X
2.0 (1.3 to 6.6)
2.9 (2.0 to 5.3)
2.5 (1.8 to 4.0)
4.2 (2.0 to 6.2)d
1.6 (1.5 to 2.3)
1.5 (1.2 to 2.9)
3.1 (1.7 to 6.6)
2.0 (1.5 to 3.7)
2.2 (1.4 to 3.0)
T/C vs C/C, P ⬍ 0.01; bA/A vs A/G, P ⬍ 0.05; cA/A vs G/G,
P ⬍ 0.01; d84/84 vs 84/X, P ⬍ 0.05; eT/C vs C/C, P ⬍ 0.01; fA/A vs
A/G, P ⬍ 0.01; gA/A vs G/G, P ⬍ 0.05.
a
we found no significant differences between MS patients,
MG patients and controls with regard to CTLA-4
mRNA levels.
CTLA-4 polymorphisms and expression of CTLA-4
protein and mRNA
When individuals were stratified by genotype at the promoter and exon 1 dimorphisms, significant differences in
both cell-surface and mRNA expression of CTLA-4 were
observed. Both in the control group and in MS patients
and controls considered together, CTLA-4 cell-surface
expression was significantly increased in individuals carrying T−318, compared to levels in individuals homozygous for C−318, and in A49 homozygotes, compared to
levels in carriers of the A49/G49 or G49/G49 genotypes
(Table 2). Both in MG patients, MS patients and controls
considered together, CTLA-4 mRNA levels were significantly higher in PBMCs from subjects carrying T−318 than
levels in cells from subjects lacking the allele (Table 3).
For analysis of the influence of the exon 4 microsatellite
alleles on CTLA-4 cell-surface expression, we subdivided
individuals into three groups, with respect to the presence or absence of the 84 base-pair (bp) allele. Although
expression was significantly higher in 84bp/84bp controls than in 84bp/Xbp controls, when controls and MS
patients were analyzed together, no significant differences between the 84bp/84bp, 84bp/Xbp and Xbp/Xbp
genotypes were found (Table 2).
CTLA-4 genotypes and CTLA-4 protein expression
Because alleles at position −318 (promoter) and 49 (exon
1) are in strong linkage disequilibrium with one another,
it is relevant to analyze the effect of combined genotypes
on CTLA-4 expression. Indeed, CTLA-4 cell-surface
expression was significantly increased in carriers of T−318
homozygous for A49 (genotype 1/3), compared to levels
in carriers of the remaining genotypes (Figure 2), for all
cells (P = 0.0026), for unsorted CD3+ cells (P = 0.001) and,
to a lesser extent, for CD4+ cells (P = 0.004). For CD8+
cells, the difference was not significant.
147
ANOVA of CTLA-4 polymorphisms and CTLA-4
protein expression
To investigate whether either one of the three polymorphic sites was most important for the observed genotype
effect on CTLA-4 expression, we performed one-way
ANOVA (data not shown) and two-way ANOVA. We
found a positive interaction between the promoter and
exon 1 polymorphisms for CTLA-4 protein expression on
PBNCs. Pair-wise mean difference analysis revealed that
differences in numbers of CTLA-4-expressing cells were
most pronounced when carriers of T−318 homozygous for
A49 (genotype 1/3) were compared to subjects homozygous for C−318 carrying A49/G49 (genotype 1/3) or
G49/G49 (genotype 2/2) (Table 4). There was no interaction between exon 4 alleles and promoter or exon 1
alleles. These results indicate that the T−318/C−318–A49/A49
Figure 2 Median values for cell-surface expression of CTLA-4 on
peripheral blood mononuclear cells (PBMCs), as determined by
flow cytometry, in patients and controls stratified according to
genotype. C/C and T/C refer to the two genotypes observed at
position −318 of the CTLA4 promoter sequence, whereas A/A, A/G
and G/G refer to the three genotypes at position 49 of the first exon
of CTLA4. Controls carrying the combined genotype T/C-A/A
(genotype 1/3) had significantly higher expression of CTLA-4 on
PBMCs than carriers of other genotypes (P = 0.022). No other differences were statistically significant.
Table 3 CTLA-4 mRNA expression as determined by competitive reverse transcriptase PCR on unstimulated peripheral blood mononuclear
cells from patients with multiple sclerosis (MS; n = 60) and myasthenia gravis (MG; n = 29) and controls (n = 26) stratified by genotype at
position −318 in the CTLA4 promoter. Median values (and interquartile ranges) are given, expressed as (CTLA-4 mRNA/␤-actin
mRNA) × 10−3
Genotype
T/C
C/C
MS
MG
Controls
All subjects
0.86 (0.64 to 1.05) (n = 12)
0.8 (0.4 to 1.2) (n = 48)
1.32 (0.84 to 6.45) (n = 4)
0.59 (0.34 to 1.06) (n = 25)
1.74 (0.73 to 2.10) (n = 6)
0.83 (0.47 to 1.23) (n = 20)
0.92 (0.75 to 1.75) (n = 22)
0.78 (0.41 to 1.19) (n = 93)
P = NS
P ⬍ 0.05
P = NS
P ⬍ 0.05
NS, not significant at 5% level.
Genes and Immunity
CTLA4 polymorphisms and gene expression
A Ligers et al
148
Table 4 Analysis of the relative importance for CTLA-4 cell-surface
expression of genotypes for dimorphisms at positions −318 (T/C
and C/C), and 49 (A/A, A/G and G/G) of CTLA4. Two-way
ANOVA matrices (A), as well as pair-wise mean differences (B)
with Bonferroni adjustments (C), are shown. The analysis reveals
not only that the −318 T/C and 49 A/A genotypes are associated
with increased CTLA-4 expression, but also that the simultaneous
presence of both genotypes confers higher expression than each of
the remaining combined genotypes
Groups
F-ratio
P
n
A.
T/C, C/C
A/A, A/G
T/C, C/C and A/A, A/G
4.427
3.228
5.087
0.041
0.079
0.029
70
50
50
Groups
T/C–A/A T/C–A/G C/C–A/A C/C–A/G C/C–G/G
B.
T/C–A/A
T/C–A/G
C/C–A/A
C/C–A/G
C/C–G/G
0.000
−0.507a
−1.621
−1.450
−1.544
0.000
−0.114
0.056
−0.037
0.000
0.171
0.077
0.000
−0.093
0.000
C.
T/C–A/A
T/C–A/G
C/C–A/A
C/C–A/G
C/C–G/G
1.000
0.032b
0.0160
⬍0.0005
⬍0.0005
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
a
Pair-wise mean differences; bP values.
genotype (also expressed as 1/3) is the only genotype
associated with increased expression of CTLA-4, and that
differences in expression are related to the combined
influence of the polymorphisms at positions −318 and 49.
Discussion
We have shown that cell-surface expression of CTLA-4
protein is influenced by common polymorphisms in the
promoter and first-exon sequences. This influence is seen
both in unsorted PBMCs, in unsorted CD3+ cells, and in
the CD4+ T-cell subtype, but not in the CD8+ T-cell subtype. Furthermore, the effect seems to be related to at
least two factors, one associated with position −318 in the
promoter and the other with position 49 of the leader
sequence.
An increase in CTLA-4 mRNA levels was observed for
individuals carrying T−318, thus lending additional support to the notion that CTLA-4 levels are influenced at
the transcriptional stage in a genotype-dependent
fashion. There are at present no reports suggesting that
the position −318 polymorphism affects any known consensus sequence in the regulatory region of the CTLA4
promoter in humans. It has been shown, however, that
the inducible expression of CTLA-4 detected during Tcell activation is controlled by this region in mice.22 The
similarity of CTLA-4 cell-surface expression levels
between MS patients and healthy controls regardless of
genotype demonstrates that the variation in levels of protein expression is associated not with disease but rather
with genotype. Indeed, its has been shown, that CTLA-4
exerts less profound inhibitory effects on proliferation for
T cells carrying G49/G49 genotype rather than for cells
Genes and Immunity
carrying A49/A49 genotype, regardless of the presence of
an autoimmune disease.23
We found no genotype-related difference in intracellular CTLA-4 protein expression. Moreover, the observed
relationship between cell-surface expression of the
CTLA-4 and specific genotypes was confined to unsorted
cells and certain T-cell phenotypes. One explanation for
this would be that mechanisms other than genotype contribute to the regulation of CTLA-4 cell-surface
expression, and that these mechanisms are differentially
operative in various T-cell subpopulations. Indeed, two
recent studies have demonstrated the existence, in mice,
of a subset of regulatory T cells whose immune-suppressive effects are dependent upon signaling via constitutively expressed CTLA-4.24,25
Genotype-conditioned regulation of CTLA-4 cellsurface expression could be explained by the presence of
a functionally important variation in the molecule’s
amino-acid sequence, possibly located in the Y201 motif
of the cytoplasmic tail. This motif has been shown to be
an important intracellular regulator of CTLA-4 protein
levels through its interaction with the clathrin adaptor
complexes AP-1 and AP-2, which mediate, respectively,
degradation and endocytosis of the CTLA-4 protein.26,27
Our results show that individuals carrying T−318
express significantly higher levels of CTLA-4 mRNA,
compared to C−318/C−318 subjects. Thus, differences in
cell-surface expression may simply reflect a situation
whereby newly synthesized CTLA-4 neither remains in
Golgi or post-Golgi compartments, nor is stored in vesicles in the cytoplasm, but rather is transported directly
to the cell surface. In fact, Alegre et al28 have by correlation of protein and mRNA levels demonstrated that
Th2 cell clones express both CTLA-4 mRNA and cellsurface CTLA-4 protein at much higher levels than Th1
clones. These results seem to suggest that differential cellsurface expression of CTLA-4 is, at least in part, regulated
by mRNA abundance.
Data concerning expression of CTLA-4 differed
between, on the one hand, all PBMCs, unsorted CD3+
cells and the CD4+ T-cell subsets, and, on the other, CD8+
T cells. CTLA-4 expression in the first three cell groups
was clearly influenced by the gene’s polymorphic loci,
whereas expression in CD8+ T cells was less genotypedependent. Mena et al19 have reported that, in the absence
of stimulation, CD8+ cells exhibit higher levels of CTLA-4
expression than CD4+ cells in untreated MS patients and
controls, but lower levels in MS patients undergoing
immunosuppressive treatment. In contrast, our results
for CTLA-4 expression in CD8+ cells did not differ greatly
between groups of subjects. Discrepancies between our
results and those of Mena et al19 may reflect the effects
in different T-cell populations of treatment and cell
stimulation on CTLA-4 expression, but it is unknown
what the exact nature of such effects might be.
It has been shown that mice depleted of CD8+ T cells
from birth develop a lymphoproliferative disorder similar in onset and severity to that seen in unmanipulated
CTLA-4−/− mice, whereas lymphoproliferation does not
occur in CTLA-4−/− mice depleted of CD4+ T cells from
birth.29 Moreover, results from experimental autoimmune
encephalomyelitis experiments suggest that CD8+ cells
may modulate the disease by eliminating CD4+ effector
T cells through direct cytotoxicity.30 Thus, as these
CTLA4 polymorphisms and gene expression
A Ligers et al
reports seem to indicate, CTLA-4 may have different
effects in vivo on CD4+ and CD8+ T cells.
Our data on CTLA-4 protein and mRNA levels in
patients and controls seem to indicate that higher mRNA
and protein levels are associated with a particular genotype and not affected with disease. However, this does
not exclude the possibility that different CTLA-4 genotypes may be associated with different clinical forms of
the same disease entity. Only a few studies evaluating the
role of the position −318 polymorphism in autoimmune
diseases have been published, and their results need
clarification.13,14,31,32 One difficulty confronting future
studies will be the low frequency of T−318—an allele that
appears to be associated with protection from autoimmune disease—which may necessitate recruitment of
considerable case-control or familial datasets for
adequately powered analysis.
Our expression data for all cells and for T cells of different phenotypes are consistent with the observation
that T−318 frequency is lower among MS patients than
controls: MS patients are less often carriers of an allele
functionally associated with higher expression of a T cell
down-regulatory protein. It would be difficult, however,
to assess whether the T−318-associated increase of CTLA4 expression in certain T-cell subsets is sufficient to suppress an autoimmune process.
The functional importance of the CTLA-4 gene has
recently been evaluated. Ligation of CTLA-4 inhibits production of IL-2,33 while blockade of CTLA-4 increases
production of IL-2, IFN-␥, IL-3 and TNF-␣ in Th1 cell
clones, and of IL-3, IL-4, IL-5 and IL-10 in Th2 cell
clones.28 Also, it has been shown that signaling via
CTLA-4 can modulate production of TGF-␤34 and suppress production of TNF-␣ and IL-1␤;18 yet, CTLA-4knockout T cells can produce TGF-␤ and IL-10.35 These
findings support the notion of an inhibitory role for
CTLA-4 and at the same time indicate that alternative
down-regulatory immune pathways may exist.
We did not find any significant associations between
dinucleotide-repeat alleles and intracellular or cellsurface protein expression. Previous reports have suggested that autoimmune diseases are associated with
longer CTLA-4 exon 4 microsatellite alleles and have
cited, as an explanation for this, the observation that the
length of AT repeats in the 3′ UTRs of transiently
expressed genes may be inversely related to mRNA stability. However, this observed association between autoimmune diseases and longer microsatellite alleles can be
better explained as a coincidence resulting from linkage
disequilibrium between specific microsatellite, exon 1
and promoter alleles.
Our data show similar levels of responsiveness to
identical mitogen concentrations in PBMCs from MS
patients and controls. Thus, there appears to be no defective expression of the CTLA-4 molecule in MS. CTLA-4
is a T cell activation marker and has inhibitory function
on T cells; increased expression of CTLA-4 on PBMCs
might contribute to the clinical improvement of MS by
mediating anergy or apoptosis of T cells.36 Our results
provide evidence that cell-surface and intracellular
CTLA-4 expression levels are tightly regulated; that CD4+
and CD8+ cells may have different roles in down-regulation of ongoing immune response in MS; that the transcription rate of CTLA-4 gene and expression levels of the
CTLA-4 protein are conditioned by specific genotypes,
one of which, by increasing CTLA-4 expression, may
decrease the risk of MS.
149
Materials and methods
Patients and controls
For flow cytometry analysis, 38 patients were diagnosed
according to the recommendations of Poser et al37 as having clinical definite MS. At the time of blood sampling,
23 patients were receiving various immunosuppressive
drug treatments. Thirty-four control specimens were
obtained either from healthy medical students and hospital employees (n = 10) or from healthy blood donors
(n = 24); control subjects were of the same ethnic background as patients.
To support observations made by flow cytometry, we
analyzed mRNA expression by competitive RT-PCR
using cDNA specimens, collected for previous studies,
from 60 MS patients,38 29 MG patients and 26 healthy
controls,39 all different from the individuals studied for
flow cytometry. At the time of blood sampling 11 MS
patients and 13 MG patients were receiving immunosuppressive drug treatment. Control specimens were
obtained from laboratory employees of the same ethnic
background as patients.
Non-overlapping groups of patients and controls were
analyzed for protein and mRNA levels. MG patients were
included particularly for mRNA analysis.
PCR-SSP for detection of CTLA-4 promoter and exon
1 polymorphisms
Genomic DNA was extracted from 5–10 ml venous blood
by a modified salting-out protocol.40 The CTLA4 promoter and exon 1 polymorphisms were genotyped by
PCR amplification with sequence-specific primers (PCRSSP); as the dimorphisms are separated by a mere 365
bases, it was possible to determine cis-located allele combinations by way of four haplotype-specific PCR amplifications for each subject. Primers were designed according to the published human CTLA-4 complementary
DNA sequence (Genebank accession number M74363).
Primer pair 5′ AGT CTC CAC TTA GTT ATC CAG ATC
CTC and 3′ CAG GGC CAG GTC CTG GT amplified the
C−318 – A49 haplotype. Primer pair 5′ AGT CTC CAC TTA
GTT ATC CAG ATC CTC and 3′ CAG GGC CAG GTC
CTG GC amplified the C−318 – G49 haplotype. Primer pair
5′ AGT CTC CAC TTA GTT ATC CAG ATC CTT and 3′
CAG GGC CAG GTC CTG GT amplified the T−318 – A49
haplotype. Primer pair 5′ AGT CTC CAC TTA GTT ATC
CAG ATC CTT and 3′ CAG GGC CAG GTC CTG GC
amplified the T−318 – G49 haplotype. Optimal PCR conditions consisted of an initial denaturation at 94°C for 2
min; followed by 10 cycles of denaturation at 94°C for
10 s and annealing at 65°C for 1 min; followed by 20
cycles of denaturation at 94°C for 10 s, annealing at 61°C
for 50 s and elongation at 72°C for 30 s. After PCR thermal cycling (GeneAmp PCR System 9600; Perkin–Elmer,
Norwalk, CT, USA), PCR reaction mixtures were loaded
in 3 mm-wide slots in 2% (w/w) ME agarose gels prestained with ethidium bromide (0.5 ␮g/ml gel) and were
run for 25–30 min at 7–8 V/cm in 0.5 × TBE buffer
(89 mM Tris base/89 mM boric acid/2 mM EDTA, ph
8.0) and gels were examined under UV illumination.
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150
Microsatellite genotyping
Individuals whose cells were to be analyzed by flow cytometry were genotyped for the microsatellite marker
located in exon 4 (CTLA-4; http://cedar.genetics.soton.
ac.uk/pub). Primers were synthesized by Scandinavian
Gene Synthesis (Köping, Sweden), and the forward
primer pair was labeled with 5′-HEX phosphoramidites.
Primer pair sequences from 5′ to 3′ were HEX-GT GAT
GCT AAA GGT TGT ATT GC and AAA ACA TAC GTG
GCT CTA TGC AC. Optimal PCR conditions consisted
of 35 cycles of denaturation at 95°C for 1 min, annealing
at 55°C for 40 s and elongation at 72°C for 1 min. Genotyping was carried out on an Applied Biosystems 377
DNA automated sequencer using the semiautomated
GeneScan/Genotyper software.41 For each sample, 1.5 ␮l
of diluted PCR product was combined with 0.5 ␮l of
internal size standard (GeneScan-350, PE Biosystems).
The accuracy and reproducibility of automated sizing of
fragments were confirmed by analysis in duplicate of
randomly chosen samples.
Competitive RT-PCR
mRNA levels of CTLA-4 were quantified by a sensitive
competitive RT-PCR method described by Huang et al38
The main principles for the method are the following: by
combining varying amounts of target cDNA with a fixed
amount of internal standard (double-stranded DNA for
the same target gene), a linear standard curve is obtained;
test samples are then co-amplified with the same amount
of internal standard, and their amounts after amplification are calculated based on the quotient of the area-
under-the-curve for target cDNA divided by that for the
internal standard using the linear regression formula
from the standard curve. To compensate for relative differences between samples, for the variable integrity of the
individual RNA samples and for variations in reverse
transcription, ␤-actin cDNA was amplified and quantified for every test sample. PCR products were detected
using an Applied Biosystems 377 DNA sequencer
equipped with the GeneScan 2.02 software, and the
results were analyzed using the Genotyper 1.1 software
(PE Biosystems).
Immunostaining and flow cytometry
PBMCs were obtained from heparinized blood by a standard Ficoll-hypaque separation method. Cells were
stimulated with Con-A (5 mg/ml) for 72 hrs. Fifty ␮l of
cell suspension (2 × 105 cells) was labeled with monoclonal antibodies (mAbs) conjugated with fluorescein isothiocyanate (FITC, green fluorescence), phycoerythrin
(PE, orange fluorescence) or peridinin chlorophyll protein (PerCP, red fluorescence). All mAbs were titrated in
preliminary experiments. Samples were incubated with
mAb for 30 min at 4°C in the dark. The cells were washed
twice with phosphate-buffered saline (PBS) containing
1% bovine serum albumin (BSA; Sigma, St Louis, MO,
USA) and centrifuged at 2200 g for 10 min.
To detect intracellular CTLA-4, cells were fixed with
4% paraformaldehyde, permeabilized with 0.1% saponin
and stained with anti-CTLA-4 mAb intracellularly. Cells
were washed twice with PBS containing 0.1% saponin,
Figure 3 Fixed and permeabilised cells were preincubated with purified anti-CTLA-4 mAb (5 ␮/106 for 30 min at 4°C). After incubation
an anti-CTLA-4 FITC-labelled mAb was added to the cell suspension. Left part of the figures shows the percentages of CD3+ cells expressing
CTLA-4 at the cell surface and CD3+ cells expressing CTLA-4 intracellularly. Right part of the figure shows the result of blocking experiment.
Genes and Immunity
CTLA4 polymorphisms and gene expression
A Ligers et al
resuspended in 250 ␮l of 1% BSA/PBS and analyzed in
a Becton Dickinson FACScan flow cytometer.
Data analysis
Flow cytometry data were processed using CellQuest
software (Becton Dickinson). Combinations of anti-CD3,
anti-CD4, anti-CD8 and anti-CTLA-4 mAbs were used to
discriminate CTLA-4 positive T cells. Ten thousand
events with light-scatter characteristics of mononuclear
cells were saved for flow cytometry analysis with five
parameters (forward scatter, reflecting cell size; side scatter, reflecting granularity; and intensity of FITC, PE and
PerCP fluorescence). To define the precise gate, background staining (ie, the number of cells with fluorescence
intensity over the defined level when stained with irrelevant mouse immunoglobulin G) was subtracted from
each specific staining. A blocking experiment with
unconjugated anti-CTLA-4 mAb was preformed to confirm staining specificity (Figure 3). Percent values
presented are the fractions of the cell populations that
stained for CTLA-4, eg, number of CD3+CTLA-4+ cells
divided by the total number of CD3+ cells.
Statistical analysis
The Mann–Whitney U test was used to compare percentages of CTLA-4+ PBMC and CTLA-4 mRNA levels. Oneand two-way ANOVA and pair-wise mean differences
were calculated from square-root values. The group carrying G49/G49 in exon 1 was excluded from two-way
ANOVA analysis due to the absence in that group of subjects carrying C−318/T−318 or 84/84 in exon 4. Statistical
calculations were performed using the Systat 9 program
(SPSS Science, SPSS Inc, Chicago, IL, USA).
Acknowledgments
We thank Dr D Svensson and Dr J Krikis for statistical
advice.
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