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Evidence for Antigen-Driven T-Cell Response
in Unstable Angina
Giuseppina Caligiuri, MD, PhD; Gabrielle Paulsson, PhD; Antonino Nicoletti, PhD;
Attilio Maseri, MD; Göran K. Hansson, MD, PhD
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Background—Activation of T cells and macrophages has been associated with unstable angina (UA), but whether this
reflects specific immune responses remains unclear.
Methods and Results—We analyzed the repertoire and the length of complementarity-determining region 3 of the T-cell
receptor (TCR) ␤-chain variable (BV) gene segments of activated lymphocytes in 23 patients with UA, 13 patients with
chronic stable angina (CSA), and 6 normal control subjects. We also tested the proliferation of systemic T cells in
response to autologous coronary plaque proteins, oxidized LDL, and Chlamydia pneumoniae as candidate antigens, in
vitro. The activated T cell–TCRBV repertoire was perturbed in 13 (57%) of 23 UA patients versus 3 (23%) of 13 CSA
patients (P⫽0.016) and was restricted to 6 (28%) of 21 expanded TCRBV families; all were significantly higher in UA
than in CSA patients. At least one monotypic or oligotypic activated TCRBV population was found in 15 (65%) of 23
UA patients and in 3 (23%) of 13 CSA patients (P⬍0.001). Finally, T cells from UA patients, but not from CSA patients
or normal control subjects, proliferated in response to autologous proteins from coronary culprit lesions and/or to
oxidized LDL.
Conclusions—Our findings suggest that the T-cell response observed in UA patients is antigen-driven and directed to
antigens contained in the culprit coronary atherosclerotic plaques. (Circulation. 2000;102:1114-1119.)
Key Words: angina 䡲 ischemia 䡲 prognosis 䡲 lymphocytes 䡲 antigens
A
commonly used as monotypic markers of T-cell populations.
We have analyzed the TCRBV antigen and mRNA repertoire
in activated T cells of patients with UA and, as control
conditions, in patients with CSA and healthy (normal control
[NC]) subjects and found that specific TCRBV types are
expanded in patients with UA. Cellular immunologic studies
have suggested that these expansions may be due to responses
to specific antigens present in atherosclerotic culprit plaques.
growing body of evidence suggests that unstable angina
(UA) is associated with local1 and systemic2,3 activation
of the immune system. Atherosclerotic plaques contain large
numbers of activated T cells, suggesting that immune mechanisms are important factors in the pathogenesis of the
atherosclerotic background.4 – 6 Indeed, inflammatory cells
infiltrate nearly all plaques,6,7 and culprit lesions of infarcted
hearts appear to be particularly enriched in activated T
lymphocytes.1 This suggests that acute T-cell activation may
play a role in plaque instability and acute clinical manifestations of coronary atherosclerosis.8 Although circulating immune markers are also chronically elevated in patients with
chronic stable angina (CSA),9 a transient burst of T-cell
activation can be detected only in patients with UA,2,3
suggesting that immune factors might precipitate plaque
complications, such as thrombus formation and vasoconstriction at the site of the culprit lesions.8 However, it has not yet
been established whether the nature of the systemic immune
response observed in UA is specific.
Antigen-driven T-cell responses are characterized by a
restricted repertoire of the highly polymorphic T-cell antigen
receptor (TCR).10 This receptor has variable portions in both
its ␣ and ␤ chains. The latter, called BV segments, are
Methods
Patients
Given the high variability of the TCRBV repertoire in the normal
population, study patients were carefully selected to form very
homogeneous groups. All patients with evidence of recent infectious
disease, erythrocyte sedimentation rate ⬎20 mm/h, fever, immunosuppressive drug therapy, immunologic disorders, known or suspected neoplastic diseases, congestive heart failure, valvular heart
disease, left bundle branch block, evidence of left ventricular
aneurysm, recent (⬍3 months) major trauma, surgery, myocardial
infarction, or coronary revascularization (coronary angioplasty or
bypass surgery) were excluded from the study. As a consequence of
such restricted selection, the sample size was relatively small;
therefore, the present findings should be considered as preliminary
in nature.
Received December 17, 1999; revision received April 6, 2000; accepted April 10, 2000.
From the Center for Molecular Medicine (G.C., G.P., A.N., G.K.H.), Karolinska Institute, Stockholm, Sweden; INSERM U460 (G.C.), Hôpital Bichat,
and INSERM U430 (A.N.), Hôpital Broussais, Paris, France; and the Cardiology Institute (A.M.), Catholic University, Rome, Italy.
Correspondence to Dr Giuseppina Caligiuri, INSERM U460, Hôpital Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail
[email protected]
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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Caligiuri et al
UA Patients
Only patients with ECG-documented new onset (⬍2 days before
admission) of severe UA11 and at least 1 coronary stenosis detected
at angiography (⬎75% reduction of lumen diameter) were admitted
to the study (n⫽23). All patients had experienced at least 2 episodes
of angina at rest or 1 episode lasting ⬎20 minutes during the last 24
hours, accompanied by transient ischemic ST-segment changes and
no detectable rise in creatine kinase-MB levels or troponin T levels
(to exclude micronecrosis, only patients with troponin T levels ⬍0.2
␮g/L were included in the study). Full medical therapy, including
␤-blockers and/or calcium antagonists, low-dose aspirin, and continuous intravenous infusion of nitrates and heparin, was introduced
on admission, and continuous ECG telemetry monitoring was applied to all patients during their stay in our Coronary Care Unit. UA
patients were divided into 2 subgroups 2 days after hospitalization:
patients responding to full medical therapy (resolving UA, n⫽13)
and patients with a more severe UA and persistence of ischemic
episodes at rest after 48 hours of full medical therapy (refractory UA,
n⫽10).
CSA Patients
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As controls for anginal instability, we selected 13 patients with
exclusively effort-related angina, stable for at least 6 months, with a
positive exercise stress test and at least 1 coronary stenosis detected
at angiography (⬎75% reduction of lumen diameter). All patients
were on low-dose aspirin and various combinations of nitrates,
␤-blockers, and/or calcium antagonists.
NC Subjects
Blood samples from 6 age-matched healthy subjects (normal ECG
and echocardiogram and no evidence of atherosclerosis by echography of the carotid arteries) were used as nonatherosclerotic controls.
Blood samples (10 mL of peripheral venous blood) were drawn in
fasting conditions, between 8:00 and 10:00 AM and within 24 hours
of hospital admission. All patients gave their written informed
consent to participate in the study, which was approved by the Ethics
Committee of our institution.
FACS Analysis of Circulating T Cells
Whole blood cells were lightly fixed, and red blood cells were lysed
and washed twice before immunostaining. Cell suspensions were
incubated with mouse anti-human CD3 or with 21 different murine
monoclonal antibodies against the human TCRBV (AV2.3, BV3,
BV5.1, BV5.2⫹5.3, BV5.3, BV6.7, BV8.1, and BV12.1, which were
generously provided by B. Olsson, MTC, Karolinska Institute,
Stockholm, Sweden, and BV2, BV9, BV11, BV13.1, BV13.6,
BV14, BV16.7, BV17, BV18, BV20, BV21.3, BV22, and BV23,
which were purchased from Immunotech), followed by incubation
with fluorescein isothiocyanate– conjugated F(ab⬘)2 fragments of
rabbit anti-mouse IgG (DAKO). Activated T cells were finally
stained by incubation with phycoerythrin-conjugated mouse anti–
HLA-DR antibodies (Immunotech). Cells (n⫽20 000) in the lymphocyte light scatter gate were acquired from each sample in a
fluorescence-activated cell sorting (FACS) flow cytometer (FACSCalibur, Becton-Dickinson). For analysis, the percentage of CD3⫹/
DR⫹ or TCRBV⫹/DR⫹ double-positive cells was calculated in the
lymphocyte gate.
TCRBV-CDR3 Fragment Length Analysis of
Enriched Blood DRⴙ Cells
Each T cell expresses a TCR of a single antigenic specificity;
therefore, only a small subset of T cells is activated by any particular
antigen (clonal selection). However, several antigen-specific T cells
may share the same BV epitope on their TCRs. Therefore, it is also
necessary to evaluate whether the expanded TCRBV populations are
oligotypic or monotypic. This can be determined by analyzing the
length of complementarity-determining region 3 (CDR3), which is
highly polymorphic because of the addition and deletion of codons
during somatic rearrangement of the TCRBV gene. To restrict the
analysis to the activated T cells, we first positively selected DR⫹
cells (including activated T cells, B cells, monocytes, and NK cells)
T-Cell Response in Unstable Angina
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from fresh whole blood by using immunomagnetic beads conjugated
with monoclonal antibodies (mouse IgM) recognizing HLA-DR
epitopes on human leukocytes (Dynabeads M-450, Dynal AS).
“Non–T-cell” negative controls were obtained by a previous negative
selection with the use of anti-CD3– coated beads, followed by the
DR⫹ positive cell selection. After repeated washing steps (recovery
⬎98% purified DR⫹ cells), DR⫹ cells were lysed, and their mRNA
was extracted (mRNA Direct Kit, Dynal AS) and reversetranscribed. A primer pair in the constant region of the TCR ␤-chain
gene served to check the effective presence of cDNA from T cells in
each sample. TCRBV-CDR3 length analysis was performed on such
cDNA preparations by following the protocol originally described by
Pannetier et al.12 Briefly, TCRBV-CDR3 regions were amplified by
using 1 fluorescent 3⬘ primer in the ␤-chain constant region and 15
different 5⬘ primers in the BV region (primers 2, 3, 5.1, 5.2, 6, 8, 9,
11, 12, 13, 14, 17, 18, 20, and 21, which were purchased from
Clontech Laboratories Inc). Polymerase chain reaction (PCR) was
performed in a total volume of 40 ␮L (1.5 mmol/L Mg2⫹). The first
cycle was at 95°C for 3 minutes, 57°C for 40 seconds, and 72°C for
1 minute; then there were 34 cycles at 95°C for 40 seconds, 57°C for
40 seconds, and 72°C for 1 minute; and the last step was at 72° for
3 minutes. After electrophoresis on an automated sequencer (ABI
377) and subsequent computer analysis (Genotyper, ABI/PerkinElmer), the differently sized peaks were separated, and their CDR3
size in codons was calculated.
Antigen-Specific T-Cell Activation
T-cell proliferation in vitro in response to candidate antigens was
analyzed in a subset of six UA and 6 CSA patients undergoing
atherectomy during the study as well as in 6 healthy controls. T-cell
proliferative responses were assessed by using peripheral blood
mononuclear cells (including T cells as well as antigen-presenting
cells, such as monocytes/macrophages, dendritic cells, and B cells)
in culture. Antigenic preparations were (1) soluble proteins from
each individual atherectomy homogenate (plaque protein at 10, 50,
and 100 ␮g/mL) or pooled together for culture with peripheral blood
mononuclear cells from NC subjects, (2) Chlamydia pneumoniae
outer membrane protein (OMP) complexes OMP-2 and MOMP13 (1,
5, and 10 ␮g/mL), and (3) copper-oxidized LDL14 (oxLDL at 1, 5,
and 10 ␮g/mL). Frozen sterile atherectomy specimens were cryohomogenized and resuspended in culture medium. After centrifugation
(14 000 rpm, 4°C, 30 minutes), the supernatants were collected and
assayed for protein content (BCA method). Human serum albumin
(10, 50 and 100 ␮g/mL) and phytohemagglutinin (1, 5, and 10
␮g/mL) served as negative and positive controls, respectively. Each
antigen was added after 2 days of culture, and proliferation was
assessed after 4 more days of culture and after addition of [3H]thymidine 18 hours before harvesting.
Statistical Analysis
Data are expressed as mean⫾SEM. To determine whether the
TCRBV repertoire of activated T cells was able to discriminate
between groups, FACS data (percentages of TCRBV⫹/DR⫹ cells)
were submitted to principal component analysis (PCA)15 by use of
IGOR software (WaveMetrics, Inc). PCA is a descriptive statistical
method that permits the simultaneous analysis of many parameters
for different groups of patients. The number of numerical variables
to be analyzed is determined by the number of subjects (in our case,
n⫽42) multiplied by the number of parameters (in our case, 21
TCRBV types). Such analysis would take into account 42⫻21⫽882
numerical variables. The aim of PCA is to obtain a representation in
a fewer-dimension space compared with the initial number of
dimensions (number of variables). For this purpose, the PCA finds
those associations of variables (called factors) that best characterize
the differences between groups. A factor is a linear combination of
related variables (such as the different TCRBV⫹ types within the
activated lymphocyte pool) that can take the place of the original
variables in further analysis. The structure of the factors (the
variables represented by each factor) is the most relevant information
that results from PCA.
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Circulation
September 5, 2000
TABLE 1. Percentages of TCRBVⴙ/DRⴙ Cells in UA Patients, CSA Patients,
and NC Subjects
TCRBV
UA
P*
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NC
CSA
a.3
0.29⫾0.04
0.63⫾0.20
0.80⫾0.11
2
0.28⫾0.07
3.89⫾1.57
2.89⫾0.61
3
0.08⫾0.01
0.28⫾0.06
2.09⫾0.66†
5.1
0.15⫾0.02
0.35⫾0.12
1.29⫾0.31†‡
0.0057
5.2⫹5.3
0.08⫾0.01
0.26⫾0.07
0.90⫾0.02†‡
0.0005
5.3
0.25⫾0.05
0.28⫾0.07
1.40⫾0.46
0.0065
6.7
0.59⫾0.19
1.36⫾0.57
1.11⫾0.34
0.7845 (NS)
8.1
0.15⫾0.03
0.85⫾0.39
0.74⫾0.11
0.0019
9
0.12⫾0.03
0.47⫾0.13
1.04⫾0.24‡
0.0021
11
0.18⫾0.04
0.53⫾0.16
0.92⫾0.27
0.0398
12.1
0.19⫾0.05
0.81⫾0.25
1.28⫾0.28‡
0.0172
13.1
0.29⫾0.07
0.56⫾0.18
1.07⫾0.36
0.2412 (NS)
13.6
0.19⫾0.06
0.39⫾0.13
1.16⫾0.34
0.0019
14
0.15⫾0.05
0.41⫾0.16
1.05⫾0.24‡
0.0018
16.1
0.34⫾0.09
0.66⫾0.10
1.78⫾0.69
0.047
17
0.26⫾0.13
0.42⫾0.13
1.21⫾0.34
0.0059
0.0071
0.0053
⬍0.0001
18
0.19⫾0.03
0.76⫾0.19
1.38⫾0.40
0.001
20
0.17⫾0.04
0.69⫾0.20
1.86⫾0.52
0.0013
21.3
0.22⫾0.10
0.78⫾0.22
0.88⫾0.16
0.0925 (NS)
22
0.10⫾0.03
0.52⫾0.13
0.68⫾0.16
0.0179
23
3.44⫾0.23
4.88⫾1.04
7.20⫾1.06
0.1277 (NS)
*Kruskal-Wallis test; †significantly different from CSA (Fisher’s PLSD); ‡significantly different from
NC (Fisher’s protected least significant difference).
Differences between groups were analyzed by nonparametric
statistical tests: Kruskal-Wallis and Fisher post hoc test for analysis
among the 3 main groups (UA, CSA, and NC) and Mann-Whitney U
test for analysis of the 2 UA subgroups (resolving and refractory
UA). StatView 4.1 software (Abacus Concept Inc) was used.
Differences between groups were considered significant at P⬍0.05.
Thirteen (57%) of 23 UA patients showed a perturbed
TCRBV⫹/DR⫹ repertoire, which was significantly different
from the one observed in NC subjects (P⬍0.05, Figure 1).
Conversely, only 3 (23%) of 13 CSA patients showed a
Results
The percentage of CD3⫹/DR⫹ cells in the FACS-gated
lymphocyte population was significantly higher in UA patients (7.8⫾1.1%, P⬍0.001) than in CSA patients
(3.8⫾0.3%) and healthy controls (2⫾0.1%). In the UA
subgroup analysis, refractory UA patients showed higher
levels of this marker compared with resolving UA patients
(9.6⫾2.2% versus 6.4⫾1%, respectively; P⬍0.05).
Activated T Cells in UA Patients Originate From
Expansion of Restricted TCRBV Families
PCA found 8 factors that could discriminate between the 3
groups (UA, CSA, and NC). The first 2 of them (factors 1 and
2) were able to explain ⬎60% of the total variance. Therefore, factor 1 and factor 2 represented the 2 most discriminating TCRBV⫹/DR⫹ repertoires among our study populations. Factor 1 was formed by the relative proportions of
TCRBV3 and TCRBV17, whereas factor 2 was composed of
these 2 isotypes together with TCRBV5.1 and TCRBV20. As
shown in Table 1, these TCRBV isotypes were overrepresented on DR⫹ cells of UA patients. Although other TCRBV
isotypes were also increased in UA patients, they did not
explain any significant proportion of the total variance.
Figure 1. Activated T cells show restricted TCRBV repertoire in
UA patients. Factor 1⫻factor 2 scatterplot from PCA on FACS
data (percentage of 21 different DR⫹/TCRBV⫹ cells) is shown.
Each individual is represented by a symbol. Discriminating
TCRBV types within factor 1 are BV3 and BV17, whereas factor
2 includes these 2 isotypes, together with BV5.1 and BV20.
These 2 factors could clearly discriminate UA patients from CSA
patients and NC subjects (P⬍0.05). Conversely, most patients
with CSA were clustered in the same areas as NC subjects
(P⫽0.26 [not significant]).
Caligiuri et al
T-Cell Response in Unstable Angina
1117
TABLE 2. Percentages of TCRBVⴙ/DRⴙ Cells in Resolving and
Refractory UA Subgroups
Resolving UA
Refractory UA
P*
a2.3
0.79⫾0.17
0.83⫾0.17
0.6418
2
1.90⫾0.35
4.20⫾1.24
0.2148
3
1.31⫾0.40
3.10⫾1.40
0.0721
5.1
1.34⫾0.45
1.23⫾0.45
0.8768
5.2⫹5.3
0.95⫾0.30
0.85⫾0.37
0.8041
5.3
1.42⫾0.53
1.39⫾0.83
0.5767
6.7
1.18⫾0.56
1.02⫾0.39
0.3685
8.1
0.66⫾0.14
0.85⫾0.18
0.4568
9
0.87⫾0.26
1.61⫾0.45
0.5351
11
1.08⫾0.44
0.70⫾0.26
0.5981
12.1
1.42⫾0.27
1.09⫾0.56
0.0628
13.1
1.05⫾0.50
1.08⫾0.54
0.3211
13.6
1.17⫾0.50
1.16⫾0.48
0.9259
14
0.85⫾0.23
1.31⫾0.47
0.5149
16.1
1.28⫾0.28
2.43⫾1.56
0.6869
17
0.86⫾0.19
1.66⫾0.75
0.9753
18
1.02⫾0.21
1.85⫾0.88
0.6642
20
2.01⫾0.68
1.65⫾0.86
0.1366
21.3
0.88⫾0.14
0.89⫾0.33
0.5351
22
0.55⫾0.13
0.84⫾0.34
0.8041
23
5.87⫾0.88
8.92⫾2.08
0.3853
TCRBV
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*Mann-Whitney U test. TCRBV 3 tended to be higher in refractory UA,
whereas TCRBV 12.1 tended to be higher in resolving UA.
perturbed TCRBV⫹/DR⫹ repertoire (P⬍0.05 versus UA patients), whereas the remaining 10 patients (77%) had a
conserved normal TCRBV⫹/DR⫹ repertoire (overlapping the
one observed in the NC subjects, Figure 1). Comparison of
the percentage of TCRBV⫹/DR⫹ cells between the UA, CSA,
and NC groups revealed that the proportion of 6 DR⫹/
TCRBV families was significantly increased in UA patients
compared with CSA patients and/or NC subjects (Table 1).
Although expanded TCRBV families were similar in the 2
UA subgroups, there was a tendency for a higher percentage
of DR⫹/TCRBV3⫹ and a lower DR⫹/TCRBV12.1⫹ in refractory compared with resolving UA patients (Table 2).
UA Is Associated With Mono-Oligotypic
Expansion of Activated T Cells
At least one monotypic or oligotypic TCRBV-CDR3 peak
(Figure 2) was detected in 15 of 23 UA patients versus 3 of
13 CSA patients (P⬍0.05). UA patients, but not CSA patients
or NC subjects, showed monotypic expansions of BV3, BV5,
BV12, BV17, and BV20 (all with P⬍0.01). Conversely,
oligotypic expansions of BV9, BV14, and BV18 were found
in 1, 2, and 3 patients with CSA, respectively, but not in UA
patients or NC subjects. Table 3 illustrates the differences
between UA and CSA patients in mono-oligotypic TCRBV
expansions. At 3 months, 8 (32%) of the 25 total TCRBVCDR3 skewed peaks were still detectable in UA patients
(BV3 in 3 [43%] of 7, BV5 in 3 [100%] of 3, and BV17 in 2
[33%] of 6); the remaining 17 (68%) of 25 TCRBV-CDR3
peaks detected on admission in UA patients were no longer
Figure 2. Example of DR⫹/TCRBV⫹ analysis by FACS and
reverse transcription PCR. Representative examples of FACS
and reverse transcription PCR analysis of DR⫹ T-cell repertoire
in patients with UA are shown. Left, FACS dot plot in fluorescent channel within lymphocyte gate. The percentage of DR⫹/
TCRBV3⫹ cells is displayed at top right of each plot. Right,
CDR3 length analysis of TCRBV3 mRNA from DR⫹-enriched
cells. Top, Representative example of monotypic expansion of
TCRBV3 in UA patients. Center, Representative example of
polyclonal expansion of TCRBV3 in UA patients. Bottom, Negative control (CD3-negative selected cells).
detected after 3 months (data not shown). In CSA patients, all
the TCRBV-CDR3 skewed peaks observed on admission
were still detectable at 3 months (data not shown).
T Cells From Patients With Refractory UA
Proliferate in Response to Plaque Antigens and
to OxLDL
The proliferative response to proteins contained in the
atherectomy homogenates was significantly higher in UA
patients than in healthy control subjects, and it tended to be
higher in UA patients than in CSA patients or NC subjects
(P⬍0.05, Figure 3). The proliferative T-cell response to
oxLDL at high doses was significantly higher in refractory
UA patients than in resolving UA patients, CSA patients, or
NC subjects (P⬍0.05, Figure 3). In contrast, the 2 patient
groups exhibited a similar proliferative response to C pneumoniae antigens (MOMP and OMP-2, Figure 3).
Discussion
An acute inflammatory state is statistically associated with a
poor clinical outcome in UA.16,17 The inflammatory response
observed in UA is not a mere consequence of plaque
disruption, thrombosis, or myocardial ischemia,18 –20 suggesting that inflammatory triggers may actually play an independent role in the pathogenesis of UA.21 The inflammatory
triggers may be of an antigenic nature, because activation of
the specific immunity is also detectable in UA and correlated
to the clinical outcome.2,3 Thus, antigen-specific immune
activation may, together with immunologically nonspecific
inflammatory stimuli, determine the outcome of acute coronary syndromes. As recently pointed out, this resembles the
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Circulation
September 5, 2000
TABLE 3.
and CSA
Monotypic and Oligotypic Expansions of DRⴙ TCRBV Families in UA
UA
CSA
Gene
Total
Mono
Oligo
Total
Mono
Oligo
P*
BV3
7
4
3
0
0
0
⬍0.0001
BV5
3
1
2
0
0
0
䡠䡠䡠
BV9
2
0
2
1
0
1
䡠䡠䡠
BV12
1
1
0
0
0
0
䡠䡠䡠
BV14
2
0
2
2
1
1
BV17
6
4
2
0
0
0
䡠䡠䡠
⬍0.0001
BV18
0
0
0
3
2
1
BV21
4
2
2
0
0
0
䡠䡠䡠
⬍0.001
Total indicates number of monotypic and oligotypic skewed CDR3 peaks; Mono, number of
monotypic CDR3 peaks; and Oligo, number of oligotypic CDR3 peaks.
*␹2 test.
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situation in rheumatoid arthritis, a disease that shares many
pathogenetic features with atherosclerotic vascular disease.22
In the present study, we found that the antigen receptor
repertoire of the activated T cells is skewed in 57% of patients
with UA versus 23% of patients with CSA, supporting the
hypothesis that an antigen-driven immune response may play
a role in the pathogenesis of clinical instability of angina
pectoris.
The antigenic triggers might be located at the site of the
culprit lesion, because a specific proliferative response to
proteins contained in the atherectomy specimens of UA
patients but not CSA patients or NC subjects was detected.
The composition of unstable plaques is complex, and the
large number of potential antigenic epitopes may explain the
Figure 3. Proliferative T-cell response to atherosclerosis candidate antigens. No differences were found for the proliferative
response to human serum albumin or phytohemagglutinin in the
3 groups (P⫽NS, data not shown). Patients with UA showed a
marked proliferative response to plaque proteins (*P⬍0.05 vs
CSA and NC groups) and to oxLDL at high doses (10 ␮g/mL,
*P⬍0.05 vs CSA and NC groups). Conversely, the proliferative
response to OMP-2 and MOMP was statistically different in UA
patients compared with NC subjects (P⬍0.05 at 5 ␮g/mL) but
not compared with CSA patients (P⫽NS).
variability in the TCR selection of activated T cells in
different patients. Indeed, it has been demonstrated that the
plaque-infiltrating T cells are polyclonal, which may reflect
the variable antigenic background in the atherosclerotic
lesions.23,24 However, during the acute phases of plaque
instability, some plaque antigens may elicit a transient,
specific, systemic immune response. Indeed, we found that
⬍5 (23%) of 21 TCRBV types are significantly expanded in
each UA patient, indicating that such dynamic T-cell activation is not polyclonal. More important, expanded activated
TCRBV⫹ cells showed monotypic or oligotypic CDR3 peaks
in 65% of UA patients. This clearly suggests activation by a
limited number of antigenic epitopes. In particular, monotypic expansions of TCRBV3 and TCRBV17 were found in 8
patients with UA (4 and 4, respectively), but none were
observed in CSA patients or NC control subjects. Interestingly, 17 (68%) of 25 total monotypic or oligotypic TCRBVCDR3 peaks were no longer detectable after 3 months from
the waning of symptoms in UA patients, suggesting that
antigen-specific T-cell populations were transiently activated
and confined to the clinically unstable phases of angina.
Conversely, expanded DR⫹/TCRBV⫹ populations were polyclonal in the majority (77%) of CSA patients, thus confirming
the greater antigenic variability of the T-cell response related
to the chronic atherosclerotic background. Interestingly
enough, the skewed TCRBV genes in CSA patients were
different from those in UA patients, suggesting that different
antigenic epitopes may be related to the chronic T-cell
activation observed in CSA patients.
Because complicated atherosclerotic plaques contain both
oxLDL and C pneumoniae, we tested the hypothesis that the
T-cell response observed in UA might be targeted to these 2
plaque antigenic proteins among the large number of potential plaque antigens.
Previous studies have shown that patients with UA present
elevated levels of oxLDL in their blood,25 and oxLDLspecific T-cell clones have been isolated from complicated
atherosclerotic plaques.14 In the present study, the proliferative response of T cells to oxLDL was significantly increased
in patients with UA compared with patients with CSA,
although data showed considerable overlap. Interestingly,
Caligiuri et al
patients with a more severe UA, refractory to the full medical
therapy, showed a marked proliferative response to oxLDL
compared with the response in patients with a more favorable
outcome (resolving UA) or with control subjects. However, a
positive response to oxLDL was usually associated with a
positive response to the other antigenic preparations also,
suggesting that plaque antigens other than oxLDL may also
be involved in the T-cell response observed in UA.
A proliferative response to C pneumoniae antigens was
detectable in stable and unstable patients, possibly reflecting
a role for a specific response to this ubiquitous pathogen in
the pathogenesis and/or the progression of atherosclerosis.
This is in line with the results of an increasing number of
serological studies.26 –30 However, no differences could be
observed between UA and CSA patients. Thus, our results do
not support a role for C pneumoniae in the pathogenesis of
plaque instability, although we cannot rule it out because of
the relatively small sample population of the present study.
Downloaded from http://circ.ahajournals.org/ by guest on August 10, 2017
Acknowledgments
This study was supported by grants from the Swedish Medical
Research Council (project 6816), the Heart & Lung and the
Blancefor-Ludovisi Foundations, and the Italian CNR. Dr Caligiuri
is the recipient of a European Society of Cardiology grant. We thank
Mary Osborne-Pellegrin for help in editing the manuscript, Berit
Olsson for providing some of the monoclonal antibodies and for help
with the FACS setting, Kerstin Höglund for collecting patient
samples and file records, Istvan Herzfeld for providing the atherectomy specimens, Ingrid Törnberg for her help in cell culture and
reverse transcription PCR, Martin Rottenberg for providing the C
pneumoniae antigens, and Anders Hamsten for providing
human LDL.
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Evidence for Antigen-Driven T-Cell Response in Unstable Angina
Giuseppina Caligiuri, Gabrielle Paulsson, Antonino Nicoletti, Attilio Maseri and Göran K.
Hansson
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Circulation. 2000;102:1114-1119
doi: 10.1161/01.CIR.102.10.1114
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