NS1 Specific CD8+ T-Cells with Effector Function and
TRBV11 Dominance in a Patient with Parvovirus B19
Associated Inflammatory Cardiomyopathy
Mathias Streitz1., Michel Noutsias2.*, Rudolf Volkmer1, Maria Rohde2, Gordon Brestrich1, Andrea
Block2, Katrin Klippert1, Katja Kotsch1, Bernhard Ay1, Michael Hummel3, Uwe Kühl2, Dirk Lassner4,
Heinz-Peter Schultheiss2, Hans-Dieter Volk1, Florian Kern1,5
1 Institute of Medical Immunology, Charité–Universitätsmedizin Berlin, Berlin, Germany, 2 Department of Cardiology and Pneumonology, Charité–Universitätsmedizin
Berlin, Berlin, Germany, 3 Institute of Pathology, Charité–Universitätsmedizin Berlin, Berlin, Germany, 4 Institute of Cardiac Diagnostic and Therapy, Berlin, Germany,
5 Division of Medicine, Brighton and Sussex Medical School, Brighton, United Kingdom
Abstract
Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients
with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the
role of B19V specific T-cells in this entity.
Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids),
peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting
B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in
PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8+
T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8+ cells) and QSALKLAIYK (10%)
and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNc secretionassay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell
receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNc, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4,
GATA3) and regulatory T-cells (FoxP3) was low.
Conclusions: Our results indicate that B19V Ag-specific CD8+ T-cells with effector function are involved in B19V associated
DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune
response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of
CD8+ T-cell responses to the identified epitopes.
Citation: Streitz M, Noutsias M, Volkmer R, Rohde M, Brestrich G, et al. (2008) NS1 Specific CD8+ T-Cells with Effector Function and TRBV11 Dominance in a Patient
with Parvovirus B19 Associated Inflammatory Cardiomyopathy. PLoS ONE 3(6): e2361. doi:10.1371/journal.pone.0002361
Editor: Douglas F. Nixon, University of California San Francisco, United States of America
Received January 7, 2008; Accepted May 3, 2008; Published June 4, 2008
Copyright: ß 2008 Streitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These investigations were supported by the DFG through the SFB TR19 (TPB1 to RVE and FK; TPB2 to MN, MH and HDV; and TP Z1 to UK and HPS).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: michel.noutsias@charite.de
. These authors contributed equally to this work.
B19V is the most commonly detected virus in endomyocardial
biopsies (EMBs) from patients presenting with acute myocarditis
(AMC), dilated cardiomyopathy (DCM) or EMBs proven
inflammatory cardiomyopathy (DCMi) [5,6,7,8].
Despite the importance of T-cells in antiviral defense, little is
known about anti-B19V specific T-cells in this entity. Using
peptide pools and intracellular IFNc secretion as read-out, antigen
specific T-cells have been identified in other diseases [9,10]. Here,
we provide evidence for antigen-specific T-cells in a patient with
EMBs proven DCMi and exceptionally high B19V viral load at
the initial presentation. We furthermore characterized T-cell
receptor Vbeta (TRBV) expression and the expression profile of
type 1, type 2, cytotoxic and regulatory T-cell markers.
Introduction
Ever since its first description [1], Parvovirus B19 (B19V)
has been associated with various human diseases, including
erythema infectiosum, hydrops fetalis, anemia, aplastic crisis and
arthritis, vasculitis [2,3,4]. B19V contains a single-stranded
DNA genome which replicates in the nucleus of infected host
cells. The icosahedral capsid of B19V consists of two structural
proteins, VP1 (83 kDa) and VP2 (58 kDa), which are encoded by
the same reading frame. VP1 differs from VP2 only by an
additional N-terminal fragment. The nonstructural protein 1
(NS1) is required for virus replication and propagation in the host
cell [2].
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B19V Specific T-Cells in DCMi
genomes. Nested PCR (nPCR) for viral genomes was carried out
as described in detail elsewhere [6]. In brief, 2 EMBs were
subjected to RNA extraction by TrizolH (Invitrogen, Karlsruhe,
Germany) for nested PCR (nPCR) of enteroviruses, and 2 EMBs
were subjected to DNA extraction (Puregene mousetail DNA
extraction kit, Qiagen, Hilden, Germany) for B19V, human
herpes virus type 6, adenovirus and Epstein-Barr virus. Each time
2 EMBs were pooled to minimize sampling error effects as a
routine procedure in our laboratory. Single EMBs were not
analyzed. For B19V nPCR and qPCR the methods are described
in brief as follows: DNA extracted from EMBs, PBMCs and
serum, respectively, was amplified using B19V-specific primers
yielding a product of 290 bp in the first and of 173 bp in the
second round. Direct sequencing was carried out with an 8capillary sequencing system CEQ 8000 (Beckman-Coulter,
Krefeld, Germany). The sequences were aligned with the archived
B19V genome (NCBI GenBank accession No. AY386330) using
the Phylogenetic Data Editor (PHYDE) Software (Institute for
Botanics, Dresden/Bonn, Germany). The primers and probe for
B19V quantitative PCR (qPCR) were designed from the B19V
VP1/2 open reading frame (sequences given in Table 2). After
initial denaturation at 95uC for 10 min, amplification was carried
out by 38 two-step cycles (15 sec at 95uC, 1 min at 57uC) on a
7900 HT Fast Real-Time PCR System (Applied Biosystems/ABI,
Darmstadt, Germany). Serial dilutions (3.5 to 3.56104 genomes
per assay) of B19V plasmid DNA (Genexpress, Berlin, Germany)
were simultaneously coamplified to generate a standard curve.
Subsequent calculation of viral loads was performed based on the
ratio of the estimated viral genome copy numbers in the TaqMan
assay over the amount of incorporated human genomic DNA for
EMBs and PBMCs, respectively, while the calculation of viral
loads in the serum was normalized to milliliters of serum volume.
Methods
Patient description
A previously healthy 44 year-old man was admitted to our
department with progressive heart failure after a prior respiratory
tract infection with febrile temperatures up to 39uC three weeks
ago without elevated biochemical markers for myocardial
ischemia. Coronary artery disease was excluded by coronary
angiography. Left ventricular ejection fraction (LVEF) assessed by
echocardiography according to the modified Simpson’s method
was 45%. This clinical presentation was consistent with the
tentative clinical diagnosis of acute myocarditis [6,11]. Cardiac
MRI for non-invasive detection of myocardial edema was not
possible in this patient because he had a hip implant owing to a
traffic accident 8 years ago. There was no evidence for rheumatic
diseases in his past medical history. The patient was treated with
heart failure medication (carvedilol, ramipril, torasemide and
spironolactone). LVEF gradually increased, and LV end-diastolic
diameter (LVEDD) gradually decreased at echocardiographic
follow-up analyses (Table 1).
Description of Investigations undertaken
Detection and quantification of B19V in EMBs, PBMCs and
serum; quantification of intramyocardial inflammation in EMBs;
detection of anti-B19V-IgG, anti-B19V-IgM and characterization
of anti-B19V humoral response
6 EMBs (1 for histological, 1 for immunohistological, and 4 for
nPCR analyses of viral genomes) were obtained from the right
ventricular septum at each time point by a standard right femoral
venous approach. Five ml of blood was obtained in VacutainerH
EDTA tubes (BD Biosciences, Heidelberg, Germany). EMBs,
PBMCs and serum were examined simultaneously for viral
Table 1. Course of clinical parameters, EMBs and serological results.
Initial presentation
6 months
12 months
LVEF (%)
45
54
70
LVEDD (mm)
64
56
54
EMBs - B19V nPCR/EMBs - B19V qPCR
positive
positive
positive
PBMCs - B19V nPCR/PBMCs - B19V qPCR
115,091
23,846
1,026
positive
negative
negative
negative
negative
143.6
141.7
184,000
Serum - B19V nPCR/Serum–B19V qPCR
positive
57,468
B19V IgG titer (ELISA)
146
B19V IgG fractions (recomLineH blot)
VP-2p; VP-N; VP-1S; VP-2r; VP-C
VP-2p; VP-N; VP-1S; VP-2r; VP-C; NS1
VP-2p; VP-N; VP-1S; VP-2r; VP-C; NS1
B19V IgM titer (ELISA)
115.7
negative
negative
B19V IgM fractions (recomLineH blot)
VP-2p; VP-N; VP-1S; VP-2r; VP-C
negative
negative
CD3/mm2
16
7.5
3.6
LFA-1/mm2
36.4
12.2
7.4
CD45R0/mm2
19
10.1
5.5
Mac-1/mm2
36.8
14.8
13.3
HLA class I AF (%)
10.4
7.4
5.1
ICAM-1 AF (%)
4.1
2.7
1.1
Evolution of echocardiographic parameters (LVEF, LVEDD: LV enddiastolic diameter), of the B19V quantification results in EMBs and PBMCs (viral copies/mg nucleic acids
in EMBs, and viral copies/ml serum, respectively) and of the DIA quantified, immunohistologically marked infiltrates and CAMs expression in EMBs. AF: fraction of area in
percent (DIA derived value for CAMs expression). Normal values for DIA quantified infiltrates and CAMs expression in EMBs: CD3: ,7/mm2, LFA-1: ,9/mm2, CD45R0:
,7/mm2, Mac-1: ,35/mm2, HLA class I: ,5.5%, ICAM-1: ,1.2%.
doi:10.1371/journal.pone.0002361.t001
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B19V Specific T-Cells in DCMi
Table 2. Primers and probes of self-designed gene expression assays.
Gene name
59 sense 39 primer
TRBC
TCCGCTGTCAAGTCCAGTTCTA
TRBV common antisense primer
59 antisense 39 primer
Fluorescence hybridization probe
GACAGGACCCCTTGCTGGTA
ACGAGTGGACCCAGGATAGGGCCAA
CTGCTTCTGATGGCTCAAACA
TRBV common probe
CACCCGAGGTCGCT
TRBV2
ACTCTGAAGATCCGGTCCACAA
TRBV3
ATCAATTCCCTGGAGCTTGGT
TRBV4
CCTGAATGCCCCAACAGC
TRBV5
GCTCTGAGCTGAATGTGAACGC
TRBV5WBL
GCTCTGAGATGAATGTGAGTGC
TRBV6
CACTGACAAAGGAGAAGTCCC
TRBV6WBL6-2
AACTGCCAAAGGAGAGGTCCC
TRBV6WBL6-4
CACTGGCAAAGGAGAAGTCCC
TRBV7
CTCTCAGGTGTGATCCAATTTCG
TRBV7WBL7-2;7-3
AGCTCAGGTGTGATCCAATTTCA
TRBV7WBL7-9
CTTTCAGGTGTGATCCAATTTCT
TRBV9
CACAACAGTTCCCTGACTTGCA
TRBV10
CATGGGCTGAGGCTGATC
TRBV11
CTGCAGAGAGGCTCAAAGGAGTAG
TRBV12
AGAACCCAGGGACTCAGCTGT
TRBV13
GAACTGAACATGAGCTCCTTGGA
TRBV14
CTGAAAGGACTGGAGGGACGTAT
TRBV15
CAGGAGGCCGAACACTTCTTT
TRBV16
GCCTCCCAAATTCACCCTGTA
TRBV18
CCAGCATCCTGAGGATCCA
TRBV19
ACTGTGACATCGGCCCAAA
TRBV20
AACCATGCAAGCCTGACCTT
TRBV23
CCCTGCAGCCTGGCAAT
TRBV24
GCTAAATTCTCCCTGTCCCTAGAGT
TRBV25
TTCCCCTGACCCTGGAGTCT
TRBV27
GGCTTAAGGCAGATCTACTATTCAATG
TRBV28
GCCAGCACCAACCAGACAT
TRBV29
AGCCGCCCAAACCTAACATT
TRBV30
CGGCAGTTCATCCTGAGTTCT
IFN?
CAGGTCATTCAGATGTAGCGGATAA
AGGAGACAATTTGGCTCTGCATT
TTTCTGTCACTCTCCTCTTTCCAATTCTTCAAA
T-bet
CAACACAGGAGGCGCACTGG
CCCCCTTGTTGTTTGTGAGCT
CACCTGTTGTGGTCCAAGTTTAATCAGCACC
FoxP3
TGGCAAACGGAGTCTGCAA
TCTCATCCAAGAGGTGATCTGCTT
AGCCGGGAGAGTTTCTCAAGCACTGC
GATA3
CCTCATTAAGCCCAAGCGAAG
TTGGCATTTCCTCCAGAGT
TCCTGTGCGAACTGTCAGACCACCAC
Perforin
GGACCAGTACAGCTTCAGCACTG
AGTCAGGGTGCAGCGGG
TGCCGCTTCTACAGTTTCCATGTGGTACAC
Granzyme B
GCGAATCTGACTTACGCCATTATT
CAAGAGGGCCTCCAGAGTCC
CCCACGCACAACTCAATGGTACTGTCG
B19V
CATTTTCCAGACAGTTTTTAATTCCA
CTTGCTGCGGGAGAAAACAC
ATGACCCAGAGCACC
Sequences of primers and fluorescence hybridization probes. For the different TRBV forward primers, one common reverse primer and one common hybridization
probe were used. * For TRBV5, 6 and 7, wobbled (WBL) forward primers were designed.
doi:10.1371/journal.pone.0002361.t002
denatured PVB19 proteins were analyzed in the serum using
recomLineH blot assays according to the manufacturer’s instructions
(Mikrogen GmbH) [14]. In brief, recomLineH strips were incubated
with serum samples (20 ml each) and subsequently anti-human IgG
or IgM conjugated with horseradish peroxidase (HRPO) were
added. In positive HRPO reactions, a dark band developed at the
corresponding locus on the strips. The positive control bands were
positive in all blots performed. Reactivities of serum antibodies
against the recombinant antigens were evaluated by comparison
Concentrations of isolated genomic human DNA amounts were
quantitatively measured using the Quantifiler Human DNA
quantification kit (ABI) according to manufacturer’s instructions.
Quantification of immunohistologically stained cells and cell
adhesion molecules (CAMs) in cryosections of EMBs was carried
out as described in detail elsewhere [12,13].
B19V IgG and IgM titers were quantified by standard ELISA
techniques according to the manufacturer’s instructions (Mikrogen
GmbH, Neuried, Germany). Antibodies against native and
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B19V Specific T-Cells in DCMi
SALK- and GLCP-reactive cells were enriched from PBMCs
after incubation with these two peptides over 6 h using the IFNc
secretion assay according to the manufacturer’s instructions
(Miltenyi Biotech, Bergisch Gladbach, Germany). PBMCs were
separated from peripheral blood obtained in VacutainerH EDTA
tubes (BD Biosciences, Heidelberg, Germany) using the LSM 1077
Lymphocyte Separation Medium (PAA, Pasching, Germany)
density gradient. Both the positively and the negatively selected
cells using the IFNc secretion assay, as well as unselected PBMCs
were lysed in RLTH buffer (Qiagen, Hilden, Germany), and total
RNA was gained using the RneasyH system (Qiagen) according to
the manufacturer’s instructions including DNAse treatment.
Retrotranscription was carried out using the High Capacity
Archive cDNA KitH (ABI).
with a cut-off band. Reactivities stronger than or equivalent to the
reference band were evaluated positive.
Analysis of B19V specific T-cells
Ex-vivo analysis of the cellular immune response was performed
10 and 12 months after the initial presentation.
Anticoagulated blood (Na-citrate) was used for preparation of
peripheral blood mononuclear cells (PBMCs). After separation by
Biocoll-gradient centrifugation (Biochrom AG, Berlin, Germany),
PBMCs were washed with sterile PBS and resuspended in RPMI
1640 medium containing 10% (v/v) fetal calf serum and 2 mM Lglutamine. The cell suspension was adjusted to a concentration of
56106 cells/ml.
T-cell responses to peptides were mapped using peptide libraries
of 10-amino-acid peptides with a shift of one amino acid, in
agreement with previous work [9,10]. Peptide sequences were
derived from the complete proteome of B19V strain ‘‘Berlin’’ [15]
and complemented by the alternative 10-amino-acid peptide
sequences of parvovirus described by Shade et al. [16].
The scan included 1,953 peptide sequences efficiently arranged
in a cross matrix system [17] resulting in 91 peptide sub-pools with
a maximum number of 40 peptides. Peptide libraries were
synthesized on cellulose membranes functionalized with a glycin
residue using the SPOT technology [18], resulting in 10-aminoacid peptides with an additional N-terminal glycin-amide.
For the screening with complete peptide libraries, 96 well round
bottom plates containing 200 ml of the adjusted cell suspension
(16106 cells) were used. Peptide sub-pools were added in 25 ml
RPMI/FCS/L-Glu medium containing 1 mg/ml (final concentration) of each peptide.
Responses to sub-pools of this library were fine mapped with 10amino-acid peptides, synthesized on solid phase support with a
purity of about 99% following HPLC purification. Additionally, a
13-amino-acid peptide sequence containing two of the CD8+ epitope
candidates with an additional CD4+ response was synthesized. For
the fine mapping and the examination of T-cell responses at the
second time point a larger number of cells were used and cell
stimulating experiments were carried out in tubes. 400 ml of cell
suspension (26106 cells) was transferred to Falcon 2052 tubes and
peptides were added in 100 ml RPMI/FCS/L-Glu medium
containing 1 mg/ml (final concentration) of each peptide. For all
experiments a negative control sample was incubated with 1 ml/ml
DMSO, a positive control sample with 1 mg/ml Staphylococcus
Enterotoxin B (SEB). Tubes and plates were incubated in a standard
incubator (5% CO2 humidified atmosphere). After 2 h, Brefeldin A
(BFA) was added. All plate wells were supplemented with 2.5 mg of
BFA in 25 ml RPMI/FCS/L-Glu and Falcon 2052 tubes with 10 mg
BFA in 500 ml RPMI/FCS/L-Glu (final concentration was 10 mg/
ml). After an incubation time of 16 h (i.e. 14 h after BFA addition),
10 mM EDTA was added and after an additional 10 min incubation
was ended by washing with cold PBS containing 0.5% BSA and
0.1% NaN3. Extracellular markers were stained with fluorochromelabeled monoclonal antibodies (mAb). Then cells were washed with
PBS/FCS/NaN3 and lysis of erythrocytes was performed (lysing
solution; Becton Dickinson). Following permeabilization (permeabilizing solution II; Becton Dickinson), intracellular staining (ICS) of
cells with fluorochrome labeled mAb was performed; finally cells
were washed in PBS containing 0.5% paraformaldehyde and
analyzed on a LSRII flow cytometer using the DIVA software (both
from Becton Dickinson).
HLA class I typing by low resolution PCR on DNA extracted
from peripheral blood (tissue typing laboratory, Charité–Universitätsmedizin Berlin) revealed the presence of HLA A*02, A*11,
B*07 in this patient.
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Preamplified Real-time RT-PCR of SALK- and GLCPreactive cells
We used self-designed (Table 2) and TaqmanH ABI inventoried
gene assays (Table 3). For the TRBV/TRBC gene assay design, we
used the gene sequences published at the Immunogenetics Database
[http://imgt.cines.fr/] according to ABI recommendations using the
Primer Express Software (version 2.0; ABI). Sequence homologies
between the various functional TRBVs were excluded by multiple
alignments using ClustalX Software (version 1.83; University of
Strasbourg, France). We chose TRBV specific forward primers and
one common reverse TRB primer on the constant TRB (TRBC)
region and a probe on the TRBC region (encompassing both human
TRBC1 and TRBC2). A further gene assay was designed on the
constant region of the TRBC (encompassing both human TRBC1
and TRBC2). For TRBV5, 6, and 7, wobbled (WBL) primers were
designed to encompass few base differences. TRBV and TRBC
minor groove binder (MGB) probes were synthesized by ABI.
Regarding the remaining self-designed gene assays, we used the
Oligo 4.1 software (Molecular Biology Insights, Cascade, USA) and
published genomic sequences (National Center for Biotechnology
Information/NCBI) according to ABI recommendations. All probes
used were FAM (carboxyfluorescein) dye labeled.
cDNA was preamplified using the PreAmpH MasterMix Kit
(ABI) according to the manufacturer’s instructions, modified by
employing half of the suggested volume of PreAmp reaction (25 ml
PreAmp reaction volume). The pooled TaqManH ABI inventoried
gene assays (including fluorescent probes) as well as the forward/
reverse primers of the self-designed gene assays were diluted with
16 Tris-EDTA (TE; 10 mmol/l Tris-HCl and 1 mmol EDTA,
pH 7.6) buffer, so that each assay was at a final concentration of
0.2 fold in the PreAmp primer/assay pool. The PreAmp reaction
Table 3. ABI inventoried TaqmanH gene expression assays.
Gene name
ABI ID number
CD3d
Hs00174158_m1
IL2
Hs00174114_m1
IL4
Hs00174122_m1
IL6
Hs00174131_m1
IL27
Hs00377366_m1
TNFa
Hs00174128_m1
NFkB
Hs00765730_m1
ABI inventoried TaqmanH gene expression assays and the respective ABI ID
numbers.
doi:10.1371/journal.pone.0002361.t003
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B19V Specific T-Cells in DCMi
involved the amplification of 1–250 ng (maximum 6.25 ml) cDNA
in a 25 ml reaction consisting of 12.5 ml TaqManH PreAmp Master
Mix and 6.25 ml pooled primer/assay mix (0.26, each assay). TPreAmp of this primer/gene assay pool was carried for 14 cycles
on a PTC-100 Programmable Thermal Controller (MJ Research,
Watertown, Massachusetts, USA) with the following program:
denaturation at 95uC for 10 min and 14 cycles of amplification
(15 sec at 95uC, 4 min at 60uC). The preamplified products were
then diluted with Tris-EDTA-buffer at a ratio 1:20 (resulting
volume 500 ml) and were used as templates for the real-time RTPCR analysis.
The real-time RT-PCR reactions were performed in a final
volume of 12.5 ml, containing 1 ml cDNA, 6.25 ml Master Mix
(TaqManTM Universal PCR Master Mix, No AmpEraseH UNG,
ABI), 0.21 ml probe (final concentration 0.05 mM) and 1.25 ml
forward and reverse primers (final concentration 0.9 mM). Reactions
were made up to a final volume of 12.5 ml with sterile water. All
experiments were performed in duplicates on a 7900 HT Fast realtime PCR System (ABI). The real-time RT-PCR protocol was as
following: Denaturation by a hot start at 95uC for 10 min, followed
by 40 cycles of a two-step program (denaturation at 95uC for 15 sec
and annealing/extension at 60uC for 1 min). TRBV gene expression
was normalized to TRBC, and effector T-cell genes were normalized
to CD3d applying the formula 22DCt. mRNA expression in
positively selected cells was compared with mRNA expression in
negatively selected cells and unselected PBMCs.
tions were in agreement with the Helsinki Declaration. Written
informed consent was obtained from the patient.
Statistical methods
Statistical analysis was performed using JMP Statistical
Discovery Software V5.1.2 (SAS Institute, Inc., Cary, NC,
USA). We compared ordinal with continuous data employing
the student’s t-test. Probability values (p) ,0.05 were considered
statistically significant.
Results
Histological analysis of EMBs revealed no evidence of active
myocarditis according to the Dallas criteria [19] at any time point
(Figure 1a, b, c). However, quantification of immunohistological
stainings of EMBs [12] by digital image analysis (DIA) revealed
increased infiltrates and expression of cell adhesion molecules
(CAMs), consistent with DCMi (Table 1; Figure 1d, g). nPCR for
cardiotropic viral genomes [6] confirmed positive B19V results in
EMBs, PBMCs and the serum. Quantitative PCR (qPCR) revealed a
B19V load of 115,091 copies/mg nucleic acids in the EMBs, and
184,000 copies/mg nucleic acids in the PBMCs, respectively. The
B19V load in the serum was quantified as 57,468 viral copies/ml.
B19V qPCR of EMBs 6 and 12 months after the initial presentation
showed substantially decreased B19V loads (23,846 and 1,026 viral
copies/mg nucleic acids, respectively). Noticeably, B19V could no
longer be tracked either in PBMCs or in the serum in these follow-up
analyses by nPCR. This was paralleled by a decreasing infiltration
and CAMs expression in the follow-up EMBs, reaching normal
values in the EMBs obtained at the third time point (Table 1;
Figure 1e, f, h, i). B19V IgM were detectable at the initial
presentation by both ELISA and recomLineH blots, but not at
Ethics
These investigations were approved by the local ethics
committee at the Charité–Universitätsmedizin Berlin in the
framework of the Sonderforschungsbereich TR19. The investiga-
Figure 1. Histological (a, b, c) and immunohistological (d–i) stainings (CD3: d–f; HLA class I expression: g–i) of the EMBs at the initial
presentation, at 6 and 12 months follow-up. Histological analyses did not reveal active or borderline myocarditis at any time point. The initially
increased CD3+ T-cells and HLA class I expression decreased at the follow-up EMBs. Original magnification a–f: 2006fold. Original magnification g–i:
1006fold.
doi:10.1371/journal.pone.0002361.g001
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B19V Specific T-Cells in DCMi
Figure 2. FACS analysis of two B19V NS1-specific CD8+ T-cell responses and one CD4+ T-cell response 10 months after the initial
presentation. The upper panels illustrate IFNc secretion, and the lower panels TNFa secretion, both 16 h after stimulation. Plots show negative
controls (a, b, g, h), CD8+ T-cell responses following stimulation with the 10-amino-acid peptides GLCPHCINVG (c, d) and SALKLAIYKA (e, f), and the
CD4+ T-cell response following stimulation with the 13-amino-acid peptide IQSALKLAIYKAT (i, k).
doi:10.1371/journal.pone.0002361.g002
Real-time RT-PCR for functional TRBV expression in SALKand GLCP-reactive T-cells demonstrated an over proportional
TRBV11 expression compared with the respective non-reactive
cells (SALK: 7.5 fold; GLCP: 4.7 fold) and non-selected PBMCs
(Figure 3). Furthermore, SALK- and GLCP-reactive T-cells
exhibited dominant expression of the type-1 T-cell markers, IFNc
(SALK: 11.9 fold; GLCP: 5 fold), IL2 (SALK: 16.6 fold; GLCP:
24.7 fold), IL27 (SALK: 8.7 fold; GLCP: 4.4 fold), T-bet (SALK:
2.3 fold; GLCP: 3.3 fold), and of the CTL markers, Perforin
(SALK: 2.3 fold; GLCP: 4 fold) and Granzyme B (SALK: 2.6 fold;
GLCP: 3.6 fold). In contrast to this, markers for type-2 (GATA3:
SALK 0.4 fold and GLCP 0.3 fold, respectively; IL4: SALK 0.3
fold and GLCP 0.2 fold, respectively) and for regulatory T-cells
(FoxP3; SALK: 0.7 fold and GLCP: 0.6 fold, respectively) were
expressed at low levels. In addition, a substantially higher
expression of general markers of inflammation was measured in
the positively selected IFNc expressing cells: IL-6 (SALK: 2.0 fold;
GLCP: 3.1 fold), TNFa (SALK: 7.5 fold; GLCP: 10.7 fold) and
NFkB (SALK: 5.9 fold; GLCP: 3 fold) (Figure 4).
follow-up analyses, consistent with seroconversion after an acute B19
infection at the initial presentation. The B19V IgG titer remained
almost stable at the three time points, however, recomLineH blot
analyses revealed the occurrence of NS1-specific antibodies 6 and 12
months after the initial presentation, indicating a persisting B19V
infection (Table 1).
Ex-vivo analysis of the cellular immune response was performed
10 and 12 months after the initial presentation. Using peptide
libraries representing the complete B19V proteome, strong CD8+
T-cell responses to at least two B19V derived peptides were
identified at both time points, with larger responses measured at
10 months (Figure 2), at which time 19.7%, and 10% of the CD8+
T-cells responded with IFNc secretion to the stimulation with the
10-amino-acid peptides, SALKLAIYKA257-266 (SALK) and
QSALKLAIYK256-265 (QSAL), respectively. The third sequence
IQSALKLAIYKAT255-267 (IQSA), a 13-amino-acid peptide,
containing the two other sequences, activated 14.6% of the
CD8+ T-cells. Similar frequencies were observed when using
TNFa as read-out instead of IFNc (Figure 2). The sequence shift
from IQSA to QSAL and SALK is only one amino acid at a time,
suggesting that they contain the same complete or partial epitope.
A small but distinct CD4+ T-cell response (0.02–0.04% of all
CD4+ T-cells) to each of these peptides was also detected (shown in
Figure 2 for the 13-amino-acid peptide). Two additional 10amino-acid peptides also stimulated CD8+ T-cell responses: The
peptide GLCPHCINVG613-622 (GLCP) was recognized by
0.71% of CD8+ T-cells, and the peptide LLHTDFEQVM276-285
(LLHT) was recognized by 0.06% of the CD8+ T-cells, both
inducing IFNc. Only the GLCP peptide induced a similar TNFa
response of similar size. Both sequences include a published HLAA2 presented epitope (GLCPHCINV [20] and LLHTDFEQV
[20]), in agreement with the HLA-Type of the donor. After two
months, the frequencies of the above described IFNc T-cell
responses had decreased to 5.34% (SALK), 4.4% (QSAL) and
0.07% (IQSA), 0.08% (GLCP) and 0.03% (LLHT). CD4+ T-cells
producing IFNc in response to stimulation with GLCP, SALK and
IQSA were also still detectable, however response were smaller
than 0.025%.
PLoS ONE | www.plosone.org
Discussion
This patient presented with signs and symptoms suggestive of
acute myocarditis [6,11]. DCMi was confirmed by immunohistology
[12,21], in the absence of active myocarditis according to the
histological Dallas criteria [19]. This is in line with the substantially
higher sensitivity of immunohistological EMBs assessment compared
with the Dallas criteria [12,22,23]. B19V is the most commonly
detected virus in patients presenting with acute myocarditis or
DCM/DCMi [5,6,7,8]. Usually, B19V genomes are confirmed in
EMBs, but not PBMCs or sera of such patients. In this particular
case, the B19V load was almost the same in EMBs and PBMCs at
the initial presentation, suggesting viremia, i.e. active viral
replication. While it is known that IgG-complexed virus particles
can be taken up by mononuclear phagocytes via the Fc-receptor or
complement-receptor mediated endocytosis, the fact that B19V
genomes were detected in the serum with a quantifiable number of
viral copies argued for the presence of viremia. At follow-up,
however, B19V genomes were confirmed in EMBs, but not in
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B19V Specific T-Cells in DCMi
Figure 3. TRBV expression of B19V NS1-specific T-cells. Bars indicate the TRBV expression normalized to TRBC in non-selected
PBMCs compared with positively and negatively selected GLCP and SALK reactive T-cells which were enriched using the IFNc
secretion assay.
doi:10.1371/journal.pone.0002361.g003
Figure 4. Expression of effector T-cell markers of B19V NS1-specific T-cells. The bars indicate marker expression (normalized to CD3d in
non-selected PBMCs) compared with positively and negatively selected GLCP and SALK reactive T-cells enriched using the IFNc secretion assay. The 3
panels show different target gene expression levels.
doi:10.1371/journal.pone.0002361.g004
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B19V Specific T-Cells in DCMi
PBMCs and serum. B19V virus load in EMBs decreased
substantially over time, however, was detectable at all time points.
LVEF increased and LVEDD decreased in parallel under
conventional heart failure medication, while intramyocardial
inflammation was found to have decreased at the second point,
and was absent at the third time point. The changes of cardiac
function data over time, and the EMBs findings are consistent with
the hypothesis that the initial antiviral immune response resulted in a
massive reduction or elimination of the virus, and subsequently
contributed to an improvement of LV function and decrement of LV
dilatation [24,25]. The time course of B19V infection is also
mirrored by the serological B19V findings: The B19V IgM titers at
the initial presentation indicate an acute B19V infection with
seroconversion at follow-up, while the occurrence of NS1-specific
IgG indicate a persisting B19V infection 6 and 12 months after the
acute event [14,26].
It was previously reported that the CD8+ T-cell response targets
multiple peptides situated mainly within the non-structural protein 1
(NS1) in adults with symptomatic B19V infection, and that these
responses can persist for more than 2 years after the acute illness
[20,27]. In agreement with this, the major T-cell responses in this
patient were directed at peptides in the NS1 protein. Consistent with
reports on the development B19V specific cellular response in adults
with an acute infection, we identified a B19V specific CD8+ T-cell
response with multiple specificities, which gradually decreased over
time [5,10]. Since responses were measured at 10 and 12 months
only, with a decrease noted at 12 months, we can only assume that
peak levels were even higher than the values measured at 10 months.
These findings are consistent with previous studies, showing that
the T-cell response increases despite resolution of clinical
symptoms and targets mainly NS1 in patients acute B19V
infections [28]). The demonstration of additional effector functions
of these B19V specific T-cells was in agreement with an increased
transcription of the type-1 and CTL markers in SALK- and
GLCP-specific cells as shown.
These data indicate that the observed sustained CD8+ T-cell
response is stimulated by B19V persistence or a delayed clearance
of the virus in the endomyocardium. The continued presence of
virus in the tissue, albeit in decreasing amounts, was a likely source
for the continued antigen exposure of the CD8+ T-cells in this
patient. High virus loads in EMBs suggests that the myocardium is
a target for B19V specific T-cells.
We hypothesize that B19V antigen-specific CD8+ T-cells were
crucial for the elimination of virus in PBMCs and the substantial
decrease of B19V viral load in EMBs. The mechanism for virus
elimination is not entirely clear, but most likely cytotoxicity, given
the type-1 and CTL dominance found in SALK- and GLCPspecific cells.
Surprisingly, not only CD8+, but also CD4+ T-cell responses
were induced by the 13-amino-acid peptide IQSALK and the
sequences included in it (SALK and QSAL). These responses were
initially identified with a 10-amino-acid peptide library, which is
not optimum for the stimulation of CD4+ T-cells. Low frequencies
of specific CD4+ T-cell responses were described previously after
acute B19V infections [29]. These results, as well as ours, suggest a
role in B19V elimination, and possibly for the pathogenesis of
autoimmune phenomena.
An additional goal of this analysis was the characterization of
B19V-reactive T-cells in regards of TRBV usage and functionality.
Our data are consistent with a highly restricted TRBV repertoire
of virus induced memory T-cells, and a type-1/CTL polarization
of the T-cell mediated antiviral immune response [28,30,31]. The
difference in type-1/type-2, CTL and Treg marker expression
most likely results from the predominance of CD8+ T-cells in the
sorted subset compared to the unselected or negatively selected
populations, and reflects the composition of the B19V reactive
population.
T-cell mediated responses are mediated by reactive TRBV
families via interactions with HLA presented antigens [30]. A
highly restricted TRBV repertoire of the CD8+ T-cell response in
persistent B19V infections has been reported by Kasprowicz et al.
(TRBV5.1 in HLA-A*2402-positive individuals) [31]. Our results
confirm a highly restricted TRVB11 repertoire of NS1-specific
CD8+ effector T-cells in this HLA A*02, A*11, B*07 patient with
B19V associated DCMi.
B19V genomes were previously shown to persist in various
human tissues, both in healthy individuals and patients [32]. As a
result of this, the biological significance of the mere presence of
B19V genomes in myocardial tissue without clinical evidence of
progressive myocardial disease and/or evidence of an antiviral
immune response is unclear, thus far. Nevertheless, the clinical
importance of B19V in DCMi was illustrated in a prospective
study, in which the LVEF of DCMi patients showing viral
elimination at follow-up EMBs improved substantially, whereas it
deteriorated over time in patients with EMBs proven viral
persistence [24]. Furthermore, viral infection, including B19V,
has an independent adverse prognostic impact in patients with
EMBs-proven myocarditis [33]. These investigations provide
indirect evidence that viral elimination is necessary for functional
improvement in DCMi. If the antiviral immune response, as
measured in the present study, was indeed a correlate of effective
B19V elimination, such a measurement early in the course of
disease may be a useful surrogate marker predicting a favorable
outcome in B19V associated DCMi.
Acknowledgments
We are indebted to Professor Susanne Modrow and Juha Lindner (Institute
for Medical Microbiology, University of Regensburg, Germany) for
performing and interpreting the B19V serological analyses. We thank the
technicians Georg Zingler (Department of Cardiology) and Annelie
Dernier (Institute of Medical Immunology) for excellent technical
assistance related to real-time RT-PCR analyses.
Author Contributions
Conceived and designed the experiments: RV HV MS MN MR MH HS
FK. Performed the experiments: RV MS MN MR GB AB KK BA.
Analyzed the data: HV MS MN MR AB UK DL FK. Contributed
reagents/materials/analysis tools: RV MN KK GB AB UK DL BA. Wrote
the paper: RV HV MS MN MR GB AB MH DL HS FK.
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