Supplementary Table 1. Amino acid sequence of heat shock proteins and their reactive T-cell and B-cell epitopes from human samples
Single T-cell epitopes
HSPs
Position
Amino acid
TCR Vβ
Specimen
Outcome
sequence
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
hHSP60, hHSP60p
HSPs
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
mHSP65, mHSP65p
GroEL, GrpELp
1-15
6-20
11-25
31-45
91-105
96-110
136-150
141-155
201-215
206-220
226-240
261-275
321-335
331-345
466-480
491-505
506-520
246-260
266-280
281-295
326-340
416-430
421-435
436-450
471-485
476-490
551-565
Position
46-60
21-35
51-65
26-40
26-40
26-40
MLRLPTVFRQMRPVS
TVFRQMRPVSRVLAP
MRPVSRVLAPHLTRA
KFGADARALMLQGVD
KNIGAKLVQDVANNT
KLVQDVANNTNEEAG
NPVEIRRGVMLAVDA
RRGVMLAVDAVIAEL
VKDGKTLNDELEIIE
TLNDELEIIEGMKFD
PYFINTSKGQKCEFQ
LEIANAHRKPLVIIA
GGAVFGEEGLTLNLE
TLNLEDVQPHDLGKV
EIIKRTLKIPAMTIA
VEKIMQSSSEVGYDA
MAGDFVNMVEKGIID
LSEKKISSIQSIVPA
AHRKPLVIIAEDVDG
EALSTLVLNRLKVGL
GEEGLTLNLEDVQPH
EKKDRVTDALNATRA
VTDALNATRAAVEEG
IVLGGGCALLRCIPA
TLKIPAMTIAKNAGV
AMTIAKNAGVEGSLI
KEEKDPGMGAMGGMG
Vβ11, Vβ9, Vβ5.2
Vβ14, Vβ8, Vβ14
Vβ1, Vβ8
Vβ20
Vβ4
Vβ21.3
Vβ5.2
Vβ12
Vβ17
Vβ3.1
Vβ4
Vβ5.1
Vβ2, Vβ12
Vβ18
Vβ22
Vβ11
Vβ7, Vβ6.7, Vβ16, Vβ11
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
LLADAVAVTMGPKGR
KTLAEAVKVTLGPKG
VAVTMGPKGRTVIIE
AVKVTLGPKGRHVVI
KVTLGPKGRNVVLEK
AVKVTLGPKGRNVII
Advanced
atherosclerotic
lesions and blood
4LL of Cp- patients+blood and 4 LL of Cp+ patients+blood.
The submolecular specificity of HSP60-specific plaquederived T-cells was analyzed and both the self and crossreactive epitopes of that autoantigen were identified (see
below).
1
Advanced and
“healthy”
atherosclerotic
lesions and blood
7 EL and 8 LL patients. Blood from all EL, LL, and two HC
groups consisting of 26 young and 14 old subjects. T-cells
derived from LL displayed a more restricted T-cell
receptor repertoire to hHSP60-derived peptides than
those isolated from EL. This data supports the concept that
hHSP60-reactive T-cells initiate atherosclerosis by
recognition of atherogenic hHSP60 epitopes.
2
Cross-reactive T-cell epitopes
TCR Vβ
Specimen
Amino acid
sequence
Vβ9
Vβ18
Ref
Advanced
atherosclerotic
lesions and blood
Outcome
4 LL of Cp- patients+blood and 4 LL of Cp+ patients+blood.
All patients with positive serology and PCR detection of
Chlamydia pneumoniae DNA had in their carotid plaques at
least two populations of hHSP60 specific T-cells: one
reactive only to self hHSP60, and the other reactive to both
the self and the Chlamydia pneumoniae analog HSP60.
Ref
1
1
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
hHSP60, hHSP60p
CpHSP60, CpHSP60p
PgHSP60/hHSP60
61-75
36-50
76-90
51-65
51-65
51-65
151-165
126-140
166-180
141-155
141-155
141-155
171-185
146-160
146-160
146-160
191-205
166-180
211-225
186-200
241-255
216-230
291-305
266-280
406-420
381-395
436-450
411-425
441-455
416-430
446-460
421-435
511-525
486-500
521-535
496-510
536-550
516-530
NP
PgHSP60
NP
TVIIEQSWGSPKVTK
RHVVIDKSFGSPQVT
DGVTVAKSIDLKDKY
DGVSIAKEIELEDPY
KDGVTVAKEIELEDK
KDGVSVAKEIELEDK
VIAELKKQSKPVTTP
VVVDELKKISKPVQH
EEIAQVATISANGDK
QIAATAAISAGDQSI
HKEIAQVATISANND
SEEVAQVGTISANGD
VATISANGDKEIGNI
TAAISAGDQSIGDLI
QVATISANNDSEIGN
QVATISANGDKQVGL
KKVGRKGVITVKDGK
MEKVGKNGSITVEEA
LEIIEGMKFDRGYIS
VLDVVEGMNFNRGYL
DAYVLLSEKKISSIQ
EDALILIYDKKISGI
LKVGLQVVAVKAPGF
RLRAGFRVCAVKAPG
VGGTSDVEVNEKKDR
VGAATEIEMKEKKDR
IVLGGGCALLRCIPA
ILPGGGTALVRCIPT
GCALLRCIPALDSLT
GTALVRCIPTLEAFL
RCIPALDSLTPANED
RCIPTLEAFLPMLAN
VNMVEKGIIDPTKVV
AYTDMIDAGILDPTK
PTKVVRTALLDAAGV
LDPTKVTRSALESAA
ASLLTTAEVVVTEIP
LLTTEALIADIPEEK
TVPGGGTTYIRAIAALEGLK
TLVVNRLRGSLKICAVKAPG
Vβ5.1
Vβ17
Vβ14
Vβ11
Vβ12
Vβ5.2
Vβ4
Vβ13.1
Vβ8
Vβ14
Vβ9
Vβ1
Vβ13.2
Vβ22
Vβ9
Vβ11
NP
Advanced
atherosclerotic
lesions, gingival
tissue, and blood
20 LL and periodontitis, 20 with periodontitis, and 20 HC.
Thirty per cent of periodontitis patients and 100% of
atherosclerosis patients reacted positively to the crossreactive peptide (TLVVNRLRGSLKICAVKAPG) from both
Pg and hHSP60. The peptide for a specific T-cell line
3
2
demonstrated the phenotype characteristic of helper Tcells (CD4+) but did not express CD25 or FOXP3 and IL-10
was elevated. Thus, this cross-reactive peptide is an
immunoreactive epitope in the periodontis-atherosclerosis
axis.
HSPs
Position
Amino acid sequence
hHSP60
1-573
(full-length)
NP
hHSP60
1-573
(full-length)
NP
hHSP60
1-573
(full-length)
NP
hHSP60, PgHSP60
NP
NP
hHSP60, GroEl,
PgHSP60
NP
hHSP60, CpHSP60
NP
TCR Vβ without amino acid sequence
TCR Vβ
Specimen
Outcome
Ref
Asym: Vβ1, Vβ2, Vβ3,
Vβ7, Vβ9, Vβ11, Bβ16,
Vβ20
Sym: Vβ3, Vβ6.2, Vβ7,
Vβ9, Vβ11, Vβ13, Vβ18,
Vβ20, Vβ21, Vβ24
AS: Vβ2, Vβ3, Vβ4, Vβ5.1,
Vβ6.1, Vβ6.2, Vβ8, Vβ11,
Bβ12, Vβ14, Vβ17, Vβ18,
Vβ21, Vβ24
Vβ2, Vβ5.2, Vβ6, Vβ8,
Vβ9, Vβ12, Vβ13.1, Vβ16,
Vβ18, Vβ19, Vβ20
Advanced
atherosclerotic
lesions and blood
10 LL, 5 with asymptomatic and 5 with symptomatic
stenosis. Blood from these patients was obtained twice, on
the date of endarterectomy and 2 weeks after surgery. 6
additional LL and blood from atherosclerotic patients were
used. Lesion derived T-cells displayed an oligoclonallyrestricted repertoire, in contrast to the polyclonal pattern
of PBMC. This indicates that HSP60 may be a major
antigenic candidate and that oligoclonal T-cell expansion
takes place in advanced human atherosclerotic lesions.
4
Advanced
atherosclerotic
lesions, gingival
tissue, and blood
5
NP
Vβ5.2-3, Vβ13.1/13.3
Advanced
atherosclerotic
lesions and blood
NP
Both CD4+ and CD8+ Tcells with a majority
bearing the αβ TCR
rather than γδ TCR.
Advanced
atherosclerotic
lesions and blood
15 LL and periodontitis, 16 with periodontitis, and 10 HC.
Antibody levels to both hHSP60 and PgHSP60 were highest
in atherosclerosis patients, followed by periodontitis
patients and healthy subjects. Clonal analysis of the T-cells
clearly demonstrated the presence of not only hHSP60 but
also PgHSP60-reactive T-cell populations in the peripheral
circulation of atherosclerosis patients. These HSP60reactive T-cells were present in atherosclerotic lesions in
some patients.
25 patients with LL and blood from 22 of these patients. A
cross-reactivity of several T-cell lines was demonstrated.
The cytokine profiles of the arterial T-cell lines specific for
hHSP60, GroEL, and PgHSP60 displayed a Th2 phenotype
predominance in CD4+ cells. A higher proportion of CD4
cells was positive for IFN-IP10 and RANTES, with low
percentages of cells positive for MCP-1 and MCP-1α,
whereas a high percentage of CD8+ cells expressed all four
chemokines. Finally, there was overexpression of the TCR
Vβ5.2-3 family in all lines.
32 patients with LL and blood. Antigen responsiveness of
T-cell lines showed that those derived using Chlamydia
organisms were more likely to respond to Chlamydia than
those isolated using other stimuli. Chlamydia-specific Tcell lines were shown to respond to OMP2 and/or hHSP60,
those recognizing CpHSP60 did not cross-react with
hHSP60, but hHSP60-responsive lines were also observed.
6
7
3
Thus, atherosclerotic plaque tissue contains a variety of
memory T-cells, and amongst these are cells capable of
recognizing Chlamydia antigens.
HSPs
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
CpHSP60p
GroELp
hHSP60p
mHSP65p
hHSP60p
mHSP65p
hHSP60p
Position
86-98
61-73
61-73
61-73
116-128
91-103
91-103
91-103
131-148
91-103
91-103
91-103
186-203
161-178
161-178
161-178
241-258
216-233
216-233
216-233
261-273
236-248
236-248
236-248
461-478
436-453
436-453
436-453
516-528
491-503
491-503
491-503
52-61
26-35
57-66
31-40
62-71
Cross-reactive B-cell epitopes
Amino acid sequence
Specimen
LKDKYKNIGAKLV
LEDPYEKIGAELV
LADKHENMGAQMV
LEDKFENMGAQMV
ATVLARSIAKEGF
ATVLAQALVREGL
ATVLAEAIYTEGL
ATVLAQAIITEGL
ISKGANPVEIRRGVMLAV
VAAGANPLGLKRGIEKAV
VTAGANPMDLKRGIDKAV
VAAGMNPMDLKRGIDKAV
ISDAMKKVGRKGVITVKD
IAEAMDKVGNEGVITVEE
IAEAMEKVGKNGSITVEE
IAEAMDKVGKEGVITVED
DAYVLLSEKKISSIQSIV
DPYILLVSSKVSTVKDLL
DALVLIYDKKISGIKDFL
SPFILLADKKISNIREML
LEIANAHRKPLVI
LEKVIGAAKPLLI
LQQVAESGRPLLI
LEAVAKAGKPLLI
QKIGIEIIKRTLKIPAMT
EATGANIVKVALEAPLKQ
EQIGARIVLKALSAPLKQ
QNVGIKVALRAMEAPLRQ
KGIIDPTKVVRTA
AGVADPVKVTRSA
AGILDPAKVTRSA
MGILDPTKVTRSA
AVTMGPKGRT
KVTLGPKGRN
PKGRTVIIEQ
PKGRNVVLEK
VIIEQSWGSP
Outcome
Ref
Blood
Sera from 5 subjects with ≥1:1280 anti-HSP antibodies and
sonographically proven atherosclerosis. Antibodies to
microbial HSP60/65 recognize specific epitopes on
hHSP60. These cross-reactive epitopes were shown to
serve as autoimmune targets in incipient atherosclerosis.
8
Blood
Purified Ig preparation from pooled plasma of >6000
healthy blood donors and a second set of samples were
collected from 12 healthy blood donors. Three epitopes
were “specific” for hHSP60 and three different epitopes
were “specific” for mHSP65. In addition, eight epitopes
9
4
mHSP65p
hHSP60p
hHSP60p
hHSP60p
mHSP65p
hHSP60p
hHSP60p
mHSP65p
hHSP60p
mHSP65p
mHSP65p
hHSP60p
mHSP65p
hHSP60p
mHSP65p
mHSP65p
mHSP65p
hHSP60p
mHSP65p
hHSP60p
mHSP65p
hHSP60p
mHSP65p
36-45
67-76
72-81
117-126
91-100
132-141
137-146
111-120
218-227
191-200
276-285
394-403
366-375
441-450
413-422
502-511
507-516
97-109
VVLEKKWGAP
SWGSPKVTKD
KVTKDGVTVA
TVLARSIAKE
TVLAQALVRE
SKGANPVEIR
PVEIRRGVML
PLGLKRGIEK
KFDRGYISPY
RFDKGYISGY
PGFGDRRKAM
LAKLSDGVAV
LAKLAGGVAV
GCALLRCIPA
GVTLLQAAPT
NAASIAGLFL
AGLFLTTEAV
ALVREGLRNVAAG
179-187
NTFGLQLEL
504-512
AASIAGLFL
mHSP60
hHSP60p
mHSP65p
mHSP60
mHSP65p
hHSP60
PgHSP60
hHSP60
PgHSP60
hHSP60
PgHSP60
hHSP60
PgHSP60
hHSP60p
PgHSP60p
hHSP60p
PgHSP60p
HpHSP60p
NP
40, 43-45, 386
40, 43-46
NP
178-181, 183
71-80
73-82
140-149
142-151
260-269
262-271
277-286
279-288
340-349
342-351
365-374
367-376
141-160
CIGSPSYNC
QGSPV
KGAPT
CSFHYQNRC
NTFGQ
VQDVANNTNE
VKEVASKTND
EEIAQVATIS
QKIEHVAKIS
LVLNRLKVGL
LVVNRLRGSL
PGFGDNRKNQ
PGFGDRRKAM
QIEKRIQEII
GIASRITQIK
NERLAKLSDG
QERLAKLAGG
EEITQVATISANSDHNIGKL
were cross-reactive in nature with a homology of 40-70%.
The presence of these “specific” epitopes may explain the
differences in epitope structure between hHSP60 and
mHSP65 observed in patients with cardiovascular disease.
Blood
Blood
Advanced
atherosclerotic
lesions and blood
Blood
High-titer sera from 10 subjects with ≥1:1280 anti-hHSP60
and anti-hHSP60 antibodies and sonographically proven
atherosclerosis. Anti-HSP60/65 antibodies from subjects
with atherosclerotic lesions react specifically with three
short, linear epitopes present in HSP60/65, which may be
involved as autoantigens in the pathogenesis of
atherosclerosis.
Sera from 5 subjects with ≥1:1280 anti-HSP antibodies and
sonographically proven atherosclerosis. Two
atherosclerosis-associated conformational HSP60 epitopes
were defined by the use of phage display and structural
alignment.
6 LL Pg+ patients and 6 HC. Blood from both patients and
HC. Six cross-reactive B-cell epitopes of PgHSP60 and
hHSP60 were defined in atherosclerosis patients with a
concomitant periodontal disease.
10
Blood from 250 CVD patients and 293 non-CVD patients.
IgG antibodies against this particular amino acid sequence
13
11
12
5
hHSP60
HSPs
PgHSP60
409-424
TSDVEVNEKKERVTEA
Cross-reactive T-cell and B-cell epitopes
Amino acid sequence
Specimen
Position
12-21
73-82
162-171
262-271
493-502
Blood
RDLLKKGVDA
VKEVASKTND
IAEAMRKVKK
LVVNRLRGSL
VIDPAKVTRV
Advanced
atherosclerotic
lesions and blood
of HpHSP60p and anti-hHSP60 predominantly appeared in
CVD patients compared to controls. Furthermore, neither
titer of HpHSP60p nor anti-hHSP60 antibodies was
correlated with the levels of high sensitive C-reactive
protein (hsCRP). Thus, IgG anti-HpHSP60p antibodies
cross-reacting with hHSP60 might be independent
diagnostic markers relevant to CVD.
Blood from 35 ACS patients (12 with UA and 23 with MI)
and 20 HC. Levels of specific serum antibodies against
hHSP60 were significantly elevated in ACS patients. One
immunodominant region was revealed corresponding to
the hHSP60(409-424) peptide. None of the seven hHSP60
sequences tested corresponded to the mHSP65 epitopes
(97-109, 179-187, and 504-512).
14
Outcome
Ref
6 LL Pg+ patients and 6 HC. Blood from both patients and
HC. Five immunodominant T-cell and B-cell epitopes of
PgHSP60 was defined in atherosclerosis patients with a
periodontal disease. This indicates that PgHSP60 might be
involved in the immunoregulatory process of
atherosclerosis.
12
hHSP60=human heat shock protein (hHSP) 60 (573 amino acid long), hHSP60p=hHSP60 peptide, mHSP65=Mycobacterium bovis heat shock protein (mHSP) 65
(540 amino acid long), mHSP65p=mHSP65 protein, CpHSP60=Chlamydia pneumonia HSP60 (544 amino acid long), PgHSP60=Porphyromonas gingivalis HSP60,
GroEL=GroELfrom Escherichia coli, HpHSP60=Helicobacter pylori HSP60, GroELp=GroEL peptide, LL=Late lesion, EL=Early lesion, Asym=Asymptomatic, Sym=
Symptomatic, AS=Atherosclerosis, CVD=cardiovascular disease, ACS=acute coronary syndrome, UA=Unstable angina, MI=myocardial infarction, NP=not performed,
HC=healthy control, IFN-IP10=Interferon-inducible protein 10, MCP-1=monocyte chemoattractant protein 1, MIP-1α=macrophage inflammatory protein
1α,*HC=healthy control with sonographically proven carotid atherosclerosis
References
1.
Benagiano, M. et al. Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques. J
Immunol 174, 6509-17 (2005).
2.
Almanzar, G. et al. Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions. J Autoimmun 39, 441-50
(2012).
3.
Choi, J., Lee, S.Y., Kim, K. & Choi, B.K. Identification of immunoreactive epitopes of the Porphyromonas gingivalis heat shock
protein in periodontitis and atherosclerosis. J Periodontal Res 46, 240-5 (2011).
6
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Rossmann, A. et al. T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor
repertoire. Exp Gerontol 43, 229-37 (2008).
Yamazaki, K. et al. T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with
atherosclerosis and periodontitis. Oral Microbiol Immunol 19, 160-7 (2004).
Ford, P. et al. Characterization of heat shock protein-specific T cells in atherosclerosis. Clin Diagn Lab Immunol 12, 259-67 (2005).
Curry, A.J., Portig, I., Goodall, J.C., Kirkpatrick, P.J. & Gaston, J.S. T lymphocyte lines isolated from atheromatous plaque contain
cells capable of responding to Chlamydia antigens. Clin Exp Immunol 121, 261-9 (2000).
Perschinka, H. et al. Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis.
Arterioscler Thromb Vasc Biol 23, 1060-5 (2003).
Uray, K., Hudecz, F., Fust, G. & Prohaszka, Z. Comparative analysis of linear antibody epitopes on human and mycobacterial 60kDa heat shock proteins using samples of healthy blood donors. Int Immunol 15, 1229-36 (2003).
Metzler, B. et al. Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis. Arterioscler Thromb
Vasc Biol 17, 536-41 (1997).
Perschinka, H. et al. Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display
and structural alignment. Atherosclerosis 194, 79-87 (2007).
Choi, J.I. et al. Epitope mapping of Porphyromonas gingivalis heat-shock protein and human heat-shock protein in human
atherosclerosis. J Dent Res 83, 936-40 (2004).
Okada, T. et al. Antibodies against heat shock protein 60 derived from Helicobacter pylori: diagnostic implications in
cardiovascular disease. J Autoimmun 29, 106-15 (2007).
Wysocki, J. et al. Human heat shock protein 60 (409-424) fragment is recognized by serum antibodies of patients with acute
coronary syndromes. Cardiovasc Pathol 11, 238-43 (2002).
7