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 sequence / TCR Vβ / Specimen / Outcome / Ref
hHSP60, hHSP60p / 1-15 / MLRLPTVFRQMRPVS / Vβ11, Vβ9, Vβ5.2 / Advanced atherosclerotic lesions and blood / 4LL of Cp- patients+blood and 4 LL of Cp+patients+blood. The submolecular specificity of HSP60-specific plaque-derived T-cells was analyzed and both the self and cross-reactive epitopes of that autoantigen were identified (see below). / 1
hHSP60, hHSP60p / 6-20 / TVFRQMRPVSRVLAP / Vβ14, Vβ8, Vβ14
hHSP60, hHSP60p / 11-25 / MRPVSRVLAPHLTRA / Vβ1, Vβ8
hHSP60, hHSP60p / 31-45 / KFGADARALMLQGVD / Vβ20
hHSP60, hHSP60p / 91-105 / KNIGAKLVQDVANNT / Vβ4
hHSP60, hHSP60p / 96-110 / KLVQDVANNTNEEAG / Vβ21.3
hHSP60, hHSP60p / 136-150 / NPVEIRRGVMLAVDA / Vβ5.2
hHSP60, hHSP60p / 141-155 / RRGVMLAVDAVIAEL / Vβ12
hHSP60, hHSP60p / 201-215 / VKDGKTLNDELEIIE / Vβ17
hHSP60, hHSP60p / 206-220 / TLNDELEIIEGMKFD / Vβ3.1
hHSP60, hHSP60p / 226-240 / PYFINTSKGQKCEFQ / Vβ4
hHSP60, hHSP60p / 261-275 / LEIANAHRKPLVIIA / Vβ5.1
hHSP60, hHSP60p / 321-335 / GGAVFGEEGLTLNLE / Vβ2, Vβ12
hHSP60, hHSP60p / 331-345 / TLNLEDVQPHDLGKV / Vβ18
hHSP60, hHSP60p / 466-480 / EIIKRTLKIPAMTIA / Vβ22
hHSP60, hHSP60p / 491-505 / VEKIMQSSSEVGYDA / Vβ11
hHSP60, hHSP60p / 506-520 / MAGDFVNMVEKGIID / Vβ7, Vβ6.7, Vβ16, Vβ11
hHSP60, hHSP60p / 246-260 / LSEKKISSIQSIVPA / NP / 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
hHSP60, hHSP60p / 266-280 / AHRKPLVIIAEDVDG / NP
hHSP60, hHSP60p / 281-295 / EALSTLVLNRLKVGL / NP
hHSP60, hHSP60p / 326-340 / GEEGLTLNLEDVQPH / NP
hHSP60, hHSP60p / 416-430 / EKKDRVTDALNATRA / NP
hHSP60, hHSP60p / 421-435 / VTDALNATRAAVEEG / NP
hHSP60, hHSP60p / 436-450 / IVLGGGCALLRCIPA / NP
hHSP60, hHSP60p / 471-485 / TLKIPAMTIAKNAGV / NP
hHSP60, hHSP60p / 476-490 / AMTIAKNAGVEGSLI / NP
hHSP60, hHSP60p / 551-565 / KEEKDPGMGAMGGMG / NP
Cross-reactive T-cell epitopes
HSPs / Position / Amino acid sequence / TCR Vβ / Specimen / Outcome / Ref
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 46-60
21-35 / LLADAVAVTMGPKGR
KTLAEAVKVTLGPKG / Vβ9 / Advanced atherosclerotic lesions and blood / 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 pneumoniaeanalog HSP60. / 1
hHSP60, hHSP60p
CpHSP60, CpHSP60p
mHSP65, mHSP65p
GroEL, GrpELp / 51-65
26-40
26-40
26-40 / VAVTMGPKGRTVIIE
AVKVTLGPKGRHVVI
KVTLGPKGRNVVLEK
AVKVTLGPKGRNVII / Vβ18
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 61-75
36-50 / TVIIEQSWGSPKVTK
RHVVIDKSFGSPQVT / Vβ5.1
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp / 76-90
51-65
51-65
51-65 / DGVTVAKSIDLKDKY
DGVSIAKEIELEDPY
KDGVTVAKEIELEDK
KDGVSVAKEIELEDK / Vβ17
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 151-165
126-140 / VIAELKKQSKPVTTP
VVVDELKKISKPVQH / Vβ14
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp / 166-180
141-155
141-155
141-155 / EEIAQVATISANGDK
QIAATAAISAGDQSI
HKEIAQVATISANND
SEEVAQVGTISANGD / Vβ11
hHSP60, hHSP60p
mHSP65, mHSP65p
CpHSP60, CpHSP60p
GroEL, GrpELp / 171-185
146-160
146-160
146-160 / VATISANGDKEIGNI
TAAISAGDQSIGDLI
QVATISANNDSEIGN
QVATISANGDKQVGL / Vβ12
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 191-205
166-180 / KKVGRKGVITVKDGK
MEKVGKNGSITVEEA / Vβ5.2
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 211-225
186-200 / LEIIEGMKFDRGYIS
VLDVVEGMNFNRGYL / Vβ4
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 241-255
216-230 / DAYVLLSEKKISSIQ
EDALILIYDKKISGI / Vβ13.1
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 291-305
266-280 / LKVGLQVVAVKAPGF
RLRAGFRVCAVKAPG / Vβ8
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 406-420
381-395 / VGGTSDVEVNEKKDR
VGAATEIEMKEKKDR / Vβ14
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 436-450
411-425 / IVLGGGCALLRCIPA
ILPGGGTALVRCIPT / Vβ9
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 441-455
416-430 / GCALLRCIPALDSLT
GTALVRCIPTLEAFL / Vβ1
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 446-460
421-435 / RCIPALDSLTPANED
RCIPTLEAFLPMLAN / Vβ13.2
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 511-525
486-500 / VNMVEKGIIDPTKVV
AYTDMIDAGILDPTK / Vβ22
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 521-535
496-510 / PTKVVRTALLDAAGV
LDPTKVTRSALESAA / Vβ9
hHSP60, hHSP60p
CpHSP60, CpHSP60p / 536-550
516-530 / ASLLTTAEVVVTEIP
LLTTEALIADIPEEK / Vβ11
PgHSP60/hHSP60
PgHSP60 / NP
NP / TVPGGGTTYIRAIAALEGLK
TLVVNRLRGSLKICAVKAPG / 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 cross-reactive peptide (TLVVNRLRGSLKICAVKAPG) from both Pg and hHSP60. The peptide for a specific T-cell line demonstrated the phenotype characteristic of helper T-cells (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. / 3
TCR Vβ without amino acid sequence
HSPs / Position / Amino acid sequence / TCR Vβ / Specimen / Outcome / Ref
hHSP60 / 1-573
(full-length) / NP / Asym: Vβ1, Vβ2, Vβ3, Vβ7, Vβ9, Vβ11, Bβ16, 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 oligoclonally-restricted 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
hHSP60 / 1-573
(full-length) / NP / Sym: Vβ3, Vβ6.2, Vβ7, Vβ9, Vβ11, Vβ13, Vβ18, Vβ20, Vβ21, Vβ24
hHSP60 / 1-573
(full-length) / NP / AS: Vβ2, Vβ3, Vβ4, Vβ5.1, Vβ6.1, Vβ6.2, Vβ8, Vβ11, Bβ12, Vβ14, Vβ17, Vβ18, Vβ21, Vβ24
hHSP60, PgHSP60 / NP / NP / Vβ2, Vβ5.2, Vβ6, Vβ8, Vβ9, Vβ12, Vβ13.1, Vβ16, Vβ18, Vβ19, Vβ20 / Advanced atherosclerotic lesions, gingival tissue, 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 HSP60-reactive T-cells were present in atherosclerotic lesions in some patients. / 5
hHSP60, GroEl, PgHSP60 / NP / NP / Vβ5.2-3, Vβ13.1/13.3 / Advanced atherosclerotic lesions and blood / 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 displayeda 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. / 6
hHSP60, CpHSP60 / NP / NP / Both CD4+ and CD8+ T-cells with a majority bearing the αβ TCR rather than γδ TCR. / Advanced atherosclerotic lesions and blood / 32 patients with LL and blood. Antigen responsiveness of T-cell lines showed that those derived using Chlamydiaorganisms were more likely to respond toChlamydia than those isolated using other stimuli.Chlamydia-specific T-cell 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. Thus, atherosclerotic plaque tissue contains a variety of memory T-cells, and amongst these are cells capable of recognizingChlamydia antigens. / 7
Cross-reactive B-cell epitopes
HSPs / Position / Amino acid sequence / Specimen / Outcome / Ref
hHSP60p
mHSP65p
CpHSP60p
GroELp / 86-98
61-73
61-73
61-73 / LKDKYKNIGAKLV
LEDPYEKIGAELV
LADKHENMGAQMV
LEDKFENMGAQMV / 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
hHSP60p
mHSP65p
CpHSP60p
GroELp / 116-128
91-103
91-103
91-103 / ATVLARSIAKEGF
ATVLAQALVREGL
ATVLAEAIYTEGL
ATVLAQAIITEGL
hHSP60p
mHSP65p
CpHSP60p
GroELp / 131-148
91-103
91-103
91-103 / ISKGANPVEIRRGVMLAV
VAAGANPLGLKRGIEKAV
VTAGANPMDLKRGIDKAV
VAAGMNPMDLKRGIDKAV
hHSP60p
mHSP65p
CpHSP60p
GroELp / 186-203
161-178
161-178
161-178 / ISDAMKKVGRKGVITVKD
IAEAMDKVGNEGVITVEE
IAEAMEKVGKNGSITVEE
IAEAMDKVGKEGVITVED
hHSP60p
mHSP65p
CpHSP60p
GroELp / 241-258
216-233
216-233
216-233 / DAYVLLSEKKISSIQSIV
DPYILLVSSKVSTVKDLL
DALVLIYDKKISGIKDFL
SPFILLADKKISNIREML
hHSP60p
mHSP65p
CpHSP60p
GroELp / 261-273
236-248
236-248
236-248 / LEIANAHRKPLVI
LEKVIGAAKPLLI
LQQVAESGRPLLI
LEAVAKAGKPLLI
hHSP60p
mHSP65p
CpHSP60p
GroELp / 461-478
436-453
436-453
436-453 / QKIGIEIIKRTLKIPAMT
EATGANIVKVALEAPLKQ
EQIGARIVLKALSAPLKQ
QNVGIKVALRAMEAPLRQ
hHSP60p
mHSP65p
CpHSP60p
GroELp / 516-528
491-503
491-503
491-503 / KGIIDPTKVVRTA
AGVADPVKVTRSA
AGILDPAKVTRSA
MGILDPTKVTRSA
hHSP60p
mHSP65p / 52-61
26-35 / AVTMGPKGRT
KVTLGPKGRN / 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 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. / 9
hHSP60p
mHSP65p / 57-66
31-40 / PKGRTVIIEQ
PKGRNVVLEK
hHSP60p
mHSP65p / 62-71
36-45 / VIIEQSWGSP
VVLEKKWGAP
hHSP60p / 67-76 / SWGSPKVTKD
hHSP60p / 72-81 / KVTKDGVTVA
hHSP60p
mHSP65p / 117-126
91-100 / TVLARSIAKE
TVLAQALVRE
hHSP60p / 132-141 / SKGANPVEIR
hHSP60p
mHSP65p / 137-146
111-120 / PVEIRRGVML
PLGLKRGIEK
hHSP60p
mHSP65p / 218-227
191-200 / KFDRGYISPY
RFDKGYISGY
mHSP65p / 276-285 / PGFGDRRKAM
hHSP60p
mHSP65p / 394-403
366-375 / LAKLSDGVAV
LAKLAGGVAV
hHSP60p
mHSP65p / 441-450
413-422 / GCALLRCIPA
GVTLLQAAPT
mHSP65p / 502-511 / NAASIAGLFL
mHSP65p / 507-516 / AGLFLTTEAV
hHSP60p
mHSP65p / 97-109 / ALVREGLRNVAAG / 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 asautoantigens in the pathogenesis of atherosclerosis. / 10
hHSP60p
mHSP65p / 179-187 / NTFGLQLEL
hHSP60p
mHSP65p / 504-512 / AASIAGLFL
mHSP60
hHSP60p
mHSP65p / NP
40, 43-45, 386
40, 43-46 / CIGSPSYNC
QGSPV
KGAPT / Blood / 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. / 11
mHSP60
mHSP65p / NP
178-181, 183 / CSFHYQNRC
NTFGQ
hHSP60
PgHSP60 / 71-80
73-82 / VQDVANNTNE
VKEVASKTND / Advanced atherosclerotic lesions and blood / 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. / 12
hHSP60
PgHSP60 / 140-149
142-151 / EEIAQVATIS
QKIEHVAKIS
hHSP60
PgHSP60 / 260-269
262-271 / LVLNRLKVGL
LVVNRLRGSL
hHSP60
PgHSP60 / 277-286
279-288 / PGFGDNRKNQ
PGFGDRRKAM
hHSP60p
PgHSP60p / 340-349
342-351 / QIEKRIQEII
GIASRITQIK
hHSP60p
PgHSP60p / 365-374
367-376 / NERLAKLSDG
QERLAKLAGG
HpHSP60p / 141-160 / EEITQVATISANSDHNIGKL / Blood / Blood from 250 CVD patients and 293 non-CVD patients. IgG antibodies against this particular amino acid sequence 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. / 13
hHSP60 / 409-424 / TSDVEVNEKKERVTEA / Blood / Blood from 35 ACS patients (12 with UA and 23 with MI) and20 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
Cross-reactive T-cell and B-cell epitopes
HSPs / Position / Amino acid sequence / Specimen / Outcome / Ref
PgHSP60 / 12-21
73-82
162-171
262-271
493-502 / RDLLKKGVDA
VKEVASKTND
IAEAMRKVKK
LVVNRLRGSL
VIDPAKVTRV / Advanced atherosclerotic lesions and blood / 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 bovisheat shock protein (mHSP) 65 (540 amino acid long), mHSP65p=mHSP65 protein, CpHSP60=Chlamydia pneumonia HSP60 (544 amino acid long), PgHSP60=Porphyromonasgingivalis HSP60, GroEL=GroELfromEscherichia 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

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2.Almanzar, G. et al. Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions. J Autoimmun39, 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 Res46, 240-5 (2011).

4.Rossmann, A. et al. T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire. Exp Gerontol43, 229-37 (2008).

5.Yamazaki, K. et al. T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis. Oral Microbiol Immunol19, 160-7 (2004).

6.Ford, P. et al. Characterization of heat shock protein-specific T cells in atherosclerosis. Clin Diagn Lab Immunol12, 259-67 (2005).

7.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 Immunol121, 261-9 (2000).

8.Perschinka, H. et al. Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis. Arterioscler Thromb Vasc Biol23, 1060-5 (2003).

9.Uray, K., Hudecz, F., Fust, G. & Prohaszka, Z. Comparative analysis of linear antibody epitopes on human and mycobacterial 60-kDa heat shock proteins using samples of healthy blood donors. Int Immunol15, 1229-36 (2003).

10.Metzler, B. et al. Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis. Arterioscler Thromb Vasc Biol17, 536-41 (1997).

11.Perschinka, H. et al. Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment. Atherosclerosis194, 79-87 (2007).

12.Choi, J.I. et al. Epitope mapping of Porphyromonas gingivalis heat-shock protein and human heat-shock protein in human atherosclerosis. J Dent Res83, 936-40 (2004).

13.Okada, T. et al. Antibodies against heat shock protein 60 derived from Helicobacter pylori: diagnostic implications in cardiovascular disease. J Autoimmun29, 106-15 (2007).

14.Wysocki, J. et al. Human heat shock protein 60 (409-424) fragment is recognized by serum antibodies of patients with acute coronary syndromes. Cardiovasc Pathol11, 238-43 (2002).

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