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Human 60-kDa Heat Shock Protein Is a Target Autoantigen of T Cells Derived from Atherosclerotic Plaques ¹

Marisa Benagiano^2,*, Mario M. D’Elios^2,*,, Amedeo Amedei^*,, Annalisa Azzurri^*, Ruurd van der Zee, Alessandra Ciervo, Gianni Rombolà, Sergio Romagnani^*,, Antonio Cassone and Gianfranco Del Prete^3,*,

^* Department of Internal Medicine, University of Florence, and Laboratory of Immunogenetics, Department of Biomedicine, Azienda Ospedaliero-Universitaria di Careggi, Florence, Italy; Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; and Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy

	Abstract

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Epidemiological studies suggest the potential importance of an inflammatory component in atherosclerosis and support the hypothesis that immune responses to Ags of pathogens cross-react with homologous host proteins due to molecular mimicry. Protein candidates involved may be the stress-induced proteins known as heat shock proteins (HSP). In this study, we report that atherosclerotic plaques harbor in vivo-activated CD4⁺ T cells that recognize the human 60-kDa HSP. Such in vivo-activated 60-kDa HSP-specific T cells are not detectable in the peripheral blood. In patients with positive serology and PCR for Chlamydia pneumoniae DNA, but not in patients negative for both, most of plaque-derived T cells specific for human 60-kDa HSP also recognized the C. pneumoniae 60-kDa HSP. We characterized the submolecular specificity of such 60-kDa HSP-specific plaque-derived T cells and identified both the self- and cross-reactive epitopes of that autoantigen. On challenge with human 60-kDa HSP, most of the plaque-derived T cells expressed Th type 1 functions, including cytotoxicity and help for monocyte tissue factor production. We suggest that arterial endothelial cells, undergoing classical atherosclerosis risk factors and conditioned by Th type 1 cytokines, express self 60-kDa HSP, which becomes target for both autoreactive T cells and cross-reactive T cells to microbial 60-kDa HSP via a mechanism of molecular mimicry. This hypothesis is in agreement with the notion that immunization with HSP exacerbates atherosclerosis, whereas immunosuppression and T cell depletion prevent the formation of arteriosclerotic lesions in experimental animals.

	Introduction

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Atherosclerosis is a multifactorial disease for which a number of different pathogenic mechanisms have been proposed. In the last two decades, attention has been given to the inflammatory processes associated with atherogenesis. In addition to classical risk factors for atherosclerosis, such as high levels of low-density lipoprotein (LDL) ⁴ cholesterol or oxidized LDL, free radicals, hyperglycemia, and genetic susceptibility to endothelial damage (1, 2), a pathogenic role for infections in atherosclerosis is suggested by the detection of pathogens (e.g., Chlamydia pneumoniae, CMV, or herpes viruses) in the arterial vessels and the association between atherosclerosis and increased Ab levels to these pathogens (3). Observations in humans and animals suggest that atherosclerotic plaques derive from specific cellular and molecular mechanisms that can be ascribed to an inflammatory disease of the arterial wall, the lesions of which consist of macrophages and T lymphocytes (4, 5, 6). Activated macrophages and T cells would be responsible for in situ production of enzymes, cytokines, and chemokines that further expand the process. If inflammation continues unabated, it results in increased numbers of plaque-infiltrating macrophages and T cells, which contribute to remodeling of the arterial wall (7). Within the T cell population of the plaque, most of the cells are activated CD4⁺ cells expressing HLA-DR and CD25 of the IL-2R (8). C. pneumoniae DNA and C. pneumoniae-specific T cell clones were found in the plaques of anti-C. pneumoniae seropositive patients. The specificity repertoire of such CD4⁺ T cells included the C. pneumoniae 60-kDa heat shock protein (CpHSP60), the 10-kDa HSP, the outer membrane protein 2, or undefined Ags of the C. pneumoniae elementary bodies. T cell clones recovered from C. pneumoniae DNA-negative plaques of anti-C. pneumoniae seronegative patients did not react to C. pneumoniae Ags (9).

In this study, we focused on the analysis of the T cell infiltrates of atherosclerotic plaques of anti-C. pneumoniae seronegative patients, making the hypothesis that human proteins expressed under stress conditions, such as the human 70-kDa HSP (hHSP70) or human 60-kDa HSP (hHSP60), might be autoantigens recognized by plaque-infiltrating T cells.

	Materials and Methods

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Patients

Carotid plaques were obtained by endoarterectomy from eight patients (five males and three females; mean age, 67 years; range, 62–73 years) with atherosclerotic arteriopathy. Four patients were seronegative for anti-C. pneumoniae Abs (Cp-neg), whereas the other four patients were seropositive for anti-C. pneumoniae Abs (Cp-pos), as shown by both commercial ELISA tests (Eurospital) and standard microimmunofluorescence assay (cutoff value, 32). Samples of PBMC were obtained from each patient. Their MHC haplotypes were as follows: HLA-A2, B7, B51, DRB1*14, and DRB1*16 in patient 1; HLA-A1, A2, B13, B18, DRB1*03, and DRB1*07 in patient 2; HLA-A24, B15, B40, DRB1*01, and DRB1*16 in patient 3; HLA-A1, A2, B18, DRB1*04, and DRB1*11 in patient 4; HLA-A24, B44.03, B58.01, DRB1*0701, and DRB1*16 in patient 5; HLA-A2, B15, B44, and DRB1*13 in patient 6; HLA-A1, B8, B27, DRB1*03, and DRB1*07 in patient 7; and HLA-A2, A24, B7, B51, DRB1*0701, and DRB1*11 in patient 8.

Detection of C. pneumoniae in atherosclerotic plaques

The presence of C. pneumoniae was investigated by nested PCR, as reported elsewhere (9, 10). Briefly, DNA was extracted from fragments of all the endoarterectomy specimens by QIAamp DNA kit (Qiagen). Nested PCR consisted of two rounds of amplification using two sets of primers, each in a 50-µl volume. On completion of primary PCR (37 cycles), 2 µl of the PCR product were added into fresh reaction mix containing the second set of primers and amplified for 25 cycles. The amplified DNA products were analyzed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and hybridized as reported previously (10). The nested PCR for C. pneumoniae included an outer primer pair (HL-1, HR-1) and an inner pair (HM-1, HR-2) that generated a product of 204 bp. The details of primers and probe are as follows: HL-1, -5'-GTTGTTCATGAAGGCCTACT-3' end; HR-1, -5'-TGCATAACCTACGGTGTGTT-3' end; HM-1, -5'-GTGTCATTCGCCAAGGTTAA-3' end; HR-2, -5'-ACCTGTCCAAGGTTCATCCT-3' end; and DNA probe, -5'-GTGTCATTCGCCAAGGTTAAAGTCTACGTT-3' end.

Generation of T cell clones from atherosclerotic plaques and peripheral blood

Fragments of atherosclerotic plaques and samples of PBMC were cultured for 7 days in RPMI 1640 medium supplemented with IL-2 to expand in vivo-activated T cells. Single T cell blasts were then cloned under limiting dilution (9, 11, 12, 13). Briefly, single T cell blasts were seeded in microwells (0.3 cells/well) in the presence of 2 x 10⁵ irradiated (5000 rad) allogeneic PBMC, PHA (0.5% vol/vol), and IL-2 (50 U/ml). At weekly intervals, irradiated allogeneic PBMC and IL-2 were added to each microculture to maintain the expansion of growing clones. Ag specificity of T cell clones was assessed by measuring [³H]thymidine uptake after 60 h of coculture with irradiated autologous PBMC in the presence of medium, recombinant hHSP70 (Sigma-Aldrich) (10 µg/ml), recombinant hHSP60 (Sigma-Aldrich) (10 µg/ml), or recombinant CpHSP60 (10 µg/ml), prepared as endotoxin-free material (14). No significant proliferation of T cell clones was found in response to irradiated autologous PBMC alone. The mitogenic index (MI) was calculated as the ratio between mean values of cpm obtained in stimulated cultures and those obtained in the presence of medium alone. MI >5 was considered positive.

T cell clones reactive to hHSP60 were also tested for proliferation in response to the recombinant HSP65 protein of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (MbHSP65) (Aalto Bio Reagents) and to the recombinant 60-kDa chaperonin GroEL of Escherichia coli (Sigma-Aldrich) (10 µg/ml).

All of the 26 plaque-derived T cell clones reactive to hHSP60 and the 18 clones reactive to both hHSP60 and CpHSP60 expressed a CD3⁺CD4⁺CD8^– phenotype and showed a single peak of fluorescence intensity. The repertoire of the TCR V chain of HSP60-specific T cell clones was analyzed with a panel of 22 mAbs specific to the following: V1, V2, V4, V7, V9, V11, V14, V16, V18, V20, V21.3, V22, and V23 (Beckman Coulter Immunotech); and V3.1, V5.1, V5.2, V5.3, V6.7, V8, V12, V13, and V17 (AMS Biotechnology); and isotype-matched nonspecific Ig were used as negative control. Data acquisition was performed in a FACSCalibur flow cytometer using the CellQuest software program (BD Biosciences). From each T cell clone, mRNA was extracted by mRNA direct isolation kit (Qiagen). For cDNA synthesis, the same amount of mRNA (50 ng) was used, and cDNA was synthesized by Moloney murine leukemia virus-reverse transcriptase (New England Biolabs) and oligo(dT) primers according to enzyme supplier’s protocol. cDNA mix of all samples was amplified under equal conditions by a 30-cycle PCR using a V TCR typing amplimer kit for V10, V15, and V19 (BD Clontech) according to the manufacturer’s instructions.

Submolecular specificity of plaque-derived T cell clones reactive to hHSP60 or to both hHSP60 and CpHSP60

To span the 573 aa sequence of hHSP60 and the 544 aa sequence of CpHSP60, 113 and 107 overlapping 15-mer peptides with a 10 (5 on each side) aa overlap, respectively, were prepared by automated, simultaneous multiple peptide synthesis, as described previously (15) Homologies between the two series of peptides were screened by using the basic local alignment search tool server of the National Center for Biotechnology Information.

Two series of 15-mer peptides corresponding to a number of sequences of the HSP65 protein of M. bovis BCG (MbHSP65) and of the 60-kDa chaperonin GroEL of E. coli (aa 21–35, 26–40, 31–45, 46–60, 51–65, 56–70, 121–136, 126–140, 131–145, 141–155, 146–160, 151–165, 161–175, 166–180, 171–185, 181–195, 186–200, 191–205, 211–225, 216–230, 221–235, 406–420, 411–425, 416–430, 421–435, and 426–440) were also prepared. Equal amounts of each component of the two series of overlapping peptides of hHSP60 and of CpHSP60 were pooled to have two series of 11 peptide pools. T cell blasts (4 x 10⁴) from each clone were cultured in triplicate for 3 days together with irradiated autologous mononuclear cells (1.5 x 10⁵) in the presence of medium, hHSP60 (10 µg/ml), CpHSP60 (10 µg/ml), or equal aliquots from each of the 22 pools in which each peptide component was present at a 10 µg/ml final concentration. After 60 h, [³H]TdR uptake was measured. T cell blasts of each clone were then retested for proliferation to the individual peptide components of the pool that had induced a MI >5.

MHC class II restriction of hHSP60 epitope recognition by plaque-derived T cell clones

The effect of anti-HLA-DR (clone G46–6) or anti-HLA-DQ (clone TU169; BD Biosciences Pharmingen) (5 µg/ml final concentration) mAbs or their isotype control (mouse IgG2a) on T cell clone proliferation induced by hHSP60 or CpHSP60 was assessed. The MHC class II restriction of the proliferative response of T cell clones to hHSP60 or CpHSP60 peptides was assessed by using irradiated allogeneic APC. To this end, PBMC from both patients and healthy donors sharing with patients one of the DRB1* alleles were stimulated with PHA followed by IL-2 to obtain polyclonal lines of activated T cells to be used as irradiated (3000 rad) allogeneic APC in coculture experiments with T cell clones in the presence of the HSP60 peptide to which they reacted.

Assessment of the cytokine profile of T cell clones

To assess the cytokine production of hHSP60-specific clones on Ag stimulation, 5 x 10⁵ T cell blasts of each clone were cocultured for 48 h in 0.5 ml of medium with 5 x 10⁵ irradiated autologous PBMC in the absence or presence of hHSP60 or CpHSP60 (10 µg/ml). At the end of culture period, duplicate samples of each supernatant were assayed for IFN-, TNF-, and IL-4 (BioSource International) (9, 13). T cell clones able to produce IFN-, but not IL-4, were categorized as Th1; clones able to produce IL-4, but not IFN-, were categorized as Th2; and clones producing both IFN- and IL-4 were categorized as Th0.

Perforin-mediated cytotoxicity and Fas-Fas ligand-mediated proapoptotic activity

Perforin-mediated cytolytic activity of T cell clones was assessed as reported previously (16). T cell blasts of hHSP60-specific clones were incubated at ratios of 10:1, 5:1, and 2.5:1 with ⁵¹Cr-labeled autologous EBV transformed B (EBV-B) cells preincubated with hHSP60 (10 µg/ml). After centrifugation, microplates were incubated for 8 h at 37°C, and 0.1 ml of supernatant was removed for measurement of ⁵¹Cr release, as reported previously (17). The ability of hHSP60-specific T cell clones to induce Fas-Fas ligand-mediated apoptosis was assessed using Fas⁺ Jurkat cells as target. T cell blasts from each clone were cocultured with ⁵¹Cr-labeled Jurkat cells at E:T ratios of 10:1, 5:1, and 2.5:1 for 18 h in the presence of PMA (10 ng/ml) and ionomycin (1 mmol/l), as reported previously (17, 18).

Assay for T cell clone helper function for monocyte tissue factor (TF) production

T cell blasts of hHSP60-specific clones (8 x 10⁵/ml) were cocultured for 16 h with autologous monocytes (4 x 10⁵/ml) in the presence of medium, hHSP70 (10 µg/ml), hHSP60 (10 µg/ml), or CpHSP60 (10 µg/ml). At the end of culture period, TF protein was measured by a specific ELISA (American Diagnostica) in duplicate samples of the supernatants obtained from cell suspensions after solubilization of membrane proteins with Triton X-100 and ultracentrifugation, as reported previously (19).

	Results

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Autoreactive CD4⁺ T cell clones specific for hHSP60 are present in atherosclerotic lesions

In vivo-activated T cells resident in the plaques or in the peripheral blood were expanded in vitro in IL-2-conditioned medium and then cloned by a procedure that has proved useful and accurate for studies of tissue-infiltrating T cells in various diseases (9, 11, 12, 13). A total number of 151 CD4⁺ and 23 CD8⁺ T cell clones were obtained from the plaques of the four Cp-neg patients, whereas 115 CD4⁺ and 21 CD8⁺ were the T cell clones derived from the plaques of the four Cp-pos patients. Nested PCR on endarterectomy specimens showed C. pneumoniae genomic material in each of the plaques obtained from the four Cp-pos patients but not in the plaques from the Cp-neg patients (data not shown).

For each patient, randomly selected CD4⁺ and CD8⁺ T cell clones derived from PBMC were matched to the corresponding plaque-derived T cell clones and assayed for proliferation in response to hHSP70, hHSP60, and CpHSP60. None of the CD8⁺ clones derived from either plaques or PBMC showed proliferation to those Ags. Likewise, none of the 266 CD4⁺ clones generated from the PBMC of either Cp-neg or Cp-pos patients showed significant proliferation to the Ags tested (Table I), although they proliferated in response to mitogen stimulation (data not shown). In contrast, a variable proportion between 11 and 35% of the CD4⁺ T cell clones generated from plaque-infiltrating T cells of either Cp-neg or Cp-pos patients proliferated significantly to hHSP60 (Table I) but not to hHSP70 Ag (MI <2). Under the same conditions, none of the 21 hHSP60-specific CD4⁺ clones from the plaques of the four Cp-neg patients proliferated significantly to CpHSP60. In contrast, in the series of the 23 hHSP60-specific CD4⁺ clones from the plaques of the four Cp-pos patients, 18 (78%) proliferated equally well to both hHSP60 and CpHSP60 (Table I). Evidence for clonality of the CD3⁺CD4⁺CD8^– T cell clones, which is critical for interpretation of data, was provided by the cytofluorimetric patterns of single TCR-V expression and the staining by only one of the TCR-V-chain-specific mAbs, with a single peak of fluorescence intensity (data not shown).

T cell blasts from each of the T cell clones reactive to hHSP60, but not to CpHSP60, were screened for proliferation in response to the 113 overlapping peptides for the hHSP60 (Table II). Each of these autoreactive T cell clones proliferated almost equally well to both the entire hHSP60 protein and to an epitope of such autoantigen. Interestingly, some hHSP60 epitopes, such as the 1–15, 6–20, and 506–520, were recognized by clones from different donors, despite their different MHC class II haplotypes or different TCR-V expression by T cell clones (Table II). Also, the five T cell clones recovered from atherosclerotic plaques of Cp-pos patients that proliferated to hHSP60, but not to CpHSP60, recognized the 1–15, 6–20, 11–25, 321–335, and 506–520 epitopes of hHSP60 (Table II). The lack of responsiveness to CpHSP60 of the 26 hHSP60-specific clones was confirmed by their inability to proliferate in response to the CpHSP60 peptide corresponding to the hHSP60 epitope to which they were reactive (data not shown), although a number of the hHSP60 epitopes recognized by this series of clones shared a few (up to 7) amino acids with the corresponding CpHSP60 peptides (Table II).

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Dose-response effect of graded Ag or peptide concentrations on the proliferative response to hHSP60 and CpHSP60 of plaque-derived T cell clones. T cell blasts from each clone were cocultured with autologous-irradiated APC in the presence of graded concentrations of hHSP60 (•), CpHSP60 (), or of the appropriate hHSP60 peptides (), or CpHSP60 peptides (). Results represent mean values of MIs measured in quadruplicate cultures of 8 representative of 26 clones reactive only to hHSP60 (upper and middle panels) and in 4 representative of 18 clones reactive to both hHSP60 and CpHSP60 (lower panels).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Proliferative response of hHSP60-specific T-clones to series of partially overlapping peptides of hHSP60, including the specific epitopes. T cell blasts from clones derived from the plaques of four patients sharing the DRB1*07 allele were cocultured with autologous-irradiated APC in the presence of series of 3 partially overlapping 15-mer peptides, including the most stimulatory one, and a couple of adjacent peptides. Results represent mean values of MIs measured in quadruplicate cultures of eight representative T cell clones.

All plaque-derived hHSP60-specific clones were assessed for their cytokine profile on Ag stimulation. In the series of hHSP60-specific clones not cross-reactive to CpHSP60, 22 (84.6%) secreted IFN- and TNF- but not IL-4 (Th1 profile), whereas in 4 clones, stimulation with hHSP60 resulted in the production of IL-4 as well (Th0 profile).

Likewise, in the series of 18 hHSP60/CpHSP60 cross-reactive clones, stimulation with either hHSP60 or CpHSP60 disclosed a Th1 profile in 15 clones (83.3%) and a Th0 profile in the other 3.

The cytolytic potential of hHSP60-specific autoreactive or cross-reactive T cell clones was assessed by using Ag-pulsed ⁵¹Cr-labeled autologous EBV-B cells as targets. At an E:T ratio of 10:1, 36 of the 37 (97%) Th1 and 5 of 7 (71%) Th0 clones lysed hHSP60-presenting autologous EBV-B cells (range of specific ⁵¹Cr release, 18–63%), whereas autologous EBV-B cells pulsed with hHSP70 (control) Ag and cocultured with the same clones were not lysed. The relative potency of Ag-induced cytotoxic activity of HSP60-specific T cell clones against autologous EBV-B cells pulsed with hHSP60 or CpHSP60 was assessed by comparison of levels of the specific ⁵¹Cr release at different E:T ratios (Fig. 3). Because activated effector T cells can also kill their targets by inducing apoptosis through Fas-Fas ligand interaction (17, 20), we evaluated the ability of activated hHSP60-specific clones to induce ⁵¹Cr release by Fas⁺ Jurkat cells undergoing apoptosis. On mitogen activation, 32 of 37 Th1 (86%) and 4 of 7 (57%) Th0 clones were able to induce apoptosis in target cells (range of specific ⁵¹Cr release, 21–59%).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Dose-response effect of graded E:T ratios on the cytotoxic activity of plaque-derived T cell clones specific for hHSP60 against Ag-pulsed autologous APC. To assess their perforin-mediated cytotoxicity, T cell clones reactive to hHSP60 (A) or to both hHSP60 and CpHSP60 (B) were cocultured at different E:T ratios with 51Cr-labeled autologous EBV-B cells pulsed with hHSP70 (10 µg/ml) () or hHSP60 (10 µg/ml) () or CpHSP60 (10 µg/ml) (), and 51Cr release was measured as index of Ag-induced specific target cell lysis. Results represent mean values of 51Cr release measured in triplicate cultures of eight representative T cell clones.

Because plaque rupture and thrombosis are notable complications of atherosclerosis, we asked whether stimulation with hHSP60 or CpHSP60 might enable plaque-infiltrating autoreactive or cross-reactive T cells to express helper function for TF production by monocytes. All clones were cocultured with autologous monocytes in the absence or presence of medium alone, hHSP70, hHSP60, or CpHSP60, and TF protein was measured. In the presence of medium alone or hHSP70, none of the 44 plaque-derived clones expressed helper function for monocyte TF production, ruling out the possibility that monocytes expressed hHSP60 suitable for T cell activation (Fig. 4). In contrast, apart from 2 Th0 clones (one autoreactive and one cross-reactive), in 42 (95%) hHSP60-specific clones, stimulation with hHSP60 resulted in the expression of substantial help for TF production by monocytes. Likewise, stimulation with CpHSP60 enabled 17 of 18 (94%) cross-reactive T cell clones to induce monocyte TF production, whereas it failed to elicit any helper function for TF production in the 25 autoreractive clones that proliferated in response to hHSP60 but not to CpHSP60 (Fig. 4).

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Monocyte TF production induced by plaque-derived T cell clones stimulated with hHSP60 or CpHSP60. T cell blasts from each clone were cocultured with autologous monocytes in the presence of hHSP70 (control Ag) or the specific Ag (hHSP60 or CpHSP60), and TF production was assessed by a specific ELISA.

	Discussion

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In this study, we demonstrate that atherosclerotic patients harbored in their carotid plaques in vivo-activated CD4⁺ T cells that reacted specifically to self HSP60. In addition, all four patients with positive serology and PCR detection of C. 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 C. pneumoniae analog HSP60. Blocking experiments with anti-DR and anti-DQ Abs and coculture of T cell clones with appropriate allogeneic APC showed that DR represents the MHC restriction element in the T cell response to either hHSP60 or CpHSP60. It is of note that, despite good viability and IL-2-induced growth, PBMC-derived T cell clones from all patients consistently failed to proliferate in response to hHSP60, CpHSP60, or hHSP70. In the absence of obvious explanations due to technical pitfalls, the reason for the inability to detect hHSP-specific T cells in the peripheral blood remains unclear. One possibility is that hHSP60-specific T cells are present in the peripheral blood in resting state, like most of circulating T cells, and are not suitable for in vitro activation and expansion induced by IL-2. Another possibility is that very low numbers, if any, of in vivo-activated hHSP60-specific T cells are present in the peripheral blood, whereas they concentrate into the lesions of arterial walls, where they find their specific Ag(s) and participate in the pathology of atherosclerosis by expressing their effector mechanisms. That such activated T cells infiltrating the atherosclerotic plaques actually participate in the disease pathogenesis is supported by the observation that the formation of arteriosclerotic lesions by immunization of rabbits with HSP65-containing material could be abolished by immunosuppression and T cell depletion with an anti-CD3 Ab plus prednisolone (27).

The presence in the plaques of Cp-positive patients of T cells reactive to CpHSP60 is in agreement with the observations by other laboratories (28, 29) and with our earlier demonstration in a similar series of patients of T cells reactive to C. pneumoniae Ags, such as sonicated elementary bodies, the 10 kDa HSP, the outer membrane protein 2, and the CpHSP60 (9). In the present study, however, the presence of plaque-derived T cell clones specific for C. pneumoniae Ags different from CpHSP60 was not investigated.

T cell recognition of either hHSP60 or CpHSP60 resulted in both proliferation and expression of functional properties by T cell clones, i.e., a predominant Th1 profile. In addition, on appropriate stimulation, the great majority of plaque-derived HSP60-specific clones induced both perforin-mediated cytolysis and Fas-Fas ligand-mediated apoptosis in target cells. Based on these findings, it is tempting to hypothesize that in the inflammatory setting of the atherosclerotic plaque in which HSP60-specific autoreactive or cross-reactive Th1 cells are activated, endothelial cells may acquire APC function for HSP60 and, together with professional APCs (30), can become targets of the cytotoxic and proapoptotic activity of HSP60-specific Th1 cells. The outcome of this process would be the expansion of the plaque and the formation of the necrotic cores characteristic of complicated and unstable atherosclerotic lesions. A linkage has been suggested between the degree of macrophage apoptosis and plaque rupture, to which apoptotic death of smooth muscle cells may also contribute (31, 32, 33). Moreover, it is reasonable to suspect that HSP60-activated Th1 cells and their cytokines can play a role in driving the up-regulation of TF production by monocytes within atherosclerotic plaques, thus contributing to the thrombogenicity of lesions (34). Indeed, the Th1 polarization of T cell responses and the poor production of Th2 cytokines occurring within the plaque may represent local risk factors of thrombosis (19), which associate with platelet adhesion to dysfunctional endothelium.

Our findings support the hypothesis that a crucial component of atherosclerosis is represented by Th1 cell-mediated immune responses to self and/or foreign Ags. More than 95% sequence homology exists between HSP60s from various bacteria, and even between bacterial and hHSP60 a 50–55% sequence homology exists, and in highly conserved regions it reaches >70% (35). The analysis of the submolecular specificity of T cell clones reactive only to hHSP60 and of clones reactive to both hHSP60 and CpHSP60 showed that the former recognized their epitope in portions of relatively poor or no homology between the two proteins, whereas the latter found their specific epitope in regions of high-sequence homology. Therefore, a number of hHSP60 T cell epitopes are "private," such as the 1–15 and 6–20 N-terminal or the 506–520 C-terminal sequences recognized by different clones of different Cp-negative patients and by a few clones of Cp-positive patients, whereas other hHSP60 epitopes are similar to, and cross-reactive with, T cell epitopes of CpHSP60. T cell clones specific for private or cross-reactive epitopes of hHSP60 do, however, express similar predominant Th1 profile and may contribute equally to inflammation in the setting of atherosclerosis.

HSP60 are released in soluble form from the surface of stressed or damaged cells and can be found in the supernatant of cell cultures in vitro or in the serum in vivo (36, 37). Whether soluble hHSP60 released in the inflammatory setting of the plaque may undergo biochemical changes, becoming the target of bona fide autoimmunity (3), remains to be investigated. Likewise, it remains to be established whether autoimmunity to hHSP60 results from a breakdown of tolerance associated with chronic inflammation and aging, and what is the role of the molecular mimicry between hHSP60 and the HSP60 of pathogens, such as C. pneumoniae, detected in the atherosclerotic lesions. The mycobacterial homologue MbHSP65 and the E. coli homologue GroEL (38) might be other candidates for cross-reactive recognition by hHSP60-reactive T cell clones in atherosclerotic plaques. In this study, however, none of the 26 T cell clones reactive to hHSP60, but not to CpHSP60, also reacted to MbHSP65 or to GroEL. Only 11 and 9 of the 18 T cell clones reactive to both hHSP60 and CpHSP60 showed poor or negligible response to MbHSP65 or GroEL, respectively, on the basis of dose-response curves. Neither MbHSP65 nor GroEL peptides that partially overlapped specific hHSP60/CpHSP60 epitopes were able to induce T cell clone proliferation at intermediate or low peptide concentrations, arguing against the hypothesis that MbHSP65 or GroEL might represent major targets of plaque-infiltrating T cells in our patients.

Data obtained in this study support the hypothesis that two major mechanisms, partially overlapping and not mutually exclusive, may be responsible for the T cell-mediated immunopathology of atherosclerosis. The first one would imply that arterial endothelial cells, undergoing the effects of classical stress factors associated with atherosclerosis and conditioned by cytokines produced by plaque-infiltrating Th1 cells, express self 60-kDa HSP. Such an autoantigen would be presented by professional APC and endothelial cells, becoming a target of autoreactive T cells specific for private epitopes of hHSP60. T cell-mediated cytotoxic and apoptotic killing of stressed endothelial cells expressing self HSP60 may activate a vicious circle of self maintenance of such Th1-mediated autoimmune mechanism of endothelial damage. The second mechanism, active in patients who failed to clear C. pneumoniae, would be mediated by plaque-infiltrating Th1 cells specific for C. pneumoniae Ags, among which CpHSP60-specific T cells that cross-recognize shared epitopes of the hHSP60 via a mechanism of molecular mimicry. The availability of self HSP60 expressed by the vascular endothelium would contribute to a second branch of the vicious circle of self maintenance of the immune response that would be mediated by CpHSP60-specific Th1 cells that cross-react to self HSP60. These possibilities are consistent with the results obtained in several experimental animal models. In these models, a central role for T cells specific for HSP60s has indeed been established. Immunization with HSP65 of LDL receptor-deficient (LDL-R^–/–) mice induced specific T cell reactivity against HSP65 as well as mammalian HSP60, and transfer into nonimmunized mice of lymphocytes or purified IgG of immunized animals enhanced the size of vascular lesions (39). In contrast, nasal or oral immunization with HSP65 of hypercholesterolemic ApoE^–/– and LDL-R^–/– mice resulted in reduced T cell reactivity to HSP and attenuated atherosclerosis (40, 41). In contrast, arthritogenic and arthritis-preventing HSP60 epitopes have been identified in the model of adjuvant arthritis in rats (42, 43) and human rheumatoid arthritis (44). The identification in present study of a number of atherosclerosis-associated T cell epitopes of HSP60 may be of importance for designing strategies on preventive or therapeutic approaches aimed to inhibit the immune and autoimmune pathogenic mechanisms of atherosclerosis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Italian Ministry of University and Research, the Ministery of Health, the University of Florence, the Associazione Italiana per la Ricerca sul Cancro, and the Istituto Superiore di Sanità.

² M.B. and M.M.D. contributed equally to this publication.

³ Address correspondence and reprint requests to Dr. Gianfranco Del Prete, Department of Internal Medicine, Viale Morgagni 85-50134 Florence, Italy. E-mail address: gdelprete{at}unifi.it

⁴ Abbreviations used in this paper: LDL, low-density lipoprotein; HSP, heat shock protein; CpHSP60, C. pneumoniae 60-kDa HSP; hHSP70, human 70-kDa HSP; hHSP60, human 60-kDa HSP; Cp-neg, seronegative for anti-C. pneumoniae Ab; Cp-pos, seropositive for anti-C. pneumoniae Ab; MI, mitogenic index; BCG, bacillus Calmette-Guérin; MbHSP65, Mycobacterium bovis BCG 65-kDa HSP; EBV-B, EBV transformed B; TF, tissue factor.

Received for publication September 17, 2004. Accepted for publication February 16, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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